BackgroundControversy 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.MethodsEAU 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.ResultsImmunostainings 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.ConclusionOur 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.Electronic supplementary materialThe online version of this article (doi:10.1186/s12974-017-0915-5) contains supplementary material, which is available to authorized users.
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.
Purpose Non infectious uveitis are characterized by the penetration of immune cells into the eye which depends on adhesion molecules expression on the blood retinal barrier (BRB). In this work, we have studied the expression of the adhesion molecules VCAM‐1 and ICAM‐1 in experimental autoimmune uveitis (EAU) by adoptive transfer and the expression of their ligands, VLA‐4 and LFA‐1, on autoreactive T‐lymphocyte (TL) Methods Autoreactive TL, obtained from the lymph node of C57BL6 mice immunized with IRBP 1‐20, were purified using CD4+ magnetic cell sorting. Those cells were studied by flow cytometry for their expression of VLA‐4 and LFA‐1. ELISAs and intracytoplasmic flow cytometry were performed to study the cytokine expression profile. Then, EAU were induced by adoptive transfer of those cells into naive mice. VCAM‐1 and ICAM‐1 histological and cellular expression was studied by immunofluorescence on eye cryosections Results Autoreactive TL were found to be from the Th1 and Th17 phenotypes. LFA‐1 was expressed on all T and non T cells and VLA‐4 mostly on non T cells. Only a minority of TL showed VLA‐4 expression. In the eye of naïve mice VCAM‐1 was absent and ICAM‐1 present only at very low level. VCAM‐1 and ICAM‐1 expression is correlated to the severity of the disease. Interestingly, VCAM‐1 was more expressed on the internal BRB and ICAM‐1 on the external BRB Conclusion This work shows that the adhesion molecules VCAM‐1 and ICAM 1 were expressed differently on the internal and external BRB and that their respective ligands VLA‐4 and LFA‐1 were also expressed differently on lymphocytes. This works complete the understanding of the major role of the adhesion molecules VCAM‐1 and ICAM‐1 in the induction uveitis in the adoptive transfer model
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