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Purpose: Bordering the central nervous system (CNS) parenchyma are the choroid (underlying the retina) and the leptomeninges (the inner layers of the meninges enveloping the brain). Although near the neural parenchyma, the choroid and leptomeninges are external to the immune privileged environment of the retina and brain and thus are distinct immune compartments. This study aimed to characterize the transcriptomic signatures of immune cells within the choroid and leptomeninges bordering the healthy adult mouse CNS., Methods: Eyes and brains were obtained from 7-week-old C57Bl/6J mice. Choroid and leptomeninges were processed for isolation of CD45+ immune cells and single cell RNA-sequencing. Additionally, single cell RNA-sequencing was performed on immune cells isolated from choroid obtained from human donor eye tissue. Immunostaining and confocal microscopy of wholemount tissue were used to validate selected immune cell populations in situ., Results: A total of 3606 cells were sequenced from mouse tissues, including 2125 CD45+ cells from choroid and 1481 CD45+ cells from leptomeninges. Clustering and differential gene expression analysis revealed heterogeneous subtypes of monocytes/macrophages, dendritic cells, T cells, and B cells. Whereas some clusters were common to both choroid and leptomeninges, others exhibited tissue-specific gene expression profiles and potential functional specializations. Analysis of 6501 CD45+ cells sequenced from human choroid identified similar immune cell populations to mouse choroid., Conclusions: This study provides a detailed characterization of the molecular signatures of immune cells within the vascular connective tissues bordering the healthy retina and brain, and their potential roles in immune protection.

Although researched extensively the understanding regarding mechanisms underlying glaucoma pathogenesis remains limited. Further, the exact mechanism behind neuronal death remains elusive. The role of neuroinflammation in retinal ganglion cell (RGC) death has been prominently theorised. This review provides a comprehensive summary of neuroinflammatory responses in glaucoma. A systematic search of Medline and Embase for articles published up to 8th March 2023 yielded 32 studies using post-mortem tissues from glaucoma patients. The raw data were extracted from tables and text to calculate the standardized mean differences (SMDs). These studies utilized post-mortem tissues from glaucoma patients, totalling 490 samples, compared with 380 control samples. Among the included studies, 27 reported glial cell activation based on changes to cellular morphology and molecular staining. Molecular changes were predominantly attributed to astrocytes (62.5%) and microglia (15.6%), with some involvement of Muller cells. These glial cell changes included amoeboid microglial cells with increased CD45 or HLA-DR intensity and hypertrophied astrocytes with increased glial fibrillary acidic protein labelling. Further, changes to extracellular matrix proteins like collagen, galectin, and tenascin-C suggested glial cells' influence on structural changes in the optic nerve head. The activation of DAMPs-driven immune response and the classical complement cascade was reported and found to be associated with activated glial cells in glaucomatous tissue. Increased pro-inflammatory markers such as interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-α) were also linked to glial cells. Glial cell activation was also associated with mitochondrial, vascular, metabolic and antioxidant component disruptions. Association of the activated glial cells with pro-inflammatory responses, dysregulation of homeostatic components and antigen presentation indicates that glial cell responses influence glaucoma progression. However, the exact mechanism triggering these responses and underlying interactions remains unexplored. This necessitates further research using human samples for an increased understanding of the precise role of neuroinflammation in glaucoma progression.

Background and Objectives: To systematically describe the clinical picture of double-antibody seronegative neuromyelitis optica spectrum disorders (DN-NMOSD) with specific emphasis on retinal involvement., Methods: Cross-sectional data of 25 people with DN-NMOSD (48 eyes) with and without a history of optic neuritis (ON) were included in this study along with data from 25 people with aquaporin-4 antibody seropositive neuromyelitis optica spectrum disorder (AQP4-NMOSD, 46 eyes) and from 25 healthy controls (HCs, 49 eyes) for comparison. All groups were matched for age and sex and included from the collaborative retrospective study of retinal optical coherence tomography (OCT) in neuromyelitis optica (CROCTINO). Participants underwent OCT with central postprocessing and local neurologic examination and antibody testing. Retinal neurodegeneration was quantified as peripapillary retinal nerve fiber layer thickness (pRNFL) and combined ganglion cell and inner plexiform layer thickness (GCIPL)., Results: This DN-NMOSD cohort had a history of [median (inter-quartile range)] 6 (5; 9) attacks within their 5 ± 4 years since onset. Myelitis and ON were the most common attack types. In DN-NMOSD eyes after ON, pRNFL ( p < 0.001) and GCIPL ( p = 0.023) were thinner compared with eyes of HCs. Even after only one ON episode, DN-NMOSD eyes already had considerable neuroaxonal loss compared with HCs. In DN-NMOSD eyes without a history of ON, pRNFL ( p = 0.027) and GCIPL ( p = 0.022) were also reduced compared with eyes of HCs. However, there was no difference in pRNFL and GCIPL between DN-NMOSD and AQP4-NMOSD for the whole group and for subsets with a history of ON and without a history of ON-as well as between variances of retinal layer thicknesses., Discussion: DN-NMOSD is characterized by severe retinal damage after ON and attack-independent retinal neurodegeneration. Most of the damage occurs during the first ON episode, which highlights the need for better diagnostic markers in DN-NMOSD to facilitate an earlier diagnosis as well as for effective and early treatments. In this study, people with DN-NMOSD presented with homogeneous clinical and imaging findings potentially suggesting a common retinal pathology in these patients.

The blood-retina barrier (BRB), which is disrupted in diabetic retinopathy (DR) and uveitis, is an important anatomical characteristic of the retina, regulating nutrient, waste, water, protein, and immune cell flux. The BRB is composed of endothelial cell tight junctions, pericytes, astrocyte end feet, a collagen basement membrane, and perivascular macrophages. Despite the importance of the BRB, retinal perivascular macrophage function remains unknown. We found that retinal perivascular macrophages resided on postcapillary venules in the superficial vascular plexus and expressed MHC class II. Using single-cell RNA-Seq, we found that perivascular macrophages expressed a prochemotactic transcriptome and identified platelet factor 4 (Pf4, also known as CXCL4) as a perivascular macrophage marker. We used Pf4Cre mice to specifically deplete perivascular macrophages. To model retinal inflammation, we performed intraocular CCL2 injections. Ly6C+ monocytes crossed the BRB proximal to perivascular macrophages. Depletion of perivascular macrophages severely hampered Ly6C+ monocyte infiltration. These data suggest that retinal perivascular macrophages orchestrate immune cell migration across the BRB, with implications for inflammatory ocular diseases including DR and uveitis.

Uveitis is characterised by breakdown of the blood-retinal barrier (BRB), allowing infiltration of immune cells that mediate intraocular inflammation, which can lead to irreversible damage of the neuroretina and the loss of sight. Treatment of uveitis relies heavily on corticosteroids and systemic immunosuppression due to limited understanding of disease pathogenesis. We performed single-cell RNA-sequencing of retinas, as well as bulk RNA-sequencing of retinal pigment epithelial (RPE) cells from mice with experimental autoimmune uveitis (EAU) versus healthy control. This revealed that the Th1/Th17-driven disease induced strong gene expression changes in response to inflammation in rods, cones, Müller glia and RPE. In particular, Müller glia and RPE cells were found to upregulate expression of chemokines, complement factors, leukocyte adhesion molecules and MHC class II, thus highlighting their contributions to immune cell recruitment and antigen presentation at the inner and outer BRB, respectively. Additionally, ligand-receptor interaction analysis with CellPhoneDB revealed key interactions between Müller glia and T cell / natural killer cell subsets via chemokines, galectin-9 to P4HB/TIM-3, PD-L1 to PD-1, and nectin-2/3 to TIGIT signalling axes. Our findings elucidate mechanisms contributing to breakdown of retinal immune privilege during uveitis and identify novel targets for therapeutic interventions., (© 2024. The Author(s).)

Age-related macular degeneration (AMD), a prevalent and progressive degenerative disease of the macula, is the leading cause of blindness in elderly individuals in developed countries. The advanced stages include neovascular AMD (nAMD), characterized by choroidal neovascularization (CNV), leading to subretinal fibrosis and permanent vision loss. Despite the efficacy of anti-vascular endothelial growth factor (VEGF) therapy in stabilizing or improving vision in nAMD, the development of subretinal fibrosis following CNV remains a significant concern. In this review, we explore multifaceted aspects of subretinal fibrosis in nAMD, focusing on its clinical manifestations, risk factors, and underlying pathophysiology. We also outline the potential sources of myofibroblast precursors and inflammatory mechanisms underlying their recruitment and transdifferentiation. Special attention is given to the potential role of mast cells in CNV and subretinal fibrosis, with a focus on putative mast cell mediators, tryptase and granzyme B. We summarize our findings on the role of GzmB in CNV and speculate how GzmB may be involved in the pathological transition from CNV to subretinal fibrosis in nAMD. Finally, we discuss the advantages and drawbacks of animal models of subretinal fibrosis and pinpoint potential therapeutic targets for subretinal fibrosis., Competing Interests: Author DG is a Co-Founder and serves as the Chief Scientific Officer for viDA Therapeutics. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Gill, Yoo, Chakravarthy, Granville and Matsubara.)

