High salt diet alleviates disease severity in native experimental autoimmune uveitis.

Background: Recent studies reported a link between high salt diet (HSD) and clinical exacerbation in mouse models of autoimmune diseases, mainly through the induction of pathogenic Th17 cells and/or HSD-induced dysbiosis. However, the topic remains controversial and not fully understood. Purpose: In this study, we investigated the effects of HSD on the development of experimental autoimmune uveitis (EAU) in C57BL/6J mice. Methods and results: Unexpectedly, our data showed a significant attenuating effect of HSD on disease severity of native EAU, induced by direct immunization with IRBP peptide. That said, HSD had no effect on EAU disease severity induced by adoptive transfer of semi-purified auto-reactive IRBP-specific T lymphocytes. Accordingly, HSD did not affect IRBP-specific systemic afferent immune response as attested by no HSD-linked changes in T lymphocytes proliferation, cytokine production and Treg proportion. Gut microbiota analysis from cecal samples in naïve and EAU mice demonstrated that HSD affected differentially α-diversity between groups, whereas ß-diversity was significantly modified in all groups. Unknown Tannerellaceae was the only taxon associated to HSD exposure in all treatment groups. Interestingly, a significantly higher abundance of unknown Gastranaerophilales, with potential anti-inflammatory properties, appeared in HSD-fed native EAU mice, only. Discussion: In conclusion, our study suggests a possible impact of HSD on gut microbiota composition and consequently on development and clinical severity of EAU. Further studies are required to investigate the potential beneficial role of Gastranaerophilales in EAU.

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

BACKGROUND: Blood-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. METHODS: Retinal 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. RESULTS: Retinal 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. CONCLUSION: Those 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.

MHC class II expression and potential antigen-presenting cells in the retina during experimental autoimmune uveitis.

BACKGROUND: Controversy exists regarding which cell types are responsible for autoantigen presentation in the retina during experimental autoimmune uveitis (EAU) development. In this study, we aimed to identify and characterize the retinal resident and infiltrating cells susceptible to express major histocompatibility complex (MHC) class II during EAU. METHODS: EAU was induced in C57BL/6 mice by adoptive transfer of autoreactive lymphocytes from IRBP1-20-immunized animals. MHC class II expression was studied by immunostainings on eye cryosections. For flow cytometry (FC) analysis, retinas were dissected and enzymatically digested into single-cell suspensions. Three MHC class II+ retinal cell populations were sorted by FC, and their RNA processed for RNA-Seq. RESULTS: Immunostainings demonstrate strong induction of MHC class II expression in EAU, especially in the inner retina at the level of inflamed vessels, extending to the outer retinal layers and the subretinal space in severely inflamed eyes. Most MHC class II+ cells express the hematopoietic marker IBA1. FC quantitative analyses demonstrate that MHC class II induction significantly correlates with disease severity and is associated with upregulation of co-stimulatory molecule expression. In particular, most MHC class IIhi cells express co-stimulatory molecules during EAU. Further phenotyping identified three MHC class II+ retinal cell populations: CD45-CD11b- non-hematopoietic cells with low MHC class II expression and CD45+CD11b+ hematopoietic cells with higher MHC class II expression, which can be further separated into Ly6C+ and Ly6C- cells, possibly corresponding to infiltrating macrophages and resident microglia. Transcriptome analysis of the three sorted populations leads to a clear sample clustering with some enrichment in macrophage markers and microglial cell markers in Ly6C+ and Ly6C- cells, respectively. Functional annotation analysis reveals that both hematopoietic cell populations are more competent in MHC class II-associated antigen presentation and in T cell activation than non-hematopoietic cells. CONCLUSION: Our results highlight the potential of cells of hematopoietic origin in local antigen presentation, whatever their Ly6C expression. Our work further provides a first transcriptomic study of MHC class II-expressing retinal cells during EAU and delivers a series of new candidate genes possibly implicated in the pathogenesis of retinal autoimmunity.