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

ABSTRACT: 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.

SUBMITTER: Lo SM

PROVIDER: S-EPMC8766412 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:PurposeTo test whether retinal pigment epithelial (RPE) cells are able to induce myeloid-derived suppressor cell (MDSC) differentiation from bone marrow (BM) progenitors.MethodsBM cells were cocultured with or without RPE cells in the presence of GM-CSF and IL-4. Numbers of resultant MDSCs were assessed by flow cytometry after 6 days of incubation. The ability of the RPE cell-induced MDSCs to inhibit T cells was evaluated by a CFSE-based T-cell proliferation assay. To explore the mechanism by which RPE cells induce MDSC differentiation, PD-L1-deficient RPE cells and blocking antibodies against TGF-β, CTLA-2α, and IL-6 were used. RPE cell-induced MDSCs were adoptively transferred into mice immunized with interphotoreceptor retinoid-binding protein in complete Freund's adjuvant to test their efficacy in suppressing autoreactive T-cell responses in experimental autoimmune uveitis (EAU).ResultsRPE cells induced the differentiation of MDSCs. These RPE cell-induced MDSCs significantly inhibited T-cell proliferation in a dose-dependent manner. PD-L1-deficient RPE cells induced MDSC differentiation as efficiently as wild-type RPE cells, and neutralizing TGF-β or CTLA-2α did not alter the numbers of induced MDSCs. However, blocking IL-6 reduced the efficacy of RPE cell-induced MDSC differentiation. Finally, adoptive transfer of RPE cell-induced MDSCs suppressed IRBP-specific T-cell responses that led to EAU.ConclusionsRPE cells induce the differentiation of MDSCs from bone marrow progenitors. Both cell surface molecules and soluble factors are important in inducing MDSC differentiation. PD-L1, TGF-β, and CTLA-2α were not measurably involved in RPE cell-induced MDSC differentiation, whereas IL-6 was important in the process. The induction of MDSCs could be another mechanism by which RPE cells control immune reactions in the retina, and RPE cell-induced MDSCs should be further investigated as a potential approach to therapy for autoimmune posterior uveitis.

Project description:Lipofuscin accumulates with age in the retinal pigment epithelium (RPE) in discrete granular organelles and may contribute to age-related macular degeneration. Because previous studies suggest that lipofuscin contains protein that may impact pathogenic mechanisms, we pursued proteomics analysis of lipofuscin. The composition of RPE lipofuscin and its mechanisms of pathogenesis are poorly understood in part because of the heterogeneity of isolated preparations. We purified RPE lipofuscin granules by treatment with proteinase K or SDS and showed by light, confocal, and transmission electron microscopy that the purified granules are free of extragranular material and associated membranes. Crude and purified lipofuscin preparations were quantitatively compared by (i) LC MS/MS proteomics analyses, (ii) immunoanalyses of oxidative protein modifications, (iii) amino acid analysis, (iv) HPLC of bisretinoids, and (v) assaying phototoxicity to RPE cells. From crude lipofuscin preparations 186 proteins were identified, many of which appeared to be modified. In contrast, very little protein ( approximately 2% (w/w) by amino acid analysis) and no identifiable protein were found in the purified granules, which retained full phototoxicity to cultured RPE cells. Our analyses showed that granules in purified and crude lipofuscin preparations exhibit no statistically significant differences in diameter or circularity or in the content of the bisretinoids A2E, isoA2E, and all-trans-retinal dimer-phosphatidylethanolamine. The finding that the purified granules contain minimal protein yet retain phototoxic activity suggests that RPE lipofuscin pathogenesis is largely independent of associated protein. The purified granules also exhibited oxidative protein modifications, including nitrotyrosine generated from reactive nitrogen oxide species and carboxyethylpyrrole and iso[4]levuglandin E(2) adducts generated from reactive lipid fragments. This finding is consistent with previous studies demonstrating RPE lipofuscin to be a potent generator of reactive oxygen species and supports the hypothesis that such species, including reactive fragments from lipids and retinoids, contribute to the mechanisms of RPE lipofuscin pathogenesis.

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

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