Loss of Chloride Transporter DRA Triggers Interleukin‐33 Secretion from Colonocytes to drive Type 2 Lamina Propria Lymphocyte Expansion

Abstract

Down Regulated in Adenoma (DRA), the major chloride transporter in the mammalian intestine has recently emerged as a critical gene contributing to the pathogenesis of Ulcerative Colitis (UC). We have previously shown that DRA knockout (KO) mice display several features of UC including compromised barrier function and dysbiosis. Further, DRA KO mice show increased susceptibility to intestinal inflammation. To elucidate the mechanisms by which loss of DRA could impact gut inflammation, gene expression between DRA KO and wild type littermate controls (WT) by RNAseq was compared. The data showed that out of 1100 genes differentially expressed in DRA KO colon, majority were related to immune responses. Upon conducting more specific immune based analysis using nanoString nCounter® Immunology Panel, we observed a significant upregulation of genes promoting effector T cell expansion, specifically Th2. Interleukin‐33 (IL‐33), an alarmin cytokine is known to be significantly upregulated in colons of UC patients and is a key driver of type 2 immune cell expansion. Since alterations in epithelial‐immune cell crosstalk can trigger inflammatory responses, we hypothesized that DRA KO mice may have aberrant epithelial‐immune cell crosstalk which could trigger changes in Lamina Propria Lymphocytes (LPLs). Following isolation of colonic LPLs and staining for various innate and adaptive immune cells, we detected a significant increase (> 2‐fold) in type 2 innate lymphoid cells (ILC2) and in Th2 cells(~8‐fold) in DRA KO mice which were both GATA3+. These observations were specific to the colon and not seen in the ileal LP or mesenteric lymph nodes. We also observed a significant induction of type 2 effector protein amphiregulin, demonstrating an increased type‐2 immune tone in DRA KO colons. Since IL‐33 is important for immune cell activation following tissue injury at mucosal surfaces we analyzed IL‐33 protein levels in the colon. IL‐33 was markedly elevated in DRA KO colons and appeared to be independent of microbiome changes as this increase in IL‐33 still persisted even after cohousing with WT mice for over 4 weeks (allowing exchange of the microbiome). Confocal imaging of colonic tissues and colonoids further confirmed the increase in epithelial IL‐33 and its dissemination from the nucleus to the cytoplasm in DRA KO colonocytes. Since IL‐33 is known to specifically expand a subset of T regs which are also GATA3+; we gated LPLs double positive for FOXP3 and GATA3 using FACS. Interestingly, total T regs and GATA3+ T regs were both markedly elevated in DRA KO colonic LP compared to WT, confirming the functional relevance of increased IL‐33. These results were further validated in human colonoid derived monolayers where DRA was significantly reduced parallel to upregulation of IL‐33 protein in UC, mirroring the results from DRA KO mice. In conclusion, loss of DRA from colonocytes triggers the release of the IL‐33 to drive a type 2 immune response. These observations emphasize the critical importance of loss of DRA and its implicati...