Background: Pharmacologic immunosuppression regimes are commonly employed in stem cell clinical trials to mitigate host immune rejection and promote survival and viability of transplanted cells. Immunosuppression and cell survival has been extensively studied in retinal and spinal tissues. The applicability of stem cell therapy is rapidly expanding to other sensory organs such as the ear and hearing. As regenerative therapy is directed to new areas, a greater understanding of immunosuppression strategies and their efficacy is required to facilitate translation to organ-specific biologic microenvironments., Objective: This systematic review appraises the current literature regarding immunosuppression strategies employed in stem cell trials of retinal and neural cells., Methods: This systematic review was performed in line with Preferred Reporting Items for Systematic Reviews and Meta-Analyses guidelines. Inclusion criteria included studies presenting data on neural or retinal cells as part of an in-human clinical trial that detailed the immunosuppression regime used. Exclusion criteria included non-English language studies, animal studies, review articles, case reports, editorials, and letters. The databases Medline, Embase, Scopus, Web of Science, and the Cochrane Library were searched from inception to February 2024. Risk of bias was evaluated using the ROBINS-I tool., Results: Eighteen articles fit the inclusion criteria. Nine articles concerned retinal cells, 5 concerned spinal cord injury, and 4 concerned amyotrophic lateral sclerosis. A multi-drug and short-term immunosuppression regime were commonly employed in the identified studies. Detected immune responses in treated patients were rare. Common immunosuppression paradigms included tacrolimus, mycophenolate mofetil and tapering doses of steroids. Local immunosuppression with steroids was employed in some studies concerning retinal diseases., Discussion: A short-term course of systemic immunosuppression seemed efficacious for most included studies, with some showing grafted cells viable months to years after immunosuppression had stopped. Longer-term follow-up is required to see if this remains the case. Side effects related to immunosuppression were uncommon., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Gowrishankar et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Objective: To examine the association between islet autoantibodies (IAbs) and the retinal neurovascular changes in type 1 diabetes mellitus (T1DM) with no diabetic retinopathy (NDR)., Methods: This cross-sectional study measured the neural retinal structure and microvascular density of 118 NDR eyes using spectral-domain optical coherence tomography angiography. Retinal structure parameters included retinal thickness (RT), inner retinal thickness (iRT), retina never fibral layer thickness (RNFL thickness), ganglion cell complex thickness (GCC thickness), and loss volume of GCC. Microvascular parameters included vessel density of superficial capillary plexus (sVD), vessel density of deep capillary plexus, and vessel density of choroid capillary plexus. Comparison and correlation analyses of these OCTA parameters were made with various IAbs, including glutamic acid decarboxylase antibody (GADA), tyrosine phosphatase-related islet antigen 2 antibody (IA2A), and zinc transporter 8 antibody (ZnT8A). A general linear model was used to understand the association of IAbs with the retina parameters., Results: The IAb positive (IAbs +) group, which included 85 patients, had thinner RT (235.20 ± 18.10 mm vs. 244.40 ± 19.90 mm at fovea, P = 0.021) and thinner iRT (120.10 ± 9.00 mm vs. 124.70 ± 6.90 mm at parafovea, P = 0.015), compared with the IAb negative (IAbs-) group comprising 33 patients. Furthermore, a more severe reduction of RT was demonstrated in the presence of multiple IAbs. Among the three IAbs, GADA was the most significant independent risk factor of all-round RT decrease (β = -0.20 vs. -0.27 at fovea and parafovea, respectively, P < 0.05), while titers of IA2A negatively affect sVD in the parafovea (β = -0.316, P = 0.003)., Conclusions: IAbs are associated with neural retinal thinning and microcirculation reduction in T1DM patients before the clinical onset of diabetic retinopathy., (© 2024. Springer-Verlag Italia S.r.l., part of Springer Nature.)

Purpose: To characterize and compare clinical and immunological features of para(p)-autoimmune retinopathy (AIR) and non-para(np)-AIR and to assess the clinical significance of the presence of serum anti-retinal antibodies (ARAs)., Methods: We retrospectively reviewed 48 Chinese patients with p-AIR or np-AIR who took comprehensive ophthalmic examinations and lab tests of the presence of serum ARAs., Results: p-AIR patients differed from np-AIR patients in terms of disease progression, ocular inflammation, findings of OCT, FFA, and presence of ARAs. No significant difference was found in the band number of serum ARAs between AIR patients and healthy controls. The prevalence of antibodies to recoverin and ɑ-enolase in the sera of p-AIR was significantly higher than that of the healthy individuals., Conclusion: While having many similar clinical signs, patients with p-AIR or np-AIR nevertheless displayed unique characteristics. Detection of ARAs subtypes, rather than their quantity, may be helpful in evaluating the conditions in the verified instances.

The roles of immune cell infiltration and ferroptosis in the progression of proliferative diabetic retinopathy (PDR) remain unclear. To identify upregulated molecules associated with immune infiltration and ferroptosis in PDR, GSE60436 and GSE102485 datasets were downloaded from the Gene Expression Omnibus (GEO). Genes associated with immune cell infiltration were examined through Weighted Gene Co-expression Network Analysis (WGCNA) and CIBERSORT algorithm. Common differentially expressed genes (DEGs) were intersected with ferroptosis-associated and immune cell infiltration-related genes. Localization of cellular expression was confirmed by single-cell analysis of GSE165784 dataset. Findings were validated by qRT-PCR, ELISA, Western blotting, and immunofluorescence staining. As a result, the infiltration of M2 macrophages was significantly elevated in fibrovascular membrane samples from PDR patients than the retinas of control subjects. Analysis of DEGs, M2 macrophage-related genes and ferroptosis-related genes identified three hub intersecting genes, TP53, HMOX1 and PPARA. qRT-PCR showed that HMOX1 was significantly higher in the oxygen-induced retinopathy (OIR) mouse model retinas than in controls. Single-cell analysis confirmed that HMOX1 was located in M2 macrophages. ELISA and western blotting revealed elevated levels of HMOX1 in the vitreous humor of PDR patients and OIR retinas, and immunofluorescence staining showed that HMOX1 co-localized with M2 macrophages in the retinas of OIR mice. This study offers novel insights into the mechanisms associated with immune cell infiltration and ferroptosis in PDR. HMOX1 expression correlated with M2 macrophage infiltration and ferroptosis, which may play a crucial role in PDR pathogenesis., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Several gaps and barriers remain for transplanting stem cells into the eye to treat ocular disease, especially diseases of the retina. While the eye has historically been considered immune privileged, recent thinking has identified the immune system as both a barrier and an opportunity for eye stem cell transplantation. Recent approaches leveraging scaffolds or cloaking have been considered in other tissues beyond immune suppression. This perspective paper outlines approaches for transplantation and proposes opportunities to overcome barriers of the immune system in stem cell transplantation in the eye., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

The complement cascade is a vital system in the human body's defense against pathogens. During the natural aging process, it has been observed that this system is imperative for ensuring the integrity and homeostasis of the retina. While this system is critical for proper host defense and retinal integrity, it has also been found that dysregulation of this system may lead to certain retinal pathologies, including geographic atrophy and diabetic retinopathy. Targeting components of the complement system for retinal diseases has been an area of interest, and in vivo, ex vivo, and clinical trials have been conducted in this area. Following clinical trials, medications targeting the complement system for retinal disease have also become available. In this manuscript, we discuss the pathophysiology of complement dysfunction in the retina and specific pathologies. We then describe the results of cellular, animal, and clinical studies targeting the complement system for retinal diseases. We then provide an overview of complement inhibitors that have been approved by the Food and Drug Administration (FDA) for geographic atrophy. The complement system in retinal diseases continues to serve as an emerging therapeutic target, and further research in this field will provide additional insights into the mechanisms and considerations for treatment of retinal pathologies.

Background: Diabetic retinopathy (DR) stands as a prominent complication of diabetes. Berberine (BBR) has reported to be effective to ameliorate the retinal damage of DR. Studying the potential immunological mechanisms of BBR on the streptozotocin (STZ) induced DR mouse model will explain the therapeutic mechanisms of BBR and provide theoretical basis for the clinical application of this drug., Methods: C57BL/6 J mice were induced into a diabetic state using a 50 mg/(kg·d) dose of STZ over a 5-day period. Subsequently, they were subjected to a high-fat diet (HFD) for one month. Following a 5-week treatment with 100 mg/(kg·d) BBR, the concentrations of inflammatory factors in the mice's peripheral blood were determined using an enzyme-linked immunosorbent assay (ELISA). Hematoxylin-eosin staining was employed to scrutinize pathological changes in the mice's retinas, while flow cytometry assessed the proportions of T-lymphocyte subsets and the activation status of dendritic cells (DCs) in the spleen and lymph nodes. CD4 + T cells and DC2.4 cell lines were utilized to investigate the direct and indirect effects of BBR on T cells under high glucose conditions in vitro., Results: Following 5 weeks of BBR treatment in the streptozotocin (STZ) mouse model of DR, we observed alleviation of retinal lesions and a down-regulation in the secretion of inflammatory cytokines, namely TNF-α, IL-1β, and IL-6, in the serum of these mice. And in the spleen and lymph nodes of these mice, BBR inhibited the proportion of Th17 cells and promoted the proportion of Treg cells, thereby down-regulating the Th17/Treg ratio. Additionally, in vitro experiments, BBR directly inhibited the expression of the transcription factor RORγt and promoted the expression of the transcription factor Foxp3 in T cells, resulting in a down-regulation of the Th17/Treg ratio. Furthermore, BBR indirectly modulated the Th17/Treg ratio by suppressing the secretion of TNF-α, IL-1β, and IL-6 by DCs and enhancing the secretion of indoleamine 2,3-dioxygenase (IDO) and transforming growth factor-beta (TGF-β) by DCs. This dual action inhibited Th17 cell differentiation while promoting Treg cells., Conclusion: Our findings indicate that BBR regulate T cell subpopulation differentiation, reducing the Th17/Treg ratio by directly or indirectly pathway. This represents a potential therapeutic avenue of BBR for improving diabetic retinopathy., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 European Federation of Immunological Societies. Published by Elsevier B.V. All rights reserved.)

