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Learn MoreBackground: 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 nave 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, lipocalin 2, 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. SOURCE: Frederick Libert (flibert@ulb.ac.be) - C2-140 ULB
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