In this study, we observed that deletion of CD226 on regulatory T cells (Tregs) precedes renal fibrosis in a mouse unilateral ureteral obstruction (UUO) model. First, we generated Treg-specific CD226 gene knockout mice (CD226^fl/fl Foxp3^YFP-Cre ). Next, CD226^fl/fl Foxp3^YFP-Cre mice and Foxp3^YFP-Cre control mice were subjected to UUO surgery. Pathologic analysis and Sirius red and Masson's trichrome staining showed that the kidneys of CD226^fl/fl Foxp3^YFP-Cre mice following UUO showed much more severe interstitial fibrosis than Foxp3^YFP-Cre control mice at days 10 and 20. Additionally, CD226^fl/fl Foxp3^YFP-Cre mice showed increased fibronectin expression, as demonstrated by immunohistochemistry (IHC) staining. Although Treg cell-restricted CD226 deficiency showed increased Foxp3⁺ expression, expression of the cell surface functional molecule CD103 was significantly reduced, indicating impaired homeostasis in the Tregs of CD226^fl/fl Foxp3^YFP-Cre mice. To better understand CD226 function, RNA sequencing (RNA-Seq) analysis was conducted in Tregs isolated from CD226^fl/fl Foxp3^YFP-Cre and Foxp3^YFP-Cre mice. RNA-Seq data showed that the helper T cell (Th) 2-related cytokines IL-4 and IL-10 were significantly up-regulated in CD226 deficient Tregs. In addition, mRNA analysis of kidney samples from the mice following UUO by qPCR also showed increased IL-4 and IL-10 expression in CD226^fl/fl Foxp3^YFP-Cre mice, as well as elevated TGF-β1 levels, indicating that CD226 deficiency in Tregs resulted in the acquisition of the ability to produce Th2 cytokines. Finally, we found that microRNA-340 (miR-340), which was down-regulated in Tregs isolated from CD226^fl/fl Foxp3^YFP-Cre mice, directly regulated IL-4 gene expression in vitro. These data suggest that the promotion of CD226 signaling on Tregs is a therapeutic target for renal disease.

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