GSDMB is increased in IBD and regulates epithelial restitution/repair independent of pyroptosis

Gasdermins are a family of structurally-related proteins originally described for their role in pyroptosis. Gasdermin B (GSDMB) is currently the least studied, and while its association with genetic susceptibility to chronic, mucosal inflammatory disorders is well-established, little is known of its functional relevance during active disease states. Herein, we report increased GSDMB in inflammatory bowel disease, with single-cell analysis identifying epithelial specificity to inflamed colonocytes/crypt top colonocytes. Surprisingly, mechanistic experiments and transcriptome profiling reveal lack of inherent GSDMB-dependent pyroptosis in activated epithelial cells and organoids, but instead, point to increased proliferation and migration during in vitro wound closure, which arrests in GSDMB-deficient cells that display hyper-adhesiveness and enhanced formation of vinculin-based actomyosin stress fibers dependent on PDGF-A-mediated FAK phosphorylation. Importantly, carriage of disease-associated GSDMB SNPs confers functional defects disrupting epithelial restitution/repair, which altogether, establishes GSDMB as a critical factor for restoration of epithelial barrier function and the resolution of inflammation. Colonic epithelial cell line, HT-29, were CRISPR cas9 targetted for GSDMB deletion: GSDMB-KO cells. GSDMB-KO cells were then rescued with overexpression of either canonical GSDMB or mutant, IBD-associated GSDMB containing 2 SNPs: glycine to arginine at position 299 and from proline to serine at position 306.