Development Of Experimental Autoimmune Uveitis Research Articles

Too Much of a Good Thing: Extended Duration of Gut Microbiota Depletion Reverses Protection From Experimental Autoimmune Uveitis.

Using the model of experimental autoimmune uveitis (EAU) induced by immunization with a retinal antigen, two studies have reported contradictory results on disease development following oral antibiotic treatment (ABX). We showed that long-term ABX did not affect EAU, but another study showed that short-term ABX was protective. We therefore studied the effects of ABX on EAU, gut microbiota, and host immune responses as a function of treatment duration. EAU-susceptible mice were treated orally with broad-spectrum antibiotics starting at least 10 weeks (long-term) or 1 week (short-term) before immunization until termination of the experiment. Gut microbiota were characterized by 16S amplicon sequencing, and host gut immune elements were studied phenotypically and functionally. Long-term ABX had no effect, whereas short-term ABX delayed EAU, as previously reported by us and others, respectively. Microbial sequencing revealed progressive reduction of gut microbiota that showed some differences in the two ABX groups. Interestingly, duration of ABX was associated with a gradual disappearance of the CD4+ and CD4+CD8+ subset of gut intraepithelial lymphocytes (IELs). This IEL subset is microbiota dependent and is absent in germ-free mice. Relative abundance of Lactobacillus reuteri correlated with the frequencies of CD4+CD8+ IELs. IELs suppressed antigen-specific activation of autoreactive T cells in culture. Gut microbiota may play dual roles in uveitis development: They promote EAU development but also help maintain IEL populations that have regulatory function against autoreactive T cells. We propose that the progressive loss of this population during long-term ABX reverses the EAU-ameliorating effects of microbiota depletion.

Mettl3 induced miR-338-3p expression in dendritic cells promotes antigen-specific Th17 cell response via regulation of Dusp16.

Pathogenic Th17 cells are critical drivers of multiple autoimmune diseases, including uveitis and its animal model, experimental autoimmune uveitis (EAU). However, how innate immune signals modulate pathogenic Th17 responses remains largely unknown. Here, we showed that miR-338-3p endowed dendritic cells (DCs) with an increased ability to activate interphotoreceptor retinoid-binding protein (IRBP)1-20 -specific Th17 cells by promoting the production of IL-6, IL-1β, and IL-23. Invivo administration of LV-miR-338-infected DCs promoted pathogenic Th17 responses and exacerbated EAU development. Mechanistically, dual-specificity phosphatase 16 (Dusp16) was a molecular target of miR-338-3p. miR-338-3p repressed Dusp16 and therefore strengthened the mitogen-activated protein kinase (MAPK) p38 signaling, resulting in increased production of Th17-polarizing cytokines and subsequent pathogenic Th17 responses. In addition, methyltransferase like 3 (Mettl3), a key m6A methyltransferase, mediated the upregulation of miR-338-3p in activated DCs. Together, our findings identify a vital role for Mettl3/miR-338-3p/Dusp16/p38 signaling in DCs-driven pathogenic Th17 responses and suggest a potential therapeutic avenue for uveitis and other Th17 cell-related autoimmune disorders.

LncRNA Neat1 targets NonO and miR-128-3p to promote antigen-specific Th17 cell responses and autoimmune inflammation

Long non-coding RNAs (lncRNAs) interaction with RNA-Binding proteins (RBPs) plays an important role in immunological processes. The generation of antigen-specific Th17 cells is closely associated with autoimmune pathogenesis. However, the function of lncRNA-RBP interactions in the regulation of pathogenic Th17 cell responses during autoimmunity remains poorly understood. Here, we found that lncRNA Neat1, highly expressed in Th17 cells, promoted antigen-specific Th17 cell responses. Both global and CD4+ T cell-specific knockdown of Neat1 protected mice against the development of experimental autoimmune uveitis (EAU). Mechanistically, Neat1 regulated RNA-Binding protein NonO, thus relieving IL-17 and IL-23R from NonO-mediated transcriptional repression and supporting antigen-specific Th17 cell responses. In addition, Neat1 also modulated miR-128-3p/NFAT5 axis to increase the expression of IL-17 and IL-23R, leading to augmented Th17 cell responses. Our findings elucidate a previously unrecognized mechanistic insight into the action of Neat1 in promoting antigen-specific Th17 responses and autoimmunity, and may facilitate the development of therapeutic targets for T cell-mediated autoimmune diseases.

