2024 Harvard Residents Course Full Program by Harvard Ophthalmology

2024 HARVARD RESIDENTS’ COURSE

The Immune System and the Eye Mass Eye and Ear I Boston, MA I January 26, 2024

Course Directors:

Isaac D. Bleicher, MD and Joan W. Miller, MD

2024 Harvard Visiting Professor: Nisha Acharya, MD, MS Professor, Department of Ophthalmology University of California, San Francisco

2024 HARVARD RESIDENTS’ COURSE

The Immune System and the Eye Friday, January 26, 2024 - Mass Eye and Ear, 8th Floor Boardroom BREAKFAST - 10:00 - 11:00 AM (7th Floor, Glass Room) 11:00 11:05 AM

Welcome and Overview - Isaac D. Bleicher, MD

11:05 11:30 AM

Paraneoplastic Syndromes in the Orbit: A Comprehensive Review - Amee D. Azad, MD and Suzanne K. Freitag, MD

11:30 11:55 AM

Non-Infectious Inflammatory Complications of Lamellar Refractive Corneal Surgery: A Review of Diffuse Lamellar Keratitis and Transient Light Sensitivity Syndrome Enchi K. Chang, MD and Roberto Pineda II, MD

11:55 12:20 PM

The Effect of Diabetes Mellitus on the Corneal Microenvironment and the Impact of the Donor Diabetic State on Corneal Transplant Outcomes Yilin Feng, MD and Reza Dana, MD, MPH, MSC

12:20 12:45 PM

Periocular Manifestations and Management of IgG4 Disease Tatiana R. Rosenblatt, MD and Michael K. Yoon, MD

LUNCH - 12:45 - 1:30 PM (7th Floor, Glass Room) 1:30 1:55 PM

Inflammatory Reaction to Intravitreal Injections Saghar Bagheri, MD, PhD and John B. Miller, MD

1:55 2:20 PM

Pediatric Non-infectious Intermediate, Posterior, and Panuveitis - Da Meng, MD, PhD and Nimesh A. Patel, MD

2:20 2:45 PM

The Role of Inflammation in the Pathogeneisis of Age-Related Macular Degeneration James M. Harris, MD, PhD and David Wu, MD, PhD

2:45 3:10 PM

The Immunopathogenesis of Adamantiades-Behçet’s Uveitis: Advances in Understanding of Genetics and Molecular Mechanisms Lindsay K. Kozek, MD, PhD and Demetrios Vavvas, MD, PhD

3:10 3:35 PM

Immune Recovery Uveitis: A Comprehensive Review Melissa Yuan, MD and Lucy Young, MD, PhD

PM BREAK & CLASS PHOTOS - 3:35 - 4:00 PM (8th Floor, Boardroom) 4:00 4:05 PM

Introduction of Dr. Nisha Acharya Lucia Sobrin, MD, MPH

4:05 5:05 PM

2024 Harvard Visiting Professor in Ophthalmology Lecture Lessons Learned from Uveitis Cases Nisha Acharya, MD, MS

5:05 5:10 PM

Conclusion - Isaac D. Bleicher, MD

RECEPTION & DINNER - 6:00 - 9:00 PM (by invitation only)

Nisha Acharya, MD, MS

Professor, Department of Ophthalmology University of California, San Francisco Dr. Acharya is the Elizabeth C. Proctor Distinguished Professor of Ophthalmology, Epidemiology & Biostatistics at the University of California, San Francisco and Director of the Uveitis and Ocular Inflammatory Disease Service at the F.I. Proctor Foundation. She is also the Associate Director and Vice Chair for Faculty Development and Mentorship in the Department of Ophthalmology at the Proctor Foundation. Dr. Acharya obtained her undergraduate and master’s degrees at Stanford University, followed by her doctorate at UCSF. She was an intern at Brigham and Women’s Hospital and completed residency at Harvard Medical School at Mass Eye and Ear. She returned to UCSF for fellowship. Dr. Acharya is a clinicianscientist with a clinical focus on uveitis and ocular inflammatory diseases. She has an NIH-funded research program that focuses on the design and implementation of international clinical trials and data analytics research on ocular inflammatory diseases. She is the immediate past-president of the American Uveitis Society and has received a Senior Achievement Award from the American Academy of Ophthalmology in recognition for her service to the profession. She has also been recognized for her mentorship of medical students with a Dean’s Mentor Award.

2024 HARVARD RESIDENTS’ COURSE

RESIDENT PAPERS

2024 Harvard Ophthalmology Residents’ Course

Paraneoplastic Syndromes in the Orbit: A Comprehensive Review Amee D. Azad, MD, MS and Suzanne K. Freitag, MD

Introduction Paraneoplastic syndromes are a group of rare, malignancy-related disorders triggered by the immune system in response to an underlying cancer. These clinical syndromes can affect any part of the body, including the eye and the orbit.1 The mechanism of action is thought be autoantibodies, cytokines, hormones, or peptides that are directed against organ systems leading to inflammation and tissue destruction.2 A recent literature review suggests that paraneoplastic syndromes occur in up to 7 to 15% of cancer patients.3 Symptoms can present before or at the same time as the cancer diagnosis and diagnosis of a paraneoplastic syndrome is defined, in part, when there is resolution of symptoms after treatment of the underlying malignancy is initiated.1,2 Therefore, it is vital for clinicians to be familiar with these syndromes to expedite diagnosis and treatment of occult malignancies in order to optimize clinical outcomes and patient quality of life. There are immunologic and non-immunologic mechanisms by which paraneoplastic syndromes cause disease. Tumor cells are inherently immunogenic and activate both cell-mediated and humoral immune systems. When activated, cytotoxic T cells attack not only the tumor cell antigens, but similar antigens in normal cells leading to disease in a process known as molecular mimicry. Non-immunologic responses can occur in the setting of aberrant production or secretion of hormones or cytokines leading to metabolic or hormonal derangements.2 Eye manifestations of paraneoplastic syndromes have limited characterization in the literature partially because they are extremely rare, with estimates suggesting an incidence of 0.01% in cancer patients.4 Ocular manifestations can occur on the anterior or posterior segment of the eye and can masquerade as more common disorders like chalazion, conjunctivitis, corneal ulcer, scleritis, episcleritis or uveitis.2,5 Some of them can lead to serious complications, including corneal perforation, panuveitis, occlusive vasculitis with retinal hemorrhages or optic nerve edema that can cause vision loss.5,6 In the orbit, paraneoplastic syndromes can manifest as eyelid skin changes, edema, orbital inflammation causing proptosis or diplopia, extraocular muscle enlargement, myositis, or perineuritis.7 (TABLE 1) Early recognition is especially valuable in these cases because intervention may significantly affect survival as well as treat the paraneoplastic syndrome.8 The purpose of this comprehensive review is to characterize orbital paraneoplastic syndromes described in the literature.

Azad, Amee

TABLE 1: Paraneoplastic Orbltal Syndromes and their Manifestations

Orbital Paraneoplastic Syndrome

Orbital and Periocular Manifestations

Associated Malignancies

Paraneoplastic pemphigus

Ulcerations on eyelids, Marginal erosions on the inferior eyelids, Symblepharon

B-cell lymphoproliferative malignancy, reticulum cell sarcoma, Castleman’s tumor, bronchogenic squamous cell carcinoma, gastric adenocarcinoma, endometrial adenocarcinoma, follicular dendritic cell sarcoma, and thymoma

Sweet syndrome

Periorbital or orbital inflammation, painful papules, plaques, or nodules, eruptions, eyelid inflammation with vesicular lesions, dacryoadenitis

Hematologic malignancy (most commonly acute myeloid leukemia), breast cancer, upper genitourinary cancer, and upper gastrointestinal cancer

Acanthosis nigricans

Papillary changes on lid margins and tarsal conjunctiva, madarosis, and eyebrows, epiphora from occlusion of the lacrimal ducts

Gastrointestinal adenocarcinoma, hepatocellular carcinoma, bronchial cancer, and breast cancer

Adult orbital xanthogranulomatous disease

Yellowish plaques and subcutaneous lesions (adult-onset xanthogranuloma) / violaceous, red-orange, or yellow lesions with subcutaneous central ulcerations and atrophy (necrobiotic xanthogranuloma)

Hematologic malignancy (chronic lymphocytic leukemia, multiple myeloma, Hodgkin, and non-Hodgkin lymphoma

Dermatomyositis

Heliotrope rash (reddish-purple eruption on the upper eyelids accompanied by eyelid edema), ophthalmoplegia, diplopia

Ovarian cancer, stomach cancer, colorectal cancer, and non-Hodgkin lymphoma

Orbital inflammation

Unilateral or bilateral periocular edema, erythema, pain, diplopia, chemosis, conjunctival injection

Renal cell carcinoma, diffuse large B-cell lymphoma, colorectal cancer, and multiple myeloma

Extraocular muscle enlargement

Ophthalmoplegia, diplopia, afferent dysfunction

Bladder cancer, breast cancer, lung cancer, bronchial carcinoma, and gastrointestinal cancer

Myositis

Acute pain, diplopia, eyelid erythema, conjunctival injection, and chemosis

non-Hodgkin lymphoma, paraganglioma, retinoblastoma, breast cancer, seminoma, lung cancer, stomach cancer, B-cell lymphoma, multiple myeloma, and renal cell carcinoma

Lambert-Eaton myasthenic syndrome

Ptosis, diplopia

Small-cell lung cancer, neuroblastoma, prostate cancer, thymoma, and lymphoproliferative disorders

Myasthenia gravis

Ptosis, diplopia

Thymoma, renal cell carcinoma, marginal zone lymphoma, mantle cell lymphoma

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Dermatologic The first record of a paraneoplastic dermatosis was by Hebra in 1868 when describing hyperpigmentation as an indicator of a visceral cancer.9 Since then, numerous dermatologic paraneoplastic syndromes have been reported, although periorbital and orbital manifestations of dermal paraneoplastic syndromes are not well documented in the literature.5 Behind endocrine and neurologic systems, the skin is the most common target of paraneoplastic syndromes.10 More than a quarter century ago, Curth proposed criteria to define the temporal relationship between malignancy and dermatologic conditions. They include 1. The malignancy and the skin disease are of concurrent onset; 2. The malignancy and the skin disease run a parallel course. Successful treatment of the tumor leads to regression of the skin disease, and recurrence of the tumor leads to a return of cutaneous signs and symptoms; 3. The relation between the skin disease and the malignancy is uniform. A specific tumor cell type or site is associated with a characteristic cutaneous eruption; 4. Based on sound case–control studies, a statistically significant association exists between the malignancy and a specific cutaneous disease, and/or 5. A genetic association exists between the malignancy and a specific cutaneous disease. The following paraneoplastic dermatoses discussed in this comprehensive review adhere to Curth’s postulates. Paraneoplastic pemphigus is most commonly associated with B-cell lymphoproliferative malignancies, but there have also been reports of presentations with reticulum cell sarcoma, Castleman’s tumor, bronchogenic squamous cell carcinoma, gastric adenocarcinoma, endometrial adenocarcinoma, follicular dendritic cell sarcoma, and thymoma.5,11,12 In 1990, Anhalt et al. published clinical criteria for paraneoplastic pemphigoid: 1. painful mucosal erosions and a polymorphous skin eruption, with papular lesions progressing to bullae evolving into erosive lesions affecting the trunk, extremities, and palms and soles, associated with confirmed or occult neoplasm; 2. cutaneous histopathologic changes – intraepidermal acantholysis, keratinocyte necrosis, and vacuolar-interface dermatitis; 3. deposition of IgG and complement in the epidermal intercellular spaces, as well as granular-linear complement deposition along the epidermal basement membrane; 4. auto-antibodies that bind the cell surface of epidermal skin cells and mucosal cells in a pemphigoid pattern; 5. a complex of four proteins (190, 210, 230, 250 kD) immunoprecipitated from keratinocytes by these auto-antibodies.13 Periocular manifestations of this disease include pseudomembranous conjunctivitis, symblepharon formation similar to ocular cicatricial pemphigoid, and ulcerative eyelid margin erosion.14 Paraneoplastic pemphigoid has also progressed to corneal melting and perforation in some cases requiring amniotic membrane grafting or penetrating keratoplasty.5,15 Sweet syndrome is also known as acute febrile neutrophilic dermatosis. Twenty percent of cases present as paraneoplastic syndromes of which 85% are associated with hematologic malignancies.5 The remaining 15% have been associated with breast, genitourinary, and gastrointestinal cancers.16 Diseasedefining major clinical criteria include acute onset of erythematous, painful papules, plaques, or nodules on the face, extremities, and upper trunk accompanied by fever. As these findings can be non-specific and seen with other dermatoses, the histopathologic findings of dense neutrophilic infiltrates without infectious or vasculitis aid in diagnosis.12 Ocular findings in Sweet syndrome includes subconjunctival hemorrhages, scleritis, episcleritis, keratitis, iritis, panuveitis, limbal nodules, and even occlusive vasculitis in the retina or papillitis.17–19 In the periorbita, Sweet syndrome can present with erythema and inflammation on the eyelids, papules, plaques, or vesicular lesions on the eyelids or dacryoadenitis.19 Necrotizing Sweet syndrome is an exceedingly rare subtype of Sweet syndrome that can masquerade as necrotizing fasciitis as both typically present with fevers and leukocytosis with neutrophilia. There are limited case reports documenting necrotizing Sweet syndrome, but two in particular involve the periorbital region.20–22 Sweet syndrome is a also a potentially vision-threatening process, therefore early recognition and aggressive treatment with topical or systemic corticosteroids and addressing the underlying malignancy is essential.5,19 Acanthosis nigricans typically presents with hyperpigmented lesions over the neck, groin, axilla, or mucosal surfaces. It is the best described cutaneous marker of internal malignancy and is most commonly associated with gastrointestinal adenocarcincoma.12,23 However, it has also been associated with hepatic, bronchial and breast cancers.5 The ocular manifestations include conjunctival injection with papillary changes, loss of eyelashes and eyebrows, as well as ectropion and epiphora from canalicular obstruction due to papillomatous lesions.24,25

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Adult orbital xanthogranulomatous diseases are rare, infiltrative disease of the eyelids and orbits and include adult-onset asthma with periocular xanthogranuloma, Erdheim-Chester disease, necrobiotic xanthogranuloma and adult-onset xanthogranuloma. These diseases are non-Langerhans histiocytoses with local and/or systemic involvement. Histologically, they share patterns characterized by infiltration of foamy histiocytes, Touton-type giant cells, lymphocytes, and fibrosis, but differ in their systemic associations and overall prognosis.26 A rare presentation of adult-onset xanthogranulomas as a paraneoplastic dermatosis associated with a hematologic malignancy has been previously reported in 43year-old man.27 He initially presented multiple yellowish plaques and subcutaneous lesions in the periorbital region and was diagnosed with chronic lymphocytic leukemia shortly thereafter.28 Necrobiotic xanthogranuloma is commonly associated with benign monoclonal gammopathy in 80% of cases, but has also been seen with multiple myeloma, Hodgkin and non-Hodgkin lymphoma and chronic lymphocytic leukemia.26,29 Necrobiotic xanthogranuloma is characterized by degeneration of collagen which leads to subcutaneous central ulcerations and atrophy that can be violaceous, red-orange, or yellow in color and involves the periorbital region in >80% of cases.30 This rare paraneoplastic dermatosis can also be vision-threatening and require multi-modal immunosuppression.28,31 Dermatomyositis is another well-recognized paraneoplastic syndrome and is classified as an idiopathic inflammatory myopathy. Dermatomyositis is a manifestation of a paraneoplastic syndrome in 15-30% of cases.32 The most common cancers associated with dermatomyositis includes ovarian, stomach, colorectal, and non-Hodgkin lymphoma.33 This disease typically presents with progressive, symmetric, proximal muscle weakness, and classically a heliotrope rash and Gottron papules. The heliotrope rash in dermatomyositis is described as a reddish-purple eruption on the upper eyelids accompanied by eyelid swelling. Ophthalmoplegia and diplopia may also occur.34 One prior case report described an initial presentation of bilateral orbital and facial edema with a heliotrope rash and proximal muscle weakness consistent with dermatomyositis associated with an ovarian teratoma.35

Orbital Inflammation Orbital inflammation in paraneoplastic syndromes can present with non-specific findings that may appear as infectious etiologies. This distinction between infectious and noninfectious orbital inflammation is vital and a thorough infectious work-up is essential prior to initiation of immunosuppressive therapy. Many cases in the literature describe paraneoplastic orbital inflammation as periorbital edema in conjunction with findings such as injection, chemosis, proptosis, and/or extraocular muscle limitations that resolve with treatment of the primary malignancy. One example in the literature is of a 60-year-old man who presented initially with unilateral periocular edema, eye pain, diplopia, joint pain, and lower extremity rash.36 Computed tomography of the orbit revealed soft tissue thickening, fat stranding, and enhancement in the right preseptal region. Initial infectious work-up was negative and pathology from a skin biopsy revealed leukocytoclastic vasculitis involving small vessels consistent with paraneoplastic vasculitis. Additional imaging revealed a small hypointense enhancing nodule in the interpolar region of the left kidney representing a renal cell carcinoma (RCC) confirmed with renal biopsy.36 While 20% of patients diagnosed with RCC present with paraneoplastic syndromes37, this case represents an uncommon etiology of paraneoplastic orbital inflammation and vasculitis. Another case report of a 58-year-old man with bilateral proptosis and significant orbital inflammation.38 He underwent an orbitotomy and incisional biopsy which revealed polyclonal lymphoplasmacytic infiltration and non-necrotizing lymphoid small vessel vasculopathy. His orbital symptoms resolved with corticosteroid. Three months later, he began having abdominal pain and was found to have a retroperitoneal mass with a lymph node biopsy confirming diffuse large B-cell lymphoma. He underwent chemotherapy, but passed 6 months later due to metastatic disease.38 IgG4-related disease has also been discussed in the literature as a possible paraneoplastic syndrome, although there is conflicting evidence on its relationship with higher risk of malignancy.39,40 One case report documents a 74-year-old man who presented with unilateral proptosis responsive to steroids,

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initially diagnosed as idiopathic orbital inflammatory syndrome.41 Computed tomography (CT) scans revealed inflammatory infiltration of the right orbital fat, the lateral and medial rectus muscles. Several months later, he presented with rectal bleeding and was found to have a tubulovillous adenoma requiring right hemicolectomy followed shortly thereafter by a development of a large abdominal mass infiltrating the mesentery, transverse colon, and mesenteric vein. Biopsies of this lesion revealed IgG4-positive plasma cell infiltration with normal serum IgG4 levels and in this context, the orbital adnexa involvement was thought to be due to IgG4-related disease. The patient’s proptosis regressed with steroids and rituximab, but he ultimately succumbed to stage IV colon adenocarcinoma.41 Another case by Teo et al. was of a 73-year-old man with bilateral relapsing and remitting orbital inflammation associated with polychondritis that did not respond to antibiotics.42 Computer tomography (CT) of the orbits showed a soft tissue mass along the roof of the orbit, which was biopsied, revealing acute on chronic inflammation. The inflammation resolved with oral corticosteroids, but one year later he was diagnosed with multiple myeloma.42 To summarize, orbital inflammation is a highly non-specific symptom and can rarely be a symptom of a paraneoplastic syndrome. While infection must always be ruled out first, paraneoplastic syndromes should be maintained on the differential for orbital inflammation especially given the risk of morbidity and mortality as evidenced by these cases.

Extraocular Muscle Enlargement In 1974, Swash reported an external ophthalmoplegia in a patient with bladder carcinoma. The patient developed necrotizing myopathy affecting the skeletal and extraocular muscles.43 Biopsy showed endomysial edema and hyaline degeneration of the muscle fibers. Since that early case, extraocular muscle enlargement has been described several times as a paraneoplastic orbital syndrome defined by resolution of extraocular muscle enlargement following treatment of the underlying malignancy and without neoplastic cells on histopathology.7 Upon review of the literature, paraneoplastic extraocular muscle enlargement is most commonly associated with solid tumors, specifically breast cancer and lung cancer but has been observed with other malignancies. Kumar and Diamond described a 71-year-old woman with a remote history of breast cancer who presented with diplopia and ophthalmoplegia.44 CT imaging demonstrated diffuse extraocular muscle enlargement. Initially, the patient was evaluated for thyroid eye disease due to these CT findings and thyroid function tests showed a low thyroid stimulating hormone in the setting of thyroxine replacement for hypothyroidism. However, she had negative antithyroid autoantibodies and further work up indicated an elevated carcinoembryonic antigen. She underwent a whole-body positron emission tomography which showed avid liver, adrenal, and skeletal lesions consistent with breast cancer recurrence. With treatment of her underlying malignancy and oral corticosteroid, her orbital symptoms normalized at nine months.44 Another case in the literature is of a 65-year-old man who presented with double vision and proptosis. CT imaging demonstrated bilateral superior rectus enlargement and he was found to have a breast mass on physical exam. Biopsy of the breast mass showed a ductal intermediate grade carcinoma in situ. After undergoing a right radical mastectomy, his proptosis and double vision resolved with improvement of superior rectus enlargement on follow-up magnetic resonance imaging.45 Similar findings were reported in a case series on extraocular muscle enlargement with 3 out of 4 patients diagnosed with paraneoplastic extraocular muscle enlargement in association with breast cancer and one with thyroid papillary carcinoma.7 Extraocular muscle enlargement has also been found as orbital paraneoplastic syndromes associated with lung cancer. One case report describes a 51-year-old woman with large cell bronchial carcinoma and bilateral proptosis with imaging findings consistent with thyroid eye disease, but without thyroid function abnormalities or positive thyroid autoantibodies. Following tumor resection, the proptosis resolved in 10 days.46 Similar presentations have been described with paraneoplastic extraocular muscle enlargement in the setting of pleomorphic carcinoma of the lung,47 as well as in a patient with eyelid swelling and afferent dysfunction secondary to compressive optic neuropathy with lung cancer.48 McKittrick-Wheelock syndrome is a rare condition of extreme electrolyte and fluid depletion caused by large distal colorectal tumors, most commonly benign villous adenomas. Another example of

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paraneoplastic extraocular muscle enlargement is demonstrated by a case of a 52-year-old female who presented with diarrhea, electrolyte derangement, and acute kidney injury with unilateral upper eyelid retraction.49 The patient had initially borderline low thyroid stimulating hormone that normalized on followup testing and equivocal anti-thyroid autoantibodies. Magnetic resonance imaging showed proptosis with bilaterally increased thickness of inferior and medial rectus muscles. She was provisionally diagnosed with euthyroid thyroid eye disease, but subsequently had four hospital admissions due to recurring diarrhea and electrolyte derangements. At the time of her fourth presentation, she had a new symptom of rectal bleeding which led to diagnosis of a large tubovillous adenoma. The electrolyte abnormalities, diarrhea, and eyelid retraction improved with resection of the tumor and follow-up imaging displayed complete resolution of rectus muscle enlargement.49 In this case, the authors speculated that, as seen in the mechanism of autoimmune thyroid eye disease, cytokines and hormones release from neoplastic cells caused similar orbital changes via action on orbital fibroblasts.49 To distinguish paraneoplastic extraocular muscle enlargement from metastatic extraocular muscle enlargement, a few of these cases demonstrated no avidity of the extraocular muscles on full body positron emission tomography-computed tomography with clinical and radiologic improvement following treatment of the underlying malignancy.7 Another important takeaway from these cases is that some of these examples of extraocular muscle enlargement preceded the cancer diagnoses by 4-10 years.7 Clinical findings of paraneoplastic syndromes may precede radiologically detectable solid tumors50, therefore patients with non-thyroid disease related extraocular muscle enlargement should be monitored at regular intervals and repeat imaging should be considered to evaluate for an underlying systemic malignancy.

Orbital Myositis Acute myositis has previously been reported as a cause for non-thyroid related extraocular muscle enlargement in 43% of cases, but a distinction is drawn between the two in this comprehensive review.51 This section focuses on cases with a predominantly inflammatory presentation relative to extraocular muscle enlargement. All types of orbital myositis typically present as acute, painful diplopia and can be associated with eyelid erythema, conjunctival injection, chemosis and respond well to systemic immunosuppression.52 Harris et al. published the first report of an orbital myositis paraneoplastic syndrome in a patient with non-Hodgkin lymphoma in 1994 which was associated with other neurologic symptoms.53 An extraocular muscle biopsy in this patient showed granulomatous inflammation and destruction of myofibers.53 Similar cases of extraocular muscle inflammation have been described in a patient with metastatic paraganglioma54 and in four cases of retinoblastoma associated with muscle inflammation but without evidence of cancer cells invading the muscle.55 The electron microscopical analysis in this study of patients with retinoblastoma showed muscle fibers with atrophy, fiber and capillary necrosis, and neutrophils around capillaries consistent with an autoimmune process or neurogenic atrophy.55 Another example of a histopathologic confirmation of paraneoplastic orbital myositis was seen in a case of a 66-year-old woman with a history of breast cancer who presented with headache, blurry vision, and diplopia. She was noted to have extraocular muscle limitations in adduction, abduction, and supraduction in the left eye and ipsilateral enlargement of all extraocular muscles and enhancement of the lacrimal gland and retroorbital fat.56 Biopsy of the lacrimal gland demonstrated evidence of breast cancer metastasis, but biopsy of the extraocular muscle revealed no tumor cells.56 A recent review on orbital myositis described a small number of cases of paraneoplastic syndromes.52 Two cases were secondary to breast cancer, two to seminomas, and the remaining were in the setting of lung carcinoma, stomach cancer, and high-grade B-cell lymphoma. In most cases, the myositis resolved or improved with removal of the primary lesion.52 Similar, more recent, cases of paraneoplastic orbital myositis have been reported with multiple myeloma57 and renal cell carcinoma.58 As the cases of paraneoplastic orbital myositis accumulate in the literature, they serve as a reminder to keep the differential diagnosis broad when approaching patients with symptoms of myositis. Thyroid eye disease remains the most common cause of extraocular muscle inflammation and should be evaluated for in unilateral or bilateral cases.52,58 A thorough history should always be taken to evaluate for drugrelated etiologies for orbital myositis, such as recent treatment with immune checkpoint inhibitors, alemtuzumab, statins, bisphosphonates, interferon alfa, or influenza vaccinations.52 Finally, proceeding

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with a comprehensive blood work-up and diagnostic protocols for a primary tumor may be indicated once more common etiologies for orbital myositis have been ruled out.

Neurologic The spectrum of paraneoplastic neurologic syndromes can affect any part of the nervous system and can manifest in the orbit as an isolated presentation or involve multiple levels of the nervous system.1 Paraneoplastic neurologic syndromes in the orbit typically cause dysfunction at the neuromuscular junction. Lambert-Eaton myasthenic syndrome (LEMS) affects about 3% of patients with small-cell lung cancer.59 To review, LEMS has lower prevalence and incidence and a higher male predilection relative to myasthenia gravis.60 In patients with paraneoplastic LEMS, there is reduced acetylcholine release from the presynaptic nerve due to antibodies directed against voltage-gated calcium channels (VGCC) produced in response to tumor antigens.61 Therefore, the anti-surface anti-VGCCA antibodies are highly specific to LEMS and detectable in 85-90% of cases.62 Seventy percent of patients with LEMS have cranial nerve involvement, and ptosis and diplopia are the most common of these cranial nerve manifestations.63 Other common symptoms of LEMS include proximal muscle weakness, dysarthria, reduced tendon reflexes, and autonomic dysfunction. Paraneoplastic LEMS has also been described in neuroblastoma in children, prostate cancer, thymoma, and lymphoproliferative disorders.64–67 Myasthenia gravis has also been previously described as a paraneoplastic syndrome and affects 15% of patients with thymoma.68 This disease is caused by antibodies directed at the postsynaptic nicotinic acetylcholine receptors in the neuromuscular junction.69 The core manifestation is fatigable muscle weakness with isolated ocular symptoms of diplopia and ptosis can seen in up to 51% of patients in a recent population-level study.70 One case of paraneoplastic myasthenia gravis in the literature presented with ptosis, ophthalmoplegia, along with systemic myopathy whose symptoms ultimately led to detection of a primary renal cell carcinoma.71,72 Other cases have been described in association with orbital marginal zone lymphoma and mantle cell lymphoma.73,74

Conclusions Orbital paraneoplastic disorders can be severe, often disabling, and in the presence of aggressive malignancy, sometimes terminal. As evidenced by this comprehensive review, these rare conditions can be caused by a wide range of solid tumors or hematologic malignancies and present with heterogenous symptoms that may masquerade as other processes. Moreover, systemic corticosteroids administered empirically can diminish the auto-inflammatory sequelae of these paraneoplastic syndromes, leading to temporary resolution of symptoms, inevitable recurrences, and progressive growth of the yet undetected primary malignancy. Since patients with paraneoplastic orbital syndromes can develop severe ophthalmoplegia, debilitating diplopia, or possible optic neuropathy due to compression of the optic nerve from extraocular muscle enlargement, it is vitally important to be familiar with these paraneoplastic syndromes and to thoroughly investigate patients for underlying malignancies when the presentation demands it.48,58 Otherwise, vision-threatening and life-threatening complications may arise.

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Zdebik A, Lantzsch H, Buhles N, Zdebik N. Ocular manifestations of dermal paraneoplastic syndromes. Postepy Dermatol Alergol. 2020;37(3):313-318. doi:10.5114/ada.2020.96167

Przeździecka-Dołyk J, Brzecka A, Ejma M, et al. Ocular Paraneoplastic Syndromes. Biomedicines. 2020;8(11):490. doi:10.3390/biomedicines8110490

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Wick MR, Patterson JW. Cutaneous paraneoplastic syndromes. Semin Diagn Pathol. 2019;36(4):211-228. doi:10.1053/j.semdp.2019.01.001

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Thiers BH, Sahn RE, Callen JP. Cutaneous manifestations of internal malignancy. CA Cancer J Clin. 2009;59(2):7398. doi:10.3322/caac.20005

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Anhalt GJ, Kim SC, Stanley JR, et al. Paraneoplastic pemphigus. An autoimmune mucocutaneous disease associated with neoplasia. N Engl J Med. 1990;323(25):1729-1735. doi:10.1056/NEJM199012203232503

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Piscopo R, Romano M, Maria AD, Vinciguerra R, Vinciguerra P. Ocular Onset of Paraneoplastic Pemphigus Presenting as Hyperemic Conjunctivitis and Massive Bilateral Eyelid Ulceration: A Case Report and Literature Review. Ocul Immunol Inflamm. 2018;26(2):265-268. doi:10.1080/09273948.2016.1203958

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Beele H, Claerhout I, Kestelyn P, Dierckxens L, Naeyaert JM, De Laey JJ. Bilateral corneal melting in a patient with paraneoplastic pemphigus. Dermatol Basel Switz. 2001;202(2):147-150. doi:10.1159/000051622

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Bilgin AB, Tavas P, Turkoglu EB, Ilhan HD, Toru S, Apaydin KC. An uncommon ocular manifestation of Sweet syndrome: peripheral ulcerative keratitis and nodular scleritis. Arq Bras Oftalmol. 2015;78(1):53-55. doi:10.5935/0004-2749.20150015

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Michel G, Lhermitte B, Cribier B, Speeg-Schatz C, Bourcier T. Sweet syndrome presenting as resistant conjunctivitis. Cornea. 2008;27(10):1189-1190. doi:10.1097/ICO.0b013e31817f8147

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Gottlieb CC, Mishra A, Belliveau D, Green P, Heathcote JG. Ocular involvement in acute febrile neutrophilic dermatosis (Sweet syndrome): new cases and review of the literature. Surv Ophthalmol. 2008;53(3):219-226. doi:10.1016/j.survophthal.2008.02.006

20.

Kroshinsky D, Alloo A, Rothschild B, et al. Necrotizing Sweet syndrome: A new variant of neutrophilic dermatosis mimicking necrotizing fasciitis. J Am Acad Dermatol. 2012;67(5):945-954. doi:10.1016/j.jaad.2012.02.024

21.

Watson SL, Kuo A, Kishi SH, Fat MN, Boxrud CA. Periorbital Necrotizing Sweet’s Syndrome: A Case Report. Ophthal Plast Reconstr Surg. Published online July 21, 2023. doi:10.1097/IOP.0000000000002463

22.

Keen JA, Fisher MD, Yu CY, Swick BL, Shriver EM. Elevated Intraocular Pressure in Periorbital Sweet’s Syndrome. Ophthal Plast Reconstr Surg. 2023;39(4):e115-e117. doi:10.1097/IOP.0000000000002373

23.

Anderson, Hudson-Peacock, Muller. Malignant acanthosis nigricans: potential role of chemotherapy. Br J Dermatol. 1999;141(4):714-716. doi:10.1046/j.1365-2133.1999.03116.x

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Tabandeh H, Gopal S, Teimory M, et al. Conjunctival involvement in malignancy-associated acanthosis nigricans. Eye Lond Engl. 1993;7 ( Pt 5):648-651. doi:10.1038/eye.1993.148

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25.

Groos EB, Mannis MJ, Brumley TB, Huntley AC. Eyelid involvement in acanthosis nigricans. Am J Ophthalmol. 1993;115(1):42-45. doi:10.1016/s0002-9394(14)73522-6

26.

Kerstetter J, Wang J. Adult Orbital Xanthogranulomatous Disease: A Review with Emphasis on Etiology, Systemic Associations, Diagnostic Tools, and Treatment. Dermatol Clin. 2015;33(3):457-463. doi:10.1016/j.det.2015.03.010

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Biswas A, Hamid B, Coupland SE, Franks A, Leonard N. Multiple periocular adult onset xanthogranulomas in a patient with chronic lymphocytic leukaemia. Eur J Dermatol EJD. 2010;20(2):211-213. doi:10.1684/ejd.2010.0858

28.

Omarjee L, Janin A, Etienne G, et al. Necrobiotic xanthogranuloma: a paraneoplastic skin lesion of haematological malignancies? Eur J Dermatol EJD. 2018;28(3):384-386. doi:10.1684/ejd.2018.3256

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Baykal C, Polat Ekinci A, Yazganoglu KD, Buyukbabani N. The clinical spectrum of xanthomatous lesions of the eyelids. Int J Dermatol. 2017;56(10):981-992. doi:10.1111/ijd.13637

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Balagula Y, Straus DJ, Pulitzer MP, Lacouture ME. Necrobiotic xanthogranuloma associated with immunoglobulin m paraproteinemia in a patient with Waldenström macroglobulinemia. J Clin Oncol Off J Am Soc Clin Oncol. 2011;29(11):e305-307. doi:10.1200/JCO.2010.32.4921

31.

Reddy VC, Salomão DR, Garrity JA, Baratz KH, Patel SV. Periorbital and Ocular Necrobiotic Xanthogranuloma Leading to Perforation. Arch Ophthalmol. 2010;128(11):1493-1494. doi:10.1001/archophthalmol.2010.254

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Luu X, Leonard S, Joseph KA. Dermatomyositis presenting as a paraneoplastic syndrome with resolution of symptoms following surgical management of underlying breast malignancy. J Surg Case Rep. 2015;2015(7):rjv075. doi:10.1093/jscr/rjv075

33.

Hill CL, Zhang Y, Sigurgeirsson B, et al. Frequency of specific cancer types in dermatomyositis and polymyositis: a population-based study. Lancet Lond Engl. 2001;357(9250):96-100. doi:10.1016/S0140-6736(00)03540-6

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Dourmishev L, Dourmishev A. Dermatomyositis:Advances in Recognition, Understanding and Management.; 2009. doi:10.1007/978-3-540-79313-7

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Yu X, Qin D, Ma D, Yao Q. Adult dermatomyositis associated with benign ovarian teratoma: A case report. Oncol Lett. 2016;11(4):2611-2614. doi:10.3892/ol.2016.4251

36.

Burgess L, Keenan M, Zhou AL, et al. Stereotactic Radiotherapy in the Treatment of Paraneoplastic Vasculitis in Oligometastatic Renal Cell Carcinoma. Curr Oncol Tor Ont. 2021;28(3):1744-1750. doi:10.3390/curroncol28030162

37.

Kim HL, Belldegrun AS, Freitas DG, et al. Paraneoplastic signs and symptoms of renal cell carcinoma: implications for prognosis. J Urol. 2003;170(5):1742-1746. doi:10.1097/01.ju.0000092764.81308.6a

38.

Diniz SB, Abalo-Lojo JM, Chahud F, Ugradar S, Cruz AAV. Systemic Diffuse Large B-Cell Lymphoma Presenting as Bilateral Orbital Vasculopathy. Ophthal Plast Reconstr Surg. 2019;35(1):e6-e8. doi:10.1097/IOP.0000000000001274

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Yamamoto M, Takahashi H, Tabeya T, et al. Risk of malignancies in IgG4-related disease. Mod Rheumatol Jpn Rheum Assoc. 2011;22:414-418. doi:10.1007/s10165-011-0520-x

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Yu T, Wu Y, Liu J, Zhuang Y, Jin X, Wang L. The risk of malignancy in patients with IgG4-related disease: a systematic review and meta-analysis. Arthritis Res Ther. 2022;24(1):14. doi:10.1186/s13075-021-02652-2

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Lemaitre S, Esquerda GM, Guardiola AC, Agustin JT, Sanda N, González-Candial M. Colon cancer and IgG4-related disease with orbital inflammation and bilateral optic perineuritis: A case report. Medicine (Baltimore). 2018;97(39):e12197. doi:10.1097/MD.0000000000012197

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Teo L, Choo CT. Orbital inflammatory disease in relapsing polychondritis. Orbit Amst Neth. 2014;33(4):298-301. doi:10.3109/01676830.2014.902479

43.

Swash M. Acute Fatal Carcinomatous Neuromyopathy. Arch Neurol. 1974;30(4):324-326. doi:10.1001/archneur.1974.00490340052012

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Kumar S, Diamond T. Paraneoplastic syndrome - a rare but treatable cause of non-thyroid-related extraocular muscle enlargement. Orbit Amst Neth. 2019;38(6):468-473. doi:10.1080/01676830.2018.1550790

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Mehta P, Chickadasarahally S, Hedley N, Ahluwalia H. Extraocular muscle enlargement as a paraneoplastic effect of breast carcinoma in a male patient. Ophthal Plast Reconstr Surg. 2011;27(6):e146-147. doi:10.1097/IOP.0b013e3182078e31

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Diacon AH, Schuurmans MM, Colesky FJ, Bolliger CT. Paraneoplastic bilateral proptosis in a case of non-small cell lung cancer. Chest. 2003;123(2):627-629. doi:10.1378/chest.123.2.627

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Kuzunishi Y, Tsuzuku A, Asano F, et al. Pleomorphic Carcinoma with Exophthalmos and a Subsequent Diagnosis of Paraneoplastic Syndrome. Intern Med Tokyo Jpn. 2021;60(4):605-609. doi:10.2169/internalmedicine.5286-20

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Yoshida M, Suda K, Oishi A, et al. Bilateral Orbital Inflammation as a Manifestation of Paraneoplastic Syndrome. Case Rep Ophthalmol. 2022;13(2):534-541. doi:10.1159/000525632

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Ahmad W, Hartley M, Singh S, et al. McKittrick-Wheelock syndrome presenting with presumed paraneoplastic syndrome extra-ocular muscle enlargement masquerading as thyroid eye disease. Endocrinol Diabetes Metab Case Rep. 2023;2023(2):22-0386. doi:10.1530/EDM-22-0386

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50.

Hart IK, Maddison P, Newsom-Davis J, Vincent A, Mills KR. Phenotypic variants of autoimmune peripheral nerve hyperexcitability. Brain J Neurol. 2002;125(Pt 8):1887-1895. doi:10.1093/brain/awf178

51.

Lacey B, Chang W, Rootman J. Nonthyroid causes of extraocular muscle disease. Surv Ophthalmol. 1999;44(3):187213. doi:10.1016/s0039-6257(99)00101-0

52.

McNab AA. Orbital Myositis: A Comprehensive Review and Reclassification. Ophthal Plast Reconstr Surg. 2020;36(2):109-117. doi:10.1097/IOP.0000000000001429

53.

Harris GJ, Murphy ML, Schmidt EW, Hanson GA, Dotson RM. Orbital myositis as a paraneoplastic syndrome. Arch Ophthalmol Chic Ill 1960. 1994;112(3):380-386. doi:10.1001/archopht.1994.01090150110032

54.

Spraul CW, Lang GE, Lang GK. [Orbital myopathy in metastatic malignant paraganglioma: a paraneoplastic syndrome?]. Klin Monatsbl Augenheilkd. 1996;209(2-3):153-157. doi:10.1055/s-2008-1035296

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Finol HJ, Márquez A, Navas E, de Navas NR. Extraocular muscle ultrastructural pathology in the paraneoplastic phenomenon associated with retinoblastoma. J Exp Clin Cancer Res CR. 2001;20(2):281-285.

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Kiziltunc P, Atilla H, Akbostanci C, Isikay C, Ataoglu O. Lacrimal gland metastasis and paraneoplastic orbital myositis due to breast carcinoma. Int J Ophthalmic Pathol. 2016;5(1).

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Hunt SV, Garrott HM, Williams ME, Ford RL. Bilateral Paraneoplastic Orbital Myositis Associated With Malignant Melanoma and Multiple Myeloma. Ophthal Plast Reconstr Surg. 2022;38(3):e72-e75. doi:10.1097/IOP.0000000000002105

58.

Herranz-Cabarcos A, Alcubierre R, Van der Veen RLP. Paraneoplastic orbital myositis as a first manifestation of renal cell carcinoma. Orbit. 2023;0(0):1-5. doi:10.1080/01676830.2023.2264916

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Sculier JP, Feld R, Evans WK, et al. Neurologic disorders in patients with small cell lung cancer. Cancer. 1987;60(9):2275-2283. doi:10.1002/1097-0142(19871101)60:9<2275::aid-cncr2820600929>3.0.co;2-3

60.

Cetin H, Vincent A. Pathogenic Mechanisms and Clinical Correlations in Autoimmune Myasthenic Syndromes. Semin Neurol. 2018;38(3):344-354. doi:10.1055/s-0038-1660500

61.

Fukunaga H, Engel AG, Osame M, Lambert EH. Paucity and disorganization of presynaptic membrane active zones in the lambert-eaton myasthenic syndrome. Muscle Nerve. 1982;5(9):686-697. doi:10.1002/mus.880050905

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Titulaer MJ, Lang B, Verschuuren JJ. Lambert–Eaton myasthenic syndrome: from clinical characteristics to therapeutic strategies. Lancet Neurol. 2011;10(12):1098-1107. doi:10.1016/S1474-4422(11)70245-9

63.

Bussat A, Langner-Lemercier S, Salmon A, Mouriaux F. Paraneoplastic syndromes in ophthalmology. J Fr Ophtalmol. 2018;41(5):e181-e185. doi:10.1016/j.jfo.2018.03.002

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de Buys Roessingh AS, Loriot MH, Wiesenauer C, Lallier M. Lambert-Eaton myasthenic syndrome revealing an abdominal neuroblastoma. J Pediatr Surg. 2009;44(8):E5-7. doi:10.1016/j.jpedsurg.2009.04.023

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Morimoto M, Osaki T, Nagara Y, Kodate M, Motomura M, Murai H. Thymoma with Lambert-Eaton myasthenic syndrome. Ann Thorac Surg. 2010;89(6):2001-2003. doi:10.1016/j.athoracsur.2009.11.041

66.

Fernandez-Torron R, Arcocha J, López-Picazo JM, et al. Isolated dysphagia due to paraneoplastic myasthenic syndrome with anti-P/Q-type voltage-gated calcium-channel and anti-acetylcholine receptor antibodies. Neuromuscul Disord NMD. 2011;21(2):126-128. doi:10.1016/j.nmd.2010.10.003

67.

Titulaer MJ, Verschuuren JJGM. Lambert-Eaton myasthenic syndrome: tumor versus nontumor forms. Ann N Y Acad Sci. 2008;1132:129-134. doi:10.1196/annals.1405.030

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69.

Dresser L, Wlodarski R, Rezania K, Soliven B. Myasthenia Gravis: Epidemiology, Pathophysiology and Clinical Manifestations. J Clin Med. 2021;10(11):2235. doi:10.3390/jcm10112235

70.

Hendricks TM, Bhatti MT, Hodge DO, Chen JJ. Incidence, Epidemiology, and Transformation of Ocular Myasthenia Gravis: A Population-Based Study. Am J Ophthalmol. 2019;205:99-105. doi:10.1016/j.ajo.2019.04.017

71.

Kurli M, Finger PT. The kidney, cancer, and the eye: current concepts. Surv Ophthalmol. 2005;50(6):507-518. doi:10.1016/j.survophthal.2005.08.003

72.

Torgerson EL, Khalili R, Dobkin BH, Reiter RE. Myasthenia gravis as a paraneoplastic syndrome associated with renal cell carcinoma. J Urol. 1999;162(1):154. doi:10.1097/00005392-199907000-00037

73.

Feng S, Dodge RE, Francis CE. Myasthenia Gravis Associated With Orbital Marginal Zone Lymphoma. J NeuroOphthalmol Off J North Am Neuro-Ophthalmol Soc. 2019;39(2):242-243. doi:10.1097/WNO.0000000000000744

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Karlin J, Peck T, Prenshaw K, Portell CA, Kirzhner M. Orbital mantle cell lymphoma presenting as myasthenia gravis. Orbit Amst Neth. 2017;36(6):365-369. doi:10.1080/01676830.2017.1337202

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Non-Infectious Inflammatory Complications of Lamellar Refractive Corneal Surgery: A Review of Diffuse Lamellar Keratitis, Central Toxic Keratopathy, and Transient Light Sensitivity Syndrome Enchi K. Chang, MD and Roberto Pineda II, MD

Introduction Corneal-based laser vision correction through lamellar refractive corneal surgery has increased in popularity with the advent of newer, safer technologies and patient preference for corrective lens independence. These surgeries alter the curvature of the central cornea with tissue ablation to change its refractive power, allowing patients to achieve good visual acuity without additional correction. An estimated 20-25 million surgeries have been performed in the last 25 years, with over 750,000 surgeries performed annually for the last 10 years.1 Currently, the most common procedures include laser-assisted in situ keratomileusis (LASIK), photorefractive keratectomy (PRK), and small incision lenticule extraction (SMILE), with LASIK and SMILE comprising the lamellar refractive surgeries. In LASIK, a femtosecond laser or microtome is used to create a hinged corneal flap, and the underlying corneal stroma is then ablated with an excimer laser. In SMILE, a corneal lenticule is created by a femtosecond laser, which is then dissected and extracted in its entirety through a small incision. Although rare, there is a risk of infectious, inflammatory, and toxic complications from lamellar refractive corneal surgery. Postoperative complications at the flap-interface junction are found in less than 0.8% and 3% of LASIK and SMILE procedures, respectively.2,3 Here, we provide an overview of three rare non-infectious, inflammatory complications of lamellar refractive corneal surgery: diffuse lamellar keratitis, central toxic keratopathy, and transient light sensitivity syndrome.

Diffuse Lamellar Keratitis Background Diffuse lamellar keratitis (DLK) was first described by Smith and Maloney in 1998 as diffuse, multifocal infiltrates confined to the flap interface that develop soon after either myopic keratomileusis or LASIK.4 Since then, DLK has also been described in patients undergoing other lamellar corneal surgeries, such as SMILE and Descemet stripping automated endothelial keratoplasty.5–7 The incidence of DLK has been reported to be 0.4-7.7% in LASIK patients, compared to 0.45-1.6% in SMILE patients.5,6,8–11 The inflammatory infiltrates in DLK manifest clinically as grainy corneal opacifications that have earned the nickname “Sands of the Sahara” syndrome.

Diagnosis DLK typically presents within several days after lamellar corneal surgery. However, depending on the inciting cause for DLK, cases have been described up to 18 years after LASIK. The diagnosis is made through careful slit lamp examination of the lamellar interface and must be distinguished from infectious keratitis. A 4-stage classification system based on the location and severity of the keratitis was described by Lineberger et al. in 2000 and is currently the most widely-used classification system.12 Stage 1 is defined by white granular cells in the periphery of the lamellar flap outside the visual axis. Stage 2 is defined by central migration of the white granular cells from Stage 1 into the the visual axis. In Stage 3, there is aggregation of dense, white, clumped cells in the central visual axis with peripheral clearing. This is typically associated with subjective hazy vision and visual acuity decline of 1-2 Snellen lines. Finally, Stage 4 is defined by severe lamellar keratitis with stromal melt, permanent scarring, and significant visual decrease.12 A modification to this classification system was proposed by Johnson et al. in 2001 to include classification as sporadic (A) or cluster (B) cases for Stages 1 and 2 (ie. IA, IB, IIA, IIB), as sporadic cases are less problematic than a cluster or outbreak of cases at a specific location.13 The

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stages are summarized in TABLE 1. The differentiation between sporadic or cluster is informative as to the cause for DLK, as clusters of cases may be associated with iatrogenic causes. TABLE 1: Four-stage classification system for diffuse lamellar keratitis (adapted from Linebarger et al. 200012) Grade 1A

Description - White granular cells in the periphery of the lamellar flap, outside the visual axis - Sporadic cases (1A) - Cluster of cases (1B)

- White granular cells in the center of the flap, involving the visual axis, in the flap periphery, or in both - Sporadic cases (2A) - Cluster of cases (2B)

- Aggregation of more dense, white, and clumped cells in the central visual axis, with relative clearing in the periphery - Visual acuity decline of 1-2 Snellen lines

- Increased density and permanent scarring, stromal melt - Significant visual decline - Hyperopic shift and irregular astigmatism

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Causes Multiple potential causes of DLK have been described and may be broadly categorized by whether they cause an individual, sporadic case or a cluster of cases. These potential triggers include corneal epithelial injury, femtosecond laser flap creation, meibomian gland secretions, corneal crosslinking, immune dysregulation, viral conjunctivitis, bacterial endotoxin, and iatrogenic causes through contamination or toxic chemicals. Corneal Epithelial Injury DLK triggered by corneal epithelial injury has been described frequently and is considered the most common cause of DLK. Corneal epithelial injury created by recurrent corneal erosion, diamond burr superficial keratectomy, foreign body injury to the cornea, and traumatic dislocation of the flap have all been described.14–18 In one case, after corneal foreign body removal, the keratitis was initially observed to be localized around the location of the prior foreign body and then extended through the entire lamellar plane.17 The impaired corneal integrity may lead to keratinocyte damage that activates inflammatory cells that then invade the stromal interface, as the potential space at the interface remains indefinitely after lamellar surgery. As such, minimizing corneal epithelial defects in patients with prior lamellar surgery, such as performing anterior stromal puncture instead of superficial keratectomy, may decrease the risk of developing DLK.14,19 Femtosecond Laser Flap Creation The use of a femtosecond laser compared to a microtome for flap creation may be associated with higher risk of DLK.20 Using a smaller suction ring number, larger optical zone, or higher energy level for flap creation with the femtosecond laser have been associated with a higher risk of developing DLK.21,22 Usually, this inflammation is mild and improves quickly with topical steroids.21 Meibomian Gland Secretions Meibomian gland secretions trapped within the flap interface has been proposed as a trigger for DLK in humans and reproduced in rabbit models.23,24 Corneal Crosslinking Corneal crosslinking with riboflavin-ultraviolet-A for corneal ectasia may trigger DLK and has been reported up to 18 years after initial LASIK surgery.7,25,26 This can occur with both epi-on or epi-off crosslinking, likely due to the epithelial injury from the epithelial permeabilization step for both procedures. Immune Dysregulation Patients with underlying autoimmune conditions or with localized ocular immune dysregulation, such as from topical immunosuppressive medications, may be predisposed to developing DLK. Case reports of unilateral DLK associated with anterior uveitis in patients with underlying spondyloarthropathy and bilateral DLK in a patient with Cogan syndrome have been described.27–29 DLK may be attributable to intrinsic factors from these patients, as McLeod et al. found no significant difference between the incidence of bilateral DLK regardless of whether surgery was performed bilaterally on the same day or sequential days.11 Interestingly, a case of DLK possibly triggered by cenegermin 0.005% ophthalmic solution for neurotrophic keratitis has been reported. It is unclear whether this medication caused DLK by increasing the pro-inflammatory effects of neurotrophic growth factor or whether the neurotrophic keratitis represents an underlying immune dysregulation resulting in a higher risk for DLK.30 Viral Conjunctivitis A case of DLK in the setting of pseudomembraneous viral conjunctivitis and corneal edema has been reported. This case resolved with topical steroids.31 Bacterial Endotoxins Endotoxins are lipopolysaccharides derived from the cell wall of gram-negative bacteria and have been shown to cause an outbreak or cluster of DLK cases when introduced to the eye. 32–34 These bacteria remain prevalent on surface biofilms when improper sterilization techniques are used. Endotoxins in the water or biofilm of autoclave or sterilization reservoirs and instrument washbasins all have been implicated.32–34 In both humans and rabbit models, Pseudomonas aeruginosa has been the most

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identified culprit bacteria, followed by Flavobacteriam, Burkholderia cepacian, and Burkholderia pickettii.24,32–35 Cases associated with gonococcal keratoconjunctivitis and staph aureus exotoxins have also been described.24,36 In these cases, proper modification of the cleaning and maintenance process for sterilization equipment has led to resolution of these clustered cases. Iatrogenic Causes Iatrogenic causes of DLK have been linked to clusters of DLK cases. Toxic residual chemicals used in autoclaving surgical instruments, such as povidone-iodine 10% solution, Palmolive ultra soap, and Klenzyme soap have all been suggested as triggers for DLK.24,37 Additionally, silicone oil contamination of the internal and external surfaces of surgical gloves and surgical marking pen ink have been implicated in outbreaks of DLK.38–40 These cases resolved after changing brands of surgical gloves or marking pen.

Pathophysiology DLK is the result of the corneal wound healing response to an initial insult or trigger that is unimpeded by the lamellar interface. The most common trigger is injury to the epithelium, which releases proinflammatory cytokines such as IL-1 and TNF-alpha that activate adjacent keratinocytes and causes an influx of chemotactic factors that allow for neutrophil migration.41 Activated keratinocytes release additional pro-inflammatory cytokines including G-CSF, MCAF, neutrophil-activating peptide (ENA-78), and monocyte-derived neutrophil chemotactic factor (MDNCF) that attract additional inflammatory cells, including macrophages and lymphocytes.42 This response may be self-perpetuating, as activated inflammatory cells release more cytokines that further attract more inflammatory cells. Confocal imaging offers additional insight into the inflammatory reaction that comprises DLK. The stromal lamellar interface in diseased corneas has many activated keratinocytes with particulate debris of various sizing and an inflammatory cell infiltrate.15,43–45 The size and amount of the debris and inflammatory cell infiltrate varies depending on the stage of DLK. Stages 1 and 2 showed a variable amount and density of inflammatory cell infiltrates in the flap interface.46 Stage 3 had rare granulocyte-like inflammatory cells but large dense clusters of a reflective granular substance, presumed to be aggregated cell remnants, in the interface.46 Stage 4 had no active inflammation but activated highly reflective keratinocytes with stromal folds.46 As such, stages 1 and 2 may reflect an active acute inflammatory response, while stages 3 and 4 represent the accumulation and decay of inflammatory cells.46

Management Treatment of DLK is comprised of frequent topical steroids for milder cases and lifting and irrigating under the LASIK flap to remove the inflammatory reaction in more severe cases.12 Topical steroids can be initiated at hourly frequency and tapered thereafter depending on response. In some cases, topical steroids have been used prior to surgery for patients at higher risk for developing DLK, such as those with severe ocular rosacea or atopy. Initiating steroids prior to surgery has also been demonstrated to be protective against DLK in rabbit models.47 If lifting the flap is necessary, debridement is usually performed with careful irrigation of both surfaces of the stromal interface. This debridement may worsen astigmatism.12 A case of recurrent DLK refractory to topical steroids and flap lift with irrigation was treated successfully with phototherapeutic keratectomy used to ablate both surfaces of the stromal interface.48 In another rabbit model study, a peribulbar injection of a platelet-activating factor antagonist was protective against DLK with minimal inflammatory response.49 However, this has not been explored in clinical practice.