Background: Diabetic retinopathy (DR) stands as a prevalent secondary complication of diabetes, notably Type 1 Diabetes Mellitus (T1D), characterized by immune system involvement potentially impacting the retinal immune response mediated by microglia. Early stages of DR witness blood-retinal barrier permeabilization, facilitating peripheral immune cell interaction with the retinal immune system. Kaempferol (Kae), known for its potent anti-inflammatory activity, presents a promising avenue in DR treatment by targeting the immune mechanisms underlying its onset and progression. Our investigation delves into the molecular intricacies of innate immune cell interaction during DR progression and the attenuation of inflammatory processes pivotal to its pathology., Methods: Employing in vitro studies, we exposed HAPI microglial and J774.A1 macrophage cells to pro-inflammatory stimuli in the presence or absence of Kae. Ex vivo and in vivo experiments utilized BB rats, a T1D animal model. Retinal explants from BB rats were cultured with Kae, while intraperitoneal Kae injections were administered to BB rats for 15 days. Quantitative PCR, Western blotting, immunofluorescence, and Spectral Domain - Optical Coherence Tomography (SD-OCT) facilitated survival assessment, cellular signaling analysis, and inflammatory marker determination., Results: Results demonstrate Kae significantly mitigates inflammatory processes across in vitro, ex vivo, and in vivo DR models, primarily targeting immune cell responses. Kae administration notably inhibits proinflammatory responses during DR progression while promoting an anti-inflammatory milieu, chiefly through microglia-mediated synthesis of Arginase-1 and Hemeoxygenase-1(HO-1). In vivo, Kae administration effectively preserves retinal integrity amid DR progression., Conclusions: Our findings elucidate the interplay between retinal and systemic immune cells in DR progression, underscoring a differential treatment response predominantly orchestrated by microglia's anti-inflammatory action. Kae treatment induces a phenotypic and functional shift in immune cells, delaying DR progression, thereby spotlighting microglial cells as a promising therapeutic target in DR management., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Age-related macular degeneration (AMD) is a degenerative ocular disease primarily affecting central vision in the elderly. Its pathogenesis is complex, involving cellular senescence and immune homeostasis dysregulation. This review investigates the interaction between these two critical biological processes in AMD pathogenesis and their impact on disease progression. Initially, cellular senescence is analyzed, with particular emphasis on retinal damage induced by senescent retinal pigment epithelial cells. Subsequently, the occurrence of immune homeostasis dysregulation within the retina and its mechanistic role in AMD areis explored. Furthermore, the paper also discusses in detail the interplay between cellular senescence and immune responses, forming a vicious cycle that exacerbates retinal damage and may influence treatment outcomes. In summary, a deeper understanding of the interrelation between cellular senescence and immune dysregulation is vital for the developing innovative therapeutic strategies for AMD., Competing Interests: The authors report no conflicts of interest in this work., (© 2024 Li et al.)

Background and Objectives : Age-related macular degeneration (AMD) is one of the leading causes of central vision loss among elderly patients, and its dry form accounts for the majority of cases. Although several causes and mechanisms for the development and progression of AMD have previously been identified, the pathogenesis of this complex disease is still not entirely understood. As inflammation and immune system involvement are strongly suggested to play a central role in promoting the degenerative process and stimulating the onset of complications, we aimed to analyze the frequency of serum anti-retinal (ARAs) and anti-endothelial cell antibodies (AECAs) in patients with dry AMD and to determine their relationship with the clinical features of the disease, notably the area of geographic atrophy (GA). Materials and Methods : This study included 41 patients with advanced-stage dry AMD and 50 healthy controls without AMD, matched for gender and age. ARAs were detected by indirect immunofluorescence using monkey retina as an antigen substrate, and the presence of AECAs was determined using cultivated human umbilical vein endothelial cells and primate skeletal muscle. Results: ARAs were detected in 36 (87.8%) AMD patients (titers ranged from 1:20 to 1:320) and in 16 (39.0%) (titers ranged from 1:10 to 1:40) controls ( p = 0.0000). Twenty of the forty-one patients (48.8%) were positive for AECAs, while in the control group, AECAs were present only in five sera (10.0%). The titers of AECAs in AMD patients ranged from 1:100 to 1:1000, and in the control group, the AECA titers were 1:100 ( p = 0.0001). There were no significant correlations between the presence of AECAs and disease activity. Conclusions : This study demonstrates a higher prevalence of circulating AECAs in patients with dry AMD; however, no correlation was found between the serum levels of these autoantibodies and the area of GA.

Introduction: Macrophage function is determined by microenvironment and origin. Brain and retinal microglia are both derived from yolk sac progenitors, yet their microenvironments differ. Utilizing single-cell RNA sequencing (scRNA-seq) data from mice, we tested the hypothesis that retinal and brain microglia exhibit distinct transcriptional profiles due to their unique microenvironments., Methods: Eyes and brains from 2-4 month wildtype mice were combined (20 eyes; 3 brains) to yield one biologically diverse sample per organ. Each tissue was digested into single cell suspensions, enriched for immune cells, and sorted for scRNA-seq. Analysis was performed in Seurat v3 including clustering, integration, and differential expression. Multi-parameter flow cytometry was used for validation of scRNA-seq results. Lymphocytic choriomeningitis virus (LCMV) Clone 13, which produces a systemic, chronic, and neurotropic infection, was used to validate scRNA-seq and flow cytometry results in vivo ., Results: Cluster analysis of integrated gene expression data from eye and brain identified 6 Tmem119 + P2ry12 + microglial clusters. Differential expression analysis revealed that eye microglia were enriched for more pro-inflammatory processes including antigen processing via MHC class I (14.0-fold, H2-D1 and H2-K1 ) and positive regulation of T-cell immunity (8.4-fold) compared to brain microglia. Multi-parameter flow cytometry confirmed that retinal microglia expressed 3.2-fold greater H2-Db and 263.3-fold more H2-Kb than brain microglia. On Day 13 and 29 after LCMV infection, CD8 + T-cell density was greater in the retina than the brain., Discussion: Our data demonstrate that the microenvironment of retina and brain differs, resulting in microglia-specific gene expression changes. Specifically, retinal microglia express greater MHC class I by scRNA-seq and multi-parameter flow cytometry, resulting in a possibly enhanced capability to stimulate CD8 + T-cell inflammation during LCMV infection. These results may explain tissue-specific differences between retina and brain during systemic viral infections and CD8 + T-cell driven autoimmune disease., Competing Interests: JL is a consultant for Line 6 Biotechnology and Genentech. JAL received research grant support from Therini Bio. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2024 Bloomfield, Gong, Droho, Makinde, Gurra, Stumpf, Kharel, Gadhvi, Winter, Cui, Cuda and Lavine.)

Diabetic retinopathy (DR) is recognized as a neurovascular complication of diabetes, and emerging evidence underscores the pivotal role of inflammation in its pathophysiology. Macrophage activation is increasingly acknowledged as a key contributor to the onset and progression of DR. Different populations of macrophages originating from distinct sources contribute to DR-associated inflammation. Retinal macrophages can be broadly categorized into two main groups based on their origin: intrinsic macrophages situated within the retina and vitreoretinal interface and macrophages derived from infiltrating monocytes. The former comprises microglia (MG), perivascular macrophages, and macrophage-like hyalocytes. Retinal MG, as the principal population of tissue-resident population of mononuclear phagocytes, exhibits high heterogeneity and plasticity while serving as a crucial connector between retinal capillaries and synapses. This makes MG actively involved in the pathological processes across various stages of DR. Activated hyalocytes also contribute to the pathological progression of advanced DR. Additionally, recruited monocytes, displaying rapid turnover in circulation, augment the population of retinal macrophages during DR pathogenesis, exerting pathogenic or protective effect based on different subtypes. In this review, we examine novel perspectives on macrophage biology based on recent studies elucidating the diversity of macrophage identity and function, as well as the mechanisms influencing macrophage behavior. These insights may pave the way for innovative therapeutic strategies in the management of DR., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Background: Dimethyl fumarate (DMF) is a fumaric acid ester that exhibits immunoregulatory and anti-inflammatory properties. However, the function of DMF in autoimmune uveitis (AU) is incompletely understood, and studies comprehensively exploring the impact of DMF on immune cells are still lacking., Methods: To explore the function of DMF in uveitis and its underlying mechanisms, we conducted single-cell RNA sequencing (scRNA-seq) on the cervical draining lymph node (CDLN) cells of normal, experimental autoimmune uveitis (EAU), and DMF-treated EAU mice. Additionally, we integrated scRNA-seq data of the retina and CDLNs to identify the potential impact of DMF on ocular immune cell infiltration. Flow cytometry was conducted to verify the potential target molecules of DMF., Results: Our study showed that DMF treatment effectively ameliorated EAU symptoms. The proportional and transcriptional alterations in each immune cell type during EAU were reversed by DMF treatment. Bioinformatics analysis in our study indicated that the enhanced expression of Pim1 and Cxcr4 in EAU was reversed by DMF treatment. Further experiments demonstrated that DMF restored the balance between effector T (Teff) /regulatory T (Treg) cells through inhibiting the pathway of PIM1-protein kinase B (AKT)-Forkhead box O1 (FOXO1). By incorporating the scRNA-seq data of the retina from EAU mice into analysis, our study identified that T cells highly expressing Pim1 and Cxcr4 were enriched in the retina. DMF repressed the ocular infiltration of Teff cells, and this effect might depend on its inhibition of PIM1 and CXCR4 expression. Additionally, our study indicated that DMF might reduce the proportion of plasma cells by inhibiting PIM1 expression in B cells., Conclusions: DMF effectively attenuated EAU symptoms. During EAU, DMF reversed the Teff/Treg cell imbalance and suppressed the ocular infiltration of Teff cells by inhibiting PIM1 and CXCR4 expression. Thus, DMF may act as a new drug option for the treatment of AU., (© 2024. The Author(s).)