Sleep loss potentiates Th17-cell pathogenicity and promotes autoimmune uveitis.

Sleep loss (SL) is a health issue associated with the higher risk of autoimmune and inflammatory disorders. However, the connection between SL, the immune system, and autoimmune diseases remains unknown. We conducted mass cytometry, single-cell RNA sequencing, and flow cytometry to analyze how SL influences immune system and autoimmune disease development. Peripheral blood mononuclear cells from six healthy subjects before and after SL were collected and analyzed by mass cytometry experiments and subsequent bioinformatic analysis to identify the effects of SL on human immune system. Sleep deprivation and experimental autoimmune uveitis (EAU) mice model were constructed, and scRNA-seq data from mice cervical draining lymph nodes were generated to explore how SL influences EAU development and related autoimmune responses. We found compositional and functional changes in human and mouse immune cells after SL, especially in effector CD4+ T and myeloid cells. SL upregulated serum GM-CSF levels in healthy individuals and in patients with SL-induced recurrent uveitis. Experiments in mice undergoing SL or EAU demonstrated that SL could aggravate autoimmune disorders by inducing pathological immune cell activation, upregulating inflammatory pathways, and promoting intercellular communication. Furthermore, we found that SL promoted Th17 differentiation, pathogenicity, and myeloid cells activation through the IL-23Th17GM-CSF feedback mechanism, thus promoting EAU development. Lastly, an anti-GM-CSF treatment rescued SL-induced EAU aggravation and pathological immune response. SL promoted Th17 cells pathogenicity and autoimmune uveitis development, especially through the interaction between Th17 and myeloid cells involving GM-CSF signaling, providing possible therapeutic targets for the SL-related pathological disorders.

MicroRNA 146a ameliorates retinal damage in experimental autoimmune uveitis

IntroductionUveitis and related intraocular inflammations are a major cause of blindness due to retinal damage caused by degeneration and loss of the photoreceptor cells. In mouse experimental autoimmune uveitis (EAU) previously we have shown mitochondrial oxidative stress with marked upregulation of αA crystallin in the inner segments of the photoreceptors. Furthermore, αA crystallin treatment prevented photoreceptor mitochondrial oxidative stress by suppressing innate and adaptive immunity in EAU.MethodsSince these immune processes are modulated by microRNAs, in this study we investigated (a) modulation of microRNAs during development of EAU by αA crystallin administration and (b) microRNA therapeutic intervention.ResultsFew microRNAs were significantly upregulated in EAU mice with intravenous injection of αA crystallin and among these, computational bioinformatic analysis revealed that the upregulated microRNA 146a targets the innate and adaptive immune responses. In EAU, intravenous as well as intravitreal administration of this microRNA prevented inflammatory cell infiltration in uvea and retina and preserved photoreceptor cells.DiscussionThis protective function suggests that microRNA146a can be a novel therapeutic agent in preventing retinal damage in uveitis.

METTL3 inhibits autoreactive Th17 cell responses in experimental autoimmune uveitis via stabilizing ASH1L mRNA.

Methyltransferase like 3 (METTL3), a primary N6-methyladenosine (m6A) methyltransferase, has been implicated in various biological and pathological processes including immune responses. However, the functions and mechanisms of METTL3 in pathogenic T helper (Th)17 cells are poorly understood. Here we found significantly decreased METTL3 expression along with reduced m6A levels in eyeballs and T cells of experimental autoimmune uveitis (EAU). Overexpression of METTL3 ameliorated the development of EAU and suppressed pathogenic Th17 cell responses in vivo and in vitro. Mechanistically, METTL3 promoted the expression of absent, small, or homeotic-like 1 (ASH1L) via enhancing its stability in a YT521-B homology domain containing 2 (YTHDC2)-dependent manner, which further decreased the expression of IL-17 and IL-23 receptor (IL-23R), resulting in reduced pathogenic Th17 responses. Together, our data reveal a pivotal role of METTL3 in regulating pathogenic Th17 responses, which may contribute to human uveitis therapy.

CDCP1 regulates retinal pigmented epithelial barrier integrity for the development of experimental autoimmune uveitis.