Central Toxic Keratopathy Background Central toxic keratopathy (CTK) was first described by Fraenkel et al. in 1989 as an inflammatory complication of LASIK surgery.50 While initially presumed to be part of the same disease spectrum as DLK, it was later categorized as its own separate clinical entity due to the lack of response to topical steroids and absence of inflammatory cells.51,52 The name “central toxic keratopathy” was later coined by Sonmez and Maloney in 2007.53 The clinical syndrome that is now known as CTK has also previously

2024 Harvard Ophthalmology Residents’ Course been called “central lamellar keratitis” or “central flap necrosis”.51,53 While initially described as a complication of LASIK, cases occurring after SMILE have also been reported.54–56 Currently, the reported incidence of CTK is between 0.016-0.052%.57,58

Diagnosis CTK typically presents 3-9 days after laser-based lamellar refractive corneal surgery with the triad of central corneal opacification, central striae, and loss of stromal bed tissue that causes a significant hyperopic shift.53,56,59,60 The central opacification appears as a focal, dense, well-demarcated, whitish opacity without associated inflammation.60 CTK is less inflammatory than DLK and can take up to 12-18 months to resolve spontaneously.59,60 Steroids are typically not indicated as they have not been shown to improve outcomes, and there is no specific guideline for treatment.53 However, steroids are often initiated for these patients as CTK commonly follows DLK, which is treated with steroids.59 The absence of an epithelial defect or inflammation also helps to distinguish CTK from an infection.60

Causes Several possible causes of CTK have been hypothesized and include reactions to surgical glove components, meibomian gland secretions, blepharitis, marker pen ink, minoxidil solution, and povidoneiodine.52,58,61,62 Other mechanisms such as a toxic reaction secondary to photoactivation of an extrinsic substance by excimer laser energy or laser energy resulting in keratinocyte apoptosis have been proposed.59 Cases associated with corneal cross-linking have been reported and may be triggered by epithelium removal, excessive application of the riboflavin solution, or the UVA irradiation itself.60,63 While toxic contaminants may trigger CTK, one study also proposed a possible underlying genetic predisposition to developing CTK after observing two twins who developed bilateral CTK after LASIK on the same day.59 The other patients undergoing refractive laser surgery on that day did not develop CTK.59

Pathophysiology The central stromal thinning the area of the opacity observed in CTK is located in the anterior stromal bed just posterior to the flap.64,65 This stromal thinning is likely a result of keratinocyte apoptosis, enzymatic destruction of the extracellular matrix, and disorganization of the collagen lamellae in the central stromal matrix.60,65,66 Specifically, neutrophil degranulation and the release of collagenase and proteolytic enzymes may cause stromal loss, and activation of fibroblasts in the same area causes corneal haze.58 The central opacity is thought to be non-inflammatory, as it lacks many features expected of inflammation under confocal microscopy.65,67 The anterior corneal curvature changes caused by anterior stromal thinning results in hyperopia and corneal flattening.58,65 The majority of corneal thinning occurs within the first postoperative week, and tissue regeneration begins around postoperative week five with associated improvement in the anterior corneal curvature.58,65

Management The management of CTK is conservative with observation only, as the disease self-resolves over months. It may take up to 18 months for CTK to resolve spontaneously.59 Steroids do not improve outcomes or to accelerate the resolution of the disease.59 However, many patients are treated initially with steroids due to concern for DLK, and they are quickly tapered once the diagnosis of CTK is made.58 Flap lift and irrigation has been proposed to remove the inciting agent for CTK and possibly improve visual outcomes; however, this has not been studied extensively.58,68

Transient Light Sensitivity Syndrome Background Transient light sensitivity syndrome (TLSS) was first described in 2001 as an acute photosensitivity with an unremarkable slit lamp examination that develops after uncomplicated corneal lamellar refractive surgery. It was subsequently described after SMILE in 2017.69 TLSS was primarily associated with refractive surgery using earlier 6 kHz and 15 kHz models of the IntraLase laser and has become less common with the newer 30 kHz and 60 kHz models, likely due to the increased energy used by earlier lasers.57 The incidence of TLSS after LASIK has been reported to be 1.1-1.3%.70,71A later study of TLSS

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in myopic LASIK, hyperopic LASIK, and myopic SMILE reported increasing incidences of 1.2% for myopic SMILE, 5.3% for myopic LASIK, and 9.0% for hyperopic LASIK. 72 However, patients with no subjective complaints but observed photosensitivity during the slit lamp examination were categorized as having TLSS for this study, likely increasing the incidence compared to other studies.

Diagnosis The diagnosis of TLSS is made based on subjective report of bilateral photophobia with a normal detailed slit lamp examination, particularly without any interface inflammation.70 The symptoms typically begin between 2-8 weeks after lamellar refractive surgery and have been reported after LASIK and SMILE.69–71

Causes TLSS has been associated with increased energy from the femtosecond laser.71,72 The incidence of TLSS has been shown to correlate directly with the amount of energy applied to the cornea, and lowering the laser energy settings has been associated with a reduction in TLSS cases.71,72 This suggests that TLSS may result from secondary effects of laser energy, such as shockwave exposure on local keratinocytes or migration of expelled gases into the peripheral cornea and episclera that could irritate the ciliary body.71 TLSS has also been reported after both epi-on and epi-off corneal collagen crosslinking in patients without prior refractive surgery, suggestive of an underlying inflammatory mechanism or reaction to electromagnetic radiation.73 Unlike DLK, TLSS is unlikely to result from endotoxin exposure, as patients who later develop DLK typically develop unilateral findings after bilateral same-day surgery, rather than bilateral findings that would be more expected from an endotoxin outbreak.70

Pathophysiology The exact pathophysiology of TLSS is unclear but is likely of an inflammatory origin. Use of the femtosecond laser compared to a mechanical keratome is associated with higher postoperative inflammation and fibrosis adjacent to the flap margin that may predispose patients to developing TLSS.70 The inflammation may be mediated by peripheral structures, such as the ciliary body, which would preclude identification with a slit lamp.69 Other proposed causes include inflammation from necrotic cell debris or byproducts of the gas bubbles, cytokines migrating from the flap interface to the perilimbal sclera and iris base, or activated keratocytes in the interface.70 In one study, confocal microscopy demonstrated activated keratinocytes in the interface surface in about half of patients with TLSS; however, activated keratinocytes can also be found on confocal microscopy of asymptomatic, uncomplicated postoperative LASIK patients as a part of the normal healing process.70,71 As such, these activated keratinocytes are of unclear clinical significance for TLSS.

Management Treatment of TLSS is with topical steroids, and symptoms typically improve within a few days.70,71 Treatment with topical cyclosporine has also been described with good results.71 Topical non-steroid antiinflammatory medications have been proposed but not yet studied.71 With regards to the femtosecond laser, using lower energy settings and decreasing the time from laser ablation to flap lifting may decrease the incidence of TLSS.70,71 It is important to perform a careful slit lamp examination to exclude DLK, as the two syndromes may require different treatment or evaluation. Confocal microscopy has also been proposed to further distinguish between TLSS and DLK, particularly in subtle cases.71

Conclusion DLK, CTK, and TLSS comprise three rare, non-infectious, inflammatory complications after corneal lamellar refractive surgery that differ in their clinical presentation, with DLK having potentially severe vision threatening outcomes. DLK is associated with clinical findings of white, granular cells in the stromal interface, and CTK is associated with the triad of central corneal opacification, central striae, and hyperopic shift. TLSS has no findings on slit lamp examination. While both DLK and TLSS are responsive to topical steroids, CTK improves without intervention. It is important to distinguish the three syndromes, as proper diagnosis may provide insight into the underlying cause of the patient’s symptoms and thus guide treatment.

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References 1.

Joffe SN. The 25th Anniversary of Laser Vision Correction in the United States. Clinical Ophthalmology 2021;Volume 15:1163–1172.

Chua D, Htoon HM, Lim L, et al. Eighteen-year prospective audit of LASIK outcomes for myopia in 53 731 eyes. Br J Ophthalmol 2019;103:1228–1234.

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Reinstein DZ, Stuart AJ, Vida RS, et al. Incidence and Outcomes of Sterile Multifocal Inflammatory Keratitis and Diffuse Lamellar Keratitis After SMILE. J Refract Surg 2018;34:751–759.

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10. Leccisotti A, Fields S V. Diffuse lamellar keratitis after LASIK with low-energy femtosecond laser. J Cataract Refract Surg 2021;47:233–237. 11. McLeod SD, Tham VM-B, Phan ST, et al. Bilateral diffuse lamellar keratitis following bilateral simultaneous versus sequential laser in situ keratomileusis. Br J Ophthalmol 2003;87:1086–7. 12. Linebarger EJ, Hardten DR, Lindstrom RL. Diffuse lamellar keratitis: diagnosis and management. J Cataract Refract Surg 2000;26:1072–7. 13. Johnson JD, Harissi-Dagher M, Pineda R, et al. Diffuse lamellar keratitis: incidence, associations, outcomes, and a new classification system. J Cataract Refract Surg 2001;27:1560–6. 14. Lin H-Y, Ho W-T. Diffuse lamellar keratitis as a rare complication of diamond burr superficial keratectomy for recurrent corneal erosion: a case report. BMC Ophthalmol 2022;22:362. 15. Harrison DA, Periman LM. Diffuse lamellar keratitis associated with recurrent corneal erosions after laser in situ keratomileusis. J Refract Surg 2001;17:463–5. 16. Iovieno A, Amiran MD, Légaré ME, Slomovic AR. Diffuse lamellar keratitis 8 years after LASIK caused by corneal epithelial defect. J Cataract Refract Surg 2011;37:418–9. 17. Weisenthal RW. Diffuse lamellar keratitis induced by trauma 6 months after laser in situ keratomileusis. J Refract Surg 2000;16:749–51. 18. Schwartz GS, Park DH, Schloff S, Lane SS. Traumatic flap displacement and subsequent diffuse lamellar keratitis after laser in situ keratomileusis. J Cataract Refract Surg 2001;27:781–3. 19. Brilakis HS, Holland EJ. Anterior stromal puncture in the treatment of loose epithelium after LASIK. J Refract Surg 2006;22:103–5. 20. Gil-Cazorla R, Teus MA, de Benito-Llopis L, Fuentes I. Incidence of diffuse lamellar keratitis after laser in situ keratomileusis associated with the IntraLase 15 kHz femtosecond laser and Moria M2 microkeratome. J Cataract Refract Surg 2008;34:28–31. 21. de Paula FH, Khairallah CG, Niziol LM, et al. Diffuse lamellar keratitis after laser in situ keratomileusis with femtosecond laser flap creation. J Cataract Refract Surg 2012;38:1014–9.

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22. Segev F, Mimouni M, Sela T, et al. Risk Factors for Sporadic Diffuse Lamellar Keratitis After Microkeratome LaserAssisted In Situ Keratomileusis: A Retrospective Large Database Analysis. Cornea 2018;37:1124–1129. 23. Fogla R, Rao SK, Padmanabhan P. Diffuse lamellar keratitis: are meibomian secretions responsible? J Cataract Refract Surg 2001;27:493–5. 24. Holzer MP, Solomon KD, Vroman DT, et al. Diffuse lamellar keratitis: evaluation of etiology, histopathologic findings, and clinical implications in an experimental animal model. J Cataract Refract Surg 2003;29:542–9. 25. Grassmeyer JJ, Goertz JG, Baartman BJ. Diffuse Lamellar Keratitis in a Patient Undergoing Collagen Corneal Cross-Linking 18 Years After Laser In Situ Keratomileusis Surgery. Cornea 2021;40:917–920. 26. Mannschreck DB, Rubinfeld RS, Soiberman US, Jun AS. Diffuse lamellar keratitis after epi-off corneal crosslinking: An under-recognized complication? Am J Ophthalmol Case Rep 2019;13:140–142. 27. Keszei VA. Diffuse lamellar keratitis associated with iritis 10 months after laser in situ keratomileusis. J Cataract Refract Surg 2001;27:1126–7. 28. Javaloy J, Barrera C, Muñoz G, et al. Spontaneous bilateral, recurrent, late-onset diffuse lamellar keratitis after LASIK in a patient with Cogan’s syndrome. J Refract Surg 2008;24:548–50. 29. Díaz-Valle D, Arriola-Villalobos P, Sánchez JMB-C, et al. Late-onset severe diffuse lamellar keratitis associated with uveitis after LASIK in a patient with ankylosing spondylitis. J Refract Surg 2009;25:623–5. 30. Dandar RA, Schiffbauer J, Cheung AY. Late-onset diffuse lamellar keratitis after treatment with cenegermin. Can J Ophthalmol 2022;57:e202–e204. 31. Gris O, Güell JL, Wolley-Dod C, Adán A. Diffuse lamellar keratitis and corneal edema associated with viral keratoconjunctivitis 2 years after laser in situ keratomileusis. J Cataract Refract Surg 2004;30:1366–70. 32. Sorenson AL, Holland S, Tran K, et al. Diffuse lamellar keratitis associated with tabletop autoclave biofilms: case series and review. J Cataract Refract Surg 2020;46:340–349. 33. Peters NT, Iskander NG, Anderson Penno EE, et al. Diffuse lamellar keratitis: isolation of endotoxin and demonstration of the inflammatory potential in a rabbit laser in situ keratomileusis model. J Cataract Refract Surg 2001;27:917–23. 34. Holland SP, Mathias RG, Morck DW, et al. Diffuse lamellar keratitis related to endotoxins released from sterilizer reservoir biofilms. Ophthalmology 2000;107:1227–33; discussion 1233-4. 35. Cox SG, Stone DU. Diffuse lamellar keratitis associated with Pseudomonas aeruginosa infection. J Cataract Refract Surg 2008;34:337. 36. Symes RJ, Catt CJ, Males JJ. Diffuse lamellar keratitis associated with gonococcal keratoconjunctivitis 3 years after laser in situ keratomileusis. J Cataract Refract Surg 2007;33:323–5. 37. Shen Y-C, Wang C-Y, Fong S-C, et al. Diffuse lamellar keratitis induced by toxic chemicals after laser in situ keratomileusis. J Cataract Refract Surg 2006;32:1146–50. 38. Hoffman RS, Fine IH, Packer M, et al. Surgical glove-associated diffuse lamellar keratitis. Cornea 2005;24:699– 704. 39. Rosman M, Chua W-H, Tseng PSF, et al. Diffuse lamellar keratitis after laser in situ keratomileusis associated with surgical marker pens. J Cataract Refract Surg 2008;34:974–9. 40. Hadden OB, McGhee CNJ, Morris AT, et al. Outbreak of diffuse lamellar keratitis caused by marking-pen toxicity. J Cataract Refract Surg 2008;34:1121–4. 41. Asano-Kato N, Toda I, Shimmura S, et al. Detection of neutrophils and possible involvement of interleukin-8 in diffuse lamellar keratitis after laser in situ keratomileusis. J Cataract Refract Surg 2003;29:1996–2000. 42. Hong JW, Liu JJ, Lee JS, et al. Proinflammatory chemokine induction in keratocytes and inflammatory cell infiltration into the cornea. Invest Ophthalmol Vis Sci 2001;42:2795–803. 43. Rana M, Adhana P, Ilango B. Diffuse Lamellar Keratitis: Confocal Microscopy Features of Delayed-Onset Disease. Eye Contact Lens 2015;41:e20-3.

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44. Chung MS, Pepose JS, El-Agha M-SH, Cavanagh HD. Confocal microscopic findings in a case of delayed-onset bilateral diffuse lamellar keratitis after laser in situ keratomileusis. J Cataract Refract Surg 2002;28:1467–70. 45. de Rojas Silva V, Rodríguez-Ares T, Díez-Feijóo E, Sánchez-Salorio M. Confocal microscopy in late-onset diffuse lamellar keratitis after laser in situ keratomileusis. Ophthalmic Surg Lasers Imaging 2003;34:68–72. 46. Bühren J, Baumeister M, Cichocki M, Kohnen T. Confocal microscopic characteristics of stage 1 to 4 diffuse lamellar keratitis after laser in situ keratomileusis. J Cataract Refract Surg 2002;28:1390–9. 47. Holzer MP, Sandoval HP, Vargas LG, et al. Evaluation of preoperative and postoperative prophylactic regimens for prevention and treatment of diffuse lamellar keratitis. J Cataract Refract Surg 2004;30:195–9. 48. Leu G, Hersh PS. Phototherapeutic keratectomy for the treatment of diffuse lamellar keratitis. J Cataract Refract Surg 2002;28:1471–4. 49. Esquenazi S, He J, Bazan HEP, Bazan NG. Prevention of experimental diffuse lamellar keratitis using a novel platelet-activating factor receptor antagonist. J Cataract Refract Surg 2004;30:884–91. 50. Fraenkel GE, Cohen PR, Sutton GL, et al. Central focal interface opacity after laser in situ keratomileusis. J Refract Surg 1998;14:571–6. 51. Lyle WA, Jin GJ. Central lamellar keratitis. J Cataract Refract Surg 2001;27:487–90. 52. Moshirfar M, Hazin R, Khalifa YM. Central toxic keratopathy. Curr Opin Ophthalmol 2010;21:274–9. 53. Sonmez B, Maloney RK. Central toxic keratopathy: description of a syndrome in laser refractive surgery. Am J Ophthalmol 2007;143:420–7. 54. Gülmez M, Fatihoglu ÖU. Central Toxic Keratopathy after Small Incision Lenticule Extraction. Klin Monbl Augenheilkd 2023;240:810–814. 55. Linghu S, Liu T, Luo H, Shi R. Central toxic keratopathy after small incision lenticule extraction surgery:A case report. Eur J Ophthalmol 2023;33:NP122–NP127. 56. Koh K, Jun I, Kim TI, et al. Central Toxic Keratopathy after Small Incision Lenticule Extraction. Korean J Ophthalmol 2020;34:254–255. 57. Stonecipher K, Ignacio TS, Stonecipher M. Advances in refractive surgery: microkeratome and femtosecond laser flap creation in relation to safety, efficacy, predictability, and biomechanical stability. Curr Opin Ophthalmol 2006;17:368–72. 58. Moshirfar M, Hall MN, West WB, et al. Five-Year Occurrence and Management of Central Toxic Keratopathy After Femtosecond Laser-Assisted LASIK. J Refract Surg 2021;37:25–31. 59. Yim CK, Zhu D. Central Toxic Keratopathy in Siblings After Laser-Assisted Keratomileusis: Case Report and Literature Review. Cornea 2022;41:640–643. 60. Dongre P, Bevara A, Deshmukh R, Vaddavalli PK. Central Toxic Keratopathy After Collagen Cross-Linking: A Case Series. Cornea 2023. 61. Marí Cotino JF, Suriano MM, De La Cruz Aguiló RI, Vila-Arteaga J. Central toxic keratopathy: a clinical case series. Br J Ophthalmol 2013;97:701–3. 62. Mohammadpour M, Khorrami-Nejad M, Heirani M, Moshirfar M. Topical Minoxidil Solution-Induced Central Toxic Keratopathy following Photorefractive Keratectomy: A Case Study. J Curr Ophthalmol 2022;34:352–356. 63. Kayabasi M, Utine CA. Central Toxic Keratopathy Following Corneal Collagen Cross-Linking. Beyoglu eye journal 2023;8:69–72. 64. Liu A, Manche EE. Anterior Segment Optical Coherence Tomography Imaging of Central Toxic Keratopathy. Ophthalmic Surg Lasers Imaging 2010:1–3. 65. Sikder S, Khalifa YM, Neuffer MC, Moshirfar M. Tomographic corneal profile analysis of central toxic keratopathy after LASIK. Cornea 2012;31:48–51.

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66. Kayabasi M, Utine CA. Central Toxic Keratopathy Following Corneal Collagen Cross-Linking. Beyoglu eye journal 2023;8:69–72. 67. Thornton IL, Foulks GN, Eiferman RA. Confocal microscopy of central toxic keratopathy. Cornea 2012;31:934–6. 68. Tu KL, Aslanides IM. Surgical intervention in central toxic keratopathy. Eur J Ophthalmol 2012:0. 69. Desautels JD, Moshirfar M, Quist TS, et al. Case of Presumed Transient Light-Sensitivity Syndrome After SmallIncision Lenticule Extraction. Cornea 2017;36:1139–1140. 70. Muñoz G, Albarrán-Diego C, Sakla HF, et al. Transient light-sensitivity syndrome after laser in situ keratomileusis with the femtosecond laser Incidence and prevention. J Cataract Refract Surg 2006;32:2075–9. 71. Stonecipher KG, Dishler JG, Ignacio TS, Binder PS. Transient light sensitivity after femtosecond laser flap creation: clinical findings and management. J Cataract Refract Surg 2006;32:91–4. 72. Reinstein DZ, Potter JG, Gupta R, et al. Transient Light Sensitivity Syndrome (TLSS) Incidence Following Femtosecond LASIK for Myopic and Hyperopic Eyes and Femtosecond SMILE for Myopic Eyes. J Refract Surg 2023;39:366–373. 73. Moshirfar M, Vaidyanathan U, Hopping GC, et al. Delayed-Onset Transient Light Sensitivity Syndrome after Corneal Collagen Cross-Linking: A Case Series. Med Hypothesis Discov Innov Ophthalmol 2019;8:250–256.

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The Effect of Diabetes Mellitus on the Corneal Microenvironment and the Impact of the Donor Diabetic State on Corneal Transplant Outcomes Yilin Feng, MD and Reza Dana, MD, MPH, MSc

Introduction Diabetes mellitus (DM) has emerged as a major global health concern with the prevalence increasing dramatically worldwide.1 Persistent hyperglycemia can cause serious systemic complications through vascular damage, delayed tissue healing, and immune regulatory dysfunction.2 A well-established ocular complication of DM is diabetic retinopathy, which can lead to devasting vision decline and even blindness. Recently, attention has been focused on diabetes-related complications in the cornea and ocular surface. In addition to dry eye disease and neurotrophic keratitis, there is evolving research suggesting that tissue from donors with diabetes may be associated with an increased risk of graft rejection in corneal transplantation.3 As a result, there appears to be increasing surgeon preference for tissues from donors without a history of diabetes.4,5,6 Currently, the impact of donor diabetes status on graft survival is widely debated and the literature lacks conclusive evidence. As such, this review aims to present the current literature on the effect of diabetes on the corneal microenvironment and the impact of the donor diabetic state on corneal transplant outcomes.

Impact of Diabetes on the Structures of the Cornea Numerous structural alterations in the cornea have been linked to diabetes based on animal and human studies. In particular, impairment of corneal sensation has been well-described, with hyperglycemia determined to be a major cause. The human cornea is the most innervated tissue in the body and is considered to be 300-600 times more sensitive than the skin.7 Corneal nerves are vital to the health of the cornea through the secretion of neuromediators that promote neuronal regeneration and wound healing.8 In DM, levels of neurotrophic factors are reduced, and the formation of advanced glycosylation end products (AGE) causes damage to the capillary endothelium and microvascular ischemia to neurons.9,10 In addition, DM leads to the recruitment of Langerhans cells and dendritic cells (DC) to corneal nerve fibers, resulting in immune-mediated corneal nerve damage early in the disease process.11,12 Reduced corneal sensitivity has been reported to be correlated with the severity of DM and corneal nerve damage has been considered a surrogate marker for peripheral diabetic polyneuropathy.6 In addition to neuronal damage, DM also causes significant disruption to the ocular surface. The tear film is critical for the protection and health of the eye. In DM, increased glucose levels and inflammatory mediators such as tumor necrosis factor-α (TNF-α) and matrix metalloproteinases in the tear film impede proper wound healing.15,16,17 Furthermore, corneal epithelial cells become pleomorphic and irregularly arranged, which increases epithelial fragility and impairs their normal barrier function, leading to superficial punctate keratitis and epithelial defects.13,18 Accumulation of AGEs in epithelial basal cells, along with decreased number of hemidesmosomes and altered epithelial adhesion found in DM, can cause recurrent corneal erosions.19,20,21 All of these factors, in combination with decreased corneal sensation, can lead to the dreaded sequelae of non-healing ulcers. DM has also been proposed to impact corneal endothelial density and function; however, the evidence in the literature remains controversial. A number of studies have shown an increased central corneal thickness (CCT) in patients with DM.22,23 This is hypothesized to be in part due to the accumulation of AGEs which leads to abnormal cross-linking of stromal collagen fibers. Another driving factor may be increased endothelial permeability, as impairment of endothelial Na+/K+ ATPase activity has been identified in DM.9 Some studies have also found lower endothelial cell density (ECD) in individuals with diabetes, and an association between a lower ECD and higher level of hemoglobin A1c has been

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reported.24,25 However, it is important to emphasize that not all studies in the literature have shown an impact of DM on corneal endothelial number and function.6,26–28 Thus, if the purported deleterious effect of DM on graft outcomes is not related to endothelial cell count or function, an alternate cause is needed to explain the apparent worse outcomes of transplantation when donor tissue is procured from donors with diabetes. Increasing evidence suggests that the diabetic state alters the immune microenvironment of the cornea, thus amplifying the capacity of resident immune cells of the cornea to sensitize the host. Below, we will summarize some of the pertinent concepts and evidence in this regard.

Ocular Immune Privilege and Allograft Survival “Ocular immune privilege” is a term that describes the generally blunted sensitization of hosts by alloantigens when grafts are placed in the ocular microenvironment.29 This term also refers to induction of a tolerogenic response, which is actively maintained in the host against foreign antigens. It is a unique evolutionary adaptation that protects the eye against inflammatory, vision-threatening responses. The eye achieves this through a combination of immunologic ignorance, systemic tolerance to novel antigens, and the development of an intraocular immunosuppressive environment that is maintained by a wide array of cytokines and neuropeptides.30 All of these mechanisms support the survival of a corneal graft. There are several methods in which the eye achieves immunologic ignorance. Firstly, corneal cells lack the expression of major histocompatibility complex (MHC) class II antigens, and the expression of MHC class I antigens is markedly reduced, with the exception of the far periphery of the cornea which is the more immunoreactive zone of the tissue.30 Sensitization to foreign antigens requires presentation of an antigen in the context of MHC molecules by antigen presenting cells (APC). Under normal conditions, corneal APCs are in an “immature” state, with negligible expression of MHC, hence they are not highly sensitizing. Secondly, the cornea is avascular and without lymphatics, which limits the trafficking of immune cells to the tissue and restricts the migration of cornea-resident APCs to secondary lymphoid tissues where T cells reside, respectively. Furthermore, the cornea expresses anti-angiogenic and antilymphangiogenic factors, such as soluble VEGFR-1, VEGFR-2, and VEGFR-3,31 which are receptors that bind bioavailable ligands and act as a sink to suppress signaling. The expression of such anti-angiogenic and anti-lymphangiogenic factors is referred to as “angiogenic privilege”, a facet of the cornea that is critical for maintaining relative immune quiescence. As such, in corneal transplantation, allograft rejection is often delayed compared to rejection of other solid organ transplants and systemic immunosuppression can be avoided. The induction of systemic tolerance from novel antigens is primarily due to a process termed anterior chamber-associated immune deviation (ACAID). ACAID has been studied extensively in animal models. When a novel antigen is exposed to the intraocular environment, intraocular APCs are activated and migrate to the spleen via the trabecular meshwork. These APCs express the F4/80 marker of macrophages and induce the generation of natural killer T (NKT) cells. Together, APCs and NKT cells create an environment that is rich in cytokines and present the foreign antigen to B cells, CD4+, and CD8+ T cells in the spleen.30,32,33 Eventually, CD4+ and CD8+ regulatory T-cells (Treg) emerge to suppress the activation and differentiation of T cells into T-helper 1 (Th1) effector cells and inhibit the expression of Th1-mediated immunity, respectively.34,35 The presence of regulatory T cells is critical in the induction of tolerance towards alloantigens, and research has shown that local delivery of regulatory T cells can promote corneal allograft survival.36,37 In addition to the mechanisms above, the aqueous humor and the cornea contain an array of immunomodulatory molecules that protect the eye from inflammation. For example, the neuropeptide alpha-melanocyte stimulating hormone (α-MSH) and the cytokine transforming growth factor (TGF)-β, both of which inhibit the activation of Th1 cells, are found in the aqueous humor.29 These immunomodulatory molecules suppress the function of pro-inflammatory cytokines in the aqueous humor and thus contribute to corneal transplant acceptance.38,39 In the cornea, programmed death ligand-1 (PDL-1) is constitutively expressed at high levels to inhibit T cell proliferation and interferon gamma (IFNγ) production, leading to prolonged allograft survival.40,41 Interleukin (IL)-1Ra is also constitutively expressed by the cornea and works to suppress APC migration.42 CD95L, found in the corneal epithelium

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and endothelium, has been known to trigger the apoptosis of CD95 expressing neutrophils and effector T cells, thereby improving allograft survival.43 All of these immunomodulatory molecules work in conjunction to maintain an immunosuppressive intraocular environment.

Diabetes and Corneal Transplant Rejection Due to the aforementioned mechanisms that shield the eye from immunologic response, corneal transplantation has high success rates when performed in noninflamed host beds. Additionally, corneal grafts do not require systemic immunosuppression or human leukocyte antigens (HLA) or ABO bloodtype matching. However, corneal transplants are not immune to allograft rejection; indeed, immunological rejection remains the leading cause of corneal transplantation failure today.44 The rejection process occurs when the mechanisms maintaining ocular immune privilege are overridden and is primarily driven by the recruitment of activated immune (primarily T) cells to the cornea following surgery. The proinflammatory cascade leads to alterations in the function of APCs, such as acquisition of MHC class II phenotypes. These APCs in turn prime naïve T cells to generate Th1 cells that target the graft epithelium, stroma, and/or endothelium via secretion of IFN-γ and other effector molecules (IL-1, TNF-α) that mediate rejection.45,46 A number of risk factors for cornea allograft rejection have been identified, including young recipient age, active host bed inflammation and neovascularization, anterior synechiae, and repeat grafting.47 Most of these risk factors are associated with a heightened inflammatory state. Given the increasing prevalence of DM globally and its association with a dysregulated inflammatory state, several studies have investigated the impact of diabetic donor status on transplant success. Recent eye banking data suggest that 30% of corneas in the donor pool are derived from donors with diabetes, a number that will continue to rise given the increasing prevalence of DM globally.46 There is also an increasing number of corneal specialists who avoid using grafts from donors with diabetes due to concerns for rejection.5,6 Thus, it is important to critically evaluate the impact of donor diabetes on cornea transplant success. The allograft rejection process is mainly initiated by DCs, which are the predominant APCs in the cornea. Their primary function is to induce and amplify inflammatory response. Many animal and human studies have shown an increased number of DCs in the diabetic cornea as a response to inflammation.12,48 In the healthy cornea, the majority of DCs reside in the periphery in their immature stage, and a small proportion of mature DCs reside in the center. In patients with type 2 DM, there is an increase in the number of mature DCs with a proportional decrease in immature DCs, suggesting a maturation process as type 2 DM progresses.49 DC density in the cornea has been shown to increase significantly during immunemediated corneal inflammation secondary to corneal graft rejection.50 As such, in addition to the structural changes in the cornea as a result of DM, the dysregulated and inflammatory microenvironment caused by DM also predisposes the cornea to allograft rejection. A number of animal studies have investigated the effect of diabetes on the cornea; however, there are few studies that directly evaluate the impact of donor diabetes status on corneal transplant immunity. A recent study in 2023 by Blanco et al. demonstrated that DM causes migration of corneal APCs and acquisition of an immunostimulatory phenotype.46 In their study, non-diabetic mice that underwent corneal transplantation using grafts from diabetic donor mice had an 100% rejection rate compared to a 50% rejection rate in mice receiving grafts from non-diabetic donors. In addition, hosts receiving diabetic grafts were found to have an increased number of migrating APCs and Th1 alloreactive cells compared to hosts receiving non-diabetic grafts. This was complemented by a decrease in the number of Treg cells in the draining lymph nodes of the recipients of diabetic corneas. Thus, these hosts were more prone to activate effector cells that can attack the graft. Interestingly, host recipients that received corneal grafts from insulin-treated diabetic mice had lower number of Th1 cells and increased Treg functionality, suggesting a recovery of tolerogenic phenotype from the use of insulin.

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Clinical Outcome Studies Several clinical studies have evaluated the outcomes of corneal grafts from diabetic donors. In 2015, a randomized controlled trial published by Lass et al. examined 1,090 patients undergoing penetrating keratoplasty (PKP) in the United States.6 In their study, the 10-year graft failure rate, baseline ECD, and 10-year ECD between recipients of a diabetic donor graft versus recipients of a non-diabetic donor graft was not significantly different. However, the assessment of donor diabetic status was limited as it was determined by eye banks and based only on historical data (i.e., without details on the severity or duration of diabetes). In the same year, a retrospective study by Vislisel et al. found similar rates of graft failure in 47 PKP and 183 Descemet stripping automated endothelial keratoplasty (DSAEK) cases when comparing tissue from donors with and without diabetes.51 Contrary to this result, however, the Cornea Preservation Time Study demonstrated that DSAEK tissue from donors with diabetes was associated with a 2.35x increased risk of primary or early failure.3 This was supported by additional evidence showing that DSAEK grafts from donors with diabetes were associated with a 2.29x greater risk of graft dislocation and lower ECD at 3-year follow up.52,53 Interestingly, a retrospective review of donor tissue at a single state eye bank revealed that tissues from donors with DM have lower mean ECD values.24 However, tissues from donors with DM remained equally likely to be included in the donor pool for keratoplasty compared to non-diabetic donor tissues, suggesting that a statistically significant difference may not be clinically significant. In addition to PKP and DSAEK, several studies have also assessed the impact of donor diabetes status on outcomes of Descemet membrane endothelial keratoplasty (DMEK). A critical and technically challenging component of DMEK is the tissue preparation process, which involves stripping the donor tissue. Although eye bank technicians can pre-strip the tissue, many surgeons prefer to prepare the tissue in the operating room, despite the risk of destroying tissue at the time of surgery.54 In 2017, Price et al. showed that donor diabetes was associated with a 5x increased risk of tissue preparation failure in DMEK grafts.55 This increased risk of tissue preparation failure in donor grafts with diabetes has been described in preparations performed by both surgeons and eye bank technicians and has been attributed to an increased adhesive interface between the Descemet’s membrane and posterior stroma in diabetic grafts.55–59 There is some evidence demonstrating a greater risk of graft preparation failure when a certain severity of DM is reached, suggesting the potential utility of stratifying donor diabetes severity and duration when evaluating for graft suitability.58 Fortunately, aside from tissue preparation failure, donor diabetic status does not appear to increase the re-bubble rate or rate of rejection after DMEK, and is not associated with increased endothelial cell loss.4,55 Although the current evidence suggests that a graft from a donor with diabetes may not impact graft rejection in DMEK outcomes, the number of studies on DMEK success rates is currently limited. Currently, a large, multicenter trial is underway to evaluate outcomes following DMEK from donors with diabetes.60

Conclusion The apparent disparities in the conclusions reached regarding the incremental risks of using diabetic donor tissue for corneal transplantation remain incompletely understood. However, in general, most authorities agree that reasons include the incomplete history of diabetes (both its severity [e.g., hemoglobin A1c level] and duration) for many donors collected by eye banks, and that the cause of death in donors is often attributed to proximal causes (e.g., cerebrovascular accident or myocardial infarction) as opposed to underlying morbidities such as DM and hypertension. DM causes multiple structural changes and a dysregulated inflammatory state in the cornea, resulting in an increased number of APCs and Th1 alloreactive cells and is thereby hypothesized to increase the risk of corneal allograft rejection. The current literature lacks conclusive evidence and additional prospective studies are needed to further elucidate this issue. Given the increasing prevalence of DM globally and thus donor corneal tissue from individuals with diabetes, there is a growing need to better characterize the impact of diabetic donor status on corneal transplantation success and inform clinical decision making.

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Chong EM, Dana MR. Graft failure IV. Immunologic mechanisms of corneal transplant rejection. Int Ophthalmol. 2008;28:209-222.

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Blanco T, Musayeva A, Singh RB, et al. The impact of donor diabetes on corneal transplant immunity. Am J Transplant Off J Am Soc Transplant Am Soc Transpl Surg. 2023;23:1345-1358.

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Aldave AJ, Terry MA, Szczotka-Flynn LB, et al. Effect of Graft Attachment Status and Intraocular Pressure on Descemet Stripping Automated Endothelial Keratoplasty Outcomes in the Cornea Preservation Time Study. Am J Ophthalmol. 2019;203:78-88.

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Terry MA, Straiko MD, Veldman PB, et al. Standardized DMEK Technique: Reducing Complications Using Prestripped Tissue, Novel Glass Injector, and Sulfur Hexafluoride (SF6) Gas. Cornea. 2015;34:845-852.

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Price MO, Lisek M, Feng MT, et al. Effect of Donor and Recipient Diabetes Status on Descemet Membrane Endothelial Keratoplasty Adherence and Survival. Cornea. 2017;36:1184-1188.

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Greiner MA, Rixen JJ, Wagoner MD, et al. Diabetes mellitus increases risk of unsuccessful graft preparation in Descemet membrane endothelial keratoplasty: a multicenter study. Cornea. 2014;33:1129-1133.

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Williams RS, Mayko ZM, Friend DJ, et al. Descemet Membrane Endothelial Keratoplasty (DMEK) Tissue Preparation: A Donor Diabetes Mellitus Categorical Risk Stratification Scale for Assessing Tissue Suitability and Reducing Tissue Loss. Cornea. 2016;35:927-931.

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Periocular Manifestations and Management of IgG4 Disease Tatiana R. Rosenblatt, MD and Michael K. Yoon, MD

Introduction Immunoglobulin G4-related disease (IgG4-RD) comprises a spectrum of inflammatory conditions that are characterized by lymphoplasmacytic infiltration by IgG4+ plasma cells, resultant tumefactive lesions at various locations in the body, fibrosis, and elevated serum IgG4 levels. (Arora, Balakrishnan, Inoue, Kubota, Deshpande, Stone). The underlying etiology of the disease remains unknown. It was first reported in 2001 in the setting of a patient with autoimmune sclerosing pancreatitis. (Hamano). Since then, IgG4-RD has been found to involve a wide range of anatomic sites, including the kidneys, biliary tract, intestinal tract, lungs, aorta, pericardium, prostate, breast, thyroid, salivary glands, lymph nodes, and skin, as well as various orbital adnexal and intracranial structures. (Stone, Kamisawa, Chaudhary, Cheuk, Mehta, Kubota, Wallace, Goto, Sato, Ebbo). Patients may experience fatigue, non-tender lymphadenopathy, and symptoms related to the specific organs involved. (Hardy) The estimated incidence of IgG4-RD in a United States insurance claims-based analysis was 0.78 per 100,000 person-years in 2015 and 1.39 per 100,000 person-years in 2019, increasing likely due to greater awareness of the disease, with an estimated prevalence of 5.3 per 100,000 persons in 2019. (Wallace incidence) The mean age at time of diagnosis is 50 – 60 years across multiple studies (Kubota, Goto clinical features, Ebbo, Hardy, Wallace incidence), and most studies have demonstrated equal prevalence among men and women (Ebbo, Goto clinical features, Hardy). The mortality rate among patients with IgG4-RD is estimated at 3.4 per 100 person-years. (Wallace incidence) In addition to the systemic findings of IgG4-RD, IgG4 disease can also affect various orbital structures, a condition termed IgG4-related ophthalmic disease (IgG4-ROD). IgG4-ROD was first identified in a patient with bilateral lacrimal gland swelling in 2004 and was subsequently demonstrated histolopathologically in 2007. (Yamamoto, Takahira dacryoadenitis) IgG4-ROD typically presents with subacute, insidious progression of symptoms including dry eye, diplopia, decreased vision, and other symptoms of orbital mass effect, often with only mild, if any, signs of inflammation. (Goto clinical features, Andrew an analysis, Zhao IgG4 ocular) Periocular manifestations of IgG4 disease can include involvement of the conjunctiva, eyelids, lacrimal glands, nasolacrimal system, extraocular muscles, nerves, and other soft tissues of the orbit, and rarely uveitis and scleritis. IgG4-ROD is bilateral in 43 – 85% of patients. (Kubota, Ebbo, Chou, Chen, Sato, Hardy, Klingenstein) It may occur in isolation, or in conjunction with systemic IgG4-RD. The most common systemic organs involved in patients with IgG4-ROD are the salivary glands, lymph nodes, and pancreas, present in 43%, 27%, and 20% of IgG4-ROD patients, respectively. (Detiger, Zhao, Andrew) The general prevalence of IgG4-ROD has historically been difficult to estimate due to few studies with varying diagnostic criteria (Andrew IgG4 idiopathic). One study of 1,014 patients with orbital lymphoproliferative disorders found that 22% of cases were due to IgG4-ROD. (Japanese study group) The prevalence of IgG4-ROD among patients with systemic IgG4-RD ranges from 4 – 34% (Ebbo Wallace ophthalmic manifestations, Uchida, Takuma). Conversely, the rate of systemic manifestations of IgG4 disease among patients with IgG4-ROD is estimated to be 48 – 80% (Goto clinical features, Ebbo), with higher likelihood of systemic disease in patients with bilateral ophthalmic findings and lacrimal gland involvement (Ebbo, Wu). This article will review the periocular manifestations of IgG4 disease and provide an update on diagnosis and treatment.

External Manifestations Periocular edema is the most common ophthalmic finding in IgG4 disease. (Deschamps, Kubota, Takano). Eyelid or periocular swelling occurs in 71 – 100% of patients with IgG4-ROD. (Andrew, Hardy,

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Wu, Kubota, Deschamps, Zhao IgG4 ocular) While it is typically painless, up to 36% of patients report periocular pain. (Kubota, Andrew IgG4 idiopathic, Hardy, Deschamps) and 25 – 36% of patients have tenderness of the eyelids. (Andrew IgG4 idiopathic, Hardy) Typically eyelid swelling progresses slowly over the course of weeks to months; however, one study reported rapid progression of eyelid edema over a period of days leading to an inability to open the eye. (Wallace) Eyelid erythema is less common, presenting in 13-25% of patients, and some patients may also develop conjunctival injection. (Andrew IgG4 idiopathic) In addition to diffuse eyelid edema, mass-like eyelid lesions have been noted in approximately 12% of patients. (Sogabe location, Goto clinical features) They are often best identified on radiographic imaging, where they appear as nodules anterior to the orbital septum. (Sogabe location)

Orbital Manifestations Over half of patients with IgG4-RD with head and neck involvement will present with orbital findings, which can include infiltration of the lacrimal gland, extraocular muscles, and nerves. (Mulholland, Ebbo) Less commonly, orbital bony infiltration may occur, noted in 11 – 23% of patients. (Ebbo, Klingenstein) Patients often present with proptosis, noted in 12 – 75% of patients with IgG4-ROD, which can be caused by extraocular muscle enlargement or other orbital soft tissue involvement. Approximately 25-33% of patients may have associated hypoglobus. (Andrew IgG4 idiopathic, Deschamps, Wu, Sogabe location, Zhao) Proptosis in these patients is typically painless and slowly progressive. (Sogabe location) Diplopia has been noted in 13 – 33% of IgG4-ROD patients (Andrew, Wu, Deschamps, Chaudhry), which is typically due to restriction from diffuse orbital lesions, though orbital myositis can less commonly contribute to diplopia in patients with IgG4-ROD. (Kubota, Goto clinical features, Sogabe location) As is the case with other manifestations of IgG4 disease, most patients with orbital involvement have an insidious onset with mild signs of inflammation, and symptoms that are predominantly due to orbital mass effect. (Andrew)

Dacryoadenitis The lacrimal gland is the most commonly involved orbital structure in IgG4-ROD. (Chou, Takahira, Chaudry, Wallace, Yu, Andrew clinical features) A large, multicenter retrospective review of 378 patients with IgG4-ROD found lacrimal gland involvement in 86% of patients. (Goto clinical features) A study of 255 patients with IgG4-ROD found lacrimal gland involvement in 98% of patients. (Zhao) Other studies have noted rates ranging from to 66 – 100%, with a higher likelihood of lacrimal gland involvement in patients with bilateral disease. (Ebbo, Kubota, Sato, Goto clinical features, Chou, Hamaoka, Sogabe location, Klingenstein, Detiger) Patients with lacrimal gland involvement tend to have associated eyelid edema, as well as symptoms and sequelae of keratoconjunctivitis sicca. (Takano recent advances, Zhao) Studies have demonstrated that patients with IgG4-ROD who have lacrimal gland findings are more likely to have systemic manifestations of IgG4-RD. (Ebbo) Patients with IgG4-related lacrimal gland enlargement may also have bilateral, symmetric salivary gland enlargement, a condition called Mikulicz disease. (Takahira, Chaudry) This condition was previously thought to be a part of Sjögren’s syndrome but has since been recognized as a separate disease that falls under the spectrum of IgG4-ROD. (Yamamoto)

Orbital Myositis Extraocular muscle enlargement has been identified in approximately 21% of patients with IgG4-ROD in a large retrospective study of 378 patients. (Goto clinical features). A study of 255 IgG4-ROD patients found extraocular muscle involvement in only 8% of patients. (Zhao) Other smaller studies have noted orbital myositis in 20 – 86% of patients. (Chou, Detiger, Ebbo, Hamaoka, Sogabe location, Hardy). The most commonly involved muscles are the inferior rectus, lateral rectus, and the superior rectus-levator complex, followed by the medial rectus and oblique muscles. (Klingenstein, Sogabe location) Extraocular muscle involvement nearly always co-occurs with other manifestations of IgG4-ROD, most commonly lacrimal gland findings. (Thompson)

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Other Orbital Soft Tissue Inflammation In addition to involvement of specific intraorbital structures, patients with IgG4-ROD may develop nonspecific orbital soft tissue lesions that can lead to proptosis and vision changes. These lesions may be mass-like or may present as diffuse orbital fat inflammation. (Sogabe location) In a review of 378 patients with IgG4-ROD, 19% of cases had isolated or diffuse orbital lesions. (Goto clinical features) Smaller studies reported focal orbital soft tissue lesions in 17 – 71% of patients (Detiger, Sogabe location, Ebbo, Hardy) and diffuse orbital fat inflammation in 23% (Sogabe location). Orbital inflammation from IgG4-ROD may frequently be misdiagnosed as orbital benign lymphoid hyperplasia or idiopathic orbital inflammation. (Andrew analysis of IgG4, Mehta, Aryasit, Sato, Matsuo) The presence of orbital inflammation can significantly impact vision, with 40% of patients reporting vision loss. (Deschamps) The majority of vision changes in IgG4-ROD patients are attributed to the compressive effect from these mass lesions causing extraocular muscle restriction and diplopia, though rarely patients can develop compressive optic neuropathy (Kubota). Approximately 12 – 30% of IgG4-ROD patients present with restricted extraocular movements, with higher rates among patients with diffuse orbital lesions compared to those with extraocular muscle enlargement. (Deschamps, Wu, Sogabe location) This suggests that the extraocular muscle restriction and ensuing diplopia in patients with IgG4-ROD is due to mass effect, rather than direct extraocular muscle infiltration, in contrast to other restrictive orbitopathies such as thyroid eye disease. (Goto clinical features, Sogabe location) In some cases, severe orbital inflammation from IgG4-ROD has resulted in optic neuropathy and even required orbital decompression to prevent permanent vision loss. (Chen successful decompression, Hardy, Hamaoka)

Nerve Involvement Both systemic IgG4-RD and IgG4-ROD can present with nerve involvement. The overall prevalence of perineural lesions in patients with IgG4-RD is approximately 6% in a study of 106 patients, with 71% of affected patients presenting with multiple perineural lesions. (Inoue) In patients with IgG4-RD, paravertebral nerves such as cervical, lumbar, and sacral spinal nerves are most frequently involved. (Inoue) Among patients with IgG4-ROD, the prevalence of perineural lesions within the orbit is estimated to be approximately 33%. (Inoue) Branches of the trigeminal nerve are most commonly involved, affecting 20 – 39% of patients with IgG4-ROD. (Goto clinical features, Inoue, Sogabe location) Lesions often occur proximally near foramina. (Inoue, Hamaoka) Other nerves that have been implicated in IgG4 disease include the optic nerve and the greater auricular nerve. (Inoue, Hamaoka, Mulholland, Takano) Lesions to nerves within the orbit are almost always associated with other findings of IgG4-ROD, especially dacryoadenitis. (Inoue) Nerve involvement is usually identified as a well-circumscribed perineural soft tissue mass on radiographic imaging. (Inoue) Typically, histopathology shows dense lymphoplasmacytic infiltration by IgG4+ plasma cells affecting the epineurium without involvement of the nerve fascicles. Sparing of the fascicles likely explains the frequent lack of neurologic deficits, though patients may have symptoms of lesion-related mass effect. (Inoue, Sogabe location, Hardy)

Infraorbital Nerve The infraorbital nerve is the most commonly involved nerve in IgG4-ROD, typically presenting with nerve enlargement on radiographic imaging. (Sogabe, Inoue, Klingenstein, Soussan) Although most patients have no neurologic deficits from these lesions, there are occasional reports of patients experiencing sensory impairment. (Mulholland, Takano) A study of 65 patients with IgG4-ROD found infraorbital nerve lesions in 32% of patients. (Sogabe location) In a study of 13 patients with IgG4-ROD, 23% were found to have infraorbital nerve enlargement. (Klingenstein) In a retrospective review of 36 orbits of 18 patients with IgG4-ROD, 5 (14%) orbits had an infraorbital nerve lesion, with bilateral lesions in two patients. (Inoue) Another study of 25 patients with IgG4-ROD found infraorbital nerve involvement in 20% of patients. (Chou) A study of 68 patients with Mikulicz disease (IgG4-ROD with lacrimal gland and salivary gland enlargement) showed infraorbital nerve enlargement in 30% of patients (Takano infraorbital). One study showed much higher rates of infraorbital nerve involvement in 20 of 28 orbits (71%). This may reflect a potential association between infraorbital nerve enlargement and other orbital lesions, as 100%

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of their cohort also had extraocular muscle enlargement. (Hardy) Infraorbital nerve enlargement has also been shown to occur more frequently in patients with inflammation of the inferior orbit, as demonstrated by a study of 15 patients with IgG4-ROD in which infraorbital nerve enlargement occurred in 80% of patients with inferior orbital inflammation and in no patients with isolated superior orbital inflammation (p < 0.001). (Soussan) One study has suggested that infraorbital nerve involvement may be a late finding of IgG4-ROD, with changes not detected until 2 to 13 years after onset of ophthalmic symptoms. (Hardy) Another study found a higher likelihood of having multiple organ lesions and disease complications among patients with infraorbital nerve lesions, as well as a significant increase in serum IgG4 levels as infraorbital nerve diameter increased. (Takano infraorbital) This further supports the notion that infraorbital nerve enlargement among patients with IgG4-ROD may reflect chronic or more severe disease. Enlargement of the infraorbital nerve can also be used to distinguish IgG4-ROD from other causes of orbital inflammation. In a study of 15 patients with IgG4-ROD and 23 patients with nonspecific orbital inflammation, infraorbital nerve enlargement was found in 53% of IgG4-ROD cases and was not present in a single case of nonspecific orbital inflammation (p < 0.0001). (Soussan) Another study evaluating infraorbital nerve enlargement among patients with various orbital lymphoproliferative disorders (16 patients with IgG4-ROD, 45 with non-Hodgkin lymphoma, 5 with reactive lymphoid hyperplasia, and 5 with idiopathic orbital inflammation) found infraorbital nerve enlargement in 41% of patients with IgG4ROD and in no patients in the other groups (p < 0.0001). (Ohshima)

Supraorbital Nerve The supraorbital nerve is also frequently involved in IgG4-ROD, though slightly less often than the infraorbital nerve. In a study of 36 orbits with IgG4-ROD, 11% of orbits had supraorbital nerve lesions (compared to 14% with infraorbital nerve lesions). (Inoue) Another study of 16 patients with IgG4-ROD found supraorbital nerve involvement in 13% of patients, while 31% had infraorbital nerve lesions. (Takahira clinical aspects) In a study of 65 IgG4-ROD patients, 17% had supraorbital nerve lesions compared to 32% with infraorbital nerve enlargement. (Sogabe location) A study of 26 orbits with IgG4ROD found supraorbital nerve involvement in 19% of orbits and infraorbital nerve involvement in 23% of orbits. (Klingenstein) Supraorbital nerve lesions may occur in isolation, but are typically associated with other perineural lesions, most commonly in the infraorbital nerve. (Inoue, Chen, Takahira clinical aspects)

Optic Nerve Optic nerve lesions in IgG4-ROD are less common than infraorbital nerve lesions but can lead to significant vision loss. (Mulholland, Takano, Takahashi) These lesions almost always occur in conjunction with supraorbital or infraorbital nerve lesions. A study of 16 patients with IgG4-ROD found 2 (13%) cases of optic nerve lesions, one of which co-occurred with an infraorbital nerve lesion and the other with both supraorbital and infraorbital lesions. (Takahira clinical aspects) Another study of 18 patients with IgG4ROD found optic nerve involvement in 22% of patients, all of whom also had infraorbital or supraorbital nerve lesions. (Inoue) One patient developed vision loss, which recovered with steroid therapy. (Inoue) A study of 56 patients with IgG4-ROD found lesions surrounding the optic nerve in 13% of patients, all of whom were male. (Hamaoka) All cases had associated lacrimal gland involvement, and 86% also had a trigeminal nerve lesion. (Hamaoka) Fifty seven percent of the patients with optic nerve lesions had vision changes (decreased visual acuity, visual field loss) consistent with optic neuropathy. Most of these patients responded well to steroid treatment but one patient experienced poor visual recovery. (Hamaoka) Another study described a patient who developed bilateral optic nerve lesions seven years after treatment for IgG4-related dacryoadenitis, highlighting that IgG4-ROD nerve lesions can be a late finding. (Hamaoka)

Other Periocular Manifestations Nasolacrimal System In addition to orbital lesions, IgG4-ROD can rarely present with nasolacrimal system involvement. (Sogabe location, Klingenstein) A study of 13 patients found nasolacrimal system and ethmoidal cell infiltration in 15% of patients. (Klingenstein) In another study of 65 patients with IgG4-ROD, only one (2%)

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patient had nasolacrimal system infiltration. (Sogabe location) These patients were relatively asymptomatic, with nasolacrimal system involvement detected on radiographic imaging. There is one reported case of a patient who presented with epistaxis and white nasal discharge who was found to have a firm sinonasal mass arising from the nasolacrimal duct, completely obstructing the left nasal vestibule. (Khoo) Biopsy was performed and showed histopathological findings consistent with IgG4 disease, and serum studies revealed elevated IgG4. (Khoo) The mass was treated with endoscopic excision and systemic steroids without evidence of recurrence. (Khoo) There are additional reports in the literature of sinonasal masses in patients with IgG4-RD, but none that appeared to emanate from the nasolacrimal system. (Khoo)

Sinuses Sinus changes can occur both in patients with IgG4-RD and IgG4-ROD. Sinus findings typically occur in association with other IgG4-related lesions, and usually more than one sinus is involved. (Khoo, Hardy) The maxillary sinus is most commonly affected, followed by the ethmoid and sphenoid sinuses. (Khoo) In a study of 20 orbits with infraorbital nerve enlargement from IgG4-ROD, 60% were found to have ipsilateral maxillary sinus involvement. (Hardy) Sinus involvement can include sinusitis, such as mucosal thickening or sinus opacification, or mass-like lesions within the sinus. (Hardy, Khoo) A literature review of sinus disease in IgG4-ROD from 2011 to 2021 identified 15 case reports of patients with IgG4-ROD and sinonasal masses, occurring in all four sinuses. (Khoo) Patients with IgG4 disease and sinus involvement may be asymptomatic or may present with nonspecific symptoms including facial pain, headache, rhinorrhea, epistaxis, or nasal obstruction. (Khoo)

Intracranial Findings Patients with IgG4 disease may rarely present with intracranial findings. A study of 97 patients with IgG4RD found pachymeningitis in 6.2% of patients, most commonly involving the transverse sinus dura and tentorium cerebelli. (Yardimci) Another study of 65 patients with IgG4-ROD found one (2%) patient with a cavernous sinus lesion. (Sogabe location) In another case, trigeminal nerve enlargement occurred within the cavernous sinus in a patient with other IgG4-ROD findings including enlarged lacrimal glands, bilateral perineural optic nerve enhancement, infraorbital and supraorbital nerve enlargement, and maxillary sinus mucosal thickening. (Chen) Another case demonstrated middle ear and middle cranial fossa lesions with associated cranial nerve II, VI, and VII palsies, with biopsy revealing histopathological findings consistent with IgG4 disease. (Wick) These cases highlight the importance of thorough evaluation and diagnostic workup in patients with suspected inflammatory lesions of unclear etiology.