Blindness or vision loss due to neuroretinal and photoreceptor degeneration affects millions of individuals worldwide. In numerous neurodegenerative diseases, including age-related macular degeneration, dysregulated immune response-mediated retinal degeneration has been found to play a critical role in the disease pathogenesis. To better understand the pathogenic mechanisms underlying the retinal degeneration, we used a mouse model of systemic immune activation where we infected mice with lymphocytic choriomeningitis virus (LCMV) clone 13. Here, we evaluated the effects of LCMV infection and present a comprehensive discovery-based proteomic investigation using tandem mass tag (TMT) labeling and high-resolution liquid chromatography-tandem mass spectrometry (LC-MS/MS). Changes in protein regulation in the posterior part of the eye, neuroretina, and RPE/choroid were compared to those in the spleen as a secondary lymphoid organ and to the kidney as a non-lymphoid but encapsulated organ at 1, 8, and 28 weeks of infection. Using bioinformatic tools, we found several proteins responsible for maintaining normal tissue homeostasis to be differentially regulated in the neuroretina and the RPE/choroid during the degenerative process. Additionally, in the organs we observed, several important protein pathways contributing to cellular homeostasis and tissue development were perturbed and associated with LCMV-mediated inflammation, promoting disease progression. Our findings suggest that the response to a systemic chronic infection differs between the neuroretina and the RPE/choroid, and the processes induced by chronic systemic infection in the RPE/choroid are not unlike those induced in non-immune-privileged organs such as the kidney and spleen. Overall, our data provide detailed insight into several molecular mechanisms of neuroretinal degeneration and highlight various novel protein pathways that further suggest that the posterior part of the eye is not an isolated immunological entity despite the existence of neuroretinal immune privilege., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Khan, Steffensen, Paskeviciute, Abduljabar, Sørensen, Vorum, Nissen and Honoré.)

SARS-CoV-2 has been shown to cause wide-ranging ocular abnormalities and vision impairment in COVID-19 patients. However, there is limited understanding of SARS-CoV-2 in ocular transmission, tropism, and associated pathologies. The presence of viral RNA in corneal/conjunctival tissue and tears, along with the evidence of viral entry receptors on the ocular surface, has led to speculation that the eye may serve as a potential route of SARS-CoV-2 transmission. Here, we investigated the interaction of SARS-CoV-2 with cells lining the blood-retinal barrier (BRB) and the role of the eye in its transmission and tropism. The results from our study suggest that SARS-CoV-2 ocular exposure does not cause lung infection and moribund illness in K18-hACE2 mice despite the extended presence of viral remnants in various ocular tissues. In contrast, intranasal exposure not only resulted in SARS-CoV-2 spike (S) protein presence in different ocular tissues but also induces a hyperinflammatory immune response in the retina. Additionally, the long-term exposure to viral S-protein caused microaneurysm, retinal pigmented epithelium (RPE) mottling, retinal atrophy, and vein occlusion in mouse eyes. Notably, cells lining the BRB, the outer barrier, RPE, and the inner barrier, retinal vascular endothelium, were highly permissive to SARS-CoV-2 replication. Unexpectedly, primary human corneal epithelial cells were comparatively resistant to SARS-CoV-2 infection. The cells lining the BRB showed induced expression of viral entry receptors and increased susceptibility towards SARS-CoV-2-induced cell death. Furthermore, hyperglycemic conditions enhanced the viral entry receptor expression, infectivity, and susceptibility of SARS-CoV-2-induced cell death in the BRB cells, confirming the reported heightened pathological manifestations in comorbid populations. Collectively, our study provides the first evidence of SARS-CoV-2 ocular tropism via cells lining the BRB and that the virus can infect the retina via systemic permeation and induce retinal inflammation., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Monu et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

To study the macroglia and microglia and the immune role in long-time light exposure in rat eyes, we performed glial cell characterization along the time-course of retinal degeneration induced by chronic exposure to low-intensity light. Animals were exposed to light for periods of 2, 4, 6, or 8 days, and the retinal glial response was evaluated by immunohistochemistry, western blot and real-time reverse transcription polymerase chain reaction. Retinal cells presented an increased expression of the macroglia marker GFAP, as well as increased mRNA levels of microglia markers Iba1 and CD68 after 6 days. Also, at this time-point, we found a higher number of Iba1-positive cells in the outer nuclear layer area; moreover, these cells showed the characteristic activated-microglia morphology. The expression levels of immune mediators TNF, IL-6, and chemokines CX3CR1 and CCL2 were also significantly increased after 6 days. All the events of glial activation occurred after 5-6 days of constant light exposure, when the number of photoreceptor cells has already decreased significantly. Herein, we demonstrated that glial and immune activation are secondary to neurodegeneration; in this scenario, our results suggest that photoreceptor death is an early event that occurs independently of glial-derived immune responses.

Noninfectious (autoimmune and immune-mediated) uveitis is one of the primary diseases leading to blindness in the world. Due to the limitation of current first-line drugs for clinical uveitis, novel drugs and targets against uveitis are urgently needed. Ganciclovir (GCV), an FDA-approved antiviral drug, is often used to treat cytomegalovirus-induced retinitis in clinical patients. Recently, GCV was found to suppress neuroinflammation via targeting STING signaling because the STING pathway plays a pivotal role in autoimmune diseases. However, until now, the effect of GCV on non-infectious uveitis has never been explored. In this work, using the rat experimental autoimmune uveitis (EAU) model, we first found STING to be highly expressed in infiltrating cells (CD68 + , CD45 + , and CD4 + ) and retinal glial cells (Iba1 + and GFAP + ) of the immunized retina. More importantly, GCV treatment can significantly suppress the initiation and progression of EAU by inhibiting infiltration of Th17 and inflammatory cells into the retina. Mechanistically, we found that GCV could reverse the levels of pro-inflammatory factors (such as IL-1β) and chemokine-related factors (such as Cxcr3), possibly via targeting the STING pathway. The present results suggest that GCV may be considered as a novel therapeutic strategy against human uveitis., (Copyright © 2022. Published by Elsevier Inc.)

Considering the fact that many retinal diseases are yet to be cured, the pathomechanisms of these multifactorial diseases need to be investigated in more detail. Among others, oxidative stress and hypoxia are pathomechanisms that take place in retinal diseases, such as glaucoma, age-related macular degeneration, or diabetic retinopathy. In consideration of these diseases, it is also evidenced that the immune system, including the complement system and its activation, plays an important role. Suitable models to investigate neuroretinal diseases are organ cultures of porcine retina. Based on an established model, the role of the complement system was studied after the induction of oxidative stress or hypoxia. Both stressors led to a loss of retinal ganglion cells (RGCs) accompanied by apoptosis. Hypoxia activated the complement system as noted by higher C3 + and MAC + cell numbers. In this model, activation of the complement cascade occurred via the classical pathway and the number of C1q + microglia was increased. In oxidative stressed retinas, the complement system had no consideration, but strong inflammation took place, with elevated TNF , IL6 , and IL8 mRNA expression levels. Together, this study shows that hypoxia and oxidative stress induce different mechanisms in the porcine retina inducing either the immune response or an inflammation. Our findings support the thesis that the immune system is involved in the development of retinal diseases. Furthermore, this study is evidence that both approaches seem suitable models to investigate undergoing pathomechanisms of several neuroretinal diseases.

Purpose: The purpose of this study was to elucidate the effects of interleukin (IL)-38 on experimental autoimmune uveitis (EAU) and its underlying mechanisms., Methods: Mice with EAU were treated with IL-38, and the retinas and cervical draining lymph nodes (CDLNs) were analyzed by flow cytometry. Single-cell RNA sequencing (scRNA-seq) was conducted to analyze the immune cell profiles of CDLNs from normal, EAU, and IL-38-treated mice., Results: Administration of IL-38 attenuated EAU symptoms and reduced the proportion of T helper 17 (Th17) and T helper 1 (Th1) cells in the retinas and CDLNs. In scRNA-seq analysis, IL-38 downregulated the IL-17 signaling pathway and reduced the expression of Th17 cell pathogenicity-related genes (Csf2 and Il23r), findings which were also confirmed by flow cytometry. In vitro, IL-38 reduced the granulocyte-macrophage colony-stimulating factor (GM-CSF) stimulation function of IL-23 and inhibited IL-23R expression in Th17 cells. Moreover, when co-cultured with Th17 cells, IL-38 prevented IL-23 production in antigen-presenting cells (APCs)., Conclusions: Our data demonstrate the therapeutic effect of IL-38 on EAU, and suggest that the effect of IL-38 may be caused by dampening of the GM-CSF/IL-23R/IL-23 feedback loop between Th17 cells and APCs.

