ANGPT1-GABARAP axis modulates NLRP3 inflammasome-mediated pyroptosis in Crohn's disease

Crohn's disease (CD) is characterized by persistent intestinal inflammation, immune dysregulation, and intestinal barrier dysfunction. Inflammasome-mediated pyroptosis is an innate immune mechanism increasingly implicated in inflammatory bowel disease (IBD); however, the upstream molecular signals associated with NLRP3-Caspase-1-GSDMD activation in CD remain insufficiently defined. Here, we explored whether the angiopoietin-1 (ANGPT1)-gamma-aminobutyric acid receptor-associated protein (GABARAP) axis is associated with CD-related pyroptotic signaling.

Protein quantitative trait locus (pQTL)-based two-sample Mendelian randomization (MR) was performed to prioritize pyroptosis-related proteins genetically associated with CD risk. Putative upstream regulators of GABARAP were then examined by two-step MR and mediation analysis. Functional validation was performed using a dextran sulfate sodium (DSS)-induced murine colitis model and LPS plus nigericin-induced cell models of NLRP3 inflammasome activation. ANGPT1-GABARAP signaling and the NLRP3-Caspase-1-GSDMD pathway were evaluated following GABARAP or ANGPT1 knockdown and exogenous recombinant human ANGPT1 (rhANGPT1) supplementation, by qRT-PCR, western blotting, ELISA, and LDH release assays.

MR analysis prioritized GABARAP as a suggestive protective candidate for CD, with genetically predicted higher GABARAP levels associated with a decreased disease risk (OR = 0.563, 95% CI 0.327-0.968, P = 0.038). Two-step MR further suggested a putative genetic association between ANGPT1 and GABARAP, and mediation analysis indicated that GABARAP may partially mediate the genetically predicted ANGPT1-CD association, with an estimated mediation proportion of 22.97%. DSS-induced colitis was associated with reduced ANGPT1 and GABARAP expression, along with increased NLRP3 expression, Caspase-1 processing, GSDMD-N accumulation, and elevated IL-1β, IL-18, and LDH levels. In vitro, silencing either GABARAP or ANGPT1 enhanced NLRP3 inflammasome-associated pyroptotic signaling under LPS plus nigericin stimulation, whereas rhANGPT1 treatment partially attenuated these responses in association with restored GABARAP expression.

These findings support a potential role for the ANGPT1-GABARAP axis in NLRP3 inflammasome-mediated pyroptosis associated with intestinal inflammation. Together, these results provide a genetically informed framework for understanding pyroptosis-related inflammatory signaling in CD and support further investigation of the potential therapeutic relevance of this axis.