Workup Imaging Radiographic imaging plays a critical role in the diagnosis of IgG4 disease. Given the insidious disease onset and typically mild, nonspecific symptoms, imaging can be used to identify findings such as infraorbital nerve enlargement that can support a diagnosis of IgG4 disease. (Klingenstein, Sogabe location) Radiographic imaging can also be used to identify potentially vision-threatening lesions in the early stages before permanent vision loss occurs. (Klingenstein, Inoue) Computed tomography (CT) and magnetic resonance imaging (MRI) are the most common imaging modalities used to evaluate IgG4 disease. (Klingenstein, Inoue, Sogabe location) Imaging can demonstrate any of the range of IgG4-ROD findings, including lacrimal gland or extraocular muscle enlargement, diffuse or nodular eyelid lesions, orbital fat inflammation, smooth-appearing perineural lesions, generalized enlargement of orbital nerves without an isolated mass lesion, bony infiltration, or other focal or diffuse orbital lesions. (Klingenstein, Andrew, Sogabe location, Kubota, Hardy, Inoue) Full body imaging, such as CT scanning of the chest, abdomen, and pelvis, is also recommended in patients with IgG4-ROD to detect systemic lesions. (Chaudry) For example, one report describes a patient who presented with IgG4-ROD with associated optic neuropathy secondary to bilateral perineural lesions, as well as lacrimal gland, extraocular muscle, and trigeminal nerve enlargement. (Hamaoka)

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Despite a lack of systemic symptoms, the patient subsequently underwent full body CT imaging that revealed numerous systemic lesions of the lungs, mediastinal lymph nodes, aorta, pituitary gland, stomach, liver, and kidneys. (Hamaoka) Positron emission tomography (PET) can also be used to identify IgG4 disease. (Inoue, Arora, Balakrishnan, Chaudry, Zhao IgG4 ocular) IgG4-related lesions demonstrate increased uptake with a variety of radiotracers, including traditional 18F-fluorodeoxyglucose and less commonly used 68GalliumDOTANOC, the latter of which binds to somatostatin receptors in inflammatory cells. (Arora, Balakrishnan, Inoue, Khosroshahi)

Laboratory Testing Serum IgG4 levels should be checked in every patient with suspected IgG4 disease. Studies suggest that elevated serum IgG4 is a reaction to inflammation, rather than a cause of inflammation. (Stone) Although serum IgG4 can be elevated in a variety of inflammatory conditions and is not unique to IgG4-RD, serum IgG4 greater than 135 mg/dL is included in multiple different sets of diagnostic criteria for IgG4-RD. (Desphande, Umehara, Soto, Chaudry) An increased ratio of IgG4/IgG is also found in most patients with IgG4-RD. (Zhao IgG4 ocular) In a study comparing 255 patients with IgG4-ROD to 179 patients with nonophthalmic IgG4-RD, serum IgG4 levels were significantly higher in IgG4-ROD patients (mean 983 mg/DL) than in those with non-ophthalmic IgG4-RD (mean 523 mg/dL) (p < 0.001). (Zhao IgG4 ocular). Patients with IgG4-ROD also demonstrated a higher IgG4/IgG ratio (mean 0.53 vs. 0.29, p < 0.001) and higher serum IgE levels (340 IU/mL vs. 259 IU/mL, p = 0.022) than those with non-ophthalmic IgG4-RD. (Zhao IgG4 ocular) This suggests that serum IgG4 may be particularly useful in the workup of patients with suspected IgG4-ROD. However, serum IgG4 levels may be normal in a minority of patients (up to 40%) with IgG4-RD, including those with IgG4-ROD, and therefore should not be the sole determinant of the presence or absence of IgG4 disease. (Hardy, Sah, Khosroshahi) Nonetheless, serum IgG4 levels can be a useful clinical marker. Higher serum IgG4 levels have been found to correlate with higher rates of bilateral disease and a greater likelihood of having lacrimal gland involvement. (Wallace, Chou) Very high serum IgG4 (greater than 900 mg/dL) has been found to correlate with higher rates of extraorbital lesions in patients with IgG4-ROD, such as salivary gland or lymph node involvement. (Kubota) IgG4 levels have also been shown to correlate with disease activity, with peak IgG4 levels prior to treatment and a subsequent decline after treatment. (Chou, Balakrishnan) While IgG4 levels do not always normalize after treatment, normal post-treatment IgG4 levels correlate with better treatment response. (Chou) After a nadir immediately post-treatment, many patients will demonstrate fluctuating IgG4 levels, which may not necessarily reflect ongoing disease activity. In a retrospective analysis of 25 patients with IgG4-ROD treated with steroids, 64% of patients had elevated serum IgG4 at most recent follow-up, including among 53% of patients who were deemed to be in disease remission without symptoms or evidence of disease on imaging. (Chou) A multicenter study of 563 patients with IgG4-related autoimmune pancreatitis found that serum IgG4 levels remained elevated in 63% of patients after steroid treatment, but only 30% of patients with persistently elevated IgG4 had disease relapse. (Kamisawa) Nevertheless, higher rates of serum IgG4 before treatment, as well as posttreatment spikes in IgG4 levels, have been shown to increase likelihood of disease recurrence. (Chou)

Biopsy All patients with suspected IgG4 disease should undergo biopsy, as this is a key component of diagnosis and can help distinguish IgG4-RD from other causes of inflammation. (Balakrishnan, Deschamps, Chen, Sato, Deshpande) The presence of IgG4+ plasma cells contributes to the diagnosis of IgG4-RD but can occur in a variety of inflammatory conditions, and therefore the histopathologic diagnosis of IgG4 disease is based on a constellation of typical findings. (Deshpande) In systemic IgG4-RD, hallmark histopathologic features of IgG4 disease are the presence of dense lymphocytic infiltrate with plasma cells rich in IgG4 (>10 IgG4+ cells per high-power field), fibrosis that assumes a whorled “storiform” pattern, and obliterative phlebitis. (Balakrishnan, Deshpande, Chaudhry, Umehara, Andrew) The presence of two of the three aforementioned histopathological features, and particularly coexistence of the first two findings, is strongly indicative of IgG4-RD. (Deshpande) Mild to moderate eosinophilia may also be present. (Balakrishnan, Wallace, Chaudhry) Histopathological analysis of biopsies from various systemic sites usually appear quite similar in IgG4-RD regardless of the tissue of origin except for salivary

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gland, lymph node, and orbital biopsies. (Desphande, Chaudhry, Sato) In these tissues, storiform fibrosis and obliterative phlebitis are often absent (Desphande, Sato). Obliterative phlebitis in particular is rarely present in orbital biopsies; a study of 21 orbital biopsies found obliterative phlebitis in only 9.5% of samples. (Sato) Another study of biopsies from 10 IgG4-ROD patients found obliterative phlebitis in only one (10%) sample. (Deschamps) A histopathological study of 7 patients with IgG4-ROD found storiform fibrosis in one (14%) patient and found no cases with obliterative phlebitis. (Andrew) It is hypothesized that the low rates of obliterative phlebitis seen in orbital samples is due to the relatively small size of orbital veins, making it challenging to identify this histopathological feature once the veins are obliterated. (Andrew) Although orbital biopsies in IgG4-ROD may not always show the classic histopathological features seen in systemic IgG4-RD, IgG4 staining can be a useful way to distinguish IgG4-ROD from other causes of orbital inflammation. (Chen, Sato, Andrew) In a retrospective study of 25 patients who had been previously diagnosed with idiopathic orbital inflammation on biopsy (without initial IgG4 staining), the subsequent addition of IgG4 staining led to a diagnosis of IgG4-ROD among 40% of the study cohort. (Deschamps) Other studies performing retrospective immunohistochemical evaluation of biopsies from patients initially diagnosed with orbital pseudotumor revealed that 25 – 50% of patients met criteria for diagnosis of IgG4-ROD. (Andrew analysis of IgG4, Mehta, Aryasit) Although IgG4+ plasma cells may be present in other orbital inflammatory conditions, higher density of IgG4+ plasma cells (>10 IgG4+ plasma cells/hpf) suggests IgG4-ROD. (Andrew, Desphande) Furthermore, biopsies in patients with IgG4-ROD have been shown to have greater amounts of lymphoplasmacytic infiltration and germinal center formation compared to biopsies of idiopathic orbital inflammation. (Andrew) Biopsies of nerve lesions often show similar findings to other tissue biopsies in IgG4-ROD. In one histopathology report from an infraorbital nerve lesion, dense lymphoplasmacytic infiltration rich in IgG4+ plasma cells (107 IgG4+ plasma cells per high-power field), scattered eosinophils, and irregular fibrosis were demonstrated without any obliterative phlebitis. (Inoue) The infiltration and fibrosis involves the epineurium with relative sparing of the perineurium and is absent in the nerve fascicles. (Sogabe)

Diagnostic Criteria IgG4-ROD can be difficult to diagnose, in part due to its varied and often nonspecific presenting features. Most experts agree that the most accurate way to diagnose IgG4-RD is multifactorial, requiring a combination of a complete history and physical exam, serum lab studies, radiologic imaging, and biopsy. (Khosroshahi) Previous diagnostic criteria for IgG4 disease are for systemic forms of IgG4-RD and include a combination of localized or diffuse tissue swelling, elevated serum IgG4 levels, and histopathology showing dense lymphoplasmacytic infiltrate with a high concentration of IgG4+ cells, storiform fibrosis, and obliterative phlebitis. (Deshpande, Umehara) In 2011, an international group of experts proposed a set of criteria specifically for the diagnosis of IgG4-ROD based solely on histopathology. (Desphande) These criteria require at least one of the hallmark histopathology findings (dense lymphoplasmacytic infiltrate, storiform fibrosis, or obliterative phlebitis) on lacrimal gland biopsy or at least two of those features on orbital biopsy, as well as a ratio of IgG4+ cells to IgG+ cells > 40% and >100 IgG4+ cells per high-power field. (Deshpande) However, even these criteria could miss cases of IgG4-ROD, since characteristic features such as storiform fibrosis and obliterative phlebitis are more likely to be absent on histopathology samples from the orbit. (Andrew, Deshpande) In 2014, an IgG4-RD study group in Japan proposed updated criteria for the diagnosis of IgG4-ROD. (Goto diagnostic) Criteria included imaging showing enlargement of the lacrimal gland, extraocular muscles, or trigeminal nerve with other associated orbital soft tissue lesions, significant lymphoplasmacytic infiltration on histopathology with possible fibrosis, and either an elevated ratio of IgG4+ cells to IgG+ cells of greater than or equal to 40% or greater than 50 IgG4+ cells per high-power field. (Goto diagnostic) A diagnosis of IgG4-ROD was considered definitive when all three criteria were met, probable when the first two criteria were met, and possible when the first and third criteria were met. (Goto diagnostic)

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Treatment There are no formal guidelines for the treatment of IgG4 disease, with most treatment data based on retrospective studies. (Chaudhry) An international panel of 42 experts convened in 2014 to attempt to assemble a unified set of recommendations for treatment of systemic IgG4-RD, but even this group could not reach consensus regarding certain aspects of management. (Khosroshahi) The aim of treatment is to address symptoms of disease, reduce inflammation, and prevent fibrosis and organ damage. (Chaudhry) All patients who are symptomatic from IgG4-RD, and some asymptomatic patients, should receive treatment. (Khosroshahi) A subset of patients who are asymptomatic may be appropriately managed with close observation without medication depending on the organs involved, such as patients who only have asymptomatic lymphadenopathy. (Khosroshahi) Steroids Steroids are widely recognized as the first-line treatment for patients with previously untreated IgG4 disease, with successful initial treatment response in 92 – 100% of patients. (Khosroshahi, Balakrishnan, Wallace, Hart, Brito-Zeron) Disease response to steroids may be slightly lower among patients with IgG4ROD. A systematic review and meta-analysis of 35 studies on treatment of IgG4-ROD found that 89% of patients responded to initial treatment with steroids. (Detiger) While studies vary on the use of intravenous versus oral corticosteroids, most experts recommend a dose of 30 – 40 mg of prednisone per day for 2 – 4 weeks, followed by a slow taper of 5 – 10 mg every two weeks. (Khosroshahi) Treatment response should be monitored using a combination of repeat serum IgG4 testing and imaging. (Balakrishnan) The goal is to discontinue steroids within 3 – 6 months, though some experts recommend maintenance dosing with low-dose corticosteroids for up to three years to prevent relapse. (Khosroshahi) There is a high risk of disease recurrence both during steroid taper and after completion of steroid treatment, with relapse rates ranging from 33 – 68% among patients with IgG4-ROD. (Detiger, Chou, Ebbo, Hong, Khosroshahi, Hardy, Brito-Zeron) Patients with disease relapse can be retreated with steroids with a gradual taper after evidence of treatment response. (Khosroshahi) Even patients on maintenance dose steroids for IgG4-RD can experience disease recurrence, with relapse rates of 10 – 23% among patients with IgG4-related autoimmune pancreatitis. (Kamisawa standard steroid, Kamisawa amendment, Hart) Steroid-Sparing Agents Given the high rates of relapse, the side effects of corticosteroids, and the potential reduced efficacy of steroids over time, many experts advocate for the use of steroid-sparing immunomodulatory medications for IgG4 disease. This may be especially important in patients who experience disease recurrence after steroid treatment. (Wallace IgG4 idiopathic) Some experts advocate for the use of steroid-sparing agents concurrently with steroids as first line treatment, which may lead to shorter steroid duration, lower rates of disease relapse, and a longer relapse-free period post-treatment. (Chou, Gan, Zhao IgG4 ocular) Others recommend the use of steroid-sparing agents as maintenance therapy after tapering off steroids or as second line treatment for patients who have relapsed. (Khosroshahi, Wallace IgG4 idiopathic, Ebbo) The most commonly used steroid-sparing agents in descending order of frequency reported are rituximab, methotrexate, cyclophosphamide, azathioprine, mycophenolate mofetil, infliximab, and cyclosporine. (Detiger) A retrospective study evaluated treatment outcomes in 255 patients with IgG4-ROD who received either corticosteroid monotherapy or corticosteroids plus a non-steroidal immunosuppressant. (Zhao IgG4 ocular) Steroid-sparing agents used in this study were rituximab, cyclophosphamide, mycophenolate mofetil, methotrexate, azathioprine, iguratimod, or leflunomide, with a median treatment duration of 21 months (range 8 – 36). Those who received steroids combined with a non-steroidal immunosuppressive medication had a 72% reduced risk of disease recurrence and a longer median time to relapse (43 months) compared to those who received steroid monotherapy (17 months) (p < 0.001). (Zhao) In a large meta-analysis of IgG4-ROD treatment, good initial treatment response was observed in 46% of patients treated with methotrexate (n = 26), 75% on cyclophosphamide (n = 16), 36% on azathioprine (n = 14), 70% on mycophenolate mofetil (n = 10), 80% on infliximab (n = 5), and 93% on rituximab (n = 57). (Detiger) However, there are few studies directly comparing the relative efficacy of these medications, with very limited prospective data. (Khosroshahi, Wallace IgG4 idiopathic, Ebbo)

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Rituximab Rituximab has thus far been shown to be the most effective of the non-steroidal medications for the treatment of IgG4 disease. It has demonstrated treatment success both as a maintenance therapy following steroid treatment and as monotherapy, including among patients who initially failed steroid therapy. (Wallace IgG4 idiopathic, Chaudry, Khosroshahi, Detiger, Brito-Zeron) Initial rituximab treatment response ranges from 93 – 100%. (Detiger, Brito-Zeron) The treatment regimen consists of two doses of intravenous rituximab, 1,000 mg per dose, given 15 days apart. (Chaudry, Carruthers, Wallace IgG4 idiopathic, Detiger) Remission rates in patients treated with rituximab, including those who relapsed after initial steroid treatment, range from to 83 – 91%. (Ebbo, Khosroshahi, Detiger) However, even patients treated with rituximab may experience disease recurrence. (Caruthers, Kubota) In a prospective study of 30 patients treated with two 1,000 mg doses of rituximab for IgG4-RD, 97% of patients demonstrated significant improvement in disease burden at six months, with 47% of patients showing complete remission at six months, and only 40% of patients maintaining remission at 12 months. (Carruthers) Radiation Radiation therapy has rarely been used as a treatment for recurrent or refractory IgG4-ROD. In a metaanalysis of 95 patients with IgG4-ROD, 6% of patients with recurrent IgG4-ROD received radiotherapy (10 – 15 fractions of 20 – 30 Gy each) in addition to treatment with corticosteroids and/or steroid-sparing agents, with a good treatment response seen in 67% of these patients. (Detiger) A case report of a patient with bilateral, sequential IgG4-related orbital inflammation refractory to steroids describes a good, lasting treatment response after radiation therapy (2,500 cGY of radiation to each orbit across 17 fractions) to the orbits. (Rutter) A case series of three patients with IgG4-ROD refractory to steroids reported a good treatment response after patients received 10 fractions of radiotherapy for a total of 2,000 cGY. (Lin) All patients were able to taper off corticosteroids following radiotherapy without evidence of remission over long-term follow-up (ranging 22 to 42 months). While these cases suggest a potential long-term benefit of radiotherapy for IgG4-ROD, one study suggested that radiotherapy, particularly in patients who had surgical debulking and oral steroids, may increase the risk of relapse. (Chen clinical features)

Discussion IgG4 disease can affect nearly any organ in the body and any orbital adnexal tissue, with a wide range of presenting signs and symptoms. Understanding the features of IgG4 disease and the importance of biopsy is especially critical for the diagnosis of IgG4-ROD, as it can be frequently misdiagnosed due to the clinical and radiologic similarities to other orbital inflammatory or lymphoproliferative conditions. This is particularly apparent in cases where biopsy is not obtained or IgG4 histopathological staining is not used. Retroactive immunohistochemical staining was consistent with IgG4-ROD in as many as 50% of patients previously diagnosed with idiopathic orbital inflammation. (Andrew, Mehta, Deschamps, Oshima, Takahira) Other studies have found that IgG4-ROD accounts for 23% of cases diagnosed as benign lymphoid hyperplasia and lymphoma, and 63% of patients diagnosed with “benign lymphoid lesions.” (Sato, Matsuo) Biopsy in patients with suspected IgG4-ROD is also important to rule out malignancy, as there can be overlap in presentation between IgG4-ROD and orbital lymphoma. A study of 13 patients with IgG4-ROD and 29 patients with ocular adnexal lymphoma found similar radiographic findings between the two groups, including comparable rates of lacrimal gland and extraocular muscle enlargement, orbital fat inflammation, and infraorbital nerve involvement. (Klingenstein) Distinguishing features of IgG4-ROD in this study were slightly higher rates of bone involvement and lacrimal system infiltration, smaller lesions, and a higher rate of disease recurrence. (Klingenstein) Interestingly, the IgG4ROD group had a longer time to diagnosis than the ocular adnexal lymphoma group. Ocular adnexal lymphoma, in particular, can be difficult to differentiate from IgG4-ROD, as approximately 9% of patients will have infiltration by IgG4+ cells on biopsy and elevated serum IgG4. (Kubota) In these cases, definitive diagnosis may require additional testing with southern blot analysis or in situ hybridization, in which ocular adnexal lymphoma will show immunoglobulin heavy chain gene rearrangements and light chain restrictions, respectively. (Kubota)

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In addition to biopsy findings, certain clinical features can increase suspicion for IgG4-ROD over other orbital pathology. IgG4-ROD tends to be bilateral, often involving the lacrimal glands, and tends to present insidiously with minimal inflammatory signs. The presence of trigeminal nerve enlargement, commonly of the infraorbital or supraorbital nerve, can also help distinguish IgG4-ROD from other orbital inflammatory conditions. (Chen, Hardy) Although isolated unilateral infraorbital nerve enlargement may occur in various tumors, such as neurogenic tumors, myofibroblastic tumors, or perineural spread of malignancies such as squamous cell carcinoma or adenoid cystic carcinoma, the lack of associated neurologic symptoms due to sparing of the nerve fascicles and the presence of bilateral nerve involvement are more likely to occur in IgG4-ROD. (Sogabe, Soussan, Takano infraorbital nerve) Properly identifying patients who have IgG4-ROD is important not only for treatment purposes, but also to initiate screening for systemic lesions, as patients with IgG4-ROD have up to an 80% chance of having IgG4-related systemic lesions that can lead to fibrosis and permanent organ damage. The presence of bilateral IgG4-ROD or lacrimal gland involvement may increase the likelihood of simultaneous systemic disease. (Ebbo, Wu) Furthermore, patients with IgG4-RD may be at increased risk of malignancy, especially non-Hodgkin lymphoma. (Andrew risk of malignancies). Some studies suggest that mucosaassociated lymphoid tissue (MALT) lymphoma may potentially arise from pre-existing IgG4-ROD with malignant transformation in approximately 10% of IgG4-ROD patients, emphasizing the importance of timely, accurate diagnosis. (Cheuk, Takahira, Sato) Patients with IgG4-ROD may also benefit from early diagnosis to prevent vision loss, which can occur via a variety of mechanisms including optic nerve involvement, extraocular muscle enlargement, or compression from orbital soft tissue inflammation. (Ebbo) Additionally, IgG4-ROD has higher rates of disease recurrence compared to other inflammatory orbital conditions, and therefore IgG4-ROD patients require extended duration of follow-up and potential adjuvant treatment in addition to corticosteroids. There are no concrete treatment guidelines for patients with IgG4-ROD, and the currently recommended first-line therapy of corticosteroids often achieves initial remission but may not be effective long term in up to 68% of patients. Recent studies suggest that rituximab may be a more effective and longer-lasting treatment than steroids for IgG4-ROD; however, randomized controlled trials evaluating both rituximab and corticosteroids are needed to better understand the relative efficacy and durability of these two medications. Patients diagnosed with IgG4 disease require extended duration follow-up given high rates of disease relapse. Risk factors for recurrence include the presence of multiple ophthalmic lesions and extraocular muscle or trigeminal nerve enlargement. (Chen clinical features) Additionally, patients receiving corticosteroid monotherapy, as opposed to steroids plus non-steroidal immunotherapy, have a higher risk of disease recurrence. Even patients receiving non-steroidal agents can have disease relapse and therefore also require extended follow-up. (Detiger) Perineural lesions of the infraorbital, supraorbital, or optic nerve can be a late finding in IgG4-ROD, with cases of nerve involvement occurring up to 13 years after initial IgG4-ROD onset, further emphasizing the importance of long-term screening and follow-up to prevent vision loss. (Hamaoka) In summary, IgG4-ROD can affect nearly any orbital tissue and typically presents subacutely with nonspecific symptoms such as eyelid edema, proptosis, extraocular movement restriction, and less commonly vision loss. It can present as isolated ophthalmic disease, which can mimic other orbital inflammatory or lymphoproliferative disorders, or in conjunction with systemic findings of IgG4-RD. Therefore, full body screening and accurate diagnosis are critical in order to prevent permanent vision loss, organ damage, and even death. Diagnosis usually requires biopsy with IgG4 staining, imaging to identify specific clinical features, and serum IgG4 testing. While steroids are the first-line treatment, IgG4ROD has high rates of disease relapse. Recent studies suggest a benefit of rituximab as adjuvant or monotherapy, though prospective trials are needed to further evaluate the optimal treatment regimen for patients with IgG4-ROD.

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Chen J, Zhang P, Ye H, et al. Clinical features and outcomes of IgG4-related idiopathic orbital inflammatory disease: From a large southern China-based cohort. Eye 2021;35:1248–1255.

Chen N, Huang TL, Hsu YH, et al. Orbital manifestations of immunoglobulin G4-related disease in bilateral lacrimal glands, optic nerves, trigeminal nerves, and maxillary sinuses. Neuro-Ophthalmology 2014;38(1):24-28.

10. Chen TS, Figueira E, Lau OC, et al. Successful "medical" orbital decompression with adjunctive rituximab for severe visual loss in IgG4-related orbital inflammatory 11. Cheuk W, Yuen HK, Chan JK. Chronic sclerosing dacryoadenitis: part of the spectrum of IgG4-related Sclerosing disease? Am J Surg Pathol 2007;31:643–5.) 12. Chou WY, Tsai CY, Tsai CC. Long-term follow-up in IgG4-related ophthalmic disease: Serum IgG4 levels and their clinical relevance. J Pers Med 2022;12:1963. 13. Deschamps R, Deschamps L, Depaz R, et al. High prevalence of IgG4-related lymphoplasmacytic infiltrative disorder in 25 patients with orbital inflammation: a retrospective case series. Br J Ophthalmol 2013;97:999-1004. 14. Desphande V, Zen Y, Chan JKC, et al. Consensus statement on the pathology of IgG4-related disease. Modern Pathology 2012;25:1181-1192. 15. Detiger SE, Faiz Karim A, Verdijk RM, et al. The treatment outcomes in IgG4-related orbital disease: a systematic review of the literature. Acta Ophthalmol 2019;97:451-459. 16. Ebbo M, Patient M, Grados A, et al. Ophthalmic manifestations in IgG4-related disease: Clinical presentation and response to treatment in a French case-series. Medicine 2017;96:10(e6205). 17. Goto H, Ueda S, Nemoto R, et al. Clinical features and symptoms of IgG4-related ophthalmic disease: a multicenter study. Jpn J Ophthalmol 2021;65:651-656. 18. Goto H, Takahira M, Azumi A, Japanese Study Group for IgG4-Related Ophthalmic Disease. Diagnostic criteria for IgG4-related ophthalmic disease. Jpn J Ophthalmol 2015;59:1-7. 19. Hamano H, Kawa S, Horiuchi A, et al. High serum IgG4 concentrations in patients with sclerosing pancreatitis. N Engl J Med 2001;344:732–8. 20. Hamaoka S, Takahira M, Kawano M, et al. Cases with IgG4-related ophthalmic disease with mass lesions surrounding the optic nerve. Am J Ophthalmol Case Rep 2022;25:101324. 21. Hardy TG, McNab AA, Rose GE. Enlargement of the infraorbital nerve: An important sign associated with orbital reactive lymphoid hyperplasia or immunoglobulin G4-related disease. Ophthalmology 2014;121(6):1297-1303.

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22. Hart PA, Kamisawa T, Brugge WR, et al. Long-term outcomes of autoimmune pancreatitis: a multicentre, international analysis. Gut 2013;62:1771-1776. 23. Inoue D, Zen Y, Sato Y, et al. IgG4-related perineural disease. Int J Rheum 2012;2012:401890-9. 24. Japanese study group of. IgG4-related ophthalmic disease. A prevalence study of IgG4-related ophthalmic disease in Japan. Jpn J Ophthalmol. 2013;57:573–9. 25. Kamisawa T, Funata N, Hayashi Y, et al. A new clinicopathological entity of IgG4- related autoimmune disease. J Gastroenterol 2003;38:982–4. 26. Kamisawa T, Shimosegawa T, Okazaki K, et al. Standard steroid treatment for autoimmune pancreatitis. Gut 2009;58:1504–1507. 27. Khoo V, Khoo HSJ, Goh LC. Nasolacrimal duct malignancy of IgG4-related disease? A curious case report of a nasal vestibular mass and review of the literature. Medeni Med J 2021;36:281-286. 28. Khosroshahi A, Wallace ZS, Crowe JL, et al. International consensus guidance statement on the management and treatment of IgG4-related disease. Arthr Rheum 2015;67(7):1688-1699. 29. Klingenstein A, Garip-Kuebler A, Priglinger S, et al. Morphologic cross-sectional imaging features of IgG4-related orbitopathy in comparison to ocular adnexal lymphoma. Clin Ophthalmol 2021;15:1119-1127. 30. Kubota T, Moritani S. Orbital IgG4-related disease: Clinical features and diagnosis. Int J Rheum 2012;2012:412896-5. 31. Lin YH, Yen SH, Tsai CC, et al. Adjunctive orbital radiotherapy for ocular adnexal IgG4-related disease: Preliminary experience in patients refractory or intolerant to corticosteroid therapy. Ocular Immun Inflamm 2015;23(2):162-167. 32. Matsuo T, Ichimura K, Sato Y, et al. Immunoglobulin G4 (IgG4)-positive or -negative ocular adnexal benign lymphoid lesions in relation to systemic involvement. J Clin Exp Hematop 2010;50:129–42 33. Mehta M, Jakobiec F, Fay A. Idiopathic fibroinflammatory disease of the face, eyelids, and periorbital membrane with immunoglobulin G4-positive plasma cells. Arch Pathol Lab Med 2009;133:1251-1255. 34. Mulholland GB, Jeffery CC, Satija P, et al. Immunoglobulin G4-related diseases in the head and neck: a systematic review. J Otolaryngol Head Neck Surg 2015;44:24. 35. Ohshima K, Sogabe Y, Sato Y. The usefulness of infraorbital nerve enlargement on MRI imaging in clinical diagnosis of IgG4-related orbital disease. Jpn J Ophthalmol 2012;56:380-382. 36. Sah RP, Chari ST. Serologic issues in IgG4-related systemic disease and autoimmune pancreatitis. Curr Opin Rheumatol 2011;23:108–113.) 37. Sato K, Notohara M, Kojima K, et al. IgG4-related disease: Historical overview and pathology of hematological disorders: review article. Pathol Int 2010;60(4):247–258. 38. Sato Y, Ohshima KI, Ichimura K, et al. Ocular adnexal IgG4-related disease has uniform clinicopathology. Pathology International 2008;58(8):465–470. 39. Sogabe Y, Ohshima K, Azumi A, et al. Location and frequency of lesions in patients with IgG4-related ophthalmic diseases. Graefes Arch Clin Exp Ophthalmol 2014;252:531-538. 40. Sogabe Y, Miyatani K, Goto R, et al. Pathological findings of infraorbital nerve enlargement in IgG4-related ophthalmic disease. Jpn J Ophthalmol 2012;56:511-514. 41. Soussan JB, Deschamps R, Sadik JC, et al. Infraorbital nerve involvement on magnetic resonance imaging in European patients with IgG4-related ophthalmic disease: a specific sign. Eur Radiol 2017;27:1335-1343. 42. Stone JH, Zen Y, Deshpande V. IgG4-related disease. N Engl J Med 2012;366:539-551. 43. Takahashi Y, Kitamura A, Kakizaki H. Bilateral optic nerve involvement in immunoglobulin G-4 related ophthalmic disease. J Neuro-Ophthalmol 2014;34:16-19.

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44. Takahira M, Ozawa Y, Kawano M, et al. Clinical aspects of IgG4-related orbital inflammation in a case series of ocular adnexal lymphoproliferative disorders. Int J Rheum 2012;2012:635473-5. 45. Takahira M, Kawano M, Zen Y, et al. IgG4-related chronic sclerosing dacryoadenitis. Arch Ophthalmol 2007;125:1575–8 46. Takano K, Yajima R, Seki N, et al. A study of infraorbital nerve swelling associated with immunoglobulin G4 Mikulicz’s disease. Modern Rheum 2014;24(5):798-801. 47. Takano K, Yamamoto M, Takahashi H, Himi T. Recent advances in knowledge regarding the head and neck manifestations of IgG4-related disease. Auris Nasus Larynx 2017;44:7-17. 48. Takuma K, Kamisawa T, Anjiki H, et al. Metachronous extrapancreatic lesions in autoimmune pancreatitis. Intern Med Tokyo Jpn 2010;49: 529–33 49. Thompson A, Whyte A. Imaging of IgG4-related disease of the head and neck. Clin Radiol. 2018;73(1):106–20. 50. Uchida K, Masamune A, Shimosegawa T, Okazaki K. Prevalence of IgG4-related disease in Japan based on nationwide survey in 2009. Int J Rheumatol. 2012;2012: 358371 51. Umehara H, Okazaki K, Masaki Y, et al. Comprehensive diagnostic criteria for IgG4- related disease (IgG4-RD). Mod Rheumatol 2012;22: 21–30. 52. Wallace ZS, Khosroshahi A, Jakobiec, et al. IgG4-related systemic disease as a cause of “idiopathic” orbital inflammation, including orbital myositis, and trigeminal nerve involvement. Surv Ophthalmol 2012;57(1):26-33. 53. Wallace ZS, Miles G, Smolkina E, et al. Incidence, prevalence, and mortality of IgG4-related disease in the USA: a claims-based analysis of commercially insured adults. Annals Rheum Dis 2023;82(7):957-962. 54. Wallace ZS, Deshpande V, Stone JH. Ophthalmic manifestations of IgG4-related disease: single-center experience and literature review. Semin Arthritis Rheum 2014;43:806–817. 55. Wick CC, Zachariah J, Manjila S, et al. IgG4-related disease causing facial nerve and optic nerve palsies: Case report and literature review. Am J Otol Head Neck Med Surg 2016;35:567-571. 56. Wu N, Sun FY. Clinical observation of orbital IgG4-related diseases. Exp Therapeutic Med 2019;17:883-887. 57. Yamamoto M, Ohara M, Suzuki C, et al. Elevated IgG4 concentrations in serum of patients with Mikulicz‘s disease. Scand J Rheumatol 2004;33:432–433 58. Yardimci GK, Arslan D, Babaoglu B, et al. IgG4-related pachymeningitis – long term follow up and outcome of six patients. Int J Rheum Dis 2023;26:1853-1860. 59. Zhao Z, Mou D, Wang Z, et al. Clinical features and relapse risks of IgG4-related ophthalmic disease: a singlecenter experience in China. Arthritis Research & Therapy 2021;23:98.

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Inflammation After Intravitreal Injections Saghar Bagheri, MD, PhD and John B. Miller, MD

Intravitreal injections, while generally safe and effective, can carry some potential complications. The risks of administering a medication intravitreally can be difficult to distinguish from the risks associated with the medication itself. Risks that have been associated with the procedure itself include ocular pain or discomfort, intraocular inflammation, cataract formation if the lens is contacted during the procedure, increased intraocular pressure, subconjunctival hemorrhage, retinal tears or detachment, infectious endophthalmitis, and allergic reactions. This review will focus on noninfectious intraocular inflammation (IOI) as a complication of intravitreal injections.

Introduction Intravitreal injections are a highly effective medical procedure used to treat various eye conditions. This technique involves the direct injection of medication into the vitreous cavity. This allows therapeutic agents to be delivered precisely to the site of pathology, increasing the bioavailability of the medication to the affected tissues while reducing systemic toxicity. This delivery mechanism, along with novel antiVEGF therapies, has revolutionized the management of retinal diseases such as age-related macular degeneration, diabetic retinopathy, and retinal vein occlusion, offering patients the possibility of improved vision and quality of life (TABLE 1). TABLE 1: Common Diseases Treated by Intravitreal Injections Neovascular Age-related macular degeneration (AMD) Diabetic Macular Edema (DME), Non-Proliferative Diabetic Retinopathy (NPDR), Proliferative Diabetic Retinopathy (PDR) Retinal Vein Occlusion (RVO) Endophthalmitis Uveitis Cystoid Macular Edema (CME), Choroidal Neovascular Membrane (CNVM) secondary to multiple retinal diseases

The history of intravitreal injections dates to the mid-20th century, with the first recorded injection in the 1980s(1,2). However, it was not until the late 20th and early 21st centuries that intravitreal injections gained widespread recognition and acceptance as a primary treatment modality(3). The development and introduction of anti-vascular endothelial growth factor (anti-VEGF) agents greatly expanded the use of intravitreal injection, particularly in the management of neovascular age-related macular degeneration (AMD), diabetic retinopathy, and other retinal vascular diseases. Intravitreal injections have become a standard and effective approach for delivering therapeutic agents precisely to the target site, minimizing systemic side effects and maximizing therapeutic benefits to the retina and vitreous(4). Continuous advancements in injection techniques and the ongoing refinement of pharmaceutical agents further contribute to the evolution of intravitreal therapy as a cornerstone in modern ophthalmic care.

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Intravitreal injections employ various techniques to deliver therapeutic agents directly into the vitreous cavity, targeting retinal and macular disorders. The approach involves inserting a fine needle through the pars plana to access the vitreous humor. This technique, known as the pars plana approach, allows for precise delivery of medications while minimizing trauma to surrounding structures. This can be done in conjunction with anterior chamber paracentesis, removal of a small amount of aqueous humor from the front of the eye, to reduce intraocular pressure before the injection (5). Recent advancements include the use of smaller gauge needles and refined injection devices, enhancing patient comfort and safety(6,7). The intravitreal injection procedure is typically performed with sterile technique, often in an outpatient clinic. As technology continues to progress, further innovations in injection techniques are likely to emerge, contributing to the continued improvement of intravitreal therapy.

Intravitreal Injection Medications Anti-VEGF Injections (eg, ranibizumab [Lucentis; Genentech] and aflibercept [Eylea; Regeneron] for approved uses; bevacizumab [Avastin; Genentech], commonly used off-label for a range of retinal diseases): These injections contain drugs that target vascular endothelial growth factor (VEGF), a protein responsible for abnormal blood vessel growth in the retina (TABLE 2). They are widely used to treat conditions such as age-related macular degeneration (AMD), diabetic retinopathy, and macular edema. Pegaptanib, approved in 2004, was initially a significant advancement in treating neovascular Age-Related Macular Degeneration (AMD), but it has largely become obsolete due to more effective anti-VEGF treatments. Bevacizumab, initially for colon cancer, has been widely used off-label for neovascular AMD since 2005, offering a cost-effective, full-length, humanized monoclonal antibody treatment. Ranibizumab, a specific ocular treatment developed later, targets all VEGFA isoforms and has shown promise but is less cost effective. Aflibercept, approved in 2011, targets VEGFA, VEGFB, and PGF. Brolucizumab (Beovu®; manufactured by Novartis) is a humanized monoclonal single-chain variable fragment (scFv) that binds and inhibits VEGF-A. Brolucizumab is a single-chain variable fragment with a molecular weight of 26 kD. Brolucizumab was FDA-approved for treatment of AMD in 2019 and for Diabetic Macular Edema in 2022. The expiration of patents for these drugs has spurred the development of anti-VEGF biosimilars, aiming to closely replicate the original drugs' effects. While they have only recently entered the market for clinical use, it is hoped they will offer more affordable options while maintaining high standards of purity, efficacy, and safety. These biosimilars include Mvasi and Zirabev for bevacizumab, FYB201, Xlucane, SB11, PF582, and Razumab for ranibizumab and YL1701, ALT-L9, FYB203, and CHS2020 for aflibercept. These biosimilars represent a significant shift in the AMD treatment landscape, potentially increasing accessibility and wider treatment options for patients(8).

Corticosteroid Injections (eg, triamcinolone, fluocinolone, dexamethasone): Intravitreal corticosteroids, notably Triamcinolone Acetonide (TA), Dexamethasone (DEX), and Fluocinolone Acetonide (FA), play a crucial role in treating Diabetic Macular Edema (DME) and other chronic noninfectious conditions including uveitis and macular edema caused by retinal vein occlusion (TABLE 2). TA has the highest potency and is effective for about three months in non-vitrectomized eyes (9,10). DEX and FA have similar anti-inflammatory potency (11). The choice of intravitreal corticosteroids depends on the patient's specific condition and previous treatment responses, offering a range of options for effective management of ocular diseases.

Antibiotic, Antiviral, or Antifungal Antibiotic (eg, vancomycin), antiviral (eg, ganciclovir [Cytovene; Hoffmann-La Roche]), or antifungal (eg. amphotericin B) injections: Intravitreal antibiotics, antivirals or antifungals are administered to treat severe eye infections, particularly endophthalmitis, which is an infection of the internal structures of the eye (TABLE 2)(12).

Immunomodulatory Injections Intravitreal immunomodulatory agents like methotrexate or rituximab are used to manage certain inflammatory eye conditions, such as non-infectious uveitis(13).

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TABLE 2: Common Intravitreal Medications. Modified from Eyewiki Drug Class

Medication

Dosing and Information

Bevacizumab (Avastin, offlabel)

1.25 mg/0.05 mL (0.675 mg/0.03 mL if for use for treatment of Zone I+ ROP in an infant)

Ranibizumab (Lucentis)

0.5 mg/0.05 mL (neovascular AMD, RVO, myopic CNVM) or 0.3 mg/0.05 mL (DR/DME)

Ranibizumab-nuna (Byooviz)

0.5 mg/0.05 mL (biosimilar for neovascular AMD, RVO, myopic CNVM)

Ranibizumab-eqrn (Cimerli)

0.5 mg/0.05 mL (biosimilar for neovascular AMD, RVO, myopic CNVM) or 0.3 mg/0.05 mL (biosimilar for DR/DME)

Aflibercept (Eylea)

2.0 mg /0.05 mL

Brolucizumab (Beovu)

6 mg/0.05 mL

Faricimab (Vabysmo)

6 mg/0.05 mL

Triamcinolone acetonide

2 mg/0.05 mL or 4 mg/0.1 mL (Triesence/ Trivaris is alcohol-free preparation FDA-approved for intraocular use)

Dexamethasone

0.4 mg/0.1 mL

Clindamycin

1 mg/0.1 mL

Vancomycin

1 mg/0.1 mL

Ceftazidime

2.25 mg/0.1 mL

Amikacin

0.4 mg/0.1 mL

Ganciclovir

4 mg/0.1 mL (administer 2 mg in 0.05 mL twice weekly for cytomegalovirus retinitis for 14 days for induction)

Foscarnet

2.4 mg/0.1 mL (administer 1.2 mg in 0.05 mL)

Fomivirsen

330 micrograms/0.05 mL

Antifungal

Amphotericin B Voriconazole

5 micrograms/0.1 mL 50-100 micrograms/0.1 mL

Immunomodulatory

Methotrexate

400 micrograms/0.1 mL

Anti-VEGF

Corticosteroid

Antibiotic

Antiviral

Inflammation After Intravitreal Injection Pathogenesis The pathogenesis of anti-VEGF associated sterile intraocular inflammation (SIOI), noninfectious intraocular inflammation that occurs after intravitreal injection, is complex and multifactorial, yet not completely understood. Anderson et al. divided the possible causes of SIOI into three main groups: factors specific to the patient, the medication, and the drug delivery platforms (14). The presence of antidrug antibodies (ADA) is a patient-specific contributing factor, occurring transiently post-administration or existing as baseline ADA. Clinical trial findings show variable ADA rates across different drugs with high

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ADA and IOI rates in brolucizumab-treated patients(15), and low ADA but high inflammation in Abicipar according to European Medicines Agency data(16). Factors related to medications include impurities in manufacturing, storage, or preparation. Contaminants from the manufacturing process, as seen with bevacizumab, may contribute to higher rates of SIOI. The presence of bacterial endotoxins, non-human particles and Fc portion of antibodies may result in induction of inflammatory response(17–20). Translational research has shown that linear epitopes shared with bacteria, non-natural surfaces due to the singlechain variable fragment format, and non-native drug species that may form over prolonged time in the eye contributed to increased immunogenicity to Brolucizumab(21). Specific to Brolucizumab-associated retinal vasculitis, hypotheses include the smaller molecular weight of brolucizumab leading to increased VEGF inhibition and deeper retinal penetration, or the presence of local anti-brolucizumab antibodies at baseline(23). The biosimilar form of ranibizumab (Razumab) initially demonstrated a high rate of inflammation, which was significantly reduced after manufacturing modifications changing the drug from lyophilized to solubilized formulation(22). Fewer studies have investigated the role of drug delivery platforms to induction of inflammatory response. Deviations from manufacturer’s guidelines on storing, handling, and administration of the medications have resulted in higher rates of inflammation(24). TABLE 3: Overview of Possible Pathogenesis of Inflammation after Intravitreal Injection (Modified from Anderson et al. Int J Retin Vitr, 2021) Patient Specific

Medication Specific

Delivery Specific

Presence of autoantibodies against drug

Bacterial endotoxins Non-human proteins Impurities Formulation Fc portion of antibody

Silicone oil-induced protein

Compromise of blood-retinal barrier (nAMD, DR) History of uveitis, autoimmune disease

aggregates Syringe agitation Shipping, handling, freeze-thawing

Clinical Features of Sterile Inflammation Intraocular inflammation is a potentially vision-threatening adverse event related to anti-VEGF pharmacological agents. It is important to remember that the overall risk of complications from intravitreal injections is relatively low, and many individuals benefit greatly from these treatments. After intravitreal injections, various types of inflammation can occur, ranging from mild conditions such as conjunctival inflammation or anterior chamber inflammation (iritis), to more serious conditions like vitreous inflammation (vitritis) or retinal vasculitis. Furthermore, intraocular inflammation (IOI) can be classified into two types: acute onset sterile IOI (SIOI) and delayed onset inflammatory vasculitis, with the latter being more commonly associated with Brolucizumab(14). These inflammatory responses occur following the intravitreal injection but are in most instances temporary and resolve with time. The incidence, character, severity, and resolution of post-injection inflammation can vary among different intravitreal injection drugs. SIOI, also known as pseudoendophthalmitis, is characterized by sudden inflammation within the eye without an infection and resolving without antibiotic treatment. The cumulative rate of SIOI per patient is reported to be 0.087% and 0.228% after 10 and 20 IVTs, respectively, and then remained stable up to 60 IVTs(25). The reported incidence of SIOI associated with anti-VEGF injections varies from 0.02% to 1.1%(26,27), occurring in all anti-VEGF agents. It has also been seen after intravitreal injection of corticosteroids(28). Onset typically occurs between 24 hours and several days after the injection(29,30). Patients present with blurred vision, floaters, ocular pain and, less commonly, photophobia (26,27,31). Blurred vision and floaters are the most common symptoms, while pain is present in up to 46% of cases and is associated with severe inflammation(31). On examination, vitritis, anterior chamber inflammation, fibrin, and keratic precipitates may be seen. Retrospective studies showed varying degrees of visual acuity at injection, presentation, and last follow-up, emphasizing the range of outcomes associated with SIOI. Visual acuity is often substantially reduced from baseline at presentation but frequently returns to pre-injection levels after inflammation resolves.

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Brolucizumab was approved by the US Food and Drug Administration (FDA) in 2019. Several studies have reported occurrences of intraocular inflammation and retinal vasculitis within the initial months following treatment with brolucizumab(32). Data from the HAWK and HARRIER trials, involving 1817 eyes treated with brolucizumab, showed a 4.6% incidence of IOI, 3.6% incidence of retinal vasculitis associated with IOI, and 2.1% incidence of retinal vascular occlusion among 50 patients(33). Conversely, the BREW study by Sharma et al., evaluated 42 patients previously on other anti-VEGF drugs and reported no adverse effects including inflammation or vasculitis. This suggests variability in the incidence of adverse effects with brolucizumab use(34). Despite limited data, review studies have highlighted a higher female predominance in developing Brolucizumab-associated retinal vasculitis(35). Retinal vasculitis and IOI after brolucizumab IVI are characterized by variable occlusion of large and/or small retinal arteries and perivenular abnormalities. It may span from peripheral vasculitis to occlusion of large retinal arteries around the optic nerve or macula with severe vision loss(36). In another study, clinical findings included a spectrum of vascular abnormalities, cotton-wool spots, Kyrieleis plaques, irregular venous calibers, and fluorescein angiography revealing delayed filling, nonperfusion, and dye leakage. These patients had significant decline in visual acuity from a baseline of 0.426 logMAR to 0.981 logMAR at the time of diagnosis(37). A case study by Kusuhara et al. showed that retinal vasculitis associated with Brolucizumab can occur even in the absence of initial abnormal findings on fluorescein angiography or indocyanine green angiography. The case suggests that the potent and prolonged inhibition of vascular endothelial growth factor by brolucizumab may severely damage retinal vascular endothelial cells leading to retinal vascular occlusions. This vascular injury may result in an intensified immune response to brolucizumab, potentially aggravated by the migration of immune cells into the vitreous cavity due to a compromised inner blood-retinal barrier. Postinjection inflammation can occur months after brolucizumab injection, so all patients receiving anti-VEGF should be thoroughly examined at every visit to rule out uveitis and occlusive vasculitis(38).

Medications Associated with Sterile Intraocular Inflammation Anti-VEGF Medications Recent studies have been inconclusive about the risks of SIOI associated with each of the anti-VEGF injections. While some studies highlighted the higher incidence of SIOI with Bevacizumab(39), other studies have either found no significant difference among medications or have reported contradictory results(40,41). A comprehensive systematic review revealed that the occurrence of sterile endophthalmitis was 0.16% with Aflibercept, in contrast to 0.10% for Bevacizumab and 0.02% for Ranibizumab(42). Another retrospective study revealed a higher rate of SIOI following Aflibercept injections compared to Ranibizumab(19). Interestingly, prior exposure to the drugs seems to affect SIOI rates differently. The potential difference in the immunogenic response to Bevacizumab compared to Ranibizumab and Aflibercept, as suggested by Cox et al., might be due to the compounding process of Bevacizumab in pharmacies. This process can introduce impurities, leading to sterile intraocular inflammation (SII) even in patients not previously exposed to the drug. In contrast, Ranibizumab and Aflibercept, distributed in manufacturer-prepared single-use vials, are less susceptible to such impurities(26). In a systematic review of 14 randomized controlled trials with 6759 eyes at baseline, there was no difference in the risk of severe sight-threatening IOI outcomes between intravitreal anti-VEGF agents and, in contrary, there was a significantly higher risk of generalized IOI after Brolucizumab relative to Aflibercept (41). In another systematic review and meta-analysis of more than 100000 injections, there was a significant difference in the incidence rate of non-infectious intraocular inflammation (SIOI) between Ranibizumab, Aflibercept and Bevacizumab. Moreover, it was shown that Bevacizumab and Aflibercept SIOI reports were present in different chronological clusters. Rate of repeated SIOI was low in all medication groups(42).

Corticosteroids Corticosteroid intravitreal injections are commonly employed to address various ocular conditions, including macular edema and uveitis itself, they carry a risk of eliciting localized inflammation within the eye(28). The introduction of a steroid into the vitreous cavity, intended to reduce inflammation and improve visual outcomes, can paradoxically trigger an inflammatory response in some cases. These injections interestingly have a higher potential for inducing post-injection inflammation compared to anti-VEGF

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drugs(10). It has been reported that the rate of sterile endophthalmitis is between 0.2% and 12.3% of triamcinolone acetonide injections and occurs within the first three days of the injection (43).

Other Agents Immunosuppressive drugs like methotrexate or rituximab, used in intravitreal immunomodulatory injections for certain inflammatory eye conditions(44,45), as well as intravitreal antibiotic injections used to treat infectious endophthalmitis, eg. vancomycin intravitreal injections (known to be associated to hemorrhagic occlusive retinal vasculitis) can also induce post-injection inflammation(46). The rate of inflammation for these drugs may vary among patients and specific conditions being treated. Intravitreal injections have recently emerged as a promising therapeutic approach for geographic atrophy (GA) aiming to inhibit pathological processes and reduce the progression of GA(47). The efficacy of intravitreal injections for GA is still under investigation, with intravitreal pegcetacoplan (SYFOVRE, Apellis Pharmaceuticals) approved by the FDA in February 2023 as the first definitive therapy for GA, and avacincaptad pegol (IZERVAY, Astellas Pharmaceuticals), granted FDA breakthrough therapy status(48,49). Trials suggest that this approach may hold promise in preserving vision and slowing the degenerative process associated with geographic atrophy (47,48,50). However, serious inflammatory complications have been reported recently with Syfovre. TABLE 4 summarizes these complications that were reported in 9 patients receiving Syfovre intravitreal injections at the time of the American Society of Retina Specialists (ASRS) meeting in Seattle on July 29, 2023. TABLE 4: Clinical Characteristics of Patients Who Received Syfovre Intravitreal Injections for Geographic Atrophy (Presented at the American Society of Retina Specialists (ASRS) meeting in Seattle on July 29, 2023) Patient #

Age

Gender

Female

Male

Female

History

Glaucoma

s/p aflibercept 88w (last aflibercept 38 days prior)

h/o COVID, OHTN, Hashimoto Thyroiditis

Latest Injection

5/1/2023

6/22/2023

4/27/2023

Injection Number

Injection Eye

VA Before

20/70

20/60

20/200

VA at Presentation

20/80

20/100

20/400

VA at Latest follow up

20/80

20/250

Interval to Symptom Onset s/p last Injection (days)

70-80

Symptoms at Onset of Issues

Found on routine visit. Floaters and blurred vision

Found on routine visit. Floaters and blurred vision (could not obtain FA due to venous access)

Lid edema, headache, decreased vision

Findings

Mild vitritis, no fibrin, no hypopyon; Could not obtain FA due to venous access

Low IOP, no fibrin, no hypopyon; Inflammation, vascular leak

High IOP, no fibrin/hypopyon, macular thickening, diffuse intraretinal hemorrhages, hyphema, vitreous hemorrhage; Peripheral nonperfusion, vascular leakage

Treatments

PST triamcinolone

Topical prednisolone q2o (first finding at 10 days); AC Tap and inject (Vanco / Ceftz / Dex) at 18 days + IVT dexamethasone implat at 20 days

Topical moxifloxacin / polytrim drops; 2 days later: topical prednisone, glaucome drops; PO prednisone, IVT aflibercept; CF; PO azithromycin

Classification

Panuveitis with No Retinal Vasculitis

Panuveitis with Retinal Nonocclusive Vasculitis

Panuveitis with Retinal Occlusive Vasculitis

Reported to ReST

7/17/2023

7/19/2023

7/12/2023

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TABLE 4 (Continued) Patient #

Age

Gender

Male

Female

History

Not available

s/p aflibercept OU q16w intervals for wet AMD (previous no IOI)

Latest Injection

6/8/2023

6/19/2023

5/30/2023

Injection Number

Not available

Injection Eye VA Before

20/150

20/50

20/30 (OD); 20/30 (OS)

VA at Presentation

20/200

20/200 (OD); 20/200 (OS)

VA at Latest follow up

NLP

20/80

Not available

Interval to Symptom Onset s/p last Injection (days)

15-Oct

Symptoms at Onset of Issues

Not available

Findings

Treatments

Classification

Male

Female

Reported to ReST

Not available

s/p aflibercept OU q16w intervals for wet AMD (previous no IOI)

Patient #

Age

Gender

Female

Male

History

Not available

h/o BRVO s/p laser, uveitis, glaucoma, s/p PPV for ERM

s/p multiple aflibercept injections; Pegccetacaoplan administered same time as Aflibercept

Latest Injection

6/20/2023

5/4/2023

4/28/2023

Injection Number

Injection Eye

Not available

VA Before

20/100

20/40

VA at Presentation

20/400

20/100

VA at Latest follow up

20/400

Interval to Symptom Onset s/p last Injection (days)

Symptoms at Onset of Issues

Not available

Findings

Corneal edema, fibrin, no hypopyon; Peripheral nonperfusion, vascular leakage

High IOP, swollen eyelid, ROV findings (intraretinal Heme, Inner retinal edema, box car, disc edema, vascular occlusion)

High IOP, corneal edema, no hypopyon / fibrin

Treatments

Topical prednisolone, PO prednisone

Topical glaucoma drops; topical prednisolone, IVT Bevacizumab

Glaucoma drops; treated with topical prednisolone, glaucoma drops

Classification

Panuveitis with Retinal Occlusive Vasculitis

Reported to ReST

7/3/2023

7/10/2023

7/14/2023

Bagheri, Saghar

Treatment Evidence for management of IOI is lacking. Management ranges from non-invasive measures, including discontinuation of the causing drug or observation, to topical and systemic corticosteroid administration, to more invasive forms of intraocular injections and surgery(51). Additional measures may be taken based on the severity of the symptoms. Based on recommendations from an expert opinion study, effective management of brolucizumab-associated intraocular inflammation involves a multifaceted approach. This includes educating patients on the importance of promptly reporting any persistent symptoms after receiving intravitreal injections (IVI, which is crucial for early intervention. In cases of suspected intraocular inflammation, comprehensive clinical examinations are necessary to evaluate for any concurrent retinal vasculitis or vascular occlusive events, utilizing multimodal imaging techniques like widefield imaging, fluorescein angiography, and optical coherence tomography (OCT). Upon confirmation of IOI, immediate discontinuation of the agents is often advised, followed by intensive treatment with potent corticosteroids, which may include topical, subtenon, intravitreal, or systemic forms, with the choice of treatment tailored according to the severity of the condition until the inflammation resolves. Additionally, ongoing care for the underlying condition such as neovascular age-related macular degeneration (nAMD) should be individualized for each patient, utilizing the standard of care available locally. These comprehensive recommendations are designed to effectively manage the range of events, as per the current knowledge and expert opinions in the field(52). Vitrectomy, intravitreal antibiotic injection, and vitreous biopsy have not been associated with improved visual outcomes compared to non-invasive treatments(53,54). Age older than 80, worse visual acuity at presentation, presence of fibrin at examination, and observation-only treatment resulted in worse visual outcomes(51,53).

