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. 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. 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. 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.

中文翻译：

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实验性自身免疫性葡萄膜炎期间视网膜内皮细胞表型改变：转录组学方法


众所周知，血视网膜屏障细胞在实验性自身免疫性葡萄膜炎（EAU）发展过程中表现出巨大的表型变化。为了在全球范围内研究血视网膜屏障（BRB）破坏的机制，我们研究了非感染性葡萄膜炎期间总视网膜细胞和视网膜内皮细胞的基因调控。通过流式细胞术在 Tie2-GFP 小鼠（CD31+ CD45− GFP+ 细胞）或野生型 C57BL/6 小鼠（CD31+ CD45− 内皮糖蛋白+ 细胞）中分离视网膜内皮细胞。通过过继转移 IRBP1-20 特异性 T 细胞在 C57BL/6 小鼠中诱导 EAU。对来自幼稚小鼠和 EAU 小鼠的总视网膜细胞和视网膜内皮细胞进行分选，并通过 RNA-Seq 比较它们的基因表达。通过视网膜整体和冷冻切片的免疫荧光以及流式细胞术验证所选基因的蛋白质表达。通过与从 Tie2-GFP 小鼠分选的视网膜内皮细胞进行比较转录组分析，验证了野生型 C57BL/6 小鼠的视网膜内皮细胞分选，Tie2-GFP 小鼠在内皮特异性受体酪氨酸激酶启动子 Tie2 的控制下表达 GFP。对总视网膜细胞的 RNA-Seq 分析主要揭示了 EAU 期间参与抗原呈递和 T 细胞激活的基因的上调。视网膜内皮细胞的特异性转录组分析使我们能够鉴定出 EAU 发育过程中视网膜内皮细胞中受调节的 82 个基因。其中 5 个基因（serpina3n、lcn2、ackr1、lrg1 和 lamc3）的蛋白表达在 BRB 细胞内部水平进行了验证。 这些数据不仅证实了已知致病分子的参与，还进一步提供了可能与非感染性后葡萄膜炎期间内部 BRB 分解有关的新候选基因和途径列表。