Abstract: About 1.3 million people suffer from IBD (chronic inflammation of the intestine) in the United States. The etiology of IBD remains elusive and preventive measures or a cure are not available. Inflammasomes and derived cytokines IL-1β are being intensely investigated as the signaling hub that is dys-regulated in inflammatory bowel disease (IBD). IL-1β is synthesized as inactive pro-forms with no secretory signal sequence. Recent studies have found that a lipid binding protein, Gasdermin D (GSDMD), is required for release of IL-1β in response to caspase-1/11 inflammasome activation. This breakthrough led to a rapid growth of literature that focuses on the pore forming and associated pyroptotic activity of GSDMD in myeloid cells. Interestingly, we discovered a novel nonpyrototic role of GSDMD in guiding the release of IL-1β containing vesicles from intestinal epithelia cells and T cells in response to Caspase 8 (Casp8) but not casp1 activation. Through unbiased proteomic analysis, we identified a set of novel GSDMD-interacting proteins in intestinal epithelial cells (IECs), including NEDD4 (an E3 ligase) and the Hsp90 co-chaperone CDC37. Ablation of GSDMD or NEDD4 abolished LPS and ATP-induced IL-1β production from IECs. Strikingly, LPS+ATP stimulation led to the polyubiquitination of pro-IL-1β, which was secreted and processed into mature IL-1β along with a complex containing full-length GSDMD, Hsp90/CDC37, NEDD4, Atg7, Casp8 but not Casp1. In vitro ubiquitination assay demonstrated that NEDD4, known to interact with LC3 and promote cargo loading into secretory vesicles, catalyzed the polyubiquitination of pro-IL-1β. Indeed, while GSDMD was associated with LC3+ vesicles; GSDMD-dependent release of extracellular vesicles (< 200 nm) were detected by electron microscopy, which contains the GSDMD/NEDD4/IL-1β complex. Moreover, inactivating mutation in the Asp276 of GSDMD, which abolishes its pore-forming and pyroptotic activity, did not impact the GSDMD-guided IL-1β secretion from IECs. In TH17 cells, a T helper cell subset highly relevant to intestinal inflammation, this GSDMD-guided secretion of polyubiquitinated pro-IL-1β complex (GSDMD, Hsp90, Casp8 and NEDD4) was also readily detected in response to ATP stimulation and TCR activation. Collectively, the data revealed a novel nonpyroptotic role for GSDMD in IL-1β release from non-myeloid cells. The pathogenic role of the GSDMD-guided IL-1β release was investigated in two mouse models of intestinal inflammation. Firstly, while polyubiquitinated pro-IL-1β, mIL-1β and the GSDMD/NEDD4-secretary complex were induced in a GSDMD-dependent manner in the intestinal explants in response to dextran sodium sulfate (DSS)-induce colitis, GSDMD-deficiency attenuated the intestinal inflammation. Secondly, GSDMD or IL-1β deficiency in naïve T cells reduced their ability to elicit intestinal inflammation. Based on these findings, we hypothesize that the GSDMD mediates distinct pathways (guided secretion and...