Conclusion The incidence, character, severity, and resolution of post-injection inflammation vary among different intravitreal injection drugs. As with any medical procedure, early detection and timely management of inflammation can help prevent potential complications and ensure better treatment outcomes. The incidence of post-injection inflammation can vary depending on several factors, including the drug used, the patient's individual response, and the condition being treated. These inflammatory responses are common side effects of the procedure but are in most instances temporary and resolve with time. It is important to remember that the overall risk of complications from intravitreal injections is relatively low, and many individuals benefit greatly from these treatments.

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References 1.

Baum U, Peyman GA, Barza M. Intravitreal administration of antibiotic in the treatment of bacterial endophthalmitis. III. Consensus. Surv Ophthalmol. 1982 Jan;26(4):204–6.

Results of the Endophthalmitis Vitrectomy Study. Archives of Ophthalmology [Internet]. 1995 Dec 1;113(12):1479. Available from: http://archopht.jamanetwork.com/article.aspx?doi=10.1001/archopht.1995.01100120009001

Grzybowski A, Told R, Sacu S, Bandello F, Moisseiev E, Loewenstein A, et al. 2018 Update on Intravitreal Injections: Euretina Expert Consensus Recommendations. Ophthalmologica. 2018;239(4):181–93.

Avery RL, Bakri SJ, Blumenkranz MS, Brucker AJ, Cunningham ET, D’Amico DJ, et al. INTRAVITREAL INJECTION TECHNIQUE AND MONITORING. Retina. 2014 Dec;34(Supplement 12):S1–18.

Doshi RR, Bakri SJ, Fung AE. Intravitreal Injection Technique. Semin Ophthalmol. 2011 May 24;26(3):104–13.

Beck KD, Rahman EZ, Ells A, Mireskandari K, Berrocal AM, Harper CA. SAFER-ROP: Updated Protocol for AntiVEGF Injections for Retinopathy of Prematurity. Ophthalmic Surg Lasers Imaging Retina. 2020 Jul;51(7):402–6.

De Stefano VS, Abechain JJ, de Almeida LF, Verginassi DM, Rodrigues EB, Freymuller E, et al. Experimental investigation of needles, syringes and techniques for intravitreal injections. Clin Exp Ophthalmol. 2011 Apr;39(3):236–42.

Kaiser SM, Arepalli S, Ehlers JP. Current and Future Anti-VEGF Agents for Neovascular Age-Related Macular Degeneration. J Exp Pharmacol. 2021 Sep;Volume 13:905–12.

Mansoor S, Kuppermann BD, Kenney MC. Intraocular Sustained-Release Delivery Systems for Triamcinolone Acetonide. Pharm Res. 2009 Apr 28;26(4):770–84.

10. Chawan-Saad J, Wu M, Wu A, Wu L. Corticosteroids for diabetic macular edema. Taiwan J Ophthalmol. 2019;9(4):233. 11. Aceves-Franco LA, Sanchez-Aguilar OE, Barragan-Arias AR, Ponce-Gallegos MA, Navarro-Partida J, Santos A. The Evolution of Triamcinolone Acetonide Therapeutic Use in Retinal Diseases: From Off-Label Intravitreal Injection to Advanced Nano-Drug Delivery Systems. Biomedicines. 2023 Jul 5;11(7):1901. 12. Radhika M, Mithal K, Bawdekar A, Dave V, Jindal A, Relhan N, et al. Pharmacokinetics of intravitreal antibiotics in endophthalmitis. J Ophthalmic Inflamm Infect. 2014 Dec 10;4(1):22. 13. Nguyen QD, Merrill PT, Sepah YJ, Ibrahim MA, Banker A, Leonardi A, et al. Intravitreal Sirolimus for the Treatment of Noninfectious Uveitis. Ophthalmology. 2018 Dec;125(12):1984–93. 14. Anderson WJ, da Cruz NFS, Lima LH, Emerson GG, Rodrigues EB, Melo GB. Mechanisms of sterile inflammation after intravitreal injection of antiangiogenic drugs: a narrative review. Int J Retina Vitreous. 2021 Dec 7;7(1):37. 15. Iyer PG, Peden MC, Suñer IJ, Patel N, Dubovy SR, Albini TA. Brolucizumab-related retinal vasculitis with exacerbation following ranibizumab retreatment: A clinicopathologic case study. Am J Ophthalmol Case Rep. 2020 Dec;20:100989. 16. European Medicines Agency. European Medicines Agency . 2020. Withdrawal Assessment Report. 17. Sharma A, Kumar N, Kuppermann BD, Bandello F, Loewenstein A. Ophthalmic biosimilars and biologics—role of endotoxins. Eye. 2020 Apr 16;34(4):614–5. 18. Koren E, Zuckerman L, Mire-Sluis A. Immune Responses to Therapeutic Proteins in Humans - Clinical Significance, Assessment and Prediction. Curr Pharm Biotechnol. 2002 Dec 1;3(4):349–60. 19. Souied EH, Dugel PU, Ferreira A, Hashmonay R, Lu J, Kelly SP. Severe Ocular Inflammation Following Ranibizumab or Aflibercept Injections for Age-Related Macular Degeneration: A Retrospective Claims Database Analysis. Ophthalmic Epidemiol. 2016 Mar 3;23(2):71–9. 20. Trivizki O, Schwartz S, Negri N, Loewenstein A, Rabina G, Shulman S. Noninfectious Inflammatory Response following Intravitreal Bevacizumab Injections: Description of a Cluster of Cases in Two Centers and a Review of the Literature. Ophthalmologica. 2018;240(3):163–6.

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21. Kearns JD, Wassmann P, Olgac U, Fichter M, Christen B, Rubic-Schneider T, et al. A root cause analysis to identify the mechanistic drivers of immunogenicity against the anti-VEGF biotherapeutic brolucizumab. Sci Transl Med. 2023 Feb;15(681). 22. Heier JS. Ranibizumab Combined With Verteporfin Photodynamic Therapy in Neovascular Age-Related Macular Degeneration. Archives of Ophthalmology. 2006 Nov 1;124(11):1532. 23. Liu L, Ammar DA, Ross LA, Mandava N, Kahook MY, Carpenter JF. Silicone Oil Microdroplets and Protein Aggregates in Repackaged Bevacizumab and Ranibizumab: Effects of Long-term Storage and Product Mishandling. Investigative Opthalmology & Visual Science. 2011 Feb 22;52(2):1023. 24. Iyer PG, Peden MC, Suñer IJ, Patel N, Dubovy SR, Albini TA. Brolucizumab-related retinal vasculitis with exacerbation following ranibizumab retreatment: A clinicopathologic case study. Am J Ophthalmol Case Rep. 2020 Dec;20:100989. 25. Daien V, Nguyen V, Essex RW, Morlet N, Barthelmes D, Gillies MC, et al. Incidence and Outcomes of Infectious and Noninfectious Endophthalmitis after Intravitreal Injections for Age-Related Macular Degeneration. Ophthalmology. 2018 Jan;125(1):66–74. 26. Cox JT, Eliott D, Sobrin L. Inflammatory Complications of Intravitreal Anti-VEGF Injections. J Clin Med. 2021 Mar 2;10(5):981. 27. Georgopoulos M, Polak K, Prager F, Prunte C, Schmidt-Erfurth U. Characteristics of severe intraocular inflammation following intravitreal injection of bevacizumab (Avastin). British Journal of Ophthalmology. 2009 Apr 1;93(4):457–62. 28. Mahjoub A, Abdesslem N Ben, Abderrazek A Ben, Zaafrane N, Mahjoub A, Aoun H, et al. Sterile endophthalmitis after intravitreal triamcinolone acetonide injection: A case report series. Annals of Medicine & Surgery. 2022 Apr;76. 29. Wickremasinghe SS, Michalova K, Gilhotra J, Guymer RH, Harper CA, Wong TY, et al. Acute Intraocular Inflammation after Intravitreous Injections of Bevacizumab for Treatment of Neovascular Age-related Macular Degeneration. Ophthalmology. 2008 Nov;115(11):1911-1915.e1. 30. Greenberg JP, Belin P, Butler J, Feiler D, Mueller C, Tye A, et al. Aflibercept-Related Sterile Intraocular Inflammation Outcomes. Ophthalmol Retina. 2019 Sep;3(9):753–9. 31. Chong DY, Anand R, Williams PD, Qureshi JA, Callanan DG. CHARACTERIZATION OF STERILE INTRAOCULAR INFLAMMATORY RESPONSES AFTER INTRAVITREAL BEVACIZUMAB INJECTION. Retina. 2010 Oct;30(9):1432–40. 32. Bodaghi B, Souied EH, Tadayoni R, Weber M, Ponthieux A, Kodjikian L. Detection and Management of Intraocular Inflammation after Brolucizumab Treatment for Neovascular Age-Related Macular Degeneration. Ophthalmol Retina. 2023 Oct;7(10):879–91. 33. Monés J, Srivastava SK, Jaffe GJ, Tadayoni R, Albini TA, Kaiser PK, et al. Risk of Inflammation, Retinal Vasculitis, and Retinal Occlusion–Related Events with Brolucizumab. Ophthalmology. 2021 Jul;128(7):1050–9. 34. Sharma A, Kumar N, Parachuri N, Sadda SR, Corradetti G, Heier J, et al. Brolucizumab—early real-world experience: BREW study. Eye. 2021 Apr 24;35(4):1045–7. 35. Witkin AJ, Hahn P, Murray TG, Arevalo JF, Blinder KJ, Choudhry N, et al. Occlusive Retinal Vasculitis Following Intravitreal Brolucizumab. J Vitreoretin Dis. 2020 Jul 1;4(4):269–79. 36. Baumal CR, Spaide RF, Vajzovic L, Freund KB, Walter SD, John V, et al. Retinal Vasculitis and Intraocular Inflammation after Intravitreal Injection of Brolucizumab. Ophthalmology. 2020 Oct;127(10):1345–59. 37. Baumal CR, Spaide RF, Vajzovic L, Freund KB, Walter SD, John V, et al. Retinal Vasculitis and Intraocular Inflammation after Intravitreal Injection of Brolucizumab. Ophthalmology. 2020 Oct;127(10):1345–59. 38. Kusuhara S, Kim KW, Miki A, Nakamura M. Angiographic findings before and after the onset of brolucizumabassociated retinal vascular occlusion and intraocular inflammation. Am J Ophthalmol Case Rep. 2022 Jun;26:101521. 39. Williams PD, Chong D, Fuller T, Callanan D. NONINFECTIOUS VITRITIS AFTER INTRAVITREAL INJECTION OF ANTI-VEGF AGENTS. Retina. 2016 May;36(5):909–13.

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40. Knickelbein JE, Chew EY, Sen HN. Intraocular Inflammation Following Intravitreal Injection of Anti-VEGF Medications for Neovascular Age-Related Macular Degeneration. Ophthalmic Epidemiol. 2016 Mar 3;23(2):69–70. 41. Patil NS, Dhoot AS, Popovic MM, Kertes PJ, Muni RH. RISK OF INTRAOCULAR INFLAMMATION AFTER INJECTION OF ANTIVASCULAR ENDOTHELIAL GROWTH FACTOR AGENTS. Retina. 2022 Nov;42(11):2134– 42. 42. Williams PD, Chong D, Fuller T, Callanan D. NONINFECTIOUS VITRITIS AFTER INTRAVITREAL INJECTION OF ANTI-VEGF AGENTS. Retina. 2016 May;36(5):909–13. 43. Durmaz Engin C, Ayhan Z, Men S, Yaman A, Saatci AO. Bilateral Severe Sterile Inflammation with Hypopyon after Simultaneous Intravitreal Triamcinolone Acetonide and Aflibercept Injection in a Patient with Bilateral Marked Rubeosis Associated with Ocular Ischemic Syndrome. Case Rep Ophthalmol Med. 2017;2017:1–5. 44. Habot-Wilner Z, Frenkel S, Pe’er J. Efficacy and safety of intravitreal methotrexate for vitreo-retinal lymphoma 20 years of experience. Br J Haematol. 2021 Jul;194(1):92–100. 45. Conrady CD, Shakoor A. Rituximab-Associated Retinal Occlusive Vasculopathy: A Case Report and Literature Review. Ocul Immunol Inflamm. 2020 May 18;28(4):622–5. 46. Witkin AJ, Chang DF, Jumper JM, Charles S, Eliott D, Hoffman RS, et al. Vancomycin-Associated Hemorrhagic Occlusive Retinal Vasculitis: Clinical Characteristics of 36 Eyes. Ophthalmology. 2017 May;124(5):583–95. 47. Heier JS, Lad EM, Holz FG, Rosenfeld PJ, Guymer RH, Boyer D, et al. Pegcetacoplan for the treatment of geographic atrophy secondary to age-related macular degeneration (OAKS and DERBY): two multicentre, randomised, double-masked, sham-controlled, phase 3 trials. The Lancet. 2023 Oct;402(10411):1434–48. 48. Antonio-Aguirre B, Arevalo JF. Treating patients with geographic atrophy: are we there yet? Int J Retina Vitreous. 2023 Nov 20;9(1):72. 49. Shughoury A, Sevgi DD, Ciulla TA. The complement system: a novel therapeutic target for age-related macular degeneration. Expert Opin Pharmacother. 2023;24(17):1887–99. 50. Shughoury A, Sevgi DD, Ciulla TA. The complement system: a novel therapeutic target for age-related macular degeneration. Expert Opin Pharmacother. 2023;24(17):1887–99. 51. Greenberg JP, Belin P, Butler J, Feiler D, Mueller C, Tye A, et al. Aflibercept-Related Sterile Intraocular Inflammation Outcomes. Ophthalmol Retina. 2019 Sep;3(9):753–9. 52. Baumal CR, Bodaghi B, Singer M, Tanzer DJ, Seres A, Joshi MR, et al. Expert Opinion on Management of Intraocular Inflammation, Retinal Vasculitis, and Vascular Occlusion after Brolucizumab Treatment. Ophthalmol Retina. 2021 Jun;5(6):519–27. 53. Hahn P, Chung MM, Flynn HW, Huang SS, Kim JE, Mahmoud TH, et al. Postmarketing Analysis of AfliberceptRelated Sterile Intraocular Inflammation. JAMA Ophthalmol. 2015 Apr 1;133(4):421. 54. Baumal CR, Spaide RF, Vajzovic L, Freund KB, Walter SD, John V, et al. Retinal Vasculitis and Intraocular Inflammation after Intravitreal Injection of Brolucizumab. Ophthalmology. 2020 Oct;127(10):1345–59.

Bagheri, Saghar

2024 Harvard Ophthalmology Residents’ Course

Pediatric Non-infectious Intermediate, Posterior, and Panuveitis: A Review Da Meng, MD PhD, Sandra Hoyek, MD, Lucia Sobrin, MD, MPH, and Nimesh A. Patel, MD

Introduction Uveitis denotes an inflammatory pathology occurring within the uveal tract of the eye, encompassing the iris, choroid, and ciliary body. This inflammatory process may selectively affect distinct ocular segments: the anterior segment, including the iris and its adjacent structures, leading to anterior uveitis; the vitreous cavity and pars plana, inducing intermediate uveitis; and the retina and choroid, resulting in posterior uveitis. Alternatively, inflammation spanning all layers of the uveal tract is termed as panuveitis. Pediatric uveitis accounts for 5–10% of all-age uveitis, with an estimated incidence of 4.3 per 100,000 children and a prevalence of 27.9 per 100,000 children.1 Although uveitis can be infectious or noninfectious, noninfectious uveitis (NIU) represents the majority of uveitis cases in developed countries,2 particularly in the pediatric population (67.2 – 93.8%).1 Despite a lower prevalence of NIU in children (29 cases per 100,000) compared with adults (121 cases per 100,000),3 the diagnosis of childhood-onset NIU is challenging due to fewer symptoms, an insidious, chronic, and recurrent course, difficulties in eye examination, resistance to treatment, and a higher risk of vision-threatening complications.4,5 These complications include amblyopia, cataract, glaucoma, and cystoid macular edema (CME). While pediatric anterior NIU has been well-studied,6–8 understanding of pediatric non-anterior NIU, including intermediate, posterior, and panuveitis, remains limited due to its low prevalence. This review will provide an overview of the demographic characteristics, risk factors and associations, clinical manifestations, diagnostic intricacies, therapeutic considerations, as well as complications associated with pediatric non-anterior NIU.

Demographic Characteristics The sex distribution of non-anterior NIU varies largely between studies. In 2016, Thorne et al. reported the prevalence of NIU in pediatric patients stratified by sex and location of inflammation. They found that the overall prevalence of NIU was higher in male patients (32 per 100,000) compared to female patients (26 per 100,000), independent from the anatomic location of uveitis. Similarly, males had a higher prevalence of all non-anterior NIU, as well as intermediate and posterior NIU, while females had a higher prevalence of panuveitis. Similar to Thorne et al, multiple studies report a male predominance in pediatric intermediate NIU, ranging from 57% to 84%.9–12 However, sex distribution for posterior and panuveitis is variable and likely dictated by the underlying etiology. In a retrospective analysis of 34 Turkish pediatric patients with Behçet uveitis (53% with panuveitis and 32% with posterior uveitis), a notable male predominance of 70% was observed.13 However, female prevalence was reported in posterior uveitis and panuveitis associated with tubulointerstitial nephritis and uveitis syndrome (TINU)14, white dot syndromes,15 and idiopathic panuveitis.16 It is notable that the female preponderance in TINU appears weaker than initial research indicated, with an increasing proportion of male patients being reported over time, possibly due to an early ascertainment bias favoring female patients in initial reports.17 Regarding the age of uveitis onset, the mean age at diagnosis of intermediate NIU ranges from 7.8 to 13.1 years,11,12,18,19 with the delay in diagnosis being attributed to the absence of symptoms at the onset of ocular inflammation.10 Mean age at pediatric panuveitis diagnosis was 10 years (range 4 – 15 years) in patients with TINU,20 and 11.1 ± 2.9 years (range 4–14 years) in patients with Vogt-Koyanagi-Harada (VKH).21 In relation to race and ethnicity, Smith et al demonstrated that the prevalence of NIU was significantly lower in Hispanic children as compared to non-Hispanics (68.5% versus 91.2%).22 They also found that

Meng, Da

Hispanics were more likely to have visual acuity of 20/50 or worse at baseline compared to non-Hispanics (P = 0.03).22 Moreover, Angeles-Han et al. showed that non-Hispanic African-American children with uveitis are more likely to be diagnosed at an older age, have lower visual acuity, and have a higher rate of complications as compared to non-Hispanic White children.23 These differences could be attributed to genetic differences between populations or to variance in health-care access leading to delay in referral and diagnosis. As a result, the authors suggested the potential need for an earlier and more aggressive treatment with systemic therapy in non-Hispanic African American children with uveitis.23

Risk Factors and Associations Non-Infectious Intermediate Uveitis Intermediate uveitis accounts for 16% to 33% of pediatric uveitis in children less than 16 years old.24–26 In childhood, the majority of cases (97%) of intermediate uveitis are idiopathic.10,27 Also known as pars planitis, idiopathic intermediate uveitis predominantly affects children and adolescents, constituting 5% to 26.7% of pediatric uveitis cases.28 Rarely, intermediate uveitis and pars planitis represent a manifestation of a more systemic disease in children. While there are occasional observations of intermediate uveitis in juvenile idiopathic arthritis (JIA) patients, anterior uveitis is a more common uveitis manifestation in JIA. In adults, intermediate uveitis can be linked to sarcoidosis, multiple sclerosis (MS), or infectious etiologies; however, in the pediatric context, it is generally idiopathic and presumably driven by autoimmune or immune-mediated mechanisms.11,24,28–30 Furthermore, a familial predisposition has also been reported.31– 33 Intermediate uveitis and pars planitis have been associated with HLA-DR2, -DR15, -B51 and DRB1*0802 haplotypes.29,30,34–36 Notably, individuals positive for HLA-DR15 can concurrently exhibit systemic manifestations seen in other HLA-DR15-related disorders like MS, optic neuritis, and narcolepsy, indicating a shared genetic background.30,36,37 Among Mexican Mestizos, more severe inflammation has been linked to HLA-B51 in female patients and to HLA-DRB10802 in male individuals.29 Nevertheless, HLA typing is not included in standard clinical tests and its clinical relevance remains uncertain.38

Non-Infectious Posterior and Panuveitis Panuveitis and posterior uveitis are commonly perceived as the least prevalent types of uveitis in children, accounting for 14% to 21% and 6% to 30% of pediatric uveitis cases, respectively.38 Noninfectious pediatric posterior and panuveitis can occur in the setting of primarily ocular conditions or in association with autoimmune systemic disorders.

Primary Ocular Diseases White dot syndromes White dot syndromes are a group of inflammatory chorioretinopathies characterized by multiple, discrete, white lesions at the level of the outer retina, retinal pigment epithelium (RPE), and choroid. It has been reported in 1% to 5% of children with uveitis.39 The specific entities that can affect children include acute posterior multifocal placoid pigment epitheliopathy (APMPPE), multiple evanescent white dot syndrome (MEWDS), multifocal choroiditis with panuveitis (MFC), and punctate inner choroiditis (PIC).39–41 APMPPE is characterized by the sudden onset of multiple, bilateral lesions that are yellow-white and placoid in appearance. These lesions are primarily located posterior to the equator and at the level of the RPE and choriocapillaris. When examined with fluorescein angiography (FA), the active placoid lesions show early hypofluorescence or blockage, followed by late, irregular hyperfluorescent staining.42 MEWDS is defined by the presence of a unilateral orange-yellow fovea with a granular appearance. Multifocal, grey-white lesions can be observed at the level of the RPE or deep retina. On FA, these lesions exhibit early punctate hyperfluoresecence in a wreath-like pattern with late staining.43 While both MFC and PIC present as bilateral chorioretinal lesions, PIC is more confined to the posterior pole with stronger association with myopia. Minimal early hyperfluorescence and late hyperfluorescent staining is typically found on FA. While MFC has moderate vitritis, PIC has minimal or no vitreous cell. Both are strongly associated with choroidal neovascularization.44

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Vogt-Koyanagi-Harada Syndrome (VKH) Vogt-Koyanagi-Harada disease is characterized by bilateral granulomatous panuveitis with or without extraocular manifestations, including neurological, integumentary, and auditory involvement.45 VKH is sometimes subdivided into two distinct clinical entities: Vogt–Koyanagi disease, primarily marked by skin changes and anterior uveitis, and Harada disease, in which neurological features and exudative retinal detachments predominate. The disease predominantly affects more pigmented individuals, such as Hispanics, Asians, Native Americans, Middle Easterners, and Asian Indians, and is associated with HLADR1 and HLA-DR4.46 Clinically, VKH is characterized by four stages: a prodromal stage resembling a viral illness lasting a few days, followed by an acute uveitic stage characterized by bilateral granulomatous anterior and multifocal posterior uveitis, a chronic stage with vitiligo, poliosis and depigmentation of the choroid, and a chronic recurrent stage defined as panuveitis with acute exacerbations of anterior uveitis and rarely posterior uveitis. VKH follows a pattern of exacerbations and remissions, with a highly variable clinical course. While VKH is most commonly diagnosed between the ages of 20 and 50, there have been a few reported cases in young children,21 with approximately 3% to 13% of VKH cases beginning in childhood.47 The prevalence of VKH among pediatric uveitis varies from 0.7% to 3% depending on the geographical location.48 Unfortunately, like other childhood uveitis, the diagnosis of VKH-associated uveitis is often delayed, and its progression tends to be more aggressive.21,49 Prominent complications that can result in vision impairment include changes in the RPE, glaucoma, cataracts, subretinal neovascularization, and phthisis bulbi.49 Sympathetic Ophthalmia Sympathetic ophthalmitis (SO) is characterized by bilateral granulomatous panuveitis, similar to VKH. The underlying mechanism of SO involves the systemic exposure of previously immune-privileged uveal antigens after trauma, triggering an autoimmune response to the uvea. This immune response leads to inflammation in both the initially injured eye and contralateral sympathizing eye. It typically occurs between 2 weeks and 3 months (range from 5 days to 66 years) following penetrating trauma or multiple intraocular surgeries.50 A study from India estimated that the incidence of SO in the pediatric age group is approximately 0.24%.51 Interestingly, late-onset cases among the pediatric patients are not uncommon, as revealed in a study by Dutta Majumder et al. In this study, SO was observed 6 months after the initial traumatic events in 35% of pediatric patients, with one child developing SO approximately 15 years after multiple vitreoretinal surgeries for retinopathy of prematurity.52 Hence, it is of utmost importance to provide thorough counseling regarding the potential risk of developing SO in the unaffected eye to parents whose children have experienced penetrating eye injuries or multiple intraocular surgeries. SO carries significant ocular morbidity, primarily due to its potential to cause vision loss in both eyes following an insult or injury to just one eye. Therefore, early recognition of the signs and symptoms of SO is critical because the condition poses a serious threat to vision. Timely and effective treatment is crucial, as many children may experience substantial vision impairment if intervention is not initiated promptly.51

Autoimmune Systemic Disorders Behçet Disease Behçet disease, a chronic and recurrent inflammatory vasculitis, is characterized by a classic triad of oral aphthous ulcers, genital ulcers, and uveitis. Systemic manifestations may involve the gastrointestinal, nervous, musculoskeletal, and cardiovascular systems. While Behçet disease typically manifests between the second and fourth decades of life, childhood onset has been reported in 3% to 26% of cases.53 Notably, the presence of the HLA-B51 gene marker is considered supportive but not definitive for diagnosis. Ocular involvement is a significant aspect of Behçet disease and can manifest in a variety of ways. The classic presentation includes hypopyon uveitis and occlusive retinal vasculitis. While Behçet uveitis can affect both the anterior and the posterior segment of the eye in isolation, panuveitis is the most frequent presentation.54 A retrospective analysis of 34 Turkish patients with pediatric Behçet uveitis revealed an average age of onset at 14 years.13 Interestingly, Kesen et al. compared childhood-onset with adult-onset Behçet uveitis, demonstrating that visual prognosis tends to be more favorable in children.55

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Tubulointerstitial Nephritis and Uveitis Syndrome Although tubulointerstitial nephritis and uveitis syndrome (TINU) usually presents as acute-onset of bilateral non-granulomatous anterior uveitis, other presentation such as intermediate, posterior, and panuveitis have also been documented.56 Median age at diagnosis has been reported to be 15 years (range 9 to 74 years). Patients are often found to have excess beta-2-microglobulin (Ub2MG) in their urine, a marker that can support this diagnosis. Human leukocyte antigen (HLA) typing has also proven to be valuable. A significant association has been observed between TINU and HLA-DRB101, HLADQB105, and HLA-DRB1*01.57 However, a definitive diagnosis of TINU can only be made with renal biopsy, which reveals eosinophilic and mononuclear cellular infiltrates with glomerular sparing. In a review of 133 reported cases of TINU syndrome, uveitis recurred or followed a chronic course in 56% of patients and persisted for several years in some cases. Despite ocular complications, including posterior synechiae, cataracts, and elevated intraocular pressure (IOP) that were reported in 21% of cases, patients generally maintained a good visual prognosis, with no cases of permanent, severe vision loss, and most patients having improved final vision to 20/25 or better.56 Pediatric Sarcoidosis Pediatric sarcoidosis is a chronic systemic granulomatous disease of unknown etiology, characterized by the formation of noncaseating granulomas. In children, there is a 3:1 ratio of Black to Caucasian cases, with an equal distribution among males and females. Most instances of pediatric sarcoidosis manifest between the ages of 8 and 15 years. This age group typically exhibit lung involvement, along with a 30 to 40% likelihood of affecting the eyes, skin, liver, and spleen. The most prevalent ocular manifestation overall is bilateral granulomatous anterior uveitis, seen in 21% to 48% of pediatric cases. Nevertheless, pediatric patients may also experience posterior uveitis, panuveitis, multifocal chorioretinitis, and optic disc edema.58 Unlike adults, retinal periphlebitis with the characteristic “candle wax drippings” is less common in children.58,59 Confirmation of the diagnosis involves a tissue biopsy of affected areas, such as a skin lesion or peripheral lymph node, which typically reveals noncaseating epithelioid cell granulomas. Given the frequent pulmonary involvement in most pediatric sarcoidosis cases, chest X-rays are commonly employed as a screening tool. Elevated levels of serum angiotensin converting enzyme (ACE), lysozyme and calcium can support the diagnosis. However, it is important to note that serum ACE levels are often physiologically elevated in children, reducing their diagnostic significance in sarcoidosis for this population. Blau Syndrome Blau syndrome, also known as familial juvenile systemic granulomatosis, is characterized by a triad of granulomatous dermatitis, arthritis, and uveitis. This condition is rare, affecting less than 1 in 1,000,000, and typically inherited in an autosomal dominant manner.60 Chronic bilateral granulomatous iridocyclitis is the common initial manifestation, but as the disease progresses over time, a significant proportion of patients with eye involvement ultimately advance to panuveitis.61 In a prospective cases series of 50 patients with Blau syndrome, the median age at onset of eye disease was 60 months, with 78% of eyes exhibiting uveitis at baseline. Panuveitis was observed in 51% of the eyes. The most common fundus finding was multifocal chorioretinal lesions (39%), followed by optic disc changes (29%).61 Repeated episodes of inflammation result in gradual deterioration in visual acuity and an increased risk of sightthreatening complications, including cataracts, glaucoma, retinal detachment, and optic atrophy.62 Systemic Lupus Erythematosus Systemic lupus erythematosus (SLE) is an autoimmune disorder that affects multiple systems in the body, characterized by presence of circulating antibodies targeting nuclear cellular components. This condition predominantly affects females, typically emerging between the ages of 20 and 30, although it can manifest from infancy to late adulthood.63 While adult and pediatric SLE share some clinical similarities, children with early-onset SLE tend to present with more active disease and have a higher prevalence of atypical manifestations.64 In a substantial retrospective study involving 852 individuals with early-onset SLE, uveitis was a rare occurrence, observed in only 0.8% of cases. Complications were reported in only two cases, with cataracts in one and retinal ischemia leading to subsequent neovascularization in the other.65 A cross-sectional study examining 52 consecutive children with SLE found that the average age

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at presentation was 11.3 years. Ocular manifestations were evident in 34.6% of these cases, which included retinal vascular lesions (5 out of 18 cases), unilateral optic neuropathy (3 out of 18 cases), and bilateral iridocyclitis (1 out of 18 cases).66

Diagnosis and Work-up A full ocular examination including age-appropriate visual acuity testing, measurement of IOP, refraction under cycloplegia, slit lamp examination, and dilated examination is indicated. For very young patients, examination under anesthesia may be required. The Standardization of Uveitis Nomenclature (SUN) grading scheme is commonly used to monitor the degree of intraocular inflammation. Ocular coherence tomography (OCT) is non-invasive tool routinely used to quantify and monitor CME. Fundus autofluorescence (FAF) is another noninvasive test that highlights areas of active inflammation (increased FAF) and previous photoreceptor and/or RPE damage (decreased FAF). Fluorescein angiography is helpful to identify the presence and extent of vascular leakage, non-perfusion, and neovascularization. Indocyanine green angiography is used to visualize choroidal inflammation. If the view of the retina is obscured by inflammation, ultrasonography can be used to evaluate vitreous traction and retinal detachment. The choice of appropriate laboratory and imaging tests is guided by the clinical features of the various types of uveitis discussed above. It is used to rule out infectious and oncological causes as well as to evaluate for associated autoimmune and inflammatory conditions. Commonly obtained tests include complete blood count with differential, chest x-ray, tuberculosis testing, syphilis testing, serum ACE (although ACE is usually elevated in children), and lysozyme. When appropriate, disease-specific IgM and IgG for toxoplasmosis, Bartonella, herpes viruses, and Lyme disease are obtained. Antinuclear antibodies (ANA), antineutrophil cytoplasmic antibodies (cANCA), and perinuclear anti-neutrophil cytoplasmic antibodies (pANCA) are obtained to evaluate for systemic vasculitides. Urinalysis, urine beta2-microglobulin, and renal biopsy, when appropriate, are used to evaluate for TINU.

Management Local therapies Topical corticosteroids such as prednisolone acetate and difluprednate are first-line in treating pediatric uveitis. However, they are often insufficient to control posterior segment disease. Local therapies such as periocular and intravitreal injections are commonly considered for non-anterior uveitis due to resistance to topical therapies. Subtenon triamcinolone injections (Kenalog®; Bristol Myers Squibb) is a reasonable option when topical therapies fail. Jung JL et al. reported improved inflammation in 85.4% of treated eyes (48 eyes in 30 patients) with resolved CME in 78% of eyes. Ocular hypertension developed in 12.5% of eyes.67 Other studies also support similar risk of IOP elevation between topical and subtenon steroids.68. No new cataract was reported. Intravitreal dexamethasone implant 0.7 mg (Ozurdex, Allergan, Irvine, CA) is a self-dissolving intravitreal implant. It can effectively control inflammation resulting in improved vision, decreased CME in pediatric patients with posterior segment-involving uveitis.69–71 88-93% of patients achieved clinical control after treatment, with each injection lasting approximately 9 months. Common side effects including elevated IOP (20-38%) and cataract formation (0-30%). Although the intravitreal dexamethasone implant has a risk of endophthalmitis, no cases were reported in these relatively small retrospective studies. Fluocinolone acetonide intravitreal implants (ILUVIEN, Alimera Sciences, UK - 0.19 mg; YUTIQ, Alimera Sciences, UK - 0.18mg, both lasting 3 years) have also been used to treat pediatric non-anterior uveitis. In a patient with JIA-associated uveitis with bilateral CME who previously had good response to the

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dexamethasone 0.7mg intravitreal implant and was a non-steroid responder with unreliable follow up, fluocinolone acetonide intravitreal implants were used both eyes with persistent reduction of CME and improved visual acuity at the end of follow up at 12 months.72

Systemic therapies Systemic corticosteroids are a standard short-term option before systemic immunomodulatory therapies (IMT) take effect. Systemic steroids are associated with growth interference, weight gain, acne, cushingoid habitus, mood changes, metabolic issues, among many other side effects. Therefore, longterm use should be avoided. Traditional Immunomodulatory Therapies (IMT) Methotrexate (MTX) is a folate analog that interrupts DNA replication and transcription, inhibiting rapidly dividing immune cells. It is considered the first line therapy for pediatric non-infectious, non-anterior uveitis not controlled on local therapy. Therefore, it is the most frequently used steroid-sparing agent. MTX was efficacious in controlling inflammation in 64.3-89% of patients with intermediate, posterior, or panuveitis and systemic steroids were successfully discontinued in 64% of patients.73 Folic acid should be supplemented while on MTX. Common side effects include liver toxicity and gastrointestinal discomfort. MTX monotherapy has been reported to control inflammation in 48% of pediatric uveitis. 74 Mycophenolate mofetil (MMF) is a pro-drug that is metabolized to mycophenolic acid and subsequently blocks the enzyme inosine-5-monophosphate dehydrogenase critical in the purine biosynthetic pathway. MMF suppresses both T and B lymphocytes. It is generally well tolerated in children but sometimes causes gastrointestinal discomfort and less commonly leukopenia, hair loss, and fatigue. 50%-80% of pediatric patients with non-infectious non-anterior uveitis achieved control of inflammation on MMF monotherapy. 75,76 Azathioprine is a purine nucleoside analog that inhibits DNA synthesis. It leads to a more significant decrease in T cell proliferation than that in B cells. Commonly reported side effects are gastrointestinal toxicities. It is moderately effective at controlling chronic, non-infectious pediatric uveitis with posterior segment involvement, achieving inactivity in 44%-69% of patients and corticosteroid-sparing in 36%-47% of patients. 77 It is not as commonly used due to more severe GI side effects than other IMTs. 1,77 Cyclosporine is a calcineurin inhibitor, suppressing T-cell proliferation and preventing release of proinflammatory cytokines. Although earlier studies suggested that cyclosporine was effective at controlling inflammation and stabilizing vision in patients with sight-threatening intermediate uveitis or panuveitis, its application is limited by major side effects such as nephrotoxicity, hypertension, gingival hyperplasia, hirsutism, and malignancy.78,79 A later multicenter retrospective study showed limited value of cyclosporine as monotherapy in controlling chronic JIA-associated pediatric uveitis with posterior complications. Only 6 of 25 (24%) patients had inactivity of uveitis while on cyclosporine alone, with increased rate of CME, vitreous opacities and retinal detachment in patients at the end of cyclosporine therapy after a mean follow up of 3.9 years. Biological Immunomodulatory Therapies Adalimumab is a fully humanized anti-tumor necrosis factor alpha (anti-TNF-α) monoclonal antibody. It is the only Food and Drug Administration (FDA) approved biologic for pediatric non-infectious uveitis. In the SYCAMORE trial, a multicenter, double-blind, randomized, placebo-controlled trial assessing the efficiency of adalimumab in the treatment of JIA-associated uveitis enrolling 90 patients80, the adalimumab + methotrexate group had lower treatment failures compared to methotrexate alone (27% vs. 60%). Serious adverse events were reported more frequently in the adalimumab group vs. MTX only group (0.29 vs. 0.19 events per patient-year) including gastrointestinal disorder and infections. More recent studies continue to support the effectiveness of adalimumab in controlling non-infectious nonanterior uveitis, leading to decreased frequency and/or severity of flare-ups, as well as decreased glucocorticoid and systemic IMT usage.8,81 There is an ongoing clinical trial assessing whether adalimumab can be safely discontinued in patients with quiescent disease led by Dr. Nisha Acharya (ClinicalTrials.gov NCT03816397).

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Infliximab is a human-murine chimeric anti-TNF-α monoclonal antibody. It has been shown to be effective and safe at treating non-infectious, non-anterior, pediatric uveitis.82,83 Some meta-analyses suggest similar therapeutic efficacy and corticosteroid sparing effects between infliximab and adalimumab, with possibly more favorable adverse effects profile for adalimumab, while others suggest superiority of adalimumab in efficacy in childhood chronic uveitis compared to infliximab as first line agents.84–86 A recent retrospective study enrolled 25 patients with chronic, non-infectious, pediatric uveitis refractory to previous biologic therapies including adalimumab who subsequently switched to infliximab.83 Four out of the 25 patients had intermediate/pan-uveitis including Blau syndrome. After switching to infliximab, 3 out of the 4 patients had improved inflammation. Rituximab is a chimeric monoclonal antibody targeting CD-20, leading to B cell inactivation. In small retrospective studies of pediatric patients with JIA-associated uveitis, both anterior and non-anterior types, that were refractory of traditional IMTs and TNF-α inhibitors, rituximab treatment resulted in uveitis inactivity in 7 out 10 patients (70%) in one study87 and 8 out 8 patients (100%) in another study88. All 4 patients with non-anterior uveitis in these two studies achieved complete control of disease with rituximab and were able to taper off and/or significantly reduce their usage of topical and systemic corticosteroids.87,88 Tocilizumab is a monoclonal antibody binding to IL-6 receptors inhibiting related pro-inflammatory pathways. In the APTITUDE study, a multicenter, single-arm, phase 2 prospective clinic trial enrolling pediatric patients with JIA-associated uveitis not responding to TNF inhibitors89, 7 out of 21 patients (33%) who received tocilizumab were responders without any severe adverse events, narrowly missing the prespecified primary endpoint (more than 7 patients responding to treatment). Of note, 3 out of 4 patients with cystoid macular edema in this cohort had complete resolution of macular edema after treatment, as reported in other studies.90 The STOP-Uveitis study, a randomized, open label phase I/II clinical trial, suggested efficacy of tocilizumab in treating noninfectious intermediate, posterior, and panuveitis in adult patients resulting in improved visual acuity, vitreous haze, and central macular thickness.91 However, pediatric patients were not included in this study. Abatacept is selective T-cell inhibitor targeting CD80 and CD86. There are mixed results for the efficacy of abatacept in treating pediatric uveitis refractory to TNF- α antagonists.92–94 Some small studies suggested that abatacept could be effective in reducing inflammation in this patient population, while larger studies failed to show convincing long-lasting effects of abatacept in the treatment of severe, longstanding, and refractory uveitis.92–94

Emerging Therapeutic Options Janus Kinase (JAK) inhibitors are small molecules targeting the JAK-STAT pathway, downstream of cytokine receptors implicated in pathology of many autoimmune and inflammatory disorders.95 Tofacitinib was approved in treating rheumatological conditions in children in 2020.96 There are case reports of promising results of JAK inhibitors in treating pediatric, non-infectious, non-anterior uveitis. For example, one patient with history of refractory JIA uveitis with posterior involvement including macular edema and retinal vasculitis previously treated with abatacept, rituximab, and tocilizumab with only partial control of disease had complete resolution of her long-standing macular edema after 6 months of oral tofacitinib monotherapy.97 There is an ongoing phase 3 clinical trial comparing the clinical effectiveness and safety of baricitinib vs. adalimumab for treatment of JIA-associated uveitis and chronic ANA-positive uveitis in pediatric patients who failed MTX without prior treatment with any biologics.98 Golimumab is another fully humanized anti-TNF-α monoclonal antibody approved in treating pediatric JIA patients in 2020. 99 In a retrospective study of 10 patients with JIA-associated uveitis refractory to adalimumab, 3 of whom with panuveitis, 5 out of 10 patients had complete persist response, while 3 other patients had initial partial response.100

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Long-term Complications and Considerations While age at diagnosis varies between studies due to different underlying diseases and sample sizes, it has been well established that younger age at uveitis onset, delayed presentation, and uveitis involving the posterior segment were associated with poorer visual outcome.101 Compared to adults, children with uveitis experience higher rates of ocular complications.5,102 While all pediatric uveitis causes complications such as band keratopathy, synechiae, cataract, ocular hypertension and glaucoma, intermediate and posterior non-infectious uveitis are more commonly associated with CME, epiretinal membrane, optic disc edema, vitreous traction, and retinal detachments.5,103,104 Pediatric patients with intermediate, posterior, and panuveitis developed ocular complications earlier in the disease course compared to anterior uveitis.5 The complications associated with uveitis of the posterior segment are more likely to lead to severe vision loss and worse visual outcome than anterior uveitis.102,5 20- 25% of posterior uveitis and panuveitis patients have severe vision loss, compared to 5%-17% in pediatric uveitis in general.102,104 In terms of monitoring, close follow up and frequent examinations are recommended until remission is achieved. During early remission and while on immunomodulatory therapy, it is recommended that the patient should be seen every 8-12 weeks based on uveitis history since flares and development of ocular complications are common. 1

Conclusions Pediatric non-infectious non-anterior uveitis are a group of diseases with low prevalence but high ocular comorbidity. They can be challenging to manage given incomplete responses to standard therapies. Novel biologic and small molecule immunomodulatory therapies are increasingly employed in the management of pediatric patients with promising results.

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Kesen MR, Goldstein DA, Tessler HH. Uveitis Associated With Pediatric Behçet Disease in the American Midwest. American Journal of Ophthalmology. 2008;146(6):819-827.e2. doi:10.1016/j.ajo.2008.05.043

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Mandeville JT, Levinson RD, Holland GN. The tubulointerstitial nephritis and uveitis syndrome. Surv Ophthalmol. 2001;46(3):195-208. doi:10.1016/s0039-6257(01)00261-2

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Levinson RD, Park MS, Rikkers SM, et al. Strong associations between specific HLA-DQ and HLA-DR alleles and the tubulointerstitial nephritis and uveitis syndrome. Invest Ophthalmol Vis Sci. 2003;44(2):653-657. doi:10.1167/iovs.020376

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Taylor SRJ, Tomkins-Netzer O, Joshi L, Morarji J, McLoone E, Lightman S. Dexamethasone Implant in Pediatric Uveitis. Ophthalmology. 2012;119(11):2412-2412.e2. doi:10.1016/j.ophtha.2012.07.025

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Sella R, Oray M, Friling R, Umar L, Tugal-Tutkun I, Kramer M. Dexamethasone intravitreal implant (Ozurdex®) for pediatric uveitis. Graefes Arch Clin Exp Ophthalmol. 2015;253(10):1777-1782. doi:10.1007/s00417-015-3124-x

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Tomkins-Netzer O, Talat L, Seguin-Greenstein S, Bar A, Lightman S. Outcome of Treating Pediatric Uveitis With Dexamethasone Implants. American Journal of Ophthalmology. 2016;161:110-115.e2. doi:10.1016/j.ajo.2015.09.036

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Ansari AS, Amir Z, Williams GS. Bilateral 0.19 mg Fluocinolone Acetonide Intravitreal Implant in the Successful Treatment of Juvenile Idiopathic Arthritis-Associated Uveitis and Secondary Macular Oedema: A Case Report and Review of Intravitreal Therapies. Ophthalmol Ther. 2021;10(1):193-200. doi:10.1007/s40123-020-00328-9

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Samson CM, Waheed N, Baltatzis S, Foster CS. Methotrexate therapy for chronic noninfectious uveitis: Analysis of a case series of 160 patients. Ophthalmology. 2001;108(6):1134-1139. doi:10.1016/S0161-6420(01)00576-0

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Chang PY, Giuliari GP, Shaikh M, Thakuria P, Makhoul D, Foster CS. Mycophenolate mofetil monotherapy in the management of paediatric uveitis. Eye (Lond). 2011;25(4):427-435. doi:10.1038/eye.2011.23

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78.

Dick AD, Azim M, Forrester JV, Dick AD, Azim M, Forrester JV. Immunosuppressive therapy for chronic uveitis: optimising therapy with steroids and cyclosporin A. British Journal of Ophthalmology. 1997;81(12):1107-1112. doi:10.1136/bjo.81.12.1107

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Liu W, Bai D, Kou L. Comparison of infliximab with adalimumab for the treatment of non-infectious uveitis: a systematic review and meta-analysis. BMC Ophthalmol. 2023;23(1):240. doi:10.1186/s12886-023-02987-1

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Maccora I, Fusco E, Marrani E, Ramanan AV, Simonini G. Changing evidence over time: updated meta-analysis regarding anti-TNF efficacy in childhood chronic uveitis. Rheumatology (Oxford). 2021;60(2):568-587. doi:10.1093/rheumatology/keaa595

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Heiligenhaus A, Miserocchi E, Heinz C, Gerloni V, Kotaniemi K. Treatment of severe uveitis associated with juvenile idiopathic arthritis with anti-CD20 monoclonal antibody (rituximab). Rheumatology (Oxford). 2011;50(8):1390-1394. doi:10.1093/rheumatology/ker107

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Miserocchi E, Modorati G, Berchicci L, Pontikaki I, Meroni P, Gerloni V. Long-term treatment with rituximab in severe juvenile idiopathic arthritis-associated uveitis. Br J Ophthalmol. 2016;100(6):782-786. doi:10.1136/bjophthalmol-2015306790

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Ramanan AV, Dick AD, Guly C, et al. Tocilizumab in patients with anti-TNF refractory juvenile idiopathic arthritisassociated uveitis (APTITUDE): a multicentre, single-arm, phase 2 trial. The Lancet Rheumatology. 2020;2(3):e135e141. doi:10.1016/S2665-9913(20)30008-4

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Vegas-Revenga N, Calvo-Río V, Mesquida M, et al. Anti-IL6-Receptor Tocilizumab in Refractory and Noninfectious Uveitic Cystoid Macular Edema: Multicenter Study of 25 Patients. Am J Ophthalmol. 2019;200:85-94. doi:10.1016/j.ajo.2018.12.019

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Sepah YJ, Sadiq MA, Chu DS, et al. Primary (Month-6) Outcomes of the STOP-Uveitis Study: Evaluating the Safety, Tolerability, and Efficacy of Tocilizumab in Patients With Noninfectious Uveitis. American Journal of Ophthalmology. 2017;183:71-80. doi:10.1016/j.ajo.2017.08.019

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Tappeiner C, Miserocchi E, Bodaghi B, et al. Abatacept in the treatment of severe, longstanding, and refractory uveitis associated with juvenile idiopathic arthritis. J Rheumatol. 2015;42(4):706-711. doi:10.3899/jrheum.140410

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Maccora I, Abu Rumeileh S, Curci F, et al. Tocilizumab and Abatacept for the Treatment of Childhood Chronic Uveitis: A Monocentric Comparison Experience. Front Pediatr. 2022;10:851453. doi:10.3389/fped.2022.851453

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Ramanan AV, Guly CM, Keller SY, et al. Clinical effectiveness and safety of baricitinib for the treatment of juvenile idiopathic arthritis-associated uveitis or chronic anterior antinuclear antibody-positive uveitis: study protocol for an open-label, adalimumab active-controlled phase 3 clinical trial (JUVE-BRIGHT). Trials. 2021;22(1):689. doi:10.1186/s13063-021-05651-5

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100. Lanz S, Seidel G, Skrabl-Baumgartner A. Golimumab in juvenile idiopathic arthritis-associated uveitis unresponsive to Adalimumab. Pediatric Rheumatology. 2021;19(1):132. doi:10.1186/s12969-021-00630-1 101. Tungsattayathitthan U, Rattanalert N, Sittivarakul W. Long-term visual acuity outcome of pediatric uveitis patients presenting with severe visual impairment. Sci Rep. 2023;13(1):2919. doi:10.1038/s41598-023-29159-x 102. Edelsten C, Reddy MA, Stanford MR, Graham EM. Visual loss associated with pediatric uveitis in english primary and referral centers. American Journal of Ophthalmology. 2003;135(5):676-680. doi:10.1016/S0002-9394(02)02148-7 103. Raveendra Murthy S, Ganesh S, C.K. M, Dubey N. Pediatric uveitis: a retrospective analysis at a tertiary eye care hospital in South India. Ophthalmol Eye Dis. 2021;13:25158414211027707. doi:10.1177/25158414211027707 104. Markomichelakis NN, Aissopou EK, Chatzistefanou KI. Pediatric Non-Infectious Uveitis: Long-Term Outcomes and Complications. Ocular Immunology and Inflammation. 2023;0(0):1-8. doi:10.1080/09273948.2022.2162422

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The role of Inflammation in Age-related Macular Degeneration James M. Harris, MD, PhD, Frances Wu, MD, and David M. Wu, MD, PhD

Abstract Age-related macular degeneration (AMD) is a common neurodegenerative disease that results in significant morbidity and economic cost to patients and society. Many genetic and environmental factors have been linked with AMD furthering our understanding of disease pathogenesis. A prominent feature of AMD, which is shared across a wide spectrum of neurodegenerative diseases, is the involvement of inflammation in disease pathogenesis. While advances in our understanding have led to breakthrough vision-saving treatments for neovascular AMD, development of therapies targeting inflammation for nonexudative (dry) AMD or refractory neovascular AMD are still in their infancy. Here we survey the evidence from patients supporting the pathogenic role of inflammation in AMD and review the efforts to treat AMD with therapeutic interventions targeting mediators of inflammation.

Introduction Age-related macular degeneration (AMD) is a neurodegenerative condition that is the leading cause of blindness in the developed world, with a global prevalence of 8.7%. In aggregate, AMD is estimated to affect ~200 million people1 and results in $300 billion in costs to society including $250 billion in direct care costs and the remainder in lost economic output.2 The prevalence and impact are likely to increase given aging population demographics, with projections reaching ~300 million affected individuals by 2040.1 Patients with AMD suffer from progressive damage and death to the macula, the central portion of the retina. The macula has the highest concentration of cone photoreceptors, responsible for high-acuity color vision, and thus their loss is particularly devastating. Photoreceptor death is thought to secondary to damage to the adjacent retinal pigment epithelial cells (RPE) and choroid, vasculature, which provide support and blood supply respectively to the neural. A variety of pathogenic mechanisms have been implicated in AMD including oxidative stress, mitochondrial dysfunction, autophagy, protein regulation, lipid metabolism, extracellular matrix signaling, and chronic inflammation.3 Many of these disease features are widely shared across neurodegenerative conditions, opening the possibility that advancement in our understanding and treatment of AMD may have implications for other neurodegenerative disease. Thus, as a neurodegenerative disease affecting the clinically observable and accessible retinal tissue, AMD may provide a unique window in pathology of central nervous system degeneration. Of these various pathogenic mechanisms, the role of inflammation has been widely observed across neurodegenerative diseases.4,5 Here we survey the genetic, molecular, and cellular evidence from human patients with AMD that implicate inflammation in the disease pathogenesis of AMD. We also review interventional clinical trials that target mediators of inflammation to assess the current state of drug development in the field and further shed light on disease mechanisms.

