Sprouty2 restricts colonic tuft and goblet cell numbers by repressing epithelial IL‐33 expression

Abstract

OBJECTIVETuft and goblet cells are secretory cell types of the intestinal epithelium that serve key roles in sensing and responding to inflammation. However, the intracellular mechanisms controlling their numbers and activity are incompletely defined. Here, we investigated the role of Sprouty2 (Spry2), an intracellular modulator of PI3K and MAPK signaling, in tuft and goblet cell development. Both PI3K and MAPK are implicated in control of differentiation in the intestine. Furthermore, both pathways have been linked to expression of IL‐33, a cytokine that indirectly influences secretory differentiation by stimulating IL‐13 release from immune cell populations. We tested the hypothesis that Spry2 controls IL‐33 production in the colon, thus altering intestinal secretory cell development.METHODSMucosal scrapings from wild type mice given dextran sodium sulphate (DSS) colitis, and colonoids treated with TNF, were subjected to qPCR analysis for Spry2 expression. Colons from mice with intestinal epithelial‐specific Spry2 deletion (Spryflox/flox;Villin‐Cre, here called Spry2KOIE) and littermate controls were collected at 8 weeks of age. Cytokine levels and epithelial marker expression were determined by immunostaining and qPCR. Signaling pathway alterations were assessed by dot blot array and confirmed by western blot. In vitro, murine colonoids were treated with IL‐33 or IL‐13 +/− signaling pathway inhibitors (LY294002, PI3K; CHIR99021, GSK3β) to determine the effects on cytokine and epithelial marker expression.RESULTSSpry2 levels were decreased in inflamed colons (59% decrease, p<0.05) and TNF‐treated colonoids (45% decrease, p<0.05), demonstrating regulation by inflammation. Spry2KOIE mice displayed increased colonic IL‐33 expression (80% increase, p<0.05), more mesenchymal IL‐13+ cells, more Dclk1+ tuft cells (2.2‐fold, p<0.01), and more Muc2+ goblet cells (1.57‐fold, p<0.01). These mice also had elevated GSK3β phosphorylation (88% increase, p<0.01) on Ser9 (an inhibitory site phosphorylated by Akt), and increased phospho‐Akt (p<0.01). In vitro, inactivation of GSK3β in murine colonoids with CHIR99021 mimicked the increased IL‐33 observed in vivo, but interestingly did not alter Dclk1 or Muc2 expression. This effect was attenuated by PI3K inhibition. IL‐13, but not IL‐33, induced Dclk1 and Muc2 in colonoids, suggesting an epithelial‐mesenchymal crosstalk circuit drives alterations in tuft and goblet cells following Spry2 loss.CONCLUSIONInflammation‐driven loss of colonic epithelial Spry2 de‐represses PI3K/Akt signals that lead to elevated IL‐33 expression. IL‐33 may act, in turn, to promote mesenchymal IL‐13 release that drives a protective expansion of tuft and goblet cells in the epithelium.Support or Funding InformationNIH R01DK095004 (Frey); Crohn's and Colitis Foundation Career Development Award (Schumacher)This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.