Diabetic retinopathy is a frequent complication of longstanding diabetes, which comprises a complex interplay of microvascular abnormalities and neurodegeneration. Zebrafish harboring a homozygous mutation in the pancreatic transcription factor pdx1 display a diabetic phenotype with survival into adulthood, and are therefore uniquely suitable among zebrafish models for studying pathologies associated with persistent diabetic conditions. We have previously shown that, starting at three months of age, pdx1 mutants exhibit not only vascular but also neuro-retinal pathologies manifesting as photoreceptor dysfunction and loss, similar to human diabetic retinopathy. Here, we further characterize injury and regenerative responses and examine the effects on progenitor cell populations. Consistent with a negative impact of hyperglycemia on neurogenesis, stem cells of the ciliary marginal zone show an exacerbation of aging-related proliferative decline. In contrast to the robust Müller glial cell proliferation seen following acute retinal injury, the pdx1 mutant shows replenishment of both rod and cone photoreceptors from slow-cycling, neurod-expressing progenitors which first accumulate in the inner nuclear layer. Overall, we demonstrate a diabetic retinopathy model which shows pathological features of the human disease evolving alongside an ongoing restorative process that replaces lost photoreceptors, at the same time suggesting an unappreciated phenotypic continuum between multipotent and photoreceptor-committed progenitors.

Confocal scanning laser ophthalmoscopy (cSLO) is a non-invasive technique for real-time imaging of the retina. We developed a step-by-step protocol for the semi-automatic evaluation of myeloid cells in cSLO images from CX3CR1 GFP mice, expressing green fluorescent protein (GFP) under control of the endogenous CX3C chemokine receptor 1 locus. We identified cSLO parameters allowing us to distinguish animals with experimental autoimmune encephalomyelitis (EAE) from sham-treated/naïve animals. Especially cell count (CC) and the total microglial area (SuA) turned out to be reliable parameters. Comparing the cSLO results with clinical parameters, we found significant correlations between the clinical EAE score and the SuA and of the inner retinal layer thickness, measured by optical coherence tomography, with the CC as well as the SuA. As a final step, we performed immunohistochemistry to confirm that the GFP-expressing cells visualized by the cSLO are Iba1 positive and validated the step-by-step protocol against manual counting. We present a semi-automatic step-by-step protocol with a balance between fast data evaluation and adequate accuracy, which is optimized by the option to manually adapt the contrast threshold. This protocol may be useful for numerous research questions on the role of microglial polarization in models of inflammatory and degenerating CNS diseases involving the retina., Competing Interests: The following financial disclosures are unrelated to the work: H-PH has received fees for serving on steering and data monitoring committees from Bayer Healthcare, Biogen, Celgene BMS, CSL Behring, GeNeuro, MedImmune, Merck, Novartis, Octapharma, Roche, Sanofi Genzyme, TG Therapeutic sand Viela Bio; fees for serving on advisory boards from Biogen, Sanofi Genzyme, Merck, Novartis, Octapharma, and Roche; and lecture fees from Biogen, Celgene BMS, Merck, Novartis, Roche, Sanofi Genzyme. SM received honoraria for lecturing and travel expenses for attending meetings from Almirall, Amicus Therapeutics Germany, Bayer Health Care, Biogen, Celgene, Diamed, Genzyme, MedDay Pharmaceuticals, Merck Serono, Novartis, Novo Nordisk, ONO Pharma, Roche, Sanofi-Aventis, Chugai Pharma, QuintilesIMS, and Teva. His research is funded by the German Ministry for Education and Research (BMBF), Deutsche Forschungsgemeinschaft (DFG), Else Kröner Fresenius Foundation, German Academic Exchange Service, Hertie Foundation, Interdisciplinary Center for Clinical Studies (IZKF) Muenster, German Foundation Neurology, and by Almirall, Amicus Therapeutics Germany, Biogen, Diamed, Fresenius Medical Care, Genzyme, Merck Serono, Novartis, ONO Pharma, Roche, and Teva. MD received speaker honoraria from Merck. PA received compensation for serving on Scientific Advisory Boards for Ipsen, Novartis, Biogen; he received speaker honoraria and travel support from Novartis, Teva, Biogen, Merz Pharmaceuticals, Ipsen, Allergan, Bayer Healthcare, Esai, UCB and Glaxo Smith Kline; he received research support from Novartis, Biogen, Teva, Merz Pharmaceuticals, Ipsen, and Roche. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Frenger, Hecker, Sindi, Issberner, Hartung, Meuth, Dietrich and Albrecht.)

Diabetic retinopathy (DR), as a major cause of blindness worldwide, is one common complication of diabetes mellitus. Inflammatory response and oxidative stress injury of endothelial cells play significant roles in the pathogenesis of DR. The study is aimed at investigating the effects of lysophosphatidylcholine (LPC) on the dysfunction of high glucose- (HG-) treated human retinal microvascular endothelial cells (HRMECs) after being cocultured with bone marrow mesenchymal stem cells (BMSCs) and the underlying regulatory mechanism. Coculture of BMSCs and HRMECs was performed in transwell chambers. The activities of antioxidant-related enzymes and molecules of oxidative stress injury and the contents of inflammatory cytokines were measured by ELISA. Flow cytometry analyzed the apoptosis of treated HRMECs. HRMECs were further treated with 10-50  μ g/ml LPC to investigate the effect of LPC on the dysfunction of HRMECs. Western blotting was conducted to evaluate levels of TLR4 and p-NF- κ B proteins. We found that BMSCs alleviated HG-induced inflammatory response and oxidative stress injury of HRMECs. Importantly, LPC offsets the protective effects of BMSCs on inflammatory response and oxidative stress injury of HRMECs. Furthermore, LPC upregulated the protein levels of TLR4 and p-NF- κ B, activating the TLR4/NF- κ B signaling pathway. Overall, our study demonstrated that LPC offsets the protective effects of BMSCs on inflammatory response and oxidative stress injury of HRMECs via TLR4/NF- κ B signaling., Competing Interests: The authors declare that there are no competing interests in this study., (Copyright © 2021 Haijun Zhao and Yanhui He.)

Endophthalmitis is a devastating infection that can cause blindness. Over half of Bacillus endophthalmitis cases result in significant loss of useful vision. Bacillus produces many virulence factors that may contribute to retinal damage and robust inflammation. We analyzed Bacillus immune inhibitor A (InhA) metalloproteases in the context of this disease, hypothesizing that InhAs contribute to Bacillus intraocular virulence and inflammation. We analyzed phenotypes and infectivity of wild-type (WT), InhA1-deficient (Δ inhA1 ), InhA2-deficient (Δ inhA2 ), or InhA1, A2, and A3-deficient (Δ inhA1-3 ) Bacillus thuringiensis. In vitro analysis of growth, proteolysis, and cytotoxicity were compared. WT and InhA mutants were similarly cytotoxic to retinal cells. The Δ inhA1 and Δ inhA2 mutants entered log-phase growth earlier than WT B. thuringiensis. Proteolysis by the Δ inhA1-3 mutant was decreased, but this strain grew similar to WT in vitro . Experimental endophthalmitis was initiated by intravitreally infecting C57BL/6J mice with 200 CFU of WT B. thuringiensis or InhA mutants. Eyes were analyzed for intraocular Bacillus and myeloperoxidase concentrations, retinal function loss, and gross histological changes. Eyes infected with the Δ inhA1 or Δ inhA2 mutant strains contained greater numbers of bacteria than eyes infected with WT throughout the infection course. Eyes infected with single mutants had inflammation and retinal function loss similar to eyes infected with the WT strain. Eyes infected with the Δ inhA1-3 mutant cleared the infection. Quantitative real-time PCR (qRT-PCR) results suggested that there may be compensatory expression of the other InhAs in the single InhA mutant. These results indicate that together, the InhA metalloproteases contribute to the severity of infection and inflammation in Bacillus endophthalmitis.

Competing Interests: AT has a sponsored research agreement and is a consultant with Palatin Technologies Inc. (Cranbury, New Jersey). HX has a sponsored research agreement with Boehringer Ingelheim and a consultant agreement with F. Hoffmann-La Roche Ltd. The remaining author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Age-related macular degeneration (AMD) is genetically associated with complement. Dendritic cells (DCs) play key roles during innate and adaptive immunity, and express complement components and their receptors. We investigated ocular DC heterogeneity and the role of DCs in the laser-induced choroidal neovascularization (CNV) model. In order to determine the function of DCs, we used two models of DC deficiency: the Flt3 -/- and Flt3l -/- mouse. We identified three types of ocular DCs: plasmacytoid DC, classical DC-1, and classical DC-2. At steady-state, classical DCs were found in the iris and choroid but were not detectable in the retina. Plasmacytoid DCs existed at very low levels in iris, choroid, and retina. After laser injury, the number of each DC subset was up-regulated in the choroid and retina. In Flt3 -/- mice, we found reduced numbers of classical DCs at steady-state, but each DC subset equally increased after laser injury between wildtype and Flt3 -/- mice. In Flt3l -/- mice, each DC subsets was severely reduced after laser injury. Neither Flt3 -/- or Flt3l -/- mice demonstrated reduced CNV area compared to wildtype mice. DCs do not play any significant role during the laser-induced CNV model of neovascular AMD., (© 2021. The Author(s).)