Genetic Link Between Inflammation and AMD Although it takes the better part of a lifetime to develop AMD, this disease has a surprisingly high genetic contribution.6 The first evidence for the significant role of genetics in this disease came from twin and family studies. Twin concordance rates were measured between 42-100%.7,8 Comparison of hundreds of monozygotic twins, who had identical genomes, to dizygotic twins, who shared only some, but not all of their genetic information, showed AMD concordance was twice as high in monozygotic twins.9 Familial aggregation studies, which measured how likely an individual was to develop late AMD if they had a family member with AMD were 4.2 times more likely to develop AMD compared to controls who had no

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relatives with AMD.10 Furthermore, having a relative with AMD was associated with earlier disease onset.11 These epidemiologic observations showed a clear and strong contribution of genetics to the incidence of AMD. Furthermore, genetics can serve as a powerful guide in the search for the causative drivers of many diseases. Principally, genetic evidence enjoys an iron-clad temporal relationship with associated diseases: genetic variants were undoubtedly present prior to disease onset. This largely eliminates the possibility that observed DNA variants were caused by the disease. When studying inflammation, which is the body’s response to tissue damage, it can be difficult to distinguish whether ongoing inflammation is the cause or consequence of disease pathology. Surprisingly, many of the linked genes turned out to be involved in inflammation broadly, with particular enrichment in components of the complement pathway.

Genetic Variation in the Complement System is Strongly Linked With AMD The complement system is named for its role in “complementing” the cell-based components of the immune system and serves as a core aspect of humoral immunity. The primary role of complement is to distinguish “self” from “non-self”. It labels microbes, pathogens, dying and diseased cells, and extracellular debris for death and clearance by phagocytosis. The complement cascade can be initiated by three distinct pathways, each with a specific task in promoting overall immunity. The “classic” pathway drives immune response to antigens recognized by antibody binding. The classic pathway is initiated by C1q binding to an immunoglobulin antibody, serum amyloid, or C-reactive protein (CRP), which promotes formation of the C1 complex and subsequent activation of C2 and C4. The second complement initiating pathway is the mannose-binding lectin pathway. Mannose-binding lectins bind to carbohydrate moieties on the surface of a wide variety of pathogens, as well as senescent and apoptotic cells. Binding of mannose-binding lectin (MBL) to a target activates MBL-associated serine proteases, MASP-1 and MASP-2, which, like the classic pathway, also activate C2 and C4. The classic and mannose-binding lectin pathways converge on C3 which is split into C3a and C3b. C3b then activates a common downstream cascade. The third and final complement activation pathway is the “alternative” pathway that results from spontaneous hydrolysis of the C3 molecule into C3a and C3b without any activating stimulus. Activated C3b interacts with factor B and factor D to create a C3 convertase, which generates more C3b, creating a positive feedback loop that amplifies the initial alternative pathway signaling. This “tic-over” mechanism provides a baseline level of complement activation throughout the body12. Thus, an environment is created in which healthy cells and tissues must express inhibitors of alternative complement attack to continue to survive, creating a cellular “dead-man switch”. These inhibitors include transmembrane proteins expressed by cells including membrane cofactor protein (MCP or CD46), decay accelerating factor (DAF or CD55), prolactin (CD59), complement C3b/C4b receptor 1 (CR1 or CD35)13 or soluble complement inhibitors that circulate in plasma and bind to various host surfaces throughout the body and include factor H, factor I, and C4-binding protein.12 Activation of any of the 3 complement pathways converge on a final common pathway of the complement system, which involves assembly of the membrane attack complex consisting of C5b, C6, C7, C8 and C9. The membrane attack complex creates a transmembrane channel leading to osmotic lysis of a targeted cell. Supplementing this primary pathway, several upstream signaling molecules are released during complement activation including the anaphylatoxins C3a and C5a, which trigger degranulation of mast cells and neutrophils, stimulate leukocyte chemotaxis and extravasation, and induce cytokine release from macrophages. C3b and C4b are opsins that bind directly to targets stimulating phagocytosis directly.14 Overall, the complement system is a powerful mechanism of removing pathogens, unhealthy cells, and cellular debris.

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FIGURE 1: The complement activation pathways including the classic, mannose-binding lectin, and alternative pathways converge on the common membrane attack complex. Components of the pathway also serve as inflammatory mediators and opsins.15

CFH Complement factor H (CFH) is one of the primary circulating complement inhibitors that protects acellular host tissues from attack by the alternative complement pathway. Factor H recruits factor I to degrade C3b, preventing complement activation and amplification. The Y402H (rs1061170) variant in CFH was the first single nucleotide polymorphism (SNP) linked to AMD because it is common in most populations and has a strong association with AMD, which allowed for effective linkage studies using DNA microarray16 and targeted approaches.17-19 This linkage has been confirmed by many subsequent studies.20-40 The high-risk Y402H variant has been associated with early age of onset and family history of AMD.36,41,42 This SNP also falls in a coding region of a functional domain of CFH, resulting in a plausible mechanistic role in mediating this genetic association. As such, the role of this variant in AMD has been investigated extensively. Barlow et al. provide a review of the structure and function of the factor H protein.43 AMD Subtypes Patients with AMD have a variety of presenting phenotypes. Early in disease, collections of lipoprotinaceous extracellular material, called drusen, can accumulate between the retinal pigment epithelium and Bruch’s membrane. At this point, visual acuity is typically not affected. End-stage or late AMD consist of two later phenotypes that result in significant vision loss. Around 80% of patients with late AMD have wet, exudative or neovascular AMD, which involves the proliferation and leakage of new blood vessels. In the remainder of patients, geographic atrophy results in the progressive death of photoreceptor cells and their neighboring retinal pigment epithelial cells. Interestingly, siblings who have advanced disease tend to have the same type of advanced disease, suggesting a genetic contribution to these advanced stages.38 Efforts have been made to identify the genetic variants linked with each of

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these AMD phenotypes to better understand their pathogenic mechanisms, with particular focus on CFH Y402H. The CFH Y402H polymorphism was associated with both early28,36,42 and late stage AMD.28,36,44-46 Drusen can be subclassified into several distinct categories including hard drusen, soft/confluent drusen, or cuticular drusen. The CFH Y402H variant increased the risk for soft drusen formation.47 A study of Swiss patients found increased risk of peripheral drusen in CFH Y402H homozygotes, but otherwise no relationship between CFH Y402H allele and drusen size, total surface area, location nasal to the optic disc, or pigmentary changes.48 A study of Hispanic patients found increased risk of bilateral intermediate to large soft drusen with this CFH variant, but no association with other early AMD disease features.49 Several studies found a stronger association of CFH Y402H with advanced AMD than early AMD50,51, though this was not the case for all studies.48 CFH Y402H has been consistently enriched in patients with exudative/neovascular AMD compared to controls.47,52-59 Baird et al. found a stronger association with the CFH Y402H risk variant and neovascular AMD compared with dry AMD42. CFH Y402H was not, however, associated with severity of CNV in an Israeli population.58 In nonexudative AMD, CFH Y402H was found to be more common in patients with geographic atrophy compared to controls.22,47,55,60,61 Population Variation While CFH Y402H was initially associated with AMD in primarily white European and American study populations, subsequent work examined the extent to which this association existed across the genetic diversity of human populations. Linkage of CFH Y402H with AMD was found for many cohorts including in UK55, Finnish62, Swiss48, Italian63, Central European52, Russian64, Israeli58, Brazilian65, Hispanic49, Indian66, and Australian cohorts.45 Interestingly, significant variation in the prevalence of this polymorphism was uncovered across populations. Caucasians and Africans had high population frequencies of Y402H, roughly 30-35%, while Chinese and Japanese populations had much lower rates of the Y402H polymorphism, around 4-7%.67-70 Within east Asia, several studies have linked Y402H to AMD in Chinese69 and Taiwanese42 populations. However, multiple studies have failed to find association of CFH Y402H with AMD including in Chinese,67,71 Korean,72 and especially Japanese70,73-77 cohorts. Outside of Asia, no significant association of Y402H with AMD was found in a South African78 cohort, though this study was small and possibly underpowered. Common Variants Variants linked to disease can be classified based on their frequency within a population. Common variants are typically defined as having a >1% frequency across a population, while rare variants have <1% frequency. From an evolutionary perspective, common variants are not likely to have highly penetrant or severe disease-causing phenotypes, since selective pressure would limit the frequency of significant deleterious variants. Highly penetrant or severe variants are more likely to be rare. While more difficult to detect, rare variants can provide convincing mechanistic insights, since they can have significant effects on protein function and thus clear causal roles in disease pathogenesis. Consistent with this theory, the majority of common disease associated variants are in noncoding genomic regions including introns and intergenic regions, while rare highly penetrant variants tend to be in gene coding regions.79 Common variants tend to be shared widely across many populations, while rare variants tend to be population specific.80 Common variants can be identified with cross-sectional approaches that randomly sample affected individuals across a study population. Rare variants are typically identified through familial studies, as they have more monogenic or Mendelian inheritance behavior, though large cross-sectional studies can also detect rare variants. In addition to the Y402H common CFH variant, several other common variants at this locus have been associated with AMD. The common I62V (rs800292) coding variant was also linked with AMD19,39, especially in east Asian54,81 cohorts. This includes Japanese74,82-84 and Korean72 populations for which the Y402H variant was less common. The protective CFH I62V variant increased binding to C3b resulting in more efficient proteolytic inhibition of the alternative pathway complement amplification85,86 and lower levels of markers of chronic complement activation including C3d and Ba.87 Many additional common variants linked with AMD were in noncoding regions including in the promoter region67,72,88 or in intronic regions.16,19,24,25,3133,36,37,39,40,72,74,88-93 .

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Interestingly, rs1410996, was found have one of the strongest associations with AMD24,25, despite its intronic position. Other common CFH variants associated with AMD are synonymous variants19,35,40,74,81,88, which did not alter the protein amino acid sequence even though the DNA sequence was altered and were likely associated due to linkage disequilibrium with causative variants (see discussion of haplotypes below).

CFHR1/3 Downstream of CFH lies CFHR1-5, a cluster of five complement factor H related genes. These genes share sequence homology to CFH and are believed to have arisen from genome duplication events. Several SNPs in CFHR2 (rs3790414) and CFHR5 (rs1759016, rs10922152, rs10922153) have been shown to be protective in AMD, while a SNP in CFHR4 increased the risk of AMD (rs1853883).39 A common deletion of CFHR1 and CFHR3 was consistently protective for AMD.25,94-100 CFHR1 and CFHR3 are made in the liver, but not in the eye. It was speculated that CFHR1 and CFHR3 may promote overall complement activity by decreasing the inhibition of CFH through competitive binding with C3,95,100 though the exact role of these proteins and their net effect on overall complement activity is still unknown.101 Haplotypes While many common SNPs at the CFH locus have been linked with AMD, it is unlikely that all or even most of them play a biological role in predisposing individuals to disease, especially given their enrichment in noncoding regions. Rather, it is more likely that most SNPs owe their disease-linkage to their correlated inheritance with a separate, biologically-significant, disease-causing variant.24 The proximity of these SNPs results in a high degree of linkage disequilibrium and they tend to be inherited together as a block of genetic information called a haplotype. Statistically grouping SNPs into haplotypes can help identify the underlying biologically-relevant disease-causing variants. These analyses revealed only a handful of haplotypes at the CFH locus including one or two risk alleles involving the CFH Y402H variant39 and two protective alleles involving I62V and the CFHR1/3 deletion.19,40,56,87,100,102-104 Additionally, a logistical regression model found that the CFHR1/3 deletion was independent of both Y402H and the highly significant synonymous variant rs2274700, implying that the CFH related genes may have a link to AMD independent of CFH.105 Furthermore, for east Asian populations including Chinese, Japanese and Korean cohorts with weaker association of CFH Y402H than in other populations, haplotype analysis revealed risk and protective CFH alleles linking this locus to AMD within these populations.67,70,72,106 Rare Variants While common variants with high frequency were the first CFH variants linked with disease, rare variants with high penetrance and effect sizes have shed light on the mechanisms of CFH in disease, even though they affect relatively few patients. Thus, a good deal of effort went into identifying highly penetrant rare variants in CFH. Geerlings et al. provided an excellent review of rare variants in the complement system in AMD.107 CFH R1210C (rs121913059) is a highly penetrant variant observed in 40 cases and one control in one study25 and with an odds ratio of 20.3-23.11 for AMD in large cohort studies.26,79,108 This polymorphism was associated with earlier onset of AMD25,108 and much higher likelihood of advanced disease, particularly geographic atrophy108. It disrupts the C-terminal ligand binding portion of CFH causing a loss of protein function. This polymorphism also leads to atypical hemolytic uremic syndrome, a disease of complement overactivation.25 Another missense variant at the same position, A1210C, similarly showed that carriers had a younger age of onset and were more likely to have a positive family history even though the variant did not segregate perfectly with AMD incidence.109 Another study deep-sequenced the CFH gene in AMD and control patients and found 65 missense, nonsense, or splice-site mutations. In five of the ten mutations that caused loss of CFH function, low levels of factor H were observed in the blood indicating haploinsufficiency. Mutations in functional domains of CFH, including R53C, D90G, and P503A, were associated with AMD. Crucially, the magnitude of AMD association was correlated with the amount of CFH disruption caused by these variants.110 R53C showed complete penetrance and D90G was present in five cases and one control in a second study.111 Both mutations resulted in functional impairment of complement inhibition by factor H.111

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P503A was also strongly linked to disease in a small cohort of Amish families with high prevalence of disease.112 Additional CFH rare variants linked to AMD include R2T, R175Q, and R303Q.113 Wagner et al. performed whole-exome sequencing on families with genetically unexplained AMD and identified four rare loss of function CFH variants. In one family, the C192F missense variant had perfect segregation in affected individuals. This variant led to decreased CFH secretion. Affected individuals had advanced AMD and drusen temporal to the macula. A family with extensive extramacular drusen was found to have the splice-site mutation IVS6+1G>A. Two other families with early onset bilateral AMD and extramacular drusen had either a R175P or R175H missense variant. CFH R175P had no CFH activity while CFH R175H was not secreted causing haploinsufficiency.114 Cuticular drusen, previously known as basal laminar drusen, have a “stars in the sky” appearance and are associated with early AMD disease onset. One study found CFH Y402H in 70% of patients with cuticular drusen (n=50) compared to 55% of patients with typical drusen (n=700) and 34% of controls (n=252).115 A second study found Y402H in all of the 30 patients with early onset cuticular drusen. Some of these patients had additional CFH mutation burden including Q408X, R1078S, c.350+6T->G, a splice site variant, R567G, a variant at a potential C3b/CRP binding site, N1050Y, and Q636N.116 These alternative CFH mutations may be independently associated with cuticular drusen presence. A study of three families with cuticular drusen found mutations in CFH including frameshift mutations I184fsX, K204fsX and c.1697-17-8del. Ten of thirteen mutation carriers had cuticular drusen, two developed CNV, and two developed membranoproliferative glomerulonephritis II, a disorder of complement hyperactivation.117 Another study employing whole exome sequencing of 14 affected family members of 6 families and 12 sporadic cases of cuticular drusen identified rare CFH variants A173G and G950H.118 Taken together, both common and rare genetic variants in CFH show this central complement regulator is clearly linked to AMD pathogenesis.

C3 All three complement initiation pathways converge on C3, and inhibition of C3b is a central function of factor H. Since C3 is a principal target of factor H and a central mediator of the complement cascade, it is a highly compelling target for investigation if complement and CFH are causative factors in AMD. Indeed, C3 genetic variants have been associated with AMD, further bolstering the role of complement in AMD pathogenesis. The most widely studied C3 variant has been R80G (mature protein sequence, also known as R102G in the proC3 sequence; rs2230199), which has been robustly associated with AMD.26,31,32 ,33,36,89,90,93,119-122 92 This common C3 variant was associated with both early AMD and all subtypes of late AMD.37,38,60,123,124 This variant may increase the rate of C3 amplification in the alternative pathway of complement activation.86 Another common C3 variant P314L (rs1047286) was also found to increase the risk of early AMD and all subtypes of late AMD.122,123 A large metanalysis confirmed this finding in Caucasian and East Asian populations.124 This metanalysis also found that an intronic variant, IVS2 (rs2250656), decreased the risk of advanced AMD in the same cohort.124 In addition to the common variants, the rare variant, K155Q (rs147859), has shown significant association with AMD.26,125 Helgason et al. found this rare variant in a cohort of Icelanders where it was associated with AMD independent of the more common C3 P314L and R102G variants. They showed that the K155Q variant had reduced binding to complement factor H31, resulting in less proteolytic inactivation of C3 by CFH and CFI.125 Furthermore, Saksens showed that AMD patients with the K155Q variant had a younger age of onset, were more likely to have a positive family history, and were more likely to have geographic atrophy rather than neovascular AMD.109 A recent study confirmed this association with GA, but not CNV.126 Whole exome sequencing revealed three additional rare variants in C3, K65G, R735W, and S1619R, associated with AMD. However, R735W and S1619R did not remain significantly associated in a larger test cohort.127 K65G decreased binding of CFH to C3b and had a slightly lower affinity for a complement

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inhibitor, membrane cofactor protein (MCP, CD46).35 Another study identified C3 R161W and its association with AMD, but it did not affect circulating C3 levels.107 The genetic linkage of both CFH and C3 strongly suggest that the complement signaling mechanism shared by these genes are a primary driver of the resulting AMD risk.

SERPING1 (C1-inhibitor) C1 inhibitor is a major inhibitor of all three of the complement initiation pathways. It is coded by the SERPING1 gene and the protein circulates in the blood. The rs2511989 polymorphism in intron 6 of SERPING1 has been best characterized and was protective in AMD.128-130 However, not all studies, including well powered studies, have detected this association.131-135 Other SNPs in SERPING1 have been identified, including rs1005510 which was associated with increased risk of neovascular AMD.130

CFI Complement factor I (CFI) is another complement inhibitor that binds to factor H, C4 binding protein, membrane cofactor protein (CD46), or complement receptor 1 (CR1; CD35) to inactivate C4b and C3b.126 Several common noncoding polymorphisms upstream of the CFI gene were strongly associated with AMD33,36 90-92,136 and neovascular AMD.38 The rs10033900 variant was associated with AMD in both Japanese and Han Chinese cohorts137,138 and rs4698775 was associated with AMD in an east Asian cohort.91 Ennis et al. found 4 marginally significant SNPs including rs10033900 that did not survive correction for multiple hypothesis testing. However, when pooling SNPs in a haplotype analysis, they found a haplotype containing this variant that was significantly associated with AMD.139 The rare CFI G119R polymorphism decreased expression and serum levels of CFI.107 This SNP showed very high penetrance and was associated with a 22 fold increase in risk of AMD.140 though subsequent studies showed that this variant was more common and less penetrant than initially thought.141 It was associated with a younger age of onset and patients with this variant were more likely to have a positive family history.109 The CFI V412M, which occurs in the CFI catalytic protease domain, was also found in a family with AMD.142 Exome sequencing of families with AMD and controls identified additional rare CFI variants P553S and L131R. These variants were highly prevalent in affected family members but did not segregate perfectly. L131R carriers had reduced serum levels of factor I and lower capacity to degrade C3b.107 Since many CFI variants are too rare for meaningful individual statistical comparisons, several studies have pooled these rare variants together to examine their effect in aggregate. Kavanagh et al. found 71 nonsynonymous CFI variants. When pooled, variants predicted to be loss of function or probably damaging were significantly related to AMD. Three SNPs, A240G, P553S, and R406H were predicted to be possibly damaging and frequent enough to be individually assessed as significantly associated with AMD. Patients with advanced AMD and any rare CFI variant had lower levels of factor I and patients with low factor I levels and rare CFI variants were more likely to have advanced AMD.143 Similarly, Seddon et al. identified 59 variants and found that the 4 loss of function variants were found exclusively in AMD cases, probably damaging variants were skewed to AMD cases (41 cases vs. 3 controls), and possibly damaging variants were also skewed to AMD cases (16 cases vs. 3 controls).125 To gain further functional insight into these rare variants, Java et al. classified CFI variants into three groups. The first group contained 18 variants in 35 patients that resulted in low factor I levels and reduced factor I activity. The second group contained six variants found in seven patients and had normal serum levels but reduced degradation of C3b. The third group consisted of 15 variants in 64 patients with normal factor I levels, but reduced efficacy of factor I function.144 Furthermore, Tan et al. discovered 20 rare nonsynonymous variants, nine of which alter CFI, six of which reduce factor I activity, and six of which were found only in AMD patients.145 Seddon et al. found that patients with low factor I levels and reduced factor I function were more likely to have a family history of AMD, higher baseline AMD grade, and more likely progression to advanced AMD, particularly GA but not neovascularization.126

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C2/CFB in MHC Class III Region Complement component 2 (C2) is involved in both the classic and mannose-binding lectin complement initiation pathways. C2 is located only 500 base pairs away from complement factor B (CFB) in the MHC class III locus, which also includes C4 (see section on HLA for more information). Factor B circulates in the blood and is a key cofactor, along with factor D, for the amplification of C3 activation in the alternative pathway. Several polymorphisms in this locus were identified as protective against AMD including CFB L9H (rs4151667) and R32Q (rs641153),32,33,36,37,81,146,147 as well as C2 E318D (rs9332739) 33,36-38,81 and a C2 intronic variant IVS10 (rs547154).24,31,147-152 These variants are in high linkage disequilibrium, so it was initially unclear which variants were causative.148 Further analysis suggested CFB R32Q (rs641153) and C2 IVS10 (rs547154) were in strong linkage disequilibrium, possibly indicating association with the same protective variant,151 while C2 E318D appeared to be independently protective.146,149 CFB 32Q decreased the ability of factor B to amplify C3 signaling in the alternative complement pathway compared to 32R.86,153 Furthermore, rare variants at this site were also identified in a Japanese population including CFB R32W and CFB R74H.154 Another study found several other variants at this locus associated with AMD, including in the C2 promoter (rs3020644), C2 intron (rs9380272)90, CFB intron (rs4151657), and the synonymous C2 A341A (rs1042663), which was in strong linkage disequilibrium with R151Q (rs438999). Additionally, an intronic variant rs429608, located in SKIV2L, a viral defense gene close to CFB, had strong linkage with AMD.36,89,91,92,93 These effects were independent of smoking, CFH genotype, and LOC387715/HTRA1 genotype.146 An additional intronic variant CFB IVS17 (rs2072633) was found to also be protective in an Indian cohort151, but not in prior studies.146,147,152 Not all studies have observed the association of these variants with AMD. C2 E318D (rs9332739),147,152 CFB R32Q (rs641153),155 and CFB L9H (rs4151667)147,152 showed no association AMD in several studies, and the C2 IVS10 (rs547154) was not associated with AMD in a Japanese cohort.83

CFD In the alternative complement pathway, C3 undergoes spontaneous hydrolysis to generate C3a and C3b. C3b interacts with factors B and D to split more C3 into C3a and C3b in an amplifying positive feedback loop. Six genomic variants were found in CFD, which codes for factor D, but they initially were not found to be associated with AMD.156 However, a subsequent study found a weak association of rs3826945 with AMD.157 This study also found that factor D levels were higher in the serum of AMD patients regardless of genotype.157

Terminal Complement Pathway The C5-9 complement proteins assemble to form the membrane attack complex. Though multiple SNPs in C5 have been identified, no clear association with AMD has emerged.158 Multiple disease-associated rare polymorphisms have been identified in the C9 gene. R95X is a nonsense mutation, causing a premature stop codon, which results in a truncated protein. This polymorphism was found most frequently in a Japanese population in which a significant 4.7-fold reduction in risk of neovascular AMD was observed. It was thought that haploinsufficiency of this gene protected against AMD.159 A different rare C9 polymorphism, P167S, was associated with AMD125 107,113,160 and patients with this variant had a younger age of onset and were more likely to have a positive family history.109 However, the functional consequences of this variant have been conflicting. In several studies, C9 levels were elevated in AMD patients107,161, while another study found lower C9 levels but more lytically active C9 function with increased polymerization, which is required for MAC formation.160 Similarly, Kremlitzka et al. also found that P167S induced greater C9 polymerization but found no effect on lytic activity. It was also observed that advanced AMD patients had elevated C5b-9 compared with mild-moderate AMD, but this was not associated with the P167S polymorphism.161 Six other C9 variants have been identified in AMD patients, however these variants were not frequent enough to test if they are significantly associated with AMD.161

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Progression While the first evidence of AMD is often the appearance of pigmentary changes and drusen, it is not until the disease progresses to advanced stages of geographic atrophy or exudative neovascularization that significant functional impairment occurs. Thus, pathways that play a role in disease progression to these late-stage forms of AMD are of particular therapeutic interest. Progression defined as the new diagnosis of exudative disease or geographic atrophy was linked with CFH Y402H.162 This variant was also associated with early disease and the association grew stronger as disease severity increased.163 Additional studies confirmed that CFH Y402H, the intronic CFH rs1410996,164 and CFH R1210C (rs121913059) were all associated with progression to advanced disease.165 Other complement pathway gene variants had inconsistent associations with AMD including C2 E318D (rs9332739),155 C3 R102G (rs2230199),61,163 CFB L9H (rs4151667).155,164,165 The genetic influence of complement variants on the rate of geographic atrophy growth was also investigated. An early study found variants in CFH, ARMS2/HTRA1, C2, and CFB as well as the presence of advanced AMD in 1 eye, and drusen size in both eyes were all independent risk factors for progression to advanced AMD over 12 years.166 Conflicting results were observed in the association of multiple complement pathway genes167 including CFH Y402H, C2, C3 R102G (rs2230199)60,168, or CFI rs17440077169,170 and the rate of geographic atrophy. For further review, Keenan et al. extensively discussed factors contributing to incidence of geographic atrophy and its rate of expansion.171 One interesting statistical approach used a multistate Markov model to predict transitions between one disease state to the next to determine factors involved in progression. This study found several factors including CHF, C3, and CFB genotype, age, smoking and BMI were all associated with progression from intermediate drusen to large drusen and from large drusen to geographic atrophy or neovascularization.172 Determining what pathways and factors lead to disease progression is critical for therapeutic strategies, though this work shows the challenges of studying disease process dynamics in patients.

Genetic Variation in Non-complement Immune-related Genes Interleukin Interleukins are cytokines that play an important role in immune system function. An investigation into five interleukin genes identified 14 SNPs and found an association of IL-8 +781 C/T SNP with neovascular AMD, but not atrophic AMD in a Taiwanese population. This study also found higher levels of IL-8 in the vitreous of patients with neovascular AMD who had the IL-8 +781 risk allele compared to controls.173

CX3CR1 CX3CR1 encodes a protein that binds to the inflammatory chemokine CXC3CL1 also known as fractalkine in humans. CX3CR1 is expressed by a wide variety of immune cells including microglia as well as Muller cells and RPE cells, and is involved in cell migration, proliferation and inflammatory signaling.174 The T280M (rs3732378)175 and V291I (rs3732379) polymorphisms were found to be a risk factor for AMD. AMD patients with the T280M polymorphism were found to have lower levels of CX3CR1, especially in the macula of both dry and neovascular AMD variants.174,176 However, one study was unable to replicate the association of T280M.135

HLA/KIR Major histocompatibility complex (MHC) proteins (also known as human leukocyte antigens or HLA in humans) are expressed by almost all cells. They are divided into three classes, I, II and III. Class I and II are transmembrane proteins that present small intracellular peptides on the external surface of the cell for display and self-recognition by immune surveillance cells. Class III MHCs are a collection of proteins that have a variety of non-antigen presenting functions and include the complement proteins C2, C4, factor B, cytokines, and heat shock proteins. HLA-C is a class-I protein that binds to inhibitory human killer

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immunoglobulin-like receptor (KIR) expressed primarily on natural killer and T-cells.177 HLA Cw*0701 alone178, or when combined with the AA haplotype of KIR, was associated with AMD.177 KIR2DL4 was also linked with AMD.113 Furthermore, the class-I HLA-B*4001 and class-II HLA-DRB1*1301 were negatively associated with AMD.178

Toll-like Receptors TLR3 belongs to the toll-like receptor family of proteins which serve as pattern recognition receptors for various common features of pathogens and induce inflammatory cytokine release and activation of myeloid cells of the innate immune system. TLR3 is a transmembrane protein that serves as a sensor for viral double-stranded DNA or RNA. While the protective association of TLR3 SNP rs3775291 L412F with AMD and GA, but not CNV179 was initially controvertial,131,135,180-183 a subsequent meta-analysis confirmed a significant association of the L412F variant with geographic atrophy.184 TLR4 is another toll-like receptor that recognizes a variety of pathogen-related molecules including lipopolysaccharide. The rs4986790 (D299G) polymorphism of TLR4 was shown to moderately increase the risk of AMD21,185,186, but additional studies have not confirmed this association.66,135,181,182,187,188 An additional variant in the TLR4 promoter, rs1927914, was also found to be associated with AMD.189

Genome-wide Associations Well-powered genome-wide association studies (GWAS) using genome sequencing and SNP microarrays have been performed with tens of thousands of participants. These studies allow comprehensive and relatively unbiased examination of the genetic architecture of AMD including both common and rare variants. Fritsche et al. examined 43,566 individuals with SNP microarrays and identified 34 genomic loci that affect susceptibility to AMD.79 Within these regions, there were 52 independently associated sets of common variants, which contained between one and >100 linked variants, of which 205 variants were potentially causal. Rare variants associated with AMD also fell exclusively within these 34 loci and rare variants in CFH, CFI, TIMP3, and SCLC16A8 were associated with AMD independent of linkage with common variants. These loci contained 368 genes that clustered in complement, collagen, lipid, and extracellular matrix pathways. Most of these variants were associated with both intermediate AMD and advanced AMD. All together, these genetic variants explained 27.2% of disease variability, 1.4% of which was due to rare variants. Interestingly, this is still only roughly half of the total genomic heritability of AMD.79 Subsequent meta-analyses linked even more inflammatory genes to AMD including C4BPA-CD55, a complement regulator, NFKBP1, and LBP190 and identified additional pathways including macrophage foam cell differentiation, humoral immune response, and MHC class II receptor activity.190 Furthermore, similar results were found with GWAS linking CFH, C9, C3, and CFI to polypoidal choroidal vasculopathy, an AMD subtype found more commonly in Asian populations.191 A recent GWAS of an even larger study population identified a total of 63 AMD loci, with the additional loci falling primarily in lipid signaling pathways.192 An additional genome-wide study of rare loss of function variants found that variants in complement pathway genes were associated with geographic atrophy and calcified drusen.193

Environmental Risk Factors During life, environmental factors affect the tissues of the body that were built from genetic blueprints. While environmental factors almost certainly play a role in disease pathogenesis, we live in extremely complex environments, and it can be difficult to control for innumerable hidden variables. Even with this limitation, clear evidence associated several pro-inflammatory environmental exposures with the risk of developing AMD. Age is a significant risk factor for AMD, as the name implies.166 The prevalence of AMD increases from 0.2% in 55-64 year-olds to 13% in people older than 85194,195 and increased age is associated with higher AMD risk.126,196 Chronic inflammation is a core feature of the aging process, though many other changes occur during aging as well.197

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Smoking is associated with an increased risk of developing AMD, a finding that has been widely reproduced.23,51,56,57,126,164,166,194,196,198-203 Current smoking was associated with higher risk of developing both geographic atrophy and neovascular AMD in an Australian cohort204, though the association with geographic atrophy was not found in a separate study.198 For men, a significant dosage effect was observed, with greater exposure to smoking leading to earlier development of AMD.205 An interesting demonstration of the contributions of both genetic and environmental factors affecting pathogenesis was found by Naj et al., who observed that nonsmokers with AMD were enriched for major genetic AMD risk variants at the CFH, ARMS2, and CFB/C2 loci compared with smokers.206 Another study found smoking potentiated the risk associated with the CFH Y402H risk allele.30 It is believed that smoking induces inflammation, with one study implicating TLR4/MyD88 and IL-1R1/MyD88 as important inflammatory mediators.207 Cigarette smoking has also been found to activate the alternative complement pathway208 and lower serum levels of CFH.209 Obesity has also been widely shown to increase risk of AMD.23,126,166,196,201-203,210-212 It was found that higher levels of visceral abdominal fat were correlated both with elevated inflammatory markers and AMD.212 Dietary fats in general211,213,214, as well as monounsaturated fats215, saturated fat, and cholesterol216 were also associated with increased rates of AMD. Infections are another interesting environmental exposure that may drive AMD pathogenesis through inflammation. Several studies have observed a correlation of Chlamydia pneumonia (C. pneumoniae) with AMD through a variety of assays including PCR of DNA from blood 217 and serum antibodies to C. pneumonia.218-220 Robman et al. found increased risk of AMD progression in those with C. pneumonia antibodies.219 Kalayoglu et al. found C. pneumoniae in four of nine CNV membranes by immunostaining and two of nine by PCR. They attributed this discrepancy to possible DNA degradation from archival specimens.221 However, other studies failed to find association of AMD with C. pneumonia antibodies in the plasma222,223 or with PCR from subretinal neovascular membranes.224 Additional pathogens, including cytomegalovirus (CMV), have also been implicated. CMV IgG titers were found to be higher in patients with exudative AMD and dry AMD compared to controls.225 Interestingly, this study did not detect an association of C. pneumonia IgG or Helicobacter pylori IgG with AMD.225 No association with Chlamydia trachomatis or Escherichia coli was found.218 Nonetheless, chronic subclinical infections driving inflammation226 may play an important role in the pathogenesis of age-related neurodegenerative diseases.227 Finally, chronic kidney disease (CKD) has been associated with early and exudative AMD, but not geographic atrophy.228,229 Patients with CKD have evidence of low-grade inflammation with elevated concentrations of C-reactive protein and fibrinogen, and lower serum albumin.230 Overall, the known environmental exposures associated with AMD all have plausible mechanisms by which they might drive disease pathogenesis via inflammation.

Cell and Molecular Evidence of Inflammation in AMD Age-related macular degeneration presents with several different phenotypic manifestations including pigmentary changes, drusen, geographic atrophy, and neovascularization. These changes occur primarily in the RPE-Bruch’s membrane-choroid complex, suggesting that AMD pathology originates within these structures. The histologic and molecular features of these disease phenotypes and the resulting changes in the RPE-Bruch’s membrane-choroid complex have been investigated extensively and show clear evidence of a tissue subjected to chronic inflammation. These studies provide critical information to help guide mechanistic understanding of the potential role of inflammation in AMD.

Bruch’s Membrane Even prior to the discovery of strong genetic evidence, the wide variety of complement proteins were found in the RPE-Bruch’s membrane-choroid complex suggested that humoral immunity played a pathogenic role in AMD. Complement factor H is made in the liver as well as the RPE and choroid and circulates through the blood, serving as an inhibitor of the alternative “tic over” complement activation

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pathway.19 Factor H coats exposed surfaces throughout the body protecting them from complement attack.13,231 In healthy people, factor H binds to heparan or dermatan sulfate moieties in dense patches on Bruch’s membrane, the RPE, the extracellular matrix between photoreceptors, and blood vessels of the choroid.13,19,232 The CFH 402H risk variant has reduced binding affinity to these glycosaminoglycans, potentially making these structures more vulnerable to chronic complement-mediated inflammatory damage.233-236 Furthermore, heparan sulfate levels were found to be 50% lower in aged eyes compared with young eyes, also decreasing the ability of factor H to bind to and protect these structures.235 Heparan sulfate may also inhibit the alternative pathway directly.235 Consistent with this model, eyes from AMD patients had less complement factor H on Bruch’s membrane compared to controls.237 Patients with the CFH 402H risk allele were found to have higher levels of the membrane attack complex in the RPE, Bruch’s membrane, and choroid,238,239 an effect that was also observed with increasing age.237

Choroid In addition to changes in Bruch’s membrane and the RPE, the choroid, which provides the blood supply to the outer retina, was also disrupted in AMD. The choriocapillaris degenerated early in AMD240, with decreased vascular density and increased presence of non-perfused, “ghost” vessels neighboring subRPE deposits.241 Accordingly, several parameters of choroidal blood flow including velocity, volume and flow rate were all lower at the fovea in AMD patients.242 AMD eyes with the lowest choroidal circulatory measurements were at highest risk for developing CNV.243 Choriocapillaris loss was greater in geographic atrophy compared with early AMD and controls,244 but there was no difference in the outer choroid.240 Thus, damage to the choroid appears to be a common feature across the spectrum of AMD pathology. Choroidal damage may be due to chronic inflammation driven by complement. C3 and C4 are present in the choriocapillaris of both controls and patients with AMD,245,246 while C3c is abundant only in the choroid of AMD patients.247 The membrane attack complex levels in the choroid increase with both aging and AMD19,237 and colocalize with CFH particularly in the macula.19 Patients with CFH risk genotype had thinner choroids237 and elevated levels of CRP in the choroidal stroma. CRP was elevated, while CFH was decreased particularly in the intercapillary septa, in patients with early and neovascular AMD.248 Interestingly, drusen are typically located over vessel walls, rather than lumens.238 It is possible that overactivation of complement that normally helps to remove cellular waste produced by the RPE causes damage and atrophy of the choroid, contributing to development or progression of AMD and potentially serving as a neovascular stimulus. Extravasated inflammatory cells in the choroid have also been associated with AMD. In the submacular choroid, early AMD patients were found to have increased levels of activated macrophages (labeled by IBA1 and HLA-DR), and neovascular AMD patients were found to have increased macrophages (labeled by HLA-DR).249 While macrophages (labeled with CD68) are present in control choroids, in early AMD, macrophages are recruited to Bruch’s membrane at sites of soft drusen, basal laminar deposits, pigmentary changes, and active disciform scarring.250 These macrophages express inducible nitric oxide synthetase (iNOS), a marker of classically activated inflammatory macrophages.250 Furthermore, mast cells are significantly more abundant in the choroid of early AMD eyes compared with controls and many of these cells appear degranulated consistent with chronic local mast cell activation.251 A comparative proteomic analysis of Bruch’s membrane and choriocapillaris between patients with AMD and controls showed that 60% of proteins that were increased in AMD patients were involved in immune responses including complement proteins (vitronectin, HLA class II DRB1-15 and DR-, C3, C5, C4-A, C6, C7, C8-, C9, CFH, and CFB) and damage-associated molecular pattern proteins (-defensins 1-3, protein S100s, crystallins, histones, and galectin-3).252 As a highly vascular tissue that supplies the photoreceptor outer segments and RPE, the choroid is a principal interface between circulating immune factors and cells with pathologic AMD tissue and a primary site of the inflammation that occurs in these disease processes.

Drusen Early features of AMD included thickening of Bruch’s membrane, accumulation of basal laminar deposits between the RPE and its basal lamina, basal linear deposits of vesicular material within Bruch’s

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membrane, drusen, and subretinal drusenoid deposits, also known as reticular pseudodrusen.253 Drusen themselves are deposits of lipoproteinacious material between the RPE and Bruch’s membrane.254 Drusen often precede the stigmata of more advanced disease, which include photoreceptor cell death, geographic atrophy or neovascularization.255-257 The risk of developing advanced disease correlated with amount of drusen in the macula258, size of drusen >63um, and confluence of drusen.259 Drusen were composed of a variety of organic materials including lipids,260 carbohydrates,261 proteins, and even cellular components.262 Many of these proteins are also present in proteinaceous aggregations found in other neurodegenerative diseases including Alzheimer’s disease.247,263 Complement pathway proteins were found in high abundance within drusen.262 Both hard and soft drusen, as well as the neighboring RPE and Bruch’s membrane, contained diffuse C3a264 and C3265, with one study localizing C3 to small, spherical substructures.266 A similar substructure appearance was found with iC3b19 and C5a.264 Drusen also contained components of the membrane attack complex including C5247,267 and C5b-9 (Figure 2).237,239,265,268-270 However, the C5b-9 complex was not present in large confluent drusen or basal deposits in one study, suggesting the MAC may be important for drusen initiation, but not propagation.237 Additionally, immunoglobulins including IgG.247,269 Ig-. and Ig-265 were present in drusen. IgG and C5 are also found in RPE cells overlying or neighboring drusen.269 While antibodies were found within drusen, C1q, the complement initiation factor that recognizes immunoglobulins, was not observed in drusen.247 In addition to complement activation molecules, several complement inhibitors were found in drusen. CFH was highly abundant in both drusen19,231,271(Figure 2) and basal laminar deposits.248 Interestingly, CFH abundance in drusen was found to be independent of the CFH Y402H risk allele.231 Vitronectin, a serum protein that inhibits the complement membrane attack complex by binding C5b,252,272 was also found in very high abundance in drusen.265,267,268,270,271,273,274 and in the cytoplasm of RPE cells neighboring drusen.266 Clusterin, another complement inhibitor that binds C5b and inhibits cell lysis272, was also found in drusen265,266,273. The cell surface complement inhibitors, complement receptor 1 (CD35) and membrane cofactor protein (CD46), were found in drusen, but only membrane cofactor protein (CD46) is found in RPE cells and small spherical substructural elements within drusen.266 In addition to inhibiting further complement activation, CD35 and CD46 help promote the clearance of complement opsonized immune complexes by phagocytosis.272,275 The presence of both complement activating molecules and complement inhibitors found in different patterns within and around drusen suggest a complex environment of complement regulation that occurs as part of drusen biogenesis. FIGURE 2: Immunohistochemistry of complement factor H (green) and the membrane attack complex (orange) within a druse (D) and choriocapillaris lumens (L). Retinal pigment epithelium (RPE), Bruch’s membrane (BM). Adapted from276.

Harris, James

Further cementing the role of complement in drusen formation and macular degeneration was the observation that clinical syndromes of complement overactivation caused retinal drusen formation. Specifically, patients with membranoproliferative glomerulonephritis type II (MPGNII), a disease of uncontrolled systemic activation of the alternative complement pathway, can develop AMD-like pathology.19,277 In MPGNII, the alternative pathway becomes overactive most commonly due to C3 nephritic factor, an activating autoantibody against the C3 convertase. However, patients with CFH or CFHR5 mutations also developed MPGNII in the absence of autoantibodies.278,279 In MPGNII, aberrantly activated complement was deposited on the glomerular basement membrane.280 These glomerular deopsits are similar to drusenoid deposits on Bruch’s membrane.281 Indeed, MPGNII patients developed subretinal drusen that were structurally and molecularly indistinguishable from those found in AMD, but occured as early as the second decade of life.279 These deposits contained MHC class II, IgG,280 IgM,282 vitronectin, C5, C5b-9, TIMP-3, amyloid-P.280 Even patients with post-streptococcal glomerulonephritis who suffered overactivation of the alternative complement system by streoptococcal antigens developed drusen.280 Subretinal nodules similar to cuticular drusen have also been observed in MPGNII patients.283 One study followed three patients prospectively who developed chronic MPGNII at ages 13, 10 and 10 years old and by ages 25, 32 and 32, respectively, all three patients developed CNV.280 In addition to CNV, patients with long-standing MPGNII can also develop geographic atrophy.283 MPGNII patients can also develop lipodystrophies suggesting a possible link between lipid metabolism and complement signaling.284 Interestingly, the CFH Y402H AMD risk allele occurs at a site responsible for binding to heparan sulfate, which plays an important role in CFH binding to structures in the eye, while a separate binding site in the factor H protein plays a role in CFH binding in the kidney. Thus, the specific glycosoaminoglycans expressed by the kidney and eye may act as a molecular zip code to direct the binding of factor H to these structures. This helps explain why the CFH Y402H allele was associated with AMD risk,13 while CFH mutations that cause MPGNII were primarily at the N-terminus and affected CFH function rather than binding.231 Other inflammatory proteins including apolipoprotein E270 and factor X were found diffusely throughout drusen while HLA-DR was found in globular subdomains within drusen.247 MHC class-II receptors were found in drusen and RPE cells, but not MHC-I, which were instead found on choriocapillaris endothelial cells.178CCL11 (eotaxin) was found in both basal linear and basal laminar deposits.285 Unbiased mass spectrometry of isolated drusen have largely confirmed the above results and also identified annexins I and VI, calgranulin A and B, psoriasin, apolipoprotein A1, and ubiquitin268 within drusen.265 Acute Phase Reactants Drusen also contained acute phase reactants,262 which are serum markers of inflammation that play a wide range of roles in mediating inflammatory activity. C-reactive protein (CRP) binds to damaged cells, nuclear constituents, and microorganisms at sites of inflammation. CRP recruited C1-4, activating the classical complement pathway, while simultaneously inhibiting the alternate and mannose-binding lectin pathways by recruiting complement factor H.286 CRP also caused mast cell degranulation and histamine release.287 CRP was found to be in high abundance in drusen and basal laminar deposits in one study248, but not a second study247. Other acute phase reactants including serum amyloid A, serum amyloid P,247,268 alpha1-antitrypsin, and fibrinogen265 have all been found within drusen. Amyloid Drusen also contained -amyloid, the major peptide deposited in Alzhiemer’s disease where it is thought to promote inflammation.288 Interestingly, immunostaining for -amyloid revealed 2-10um vesicle-like subdomains within drusen in which -amyloid aggregated in concentric ring-like structures surrounding a central core.268,289,290 Similar structures were found in Alzhiemier’s disease and thought to be from an endosomal or lysosomal origin.289 In AMD, -amyloid is deposited in drusen in an oligomeric form, and not the fibrillar form that is characteristic of Alzheimer’s plaques.291 The fibrillar form consists of 6-15nm rod-like structures that form -sheets and exhibit birefringence with polarized light.268 While the fibril form is a pathologic hallmark of Alzheimer’s disease, it is the oligomeric form that is now believed to be the primary toxic molecule in Alzhiemer’s disease.291 While one study found fibrillar -amyloid in addition to the oligomers in drusen,292 fibrillar -amyloid has not been widely observed in AMD.

2024 Harvard Ophthalmology Residents’ Course The -amyloid vesicular substructures are typically found centrally within a druse, neighboring the RPE basement membrane.291 It is believed the -amyloid rich subdomains are likely by-products of overlying RPE cell degeneration, since -amyloid immunoreactivity is seen in the perinuclear cytoplasm, endosomal-lysosomal system, Golgi apparatus, and endoplasmic reticulum of RPE cells, especially those above drusen.289 Amyloid precursor protein was also observed in the basolateral aspect of RPE cells in increasing quantity with age.239 The -amyloid vesicles and granules were found to be most numerous in eyes with geographic atrophy, especially at the border of atrophic lesions.290 Though the -amyloid rich substructures are devoid of many of the other inflammatory proteins that are commonly found in drusen,268,291 -amyloid has been shown to be proinflammatory. -amyloid binds complement factor I, inhibiting its ability to inactivate C3b.293 Furthermore, injecting -amyloid 1-40 protein into rats resulted in upregulation of many inflammatory genes including IL-6, TNF-α, IL-1β, IL-18, caspase-1, NLRP3, and XAF1 with increased protein expression of IL-1B and IL-6 in the RPE, choroid, neuroretina, and vitreous.294 The presence of amyloid within drusen may serve as an inflammatory nidus and suggests common pathogenic mechanisms between AMD and Alzheimer’s disease. Additional Components of Drusen In addition to inflammatory proteins, drusen had abundant modified organic molecules including oxidized proteins and lipids, advanced glycation end products, and abnormal protein crosslinking that likely arose from oxidative damage and metabolic dysfunction.265,273 Carboxyethylpyrrole (CEP) is a protein adduct derived from docosahexaenoate-containing lipids and was more abundant in AMD than normal tissues.265,273 Accumulation of CEP in drusen may function as a neoantigen as autoantibodies against this molecule were found in 40% of patients with AMD.295 Furthermore, CEP levels and autoantibodies were elevated in patients with risk alleles in HTRA1 and ARMS2, which code for heat shock and mitochondrial proteins respectively that are related to protein metabolism and oxidative stress.296 Mice systemically injected with CEP generated antibodies against this molecule and fixed C3 to Bruch’s membrane causing drusenoid deposits to accumulate beneath the RPE during aging. Some even developed degenerative lesions similar to geographic atrophy.297 Other oxidatively crosslinked proteins in drusen included MMP3 and vitronectin.265 Malondialdehyde (MDA) is another product of oxidative stress found in drusen. This proinflammatory molecule results from lipid peroxidation. CFH bound to MDA, decreasing inflammation and reducing the uptake of this molecule by macrophages.298 Risk variants in CFH decreased binding to MDA, increasing inflammation.298 Identifying the cellular origin of the material found in drusen provided further insight into disease mechanisms. There appeared to be a substantial contribution of drusen material from RPE cells, supporting a model where RPE cells become unhealthy and start to suffer complement attack leading to death and deposition of cellular material along Bruch’s membrane. These deposits then acted as a nidus for chronic inflammation and a nucleation site for drusen, initiating the AMD disease process.262,266 Evidence for this potential mechanism was based on several observations. RPE cells neighboring drusen show histologic abnormalities including irregular size, shape, pigment distribution, changes in nuclei, proliferation, and cytoplasmic changes indicative of dysfunction and cellular stress. Ultrastructural investigations also demonstrated changes neighboring RPE including organelle distribution, content, and pigmentation. Many of the proteins and lipids found within drusen were also observed in neighboring, unhealthy appearing RPE cells. 247,269,299 Furthermore, early drusen also contained cellular material including RPE-derived basal laminae, organelles, cellular fragments, and even whole cells. RPE neighboring drusen extended blebs into drusen and predrusenoid spaces.300 Additionally, lipofusin and melanin, constitutes of RPE cells, were found in drusen.262 The cell surface complement inhibitor CD46 was typically distributed basolaterally in the RPE of unaffected controls, but was decreased in areas of early AMD, potentially exposing these cells to complement attack.301 Interestingly, drusen have resolved spontaneously or with laser treatment, however this was typically associated with death of the overlying RPE. This finding was consistent with the RPE being a significant source of drusenoid material such that once the RPE was no longer present, homeostatic mechanisms cleared the drusen deposits.259

Harris, James

Furthermore, choroidal dendritic cells, which are potent antigen presenting cells have also been implicated in drusen formation and may contribute material to drusen. Choroidal dendritic cells have been observed extending processes across Bruch’s membrane into drusen, where they associate with the RPE blebs. It was speculated that the core-like structures in drusen may have a dendritic cell origin.262 In addition to classic drusen, which occur between the RPE and Bruch’s membrane, subretinal drusenoid deposits, also known as reticular pseudodrusen, are deposits between the RPE and photoreceptor outer segments. These reticular pseudodrusen were highly correlated with the presence and expansion of geographic atrophy302,303 and associated with type 3 neovascularization,304 in which aberrant vessels grow from the circulation of the inner retina, rather than the choroid as is the case for classic CNV. Interestingly, eyes with subretinal drusenoid deposits showed decreased contrast sensitivity and slow dark adaption, unlike traditional drusen.304 Similar to classic drusen, subretinal drusenoid deposits contained apolipoprotein E, complement factor H, and vitronectin.271,305 Advanced deposits contained fragments of outer segments, RPE organelles, and cell bodies,306 but these cellular components were initially absent.271 Though one study did not observe associated immune cells,306 another found microglia/macrophages (labeled with Iba1) associated with subretinal drusenoid deposits.305 Interestingly, no -amyloid, C3 or C5a were observed in subretinal drusenoid deposits, although only two eyes were processed in this study.305

Choroidal Neovascularization The cellular and molecular features of neovascular tissue found in wet AMD have also revealed evidence of chronic inflammation. Examination of the molecular components of subretinal neovascular membranes associated with AMD showed an abundance of IgG, IgA, IgE, C1q, C3c, and C3d within the connective stroma of new blood vessel walls.307 Complement factor H was found at the peripheral aspect of the inner and outer surface of basolaminar deposits and colocalized with C3, C5b-9, and vitronectin.308 HLA-DR and HLA-DQ, antigen presenting MHC molecules, were expressed by glial cells, RPE, and vascular endothelial cells within CNV.307 Immune cells including monocytes, macrophages,307,309-312 and foreign body giant cells were found throughout neovascular tissue255,313-316 including the leading edge,317 consistent with a chronic inflammatory environment311. These inflammatory cells were often observed near segments of thin or broken Bruch’s membrane313,316,318,319 where active neovascular buds projected from the choroid into CNV sites. Inflammatory cells, including perivascular macrophages (labeled with CD68), were also found in the subretinal and subRPE spaces and attached to the RPE basement membrane.320-322 Macrophages exist in at least two states including a pro-inflammatory M1 state, marked by CXCL11 expression, or a pro-angiogenic M2 state, marked by CCL22 expression. Pro-angiogenic CCL22+ M2 macrophages were enriched in CNV.323 Aqueous humor sampling in patients with neovascular AMD also showed elevated CCL22/CXCL11 protein ratios compared to controls as well as in patients with recurrent neovascular disease after anti-VEGF therapy compared to treatment-naïve patients.324 Macrophages appeared to be recruited by the RPE, which expressed monocyte chemotactic protein.325 Elevated levels of monocyte chemotactic protein were found in the aqueous humor of patients with neovascular AMD, along with soluble intercellular adhesion molecule 1 (sICAM-1), and soluble vascular cell adhesion molecule 1 (sVCAM1), which helped recruit leukocytes.326 Neovascular vessels also contained proinflammatory cytokines including IL-1B and TNF-, which were thought to originate from local macrophages.321 In addition to circulating, blood-derived macrophages, changes in microglia, the resident tissue phagocytes of the retina were observed in neovascular AMD.327 In control eyes, resident phagocytic cells presumed to be microglia were located in the ganglion cell layer and inner plexiform layer in a quiescent state with a small stellate appearance and perivascular distribution.328 These cells were positive for microglial markers CX3CR1 and CD18. In eyes from patients with AMD, these cells were found ectopically in the outer retina and subretinal space at sites of RPE disruption and photoreceptor degeneration, as well as sites neighboring CNV.175 The CX3CR1 polymorphism, T280M (discussed

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above), impaired microglial migration, potentially trapping microglia in the subretinal space.175. It was speculated that these trapped microglia may break down Bruch’s membrane and promote angiogenesis. Other inflammatory cells including lymphocytes307 and mast cells have also been observed in neovascular tissue. These immune cells also may play a role in breaking through Bruch’s membrane, which is widely believed to be a precondition for neovascularization.329,330 Mast cells were found around or within Bruch’s membrane, occasionally present at breaks in this membrane. Mast cells interacted with endothelial cells to induce angiogenesis.251 However, while CCR3 and its ligand CCL11 (eotaxin-1) were specifically expressed in choroidal neovascular endothelial cells,285 no mast cells or eosinophils were found within CNV.331 CXCL10 (IP-10) was also present in neovascular endothelial cells.285 One study observed plasma cells in subfoveal neovascular membranes,332 but overall B-cells, natural killer cells and cytotoxic T-cells were rare within this tissue.307,311 Overall, neovascularization associated with AMD clearly corresponds with a complex inflammatory environment. However, it is not clear whether this inflammation plays a causal, neutral or protective role in neovascularization and it is certainly possible that different aspects of inflammation fall into each of these categories.

