Uveitis, a vision-threatening inflammatory disease worldwide, is closely related to resident microglia. Retinal microglia are the main immune effector cells with strong plasticity, but their role in uveitis remains unclear. N6-methyladenosine (m⁶A) modification has been proven to be involved in the immune response. Therefore, we in this work aimed to identify the potentially crucial m⁶A regulators of microglia in uveitis. Through the single-cell sequencing (scRNA-seq) analysis and experimental verification, we found a significant decrease in the expression of fat mass and obesity-associated protein (FTO) in retinal microglia of uveitis mice and human microglia clone 3 (HMC3) cells with inflammation. Additionally, FTO knockdown was found to aggravate the secretion of inflammatory factors and the mobility/chemotaxis of microglia. Mechanistically, the RNA-seq data and rescue experiments showed that glypican 4 (GPC4) was the target of FTO, which regulated microglial inflammation mediated by the TLR4/NF-κB pathway. Moreover, RNA stability assays indicated that GPC4 upregulation was mainly regulated by the downregulation of the m⁶A “reader” YTH domain family protein 3 (YTHDF3). Finally, the FTO inhibitor FB23-2 further exacerbated experimental autoimmune uveitis (EAU) inflammation by promoting the GPC4/TLR4/NF-κB signaling axis, and this could be attenuated by the TLR4 inhibitor TAK-242. Collectively, a decreased FTO could facilitate microglial inflammation in EAU, suggesting that the restoration or activation of FTO function may be a potential therapeutic strategy for uveitis.

葡萄膜炎是一种世界范围内威胁视力的炎症性疾病，与常驻小胶质细胞密切相关。视网膜小胶质细胞是主要的免疫效应细胞，可塑性强，但其在葡萄膜炎中的作用尚不清楚。N6-甲基腺苷（m ⁶ A）修饰已被证明参与免疫反应。因此，我们在这项工作中旨在确定葡萄膜炎中小胶质细胞的潜在关键 m ^{6 A 调节因子。}通过单细胞测序（scRNA-seq）分析和实验验证，我们发现葡萄膜炎小鼠视网膜小胶质细胞和人小胶质细胞克隆3（HMC3）细胞中脂肪量和肥胖相关蛋白（FTO）的表达显着下降有炎症。此外，发现FTO敲低会加剧炎症因子的分泌和小胶质细胞的移动性/趋化性。从机制上讲，RNA-seq数据和救援实验表明磷脂酰肌醇蛋白聚糖4（GPC4）是FTO的靶标，FTO调节TLR4/NF-κB通路介导的小胶质细胞炎症。^{此外，RNA稳定性测定表明GPC4的上调主要是通过m 6} A“阅读器”YTH结构域家族蛋白3（YTHDF3）的下调来调节的。最后，FTO 抑制剂 FB23-2 通过促进 GPC4/TLR4/NF-κB 信号轴进一步加剧实验性自身免疫性葡萄膜炎 (EAU) 炎症，而 TLR4 抑制剂 TAK-242 可以减弱这种情况。总的来说，FTO 减少可能会促进 EAU 中的小胶质细胞炎症，这表明 FTO 功能的恢复或激活可能是葡萄膜炎的潜在治疗策略。