The ocular tissue microenvironment is immune privileged and uses several mechanisms of immunosuppression to prevent the induction of inflammation. Besides being a blood-barrier and source of photoreceptor nutrients, the retinal pigment epithelial cells (RPE) regulate the activity of immune cells within the retina. These mechanisms involve the expression of immunomodulating molecules that make macrophages and microglial cells suppress inflammation and promote immune tolerance. The RPE have an important role in ocular immune privilege to regulate the behavior of immune cells within the retina. Reviewed is the current understanding of how RPE mediate this regulation and the changes seen under pathological conditions., Competing Interests: AT is a scientific advisor and recipient of a sponsored research agreement from Palatin Technologies Inc. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Taylor, Hsu and Ng.)

Retinitis pigmentosa (RP) is a hereditary disease of the retina that results in complete blindness. Currently, there are very few treatments for the disease and those that exist work only for the recessively inherited forms. To better understand the pathogenesis of RP, multiple mouse models have been generated bearing mutations found in human patients including the human Q344X rhodopsin knock-in mouse. In recent years, the immune system was shown to play an increasingly important role in RP degeneration. By way of electroretinography, optical coherence tomography, funduscopy, fluorescein angiography, and fluorescent immunohistochemistry, we show degenerative and vascular phenotypes, microglial activation, photoreceptor phagocytosis, and upregulation of proinflammatory pathway proteins in the retinas of the human Q344X rhodopsin knock-in mouse. We also show that an FDA-approved pharmacological agent indicated for the treatment of rheumatoid arthritis is able to halt activation of pro-inflammatory signaling in cultured retinal cells, setting the stage for pre-clinical trials using these mice to inhibit proinflammatory signaling in an attempt to preserve vision. We conclude from this work that pro- and autoinflammatory upregulation likely act to enhance the progression of the degenerative phenotype of rhodopsin Q344X-mediated RP and that inhibition of these pathways may lead to longer-lasting vision in not only the Q344X rhodopsin knock-in mice, but humans as well.

Purpose: In spite of clear differences in tissue function and significance to ocular disease, little is known about how immune responses differ between the retina and uveal tract. To this end we compared the effects of acute systemic inflammation on myeloid cells within the mouse retina, iris-ciliary body, and choroid., Methods: Systemic inflammation was induced in Cx3cr1gfp/gfp and CD11c-eYFP Crb1wt/wtmice by intraperitoneal lipopolysaccharide (LPS). In vivo fundus imaging was performed at two, 24, and 48 hours after LPS, and ocular tissue wholemounts were immunostained and studied by confocal microscopy. Flow cytometry was used to investigate the expression of activation markers (MHC class II, CD80, CD86) on myeloid cell populations at 24 hours. For functional studies, retinal microglia were isolated from LPS-exposed mice and cocultured with naïve OT-II CD4+ T-cells and ovalbumin peptide. T-cell proliferation was measured by flow cytometry and cytokine assays., Results: Systemic LPS altered the density and morphology of retinal microglia; however, retinal microglia did not upregulate antigen presentation markers and failed to stimulate naïve CD4+ T-cell proliferation in vitro. In contrast, uveal tract myeloid cells displayed a phenotype consistent with late-activated antigen-presenting cells at 24 hours. Systemic LPS induced remodeling of myeloid populations within the uveal tract, particularly in the choroid, where dendritic cells were partially displaced by macrophages at 24 hours., Conclusions: The disparate myeloid cell responses in the retina and uveal tract after systemic LPS highlight differential regulation of innate immunity within these tissue environments, observations that underpin and advance our understanding of ocular immune privilege.

Interleukin-1α (IL-1α) is an alarmin involved in the recruitment of macrophages and neutrophils during tissue inflammation. IL-1α can undergo cleavage by proteases, such as calpain-1, that enhances IL-1α binding to its receptor, although proteolytic cleavage is not necessary for biological activity. Macrophages and neutrophils are involved in the retinal inflammation associated with development of AIDS-related human cytomegalovirus (HCMV) retinitis. We therefore performed studies to test the hypothesis that IL-1α gene expression is stimulated intraocularly during retinitis development using two mouse models of murine cytomegalovirus (MCMV) retinitis that differ in method of immunosuppression, one by retrovirus-induced immunosuppression (MAIDS) and the other by corticosteroid-induced immunosuppression. MCMV-infected eyes of groups of retinitis-susceptible mice with MAIDS of 10 weeks duration (MAIDS-10 mice) and retinitis-susceptible corticosteroid-treated mice showed significant stimulation of IL-1α mRNA. Western blot analysis confirmed IL-1α protein production within the MCMV-infected eyes of MAIDS-10 mice. Whereas significant intraocular calpain-1 mRNA and protein production were also observed within MCMV-infected eyes of MAIDS-10 mice, the MCMV-infected eyes of retinitis-susceptible corticosteroid-treated mice showed a pattern of mRNA synthesis equivalent to that found within the MCMV-infected eyes of healthy mice that fail to develop retinitis. Our findings suggest a role for the alarmin IL-1α in the pathogenesis of MCMV retinitis in immunosuppressed mice. These findings may extend to the pathogenesis of HCMV retinitis in patients with AIDS or other forms of immunosuppression., (Copyright © 2021 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Autoimmune retinopathy (AIR) is a rare immune-mediated inflammation of the retina. The autoantibodies against retinal proteins and glycolytic enzymes were reported to be involved in the pathogenesis. This retrospective cohort study assessed the antiretinal autoantibody profiles and their association with clinical outcomes of AIR patients in Thailand. We included 44 patients, 75% were females, with the overall median age of onset of 48 (17-74, IQR 40-55.5) years. Common clinical presentations were nyctalopia (65.9%), blurred vision (52.3%), constricted visual field (43.2%), and nonrecordable electroretinography (65.9%). Underlying malignancy and autoimmune diseases were found in 2 and 12 female patients, respectively. We found 41 autoantibodies, with anti-α-enolase (65.9%) showing the highest prevalence, followed by anti-CAII (43.2%), anti-aldolase (40.9%), and anti-GAPDH (36.4%). Anti-aldolase was associated with male gender (P = 0.012, OR 7.11, 95% CI 1.54-32.91). Anti-CAII showed significant association with age of onset (P = 0.025, 95% CI - 17.28 to - 1.24), while anti-α-enolase (P = 0.002, OR 4.37, 95% CI 1.83-10.37) and anti-GAPDH (P = 0.001, OR 1.87, 95% CI 1.32-2.64) were significantly associated with nonrecordable electroretinography. Association between the antibody profiles and clinical outcomes may be used to direct and adjust the treatment plans and provide insights in the pathogenesis of AIR., (© 2021. The Author(s).)

Purpose : To detect circulating retina-specific autoreactive CD4 + T-cells and antiretinal antibodies (ARA) in latent tuberculosis (TB)-associated uveitis or sarcoid uveitis patients. Methods : The presence of crude retinal extract (RE) autoreactive CD4 + T-cells was determined by a highly sensitive flowcytometric-based technique examining co-expression of CD25 and CD134 (OX40) on RE stimulated PBMC. The presence of ARA in available matched serum samples was assessed by indirect immunofluorescence. Results : No autoreactive CD4 + T-cells against RE could be detected in either latent TB-associated uveitis or sarcoid uveitis patients, while ARA were detected in the serum of the majority (5/6) of latent TB-associated uveitis and all (3/3) sarcoid uveitis patients. Conclusion : Even with the use of this highly sensitive flowcytometric technique circulating retina-specific autoreactive CD4 + T-cells could not be detected. In contrast, ARA were detected in the majority of patients indicating an adaptive humoral immune response toward retinal antigens had occurred.

Neuroinflammation has been put forward as a mechanism triggering axonal regrowth in the mammalian central nervous system (CNS), yet little is known about the underlying cellular and molecular players connecting these two processes. In this study, we provide evidence that MMP2 is an essential factor linking inflammation to axonal regeneration by using an in vivo mouse model of inflammation-induced axonal regeneration in the optic nerve. We show that infiltrating myeloid cells abundantly express MMP2 and that MMP2 deficiency results in reduced long-distance axonal regeneration. However, this phenotype can be rescued by restoring MMP2 expression in myeloid cells via a heterologous bone marrow transplantation. Furthermore, while MMP2 deficiency does not affect the number of infiltrating myeloid cells, it does determine the coordinated expression of pro- and anti-inflammatory molecules. Altogether, in addition to its role in axonal regeneration via resolution of the glial scar, here, we reveal a new mechanism via which MMP2 facilitates axonal regeneration, namely orchestrating the expression of pro- and anti-inflammatory molecules by infiltrating innate immune cells.

Neurodegeneration mediates neurological disability in inflammatory demyelinating diseases of the CNS. The role of innate immune cells in mediating this damage has remained controversial with evidence for destructive and protective effects. This has complicated efforts to develop treatment. The time sequence and dynamic evolution of the opposing functions are especially unclear. Given limits of in vivo monitoring in human diseases such as multiple sclerosis (MS), animal models are warranted to investigate the association and timing of innate immune activation with neurodegeneration. Using noninvasive in vivo retinal imaging of experimental autoimmune encephalitis (EAE) in CX3CR1GFP/+-knock-in mice followed by transcriptional profiling, we are able to show 2 distinct waves separated by a marked reduction in the number of innate immune cells and change in cell morphology. The first wave is characterized by an inflammatory phagocytic phenotype preceding the onset of EAE, whereas the second wave is characterized by a regulatory, antiinflammatory phenotype during the chronic stage. Additionally, the magnitude of the first wave is associated with neuronal loss. Two transcripts identified - growth arrest-specific protein 6 (GAS6) and suppressor of cytokine signaling 3 (SOCS3) - might be promising targets for enhancing protective effects of microglia in the chronic phase after initial injury.