Geographic Atrophy As with early and neovascular AMD, complement dysfunction has been implicated in geographic atrophy. The transmembrane complement inhibitor, membrane cofactor protein (MCP, CD46), is progressively lost from RPE cells early in geographic atrophy prior to the onset of morphologic RPE changes. Furthermore, increasing grades of RPE degeneration are associated with further reductions in membrane cofactor protein.333 Another complement inhibitor, CD59 or membrane inhibitor of reactive lysis, is increased in regions of uninvolved RPE in early AMD but lower in areas of geographic atrophy.301 Photoreceptor outer segments at the border of GA lesions were found to be opsonized with C3 and had higher levels of CFB, both of which were absent from controls.245 CRP and CFH were decreased in areas of geographic atrophy.248 Further characterization of the distribution of factor H revealed reduced levels in the choroid neighboring areas of medium-sized geographic atrophy but increased in small- and large-sized geographic atrophy.334 The membrane attack complex, C5b-9, was increased in Bruch’s membrane and the basal surface and cytoplasm of the RPE neighboring sites of geographic atrophy.334 The cellular milleu of geographic atrophy also shows evidence of chronic inflammation. In patients with geographic atrophy, microglia were found in outer nuclear layers associated with degenerating rods. Balloon-shaped microglia contained rhodopsin and 7G6 cytoplasmic inclusions in the outer nuclear layer and subretinal space suggestive of rod and cone phagocytosis respectively.328 Presumed microglia were observed in atrophic Henle’s layer335 and outer retina in areas of small- and medium-sized geographic atrophy in an ameboid shape,334 In contrast, large-sized areas of geographic atrophy showed far fewer presumed microglia (labeled with Iba1).334 Circulating macrophages (labeled with CD68) showed a similar pattern with increased numbers in small- and medium sized atrophic lesions in the nerve fiber layer, ganglion cell layer, and the outer segments, but reduced numbers in large atrophic lesions.334 Within the lesion, the outer plexiform layer was in direct contact with residual pigmented material and macrophages on Bruch’s membrane. It was speculated that in basal laminar deposits, undegraded RPE pigment or Bruch’s membrane recruited and stimulated these macrophages.336 CD163 immunostaining, which labels primarily bone-marrow derived macrophages, revealed an increase in the number and size of these peripheral macrophages in geographic atrophy compared to controls, primarily in the outer retina and neighboring the RPE.312 CCL2, also known as monocyte chemoattractant protein-1, binds to CCR2 on macrophages causing macrophage accumulation.337 Intraocular CCL2 levels were higher in eyes with geographic atrophy compared with controls and subretinal CCR2+ inflammatory monocytes were found infiltrating the retina in these patients.338 Furthermore, giant cells, derived from macrophage fusion, are an indicator of chronic granulomatous inflammation and were observed at the leading edge of geographic atrophy.250,336 These cells bordered areas of progressive photoreceptor atrophy and contained pigment clumps, likely representing melaninderived degenerated RPE cells, in secondary lysosomes. Degranulated mast cells were also observed in Sattler’s layer in regions of RPE and choriocapillaris atrophy and the number of these cells positively correlated with the extent of choriocapillaris degeneration.251

Harris, James

Transcriptomics In addition to proteomic evidence of inflammation in AMD, transcriptomic differences have been observed. Studies in mice have found that aging increased expression of genes related to inflammation in the complement cascades, leukocyte activation, phagocytosis and IL-10 signaling in both the RPE and choroid,339 as well as the neurosensory retina.340 Comparison of human peripheral retina to the macula showed variation in the inflammatory genes including CCL19, CCL26, and CXCL14,341 which may contribute to the anatomic localization of AMD in the macula. Additional transcriptomic studies using microarrays showed RPE-choroids from AMD at every stage (early, CNV or geographic atrophy), upregulated a module of genes involved in cell-based inflammatory responses. This was one of the earliest signs of disease, prior to onset of vision loss as predicted by a machine learning algorithm. Upregulated disease-associated transcripts included chemokines such as CXCL1, CXCL2, CXCL9, CXCL10, CXCL11, CCL2, and CCL8, which recruit macrophages, dendritic cells, granulocytes and lymphocytes. Markers of these immune cells such as CD86 in dendritic cells, CD69 in leukocytes and IL141 and CTSL2 in activated macrophages were also elevated in AMD patients. Elevated immunoglobulin gene expression was also observed. Complement genes including C3, C4, C1S, CFI, and SERPING1 were also higher.342 Recently, single-cell RNA sequencing of retinas with macular degeneration has further advanced our understanding of the inflammatory mechanisms in AMD. Interestingly, the microglial activation signatures in early AMD, which include upregulation of HLA-DR, C1QB, C1QC, APOE, and B2M, are shared with the early phases of other neurodegenerative diseases including Alzheimer’s disease and multiple sclerosis. As AMD progressed to neovascular stages, microglia upregulated inflammasomal signaling including IL1B, NOD2, and NFKB1. Interaction analysis showed that microglial upregulation of IL-1B and IL-6 were highly correlated with VEGFA expression in astrocytes.343

Serum Studies Environmental risk factors of smoking, aging, and obesity, as well as the strong link of the complement system to AMD sparked interest in systemic factors circulating within the blood that may contribute to AMD. There has been extensive work studying changes in these circulating factors that might help shed light on disease pathogenesis and even serve as diagnostic or prognostic biomarkers to help guide precision medicine efforts.344 A caveat of these studies is that measurement of the levels of systemic inflammatory factors are subject to a wide range of potentially confounding sources of chronic inflammation that may be hard to control for and thus can result in spurious or difficult to detect signals within a noisy background. That said, in aggregate, these studies can provide useful insight into the systemic factors that accompany AMD. Complement Given the strong genetic linkage of complement factors with AMD, there has been much interest in assessing the levels of circulating complement factors in both AMD patients and those with genetic predispositions. The majority of studies showed increased levels of complement activation products including Bb,345, C3a,346 C3b, C5a,345 and C5-9 in patients with AMD or CFH risk genotypes.347 C3a346,348, C3b, C4a, and C5a were also elevated in patients with neovascular disease.349 Rare exceptions to this trend included C3a, which was not associated with CFH risk genotype in one study346 and no association of C5a with AMD was found in another87. Markers of chronic complement activation including Ba and C3d were also elevated in patients with AMD or the risk haplotypes of CFH.87 Similarly, risk haplotypes of C3 and C2/CFB were also associated with elevated Ba and C3d levels respectively.87 However, levels of factor D, another marker of chronic complement activation was associated with AMD or CFH risk variant in some studies, but not others.87,347,350 The C3d/C3 ratio, a marker of alternative pathway activation was elevated in both patients with AMD and the CFH risk genotype.351 In contrast, serum levels of the substrate proteins C3 and C4 were not correlated with AMD347 and C3 was not associated with the CFH risk genotype.350 The regulatory proteins CFH, CFI and CFB had mixed association with AMD with some studies showing no correlation,87,347,350 others finding reduced CFH levels345,350 or increased CFB351 or CFI350 levels in AMD patients or patients with the CFH risk variant87. C3, CFB, CFH, iC3b, and C3a levels were all positively associated with BMI and C5a was elevated in patients with C3 and ARMS2 risk

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genotypes.345 The variability in these serum measurements show the complex interaction of genetics, environment, and disease pathogenesis that result in changes in the overall landscape of circulating complement factors. Acute Phase Reactants Multiple studies have found elevated circulating CRP levels in AMD patients,34,51,81,212,352, including in early, intermediate, and advanced AMD.353-356 Several prospective studies found elevated CRP increased the risk of developing AMD.223,357 CRP was correlated with AMD risk factors including current smoking, obesity,358-360 and negatively correlated with protective factors including serum antioxidants, vitamin C, lutein, zeaxanthin and higher fish intake.360 Elevated CRP was also associated with progression of AMD.354,359 While genetic variants in CRP associated with increased levels of circulating CRP were not associated with AMD individually,29 when combined into haplotypes, these variants slightly increased risk of developing AMD.51 These CRP haplotypes also potentiated the effect of the CFH Y402H polymorphism on AMD risk,51 though there are conflicting results as to whether serum CRP levels correlated with the CFH Y402H risk allele.350,361 However, not all studies have not found elevated levels of CRP in AMD215,223,362-365. The CFH Y402H risk variant occurs at a binding site for C-reactive protein (CRP), heparin,19,366,367 and group A streptococcus.368 CRP circulates in the blood in a pentameric form. When CRP binds to damaged or necrotic cells, it switches to a monomeric form369 and monomeric CRP was observed at sites of necrotic retinal pigment epithelium in AMD patients.370 Studies of the CFH risk variant show reduced binding affinity for monomeric CRP,368,371-373 but not mast-cell derived heparin374,375 or pentameric CRP.373 Risk variants of factor H have reduced binding to this monomeric form, resulting in increased complement activation, enhanced phagocytosis, and release of proinflammatory TNF- and IL-8.370 Perhaps the reduced binding of factor H to group A streptococcus contributes to immunity against this pathogen, conferring an evolutionary advantage and explaining the high frequency of this risk allele in certain populations. Serum levels of other acute phase reactants have also been studied. An early study of AMD patients found elevated levels of the alpha-2 globulin plasma fraction, which contains the acute phase reactants ceruloplasmin, alpha-2-macroglobulin, and haptoglobin.376 Elevated levels of plasminogen activator inhibitor-1,365 and amyloid- 1-42, were associated with AMD. Plasma fibrinogen levels have been studied in AMD patients with conflicting results with some studies finding elevated levels196,201 and others finding no correlation215,363,365 as was the case for serum amyloid-A.212,223 No association was observed between von Willebrand factor,365 leptin, or amyloid- 1-40212 and AMD. Antibodies Autoantibodies against retinal tissue have also been consistently observed in patients with AMD. These autoantibodies were found in all forms of AMD and bind to various retinal structures including the photoreceptor outer segments,376,377 nuclear layers,378 or exhibit diffuse staining.376 Autoantibodies exhibited overlap with glial fibrillary acid protein (GFAP), a marker of astrocytes, suggesting that this protein or other astrocytic antigens may be a target of autoantibodies.379 Retinal targeting antibodies were found in 66% of AMD patients vs 18% in controls or 24% in patients with other retinal diseases.380 Furthermore, patients with disrupted RPE were found to have higher serum retinal autoantibodies.376 A prospective study found autoantibodies against the retina to be higher in early AMD, but found no association of baseline autoantibody titer with the progression to advanced AMD after 10 years.381 A separate study measured retinal autoantibody levels before and after treatment with intravitreal bevacizumab and found lower titers following treatment.382 Cytokines Circulating levels of cytokines have shown mixed results in AMD patients. One study found elevated levels of TGF-1 and MCP-1 in the urine of patients with AMD and elevated MCP-1 in geographic atrophy.344 Another study of patients with dry AMD found elevated levels of IL-6, IL-18, and TNF- in patients with a CFH Y402H risk genotype.383 A prospective analysis of 975 Beaver Dam Eye study patients found that elevated levels of TNF-R2 and IL-6 were associated with incidence of early AMD.223

Harris, James

Elevated IL-6 was also associated with progression of AMD, elevated BMI, and current smoking status.359 However, several other studies found no association of AMD with IL-6,365 TNF-223 212 or IL-1B.383 No difference in CXCL9-11 plasma levels were found in AMD patients.384 A screen of 27 serum cytokines found levels of the eosinophil attractant, eotaxin-1 (CCL11) and IP-10 (CXCL10) were elevated in all stages of AMD including geographic atrophy and neovascular disease, except for eotaxin and neovascular disease.285 Circulating Cells In addition to serum proteins, the cellular composition of the blood of patients with AMD has also been analyzed. Most studies have not found an association with total white blood cell count with AMD.215,223,365 However, a prospective study of 3654 participants found that elevated baseline WBC count independent of smoking was associated with a variety of early AMD phenotypes including pigmentary change, soft drusen, and reticular drusen, but was not associated with late AMD.385 Examination of subsets of leukocytes has been more informative. Several studies have found elevated neutrophil to leukocyte 386 or platelet to leukocyte ratios387 in AMD patients. Since prolonged inflammation results in increased neutrophil and macrophage levels and leukocyte apoptosis, elevation in these ratios are proxies for chronic inflammation.387 Further supporting this finding, CD11b+ and CD16hi, HLA-DRneutrophils were increased,388 while CD8+ CXCR3+ T cells were reduced in neovascular AMD patients, consistent with the function of CXCR3 as an inhibitor of angiogenesis.384 Examination of patients with fundus autofluorescence patterns that predict high risk of geographic atrophy progression revealed that circulating monocytes had elevated levels of CD200, a protein that is believed to decrease neuroinflammation.389 CD200+ monocytes were also elevated in patients with neovascular AMD.390 Another study found decreased levels of the cell surface complement inhibitors CD46 and CD59 on monocytes in patients with neovascular AMD compared to controls.390 CD35, another cell surface complement inhibitor that also promotes complement mediated phagocytosis, was elevated on monocytes, lymphocytes and granulocytes.275 Within lymphocytes, lower levels of CD4+ T helper cells were found in both control and AMD patients carrying the CFH Y402H risk variant as well as older individuals.361 However, no difference in CD4+ T helper cells was found in patients with neovascular AMD compared with controls.361 Another study found T helper 1 cells were lower in patients with exudative AMD.391 There was no difference in proinflammatory T helper 17 cells in these groups.391 T cells expressed higher levels of CD56, a marker of immunosenescence, in patients with AMD and the Y402H risk variant392 and lower levels of the cell surface complement inhibitor CD46 in patients with neovascular AMD with fibrosis.390

Human Clinical Trials While the preceding genetic and molecular evidence makes a strong case for the role of inflammation in AMD, disease-modifying treatments that target the inflammatory pathways outlined above would confirm the importance of these pathways in disease mechanisms with significant benefits to patients. Thus far, our progress in developing inflammation-based AMD treatments inspires cautious optimism.

NSAIDs Among the most used immunomodulatory therapies in clinical practice are corticosteroid and nonsteroidal anti-inflammatory (NSAID) medications. Both medications have been investigated in the context of AMD. NSAIDs have largely failed to provide therapeutic benefits in AMD. A large study of close to 40,000 females who were given low dose aspirin showed no effect on self-reported AMD rates at 10-year follow up.393 A separate large observational study found no association of NSAID use with early or late AMD after 5 years.394 Furthermore, in 22 eyes that were refractory to anti-VEGF therapy, treatment with topical bromfenac twice daily for 2 months showed no benefit.395 Similarly, 61 patients assigned to diclofenac or placebo after photodynamic therapy also showed no benefit.396 Thus, NSAIDs do not appear to modify AMD disease course.

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Corticosteroids Prior to the widespread use of anti-vascular endothelial growth factor (VEGF) for exudative macular degeneration, corticosteroids were extensively investigated as potential AMD therapies. Initial uncontrolled studies showed intravitreal injection of triamcinolone improved exudation and vision in patients with neovascular AMD over a short time ranging from 2 weeks to several months.397,398 Intravitreal triamcinolone resolved serous pigment epithelial detachments and occult CNV in two patients several months after treatment.399 One study examined the eyes of a patient, who unfortunately died for unrelated reasons, with bilateral neovascular AMD 6 weeks after intravitreal triamcinolone injection of one eye. This patient had improvement in vision several days following injection. Immunohistochemical analysis showed significantly decreased MHC-II expression and microglial condensation in the treated eye compared with the contralateral eye.400 Single intravitreal injection of 4mg triamcinolone improved visual acuity at 3 and 6 months in patients with exudative AMD compared to controls.401 Higher steroid doses including 25mg of triamcinolone also showed improved visual acuity compared to controls after 1 and 3 months402 and after repeat injection from 3.1 to 18 months.403 However, despite the short-term vision benefits, the treatment effects appeared to wear off or cause complications after longer periods of time. One study showed a significant antiangiogenic benefit at 3 months after a single injection of 4mg triamcinolone in patients with CNV from AMD, but no benefit for visual acuity after one year.404 Several well powered studies of triamcinolone intravitreal injection found no benefit in vision after 1-1.5 years compared to controls who got short-acting dexamethasone.405,406 While single intravitreal injection of 4mg triamcinolone reduced the rate of severe vision loss in patients with choroidal neovascularization not suitable for laser treatment compared to historic controls up to 18 months after treatment, compilations including steroid-induced intraocular pressure elevation and cataract formation were observed.407 A trial of fluocinolone acetonide inserts for bilateral geographic atrophy from AMD was terminated and the results were not published.408 Several long-term observational studies of large cohorts examined whether exposure to anti-inflammatory medications affected rates of AMD. The AREDS prospective study examined 2506 participants without AMD in either eye and 788 participants with AMD in just 1 eye and found that the incidence of central geographic atrophy was lower with exposure to any anti-inflammatory medication.202 However, a retrospective study of 3654 patients in the Blue Mountains Eye study found no association of the use of corticosteroids or NSAIDs with either early or late AMD at baseline or after 5 years.394 Additional studies examined the use of corticosteroids in combination with photodynamic therapy (PDT) or anti-VEGF therapy. Intravitreal injection of triamcinolone reduced the frequency of PDT retreatment and preserved vision.409,410 However, side effects including elevated intraocular pressure and cataract progression were seen with the combined treatment.410 An additional study found patients receiving intravitreal triamcinolone had a greater rate of full resolution of angiogenesis, but no difference in final visual outcome.411 A study of sub-Tenon’s triamcinolone injection in combination with PDT showed no difference on fluorescein angiography 3 and 6 months after treatment.412 While anti-VEGF therapy was shown to be superior to corticosteroids for exudative AMD413, several studies have compared the combined treatment of both anti-VEGF and corticosteroids to anti-VEGF monotherapy. Either intravitreal triamcinolone or dexamethasone releasing implants reduced the frequency of required anti-VEGF injections over a period of 6 months.414,415 However, no difference in vision was observed with the additional corticosteroid treatment.415,416 Rezar-Dreindl et al. measured cytokine profiles from intravitreal samples of 40 patients receiving ranibizumab or combined ranibizumab and intravitreal dexamethasone implant. Prior to treatment, patients with neovascular AMD had elevated levels of macrophage chemoattractant protein (MCP-1), monokine induced by -interferon (MIG) and lipocalin-2 neutrophil gelatinase-associated lipocalin (NGAL), while tumor necrosis factor (TNF)-, interleukin (IL)-12p70 and secreted protein acidic and rich in cysteine (SPARC) were lower compared to healthy controls. Interestingly, they did not measure a difference in VEGF levels at baseline. Following combined therapy, VEGF, MIG, PDGF-AA and TGF were reduced, while no differences in cytokines were observed with just ranibizumab treatment. Interleukin-6 and PDGF-AA levels correlated with central retinal thickness.416 Overall, corticosteroid treatment may confer some short term benefit for neovascular

Harris, James

AMD, though it is not as effective as anti-VEGF therapy and has potential side effects of steroid-response ocular hypertension and cataract progression.

Targeted Immunomodulatory Drugs In addition to corticosteroids and NSAIDs, which are broadly immunosuppressive and anti-inflammatory, more targeted immunomodulatory agents have been studied in AMD both for neovascular AMD refractory to anti-VEGF treatment and for geographic atrophy. A series of case studies investigated the effect of treatment with the mouse-human hybrid TNF- inhibitor antibody, infliximab, on neovascular AMD. An initial study observed patients with subretinal choroidal neovascular membrane (CNVM) receiving systemic infliximab treatment for inflammatory arthritis. One patient had their CNVM resolve and two other patients had improvement in their CNVM after 6 months.417 Furthermore, in another study, two intravitreal injections of infliximab resulted in improvement in best corrected visual acuity after seven months in all three patients receiving treatment, though one patient relapsed following treatment.418 However, a subsequent study that injected infliximab found that the intravitreal infliximab injection was not tolerated in four patients. Two patients had clinical evidence of retinal toxicity and all four patients had decreased combined response on electroretinography. Additionally, anti-infliximab antibodies were observed in three patients.419 A study of four patients with neovascular AMD non-responsive to anti-VEGF received a single intravitreal injection of infliximab. No improvement in visual acuity or subretinal or intraretinal fluid was found.420 Adalimumab is a fully humanized monoclonal TNF- inhibitor that was also investigated in the context of neovascular AMD. In one retrospective pilot study, 26 eyes with neovascular AMD and suboptimal response to anti-VEGF agents were injected intravitreally with 1 or 2mg infliximab, 2mg adalimumab, or 1.25mg bevacizumab. Neither infliximab nor adalimumab significantly improved the best corrected visual acuity or central macular thickness after 3 months compared to continued bevacizumab injection.421 Adalimumab was further studied in trial NCT01136252, which injected 0.05mg intravitreally in patients with neovascular AMD, but results were never published.422 Methotrexate, a folic acid inhibitor and immunosuppressive agent, was also piloted in neovascular AMD patients. Two patients who had failed anti-VEGF therapy received intravitreal methotrexate and had improvement on OCT and improved visual acuity in one patient.423 Sirolimus is a mechanistic target of rapamycin (mTOR) kinase inhibitor and potent immunosuppressive that has been studied for both neovascular AMD and geographic atrophy. In an initial study, 13 patients receiving anti-VEGF treatment were randomized to also receive systemic sirolimus, daclizumab (an IL-2 receptor blocking antibody), or infliximab. Fewer anti-VEGF injections were required for daclizumab and sirolimus, but not infliximab.424 A recent study randomized 20 patients with persistent exudative AMD and a history of repeated anti-VEGF treatments to either continued anti-VEGF treatment or intravitreal sirolimus. This study found a 40um decrease in central macular thickness in the sirolimus treated patients compared to a 20um increase in the anti-VEGF treated patients. However, there was no difference in visual acuity following treatment.425 Another study examined subconjunctival sirolimus injections in 11 participants with geographic atrophy and observed no change in geographic atrophy area, drusen, central retinal thickness or macular sensitivity, though visual acuity worsened.426 A second study randomly assigned 6 patients with bilateral geographic atrophy to intravitreal sirolimus injection in one eye every two months. Unfortunately two patients had accelerated retinal thinning and one had accelerated expansion of geographic atrophy.427 A phase two trial of 52 participants randomized patients to monthly intravitreal sirolimus or sham treatment. The 27 eyes receiving sirolimus had no change in the growth rate of geographic atrophy or in visual acuity compared to controls. The trial was stopped early due to sterile endophthalmitis in three of the treated patients.428 Additionally, while not strictly an immunomodulator, the anti--amyloid antibody BAM114341/ GSK933776 was studied for geographic atrophy in a phase II trial. 191 patients were randomized to 3 separate

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intravenous doses of GSK933776 and a placebo arm. There was no reduction in the rate of geographic atrophy enlargement for any treatment group.429

Complement Inhibition There have been a variety of trials investigating novel and preexisting molecules that target various points in the complement pathway. Given the efficacy of anti-VEGF treatment for exudative AMD, complement inhibition has focused on treating patients with geographic atrophy, for which no other treatments currently exist. Therapeutic targets included inhibitors of factor D, C3, and C5. Factor D Inhibitors Lampalizumab is an antigen binding fab fragment of a human monoclonal antibody that inhibits complement factor D, selectively blocking the alternative complement amplification pathway but not the classic or mannose-binding lectin pathways. The phase II MAHALO study treated 42 patients intravitreally either monthly or every other month with lampalizumab and measured a change in geographic atrophy lesion area assessed using fundus autofluorescence from baseline to 18 months. Monthly treated patients had a 20% reduction in the progression of the lesion area compared to 41 sham controls. Furthermore, carriers of the intronic complement factor I variant rs17440077 showed a 44% reduction in geographic atrophy lesion expansion with monthly treatment and had lower factor I levels.170 Despite these initial promising results, the identically-designed Chroma and Spectri follow up phase III trials studied 1881 patients and gave lampalizumab either every 4 or 6 weeks for 96 weeks. These trials detected no change in geographic atrophy progression in either trial, nor found any association of CFI genetic variation with progression.430 C3 Inhibitors C3 is a central mediator downstream of all three complement initiation pathways. Pegcetacopan is a pegylated synthetic molecule that selectively inhibits C3. The FILLY phase II trial was a prospective, multicenter, randomized, sham-controlled trial of 246 patients with geographic atrophy. They observed a 29% and 20% reduction in GA growth as measured by fundus autofluorescence with monthly or everyother month injection of 15mg pegcatacoplan, respectively. There was no change in measures of visual function including BCVA. However, treated patients developed exudative AMD at higher rates. It was speculated that C3-mediated signaling may promote the proinflammatory M1 microglial phenotype responsible for geographic atrophy. By inhibiting C3, treatment may shift this microglial population to the proangiogenic M2 microglial phenotype.431 Several post-hoc studies of FILLY participants found further structural evidence of a potential benefit of pegcetcopan in geographic atrophy. One post-hoc analysis of 167 patients found reduction in the rates of progression of incomplete to complete RPE and outer retinal atrophy (50% in monthly treated patients vs. 81.8% in sham controls).432 A second post-hoc study found 29% and 26% thicker outer nuclear layer measured at the 5.16 degree contour line at month 12.433 The OAKS and DERBY trials were 2-year, multicenter, randomized, double-masked, sham-controlled, phase 3 studies of pegcetacoplan with a combined total of 1258 participants. Intravitreal injection of 0.1mL of pegcetacoplan was performed either monthly or every other month. In the OAKS trial, treatment slowed GA by 21% and 16% for monthly and every-other monthly dosing after 1 year and 22% and 18% respectively after 2 years. In the DERBY trial, there was a nonsignificant trend to slower GA growth after one year, that reached significance by 24 months with similar magnitude of effect as the OAKS trial. However, there was no difference in any measure of visual function including monocular maximum reading speed, change from baseline in mean functional reading independence index score, change from baseline in normal luminance best corrected visual acuity, and change from baseline in mean threshold sensitivity in either study. Both studies observed increased rates of CNV.434 The increase in neovascularization with complement inhibition is an interesting finding. A mouse study similarly found that C3 or C5aR deficiency led to neovascularization in a macrophage-dependent mechanism, by which C5a promotes an anti-angiogenic macrophage phenotype.435 In 2023, pegcatacopan became the first FDAapproved treatment for GA and a 3-year monitoring study (GALE) is currently underway.

Harris, James

Intravitreal injection of an anti-C3 monoclonal antibody, NGM621, was studied in the phase 2 CATALINA trial and failed to slow GA area growth rates over 1 year with every 4 or 8-week injections. However, a subgroup with a specific GA area size did have a modest reduction in GA growth and further investigation is ongoing.436 C5 inhibitors C5 is the first molecule activated in the terminal complement pathway that leads to assembly of the membrane attack complex. Eculizumab (Soliris) is a humanized anti-C5 inhibitory antibody approved for paroxysmal nocturnal hemoglobinuria. The COMPLETE trial (NCT00935883) studied systemic eculizumab in a dose escalation design compared with placebo and measured the rate of geographic atrophy area enlargement after 26 weeks in 30 eyes from 30 patients. No difference was found for any treatment groups.437 Tesidolumab/LFG316 is another IgG1 that inhibits C5 and similarly no difference of GA growth rate or visual acuity was found with treatment in a phase II trial.438 ARC1905 is an avacincaptad pegol comprised of a pegylated RNA aptamer designed to inhibit C5 activation. The GATHER1 phase II/III study enrolled 286 patients with geographic atrophy and measured the rate of growth over 18 months using fundus autofluoresence. An average of 27.4-27.8% reduction in the rate of growth of geographic atrophy was observed with monthly intravitreal injection of either 2 or 4mg of drug.439 However, there was no improvement in BCVA or patient survey scores, and an increased rate of CNV was observed.439 This medication is also FDA approved for the treatment of geographic atrophy. Other complement targets An anti-C1q antibody fragment (ANX007) showed slower loss of vision but no significant change in GA area growth in a phase 2 ARCHER trial.436 An anti-properdin antibody, CLG561, also failed to slow progression of GA in a phase 2 study.440 The C3 inhibitor, POT-4 (AL-78898A), was terminated prematurely due to 54.14% of patients getting drug deposits in the eye.441 Other therapies, including C5aR inhibitor JPE-1375, have been stopped due to acquisitions or discontinuation of operations of the sponsoring companies.

Immunomodulatory Therapies in Early Development Additional complement modifying treatments are currently in development.436 Minocycline, which had been shown to inhibit microglial activation in preclinical models, failed to improve vision or slow GA progression in a phase II trial.442 Other treatments include overexpression constructs of CD59 (AAVCAGsCD59/HMR59) and CFI (GT005), recombinant protective form of CFH (GEM103), CFH and C3b binding enhancers (AVD-104), and inhibitors of CFD (danicopan/ALXN2040), CFB (IONIS-FBLRX/Isis 696844436,441 and Iptacopan/LNP023), MASP-2 (narsoplimab/OMS721), MASP-3 (OMS906) and C3 (CB 2782-PEG and KNP-301). Other anti-inflammatory therapies outside of the complement system include modulators of macrophage transcription (TMi-018). The results from these studies will provide key mechanistic insight into the role of inflammation in the pathogenesis of AMD in humans and will hopefully provide significant benefits to patients.

Discussion and Conclusion A large body of work has convincingly implicated chronic inflammation in the pathogenesis of early and late AMD in humans. This evidence includes the highly reproducible genetic linkage of AMD and multiple proteins in inflammatory signaling pathways and complement cascades, histologic evidence of molecular and cellular findings of chronic inflammation in human eyes with AMD, and the clinical comorbidity of AMD disease features in diseases of chronic complement activation (MPGNII and aHUS). Adding further support to the role of inflammation in AMD is more circumstantial evidence including the robust associations of proinflammatory environmental risk factors including aging, smoking, and obesity, and the linkage of serum markers of chronic inflammation with AMD.

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Despite this clear body of evidence linking inflammation and AMD, there has been limited success in developing effective therapeutics targeting these pathways. Key questions remain. It is likely that not all of the inflammatory associations found with AMD are directly driving pathogenesis. Some of these changes may be incidental or secondary to the underlying disease pathology. Alternatively, some inflammatory reactions may be compensatory, helping to limit disease progression. The development of neovascularization in some patients treated with complement inhibitors may be an example of such protective mechanisms. A better understanding of the complex interaction of cellular and humoral immunity with tissue specific factors in the retina will help focus future therapeutic efforts. Furthermore, as we develop deeper understanding of these systems, new biologic paradigms will likely come to light. For instance, the complement system, which has largely been thought of as an extracellular mediator of inflammation, has recently been recognized as playing an important role in intracellular homeostatic mechanisms.443 It is possible that this novel biological role for complement is involved in AMD, an area that has not been explored. Furthermore, while genetic or environmental factors that create a chronic inflammatory environment increase the risk of developing early and late AMD, the specific molecular events that occur at the transition points of disease pathogenesis are less clear. For instance, while a person carrying a CFH risk allele may experience a lifetime of slightly increased complement activation, what triggers the formation of drusen or the progression from early disease to advanced stages? To identify patients most likely to benefit from intervention and to develop therapies to slow or halt disease progression requires a better understanding of the molecular mechanisms of these transition points. New molecular and computational tools including cryogenic electron microscopy, improved proteomic sequencing methods, spatial transcriptomics, and additional single-cell RNA sequencing studies will allow better understanding of the precise molecular and cellular events that occur during AMD disease progression. Doing so will facilitate the development of more selective anti-inflammatory agents targeted at specific diseases processes using the expanding therapeutic armamentarium of small molecules, antibodies and their derivatives,, and increasingly gene-based therapies. These advancements in this fascinating disease will allow us to better serve patients and will hopefully provide insight into the pathologic mechanisms of neurodegenerative diseases more broadly, including those in the darker recesses of the central nervous system.

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42. Baird, P. N. et al. Analysis of the Y402H variant of the complement factor H gene in age-related macular degeneration. Invest Ophthalmol Vis Sci 47, 4194-4198 (2006). https://doi.org:10.1167/iovs.05-1285 43. Barlow, P. N., Hageman, G. S. & Lea, S. M. Complement factor H: using atomic resolution structure to illuminate disease mechanisms. Adv Exp Med Biol 632, 117-142 (2008). 44. Narayanan, R. et al. Complement factor H polymorphism in age-related macular degeneration. Ophthalmology 114, 1327-1331 (2007). https://doi.org:10.1016/j.ophtha.2006.10.035 45. Xing, C. et al. Complement factor H polymorphisms, renal phenotypes and age-related macular degeneration: the Blue Mountains Eye Study. Genes Immun 9, 231-239 (2008). https://doi.org:10.1038/gene.2008.10 46. Sofat, R. et al. Complement factor H genetic variant and age-related macular degeneration: effect size, modifiers and relationship to disease subtype. Int J Epidemiol 41, 250-262 (2012). https://doi.org:10.1093/ije/dyr204 47. Magnusson, K. P. et al. CFH Y402H confers similar risk of soft drusen and both forms of advanced AMD. PLoS Med 3, e5 (2006). https://doi.org:10.1371/journal.pmed.0030005 48. Droz, I. et al. Genotype-phenotype correlation of age-related macular degeneration: influence of complement factor H polymorphism. Br J Ophthalmol 92, 513-517 (2008). https://doi.org:10.1136/bjo.2007.127811 49. Tedeschi-Blok, N., Buckley, J., Varma, R., Triche, T. J. & Hinton, D. R. Population-based study of early age-related macular degeneration: role of the complement factor H Y402H polymorphism in bilateral but not unilateral disease. Ophthalmology 114, 99-103 (2007). https://doi.org:10.1016/j.ophtha.2006.07.043 50. Goverdhan, S. V. et al. An analysis of the CFH Y402H genotype in AMD patients and controls from the UK, and response to PDT treatment. Eye (Lond) 22, 849-854 (2008). https://doi.org:10.1038/sj.eye.6702830 51. Despriet, D. D. et al. Complement factor H polymorphism, complement activators, and risk of age-related macular degeneration. JAMA 296, 301-309 (2006). https://doi.org:10.1001/jama.296.3.301 52. Weger, M. et al. Association of the HTRA1 -625G>A promoter gene polymorphism with exudative age-related macular degeneration in a Central European population. Mol Vis 13, 1274-1279 (2007). 53. Wegscheider, B. J. et al. Association of complement factor H Y402H gene polymorphism with different subtypes of exudative age-related macular degeneration. Ophthalmology 114, 738-742 (2007). https://doi.org:10.1016/j.ophtha.2006.07.048 54. Hayashi, H. et al. CFH and ARMS2 variations in age-related macular degeneration, polypoidal choroidal vasculopathy, and retinal angiomatous proliferation. Invest Ophthalmol Vis Sci 51, 5914-5919 (2010). https://doi.org:10.1167/iovs.10-5554 55. Sepp, T. et al. Complement factor H variant Y402H is a major risk determinant for geographic atrophy and choroidal neovascularization in smokers and nonsmokers. Invest Ophthalmol Vis Sci 47, 536-540 (2006). https://doi.org:10.1167/iovs.05-1143 56. Hughes, A. E. et al. Neovascular age-related macular degeneration risk based on CFH, LOC387715/HTRA1, and smoking. PLoS Med 4, e355 (2007). https://doi.org:10.1371/journal.pmed.0040355 57. DeAngelis, M. M. et al. Cigarette smoking, CFH, APOE, ELOVL4, and risk of neovascular age-related macular degeneration. Arch Ophthalmol 125, 49-54 (2007). https://doi.org:10.1001/archopht.125.1.49 58. Chowers, I. et al. Association of complement factor H Y402H polymorphism with phenotype of neovascular age related macular degeneration in Israel. Mol Vis 14, 1829-1834 (2008). 59. Souied, E. H. et al. Y402H complement factor H polymorphism associated with exudative age-related macular degeneration in the French population. Mol Vis 11, 1135-1140 (2005). 60. Scholl, H. P. et al. CFH, C3 and ARMS2 are significant risk loci for susceptibility but not for disease progression of geographic atrophy due to AMD. PLoS One 4, e7418 (2009). https://doi.org:10.1371/journal.pone.0007418 61. Schmitz-Valckenberg, S. et al. Progression of Age-Related Macular Degeneration Among Individuals Homozygous for Risk Alleles on Chromosome 1 (CFH-CFHR5) or Chromosome 10 (ARMS2/HTRA1) or Both. JAMA Ophthalmol 140, 252-260 (2022). https://doi.org:10.1001/jamaophthalmol.2021.6072 62. Seitsonen, S. et al. Analysis of variants in the complement factor H, the elongation of very long chain fatty acidslike 4 and the hemicentin 1 genes of age-related macular degeneration in the Finnish population. Mol Vis 12, 796801 (2006). 63. Simonelli, F. et al. Polymorphism p.402Y>H in the complement factor H protein is a risk factor for age related macular degeneration in an Italian population. Br J Ophthalmol 90, 1142-1145 (2006). https://doi.org:10.1136/bjo.2006.096487

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64. Fisher, S. A. et al. Assessment of the contribution of CFH and chromosome 10q26 AMD susceptibility loci in a Russian population isolate. Br J Ophthalmol 91, 576-578 (2007). https://doi.org:10.1136/bjo.2006.105577 65. Teixeira, A. G. et al. Association of complement factor H Y402H polymorphism and age-related macular degeneration in Brazilian patients. Acta Ophthalmol 88, e165-169 (2010). https://doi.org:10.1111/j.17553768.2010.01932.x 66. Kaur, I. et al. Analysis of CFH, TLR4, and APOE polymorphism in India suggests the Tyr402His variant of CFH to be a global marker for age-related macular degeneration. Invest Ophthalmol Vis Sci 47, 3729-3735 (2006). https://doi.org:10.1167/iovs.05-1430 67. Chen, L. J. et al. Association of complement factor H polymorphisms with exudative age-related macular degeneration. Mol Vis 12, 1536-1542 (2006). 68. Grassi, M. A. et al. Ethnic variation in AMD-associated complement factor H polymorphism p.Tyr402His. Hum Mutat 27, 921-925 (2006). https://doi.org:10.1002/humu.20359 69. Lau, L. I. et al. Association of the Y402H polymorphism in complement factor H gene and neovascular age-related macular degeneration in Chinese patients. Invest Ophthalmol Vis Sci 47, 3242-3246 (2006). https://doi.org:10.1167/iovs.05-1532 70. Okamoto, H. et al. Complement factor H polymorphisms in Japanese population with age-related macular degeneration. Mol Vis 12, 156-158 (2006). 71. Xu, Y. et al. Association of CFH, LOC387715, and HTRA1 polymorphisms with exudative age-related macular degeneration in a northern Chinese population. Mol Vis 14, 1373-1381 (2008). 72. Kim, N. R. et al. Association between complement factor H gene polymorphisms and neovascular age-related macular degeneration in Koreans. Invest Ophthalmol Vis Sci 49, 2071-2076 (2008). https://doi.org:10.1167/iovs.07-1195 73. Uka, J. et al. No association of complement factor H gene polymorphism and age-related macular degeneration in the Japanese population. Retina 26, 985-987 (2006). https://doi.org:10.1097/01.iae.0000244068.18520.3e 74. Mori, K. et al. Coding and noncoding variants in the CFH gene and cigarette smoking influence the risk of agerelated macular degeneration in a Japanese population. Invest Ophthalmol Vis Sci 48, 5315-5319 (2007). https://doi.org:10.1167/iovs.07-0426 75. Fuse, N. et al. Polymorphisms in Complement Factor H and Hemicentin-1 genes in a Japanese population with dry-type age-related macular degeneration. Am J Ophthalmol 142, 1074-1076 (2006). https://doi.org:10.1016/j.ajo.2006.07.030 76. Tanimoto, S. et al. A polymorphism of LOC387715 gene is associated with age-related macular degeneration in the Japanese population. Neurosci Lett 414, 71-74 (2007). https://doi.org:10.1016/j.neulet.2006.12.011 77. Gotoh, N. et al. No association between complement factor H gene polymorphism and exudative age-related macular degeneration in Japanese. Hum Genet 120, 139-143 (2006). https://doi.org:10.1007/s00439-006-0187-0 78. Ziskind, A., Bardien, S., van der Merwe, L. & Webster, A. R. The frequency of the H402 allele of CFH and its involvement with age-related maculopathy in an aged Black African Xhosa population. Ophthalmic Genet 29, 117119 (2008). https://doi.org:10.1080/13816810802216472 79. Fritsche, L. G. et al. A large genome-wide association study of age-related macular degeneration highlights contributions of rare and common variants. Nat Genet 48, 134-143 (2016). https://doi.org:10.1038/ng.3448 80. Genomes Project, C. et al. A global reference for human genetic variation. Nature 526, 68-74 (2015). https://doi.org:10.1038/nature15393 81. Huang, L. Z. et al. Whole-exome sequencing implicates UBE3D in age-related macular degeneration in East Asian populations. Nat Commun 6, 6687 (2015). https://doi.org:10.1038/ncomms7687 82. Arakawa, S. et al. Genome-wide association study identifies two susceptibility loci for exudative age-related macular degeneration in the Japanese population. Nat Genet 43, 1001-1004 (2011). https://doi.org:10.1038/ng.938 83. Goto, A. et al. Genetic analysis of typical wet-type age-related macular degeneration and polypoidal choroidal vasculopathy in Japanese population. J Ocul Biol Dis Infor 2, 164-175 (2009). https://doi.org:10.1007/s12177-0099047-1 84. Kondo, N., Honda, S., Kuno, S. & Negi, A. Coding variant I62V in the complement factor H gene is strongly associated with polypoidal choroidal vasculopathy. Ophthalmology 116, 304-310 (2009). https://doi.org:10.1016/j.ophtha.2008.11.011

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85. Tortajada, A. et al. The disease-protective complement factor H allotypic variant Ile62 shows increased binding affinity for C3b and enhanced cofactor activity. Hum Mol Genet 18, 3452-3461 (2009). https://doi.org:10.1093/hmg/ddp289 86. Heurich, M. et al. Common polymorphisms in C3, factor B, and factor H collaborate to determine systemic complement activity and disease risk. Proc Natl Acad Sci U S A 108, 8761-8766 (2011). https://doi.org:10.1073/pnas.1019338108 87. Hecker, L. A. et al. Genetic control of the alternative pathway of complement in humans and age-related macular degeneration. Hum Mol Genet 19, 209-215 (2010). https://doi.org:10.1093/hmg/ddp472 88. Liao, X., Lan, C. J., Cheuk, I. W. & Tan, Q. Q. Four complement factor H gene polymorphisms in association with AMD: A meta-analysis. Arch Gerontol Geriatr 64, 123-129 (2016). https://doi.org:10.1016/j.archger.2016.01.011 89. Edwards, A. O. et al. Evaluation of clustering and genotype distribution for replication in genome wide association studies: the age-related eye disease study. PLoS One 3, e3813 (2008). https://doi.org:10.1371/journal.pone.0003813 90. Chen, W. et al. Genetic variants near TIMP3 and high-density lipoprotein-associated loci influence susceptibility to age-related macular degeneration. Proc Natl Acad Sci U S A 107, 7401-7406 (2010). https://doi.org:10.1073/pnas.0912702107 91. Cheng, C. Y. et al. New loci and coding variants confer risk for age-related macular degeneration in East Asians. Nat Commun 6, 6063 (2015). https://doi.org:10.1038/ncomms7063 92. Fritsche, L. G. et al. Seven new loci associated with age-related macular degeneration. Nat Genet 45, 433-439, 439e431-432 (2013). https://doi.org:10.1038/ng.2578 93. Colijn, J. M. et al. Genetic Risk, Lifestyle, and Age-Related Macular Degeneration in Europe: The EYE-RISK Consortium. Ophthalmology 128, 1039-1049 (2021). https://doi.org:10.1016/j.ophtha.2020.11.024 94. Hughes, A. E. et al. A common CFH haplotype, with deletion of CFHR1 and CFHR3, is associated with lower risk of age-related macular degeneration. Nat Genet 38, 1173-1177 (2006). https://doi.org:10.1038/ng1890 95. Hageman, G. S. et al. Extended haplotypes in the complement factor H (CFH) and CFH-related (CFHR) family of genes protect against age-related macular degeneration: characterization, ethnic distribution and evolutionary implications. Ann Med 38, 592-604 (2006). 96. Kubista, K. E. et al. Copy number variation in the complement factor H-related genes and age-related macular degeneration. Mol Vis 17, 2080-2092 (2011). 97. Raychaudhuri, S. et al. Associations of CFHR1-CFHR3 deletion and a CFH SNP to age-related macular degeneration are not independent. Nat Genet 42, 553-555; author reply 555-556 (2010). https://doi.org:10.1038/ng0710-553 98. Hughes, A. E., Orr, N., Cordell, H. J. & Goodship, T. Reply to “Associations of CFHR1–CFHR3 deletion and a CFH SNP to age-related macular degeneration are not independent”. Nature Genetics 42, 555-556 (2010). https://doi.org:10.1038/ng0710-555 99. Schmid-Kubista, K. E. et al. Contribution of copy number variation in the regulation of complement activation locus to development of age-related macular degeneration. Invest Ophthalmol Vis Sci 50, 5070-5079 (2009). https://doi.org:10.1167/iovs.09-3975 100. Spencer, K. L. et al. Deletion of CFHR3 and CFHR1 genes in age-related macular degeneration. Hum Mol Genet 17, 971-977 (2008). https://doi.org:10.1093/hmg/ddm369 101. Cserhalmi, M., Papp, A., Brandus, B., Uzonyi, B. & Jozsi, M. Regulation of regulators: Role of the complement factor H-related proteins. Semin Immunol 45, 101341 (2019). https://doi.org:10.1016/j.smim.2019.101341 102. Pappas, C. M. et al. Protective chromosome 1q32 haplotypes mitigate risk for age-related macular degeneration associated with the CFH-CFHR5 and ARMS2/HTRA1 loci. Hum Genomics 15, 60 (2021). https://doi.org:10.1186/s40246-021-00359-8 103. Ennis, S. et al. Fine-scale linkage disequilibrium mapping of age-related macular degeneration in the complement factor H gene region. Br J Ophthalmol 91, 966-970 (2007). https://doi.org:10.1136/bjo.2007.114090 104. Sivakumaran, T. A. et al. A 32 kb critical region excluding Y402H in CFH mediates risk for age-related macular degeneration. PLoS One 6, e25598 (2011). https://doi.org:10.1371/journal.pone.0025598

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105. Fritsche, L. G. et al. An imbalance of human complement regulatory proteins CFHR1, CFHR3 and factor H influences risk for age-related macular degeneration (AMD). Hum Mol Genet 19, 4694-4704 (2010). https://doi.org:10.1093/hmg/ddq399 106. Ng, T. K. et al. Multiple gene polymorphisms in the complement factor h gene are associated with exudative agerelated macular degeneration in chinese. Invest Ophthalmol Vis Sci 49, 3312-3317 (2008). https://doi.org:10.1167/iovs.07-1517 107. Geerlings, M. J., de Jong, E. K. & den Hollander, A. I. The complement system in age-related macular degeneration: A review of rare genetic variants and implications for personalized treatment. Mol Immunol 84, 65-76 (2017). https://doi.org:10.1016/j.molimm.2016.11.016 108. Ferrara, D. & Seddon, J. M. Phenotypic Characterization of Complement Factor H R1210C Rare Genetic Variant in Age-Related Macular Degeneration. JAMA Ophthalmol 133, 785-791 (2015). https://doi.org:10.1001/jamaophthalmol.2015.0814 109. 109 Saksens, N. T. et al. Rare Genetic Variants Associated With Development of Age-Related Macular Degeneration. JAMA Ophthalmol 134, 287-293 (2016). https://doi.org:10.1001/jamaophthalmol.2015.5592 110. Triebwasser, M. P. et al. Rare Variants in the Functional Domains of Complement Factor H Are Associated With Age-Related Macular Degeneration. Invest Ophthalmol Vis Sci 56, 6873-6878 (2015). https://doi.org:10.1167/iovs.15-17432 111. Yu, Y. et al. Whole-exome sequencing identifies rare, functional CFH variants in families with macular degeneration. Hum Mol Genet 23, 5283-5293 (2014). https://doi.org:10.1093/hmg/ddu226 112. Hoffman, J. D. et al. Rare complement factor H variant associated with age-related macular degeneration in the Amish. Invest Ophthalmol Vis Sci 55, 4455-4460 (2014). https://doi.org:10.1167/iovs.13-13684 113. Ratnapriya, R. et al. Family-based exome sequencing identifies rare coding variants in age-related macular degeneration. Hum Mol Genet 29, 2022-2034 (2020). https://doi.org:10.1093/hmg/ddaa057 114. Wagner, E. K. et al. Mapping rare, deleterious mutations in Factor H: Association with early onset, drusen burden, and lower antigenic levels in familial AMD. Sci Rep 6, 31531 (2016). https://doi.org:10.1038/srep31531 115. Grassi, M. A. et al. Complement factor H polymorphism p.Tyr402His and cuticular Drusen. Arch Ophthalmol 125, 93-97 (2007). https://doi.org:10.1001/archopht.125.1.93 116. Boon, C. J. et al. Basal laminar drusen caused by compound heterozygous variants in the CFH gene. Am J Hum Genet 82, 516-523 (2008). https://doi.org:10.1016/j.ajhg.2007.11.007 117. van de Ven, J. P. et al. Clinical evaluation of 3 families with basal laminar drusen caused by novel mutations in the complement factor H gene. Arch Ophthalmol 130, 1038-1047 (2012). https://doi.org:10.1001/archophthalmol.2012.265 118. Duvvari, M. R. et al. Whole Exome Sequencing in Patients with the Cuticular Drusen Subtype of Age-Related Macular Degeneration. PLoS One 11, e0152047 (2016). https://doi.org:10.1371/journal.pone.0152047 119. Yates, J. R. et al. Complement C3 variant and the risk of age-related macular degeneration. N Engl J Med 357, 553-561 (2007). https://doi.org:10.1056/NEJMoa072618 120. Maller, J. B. et al. Variation in complement factor 3 is associated with risk of age-related macular degeneration. Nat Genet 39, 1200-1201 (2007). https://doi.org:10.1038/ng2131 121. Francis, P. J., Hamon, S. C., Ott, J., Weleber, R. G. & Klein, M. L. Polymorphisms in C2, CFB and C3 are associated with progression to advanced age related macular degeneration associated with visual loss. J Med Genet 46, 300-307 (2009). https://doi.org:10.1136/jmg.2008.062737 122. Thakkinstian, A. et al. Systematic review and meta-analysis of the association between complement component 3 and age-related macular degeneration: a HuGE review and meta-analysis. Am J Epidemiol 173, 1365-1379 (2011). https://doi.org:10.1093/aje/kwr025 123. Despriet, D. D. et al. Complement component C3 and risk of age-related macular degeneration. Ophthalmology 116, 474-480 e472 (2009). https://doi.org:10.1016/j.ophtha.2008.09.055 124. Zhang, J., Li, S., Hu, S., Yu, J. & Xiang, Y. Association between genetic variation of complement C3 and the susceptibility to advanced age-related macular degeneration: a meta-analysis. BMC Ophthalmol 18, 274 (2018). https://doi.org:10.1186/s12886-018-0945-5 125. Seddon, J. M. et al. Rare variants in CFI, C3 and C9 are associated with high risk of advanced age-related macular degeneration. Nat Genet 45, 1366-1370 (2013). https://doi.org:10.1038/ng.2741

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126. Seddon, J. M. et al. Rare Dysfunctional Complement Factor I Genetic Variants and Progression to Advanced AgeRelated Macular Degeneration. Ophthalmol Sci 3, 100265 (2023). https://doi.org:10.1016/j.xops.2022.100265 127. Duvvari, M. R. et al. Analysis of rare variants in the C3 gene in patients with age-related macular degeneration. PLoS One 9, e94165 (2014). https://doi.org:10.1371/journal.pone.0094165 128. Ennis, S. et al. Association between the SERPING1 gene and age-related macular degeneration: a two-stage case-control study. Lancet 372, 1828-1834 (2008). https://doi.org:10.1016/S0140-6736(08)61348-3 129. Gibson, J., Cree, A., Collins, A., Lotery, A. & Ennis, S. Determination of a gene and environment risk model for age-related macular degeneration. Br J Ophthalmol 94, 1382-1387 (2010). https://doi.org:10.1136/bjo.2010.182568 130. Lee, A. Y. et al. The effect of genetic variants in SERPING1 on the risk of neovascular age-related macular degeneration. Br J Ophthalmol 94, 915-917 (2010). https://doi.org:10.1136/bjo.2009.172007 131. Allikmets, R. et al. The SERPING1 gene and age-related macular degeneration. Lancet 374, 875-876; author reply 876-877 (2009). https://doi.org:10.1016/S0140-6736(09)61618-4 132. Park, K. H., Ryu, E., Tosakulwong, N., Wu, Y. & Edwards, A. O. Common variation in the SERPING1 gene is not associated with age-related macular degeneration in two independent groups of subjects. Mol Vis 15, 200-207 (2009). 133. Carter, J. G. & Churchill, A. J. Analysis of SERPING1 and its association with age-related macular degeneration. Acta Ophthalmol 89, e212-213 (2011). https://doi.org:10.1111/j.1755-3768.2009.01788.x 134. Lu, F. et al. An association study of SERPING1 gene and age-related macular degeneration in a Han Chinese population. Mol Vis 16, 1-6 (2010). 135. Neale, B. M. et al. Genome-wide association study of advanced age-related macular degeneration identifies a role of the hepatic lipase gene (LIPC). Proc Natl Acad Sci U S A 107, 7395-7400 (2010). https://doi.org:10.1073/pnas.0912019107 136. Fagerness, J. A. et al. Variation near complement factor I is associated with risk of advanced AMD. Eur J Hum Genet 17, 100-104 (2009). https://doi.org:10.1038/ejhg.2008.140 137. Kondo, N., Bessho, H., Honda, S. & Negi, A. Additional evidence to support the role of a common variant near the complement factor I gene in susceptibility to age-related macular degeneration. Eur J Hum Genet 18, 634-635 (2010). https://doi.org:10.1038/ejhg.2009.243 138. Qian, D. et al. Common variant rs10033900 near the complement factor I gene is associated with age-related macular degeneration risk in Han Chinese population. Eur J Hum Genet 22, 1417-1419 (2014). https://doi.org:10.1038/ejhg.2014.37 139. Ennis, S., Gibson, J., Cree, A. J., Collins, A. & Lotery, A. J. Support for the involvement of complement factor I in age-related macular degeneration. Eur J Hum Genet 18, 15-16 (2010). https://doi.org:10.1038/ejhg.2009.113 140. van de Ven, J. P. et al. A functional variant in the CFI gene confers a high risk of age-related macular degeneration. Nat Genet 45, 813-817 (2013). https://doi.org:10.1038/ng.2640 141. Alexander, P., Gibson, J., Cree, A. J., Ennis, S. & Lotery, A. J. Complement factor I and age-related macular degeneration. Mol Vis 20, 1253-1257 (2014). 142. Pras, E. et al. Rare genetic variants in Tunisian Jewish patients suffering from age-related macular degeneration. J Med Genet 52, 484-492 (2015). https://doi.org:10.1136/jmedgenet-2015-103130 143. Kavanagh, D. et al. Rare genetic variants in the CFI gene are associated with advanced age-related macular degeneration and commonly result in reduced serum factor I levels. Hum Mol Genet 24, 3861-3870 (2015). https://doi.org:10.1093/hmg/ddv091 144. Java, A. et al. Functional Analysis of Rare Genetic Variants in Complement Factor I (CFI) using a Serum-Based Assay in Advanced Age-related Macular Degeneration. Transl Vis Sci Technol 9, 37 (2020). 145. Tan, P. L. et al. Systematic Functional Testing of Rare Variants: Contributions of CFI to Age-Related Macular Degeneration. Invest Ophthalmol Vis Sci 58, 1570-1576 (2017). https://doi.org:10.1167/iovs.16-20867 146. McKay, G. J. et al. Further assessment of the complement component 2 and factor B region associated with agerelated macular degeneration. Invest Ophthalmol Vis Sci 50, 533-539 (2009). https://doi.org:10.1167/iovs.08-2275 147. Richardson, A. J., Islam, F. M., Guymer, R. H. & Baird, P. N. Analysis of rare variants in the complement component 2 (C2) and factor B (BF) genes refine association for age-related macular degeneration (AMD). Invest Ophthalmol Vis Sci 50, 540-543 (2009). https://doi.org:10.1167/iovs.08-2423

