Characterization of molecular and cellular mechanisms regulating NLRP3 inflammasome activity

Abstract

The NLRP3 protein is a key initiator of inflammation in humans. NLRP3 becomes activated by a multitude of danger signals, including microbial infection, metabolic dysfunction, and cell-internalized particulates. Upon activation, NLRP3 nucleates formation of a multiprotein complex called the inflammasome, in which caspase-1 activity mediates processing of the pro-inflammatory cytokines IL-1β and IL-18 and induces pyroptosis, a pro-inflammatory form of cell death. While multiple regulators of the NLRP3 inflammasome have been described, specific ligands of NLRP3 and a comprehensive mechanism for its activation remain largely unknown. We performed a proteomics screen using co-immunoprecipitation (IP) and mass spectrometry to identify cellular proteins that bind NLRP3. Using this screen, we identified multiple NLRP3 interactors, including a family of mitochondrial solute carrier proteins. Co-IP experiments verified that four of these carriers, namely CTP, ANT3, OGC, and Citrin, specifically interact with NLRP3. Because of its linkage with metabolic and inflammatory diseases, we further assessed the role of Citrin, a calcium-binding aspartate/glutamate carrier, in inflammasome function. When overexpressed, Citrin induced IL-1β release and inflammasome nucleation, while CRISPR/Cas9 knockout of Citrin from THP-1 macrophages significantly abrogated NLRP3-dependent pyroptosis, IL-1β release, and ASC speck formation. Citrin’s ability to promote NLRP3 inflammasome activation was dependent on its calcium-sensing N-terminal domain. Importantly, Citrin’s regulatory function was specific to NLRP3 and did not affect the AIM2 inflammasome or other cell death pathways. Altogether, our studies identify Citrin as a new mitochondrial regulator of the NLRP3 inflammasome.