Dissecting priming and activation mechanisms of NLRP3 against inflammatory diseases

About This Grant

PROJECT SUMMARY Sterile inflammation underlies a broad range of human diseases, including atherosclerosis and monogenic autoinflammatory disorders like Cryopyrin-Associated Periodic Syndromes (CAPS). A central driver of sterile inflammation is the NLRP3 inflammasome, a cytosolic multiprotein complex that mediates the release of the proinflammatory cytokine IL-1β. While inflammasome activation is protective in infections, dysregulated activation contributes to diseases. The classical model of NLRP3 activation involves a two-step process: priming, typically provided by microbial ligands through NF-κB signaling and posttranslational modifications, and activation by danger signals. However, the mechanisms driving inflammasome activation in sterile inflammatory diseases remain poorly defined. In my K99 phase, I will investigate how oxidized phospholipids (oxPAPC), which accumulate in atherosclerotic plaques, function as “all-in-one” sterile signals to both prime and activate the NLRP3 inflammasome in macrophages. Our preliminary data show that oxPAPC drives NRF2-dependent metabolic rewiring, leading to glycosylation of NLRP3 – a novel regulatory mechanism required for inflammasome priming and activation. I will dissect these mechanisms using a combination of structural biology, glycoproteomics, and spatial transcriptomics. This work will identify key posttranslational modifications controlling inflammasome activity and map the spatial organization of IL-1β–producing macrophages within atherosclerotic lesions. In the R00 phase, I will apply the conceptual and technical framework developed during the K99 to study CAPS, a group of rare autoinflammatory diseases caused by NLRP3 gain-of-function mutations. I hypothesize that CAPS-associated variants mimic or bypass the priming step through loss of inhibitory phosphorylation and competition with glycosylation. I will generate a novel inducible knock-in mouse model to investigate how these mutations alter inflammasome activation and tissue inflammation in vivo, combining single-cell transcriptomics and immunofluorescence to uncover immune–stromal crosstalk in inflamed skin and joints. By integrating studies of metabolic inflammation and monogenic autoinflammation, this project will uncover shared checkpoints of NLRP3 regulation, with the ultimate goal of identifying new therapeutic targets that suppress pathological inflammation while preserving host defense.