Cub domain-containing protein 1 (CDCP1) is a protein that is highly expressed on the surface of many cancer cells. However, its distribution in normal tissues and its potential roles in nontumor cells are poorly understood. We found that CDCP1 is present on both human and mouse retinal pigment epithelial (RPE) cells. CDCP1-KO mice developed attenuated retinal inflammation in a passive model of autoimmune uveitis, with disrupted tight junctions and infiltrating T cells detected in RPE flat mounts from WT but not CDCP1-KO mice during EAU development. Mechanistically, we discovered that CDCP1 on RPE cells was upregulated by IFN-γ in vitro and after EAU induction in vivo. CD6 stimulation induced increased RPE barrier permeability of WT but not CDCP1-knockdown (CDCP1-KD) RPE cells, and activated T cells migrated through WT RPE monolayers more efficiently than the CDCP1-KD RPE monolayers. In addition, CD6 stimulation of WT but not the CDCP1-KD RPE cells induced massive stress fiber formation and focal adhesion disruption to reduce cell barrier tight junctions. These data suggest that CDCP1 on RPE cells interacts with CD6 on T cells to induce RPE cytoskeleton remodeling and focal adhesion disruption, which open up the tight junctions to facilitate T cell infiltration for the development of uveitis.

A Novel Tree Shrew Model of Chronic Experimental Autoimmune Uveitis and Its Disruptive Application.

BackgroundPrevious studies have established several animal models for experimental autoimmune uveitis (EAU) in rodents without the fovea centralis in the human retina. This study aimed to develop and explore the application of a novel EAU model in tree shrews with a cone-dominated retina resembling the human fovea.MethodsTree shrews were clinically and pathologically evaluated for the development and characteristics of EAU immunized with six inter-photoreceptor retinoid-binding proteins (IRBPs). IRBP-specific T-cell proliferation and serum cytokine of tree shrews were evaluated to determine the immune responses. Differentially expressed genes (DEGs) were identified in the eyes of tree shrews with EAU by RNA-sequencing. The disruptive effects of the DEG RGS4 inhibitor CCG 203769 and dihydroartemisinin on the EAU were investigated to evaluate the potential application of tree shrew EAU.ResultsIRBP1197–1211 and R14 successfully induced chronic EAU with subretinal deposits and retinal damage in the tree shrews. The immunological characteristics presented the predominant infiltration of microglia/macrophages, dendritic cells, and CD4-T-cells into the uvea and retina and pathogenic T helper (Th) 1 and Th17 responses. The subretinal deposits positively expressed amyloid β-protein (Aβ), CD8, and P2Y purinoceptor 12 (P2RY12). The crucial DEGs in R14-induced EAU, such as P2RY2 and adenylate cyclase 4 (ADCY4), were enriched for several pathways, including inflammatory mediator regulation of transient receptor potential (TRP) channels. The upregulated RGS4 in IRBP-induced EAU was associated with mitogen-activated protein kinase (MAPK) activity. RGS4 inhibition and dihydroartemisinin could significantly alleviate the retinal pathological injuries of IRBP1197-1211-induced EAU by decreasing the expression of CD4 T-cells.ConclusionOur study provides a novel chronic EAU in tree shrews elicited by bovine R14 and tree shrew IRBP1197-1211 characterized by retinal degeneration, retinal damage with subretinal Aβ deposits and microglia/macrophage infiltration, and T-cell response, probably by altering important pathways and genes related to bacterial invasion, inflammatory pain, microglial phagocytosis, and lipid and glucose metabolism. The findings advance the knowledge of the pathogenesis and therapeutics of the fovea-involved visual disturbance in human uveitis.

Succinic acid exacerbates experimental autoimmune uveitis by stimulating neutrophil extracellular traps formation via SUCNR1 receptor

AimsTo investigate the effect of succinic acid on the development of experimental autoimmune uveitis (EAU) and the underlying mechanism.MethodsSuccinic acid was administrated intraperitoneally to evaluate its effects on immune response...

Inhibiting TLR7 Expression in the Retinal Pigment Epithelium Suppresses Experimental Autoimmune Uveitis.