This study focuses on gene expression differences between early retinal states that ultimately lead to normal development, late onset retinoblastoma, or rapid bilateral retinoblastoma tumors. The late-onset and early-onset retinoblastoma tumor cells are remarkably similar to normally proliferating retinal progenitor cells, but they fail to properly express differentiation markers associated with normal development. Further, early-onset retinoblastoma tumor cells express a robust immune gene expression signature followed by accumulation of dendritic, monocyte, macrophage, and T-lymphocyte cells in the retinoblastoma tumors. This characteristic was not shared by either normal retinae or late-onset retinoblastomas. Comparison of our data with other human and mouse retinoblastoma tumor gene expression significantly confirmed, that the immune signature is present in tumors from each species. Strikingly, we observed that the immune signature in both mouse and human tumors was most highly evident in those with the lowest proliferative capacity. We directly assessed this relationship in human retinoblastoma tumors by co-analyzing proliferation and immune cell recruitment by immunohistochemistry, uncovering a significant inverse relationship between increased immune-cell infiltration in tumors and reduced tumor cell proliferation. Directly inhibiting proliferation with a PI3K/mTOR inhibitor significantly increased the number of CD45 + immune cells in the retina. This work establishes an in vivo model for the rapid recruitment of immune cells to tumorigenic neural tissue.

Purpose: For this study we aimed to understand if retinal pigment epithelial (RPE) cells express antimicrobial peptide lysozyme as a mechanism to protect the neuroretina from blood-borne pathogens., Methods: The expression of lysozyme in human and mouse RPE cells was examined by RT-PCR or immune (cyto)histochemistry in cell cultures or retinal sections. RPE cultures were treated with different concentrations of Pam3CSK4, lipopolysaccharides (LPS), staphylococcus aureus-derived peptidoglycan (PGN-SA), Poly(I:C), and Poly(dA:dT). The mRNA expression of lysozyme was examined by qPCR and protein expression by ELISA. Poly(I:C) was injected into the subretinal space of C57BL/6J mice and eyes were collected 24 hours later and processed for the evaluation of lysozyme expression by confocal microscopy. Bactericidal activity was measured in ARPE19 cells following LYZ gene deletion using Crispr/Cas9 technology., Results: The mRNA and protein of lysozyme were detected in mouse and human RPE cells under normal conditions, although the expression levels were lower than mouse microglia BV2 or human monocytes THP-1 cells, respectively. Immunohistochemistry showed punctate lysozyme expression inside RPE cells. Lysozyme was detected by ELISA in normal RPE lysates, and in live bacteria-treated RPE supernatants. Treatment of RPE cells with Pam3CSK4, LPS, PGN-SA, and Poly(I:C) enhanced lysozyme expression. CRISPR/Cas9 deletion of lysozyme impaired bactericidal activity of ARPE19 cells and reduced their response to LPS and Poly(I:C) stimulation., Conclusions: RPE cells constitutively express antimicrobial peptide lysozyme and the expression is modulated by pathogenic challenges. RPE cells may protect the neuroretina from blood-borne pathogens by producing antimicrobial peptides, such as lysozyme.

Background: To investigate age-related severity, patterns of retinal structural damage, and functional visual recovery in pediatric and adult cohorts of myelin oligodendrocyte glycoprotein antibody-associated disease (MOGAD) optic neuritis (ON)., Methods: All MOGAD patients from the 5 participating centers were included. Patients with initial manifestation <18 years were included in the pediatric (MOGAD ped ) cohort and patients with ≥18 years in the adult (MOGAD adult ) cohort. For patients with MOGAD ON, examinations at least ≥6 months after ON onset were included in the analyses. Using spectral domain optical coherence tomography (SD-OCT), we acquired peripapillary retinal nerve fiber layer thickness (pRNFL) and volumes of combined ganglion cell and inner plexiform layer (GCIPL). High- and 2.5% low-contrast visual acuity (HCVA, LCVA) and visual-evoked potentials (VEP) were obtained., Results: Twenty MOGAD ped (10.3±3.7 years, 30 MOGAD ON eyes) and 39 MOGAD adult (34.9±11.6 years, 42 MOGAD ON eyes) patients were included. The average number of ON episodes per ON eye was similar in both groups (1.8±1.3 and 2.0±1.7). In both pediatric and adult MOGAD, ON led to pronounced neuroaxonal retinal atrophy (pRNFL: 63.1±18.7 and 64.3±22.9 μm; GCIPL: 0.42±0.09 and 0.44±0.13 mm 3 , respectively) and moderate delay of the VEP latencies (117.9±10.7 and 118.0±14.5 ms). In contrast, visual acuity was substantially better in children (HCVA: 51.4±9.3 vs. 35.0±20.6 raw letters, p=0.001; LCVA: 22.8±14.6 vs. 13.5±16.4, p=0.028). Complete visual recovery (HCVA-logMAR 0.0) occurred in 73.3% of MOGAD ped and 31% MOGAD adults ON eyes, while 3.3% and 31% demonstrated moderate to severe (logMAR > 0.5) visual impairment. Independent of retinal atrophy, age at ON onset significantly correlated with visual outcome., Conclusion: Pediatric MOGAD ON showed better visual recovery than adult MOGAD ON despite profound and almost identical neuroaxonal retinal atrophy. Age-related cortical neuroplasticity may account for the substantial discrepancy between structural changes and functional outcomes.

Autoimmune uveitis is a sight-threatening ocular inflammatory condition in which the retina and uveal tissues become a target of autoreactive immune cells. The CD47 is a ubiquitously expressed transmembrane protein which plays multiple roles in fundamental cellular functions including phagocytosis, proliferation, and adhesion. Signal regulatory protein alpha (SIRPα), one of the CD47 ligands, is predominantly expressed in myeloid lineage cells such as dendritic cells (DCs) or macrophages, and CD47-SIRPα signaling pathway is implicated in the development of autoimmune diseases. Our current study demonstrates how CD47 depletion is effective in the prevention of experimental autoimmune uveitis (EAU), an animal model of human autoimmune uveitis, in animals deficient of CD47 ( CD47 -/- ). Systemic suppression of SIRPα + DCs in animals deficient in CD47 resulted in the inability of autoreactive CD4 + T cells to develop, which is crucial to induction of EAU. Of interest, retinal microglia, the resident immune cell of the retina, express SIRPα, however these cells were not operative in EAU suppression in response to CD47 depletion. These results identify CD47 as a significant regulator in the development of SIRPα + DCs that is vital to disease induction in EAU., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Okunuki, Tabor, Lee and Connor.)

Purpose: Agonistic β2-adrenergic receptor autoantibodies (β2-agAAb) have been observed in sera of patients with ocular hypertension and open-angle glaucoma (OAG). They target the β2-receptors on trabecular meshwork, ciliary body and pericytes (Junemann et al. 2018; Hohberger et al. 2019). In addition to their influence on the intraocular pressure, an association to retinal microcirculation is discussed. This study aimed to investigate foveal avascular zone (FAZ) characteristics by en face OCT angiography (OCT-A) in glaucoma suspects and its relationship to β2-agAAb status in patients with OAG., Material and Methods: Thirty-four patients (28 OAG, 6 glaucoma suspects) underwent standardized, clinical examination including sensory testing as white-on-white perimetry (Octopus G1, mean defect, MD) and structural measures as retinal nerve fibre layer (RNFL) thickness, neuroretinal rim width (BMO-MRW), retinal ganglion cell layer (RGCL) thickness, and inner nuclear layer (INL) thickness with high-resolution OCT. FAZ characteristics were measured by OCT-A scans of superficial vascular plexus (SVP), intermediate capillary plexus (ICP), and deep capillary plexus (DCP). FAZ-R was calculated (area FAZ (SVP)/area FAZ (ICP)). Using cardiomyocyte bioassays we analysed serum samples for the presence of β2-agAAb., Results: (I) Total mean FAZ area [mm2]: 0.34±0.16 (SVP), 0.24±0.12 (ICP), and 0.49±0.24 (DCP); mean FAZ-R 1.58±0.94. No correlation was seen for FAZ-R with MD, RNFL, BMO-MRW, RGCL thickness and INL thickness (p>0.05). (II) ß2-agAAb have been observed in 91% patients and showed no correlation with MD, RNFL, BMO-MRW, RGCL thickness and INL thickness (p>0.05). (III) FAZ-R correlated significantly with the β2-agAAb-induced increase of the beat rate of cardiomyocyte (p = 0.028)., Conclusion: FAZ characteristics did not correlate with any glaucoma associated functional and morphometric follow-up parameter in the present cohort. However, level of β2-agAAb showed a significantly correlation with FAZ-ratio. We conclude that β2-agAAb might be a novel biomarker in glaucoma pathogenesis showing association to FAZ-ratio with OCT-A., Competing Interests: Hohberger B, none; Hosari S, none; Wallukat G, Berlin Cures (emblyoyed), patent EP1832600A1: Peptides against autoantibodies associated with glaucoma and use of these peptides; Kunze R, patent EP1832600A1: Peptides against autoantibodies associated with glaucoma and use of these peptides; Krebs J, none; Müller M,none; Hennig T, none; Lämmer R, none; Horn F, none; Muñoz L.E., none; Herrmann M, patent EP1832600A1: Peptides against autoantibodies associated with glaucoma and use of these peptides; Mardin C, none. This does not alter our adherence to PLOS ONE policies on sharing data and materials.

