Advancing inflammasome research: No-wash immunoassays for active IL-18 and gasdermin D-mediated pyroptosis detection 3596

Abstract Description Effective tools for monitoring inflammasome activation are crucial for understanding its role in immune function and advancing drug discovery efforts. We previously developed high throughput-amenable assays for caspase-1 activation, IL-1β release and NLRP3 target engagement. Here, we expand capabilities for researchers with new assays for two additional inflammasome activation markers: active IL-18 and gasdermin D (GSDMD)-mediated pyroptosis. Both assays utilize an optimized ternary NanoLuc® luciferase, consisting of a thermally stable polypeptide and two small peptide tags (α and β). To selectively measure active IL-18, we labeled human IL-18 binding protein and an IL-18 antibody with the peptide tags, enabling effective capture and stabilization of active IL-18 in cell culture, where it is otherwise unstable at 37 °C. For pyroptosis detection, we labeled GSDMD antibodies—including one specific for the cleaved N-terminal domain—with the peptide tags. This GSDMD assay showed strong signal in THP-1 cells treated with an inflammasome inducer, while signal was absent in THP-1 GSDMD knockout cells, that undergo delayed GSDME-mediated cell death. This demonstrated specificity for GSDMD-mediated pyroptosis. Apoptotic cells do not show active IL-18 release or cleaved GSDMD. These new tools enable studies of pyroptosis in epithelial cells that express IL-18. Together, these novel assays will enhance the toolkit available for inflammasome research and therapeutic development. Funding Sources None Topic Categories Innate Immune Responses and Host Defense: Molecular Mechanisms (INM)