Experimental autoimmune uveitis (EAU), a model of human uveitis, is an organ-specific, T cell-mediated autoimmune disease. Autoreactive T cells can penetrate the blood-retinal barrier, which is a physical defense composed of tight junction-linked retinal pigment epithelial (RPE) cells. RPE cells serve as antigen-presenting cells (APCs) in the eye since they express MHC class I and II and Toll-like receptors (TLRs). Although previous studies have shown that supplementation with TLR agonists exacerbates uveitis, little is known about how TLR signaling in the RPE contributes to the development of uveitis. In this study, we isolated the RPE from EAU mice, which were induced by active immunization (aEAU) or adoptive transfer of antigen-specific T cells (tEAU). The expression of TLRs on RPE was determined, and both aEAU and tEAU mice exhibited induced tlr7 expression. The TLR7 agonist R848 was shown to induce aggressive disease progression, along with significantly elevated levels of the uveopathogenic cytokine IL-17. Furthermore, not only IL-17 but also R848 appeared to enhance the inflammatory response and to impair the barrier function of the RPE, indicating that TLR7 signaling is involved in the pathogenesis of EAU by affecting the behaviors of the RPE and consequently allowing the infiltration of autoreactive T cells intraocularly. Finally, local application of shRNA against TLR7 delivered by recombinant AAV effectively inhibited disease severity and reduced IFN-γ and IL-17. Our findings highlight an immunomodulatory role of RPE TLR7 in EAU development and provide a potential therapeutic strategy for autoimmune uveitis.

CD4+ T-Cell Plasticity in Non-Infectious Retinal Inflammatory Disease.

Non-infectious uveitis (NIU) is a potentially sight-threatening disease. Effector CD4+ T cells, especially interferon-γ-(IFNγ) producing Th1 cells and interleukin-17-(IL-17) producing Th17 cells, are the major immunopathogenic cells, as demonstrated by adoptive transfer of disease in a model of experimental autoimmune uveitis (EAU). CD4+FoxP3+CD25+ regulatory T cells (Tregs) were known to suppress function of effector CD4+ T cells and contribute to resolution of disease. It has been recently reported that some CD4+ T-cell subsets demonstrate shared phenotypes with another CD4+ T-cell subset, offering the potential for dual function. For example, Th17/Th1 (co-expressing IFNγ and IL-17) cells and Th17/Treg (co-expressing IL-17 and FoxP3) cells have been identified in NIU and EAU. In this review, we have investigated the evidence as to whether these ‘plastic CD4+ T cells’ are functionally active in uveitis. We conclude that Th17/Th1 cells are generated locally, are resistant to the immunosuppressive effects of steroids, and contribute to early development of EAU. Th17/Treg cells produce IL-17, not IL-10, and act similar to Th17 cells. These cells were considered pathogenic in uveitis. Future studies are needed to better clarify their function, and in the future, these cell subsets may in need to be taken into consideration for designing treatment strategies for disease.

Timing Effect of Adenosine-Directed Immunomodulation on Mouse Experimental Autoimmune Uveitis.

Adenosine is an important regulatory molecule of the immune response. We have previously reported that treatment of experimental autoimmune uveitis (EAU)-prone mice with an adenosine-degrading enzyme (adenosine deaminase) prohibited EAU development by inhibiting Th17 pathogenic T cell responses. To further validate that the targeting of adenosine or adenosine receptors effectively modulates Th17 responses, we investigated the effect of adenosine receptor antagonists. In this study, we show that the A2AR antagonist SCH 58261 (SCH) effectively modulates aberrant Th17 responses in induced EAU. However, timing of the treatment is important. Whereas SCH inhibits EAU when administered during the active disease stage, it did not do so if administered during quiescent disease stages, thus implying that the existing immune status influences the therapeutic effect. Mechanistic studies showed that inhibition of γδ T cell activation is crucially involved in adenosine-based treatment. Adenosine is an important costimulator of γδ T cell activation, which is essential for promoting Th17 responses. During ongoing disease stages, adenosine synergizes with existing high levels of cytokines, leading to augmented γδ T cell activation and Th17 responses, but in quiescent disease stages, when existing cytokine levels are low, adenosine does not enhance γδ T cell activation. Our results demonstrated that blockade of the synergistic effect between adenosine and inflammatory cytokines at active disease stages can ameliorate high-degree γδ T cell activation and, thus, suppress Th17 pathogenic T cell responses.

