Single-cell RNA sequencing reveals CD74high CCL5high retinal microglia initiating immune cells infiltration in autoimmune uveoretinitis. Single-cell RNA sequencing reveals CD74high CCL5high retinal microglia initiating immune cells infiltration in autoimmune uveoretinitis

In this study, we found that microglia have a considerable number of cells compared to T cells, indicating an equally critical role of microglia in the progression of autoimmune uveitis. We further identified a specific microglial subpopulation expressed with high levels of CD74 and CCL5, which may be directly related to inflammation regulation in autoimmune uveitis and named inflammation-associated microglia (IAMs). Decreasing the number of IAMs by gene regulation methods or CD74/CCL5 neutralizing antibodies effectively reduced inflammation in EAU mice and delayed disease progression. A mechanistic study indicated that the CD74/CCL5 axis was mainly responsible for the regulation of the immune response in autoimmune uveitis. The intracytoplasmic domain of CD74 (CD74–ICD) may be cleaved by the SPPL2A protease and then activate the NF-kB-dependent inflammation pathway in IAM, resulting in the production of CCL5, which recruits peripheral T cells into the retina and causes an inflammatory burst in autoimmune uveitis mice. Decreasing the level of CD74 or CCL5 could effectively reduce uveitogenic T cell infiltration and relieve the autoimmune response in EAU mouse models, indicating the potential therapeutic value of CD74 and CCL5 in autoimmune uveitis. Overall design: We used single-cell RNA sequencing to explore the pathogenesis of EAU during the course of disease development. We continually detected the clinical manifestations and pathologic changes of uveitis after mice were immunized with IRBP for different days. The slit-lamp results showed that the inflammatory signs of EAU mice were most obvious at day 14, including conjunctival hyperemia and posterior synechiae. The clinical score results suggested that the inflammatory response may begin at day 10 after immunization, reach a peak at 14 days, and gradually recover over the course of disease. Thus, we choosed day 0, day 14, day 21 and day 28.

本研究发现，相较于T细胞，小胶质细胞（microglia）的细胞丰度显著更高，这提示小胶质细胞在自身免疫性葡萄膜炎的病程进展中同样扮演着至关重要的角色。我们进一步鉴定出一类特异性小胶质细胞亚群，该亚群高表达CD74与CCL5，其可能与自身免疫性葡萄膜炎的炎症调控直接相关，我们将其命名为炎症相关小胶质细胞（inflammation-associated microglia, IAMs）。通过基因调控手段或CD74/CCL5中和抗体减少IAMs的数量，可有效减轻实验性自身免疫性葡萄膜炎（experimental autoimmune uveitis, EAU）小鼠的炎症反应，并延缓疾病进程。机制研究表明，CD74/CCL5轴主要负责调控自身免疫性葡萄膜炎中的免疫应答。CD74的胞内结构域（CD74–ICD）可被SPPL2A蛋白酶切割，随后激活IAM中的核因子κB（NF-κB）依赖性炎症通路，进而促进CCL5的产生，招募外周T细胞浸润至视网膜，最终引发自身免疫性葡萄膜炎小鼠的炎症爆发。降低CD74或CCL5的表达水平，可有效减少致葡萄膜炎性T细胞的浸润，并缓解EAU小鼠模型中的自身免疫应答，这表明CD74与CCL5在自身免疫性葡萄膜炎中具有潜在的治疗价值。【实验总体设计】我们采用单细胞RNA测序（single-cell RNA sequencing, scRNA-seq）探究EAU在疾病发展进程中的发病机制。在小鼠经IRBP免疫后的不同天数，我们持续监测葡萄膜炎的临床表现与病理变化。裂隙灯检查结果显示，EAU小鼠的炎症体征在免疫后第14天最为显著，表现为结膜充血与虹膜后粘连。临床评分结果提示，炎症反应可于免疫后第10天启动，于第14天达到峰值，并随病程进展逐渐缓解恢复。因此，我们选取免疫后第0天、第14天、第21天及第28天作为核心观测时间点。