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.