Autoimmunity to neuroretina in the concurrent absence of IFN-γ and IL-17A is mediated by a GM-CSF-driven eosinophilic inflammation

IFN-γ and IL-17A can each elicit ocular autoimmunity independently of the other. Since absence of IFN-γ or IL-17A individually failed to abolish pathology of experimental autoimmune uveitis (EAU), we examined EAU development in the absence of both these cytokines. Ifng−/−Il17a−/− mice were fully susceptible to EAU with a characteristic eosinophilic ocular infiltrate, as opposed to a mononuclear infiltrate in WT mice. Retinal pathology in double-deficient mice was ameliorated when eosinophils were genetically absent or their migration was blocked, supporting a pathogenic role for eosinophils in EAU in the concurrent absence of IFN-γ and IL-17A. In EAU-challenged Ifng−/−Il17a−/− mice, ocular infiltrates contained increased GM-CSF-producing CD4+ T cells, and supernatants of retinal antigen-stimulated splenocytes contained enhanced levels of GM-CSF that contributed to activation and migration of eosinophils in vitro. Systemic or local blockade of GM-CSF ameliorated EAU in Ifng−/−Il17a−/− mice, reduced eosinophil peroxidase levels in the eye and in the serum and decreased eosinophil infiltration to the eye. These results support the interpretation that, in the concurrent absence of IFN-γ and IL-17A, GM-CSF takes on a major role as an inflammatory effector cytokine and drives an eosinophil-dominant pathology. Our findings may impact therapeutic strategies aiming to target IFN-γ and IL-17A in autoimmune uveitis.

A combination of inhibiting microglia activity and remodeling gut microenvironment suppresses the development and progression of experimental autoimmune uveitis

Noninfectious (autoimmune and immune-mediated) uveitis is an ocular inflammatory disease which can lead to blindness in severe cases. Due to the potential side effects of first-line drugs for clinical uveitis, novel drugs and targets against uveitis are still urgently needed. In the present study, using rat experimental autoimmune uveitis (EAU) model, we first found that minocycline treatment can substantially inhibit the development of EAU and improve the retinal function by suppressing the retinal microglial activation, and block the infiltration of inflammatory cells, including Th17, into the retina by decreasing the major histocompatibility complex class II (MHC II) expression in resident and infiltrating cells. Moreover, we demonstrated that minocycline treatment can remodel the gut microenvironment of EAU rats by restoring the relative abundance of Ruminococcus bromii, Streptococcus hyointestinalis, and Desulfovibrio sp. ABHU2SB and promoting a functional shift in the gut via reversing the levels of L-proline, allicin, aceturic acid, xanthine, and leukotriene B4, and especially increasing the production of propionic acid, histamine, and pantothenic acid. At last, we revealed that minocycline treatment can significantly attenuate the progression of EAU after inflammation onset, which may be explained by the role of minocycline in the remodeling of the gut microenvironment since selective elimination of retinal microglia on the later stages of EAU was shown to have little effect. These data clearly demonstrated that inhibition of microglial activation and remodeling of the gut microenvironment can suppress the development and progression of experimental autoimmune uveitis. Considering the excellent safety profile of minocycline in multiple clinical experiments, we suggest that minocycline may have therapeutic implications for clinical uveitis.

Parental Uveitis Influences Offspring With an Increased Susceptibility to the Experimental Autoimmune Uveitis.

Purpose: Previous studies have shown that parental abnormal physiological conditions such as inflammation, stress, and obesity can be transferred to offspring. The purpose of this study was to investigate the impact of parental uveitis on the development and susceptibility to experimental autoimmune uveitis (EAU) in offspring.Methods: Parental male and female B10RIII mice were immunized with interphotoreceptor retinoid binding protein (IRBP) 161–180 in complete Freund's adjuvant and were immediately allowed to mate. Gross examination of the offspring gestated with EAU was performed to determine the influence of parental uveitis on offspring development after birth. Gene expression profiles were analyzed in the affected eyes of offspring under EAU to identify differentially expressed genes (DEGs). Adult offspring were given 5, 25, and 50 μg IRBP161−180 to compare their susceptibility to EAU. Immunized mice were clinically and pathologically evaluated for the development of EAU. Ag-specific T-cell proliferation and IL-17 production from spleens and lymph nodes were evaluated on day 14 or 35 after immunization.Results: Hair loss, delay of eye opening, and swollen spleens in the offspring from parents with uveitis were observed from day 14 to 39 after birth. DEGs were involved in the immune system process, muscle system process, and cell development. The altered antigen processing and presentation, cell adhesion molecules, and phagosome in the eyes of the offspring from uveitis-affected parents were enriched. Offspring gestated with EAU showed a susceptibility to EAU and an earlier onset and higher severity of EAU compared to the control group mice. IRBP-specific lymphocyte proliferation and IL-17 production were observed in the EAU offspring with exposure to parental uveitis.Conclusions: The results suggest that mouse parents with uveitis can increase their offspring's susceptibility to EAU, probably through altering cell adhesion molecules and antigen processing and presentation related to the T-cell proliferation and Th17 response.