ESC- and iPSC-derived retinal transplantation is a promising therapeutic approach for disease with end-stage retinal degeneration, such as retinitis pigmentosa and age-related macular degeneration. We previously showed medium- to long-term survival, maturation, and light response of transplanted human ESC- and iPSC-retina in mouse, rat, and monkey models of end-stage retinal degeneration. Because the use of patient hiPSC-derived retina with a disease-causing gene mutation is not appropriate for therapeutic use, allogeneic transplantation using retinal tissue/cells differentiated from a stocked hESC and iPSC line would be most practical. Here, we characterize the immunological properties of hESC- and iPSC-retina and present their three major advantages: (1) hESC- and iPSC-retina expressed low levels of human leukocyte antigen (HLA) class I and little HLA class II in vitro, (2) hESC- and iPSC-retina greatly suppressed immune activation of lymphocytes in co-culture, and (3) hESC- and iPSC-retina suppressed activated immune cells partially via transforming growth factor β signaling. These results support the use of allogeneic hESC- and iPSC-retina in future clinical application., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Purpose: Researches have confirmed that the retinal and choroidal thickness in patients with autoimmune disease-associated uveitis displays significant changes. However, the relationships between rheumatoid factor (RF) and thickness of the retina and choroid in individuals without ocular manifestations remain unclear. The aim of this study is to assess the associations of RF with retinal and choroidal thickness., Methods: The individuals enrolled in the cross-sectional research received full ocular examinations. The participants were classified as the RF (+) group (RF ≥ 15.0 IU/ml) and the RF (-) group (RF < 15.0 IU/ml) according to the serum RF titers. The thickness of the retina and choroid was measured by swept-source optical coherence tomography (SS-OCT)., Results: The study covered 65 right eyes of 65 individuals that are RF-positive and 130 right eyes of 130 age- and sex-matched individuals that are RF-negative. The RF (+) group showed decreased choroidal thickness that achieved statistical significance only in the outer inferior and outer temporal sectors, as compared to the RF (-) group. There was no statistically significant difference regarding the retinal thickness between the two groups. Pearson's correlation analysis revealed that the RF was significantly negatively related to the choroidal thickness in all areas. However, there was no significant correlation between the RF and the retinal thickness., Conclusions: Serum RF titers are closely linked with choroidal thickness before the emergence of ocular symptoms. Research into the relationships may improve our understanding of the role of serum RF in the pathogenesis of uveitis., Competing Interests: None of the authors has a financial or proprietary interest in any material or method mentioned., (Copyright © 2021 Qingjian Li et al.)

Retinal Müller glial cells (RMG) are involved in virtually every retinal disease; however, the role of these glial cells in neuroinflammation is still poorly understood. Since cell surface proteins play a decisive role in immune system signaling pathways, this study aimed at characterizing the changes of the cell surface proteome of RMG after incubation with prototype immune system stimulant lipopolysaccharide (LPS). While mass spectrometric analysis of the human Müller glia cell line MIO-M1 revealed 507 cell surface proteins in total, with 18 proteins significantly more abundant after stimulation (ratio ≥ 2), the surfaceome of primary RMG comprised 1425 proteins, among them 79 proteins with significantly higher abundance in the stimulated state. Pathway analysis revealed notable association with immune system pathways such as "antigen presentation", "immunoregulatory interactions between a lymphoid and a non-lymphoid cell" and "cell migration". We could demonstrate a higher abundance of proteins that are usually ascribed to antigen-presenting cells (APCs) and function to interact with T-cells, suggesting that activated RMG might act as atypical APCs in the course of ocular neuroinflammation. Our data provide a detailed description of the unstimulated and stimulated RMG surfaceome and offer fundamental insights regarding the capacity of RMG to actively participate in neuroinflammation in the retina.

Diabetic retinopathy (DR) is one of the most common complications of Diabetes Mellitus (DM) and is directly associated with inflammatory processes. Currently, neuro-inflammation is considered an early event in DR and proceeds via microglia polarization. A hallmark of DR is the presence of retinal reactive gliosis. Here we report the beneficial effect of ( S S ,1 R )-1-docecylsulfiny-5 N ,6 O -oxomethylidenenojirimycin (( Ss )-DS-ONJ), a member of the sp 2 -iminosugar glycolipid (sp 2 -IGL) family, by decreasing iNOS and inflammasome activation in Bv.2 microglial cells exposed to pro-inflammatory stimuli. Moreover, pretreatment with ( Ss )-DS-ONJ increased Heme-oxygenase (HO)-1 as well as interleukin 10 (IL10) expression in LPS-stimulated microglial cells, thereby promoting M2 (anti-inflammatory) response by the induction of Arginase-1. The results strongly suggest that this is the likely molecular mechanism involved in the anti-inflammatory effects of ( S S )-DS-ONJ in microglia. ( S S )-DS-ONJ further reduced gliosis in retinal explants from type 1 diabetic BB rats, which is consistent with the enhanced M2 response. In conclusion, targeting microglia polarization dynamics in M2 status by compounds with anti-inflammatory activities offers promising therapeutic interventions at early stages of DR., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Cano-Cano, Alcalde-Estévez, Gómez-Jaramillo, Iturregui, Sánchez-Fernández, García Fernández, Ortiz Mellet, Campos-Caro, López-Tinoco, Aguilar-Diosdado, Valverde and Arroba.)

Ocular toxoplasmosis is the leading cause of posterior uveitis worldwide. We conducted an observational study of 262 consecutive individuals (n = 344 eyes) with ocular toxoplasmosis who were followed over a 34-month period. Most subjects were T. gondii IgG + /IgM- (n = 242; 92.4%; 317 eyes), and 140 eyes (40.7%) had active lesions. For eyes in which retinal lesions were active at recruitment and best-corrected visual acuity (BCVA) could be measured (n = 133), 21.0% (n = 28) remained blind (BCVA below 20/400) after inflammation resolved. In these eyes, atypical ocular toxoplasmosis (OR 4.99; 95% CI 1.14-22.85; p = 0.0330), macular lesion (OR 9.95; 95% CI 2.45-47.15; p = 0.0019) and any complication (OR 10.26; 95% CI 3.82-30.67; p < 0.0001) were associated with BCVA below 20/200. For eyes with only inactive lesions at recruitment and BCVA measured (n = 178), 28.1% (n = 50) were blind. In these eyes, having at least one lesion larger than one disc-diameter (OR 6.30; 95% CI 2.28-22.46; p = 0.0013) and macular lesion (OR 5.69; 95% CI 2.53-13.54; p < 0.0001) were associated with BCVA below 20/200. Older age (OR 1.02; 95% CI 1.00-1.05; p = 0.0493) and active disease at presentation (OR 4.74; 95% CI 1.95-12.91; p = 0.0011) were associated with recurrences. Additional clinical attention should be directed towards patients with risk factors for poor visual outcome.

Objective: Microglia/macrophage activation is previously reported to be involved in various ocular diseases. However, the separate role of M1/M2 phenotype microglia/macrophage in the pathological process of oxygen-induced retinopathy (OIR) remains unknown. In this research, we explored the role and regulatory mechanism of M1/M2 microglia/macrophage in OIR in C57BL/6J mice. Furthermore, we demonstrated the time phase of M1/M2 shifting of microglia/macrophage during the natural process of OIR, which is very essential for further investigations., Materials and Methods: C57BL/6j pups were exposed to hyperoxia environment from postnatal 7(P7) to P12 then returned to normoxia. The mice were then euthanized, and the eyes were harvested at a series of time points for further investigation. The M1/M2 phenotype microglia/macrophage activity was presented by immunofluorescent staining and real-time quantitative polymerase chain reaction (qPCR). The NF-κb-STAT3 signaling and IL-4-STAT6-PPAR-γ signaling pathway activity was examined by western blot analysis., Results: The microglia/macrophage were activated when the OIR model was set up after P12. The M1 microglia/macrophage activation was found in neovascularization (NV) tufts in both central and peripheral retina, which started from P12 when the mice were returned to normoxia environment and peaked at P17. During this period of time, the NF-κb-STAT3 signaling pathway was activated, resulting in the upregulated M1 phenotype microglia/macrophage polarization, along with the enhanced inflammatory cytokine expression including tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and IL-1β. Consequently, the NV tufts were observed from P12 and the volume continued to increase until P17. However, the M2 phenotype microglia/macrophage activity took over during the late phase of OIR started from P17. The IL-4-STAT6-PPAR-γ signaling activity was upregulated from P17 and peaked at P20, inducing M2 phenotype microglia polarization, which consequently led to the inhibition of inflammatory cytokines and spontaneous regression of NV tufts., Conclusions: Microglia/macrophage participate actively in the natural process of OIR in mice, and two phenotypes exert different functions. Treatment modulating microglia/macrophage polarize toward M2 phenotype might be a novel and promising method for ocular neovascular diseases such as retinopathy of prematurity (ROP), wet age-related macular degeneration (wAMD), and diabetic retinopathy (DR).