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-1? are being intensely investigated as the signaling hub that is dys-regulated in inflammatory bowel disease (IBD). IL-1? 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-1? 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-1? 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-1? production from IECs. Strikingly, LPS+ATP stimulation led to the polyubiquitination of pro-IL-1?, which was secreted and processed into mature IL-1? 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-1?. 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-1? complex. Moreover, inactivating mutation in the Asp276 of GSDMD, which abolishes its pore-forming and pyroptotic activity, did not impact the GSDMD-guided IL-1? secretion from IECs. In TH17 cells, a T helper cell subset highly relevant to intestinal inflammation, this GSDMD-guided secretion of polyubiquitinated pro-IL-1? 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-1? release from non-myeloid cells. The pathogenic role of the GSDMD-guided IL-1? release was investigated in two mouse models of intestinal inflammation. Firstly, while polyubiquitinated pro-IL-1?, mIL-1? 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-1? deficiency in nave T cells reduced their ability to elicit intestinal inflammation. Based on these findings, we hypothesize that the GSDMD mediates distinct pathways (guided secretion and pyroptosis) for IL-1? release in non-myeloid and myeloid cells, which jointly contribute to the pathogenesis of intestinal inflammation in a coordinated manner. We will test this hypothesis through the following aims: (1) Investigate the molecular mechanism for GSDMD-guided secretory pathway for IL-1? release; (2) Investigate the cell-type and pathway-specific role of GSDMD-IL-1? axis in intestinal inflammation, including GSDMD-mediated pyroptotic versus GSDMD-guided IL-1? secretion on the intestinal inflammation.

Public Health Relevance

About 1.3 million people suffer from inflammatory bowel disease (IBD) in the United States. With cause of the disease still elusive, there is currently no cure or preventive measure for the condition. Interluekin-1? (IL-1?) is a potent pro-inflammatory soluble protein that is highly upregulated in the inflamed intestinal tissues in patients with Ulcerative Colitis and Crohn? Disease. It is currently unclear how IL-1? is released by various cell types in the intestine. In this application, we propose to delineate the biochemical events-mediated by Gasdermin D, a novel protein participating in driving IL-1 ? production and intestinal inflammation. Completion of this project will provide novel therapeutic strategies to break the pathogenic cross-talk between immune and structural cells and terminate the non-resolving inflammation.

National Institute of Health (NIH)
National Institute of Allergy and Infectious Diseases (NIAID)
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Special Emphasis Panel (ZAI1)
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Case Western Reserve University
United States
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