Laquinimod arrests development of experimental autoimmune uveitis (EAU) and inhibits related immune processes, in the context of altered gut microbiota

Abstract EAU is an animal model for autoimmune uveitis in humans and is used to study mechanisms and potential therapeutics for this blinding disease. Here, we tested the immunosuppressive efficacy of Laquinimod (LAQ), a potent AhR (aryl hydrocarbon receptor) ligand on development of EAU. C57BL/6J mice were challenged for EAU with 300ug of the uveitogenic antigen, IRBP 651–670 and were treated from day 0 or day 7, relative to immunization, with 25mg/Kg LAQ, or PBS, by oral gavage. EAU progression was monitored by fundus examination and confirmed by histology on day 14 post immunization (p.i.). Treatment with LAQ from day 0 p.i. completely prevented EAU development and inhibited proliferation and production of pro-inflammatory cytokines to peptide 651–670. Treatment with LAQ from day 7 p.i. also significantly inhibited the development of EAU and cellular immune responses, but to a lesser degree than in day 0 mice. In contrast to its inhibitory effect on pro-inflammatory processes, LAQ treatment increased the proportions of T-regulatory FoxP3+CD4+ T cells in treated mice, as compared to the control group. Metagenomic analyses revealed that immunized mice treated with LAQ had altered composition of gut microbiota when compared with PBS controls, with an increased population of Bifidobacteriaceae, and decreased Clostridaceae 1, Deflubiitaleaceae and Anaeroplasmataceae bacteria. Our results demonstrate that LAQ, a water-soluble AhR ligand, is a potent inhibitor of EAU development and exerts its effect primarily during the inductive phase of the disease. The connection of the observed changes in gut microbiota to its inhibitory effects on disease is under investigation.

More roles of IL-4 than being as an antagonist of IFN-γ in experimental autoimmune uveitis

Abstract IL-4 appears to be beneficial for some autoimmune diseases. Despite its role in antagonizing pathogenic Th1 response, our recent work has demonstrated IL-4 possesses functions affecting the regulatory T (Treg) cells. Here we aimed to identify the role of IL-4 in the development of experimental autoimmune uveitis (EAU). EAU was induced in WT and IL-4 KO mice via active interphotoreceptor retinoid binding protein (IRBP) immunization, and the disease development was monitored by funduscopic and histology analysis as well as the IRBP-specific immune responses by flow cytometric analysis and ELISA. It showed that the onset of disease, if the clinical score equals to 1, was day 14 and 20 in IL-4KO and WT mice, respectively. The fundoscopic images demonstrated the more and bigger size of lesions, increased leukocyte infiltration, vasculitis and neovascularization in IL-4KO mice. The histological analysis also confirmed the mononuclear cell infiltration. Moreover, Th1 effector is known as the dominant pathogenic mediators, whereas Treg cells play an important and indispensable role in control of EAU progression and disease remission. The splenic T cells from IL-4 KO EAU animals produced significantly elevated level of IFN-γ in responding to IRBP stimulation. However, there seems no significant but some alternation in the presence of Treg cells (Foxp3+) among CD4+CD25+ T cells in the spleens and lymph nodes of IL-4 KO and WT mice. These results demonstrate the lack of IL-4 disrupting the induction of EAU and leading to an increased disease severity through not only antagonizing IFN-γ but also involved mechanisms of immune-regulation. Therefore, IL-4 might play more roles than being just an antagonist of IFN-γ in autoimmune uveitis.

