Epithelial-derived gasdermin D mediates nonlytic IL-1β release during experimental colitis
Katarzyna Bulek, … , Theresa T. Pizarro, Xiaoxia Li
Katarzyna Bulek, … , Theresa T. Pizarro, Xiaoxia Li
Published June 29, 2020
Citation Information: J Clin Invest. 2020;130(8):4218-4234. https://doi.org/10.1172/JCI138103.
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Research Article Gastroenterology Inflammation

Epithelial-derived gasdermin D mediates nonlytic IL-1β release during experimental colitis

Abstract

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-1β. Here, we report a nonpyroptotic role of full-length GSDMD in guiding the release of IL-1β–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-1β, serving as a signal for cargo loading into secretory vesicles. GSDMD and IL-1β colocalize with the exosome markers CD63 and ALIX intracellularly, and GSDMD and NEDD4 are required for release of CD63+ sEVs containing IL-1β, 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-1β–containing sEVs is detected in cultured colonic explants from colitic mice, GSDMD deficiency substantially attenuates disease severity, implicating GSDMD-mediated release of IL-1β sEVs in the pathogenesis of intestinal inflammation, such as that observed in IBD.

Authors

Katarzyna Bulek, Junjie Zhao, Yun Liao, Nitish Rana, Daniele Corridoni, Agne Antanaviciute, Xing Chen, Han Wang, Wen Qian, William A. Miller-Little, Shadi Swaidani, Fangqiang Tang, Belinda B. Willard, Keith McCrae, Zizhen Kang, George R. Dubyak, Fabio Cominelli, Alison Simmons, Theresa T. Pizarro, Xiaoxia Li

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Figure 7

Polyubiquitination of pro–IL-1β by NEDD4 facilitates its secretion via caspase-8–dependent GSDMD-guided nonlytic pathway.

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Polyubiquitination of pro–IL-1β by NEDD4 facilitates its secretion via c...
YAMC cells were lentivirally transduced with either scramble (Scr) or NEDD4-targeting shRNA (Nedd4-KD), and stable clones treated as indicated; cells with no guide RNA were used as controls (Ctr). Representative Western blots of (A) total protein from supernatants pulled down with anti–IL-1β, and (B) in vitro ubiquitination assays with recombinant human proteins E1 (Ube1), E2 (UbcH5c/Ube2D3), E3 (NEDD4), and IL-1β as a substrate followed by mass spectometry analysis (Supplemental Table 3). (C) IL-1β–deficient YAMC cells were restored with either WT or mutant (IL-1β3KA) cDNA. Cells were treated as indicated, and total protein from supernatants was directly analyzed or subjected to IL-1β IP followed by Western blotting. (D) WCLs and protein extracts from supernatants were probed for indicated proteins. Except for mass spectrometry analysis, all experiments were repeated 3 times and yielded consistent results.