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148. Gold, B. et al. Variation in factor B (BF) and complement component 2 (C2) genes is associated with age-related macular degeneration. Nat Genet 38, 458-462 (2006). https://doi.org:10.1038/ng1750 149. Spencer, K. L. et al. Protective effect of complement factor B and complement component 2 variants in age-related macular degeneration. Hum Mol Genet 16, 1986-1992 (2007). https://doi.org:10.1093/hmg/ddm146 150. Thakkinstian, A. et al. The association between complement component 2/complement factor B polymorphisms and age-related macular degeneration: a HuGE review and meta-analysis. Am J Epidemiol 176, 361-372 (2012). https://doi.org:10.1093/aje/kws031 151. Kaur, I. et al. The involvement of complement factor B and complement component C2 in an Indian cohort with age-related macular degeneration. Invest Ophthalmol Vis Sci 51, 59-63 (2010). https://doi.org:10.1167/iovs.094135 152. Jakobsdottir, J., Conley, Y. P., Weeks, D. E., Ferrell, R. E. & Gorin, M. B. C2 and CFB genes in age-related maculopathy and joint action with CFH and LOC387715 genes. PLoS One 3, e2199 (2008). https://doi.org:10.1371/journal.pone.0002199 153. Montes, T., Tortajada, A., Morgan, B. P., Rodriguez de Cordoba, S. & Harris, C. L. Functional basis of protection against age-related macular degeneration conferred by a common polymorphism in complement factor B. Proc Natl Acad Sci U S A 106, 4366-4371 (2009). https://doi.org:10.1073/pnas.0812584106 154. Momozawa, Y. et al. Low-frequency coding variants in CETP and CFB are associated with susceptibility of exudative age-related macular degeneration in the Japanese population. Hum Mol Genet 25, 5027-5034 (2016). https://doi.org:10.1093/hmg/ddw335 155. Farwick, A. et al. Variations in five genes and the severity of age-related macular degeneration: results from the Muenster aging and retina study. Eye (Lond) 23, 2238-2244 (2009). https://doi.org:10.1038/eye.2008.426 156. Zeng, J. et al. Lack of association of CFD polymorphisms with advanced age-related macular degeneration. Mol Vis 16, 2273-2278 (2010). 157. Stanton, C. M. et al. Complement factor D in age-related macular degeneration. Invest Ophthalmol Vis Sci 52, 8828-8834 (2011). https://doi.org:10.1167/iovs.11-7933 158. Baas, D. C. et al. The complement component 5 gene and age-related macular degeneration. Ophthalmology 117, 500-511 (2010). https://doi.org:10.1016/j.ophtha.2009.08.032 159. Nishiguchi, K. M. et al. C9-R95X polymorphism in patients with neovascular age-related macular degeneration. Invest Ophthalmol Vis Sci 53, 508-512 (2012). https://doi.org:10.1167/iovs.11-8425 160. McMahon, O. et al. The rare C9 P167S risk variant for age-related macular degeneration increases polymerization of the terminal component of the complement cascade. Hum Mol Genet 30, 1188-1199 (2021). https://doi.org:10.1093/hmg/ddab086 161. Kremlitzka, M. et al. Functional analyses of rare genetic variants in complement component C9 identified in patients with age-related macular degeneration. Hum Mol Genet 27, 2678-2688 (2018). https://doi.org:10.1093/hmg/ddy178 162. Seddon, J. M. et al. Association of CFH Y402H and LOC387715 A69S with progression of age-related macular degeneration. JAMA 297, 1793-1800 (2007). https://doi.org:10.1001/jama.297.16.1793 163. Farwick, A., Wellmann, J., Stoll, M., Pauleikhoff, D. & Hense, H. W. Susceptibility genes and progression in agerelated maculopathy: a study of single eyes. Invest Ophthalmol Vis Sci 51, 731-736 (2010). https://doi.org:10.1167/iovs.09-3953 164. Seddon, J. M. et al. Prediction model for prevalence and incidence of advanced age-related macular degeneration based on genetic, demographic, and environmental variables. Invest Ophthalmol Vis Sci 50, 2044-2053 (2009). https://doi.org:10.1167/iovs.08-3064 165. Seddon, J. M., Silver, R. E., Kwong, M. & Rosner, B. Risk Prediction for Progression of Macular Degeneration: 10 Common and Rare Genetic Variants, Demographic, Environmental, and Macular Covariates. Invest Ophthalmol Vis Sci 56, 2192-2202 (2015). https://doi.org:10.1167/iovs.14-15841 166. Seddon, J. M., Reynolds, R., Yu, Y., Daly, M. J. & Rosner, B. Risk models for progression to advanced age-related macular degeneration using demographic, environmental, genetic, and ocular factors. Ophthalmology 118, 22032211 (2011). https://doi.org:10.1016/j.ophtha.2011.04.029 167. Grassmann, F. et al. Assessment of Novel Genome-Wide Significant Gene Loci and Lesion Growth in Geographic Atrophy Secondary to Age-Related Macular Degeneration. JAMA Ophthalmol 137, 867-876 (2019). https://doi.org:10.1001/jamaophthalmol.2019.1318

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355. Boekhoorn, S. S., Vingerling, J. R., Witteman, J. C., Hofman, A. & de Jong, P. T. C-reactive protein level and risk of aging macula disorder: The Rotterdam Study. Arch Ophthalmol 125, 1396-1401 (2007). https://doi.org:10.1001/archopht.125.10.1396 356. 356 Kikuchi, M. et al. Elevated C-reactive protein levels in patients with polypoidal choroidal vasculopathy and patients with neovascular age-related macular degeneration. Ophthalmology 114, 1722-1727 (2007). https://doi.org:10.1016/j.ophtha.2006.12.021 357. Mitta, V. P. et al. C-reactive protein and the incidence of macular degeneration: pooled analysis of 5 cohorts. JAMA Ophthalmol 131, 507-513 (2013). https://doi.org:10.1001/jamaophthalmol.2013.2303 358. Greenfield, J. R. et al. Obesity is an important determinant of baseline serum C-reactive protein concentration in monozygotic twins, independent of genetic influences. Circulation 109, 3022-3028 (2004). https://doi.org:10.1161/01.CIR.0000130640.77501.79 359. Seddon, J. M., George, S., Rosner, B. & Rifai, N. Progression of age-related macular degeneration: prospective assessment of C-reactive protein, interleukin 6, and other cardiovascular biomarkers. Arch Ophthalmol 123, 774782 (2005). https://doi.org:10.1001/archopht.123.6.774 360. Seddon, J. M., Gensler, G., Klein, M. L. & Milton, R. C. C-reactive protein and homocysteine are associated with dietary and behavioral risk factors for age-related macular degeneration. Nutrition 22, 441-443 (2006). https://doi.org:10.1016/j.nut.2005.12.004 361. Nielsen, M. K. et al. Complement factor H Y402H polymorphism results in diminishing CD4(+) T cells and increasing C-reactive protein in plasma. Sci Rep 13, 19414 (2023). https://doi.org:10.1038/s41598-023-46827-0 362. McGwin, G., Hall, T. A., Xie, A. & Owsley, C. The relation between C reactive protein and age related macular degeneration in the Cardiovascular Health Study. Br J Ophthalmol 89, 1166-1170 (2005). https://doi.org:10.1136/bjo.2005.067397 363. Dasch, B. et al. Inflammatory markers in age-related maculopathy: cross-sectional analysis from the Muenster Aging and Retina Study. Arch Ophthalmol 123, 1501-1506 (2005). https://doi.org:10.1001/archopht.123.11.1501 364. Boey, P. Y. et al. C-reactive protein and age-related macular degeneration and cataract: the singapore malay eye study. Invest Ophthalmol Vis Sci 51, 1880-1885 (2010). https://doi.org:10.1167/iovs.09-4063 365. Wu, K. H. et al. Circulating inflammatory markers and hemostatic factors in age-related maculopathy: a populationbased case-control study. Invest Ophthalmol Vis Sci 48, 1983-1988 (2007). https://doi.org:10.1167/iovs.06-0223 366. Giannakis, E. et al. A common site within factor H SCR 7 responsible for binding heparin, C-reactive protein and streptococcal M protein. Eur J Immunol 33, 962-969 (2003). https://doi.org:10.1002/eji.200323541 367. Herbert, A. P. et al. Structure shows that a glycosaminoglycan and protein recognition site in factor H is perturbed by age-related macular degeneration-linked single nucleotide polymorphism. J Biol Chem 282, 18960-18968 (2007). https://doi.org:10.1074/jbc.M609636200 368. Sjoberg, A. P. et al. The factor H variant associated with age-related macular degeneration (His-384) and the nondisease-associated form bind differentially to C-reactive protein, fibromodulin, DNA, and necrotic cells. J Biol Chem 282, 10894-10900 (2007). https://doi.org:10.1074/jbc.M610256200 369. Mihlan, M. et al. Monomeric C-reactive protein modulates classic complement activation on necrotic cells. FASEB J 25, 4198-4210 (2011). https://doi.org:10.1096/fj.11-186460 370. Lauer, N. et al. Complement regulation at necrotic cell lesions is impaired by the age-related macular degeneration-associated factor-H His402 risk variant. J Immunol 187, 4374-4383 (2011). https://doi.org:10.4049/jimmunol.1002488 371. Skerka, C. et al. Defective complement control of factor H (Y402H) and FHL-1 in age-related macular degeneration. Mol Immunol 44, 3398-3406 (2007). https://doi.org:10.1016/j.molimm.2007.02.012 372. Laine, M. et al. Y402H polymorphism of complement factor H affects binding affinity to C-reactive protein. J Immunol 178, 3831-3836 (2007). https://doi.org:10.4049/jimmunol.178.6.3831 373. Hakobyan, S. et al. Complement factor H binds to denatured rather than to native pentameric C-reactive protein. J Biol Chem 283, 30451-30460 (2008). https://doi.org:10.1074/jbc.M803648200 374. Ormsby, R. J. et al. Functional and structural implications of the complement factor H Y402H polymorphism associated with age-related macular degeneration. Invest Ophthalmol Vis Sci 49, 1763-1770 (2008). https://doi.org:10.1167/iovs.07-1297

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375. Yu, J. et al. Biochemical analysis of a common human polymorphism associated with age-related macular degeneration. Biochemistry 46, 8451-8461 (2007). https://doi.org:10.1021/bi700459a 376. Penfold, P. L., Provis, J. M., Furby, J. H., Gatenby, P. A. & Billson, F. A. Autoantibodies to retinal astrocytes associated with age-related macular degeneration. Graefes Arch Clin Exp Ophthalmol 228, 270-274 (1990). https://doi.org:10.1007/BF00920033 377. Gurne, D. H., Tso, M. O., Edward, D. P. & Ripps, H. Antiretinal antibodies in serum of patients with age-related macular degeneration. Ophthalmology 98, 602-607 (1991). https://doi.org:10.1016/s0161-6420(91)32252-8 378. Patel, N. et al. Circulating anti-retinal antibodies as immune markers in age-related macular degeneration. Immunology 115, 422-430 (2005). https://doi.org:10.1111/j.1365-2567.2005.02173.x 379. Joachim, S. C., Bruns, K., Lackner, K. J., Pfeiffer, N. & Grus, F. H. Analysis of IgG antibody patterns against retinal antigens and antibodies to alpha-crystallin, GFAP, and alpha-enolase in sera of patients with "wet" age-related macular degeneration. Graefes Arch Clin Exp Ophthalmol 245, 619-626 (2007). https://doi.org:10.1007/s00417006-0429-9 380. Chen, H., Wu, L., Pan, S. & Wu, D. Z. An immunologic study on age-related macular degeneration. Yan Ke Xue Bao 9, 113-120 (1993). 381. Cherepanoff, S., Mitchell, P., Wang, J. J. & Gillies, M. C. Retinal autoantibody profile in early age-related macular degeneration: preliminary findings from the Blue Mountains Eye Study. Clin Exp Ophthalmol 34, 590-595 (2006). https://doi.org:10.1111/j.1442-9071.2006.01281.x 382. Kubicka-Trzaska, A., Wilanska, J., Romanowska-Dixon, B. & Sanak, M. Circulating antiretinal antibodies predict the outcome of anti-VEGF therapy in patients with exudative age-related macular degeneration. Acta Ophthalmol 90, e21-24 (2012). https://doi.org:10.1111/j.1755-3768.2011.02237.x 383. Cao, S. et al. Relationship between systemic cytokines and complement factor H Y402H polymorphism in patients with dry age-related macular degeneration. Am J Ophthalmol 156, 1176-1183 (2013). https://doi.org:10.1016/j.ajo.2013.08.003 384. Falk, M. K. et al. Blood expression levels of chemokine receptor CCR3 and chemokine CCL11 in age-related macular degeneration: a case-control study. BMC Ophthalmol 14, 22 (2014). https://doi.org:10.1186/1471-241514-22 385. Shankar, A., Mitchell, P., Rochtchina, E., Tan, J. & Wang, J. J. Association between circulating white blood cell count and long-term incidence of age-related macular degeneration: the Blue Mountains Eye Study. Am J Epidemiol 165, 375-382 (2007). https://doi.org:10.1093/aje/kwk022 386. Ilhan, N. et al. Assessment of Neutrophil/Lymphocyte Ratio in Patients with Age-related Macular Degeneration. Ocul Immunol Inflamm 23, 287-290 (2015). https://doi.org:10.3109/09273948.2014.921715 387. Sengul, E. A. et al. Correlation of neutrophil/lymphocyte and platelet/lymphocyte ratio with visual acuity and macular thickness in age-related macular degeneration. Int J Ophthalmol 10, 754-759 (2017). https://doi.org:10.18240/ijo.2017.05.16 388. Lechner, J. et al. Alterations in Circulating Immune Cells in Neovascular Age-Related Macular Degeneration. Sci Rep 5, 16754 (2015). https://doi.org:10.1038/srep16754 389. Krogh Nielsen, M. et al. Patients with a fast progression profile in geographic atrophy have increased CD200 expression on circulating monocytes. Clin Exp Ophthalmol 47, 69-78 (2019). https://doi.org:10.1111/ceo.13362 390. Singh, A., Falk, M. K., Hviid, T. V. & Sorensen, T. L. Increased expression of CD200 on circulating CD11b+ monocytes in patients with neovascular age-related macular degeneration. Ophthalmology 120, 1029-1037 (2013). https://doi.org:10.1016/j.ophtha.2012.11.002 391. Singh, A. et al. Systemic frequencies of T helper 1 and T helper 17 cells in patients with age-related macular degeneration: A case-control study. Sci Rep 7, 605 (2017). https://doi.org:10.1038/s41598-017-00741-4 392. Faber, C. et al. Age-related macular degeneration is associated with increased proportion of CD56(+) T cells in peripheral blood. Ophthalmology 120, 2310-2316 (2013). https://doi.org:10.1016/j.ophtha.2013.04.014 393. Christen, W. G., Glynn, R. J., Chew, E. Y. & Buring, J. E. Low-dose aspirin and medical record-confirmed agerelated macular degeneration in a randomized trial of women. Ophthalmology 116, 2386-2392 (2009). https://doi.org:10.1016/j.ophtha.2009.05.031 394. Wang, J. J. et al. Systemic use of anti-inflammatory medications and age-related maculopathy: the Blue Mountains Eye Study. Ophthalmic Epidemiol 10, 37-48 (2003). https://doi.org:10.1076/opep.10.1.37.13776

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395. Zweifel, S. A., Engelbert, M., Khan, S. & Freund, K. B. Retrospective review of the efficacy of topical bromfenac (0.09%) as an adjunctive therapy for patients with neovascular age-related macular degeneration. Retina 29, 15271531 (2009). https://doi.org:10.1097/IAE.0b013e3181b32f4c 396. Adjunctive Diclofenac with Verteporfin Study, G. et al. Effect of adjunctive diclofenac with verteporfin therapy to treat choroidal neovascularization due to age-related macular degeneration: phase II study. Retina 27, 693-700 (2007). https://doi.org:10.1097/IAE.0b013e318030e519 397. Penfold, P. L., Gyory, J. F., Hunyor, A. B. & Billson, F. A. Exudative macular degeneration and intravitreal triamcinolone. A pilot study. Aust N Z J Ophthalmol 23, 293-298 (1995). https://doi.org:10.1111/j.14429071.1995.tb00179.x 398. Jonas, J. B. et al. Intravitreal triamcinolone acetonide for exudative age related macular degeneration. Br J Ophthalmol 87, 462-468 (2003). https://doi.org:10.1136/bjo.87.4.462 399. Nicolo, M., Ghiglione, D., Lai, S. & Calabria, G. Intravitreal triamcinolone in the treatment of serous pigment epithelial detachment and occult choroidal neovascularization secondary to age-related macular degeneration. Eur J Ophthalmol 15, 415-419 (2005). 400. Penfold, P. L., Wong, J. G., Gyory, J. & Billson, F. A. Effects of triamcinolone acetonide on microglial morphology and quantitative expression of MHC-II in exudative age-related macular degeneration. Clin Exp Ophthalmol 29, 188-192 (2001). https://doi.org:10.1046/j.1442-9071.2001.00407.x 401. Danis, R. P., Ciulla, T. A., Pratt, L. M. & Anliker, W. Intravitreal triamcinolone acetonide in exudative age-related macular degeneration. Retina 20, 244-250 (2000). 402. Jonas, J. B., Degenring, R. F., Kreissig, I., Friedemann, T. & Akkoyun, I. Exudative age-related macular degeneration treated by intravitreal triamcinolone acetonide. A prospective comparative nonrandomized study. Eye (Lond) 19, 163-170 (2005). https://doi.org:10.1038/sj.eye.6701438 403. Jonas, J. B., Akkoyun, I., Budde, W. M., Kreissig, I. & Degenring, R. F. Intravitreal reinjection of triamcinolone for exudative age-related macular degeneration. Arch Ophthalmol 122, 218-222 (2004). https://doi.org:10.1001/archopht.122.2.218 404. Gillies, M. C. et al. A randomized clinical trial of a single dose of intravitreal triamcinolone acetonide for neovascular age-related macular degeneration: one-year results. Arch Ophthalmol 121, 667-673 (2003). https://doi.org:10.1001/archopht.121.5.667 405. Lee, J., Freeman, W. R., Azen, S. P., Chung, E. J. & Koh, H. J. Prospective, randomized clinical trial of intravitreal triamcinolone treatment of neovascular age-related macular degeneration: one-year results. Retina 27, 1205-1213 (2007). https://doi.org:10.1097/IAE.0b013e31815ec367 406. Jonas, J. B., Spandau, U. H., Kamppeter, B. A. & Harder, B. Follow-up after intravitreal triamcinolone acetonide for exudative age-related macular degeneration. Eye (Lond) 21, 387-394 (2007). https://doi.org:10.1038/sj.eye.6702222 407. Challa, J. K. et al. Exudative macular degeneration and intravitreal triamcinolone: 18 month follow up. Aust N Z J Ophthalmol 26, 277-281 (1998). https://doi.org:10.1111/j.1442-9071.1998.tb01330.x 408. Taskintuna, I., Elsayed, M. E. & Schatz, P. Update on Clinical Trials in Dry Age-related Macular Degeneration. Middle East Afr J Ophthalmol 23, 13-26 (2016). https://doi.org:10.4103/0974-9233.173134 409. Ruiz-Moreno, J. M., Montero, J. A. & Zarbin, M. A. Photodynamic therapy and high-dose intravitreal triamcinolone to treat exudative age-related macular degeneration: 2-year outcome. Retina 27, 458-461 (2007). https://doi.org:10.1097/IAE.0b013e318030c77c 410. Chaudhary, V., Mao, A., Hooper, P. L. & Sheidow, T. G. Triamcinolone acetonide as adjunctive treatment to verteporfin in neovascular age-related macular degeneration: a prospective randomized trial. Ophthalmology 114, 2183-2189 (2007). https://doi.org:10.1016/j.ophtha.2007.02.013 411. Chan, A. et al. Photodynamic therapy with and without adjunctive intravitreal triamcinolone acetonide: a retrospective comparative study. Ophthalmic Surg Lasers Imaging 40, 561-569 (2009). https://doi.org:10.3928/15428877-20091030-05 412. Neovascular Age-Related Macular Degeneration, P. C. et al. Periocular triamcinolone and photodynamic therapy for subfoveal choroidal neovascularization in age-related macular degeneration. Ophthalmology 114, 1713-1721 (2007). https://doi.org:10.1016/j.ophtha.2007.03.071 413. Jonas, J. B. et al. Intravitreal bevacizumab versus triamcinolone acetonide for exudative age-related macular degeneration. Ophthalmic Res 41, 21-27 (2009). https://doi.org:10.1159/000162113

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414. Kuppermann, B. D. et al. Dexamethasone Intravitreal Implant as Adjunctive Therapy to Ranibizumab in Neovascular Age-Related Macular Degeneration: A Multicenter Randomized Controlled Trial. Ophthalmologica 234, 40-54 (2015). https://doi.org:10.1159/000381865 415. Ahmadieh, H. et al. Intravitreal bevacizumab versus combined intravitreal bevacizumab and triamcinolone for neovascular age-related macular degeneration: six-month results of a randomized clinical trial. Retina 31, 18191826 (2011). https://doi.org:10.1097/IAE.0b013e31820d58f2 416. Rezar-Dreindl, S. et al. The Intraocular Cytokine Profile and Therapeutic Response in Persistent Neovascular AgeRelated Macular Degeneration. Invest Ophthalmol Vis Sci 57, 4144-4150 (2016). https://doi.org:10.1167/iovs.1619772 417. Markomichelakis, N. N., Theodossiadis, P. G. & Sfikakis, P. P. Regression of neovascular age-related macular degeneration following infliximab therapy. Am J Ophthalmol 139, 537-540 (2005). https://doi.org:10.1016/j.ajo.2004.09.058 418. Theodossiadis, P. G., Liarakos, V. S., Sfikakis, P. P., Vergados, I. A. & Theodossiadis, G. P. Intravitreal administration of the anti-tumor necrosis factor agent infliximab for neovascular age-related macular degeneration. Am J Ophthalmol 147, 825-830, 830 e821 (2009). https://doi.org:10.1016/j.ajo.2008.12.004 419. Giganti, M. et al. Adverse events after intravitreal infliximab (Remicade). Retina 30, 71-80 (2010). https://doi.org:10.1097/IAE.0b013e3181bcef3b 420. Arias, L. et al. Intravitreal infliximab in patients with macular degeneration who are nonresponders to antivascular endothelial growth factor therapy. Retina 30, 1601-1608 (2010). https://doi.org:10.1097/IAE.0b013e3181e9f942 421. Wu, L. et al. Intravitreal tumor necrosis factor-alpha inhibitors for neovascular age-related macular degeneration suboptimally responsive to antivascular endothelial growth factor agents: a pilot study from the Pan American Collaborative Retina Study Group. J Ocul Pharmacol Ther 29, 366-371 (2013). https://doi.org:10.1089/jop.2012.0203 422. Volz, C. & Pauly, D. Antibody therapies and their challenges in the treatment of age-related macular degeneration. Eur J Pharm Biopharm 95, 158-172 (2015). https://doi.org:10.1016/j.ejpb.2015.02.020 423. Kurup, S. K., Gee, C. & Greven, C. M. Intravitreal methotrexate in therapeutically resistant exudative age-related macular degeneration. Acta Ophthalmol 88, e145-146 (2010). https://doi.org:10.1111/j.1755-3768.2009.01560.x 424. Nussenblatt, R. B. et al. A randomized pilot study of systemic immunosuppression in the treatment of age-related macular degeneration with choroidal neovascularization. Retina 30, 1579-1587 (2010). https://doi.org:10.1097/IAE.0b013e3181e7978e 425. Minturn, R. J. et al. Intravitreal sirolimus for persistent, exudative age-related macular degeneration: a Pilot Study. Int J Retina Vitreous 7, 11 (2021). https://doi.org:10.1186/s40942-021-00281-0 426. Wong, W. T. et al. Treatment of geographic atrophy with subconjunctival sirolimus: results of a phase I/II clinical trial. Invest Ophthalmol Vis Sci 54, 2941-2950 (2013). https://doi.org:10.1167/iovs.13-11650 427. Petrou, P. A. et al. Intravitreal Sirolimus for the Treatment of Bilateral Geographic Atrophy Associated with AgeRelated Macular Degeneration: Results of a Phase I/II Trial. Investigative Ophthalmology & Visual Science 55, 5892-5892 (2014). 428. Gensler, G. et al. Treatment of Geographic Atrophy with Intravitreal Sirolimus: The Age-Related Eye Disease Study 2 Ancillary Study. Ophthalmol Retina 2, 441-450 (2018). https://doi.org:10.1016/j.oret.2017.08.015 429. Rosenfeld, P. J. et al. A Randomized Phase 2 Study of an Anti-Amyloid beta Monoclonal Antibody in Geographic Atrophy Secondary to Age-Related Macular Degeneration. Ophthalmol Retina 2, 1028-1040 (2018). https://doi.org:10.1016/j.oret.2018.03.001 430. Holz, F. G. et al. Efficacy and Safety of Lampalizumab for Geographic Atrophy Due to Age-Related Macular Degeneration: Chroma and Spectri Phase 3 Randomized Clinical Trials. JAMA Ophthalmol 136, 666-677 (2018). https://doi.org:10.1001/jamaophthalmol.2018.1544 431. Liao, D. S. et al. Complement C3 Inhibitor Pegcetacoplan for Geographic Atrophy Secondary to Age-Related Macular Degeneration: A Randomized Phase 2 Trial. Ophthalmology 127, 186-195 (2020). https://doi.org:10.1016/j.ophtha.2019.07.011 432. Nittala, M. G. et al. Association of Pegcetacoplan With Progression of Incomplete Retinal Pigment Epithelium and Outer Retinal Atrophy in Age-Related Macular Degeneration: A Post Hoc Analysis of the FILLY Randomized Clinical Trial. JAMA Ophthalmol 140, 243-249 (2022). https://doi.org:10.1001/jamaophthalmol.2021.6067

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433. Pfau, M. et al. Association of complement C3 inhibitor pegcetacoplan with reduced photoreceptor degeneration beyond areas of geographic atrophy. Sci Rep 12, 17870 (2022). https://doi.org:10.1038/s41598-022-22404-9 434. Heier, J. S. et al. Pegcetacoplan for the treatment of geographic atrophy secondary to age-related macular degeneration (OAKS and DERBY): two multicentre, randomised, double-masked, sham-controlled, phase 3 trials. Lancet 402, 1434-1448 (2023). https://doi.org:10.1016/S0140-6736(23)01520-9 435. Langer, H. F. et al. Complement-mediated inhibition of neovascularization reveals a point of convergence between innate immunity and angiogenesis. Blood 116, 4395-4403 (2010). https://doi.org:10.1182/blood-2010-01-261503 436. Shughoury, A., Sevgi, D. D. & Ciulla, T. A. The complement system: a novel therapeutic target for age-related macular degeneration. Expert Opin Pharmacother 24, 1887-1899 (2023). https://doi.org:10.1080/14656566.2023.2257604 437. Yehoshua, Z. et al. Systemic complement inhibition with eculizumab for geographic atrophy in age-related macular degeneration: the COMPLETE study. Ophthalmology 121, 693-701 (2014). https://doi.org:10.1016/j.ophtha.2013.09.044 438. Yu, H. J. & Wykoff, C. C. Investigational Agents in Development for the Treatment of Geographic Atrophy Secondary to Age-Related Macular Degeneration. BioDrugs 35, 303-323 (2021). https://doi.org:10.1007/s40259021-00481-y 439. Pfau, M. Re: Jaffe et al.: C5 inhibitor avacincaptad pegol for geographic atrophy due to age-related macular degeneration (Ophthalmology. 2021;128:576-586). Ophthalmology 128, e219 (2021). https://doi.org:10.1016/j.ophtha.2021.08.018 440. Kassa, E., Ciulla, T. A., Hussain, R. M. & Dugel, P. U. Complement inhibition as a therapeutic strategy in retinal disorders. Expert Opin Biol Ther 19, 335-342 (2019). https://doi.org:10.1080/14712598.2019.1575358 441. Patel, P. N. et al. Targeting the Complement Cascade for Treatment of Dry Age-Related Macular Degeneration. Biomedicines 10 (2022). https://doi.org:10.3390/biomedicines10081884 442. Keenan, T. D. L. et al. A Phase II Trial Evaluating Oral Minocycline in the Treatment of Geographic Atrophy in AgeRelated Macular Degeneration. Investigative Ophthalmology & Visual Science 64, 5058-5058 (2023). 443. West, E. E. & Kemper, C. Complosome - the intracellular complement system. Nat Rev Nephrol 19, 426-439 (2023). https://doi.org:10.1038/s41581-023-00704-1

Kozek, Lindsay

The Immunopathogenesis of Adamantiades-Behçet’s Uveitis: Advances in Understanding of Genetics and Molecular Mechanisms Lindsay Klofas Kozek, MD, PhD and Demetrios Vavvas, MD, PhD

Abstract Adamantiades-Behçet’s disease (ABD) is a chronic inflammatory disorder manifesting as a systemic vasculitis, characterized by recurrent oral and genital ulcers, skin lesions, and uveitis. Much has been elucidated regarding the immunopathogenesis since the initial identification of a strong genetic susceptibility related to HLA-B51 variants. Current understanding of the disease involves a complex interplay between defective antigen processing, environmental triggers, and an altered balance between different components of the immune system. This review aims to comprehensively synthesize the immunogenetics and immunopathology of ABD, particularly concerning ocular involvement, providing a current understanding of the molecular mechanisms underlying the disease.

Introduction Adamantiades-Behçet’s disease (ABD) is a chronic inflammatory disorder affecting multiple organ systems. ABD manifests as a systemic vasculitis classically characterized by recurrent oral and genital ulcers, skin lesions, and uveitis, along with numerous other systemic issues. The underlying pathogenesis is believed to arise from a dysregulated autoimmune/autoinflammatory response, occurring in genetically predisposed individuals and potentially influenced or triggered by environmental factors. Although ABD has its highest prevalence along the historic Silk Road, spanning East Asia to the Mediterranean Basin, it can be found globally. Typically surfacing in the third or fourth decade of life, diagnosis relies on clinical evaluation, given that specific biomarkers have not yet been identified. Nevertheless, contemporary advances, such as cost-effective genetic sequencing, allow for the identification of genetic predisposition, with HLA-B51 most strongly linked. Ocular involvement is a prominent facet of ABD, occurring in as many as 70% of patients, and may be the first manifestation of disease in up to 20%.1 The most common ocular manifestation is bilateral uveitis; however, a wide range of manifestations has been reported, spanning the entirety of the eye. Uveitis in ABD most commonly presents as a relapsing-remitting, non-granulomatous posterior or panuveitis, often associated with an occlusive retinal vasculitis. Despite improved prognoses with new biologic treatments, vision loss remains a significant source of morbidity in ABD. This review aims to comprehensively elucidate the immunogenetics and immunopathology of ABD, particularly concerning ocular involvement, providing a synthesized understanding of the complexities surrounding this disease.

Historical Underpinnings The disease was originally described in 1930 during the annual meeting of the Medical Association of Athens by Dr. Benediktos Adamantiades, a Greek ophthalmologist.2 The history of early descriptions of ABD, including reports as far back as Hippokrates, is nicely summarized by Zouboulis & Keitel. Adamantiades described a patient with recurrent uveitis in both eyes, oral and genital ulcers, and sterile arthritis of the knees, connecting the constellation of symptoms as a single disease, and eventually proposing the first diagnostic criteria. Hulsi Behçet, a Turkish dermatologist, described a series of similar cases, the first of which in 1937, which he termed “triple symptom complex,” referring to the triad of recurrent oral aphthous ulcers, genital ulcers, and eye involvement, and referencing Adamantiades’s work in his manuscripts.

2024 Harvard Ophthalmology Residents’ Course The term Behçet’s syndrome was first coined by T. Jensen, a dermatologist from Denmark who established the pathergy test, and was further popularized in 1946 by Helen Ollendorff-Curth, a GermanAmerican dermatologist. While Ollendorff-Curth referenced Adamantiades’s work, it seems likely that she did not have detailed knowledge of his findings due to their initial publication occurring in the French language. We use the term Adamantiades-Behçet’s disease (ABD) in our manuscript to honor both pioneering clinicians, and acknowledge some groups find this controversial.3–5

Immunogenetics Major Histocompatibility Class (MHC) and HLA-B51 The association of ABD with HLA-B51 was first reported by a Japanese group in 1973.6 It has since been confirmed by numerous studies in multiple different populations, including two separate genome-wide association studies (GWAS).7,8 Interestingly, there is an increased prevalence of HLA-B51 in Silk Road nations compared to the United States and Northern Europe, explaining at least some of the location variability.9,10 In a meta-analysis of various ethnic groups, the overall odds-ratio for ABD susceptibility associated with HLA-B51 was 5.78.11 Moreover, HLA-B51 may have a prognostic value, correlating with a higher degree of ocular involvement.12–14 HLA-B51 is a member of the MHC class I family of molecules. MHC class I molecules present peptide fragments, such as those derived from intracellular viral and bacterial pathogens, to cytotoxic T lymphocytes, also known as CD8+ T cells. These lymphocytes bind peptide-MHC-I complexes via their T cell receptors (TCRs). In humans, the human leukocyte antigens (HLA) corresponding to the MHC class I include HLA-A, HLA-B, and HLA-C. Conversely, MHC class II molecules (consisting of HLAs DP, DM, DO, DQ, and DR) present exogenous antigens to T cells. The presentation of intracellular peptides by MHC class I molecules allows cytotoxic T cells to survey the interior of host cells for signs of infection or other abnormalities, stimulating either immune activation or tolerance induction. For as-of-yet unknown reasons, specific dysfunctional presentation or creation of these MHC I shapes drives the immune system towards ABD. Despite this association, -B51 alone contributes only 19% of the overall genetic susceptibility to ABD, suggesting that other genetic factors are also at play.15 Several other HLA genes show disease susceptibility or protection in different populations, however none are as strongly linked as HLA-B51.16,17 Patients who were HLA-A26 carriers in particular had higher risk for ocular involvement, including chorioretinitis.17 A recent paradigm shift in understanding Adamantiades-Behçet’s disease places it in a category of inflammatory conditions coined “MHC-I-opathies.” This designation denotes a cluster of conditions characterized by shared clinical features and genetic connections to the MHC-I antigen presentation pathway.18 In addition to ABD, the uveitides birdshot uveitis and HLA-B27-associated anterior uveitis are also considered to be MHC-I-opathies. Psoriasis, psoriatic arthritis, and spondyloarthritis round out the group of classical diseases. For unclear reasons, uveitis is a feature of all MHC-I-opathies to varying degrees, ranging from a defining feature of the disease to a less common manifestation. HLA-B51 drives disease via specific amino acid residues that alter the MHC I complex interactions. Several of the polymorphisms consistently associated with ABD occur in amino acid residues that lie in the antigen-binding groove and are critical to defining peptide specificity of HLA-B51.19 One of these amino acids, residue 67, is one of the only two amino acids that differ between HLA-B51 and HLA-B52, which confers no additional risk for ABD.20 Another GWAS further advanced understanding of ABD by identifying epistasis between HLA-B51 and endoplasmic reticulum (ER)-associated aminopeptidase (ERAP) 1, another member of the MHC class I antigen presentation pathway.21 The product of the ERAP1 gene is a peptidase that serves as the final editor of antigenic peptides.22 Intracellular antigens are thought to be initially processed by proteasomes, then translocated into the ER by the transporter of antigen presentation (TAP) peptide transporter.

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ERAP1 and its relative ERAP2 trim these processed antigenic peptides to prepare them for loading onto MHC-I molecules. A single nucleotide polymorphism (SNP) in ERAP1 (p.Arg725Gln) was found to convey risk not only for ABD in individuals with HLA-B51, but also independently for ABD uveitis in these carriers, in a recessive fashion.21 A separate group identified a specific haplotype of ERAP1, ERAP1-Hap10, that contained the p.Arg725Gln allele and conveyed increased odds of developing ABD only in the presence of HLA-B51.23 For individuals who were homozygous for ERAP1-Hap10, Takeuchi et al., found a 10-fold increased odds of ABD in carriers of HLA-B51, but no increase in disease odds in the absence of HLA-B51. While this is an impressive effect size, ERAP1-Hap10 occurred at a relatively low overall frequency in the Turkish population that was studied. Despite this, important mechanistic information can be gleaned from this study by examining the effects of the ERAP1-Hap10 haplotype on overall function of the MHC pathway. The interdependence of HLA-B51 and ERAP1 in selecting and presenting peptides to the immune system for local immune activation decisions likely has mechanistic implications. The full library of peptides that an MHC molecule can bind is referred to as the immunopeptidome.24 ERAP1 variants may alter the peptidome either by diminishing the production of disease-protective peptides or by failing to select out disease-promoting peptides. When expressed in an HLA-B51:01 background, the ABD-ERAP1 variant altered the balance of the two HLA-B51 subpeptidomes.25 Experimentally mimicking ERAP1-Hap10 by silencing or knockout of ERAP1 increases the binding of unconventional peptides by HLA-B51:01, specifically resulting in binding of longer-than-usual peptides consisting of nine amino acids or longer, rather than classic octamers.26,27 Many of these longer peptides exhibited sequence homology with microbial epitopes, possibly causing normal MHC I presentations to mimic those of infected cells and stimulate immune overactivation. In addition to altering the peptidome, the level of ERAP1 expression has been shown to alter cell surface expression of MHC class I molecules.28 Another group also found that experimental silencing of ERAP1 alters cell-type specific surface expression of HLA-B51, potentially explaining why certain tissues and not others are affected in ABD.27 Finally, individuals with ABD who carried HLA-B51 and ERAP1-Hap10 had increased proportions of activated CD8+ T cells in peripheral blood and specifically had an increased population of an oligocloncal T cell population known to have undergone repetitive cycles of antigen stimulation.26 This illustrates how such individuals may an altered immune system overall, and the organs that first succumb to this underlying milieu unfortunately include the eye. While the mechanistic underpinnings of alterations in the antigen presentation pathway caused by the HLA-B51/ERAP1 interaction begin to illustrate a fascinating immune-driven story, these genetic variations only offer a partial explanation for ABD. Indeed, a 2021 study assessed the percentage of variance that could be explained by known genetic susceptibility loci, identified by GWAS with a high level of predetermined significance.29 21 loci, including HLA genes, ERAP1, and various interleukins, were included but were found to only explain about 60% of the genetic contribution to Behçet’s disease. This implies that the remaining 40% of heritability has yet to be uncovered. Possible contributions to the remainder of the heritability will be discussed below.

Outside the HLA-Box Polymorphisms in multiple other immune regulatory genes, including interleukin (IL)-10 (IL-10), IL12A, IL21 receptor (IL-21R), IL-23 receptor (IL-23R), C-C motif chemokine receptor 1 (CCR1), CCR3, and others have been associated with ABD and are thoroughly reviewed by multiple other sources, including by Deng et al.30,31 Serum vitamin D plays a role in multiple distinct cellular processes, including immune regulation.32 Multiple groups have evaluated the impact of serum vitamin D levels in the susceptibility, pathogenesis, and clinical activity of ABD, the results of which were summarized in a recent review by Melikoglu et al.33 Variants in components of the vitamin D axis have been identified as enriched in patients with ABD. For example, in a case-control study in a Turkish population, Dal et al. found that polymorphisms in the

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vitamin D receptor (VDR) were enhanced in patients with ABD, with one polymorphism specifically enriched in patients with ocular involvement.34 A SNP in another vitamin D pathway member, 7dehydrocholesterol reductase (DHCR7), which regulates vitamin D production in response to UV light, was found to have a higher frequency in ocular ABD patients in a Chinese population.35 A recent GWAS of 436 Turkish patients with ABD identified a significant association between ocular involvement and SLCO4A1, a member of the organic anion transporting polypeptide (OATP) family previously implicated in inflammation responses.12,36 Interestingly, SLCO4A1 was found to be downregulated in a transcriptomic analysis of human retinal detachment and expression levels were correlated with photoreceptor death.37 Other novel loci recently identified include a locus within IFNGR1, a subunit of the interferon gamma receptor, and a locus within the intergenic region between LNCAROD/DKK1, both of which antagonize Wnt signaling.38 While these other genetic loci may contribute to the progression or initiation of ABD, none of these additional mechanisms have been shown to have odds ratios as high as those in the HLA-B51 studies above. However, it is possible to develop ABD without the HLA-B51 variant, suggesting that disease may be based on the accumulation of multiple genetic susceptibilities.

Role of Environmental Triggers One theory of ABD pathogenesis is that specific peptides may trigger an autoimmune and/or autoinflammatory response, the “arthritogenic peptide theory.” This hypothesis has been proposed for another MHC-I-opathy, spondyloarthritis, as a possible explanation for its association with HLA-B27.39 This hypothesis is based on the concept of molecular mimicry, namely that activation of T cell responses by an extrinsic antigen may spur the development of autoimmunity. In ABD, certain infections have been implicated as potential triggers, particularly bacteria including Streptococcus, Helicobacter pylori, and Mycolasma, and viral infections including herpes simplex virus 1 (HSV-1), cytomegalovirus (CMV), Epstein-Barr virus (EBV), hepatitis, parvovirus B19, and varicella zoster virus. 40 Because of the oral aphthous and genital ulcers, members of the Herpesviridae have been the subject of considerable interest and suspicion.41 While HSV-1 DNA has been detected in peripheral immune cells of ABD patients, this is confounded by the relatively global prevalence of anti-HSV immunity.42–45 Moreover, a randomized clinical trial was conducted to investigate whether acyclovir, an antiviral medication commonly employed in the treatment of HSV, could ameliorate orogenital ulcers in ABD patients.46 Despite the promising hypothesis, the trial did not demonstrate any significant therapeutic effect. These data, combined with the high global prevalence of HSV yet low prevalence of ABD, argue against a significant role for HSV in ABD pathogenesis. Differences in oral hygiene and the oral microbiome as well as certain dental procedures have been suggested as potential triggers for ABD.47,48 Because Streptococcus species are heavily represented in the oral microbiome, they are among the first and best studied microbes.40 In particular, ABD patients have been shown to have higher antibody titers for Streptococcus sanguinis.49 DNA from S. sanguniis has been identified in macrophages isolated from skin lesions in patients with ABD.50 Mice inoculated orally with S. sanguinis, either alone or after mechanical or thermal disruption of the oral mucosa, developed oral inflammation, but also surprisingly developed mild anterior segment inflammation consistent with iridocyclitis.51 Topical and systemic antibiotics have been investigated for treatment of oral lesions in ABD patients with some success, though the mechanism of action is not clear; however, unlike in H. pylori infection, there is not data to support systemic antibiotic treatment for eradication of specific microbiome species.52 In addition to the role of microbes, there are several potential autoantigens that have been described in ABD: heat shock protein 60 (HSP60), retinal S antigen, and interphotoreceptor retinoid-binding protein (IRBP). S. sanguinis contains DNA, specifically the bes-1 gene, that has high homology with HSP60 as well as Brn3b, a transcription factor expressed by retinal ganglion cells.50,53,54

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However, the possible contribution of HSPs to the pathogenesis of ABD was recently reviewed by Aydin & Hatemi, who concluded that definitive evidence for a causative role or contributory role of HSPs in ABD is lacking. 55 Two eye-specific antigens, retinal S antigen (S-Ag) and interphotoreceptor retinoid-binding protein (IRBP), have been investigated for their potential roles in triggering ABD. S-Ag, also known as arrestin, binds to rhodopsin in retinal rod outer segments after rhodopsin has been phosphorylated by photoactivation.56 S-Ag has a well-established role in autoimmune uveitis, both in experimental autoimmune uveitis (EAU) in animal models as well as autoimmune retinopathies in humans, such as cancer-associated retinopathy (CAR) and autoimmune retinopathy (AIR).57–60 S-Ag is included on some CAR and AIR immunoblot panels for clinical diagnosis. In a small series of 8 patients with ABD, the responses of Th1 cells to S-Ag peptides were highly variable depending on the individual as well as the duration of uveitis.61 A larger study by the same group found the existence of S-Ag-specific T cells, mostly activated memory CD4+ T cells, in just over half of patients with active ABD as well as in some patients with inactive ABD.62 IRBP is secreted by photoreceptors and facilitates the transfer of retinoids between the retina and retinal pigment epithelium (RPE) during phototransduction (reviewed by Zeng et al.).63 IRBP consists of four repeating units, the fourth of which is highly uveitogenic. IRBP also has a wellknown role in EAU models and is less commonly associated with CAR.58,64 In an early study, lymphocytes isolated from ABD patients with active uveitis tended to have greater proliferative responses to stimulation with S-Ag and/or IRBP than those from patients without uveitis.65 A more recent study evaluated immune responses of peripheral blood mononuclear cells (PBMCs) of ABD patients with or without uveitis versus healthy controls when stimulated with IRBP or S-antigen.66 IL-6, IFN-γ, and IL-17 production in response to IRBP stimulation was higher in ABD patients than in healthy controls, and IFN-γ production was higher in ABD patients with active uveitis than in ABD patients with uveitis in remission. IRBP stimulated higher levels of IL-17 and IFN-γ production than S-Ag, while S-Ag stimulated comparatively higher IL-6 production. Several patients in the former study had responses to both S-Ag and IRBP, suggesting that autoimmunity to retinal antigens may occur over the course of the disease once the blood-retina barrier has been breached, rather than as the inciting event of the disease.65 Additionally, in the study by Takeuchi et al., immune responses to IRBP and S-antigen were observed in PMBCs from healthy controls, underscoring the importance of the immune privilege of the eye.66 To conclude the discussion on environmental triggers, Behçet's disease likely arises from a synergistic interplay between genetic predisposition and environmental triggers. While no single infectious agent has been identified as causative, there is a consensus that environmental factors, whether infectious or integral to the microbiome, contribute significantly to the development of immune dysfunction. Further research is needed to fully understand the complex interplay between environment and genetic susceptibility in the development and progression of the disease.

Role of Specific Immune Cell Populations ABD occurs due to an uncontrolled autoinflammatory response. As such, it may be better described as a predominantly autoinflammatory disease than an autoimmune disease, though it has components of both.67 Autoinflammatory diseases have their origins within malfunctioning of the innate immune system, whereas autoimmune diseases originate from the adaptive immune system. The following section will address the roles of the various components of the innate and adaptive immune systems in the immunopathogenesis of ABD.

Innate Immune Cells Neutrophil Hyperactivation May Play a Role in the Pathogenesis of ABD The primary function of neutrophils is to engulf and neutralize invading pathogens through phagocytosis and release of antimicrobial substances as well as reactive oxygen species. Additionally, neutrophils can trap and kill bacteria through the formation of neutrophil extracellular traps (NETs), a function of neutrophils first identified in 2004.68 74 NETs are web-like structures composed of DNA, histones, and antimicrobial proteins that act as traps to ensnare and immobilize pathogens. However, an imbalance in

2024 Harvard Ophthalmology Residents’ Course NET formation (termed “NETosis”) and NET degradation has been implicated in various autoimmune and inflammatory disorders, including ABD.69,70 Circulating neutrophils isolated from the sera of ABD patients were found to release significantly higher amounts of NETs and have associated increased expression of enzymes involved in NETosis.71–74 Moreover, when neutrophils from healthy individuals were stimulated with serum from patients with active ABD, NET release was significantly increased.73 NETs isolated from ABD patients stimulated cultured macrophages to produce more proinflammatory cytokines than NETs from healthy controls, specifically increasing increased expression of proinflammatory markers IL-6, TNFa, and COX2.72,74 ABD NETs were also found to promote Th1 cell differentiation, increase the frequency of Th1/Th17 cells, and promote secretion of the Th17-related cytokines IL-17 and IFN-γ.72,74 Finally, ABD NETs contained more oxidized DNA than healthy controls, likely due to increased production of reactive oxygen species (ROS) by ABD neutrophils.72 The mechanisms underlying altered NET formation and composition in ABD remain largely undefined. Shu et al. described a role for IL-8 in inducing NET formation via NADPH oxidase-mediated ROS production and the MAPK pathway, specifically in ABD patients with active ocular disease.74 IL-8 levels have been shown to be correlated with disease activity and clinical manifestations of ABD, including eye involvement.75,76 IL-8 levels have even been shown to better correlate with disease activity than the classical inflammatory laboratory tests, the erythrocyte sedimentation rate (ESR) and C-reactive protein (CRP) values.77 IL-8, also known as neutrophil activating factor, upregulates neutrophil chemotaxis. IL-8 has been proposed to be secreted at least in part by small vessel endothelial cells, with supporting experimental evidence in cultured human endothelial cells.78 Indeed, high serum levels of IL-8 were found in ABD patients with vascular involvement, including deep venous thrombosis and superficial thrombophlebitis.75 ABD patients exist in a prothrombotic state, the origin of which is not well-defined, but may be related to increased levels of procoagulants or abnormalities of vascular endothelial cells.79,80 NETs contain factors that make them procoagulants, and evidence is mounting for their role in pathological thrombus formation in humans.81 Infiltrating NETs have been histologically identified in areas of vasculitis and microthrombi in tissues from patients with ABD.71 77 NETs isolated from ABD patients triggered death of cultured endothelial cells, suggesting a fundamental role of NETs in endothelial damage.73 NETs from ABD patients also resulted in a higher peak thrombin concentration during coagulation assays.71 In summary, NETs in ABD may alter the delicate balance of immune regulation to promote inflammation and likely play a role in vascular damage and thrombosis. NETs may contribute to disruption of the bloodbrain barrier and, by extrapolation, could plausibly disrupt the blood-retina barrier as well.82 In a mouse model of uveitis, intravitreal injection of an IL-8 receptor (CXCR2) antagonist, SB225002, suppressed NET formation and resulted in decreased phosphorylation of ERK1/2 and p38, indicating suppression of MAPK activity.74 This suggests that IL-8 induction of NET formation is controlled at least partially via the MAPK pathway, implying new therapeutic possibilities through modulation of IL-8 or the MAPK pathway. Clinical development is occurring on this arm of immunology; an anti-IL-8 monoclonal antibody was recently shown to be safe and well-tolerated in a Phase I trial in patients with solid tumors.83 While aberrant NET formation may not be the initiating event in the development of ABD, it is possible that NET modulation may provide therapeutic benefit. Finally, because of the role neutrophil hyperactivity is thought to play in the pathogenesis of ABD, the neutrophil-to-lymphocyte ratio (NLR) has been evaluated as an indicator of disease activity, and it has even been proposed as a diagnostic criterion.84–89 While one study did not find an association with ocular activity, a larger study found a significant correlation with a higher maximum NLR and worse visual outcomes.86,87 Dysregulation of Natural Killer Cells Natural killer (NK) cells’ primary function is to detect and eliminate abnormal cells without prior sensitization or activation. NK cells patrol the body and when they encounter cells that deviate from normal, they can directly induce apoptosis in the abnormal cell by release of cytotoxic granules. NK cells

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also modulate immune responses by interacting with other immune cells, such as dendritic cells, T cells, and macrophages. The activity of NK cells is tightly regulated to prevent attacks on healthy cells. They achieve this balance by integrating signals from activating and inhibitory receptors, which recognize specific molecules on the surface of target cells. The specific role of NK cells in ABD has yet to be precisely determined, however many potential roles have been posited.90 While the importance of HLA-B51 in ABD suggests a main role for CD8+ T cell dysregulation, MHC-I molecules also have non-classical interactions with NK cells. The integration of activating and inhibitory signals occurs via NK receptors such as killer inhibitory receptors (KIRs). KIR function is dependent on the peptide bound to the MHC class I molecule.91 Peptidomes with reduced affinity, such as those related to the pathogenic ERAP1 variant, may fail to generate an KIR-mediated inhibitory response to a self-peptide presented by an MHC. This might amplify the susceptibility to lysis by natural killer (NK) cells and stimulate disease.92 Patients with ocular ABD had abnormal KIR expression as well as increased numbers of NK cells in aqueous humor compared to other uveitis syndromes.93,94 Additionally, through their immunomodulatory role, NK cells may disrupt T cell homeostasis in ABD. NK cells can be divided into two functional subsets, NK1 and NK2 cells. NK1 cells mainly produce IFN-γ while NK2 cells produce IL-5 and IL-13.95 The balance of NK1/NK2 can modify T cell responses directly by cytokine expression profiles or indirectly by modulation of antigen-presenting cells.96 Cytokine secretions of NK cells from patients with ABD uveitis were measured during periods of disease relapse and remission. During the relapse period, NK cells had an NK1 profile with increased secretion of Th1 type cytokines: TNF-α, IFN-γ and IL-2, whereas during remission, secretion of the Th2 cytokine IL-4 was increased.97 These results reinforced findings from an earlier study that also demonstrated that the balance of NK cells plays a role in relapse versus remission by modulating a Th1 response.98 Imbalance of Monocytes and Macrophages Monocytes, another component of the innate immune system, are precursors to macrophages and dendritic cells. In response to infectious stimuli, they are recruited to the site of microbial invasion, where they can differentiate into tissue-specific macrophages. There is an altered balance of subpopulations of monocytes in patients with ABD compared to healthy controls.99,100 In a single cell analysis of PBMCs from ABD patients, a specific population identified as C1qhi monocytes were found to enhance secretion of proinflammatory cytokines, such as IL-6 and TNFa, possibly in an IFN-γ-dependent manner.101 Macrophages have numerous essential immune functions, including phagocytosis, antigen presentation, and release of cytokines and growth factors that regulate immune responses and aid in tissue repair and remodeling.102 The ability of macrophages to switch between different functional states in response to environmental cues is known as macrophage polarization. M1 macrophages are generally proinflammatory and involved in host defense, while M2 macrophages are associated with anti-inflammatory responses and tissue repair.103 Treating control macrophages with serum isolated from ABD patients induced a pro-inflammatory M1-like polarization state that was dependent on NF-κB signaling.104,105 The predominance of M1 macrophages in ABD has been mechanistically linked to GWAS-identified polymorphisms in C-C chemokine receptor 1 (CCR1) and IL-10.103,106 Overall, while it seems unlikely that monocytes and macrophages are the driving force behind the immune dysregulation in ABD, the altered balance of subpopulations may play a role in disease perpetuation.

