Single cell-RNA sequencing analysis of T cells in diabetic Akita mouse inducing experimental autoimmune uveoretinitis

Abstract

Purpose : Several studies have indicated that inflammatory process is involved in the pathogenesis of diabetes, however little is known for an effect of immune responses impaired in diabetic condition on development of autoimmune diseases. We have recently indicated that experimental autoimmune uveitis (EAU) is suppressed in Ins2^Akita (Akita) mice, which spontaneously develop Type 1 diabetes mellitus by a mutation in the insulin 2 gene. This study was conducted to investigate the landscape of transcriptional heterogeneity of T cells in Akita mice by Single cell-RNA sequencing (scRNAseq).

Methods : EAU was induced in 10 C57BL/6 (WT) mice and 10 Akita mice by subcutaneous immunization with 200 µL of 2 mg/mL human IRBP 1-20 (1:1 wt/vol) and CFA containing M. tuberculosis strain H37RA (2.5 mg/mL). Administration of 0.5 µg pertussis toxin (i.p.) was also added. Splenocytes were obtained on day 21 post immunization. T cells were purified by magnetic negative selection using MACS cell separation, and pooled in WT-EAU mice or Akita-EAU mice individually. The number of purified T cells applied to scRNAseq analysis was 4230 cells in WT-EAU mice and 4712 cells in Akita mice, but the number of filtered cells by quality control was 2918 cells in WT-EAU mice and 3782 cells in Akita-EAU mice. Cluster analysis was performed on these cells and the expression levels of genes were compared by Differential expressed genes (DEGs) analysis.

Results : DEGs analysis was performed focusing on whole cells, Th1, Th17, and Treg cells from the cell population obtained by cluster analysis. The most changed gene between WT-EAU and Akita-EAU mice was Jun, the transcription factor Activator Protein-1 (AP-1) component, which was significantly decreased in Akita-EAU mice compared to WT-EAU mice. In Th1 cells, the AP-1 components Jun, Jund, Fos, and Fosb were significantly decreased in Akita-EAU mice compared to WT-EAU mice, and were detected as the higher DEGs. Jun, Fos, and Fosb were also at the upper end of the DEGs in Treg cells, and were significantly reduced in Akita-EAU mice compared to WT-EAU mice. However, Jun was also reduced Th17 cells of Akita-EAU mice compared with WT-EAU mice, but was not significantly.

Conclusions : It was indicated that AP-1 signaling pathway was inhibited in T cells of Akita mice inducing EAU, especially in Th1 and Treg cells, which would be related to suppression of EAU in Akita mice.

This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.