Epithelial-Derived Gasdermin D Mediates Nonlytic Il-1 Beta Release During Experimental Colitis

Gasdermin D (GSDMD) induces pyroptosis via the pore-forming activity of its N-terminal domain, cleaved by activated caspases associated with the release of IL-1 beta. Here, we report a nonpyroptotic role of full-length GSDMD in guiding the release of IL-1 beta-containing small extracellular vesicles (sEVs) from intestinal epithelial cells (IECs). In response to caspase-8 inflammasome activation, GSDMD, chaperoned by Cdc37/Hsp90, recruits the E3 ligase, NEDD4, to catalyze polyubiquitination of pro-IL-1 beta, serving as a signal for cargo loading into secretory vesicles. GSDMD and IL-1 beta colocalize with the exosome markers CD63 and ALIX intracellularly, and GSDMD and NEDD4 are required for release of CD63(+) sEVs containing IL-1 beta, GSDMD, NEDD4, and caspase-8. Importantly, increased expression of epithelial-derived GSDMD is observed both in patients with inflammatory bowel disease (IBD) and those with experimental colitis. While GSDMD-dependent release of IL-1 beta-containing sEVs is detected in cultured colonic explants from colitic mice, GSDMD deficiency substantially attenuates disease severity, implicating GSOMD-mediated release of IL-1 beta sEVs in the pathogenesis of intestinal inflammation, such as that observed in IBD.