Adaptive Immune Cells T lymphocytes play a central role in adaptive immunity. T cells are characterized by the presence of T cell receptors (TCRs) on their surfaces, which enable them to identify antigens presented by other cells. There are two broad categories of T cells: helper T cells (CD4+), which coordinate immune responses by activating other immune cells and facilitating antibody production, and cytotoxic T cells (CD8+), which directly target and destroy infected or abnormal cells. The ability of T cells to distinguish between self and non-self and their capacity to generate diverse responses make them indispensable in maintaining immune surveillance and ensuring the body's defense against various pathogens. This system can become dysregulated in two general ways: erroneous antigen presentation by MHC molecules such as HLA-B51, or malfunctioning of specific T cells which no longer behave as programmed. In ABD, the clear role of HLA-B51 suggests that antigen presentation goes awry on the side of the presenting cell. Aberrant

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T cells would then identify these erroneously presented self-antigens as pathogenic, inducing immune attack of cells containing these self-antigens. Helper T Cells CD4 cells encompass several different subtypes, including Th1, Th2, Th17, Th22, Treg, and Tfh cells.107 Th1 cells are involved in cellular immunity and defense against intracellular pathogens, such as viruses and certain bacteria. They produce cytokines like interferon-gamma (IFN-γ) and tumor necrosis factorbeta (TNF-β) and activate cytotoxic T cells and macrophages. Th2 cells are associated with humoral immunity and defense against extracellular parasites and allergens. They produce cytokines such as interleukin-4 (IL-4), interleukin-5 (IL-5), and interleukin-13 (IL-13), which stimulate B cells to produce antibodies and activate eosinophils. Th17 cells play a role in inflammatory responses and defense against extracellular bacteria and fungi. They produce cytokines like interleukin-17 (IL-17) and interleukin-22, contributing to the recruitment of neutrophils and the maintenance of mucosal barriers. Treg cells are crucial for immune tolerance and the prevention of excessive immune responses. They suppress the activity of other immune cells, preventing autoimmune reactions and maintaining immune homeostasis. Disruption of T cell homeostasis is thought to play a key role in the pathogenesis of ABD, with expansion of Th1 and Th17 populations and a decline in Tregs.108 In the initial stages of EAU, autoantigen-specific Th1 cells are thought to initiate disease, followed by autoantigen-specific Th17 cells that either join or replace the Th1 cells later in the course of the disease.109–111 Analysis of peripheral lymphocytes in ABD patients with and without uveitis found the percentage of Th1 cells was significantly higher in patients with active uveitis than in those with ABD but no prior ocular symptoms.112 Aqueous humor from patients with ABD uveitis also shows Th1 polarization, with elevated levels of IFN-γ and TNFa and undetectable levels of IL-10.113 Mucocutaneous ABD lesions also show a preponderance of Th1 cytokines over Th2 cytokines, suggesting that dysregulation of cellular immunity has an important role in ABD.114 Contrasting data have been reported regarding the role of Th2 cells, with some groups finding Th2 profile dominance during the active phase of ABlD, including in a group with ABD uveitis.115,116 More recently, evidence is mounting to support that Th17 cells also have a major pathogenic role in ABD.117 Dysregulation of the IL-23/Th17 axis has been described in various other autoimmune diseases.118 IL-23 induces and expands the population of Th17 cells and augments their production of IL17, a pro-inflammatory cytokine. GWAS analyses revealed specific SNPs in IL-23R that occur at higher frequencies in patients with ABD.7,8,119 Patients with active ABD, including active uveitis, had a higher percentage of circulating Th17 cells compared to healthy controls, as well as increased levels of IL-17A and IL-23 expression during active stages of the disease and decreased levels during periods of remission.120–122 Patients with active ABD had markedly increased levels of IL-21, a cytokine that promotes IL-17A production, compared to patients with ABD in remission.121 Once disease was in remission, the frequency of IL-17-expressing cells diminished.123 Another distinct population of helper T cells, Th22 cells, produces IL-22 and TNF-α and is often associated with immunity in mucosal and barrier surfaces. T cells from aqueous humor samples from ABD patients secreted large amounts of IL-22 and TNF-α.124 When treated with the anti-TNF-α molecule infliximab, production of IL-22 and TNF-α was attenuated. ABD patients with active uveitis also showed higher expression of IL-22 by PBMCs, and a high level of IL-22 correlated with the presence of small vessel inflammation including retinal vasculitis and erythema nodosum.125 Finally, the data describing the impact of Treg cells on ABD are conflicting. One group found that Tregspecific transcription factor expression was increased in the cerebrospinal fluid of patients with ABD.126 One study found decreased Tregs in peripheral blood of ABD patients, whereas another group found the contrary.121,127 Experimentally, upregulation of Treg cells ameliorates inflammatory symptoms in EAU.128 Ultimately, regulatory T cells may have tissue-specific or temporal roles in the pathogenesis and modulation of ABD.

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Cytotoxic T Cells The major role of MHC-I mutations implicates the involvement of CD8 T cells in the pathogenesis of ABD. Some groups argue for a key role of CD8+ T cells in activating neutrophils and driving inflammation in ABD, while others argue that evidence for CD8+ T cell involvement is weak and propose that nonclassical interactions of HLA-B51 may be pathogenic instead.20,129 Overall, there is a relative paucity of studies related to the role of CD8+ T cells in ABD. An enucleated eye from a patient with ABD with recurrent retinal vasculitis ultimately complicated by rhegmatogenous retinal detachment and severe vision loss only demonstrated nongranulomatous uveitis with a CD4+/CD8+ ratio of 1.5:1.0.130 However, due to the severity of the ocular disease, this may not reflect the infiltrating immune cell population in earlier, less severe forms of ABD, and retinal detachment alone can stimulate intraocular inflammation. CD8+ T cells were significantly increased in the peripheral blood of patients with active ABD.131 Aqueous humor from ABD patients showed a predominance of CD8+ T cells compared to other uveitides such as VKH and recurrent anterior uveitis, which had a CD4+ predominance.94,113 A specific population of T cells, CD8brightCD56+ T cells, had cytotoxic and cytolytic activity against vascular endothelial cells.132 Single-cell analysis of immune cells in aqueous humor from patients with Vogt-Koyanagi-Harada disease (VKHD) and ABD uveitis demonstrated T cell clonal expansion in both diseases. However, in contrast to VKHD, which was dominated by CD4+ T cells, ABD uveitis showed a preferential expansion of CD8+ T cell clones.133 While this study had a small sample size, it provides evidence that CD8+ T cells should not be discounted. Given the unique immune environment of the eye, it is also possible that T cell profiles may be different in the eye versus other areas of the body. For example, in HLA-27-related anterior uveitis, expansion of T cell receptors was 10-100 times greater in the eye than in blood, suggesting eyespecific recruitment or expansion.134

Endothelial Dysfunction and the Immune Privilege of the Eye Immune tolerance is established and finely regulated in the eye, to preserve vision and potentially maintain the circadian rhythm.135 Loss of immune privilege, such as in sympathetic ophthalmia, sensitizes the immune system to ocular antigens, subsequently resulting in intraocular inflammation. The predominant vasculitis component of ABD causes compromise of the blood-retinal barrier, as evidenced by vascular leakage seen on fluorescein angiography, which may unmask ocular antigens and open the door for intraocular inflammation. Endothelial dysfunction is well-described in ABD.136 Suppression of adhesion molecules, such as Ecadherin, intercellular adhesion molecule 1 (ICAM-1) and vascular cell adhesion molecule 1 (VCAM-1), has been postulated to be protective against EAU by maintaining the blood-retina barrier.137 Multiple studies show abnormalities of adhesion molecule function and expression in ABD. Histopathology from an enucleated eye from a patient with ABD showed abundant expression of adhesion molecules on vascular endothelial cells.130 Levels of circulating ICAM-1 in the plasma of patients with ABD has been found to be elevated compared to controls.138 Experimentally, serum derived from patients with active ABD induced expression of adhesion molecule mRNA in vascular endothelial cell cultures, which was reversed with neutralization of IL-17A.122 Additionally, direct damage to endothelial cells can also compromise barrier integrity. The combination of increased adhesion molecule expression and direct damage to endothelial cells may compromise the blood-retinal barrier, allowing for immune recognition and attack of previously sequestered ocular antigens. Local modulation of adhesion molecules or endothelial protection represents a possible therapeutic strategy.139

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Brief Summary of Current Treatments and Potential for Novel Targets The management of ABD involves a multidisciplinary approach tailored to individual symptoms and organ involvement. Topical or systemic corticosteroids are often employed, as well as conventional immunosuppressive agents, including azathioprine, methotrexate, and cyclosporine. In cases of refractory or severe ABD, biologic agents such as TNF inhibitors (including infliximab, adalimumab, golimumab) or interleukin inhibitors (tocilizumab, ustekinumab, secukinumab) may be considered. With the emergence of biologic agents, overall prognosis and visual outcomes for patients with ABD have greatly improved. Current treatments have been comprehensively reviewed by other authors.140,141 New pharmacologic targets continue to emerge as more is understood about the immunopathogenesis of ABD.

Conclusion Based on existing knowledge, the pathogenesis of ABD uveitis appears to rely on an initial genetic predisposition to dysregulated antigen processing, with a concomitant or extrinsic vascular injury, thereby inducing abnormal vessel permeability. The initiating event may be an environmental trigger, such as microbial exposure through infection or the microbiome. Damaged vascular endothelium disrupts the blood-retina barrier, culminating in the compromise of immune privilege of the ocular environment. The combination of abnormal antigen processing and compromised barrier integrity contributes to an autoinflammatory response within the eye, giving rise to posterior uveitis and other ocular manifestations. Many questions of ABD pathogenesis remain to be answered. Defining the initiating event, whether it be a microbial trigger or stochastic genetic event, will allow treatment to begin earlier in the disease course and mitigate morbidity. Further defining the complex interplay between the various aspects of the immune system will help optimize therapeutic targets. Finally, elucidating the role of MHC class I molecules in triggering or exacerbating inflammation in Adamantiades-Behçet’s disease will have implications for related diseases, such as HLA-B27 AAU, birdshot uveitis, and the spondyloarthropathies. In conclusion, this review highlights the latest findings in the intricate landscape of AdamantiadesBehçet’s uveitis, shedding light on its multifaceted pathogenesis, clinical manifestations, and therapeutic considerations. ABD uveitis represents a complex interplay of genetic susceptibility, aberrant antigen processing, vascular dysfunction, and immune dysregulation. Though the advent of new biologic therapies targeted at modulating the immune system has improved disease control and prognosis, challenges persist in achieving sustained control and prevention of disease progression. Future study is essential for developing targeted therapies and personalized approaches for treatment, and will have broader implications for understanding the ocular immune environment and uveitis.

Kozek, Lindsay

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Kucuksezer UC, Aktas-Cetin E, Bilgic-Gazioglu S, Tugal-Tutkun I, Gül A, Deniz G. Natural killer cells dominate a Th-1 polarized response in Behçet’s disease patients with uveitis. Clin Exp Rheumatol. 2015;33(6 Suppl 94):S2429.

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Gazzito Del Padre TC, Belem JMFM, de Aguiar MF, et al. Distribution of monocytes subpopulations in the peripheral blood from patients with Behçet’s disease - Impact of disease status and colchicine use. Clin Immunol. 2021;231:108854. doi:10.1016/j.clim.2021.108854

100. Li C, Liu J, Yu X, et al. Aberrant monocyte subsets in patients with Behçet’s disease. Clin Immunol Orlando Fla. 2021;225:108683. doi:10.1016/j.clim.2021.108683 101. Zheng W, Wang X, Liu J, et al. Single-cell analyses highlight the proinflammatory contribution of C1q-high monocytes to Behçet’s disease. Proc Natl Acad Sci U S A. 2022;119(26):e2204289119. doi:10.1073/pnas.2204289119 102. Austermann J, Roth J, Barczyk-Kahlert K. The Good and the Bad: Monocytes’ and Macrophages’ Diverse Functions in Inflammation. Cells. 2022;11(12):1979. doi:10.3390/cells11121979 103. Hirahara L, Takase-Minegishi K, Kirino Y, et al. The Roles of Monocytes and Macrophages in Behçet’s Disease With Focus on M1 and M2 Polarization. Front Immunol. 2022;13:852297. doi:10.3389/fimmu.2022.852297 104. Alpsoy E, Kodelja V, Goerdt S, Orfanos CE, Zouboulis CC. Serum of patients with Behçet’s disease induces classical (pro-inflammatory) activation of human macrophages in vitro. Dermatol Basel Switz. 2003;206(3):225232. doi:10.1159/000068888 105. Wu X, Wang Z, Shi J, et al. Macrophage polarization toward M1 phenotype through NF-κB signaling in patients with Behçet’s disease. Arthritis Res Ther. 2022;24(1):249. doi:10.1186/s13075-022-02938-z

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106. Nakano H, Kirino Y, Takeno M, et al. GWAS-identified CCR1 and IL10 loci contribute to M1 macrophagepredominant inflammation in Behçet’s disease. Arthritis Res Ther. 2018;20(1):124. doi:10.1186/s13075-0181613-0 107. Sun L, Su Y, Jiao A, Wang X, Zhang B. T cells in health and disease. Signal Transduct Target Ther. 2023;8(1):150. doi:10.1038/s41392-023-01471-y 108. Hu D, Guan JL. The roles of immune cells in Behçet’s disease. Adv Rheumatol. 2023;63(1):49. doi:10.1186/s42358-023-00328-w 109. Sun B, Rizzo LV, Sun SH, et al. Genetic susceptibility to experimental autoimmune uveitis involves more than a predisposition to generate a T helper-1-like or a T helper-2-like response. J Immunol Baltim Md 1950. 1997;159(2):1004-1011. 110. Taylor AW. Ocular immune privilege. Eye. 2009;23(10):1885-1889. doi:10.1038/eye.2008.382 111. Yoshimura T, Sonoda KH, Miyazaki Y, et al. Differential roles for IFN-γ and IL-17 in experimental autoimmune uveoretinitis. Int Immunol. 2008;20(2):209-214. doi:10.1093/intimm/dxm135 112. İlhan F, Demir T, Türkçüoğlu P, Turgut B, Demir N, Gödekmerdan A. Th1 polarization of the immune response in uveitis in Behçet’s disease. Can J Ophthalmol. 2008;43(1):105-108. doi:10.3129/i07-179 113. Ahn JK, Yu HG, Chung H, Park YG. Intraocular cytokine environment in active Behçet uveitis. Am J Ophthalmol. 2006;142(3):429-434. doi:10.1016/j.ajo.2006.04.016 114. Ben Ahmed M, Houman H, Miled M, Dellagi K, Louzir H. Involvement of chemokines and Th1 cytokines in the pathogenesis of mucocutaneous lesions of Behçet’s disease. Arthritis Rheum. 2004;50(7):2291-2295. doi:10.1002/art.20334 115. Aridogan BC, Yildirim M, Baysal V, Inaloz HS, Baz K, Kaya S. Serum Levels of IL-4, IL-10, IL-12, IL-13 and IFNgamma in Behçet’s disease. J Dermatol. 2003;30(8):602-607. doi:10.1111/j.1346-8138.2003.tb00442.x 116. Cingu AK, Turkcu FM, Aktas S, Sahin A, Ayyildiz O. Serum IL-4, IL-12, IL-13, IL-27, and IL-33 levels in active and inactive ocular Behcet’s disease. Int Ophthalmol. 2020;40(12):3441-3451. doi:10.1007/s10792-020-01530-1 117. Nanke Y, Yago T, Kotake S. The Role of Th17 Cells in the Pathogenesis of Behcet’s Disease. J Clin Med. 2017;6(7):74. doi:10.3390/jcm6070074 118. Xiong DK, Shi X, Han MM, et al. The regulatory mechanism and potential application of IL-23 in autoimmune diseases. Front Pharmacol. 2022;13:982238. doi:10.3389/fphar.2022.982238 119. Yenmis G, Sabancelebi S, Atak E, Yalinkilic SB, Soydas T, Sadikoglu T. Association of IL-23R and IL-10 variations with Behçet disease: a genetic analysis study. Immunol Res. Published online November 13, 2023. doi:10.1007/s12026-023-09433-w 120. Chi W, Zhu X, Yang P, et al. Upregulated IL-23 and IL-17 in Behçet patients with active uveitis. Invest Ophthalmol Vis Sci. 2008;49(7):3058-3064. doi:10.1167/iovs.07-1390 121. Geri G, Terrier B, Rosenzwajg M, et al. Critical role of IL-21 in modulating TH17 and regulatory T cells in Behçet disease. J Allergy Clin Immunol. 2011;128(3):655-664. doi:10.1016/j.jaci.2011.05.029 122. Hamzaoui K, Bouali E, Ghorbel I, Khanfir M, Houman H, Hamzaoui A. Expression of Th-17 and RORγt mRNA in Behçet’s Disease. Med Sci Monit Int Med J Exp Clin Res. 2011;17(4):CR227-234. doi:10.12659/msm.881720 123. Na SY, Park MJ, Park S, Lee ES. Up-regulation of Th17 and related cytokines in Behçet’s disease corresponding to disease activity. Clin Exp Rheumatol. 2013;31(3 Suppl 77):32-40. 124. Sugita S, Kawazoe Y, Imai A, et al. Role of IL-22- and TNF-α-producing Th22 cells in uveitis patients with Behcet’s disease. J Immunol Baltim Md 1950. 2013;190(11):5799-5808. doi:10.4049/jimmunol.1202677 125. Cai T, Wang Q, Zhou Q, et al. Increased expression of IL-22 is associated with disease activity in Behcet’s disease. PloS One. 2013;8(3):e59009. doi:10.1371/journal.pone.0059009 126. Hamzaoui K, Borhani Haghighi A, Ghorbel IB, Houman H. RORC and Foxp3 axis in cerebrospinal fluid of patients with neuro-Behçet’s disease. J Neuroimmunol. 2011;233(1-2):249-253. doi:10.1016/j.jneuroim.2011.01.012 127. Hamzaoui K, Hamzaoui A, Houman H. CD4+CD25+ regulatory T cells in patients with Behçet’s disease. Clin Exp Rheumatol. 2006;24(5 Suppl 42):S71-78. 128. Shim J, Lee ES, Park S, Bang D, Sohn S. CD4(+) CD25(+) regulatory T cells ameliorate Behcet’s disease-like symptoms in a mouse model. Cytotherapy. 2011;13(7):835-847. doi:10.3109/14653249.2011.571245 129. Giza M, Koftori D, Chen L, Bowness P. Is Behçet’s disease a ‘class 1‐opathy’? The role of HLA‐B*51 in the pathogenesis of Behçet’s disease. Clin Exp Immunol. 2018;191(1):11-18. doi:10.1111/cei.13049

Kozek, Lindsay 130. George RK, Chan CC, Whitcup SM, Nussenblatt RB. Ocular immunopathology of Behçet’s disease. Surv Ophthalmol. 1997;42(2):157-162. doi:10.1016/s0039-6257(97)00026-x 131. Freysdottir J, Lau S, Fortune F. Gammadelta T cells in Behçet’s disease (BD) and recurrent aphthous stomatitis (RAS). Clin Exp Immunol. 1999;118(3):451-457. doi:10.1046/j.1365-2249.1999.01069.x 132. Ahn JK, Chung H, Lee D sup, Yu YS, Yu HG. CD8brightCD56+ T cells are cytotoxic effectors in patients with active Behcet’s uveitis. J Immunol Baltim Md 1950. 2005;175(9):6133-6142. doi:10.4049/jimmunol.175.9.6133 133. Kang H, Sun H, Yang Y, et al. Autoimmune uveitis in Behçet’s disease and Vogt-Koyanagi-Harada disease differ in tissue immune infiltration and T cell clonality. Clin Transl Immunol. 2023;12(9):e1461. doi:10.1002/cti2.1461 134. Yang X, Garner LI, Zvyagin IV, et al. Autoimmunity-associated T cell receptors recognize HLA-B*27-bound peptides. Nature. 2022;612(7941):771-777. doi:10.1038/s41586-022-05501-7 135. Niederkorn JY. The Eye Sees Eye to Eye With the Immune System: The 2019 Proctor Lecture. Invest Ophthalmol Vis Sci. 2019;60(13):4489-4495. doi:10.1167/iovs.19-28632 136. Butta NV, Fernández-Bello I, López-Longo FJ, Jiménez-Yuste V. Endothelial Dysfunction and Altered Coagulation As Mediators of Thromboembolism in Behçet Disease. Semin Thromb Hemost. 2015;41(6):621-628. doi:10.1055/s-0035-1556727 137. Kim J, Chun J, Ahn M, Jung K, Moon C, Shin T. Blood-retina barrier dysfunction in experimental autoimmune uveitis: the pathogenesis and therapeutic targets. Anat Cell Biol. 2022;55(1):20-27. doi:10.5115/acb.21.227 138. Saglam K, Yilmaz MI, Saglam A, Ulgey M, Bulucu F, Baykal Y. Levels of circulating intercellular adhesion molecule-1 in patients with Behçet’s disease. Rheumatol Int. 2002;21(4):146-148. doi:10.1007/s00296-001-01489 139. Chen YH, Lightman S, Eskandarpour M, Calder VL. Adhesion Molecule Targeted Therapy for Non-Infectious Uveitis. Int J Mol Sci. 2022;23(1):503. doi:10.3390/ijms23010503 140. Gueudry J, Leclercq M, Saadoun D, Bodaghi B. Old and New Challenges in Uveitis Associated with Behçet’s Disease. J Clin Med. 2021;10(11):2318. doi:10.3390/jcm10112318 141. Zhong Z, Su G, Yang P. Risk factors, clinical features and treatment of Behçet’s disease uveitis. Prog Retin Eye Res. 2023;97:101216. doi:10.1016/j.preteyeres.2023.101216

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Immune Recovery Uveitis: A Comprehensive Review Melissa Yuan, MD and Lucy H. Young, MD, PhD

Abstract Immune reconstitution inflammatory syndrome (IRIS) describes a hyper-inflammatory state, often manifesting as paradoxical worsening of opportunistic or latent infections, in patients with previously suppressed immune systems who undergo treatment. Ocular IRIS is known as immune recovery uveitis (IRU), a challenging complication that can occur in patients with a compromised immune system, particularly those with human immunodeficiency virus (HIV) infection who initiate highly active antiretroviral therapy (HAART). IRU primarily manifests as posterior segment inflammation with vitritis, resulting in reduced vision and floaters in the affected eye. This review article aims to provide a comprehensive overview of immune recovery uveitis, encompassing its pathophysiology, clinical manifestations, diagnostic approaches, management strategies, and future prospects. Understanding IRU is crucial for ophthalmologists, infectious disease specialists, and other healthcare professionals involved in the care of individuals with HIV/AIDS, as timely intervention can help prevent vision-threatening complications and improve patients' quality of life.

Introduction Background The human immunodeficiency virus (HIV) targets the immune system by reducing the population of CD4+ T lymphocytes, causing patients to be more susceptible to opportunistic infections. Acquired immunodeficiency syndrome (AIDS) is the severe manifestation of HIV, defined as a CD4+ T cell count of less than 200 or a specific AIDS-defining illness (one of several opportunistic infections, malignancies, or other conditions that occur in the setting of profound immunosuppression).1 Cytomegalovirus (CMV) retinitis is commonly observed in individuals with advanced or late-stage HIV/AIDS and low CD4+ T cell counts, typically <50 cells/µL. CMV retinitis became increasingly prevalent in the 1980s and 1990s as the prevalence of HIV increased.2,3 While 50-90% of adults are seropositive for CMV, the virus typically remains latent but can become active during immunosuppression, most frequently due to HIV/AIDS but also in the setting of organ/bone marrow transplantation, chemotherapy, or malignancy. 3 This is typically experienced as viremia but can cause end-organ manifestations in GI tract, lung, liver, peripheral and central nervous systems. Retinitis is one of these end-organ manifestations, thought to be due to hematogenous spread of the virus to the retina.3 Because it occurs in the setting of suppressed immunity, CMV retinitis is associated with very minimal or no anterior chamber and vitreous inflammation. The introduction of protease inhibitors in 1995 marked a significant breakthrough in HIV treatment. These drugs, which were later approved by the US FDA in 1996, became the cornerstone for highly active antiretroviral therapy (HAART). HAART involves the use of a combination of three or more antiretroviral drugs of different classes, typically protease inhibitors and nonnucleoside reverse transcriptase inhibitors. This treatment regimen leads to a substantial reduction in HIV replication and allows the recovery of CD4+ T cells. The implementation of HAART brought about significant improvements in health outcomes in patients with HIV. HAART enables the recovery of CD4+ T lymphocytes and, as such, leads to recovery of the normal immune response against pathogens. The recovery of CD4+ T cell counts is typically most rapid in the first 3 months, followed by more gradual increases over the following months to years.4 As HAART became more widely used, there was a concurrent decline in mortality rates and a decrease in opportunistic infections in patients with HIV. In particular, the incidence of CMV retinitis was substantially reduced, with a 55% to 95% reduction incidence,5 and the prognosis of those affected was improved.6

Yuan, Melissa

This progress in HIV therapy has played a crucial role in managing the disease and improving the quality of life for individuals living with HIV. Systemically, HAART therapy may precipitate immune reconstitution inflammatory syndrome (IRIS), a hyper-inflammatory, dysregulated immune reaction often against opportunistic infections or other antigens. This syndrome typically arises within the initial six months of HAART treatment for HIV/AIDS, but can also occur in other scenarios when a previously suppressed immune system regains function. IRIS can manifest in various ways, including the exacerbation of infectious and non-infectious diseases.7,8 It is thought that IRIS occurs after reversal of immunosuppression by HAART due to the recovery of immune responses against specific antigens.7 This then can cause paradoxical worsening of treated opportunistic infections or the unmasking of previously subclinical, untreated infections in patients with previously suppressed immune systems. Ocular IRIS is referred to as immune recovery uveitis (IRU) and can cause significant morbidity.9,10 IRU was first described in the late 1990s by two groups - Karavellas et al., and Zegans et al. - when patients with AIDS and CMV retinitis were noted to develop transient intraocular inflammation associated with initiation of HAART.11,12 Karavellas et al. observed 130 patients with AIDS and CMV retinitis over 15 months and found that 5 developed symptomatic vitritis and optic nerve edema with cystoid macular edema (CME) or epiretinal membrane (ERM) formation. All affected patients were found to have inactive CMV retinitis which was asymptomatic prior to the onset of inflammation and had had a recovery in CD4+ T cell counts with HAART. The patients were treated with systemic corticosteroids with improvement in the intraocular inflammation and without a reactivation of the retinitis. When 509 patients from the 11 years prior to the use of protease inhibitors were evaluated retrospectively, there were no comparable cases of intraocular inflammation. As such, Karavellas et al. concluded that this syndrome of posterior segment inflammation (now known as IRU) was related to increased immunocompetence due to combined antiretroviral treatment in patients with a history of CMV retinitis.11 Around the same time, Zegans et al. reported 8 patients with AIDS and CMV retinitis who developed vitreous inflammation after CD4+ T-lymphocyte recovery from combination antiretroviral therapy. In this cohort, 6 of these patients had unilateral CMV retinitis prior to HAART, and the inflammatory reaction after starting combination antiretroviral therapy was isolated to the eye with CMV retinitis. Treatment included periocular and topical steroids, as well as systemic antivirals. The authors felt that the intraocular inflammation was reflective of an improved immune response against CMV.12 Over the subsequent years, more reports have been published regarding the clinical features, epidemiology, pathogenesis, diagnosis, and complications of IRU. We seek to summarize these in this review.

Objective IRU is caused by has the potential for vision-threatening complications and is caused by unique pathophysiologic mechanisms. Despite remarkable advances in HIV management, IRU remains a pertinent concern for these patients. The aim of this review is to provide a summary of the biology, pathogenesis, prevalence, clinical manifestations, diagnostic criteria, prognosis, and management options for IRU. Our hope is to provide context and guidance for ophthalmologists when they encounter this rare entity.

Biology and Pathogenesis The pathogenesis of IRU is multifactorial. It primarily occurs due to the restoration of CD4+ T cells in previously immunocompromised individuals, usually those with HIV/AIDS commencing HAART.9,13 HAART causes polyclonal expansion of T-lymphocytes and improvement in CD4+ T cell function.14 As the CD4+ T cell counts rise, there is a robust response to various opportunistic infections or antigens that the

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patient was previously unable to mount a sufficient immune response against. This excessive immune response can lead to ocular inflammation and the development of IRU.15 While IRU is almost always found in the setting of increased CD4+ T cells, there are rare reports in which this is not the case.16,17 Additionally, aside from HIV/AIDS, IRU has also been reported in the setting of immunosuppression in other conditions, including rheumatoid arthritis, systemic lupus erythematosus, leukemia, and after stem cell transplant for lymphoma.16,18–20 Several mechanisms contribute to the development of IRU. One such mechanism is antigenic persistence, which refers to the persistence of microbial antigens within the eye even after the primary infection has been treated. The persistence of CMV antigens within the eye then triggers an immune response when the immune system becomes reconstituted.10 CMV antigens, in particular, are thought to play a role in the pathogenesis of IRU. First, IRU does not occur in eyes that do not have a pre-existing history of CMV retinitis. It has also been proposed that in the development of IRU, CMV retinitis causes breakdown in the blood-ocular barrier; thus, after immune recovery, CMV antigens that enter the systemic circulation trigger a robust immune response to attack the antigens in the eye, which is normally an immune-privileged site.21 Immunohistochemical studies of epiretinal membranes in IRU reveals evidence of a chronic inflammatory process and a T lymphocyte population, suggesting a possible T cell mediated immune reaction to latent CMV antigens in the setting of inactive CMV retinitis.22 IRU as a CMV antigenmediated disease is further supported by evidence that increased CMV retinitis lesion size is associated with greater risk for developing IRU.21 Additionally, delaying HAART initiation until completion of CMV retinitis treatment decreases the rate of IRU.23 This may minimize exposure to CMV antigens during the critical phase of immune recovery in the eye.24 Finally, more aggressive anti-CMV therapy with the ganciclovir implant may reduce the risk of IRU.9 Of note, studies have generally found no evidence of CMV DNA in the peripheral blood nor in the eye in patients with IRU, indicating no active replication of the infectious virus; this does not contradict the possibility of antigenic persistence, which can occur in the absence of viral replication.21,25 However, not all patients who have CMV retinitis and immune recovery will ultimately develop IRU. This indicates there may be genetic, environmental, and other factors involved. In fact, even in patients with two affected eyes with CMV retinitis, the development of IRU may be unilateral or asymmetric.21 NonCD4+ related immune dysfunction may contribute to IRU development. Hartigan et al. found that patients with prior CMV retinitis who developed IRU had significantly lower levels of Th17 cells compared to those who did not develop IRU.26 This study also showed that patients with IRU had poor systemic CMVspecific T cell responses, which play a role in controlling the primary infection, while CD8+ T cell responses were similar. These findings suggest that the depletion of Th17 cells and poor systemic CMVspecific T cell responses are associated with the development of IRU in patients with CMV retinitis. Thus, IRU may be most likely to develop in patients with greater immune dysfunction prior to HAART intiation.26 Furthermore, there is evidence to suggest that IRU may have an autoimmune component. Crossreactivity between CMV antigens and host antigens could potentially trigger an autoimmune response within the eye, leading to inflammation. In support of this, Schrier et al. found that IRU eyes contain elevated IL-12, but reduced IL-6 and interferon gamma – seen in antigen-specific T cell responses. Selfantigens do not cause an interferon gamma response, so the antigen stimulus in IRU may be a selfantigen. Alternatively, it is possible that there are CMV proteins eliciting less interferon gamma than intact virions.25 Genetic predisposition may also play a role in susceptibility to IRU. One small study of 4 patients with IRU in Italy were positive for human leukocyte antigen (HLA) allele HLA B18-8. While this study was limited, the fact that a specific HLA allele has been associated with an increased risk of IRU, highlighting a potential interplay between genetics and IRU.27 Of note, not all cases are associated with a significant increase in CD4 counts. Although most studies have required evidence of immune recovery as indicated by an increased CD4+ T cell count to diagnose IRU, there have been reported cases of IRU that occurred in the absence of CD4+ T cell increases.15–17 This indicates that a large rise in CD4+ T cell counts may not be necessary, and this measure alone likely does not specifically capture the recovery of anti-CMV immunity. Specific assays for immune activity

Yuan, Melissa

against CMV may be a more precise predictor of the risk of IRU, but these assays are not widely available.27,28 To summarize, the pathophysiology of IRU is characterized by the restoration of immune function, typically in the setting of increased CD4+ T cell counts, resulting in an excessive immune response in an eye with previously treated CMV retinitis. It is largely thought to be a CMV antigen-related inflammatory disorder, but dysregulation of cytokines and potential autoimmune responses also contribute to its development. Moreover, genetic predisposition may further influence susceptibility to IRU. This complex interplay between prior CMV retinitis, CD4+ T cell counts, innate immune responses, and the immunocompetence achieved through antiretroviral therapy underlies the pathogenesis of IRU.

Risk Factors In addition to the previously stated necessary precipitating factors of prior CMV retinitis and immune recovery, are other risk factors are associated with increased risk of IRU. One risk factor is a low CD4+ T count (≤50 cells/microliter) at the time of initiation of HAART.21 IRU complications are also more likely with a greater increase in CD4+ T cell count from the nadir to the time of IRU diagnosis, possibly due to a greater inflammatory response in these patients.29 Larger areas of affected retina in CMV retinitis has also been associated with increased risk for IRU. Specifically, >25% retinal area involvement had an odds ratio of 2.72 of developing IRU in one study,21 and in another study, >30% retinal area involvement had a 4.5-times higher risk of developing IRU compared to an area smaller than 18%.22 The effects of lesion size may be explained by a larger extent of blood-retinal barrier breakdown but may also indicate a higher antigen load in larger lesions.22 Of note, Arevalo et al. found that eyes with IRU had an average CMV surface area of 31.7% and eyes without IRU had an average CMV surface area of 35%, showing no significance in CMV retinitis surface area in terms of developing IRU in that study.30 Yeo et al. similarly found no association between the area affected by CMV retinitis and IRU risk, though their cohort was primarily composed of patients with <25% retinal area affected by CMV retintiis.29 The use of antiviral medication cidofovir for treatment of CMV is a notable modifiable risk factor for IRU. both intravenous and intravitreal. Song et al. found that the use of cidofovir increased the risk of subsequent development of IRU in patients who had recovered from CMV retinitis.31 Specifically, patients who were continued to be treated with any anti-CMV antiviral had a 3.8-fold increase in the odds of developing IRU with anti-CMV therapy compared with no treatment after immune recovery. Subgroup analyses revealed that this effect was due to cidofovir. The odds of eyes treated with cidofovir developing immune recovery uveitis were 3.3 greater than if treated with an alternative regimen, 4.1 greater if treated intravenously rather than intravitreally, and 5.2 greater than if not treated at all after immune recovery.31 This inflammatory effect of cidofovir was also noted by Kempen et al, who found that intravitreal cidofovir was associated with a 19-fold higher risk of IRU.21 Cidofovir itself can cause anterior uveitis itself possibly due to toxicity to the ciliary body.32,33 The increased risk of IRU in cidofovir-treated patients may be related to these toxic and inflammatory effects. In terms of factors that are negatively associated with IRU development, posterior pole involvement of CMV retinitis was found to be protective against IRU.21 Additionally, male sex was found to be negatively associated with IRU.21

Epidemiology Prevalence The prevalence of IRU after HAART initiation in patients with a history of CMV retinitis is estimated around 20%.34 However, the exact prevalence is challenging to determine due to variations in patient populations and definitions, with reported prevalence ranging from 1.5% to 63% . In US-based studies, the prevalence has been noted to be 9.6%,35 18.2%,6 and 63.3%,21 in different case series. In international studies, the prevalence has been noted to be 37.5% in a Venezuelan study,30 53.5% in a

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Mexican study,23 17.4% in an Indian study,36 and 1.5% in a Japanese study.37 One reason for the difference in prevalence may be differences in timing of initiation of HAART relative to treatment of CMV retinitis. Ortega et al. found a lower prevalence and severity of IRU when HIV-infected patients started HAART after completing CMV retinitis treatment than when treatments were concomitant.23

Incidence Similar to the prevalence, incidence also has varied between studies. The early cohorts of patients with IRU, observed in the years immediately after HAART was approved and became widespread, had incidences ranging from 0.109 per person-year to 0.82 per person-year.6,22 A few years after these initial report, Jabs et al. then reported an incidence of IRU of 2.7–3.6/100 person-years.2 While the rates of IRU vary widely, these reports indicate that IRU is an important consideration in patients with a history of CMV retinitis who subsequently undergo immune recovery. Contributing to the variable incidences and prevalences reported in the literature is the heterogeneity in study definitions; for example, vitritis is a part of the definition in most studies, but CME and ERM are more persistent complications that are variably included in definitions.38

Timing of Onset The time from HAART initiation to the development of IRU ranges in the literature, from as short as weeks,11,39 to 4-5 months, and up to 128 months.26 Median time of onset is typically around 20-43 weeks (5-9 months).2 To highlight some specific studies: Zegans et al. found IRU set in on average 8 months after addition of a protease inhibitor,12 Karavellas et al. found a median time from HAART initiation to IRU of 43 weeks (9.9 months),35 Sudharshan et al. found an average of 4 months to 2.5 years between the start of HAART and onset of IRU,36 and Henderson et al. found an average of 5.5 months (range 1-14 months) between protease inhibitor initiation and vitreous inflammation.40 These discrepancies may be due to some studies including only symptomatic patients with others including all patients with clinically evident intraocular inflammation regardless of symptoms. Yeo et al. found that age was negatively correlated with duration of time to develop IRU after HAART initiation. Specifically, the median duration from initiation of HAART to onset of IRU was 763 days in those under 50 years old and 161 days in those over 50 years old.29

Clinical Features IRU can affect a variety of ocular structures, though most commonly presents with vitreous inflammation. As such, patients with IRU most commonly report symptoms such as decreased vision and floaters. IRU can have a component of anterior uveitis with associated symptoms such as photophobia and eye pain. Occasionally, IRU can be asymptomatic. IRU may follow an acute and self-limiting course, or a chronic inflammatory course with long-term sequelae.10 Complications of IRU can include vitreomacular traction, retinal neovascularization, and epiretinal membrane formation, which can lead to vision loss if not managed appropriately. Retinal detachment is another potential complication, with rates varying depending on the presence of previously-diagnosed or newly-diagnosed retinitis. Proliferative vitreoretinopathy, although rare in CMV retinitis-associated retinal detachment, may be encountered in retinal detachments associated with IRU. To further detail some of the manifestations of IRU: Anterior Manifestations IRU can be associated with anterior segment inflammation. In one study, about half of eyes had both anterior uveitis and vitritis.29 Kempen et al. found that 58% of IRU eyes had AC cells, while 70% had vitreous haze.21 This inflammation, which can sometimes be persistent, can cause posterior subcapsular cataracts, anterior subcapsular cataract, and inflammatory deposits on the lens.41 Rarely, the anterior chamber inflammation can be significant enough to cause a hypopyon, as reported in 2 case reports.42,43

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Posterior synechiae, adhesions between the iris and lens, are common in almost all types of uveitis. However, posterior synechiae were found in one cohort not to differ significantly between patients with IRU when compared to those with a history of CMV retinitis but no IRU (6.0% vs 5.7%).21 Posterior Manifestations Vitreous Vitritis is one of the most common findings in IRU. This finding can be either asymptomatic or symptomatic, and either transient or persistent.12,21,41 Henderson and Mitchell found that in 80 patients with inactive CMV retinitis who received HAART, most of these patients developed a mild transient vitritis, often not requiring treatment. Clinically evident IRU with decreased visual acuity requiring therapy developed in only 7 patients.40 Vitritis is often associated with decreased vision. In Henderson’s cohort, the eyes with significant IRU had a mean visual acuity loss of 2.8 Snellen lines.40 Robinson et al. found that vitreous haze was significantly associated with lower visual acuity.44 Retina Epiretinal membranes (ERMs) are nonvascularized, fibrocellular membranes that form on the inner surface of the retina. These can develop idiopathically or in the setting of intraocular inflammation, diabetic retinopathy, trauma, prior surgery, or retinal detachment. The pathophysiology of ERM involves tissue damage and subsequent reparative processes, driving the proliferation of glial and retinal pigment epithelium cells on the surface of the inner limiting membrane. ERM is found frequently in IRU. Kempen et al. found ERM in 48.9% of IRU eyes vs 13.3% of eyes of patients with a history of CMV retinitis but no IRU, indicating a 3.7 times increased risk.21 In Karavellas’s cohort, 8 of 26 developed ERM. This incidence was much higher than in a cohort of patients with CMV retinitis prior to the age of HAART, in which only 3 of 803 eyes had ERM, and all in the setting of CMV retinitis in the macula.22 Immunohistological examination of ERMs removed from eyes with IRU show evidence of chronic inflammation with a predominant T-lymphocyte population.22 Cystoid macular edema (CME), characterized by intraretinal edema causing cystic retinal thickening in the macula, can occur in the setting of intraocular inflammation. IRU significantly increases the risk for CME, while CME is not commonly seen in CMV retinitis. One study showed a 12.3 times increased risk of CME in IRU – with CME found in 45.5% of IRU eyes vs 3.7% of eyes of patients with a history of CMV retinitis but no IRU.21 Karavellas et al. found CME in 35% of 26 eyes with IRU.22 Guzak et al. reported a case of CME in IRU that was successfully treated with topical NSAIDs.45 Interestingly, in Yeo et al.’s cohort of 30 patients with IRU, there were no cases of CME. This was proposed to be related to earlier treatment of the inflammation in IRU.29 Macular holes are usually the result of tractional forces on the retina, and similar to ERM, can be idiopathic or secondary to other factors such as trauma, vitreomacular traction, retinal detachment, or inflammatory processes.46 There has been one reported case of a full-thickness macular hole with subretinal fluid associated with IRU.30 Frosted branch angiitis is an uncommon form of severe retinal perivasculitis with prominent perivascular sheathing of arterioles and venules, which can be seen in CMV retinitis. It has rarely been described in IRU as well, in 3 case reports.17,39,47 Retinal detachments can occur in IRU, with or without proliferative vitreoretinopathy (PVR). The rate of retinal detachment in IRU was 2.3/100 eye-years in one study.2 PVR, characterized by the growth and contraction of cellular membranes within the vitreous and retina, is an abnormal healing response driven by inflammatory, retinal, and retinal pigment epithelial cells. It typically occurs in the setting of complex retinal detachments. PVR is rare in CMV retinitis-associated retinal detachment in patients with AIDS due to the lack of immune/inflammatory response. However, PVR is more commonly encountered in retinal detachments associated with IRU.48 In one cohort, PVR was seen in one third of patients. In one eye, spontaneous vitreous hemorrhage from avulsion of a blood vessel after contraction of the inflamed vitreous was seen. PVR recurred in all cases after surgery, severely compromising the visual outcome.41 In another cohort, 3 patients (16%) had retinal detachments, 2 of which were complicated by PVR.30

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Retinal neovascularization typically occurs in the setting of an imbalance between pro-angiogenic and anti-angiogenic factors, often in the setting of retinal hypoxia/ischemia. However, retinal neovascularization is also sometimes associated with uveitis.49 Wright et al. reported extensive peripheral retinal neovascularization as a late finding of IRU in 3 patients out of a 15-patient cohort with IRU.50 The neovascularization was so severe in one case that it required vitrectomy for recurrent vitreous hemorrhage. Neovascularization in IRU has been reported in the setting of nonperfusion, where the pathophysiology of neovascularization is clear, but also in cases without retinal ischemia. Neovascularization in this setting seems to be related to the inflammatory milieu with proinflammatory growth factors and cytokines.50,51 Optic nerve Optic nerve edema, also known as papillitis, may occur in the setting of intraocular inflammation of all types – anterior uveitis, intermediate uveitis, posterior uveitis, or panuveitis.52 Papillitis is relatively frequently seen in IRU, and may be associated with the severity and duration of inflammation. In one of the original reports of IRU, papillitis was seen in all 5 patients in that cohort.11 Optic nerve head neovascularization has also been reported in a few patients with IRU. Sanislo et al. reported a patient with IRU who developed prominent optic nerve head neovascularization without nonperfusion on fluorescein angiography. Similar to the retinal neovascularization discussed above, this optic nerve head neovascularization was thought to occur in the setting of intraocular inflammation and not nonperfusion.51 Postelmans et al. reported an atypical case of IRU associated with bilateral peripheral occlusive vasculopathy and bilateral neovascularization of the optic disc.53 Additionally, one patient in Nguyen et al.’s cohort was noted to have optic disc neovascularization, which resolved spontaneously.6

Diagnosis IRU is a clinical diagnosis based on the proper clinical scenario and exam as described above, in the absence of other causes of intraocular inflammation. There are no formal diagnostic criteria established for IRU. The NIH-sponsored AIDS Clinical Trial Group defined IRU as a decrease in vision with at least two of the following signs in the absence of active CMV retinitis: (a) presence of >2+ inflammatory cells in vitreous by slit lamp examination, (b) CME, or (c) epiretinal membrane formation in patients receiving potent antiretroviral therapy with evidence of immune reconstitution.1 A different NIH-sponsored group, the Studies of the Ocular Complications of AIDS, defined IRU as “the occurrence of intermediate uveitis in patients with CMV retinitis who have evidence of immune reconstitution, as shown by a rise in CD4 T cell counts after initiation of HAART.” 1Neither of these definitions is considered to be a definitive set of criteria in current practice.1 As such, IRU is clinically identified by a new or heightened intraocular inflammatory reaction in individuals with HIV/AIDS and a prior diagnosis of CMV retinitis, typically occurring weeks after commencing HAART with associated immune reconstitution. CD4+ T cell count recovery to more than 100 cells per microliter and at least 50 cells per microliter greater than their nadir has been used as the definition of immune recovery in several studies on IRU.6,21,29 Sampling of the aqueous and vitreous of patients with IRU can also help to differentiate between IRU, active CMV retinitis, and controls. Schrier et al. examined cytokines IL-6, IL-12, interferon gamma using enzyme-linked immunosorbent assays, and CMV DNA by polymerase chain reaction, in the aqueous and vitreous of these groups. This study found that IRU eyes had higher levels of IL-12 compared to controls and negative CMV DNA. All of the active CMV retinitis eyes had positive CMV DNA and had higher levels of IL6 than controls and IRU eyes. Interferon gamma did not differ significantly between CMV retinitis and IRU. As such, IRU can be differentiated from active CMV retinitis by the presence of IL-12, diminished IL6, and absence of detectable CMV DNA.25 Recent research has explored the potential of microRNA (miRNA) as biomarkers for diagnosing IRU. A study by Duraikkanu et al. investigated the expression of miRNA-192 and miRNA-543 in HIV-infected patients with ocular manifestations. Results indicated that miRNA-192-5p and miRNA-543 were

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downregulated in the peripheral blood of patients with ocular manifestations. These miRNAs showed promise in distinguishing HIV patients with IRU, as they target multiple genes involved in inflammatory pathways.54

Differential Diagnosis Distinguishing IRU from other forms of uveitis is crucial to initiate appropriate treatment. Conditions that may mimic IRU include infectious uveitis (e.g., reactivation of CMV retinitis, syphilis, toxoplasmosis, tuberculosis, fungal infections), non-infectious uveitis (e.g., sarcoidosis, Behçet's, drug-induced uveitis, pars planitis), and masquerade syndromes (e.g., intraocular lymphoma). It is particularly important to exclude infectious conditions including syphilis and herpetic infection, as well as drug toxicity (particularly cidofovir), in these patients. A meticulous clinical evaluation and history, along with laboratory and imaging studies, can help differentiate IRU from its mimickers. Aqueous or vitreous sampling can be helpful to rule out infectious etiologies in unclear cases.

Prognosis The prognosis of IRU is not well defined in large studies. Some patients experience complete resolution of inflammation, while others face persistent disease or vision loss from complications. Despite improvement of inflammation, IRU patients remain at risk of complications including cataracts, secondary glaucoma, optic neuropathy, cystoid macular edema, epiretinal membrane, and retinal detachment. In a 5-year outcomes study, 26% lost one or more Snellen lines of visual acuity at 6 months, while most patients maintained or improved vision. Complications included cataract (66.7 %), glaucoma and ocular hypertension (33.3 %). The risk of complications was associated with the absolute difference in CD4 counts between time of IRU onset and HAART commencement (p = 0.041).29 In a study on periocular steroids for IRU, all of the treated eyes regained vision to within one Snellen line of pre-IRU visual acuity.40 Kempen et al. reported that 50% of 50 eyes with IRU developed moderate vision loss at 20/50 or worse.21 Even after recovery of visual acuity and resolution of inflammation, there may be persistent retinal dysfunction. In two patients with IRU who were followed serially with OCT and ERG, chronic retinal injury was evidenced by ellipsoid line loss in one case and gradual optic disc cupping despite normal IOP in the other. ERG in both cases revealed generalized retinal dysfunction, with delayed, subnormal ERGs in the eyes with IRU. In one patient who received serial ERGs, there was improvement over time with treatment.55

Management The management of immune recovery uveitis (IRU) is similar to that of other noninfectious uveitis and is tailored to the severity of inflammation and clinical manifestations. The primary goals of treatment are to control intraocular inflammation, improve visual acuity, and prevent long-term complications. The management of IRU involves a combination of topical and systemic immunosuppression, typically with corticosteroids. If asymptomatic, some cases are transient and may even self-resolve. For mild to moderate cases of IRU, topical corticosteroids can be used to reduce anterior chamber inflammation. Earlier treatment of the inflammation may prevent later complications, such as CME.29 Periocular corticosteroids may also be beneficial, shown to be efficacious for the intraocular inflammation.40 In more severe cases, systemic corticosteroids may be considered. In the initial case series reported by Karavellas et al. and Zegans et al., most patients were treated with systemic steroids.11,12 In cases of recurrent or refractory IRU, it may be reasonable to consider steroid-sparing immunosuppressive agents such as methotrexate or mycophenolate, though these are not commonly used in IRU.

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Systemic antiviral therapy generally does not appear to improve outcomes in IRU.21,31,56 Cidofovir, a previously frequently used treatment for CMV, has also been identified as a primary risk factor for the subsequent development of IRU. Ongoing treatment of healed CMV retinitis after immune recovery does not seem to protect against the development of IRU. Additionally, Wohl et al. found no difference in occurrence of IRU in patients with CMV retinitis who continued or discontinued maintenance CMV retinitis therapy (most frequently with oral ganciclovir in their cohort).57 However, Kosobucki et al. found that valganciclovir improved visual outcomes in patients with CME in the setting of IRU.58 In general, additional antiviral treatment does not play a role in IRU given the overall evidence that these do not typically improve outcomes.

Management of CME Eyes with IRU-associated CME can maintain good vision and may spontaneously improve, As such, mild cases may be amenable to observation or treated with topical therapy.59,60 For example, Guzak et al. reported a case of CME in IRU that improved with topical NSAIDs only.45 Sub-tenon’s injections of steroids are modestly effective for the CME seen in IRU.22 In Karavellas et al.’s cohort, corticosteroid treatment, used only in more severe cases of IRU with CME and decreased vision of 2+ lines, led to improved vision in the patients treated with steroids. In patients not treated with steroids, the macular edema persisted or worsened. Thus, local and/or systemic steroid treatment for IRU may improve visual acuity compared to observation.22 In Nguyen et al.’s cohort, 4 of 6 eyes with IRU were reported to have CME and 2 of the eyes improved with steroid treatment. In the other two patients, the CME persisted despite aggressive therapy with topical, periocular, and systemic corticosteroids.6 Morrison et al. found that high-dose intravitreal triamcinolone was effective for CME in IRU. In 8 eyes with an average of 51 months of IRU prior to the first triamcinolone injection, vision and macular thickness improved significantly, with only 3 eyes requiring repeat injections.61 A fluocinolone acetonide implant has also been shown to be effective and safe in IRU-associated CME.62 Of note, the patients who received a fluocinolone implant also received treatment for CMV retinitis (ganciclovir implant or oral valganciclovir) in order to minimize the risk of recurrence of CMV retinitis.

Surgical Management Surgical interventions, particularly vitrectomy, are uncommon after IRU but may be necessary to manage complications like ERM or retinal detachment. Surgical repair of retinal detachment associated with CMV retinitis has been well-described due to its complexity, and typically involves pars plana vitrectomy with silicone oil tamponade.3,63,64 These CMV retinitis-related detachments, often with atrophic areas and numerous breaks, may be further complicated in the inflammatory milieu of eyes with IRU; in Karavellas et al.’s cohort, 2 of 29 eyes developed rhegmatogenous retinal detachments and proliferative vitreoretinopathy, requiring vitrectomy, membrane peeling, silicone oil tamponade, and scleral buckling. The postoperative course was complicated for these patients, with epiretinal and subretinal reproliferation. One patient developed a tractional retinal detachment after silicone oil removal.22 While retinal detachments have been reported in other cohorts of IRU,21,30 there is a shortage of literature on the surgical management and clinical course of these detachments.

Illustrative Case A 34-year-old woman with a history of HIV/AIDS and prior treatment for CMV retinitis, now on HAART, presents with gradual loss of vision in both eyes. Visual acuity was 20/200 in both eyes. Imaging from her initial presentation (FIGURE 1) demonstrates optic nerve hyperemia and late leakage, areas of chorioretinal scarring/atrophy and petaloid leakage in the macula, consistent with cystoid macular edema.

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FIGURE 1: Representative case of IRU. At presentation, color photography of the right (A) and left (D) eyes and fluorescein angiography in the right (B-C) and left (E-F) eyes demonstrate optic nerve hyperemia and late leakage, areas of chorioretinal scarring/atrophy and petaloid leakage in the macula, consistent with cystoid macular edema.

At follow-up 16 years later, the patient's exam was stable (FIGURE 2). There was extensive chorioretinal scarring in both eyes with a few peripheral hemorrhages and diffuse atrophy with multiple areas of ellipsoid zone disruption on OCT. Visual acuity was 20/80 in the right eye and 20/250 in the left eye. FIGURE 2: At follow-up 16 years later, ultra-widefield fundus photography shows extensive chorioretinal scarring in both eyes with a few peripheral hemorrhages (A-B). OCT (C-D) showed architectural changes with multiple areas of ellipsoid zone disruption, left more than right, consistent with prior CME.

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Future Directions Advances in the understanding of IRU's pathophysiology and the development of targeted therapies hold promise for improving the management of this condition. Further research into biomarkers that predict risk for IRU may assist clinicians in identifying patients who may need closer monitoring. Despite the advent of new therapies for noninfectious uveitis, there has not been significant research into the applications of these therapies for IRU. Biologics and novel immunomodulatory agents may offer more targeted and safer alternatives to traditional corticosteroid-based therapies. Additionally, further research is needed on the surgical management of IRU-associated retinal detachments, which have the potential to be complicated by PVR.

Conclusion While HAART has significantly improved the prognosis and life expectancy of patients with HIV/AIDS and decreased the incidence of CMV retinitis, IRU remains a challenging and potentially vision-threatening entity. IRU typically occurs weeks to months after immune recovery, but may even occur years later, and is thought to be a CMV antigen-related inflammatory condition. It is important to rule out infectious etiologies of uveitis when managing IRU. IRU can be asymptomatic or minimally symptomatic, improving with topical or periocular treatment only. However, there are also cases of persistent CME or severe PVR in the setting of IRU. A comprehensive understanding of the biology, clinical presentation, diagnostic criteria, and management strategies of IRU is crucial for all ophthalmologists. Further research and clinical advancements hold the potential to improve the diagnosis and management of this complex condition.

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2024 Harvard Ophthalmology Residents’ Course

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Yuan, Melissa

2024 HARVARD RESIDENTS’ COURSE

The Immune System and the Eye Course Directors:

Isaac D. Bleicher, MD and Joan W. Miller, MD

2024 Harvard Visiting Professor: Nisha Acharya, MD, MS Professor, Department of Ophthalmology University of California, San Francisco

Presenting Residents: Amee D. Azad, MD Saghar Bagheri, MD, PhD Enchi K. Chang, MD Yilin Feng, MD James M. Harris, MD, PhD Lindsay K. Kozek, MD, PhD Da Meng, MD, PhD Tatiana R. Rosenblatt, MD Melissa Yuan, MD

Faculty Preceptors: Reza Dana, MD, MPH, MSC Suzanne K. Freitag, MD John B. Miller, MD Nimesh A. Patel, MD Roberto Pineda II, MD Demetrios Vavvas, MD, PhD David Wu, MD, PhD Michael K. Yoon, MD Lucy Young, MD, PhD

About the Course: The theme for the 2024 Harvard Ophthalmology and Mass Eye and Ear Residents’ Course is “The Immune System and the Eye”. Third-year residents from the Department of Ophthalmology will present their review articles featuring topics related to this theme. This year’s Visiting Professor in Ophthalmology is Nisha Acharya, MD, MS from the Department of Ophthalmology at the University of California, San Francisco. Our distinguished guest will share her extensive experience and expertise with the residents and will also present the 2024 Harvard Visiting Professor in Ophthalmology Lecture.