GM-CSF mediates development of eosinophil-dominant autoimmune uveitis in the absence of both IFN-γ and IL-17A

Abstract Autoimmune uveitis is a group of blinding diseases triggered by activated retina-specific T cells. Studies in uveitis patients and experiments in animal models of experimental autoimmune uveitis (EAU) support the notion that Th1 and Th17 cells are both pathogenic effectors and each can elicit ocular autoimmunity independently of the other. Since absence of IFN-γ or IL-17A individually failed to abolish pathology of EAU, we examined EAU development in the absence of both cytokines. Ifng−/−Il17a−/− mice were fully susceptible to EAU and displayed eosinophil-dominant ocular infiltrates, as opposed to mononuclear infiltrates in WT mice. EAU was ameliorated in double-deficient mice when eosinophils were genetically absent or their migration was blocked, supporting a pathogenic role of eosinophils in the absence of both IFN-γ and IL-17A. In Ifng−/−Il17a−/− mice immunized for EAU, ocular infiltrates contained increased GM-CSF-producing CD4 T cells, and supernatants of antigen-recalled splenocytes contained enhanced levels of GM-CSF that contributed to activation and migration of eosinophils in vitro. Systemic or local blockade of GM-CSF ameliorated EAU in Ifng−/−Il17a−/− mice. Neutralization of GM-CSF during the induction phase of EAU decreased eosinophil infiltration to the eye and eosinophil peroxidase levels in the eye and in the serum. These results support the interpretation that, in the concurrent absence of IFN-γ and IL-17A, GM-CSF takes on a major role as a pathogenic effector cytokine and induces an eosinophil-dominant pathology. This may impact therapeutic strategies aiming to target IFN-γ and IL-17A in autoimmune uveitis.

Retinal endothelial cell phenotypic modifications during experimental autoimmune uveitis: a transcriptomic approach

BackgroundBlood-retinal barrier cells are known to exhibit a massive phenotypic change during experimental autoimmune uveitis (EAU) development. In an attempt to investigate the mechanisms of blood-retinal barrier (BRB) breakdown at a global level, we studied the gene regulation of total retinal cells and retinal endothelial cells during non-infectious uveitis.MethodsRetinal endothelial cells were isolated by flow cytometry either in Tie2-GFP mice (CD31+ CD45− GFP+ cells), or in wild type C57BL/6 mice (CD31+ CD45− endoglin+ cells). EAU was induced in C57BL/6 mice by adoptive transfer of IRBP1–20-specific T cells. Total retinal cells and retinal endothelial cells from naïve and EAU mice were sorted and their gene expression compared by RNA-Seq. Protein expression of selected genes was validated by immunofluorescence on retinal wholemounts and cryosections and by flow cytometry.ResultsRetinal endothelial cell sorting in wild type C57BL/6 mice was validated by comparative transcriptome analysis with retinal endothelial cells sorted from Tie2-GFP mice, which express GFP under the control of the endothelial-specific receptor tyrosine kinase promoter Tie2. RNA-Seq analysis of total retinal cells mainly brought to light upregulation of genes involved in antigen presentation and T cell activation during EAU. Specific transcriptome analysis of retinal endothelial cells allowed us to identify 82 genes modulated in retinal endothelial cells during EAU development. Protein expression of 5 of those genes (serpina3n, lcn2, ackr1, lrg1 and lamc3) was validated at the level of inner BRB cells.ConclusionThose data not only confirm the involvement of known pathogenic molecules but further provide a list of new candidate genes and pathways possibly implicated in inner BRB breakdown during non-infectious posterior uveitis.

I-alowCD11bhigh DC Regulates the Immune Response in the Eyes of Experimental Autoimmune Uveitis.

Regulatory dendritic cells (DCreg) have been reported to be a negative regulator in the immune response. These cells are widely distributed in the liver, spleen, and lung. However, the status and function of DCreg in the eyes and disease are still not very clear. Herein, we found that the number of I-alowCD11bhigh DC increased in the eye and spleen at the recovery stage of experimental autoimmune uveitis (EAU), which is a mouse model for autoimmune uveitis. These cells expressed lower levels of CD80, CD86, and CD54 than the mature DCs and expressed interleukin 10 (IL-10), indoleamine 2,3-dioxygenase (IDO), and transforming growth factor beta (TGF-β) as well. Moreover, these DCreg can regulate the development of EAU by promoting CD4+CD25+Foxp3+ regulatory T cells. The increased interferon-gamma (IFN-γ) in the aqueous humor of EAU participates in inducing DCreg to alleviate the symptom of EAU. Furthermore, DCreg was found to exist in the eyes of normal mice. Aqueous humor, containing a certain concentration of IL-10, TGF-β, prostaglandin E2 (PGE2), IDO, and nitric oxide (NO), induced the tolerance of DCreg in normal eyes. It can be concluded that DCreg exists in the eyes and plays a protective role in inflamed eyes. These DCreg induced by IFN-γ might be used as a strategy to develop therapy for EAU management.