Immunosuppression in stem cell clinical trials of neural and retinal cell types: A systematic review.

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.

Upregulation of HMOX1 associated with M2 macrophage infiltration and ferroptosis in proliferative diabetic retinopathy.

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.

Retinal transplant immunology and advancements.

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.

Immunomodulatory effects of Kaempferol on microglial and Macrophage cells during the progression of diabetic retinopathy.

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.

Beneficial mechanisms of dimethyl fumarate in autoimmune uveitis: insights from single-cell RNA sequencing.

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.

Macrophage activation contributes to diabetic retinopathy.

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.

Glial cell response to constant low light exposure in rat retina.

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.

Therapeutic Effect of IL-38 on Experimental Autoimmune Uveitis: Reprogrammed Immune Cell Landscape and Reduced Th17 Cell Pathogenicity.

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.

Lysophosphatidylcholine Offsets the Protective Effects of Bone Marrow Mesenchymal Stem Cells on Inflammatory Response and Oxidative Stress Injury of Retinal Endothelial Cells via TLR4/NF-κB Signaling.

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 µg/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-κB 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-κB, activating the TLR4/NF-κB 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-κB signaling.

The Role of Retinal Pigment Epithelial Cells in Regulation of Macrophages/Microglial Cells in Retinal Immunobiology.

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.

Myeloid Cells in the Mouse Retina and Uveal Tract Respond Differently to Systemic Inflammatory Stimuli.

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.

MMP2 Modulates Inflammatory Response during Axonal Regeneration in the Murine Visual System.

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.

Proinflammatory Pathways Are Activated in the Human Q344X Rhodopsin Knock-In Mouse Model of Retinitis Pigmentosa.

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.

Autoantibody profiles and clinical association in Thai patients with autoimmune retinopathy.

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.

Evidence for the involvement of interleukin-1α during development of experimental cytomegalovirus retinitis in immunosuppressed mice.

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.

Low Immunogenicity and Immunosuppressive Properties of Human ESC- and iPSC-Derived Retinas.

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 ß signaling. These results support the use of allogeneic hESC- and iPSC-retina in future clinical application.

Retinoblastoma tumor cell proliferation is negatively associated with an immune gene expression signature and increased immune cells.

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.

The phase changes of M1/M2 phenotype of microglia/macrophage following oxygen-induced retinopathy in mice.

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-1ß. 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).

No Evidence for Circulating Retina Specific Autoreactive T-cells in Latent Tuberculosis-associated Uveitis and Sarcoid Uveitis.

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.

Macrophage to myofibroblast transition contributes to subretinal fibrosis secondary to neovascular age-related macular degeneration.

BACKGROUND: Macular fibrosis causes irreparable vision loss in neovascular age-related macular degeneration (nAMD) even with anti-vascular endothelial growth factor (VEGF) therapy. Inflammation is known to play an important role in macular fibrosis although the underlying mechanism remains poorly defined. The aim of this study was to understand how infiltrating macrophages and complement proteins may contribute to macular fibrosis. METHODS: Subretinal fibrosis was induced in C57BL/6J mice using the two-stage laser protocol developed by our group. The eyes were collected at 10, 20, 30 and 40 days after the second laser and processed for immunohistochemistry for infiltrating macrophages (F4/80 and Iba-1), complement components (C3a and C3aR) and fibrovascular lesions (collagen-1, Isolectin B4 and α-SMA). Human retinal sections with macular fibrosis were also used in the study. Bone marrow-derived macrophages (BMDMs) from C57BL/6J mice were treated with recombinant C3a, C5a or TGF-ß for 48 and 96 h. qPCR, Western blot and immunohistochemistry were used to examine the expression of myofibroblast markers. The involvement of C3a-C3aR pathway in macrophage to myofibroblast transition (MMT) and subretinal fibrosis was further investigated using a C3aR antagonist (C3aRA) and a C3a blocking antibody in vitro and in vivo. RESULTS: Approximately 20~30% of F4/80+ (or Iba-1+) infiltrating macrophages co-expressed α-SMA in subretinal fibrotic lesions both in human nAMD eyes and in the mouse model. TGF-ß and C3a, but not C5a treatment, significantly upregulated expression of α-SMA, fibronectin and collagen-1 in BMDMs. C3a-induced upregulation of α-SMA, fibronectin and collagen-1 in BMDMs was prevented by C3aRA treatment. In the two-stage laser model of induced subretinal fibrosis, treatment with C3a blocking antibody but not C3aRA significantly reduced vascular leakage and Isolectin B4+ lesions. The treatment did not significantly alter collagen-1+ fibrotic lesions. CONCLUSIONS: MMT plays a role in macular fibrosis secondary to nAMD. MMT can be induced by TGF-ß and C3a but not C5a. Further research is required to fully understand the role of MMT in macular fibrosis. Macrophage to myofibroblast transition (MMT) contributes to subretinal fibrosis. Subretinal fibrosis lesions contain various cell types, including macrophages and myofibroblasts, and are fibrovascular. Myofibroblasts are key cells driving pathogenic fibrosis, and they do so by producing excessive amount of extracellular matrix proteins. We have found that infiltrating macrophages can transdifferentiate into myofibroblasts, a phenomenon termed macrophage to myofibroblast transition (MMT) in macular fibrosis. In addition to TGF-ß1, C3a generated during complement activation in CNV can also induce MMT contributing to macular fibrosis. RPE = retinal pigment epithelium. BM = Bruch's membrane. MMT = macrophage to myofibroblast transition. TGFB = transforming growth factor ß. a-SMA = alpha smooth muscle actin. C3a = complement C3a.