Citrin, a mitochondrial transporter, interacts with NLRP3 and regulates 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 its mechanism of 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, OGCP, and Citrin, specifically interact with NLRP3. Because of its linkage with known metabolic disease, we further assessed the role of Citrin, a calcium-binding aspartate/glutamate carrier, in inflammasome function. When ectopically expressed, Citrin induced IL-1β release in cells, while CRISPR/Cas9 knockout of Citrin from THP-1 macrophages significantly abrogated NLRP3-dependent pyroptosis, IL-1β release, and ASC speck formation. This regulatory activity of Citrin was dependent on its calcium-sensing N-terminal domain. Importantly, this regulation was specific to NLRP3 and did not affect the AIM2 inflammasome or other cell death pathways. Altogether, our studies reveal a novel role for Citrin in regulation of the NLRP3 inflammasome and inflammatory cytokine release.