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

Year: 2025
Type: article
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)
DOI: 10.1093/jimmun/vkaf283.1377
Language: English
Open Access status: bronze
OpenAlex ID: W4416446143
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