271 - Emerging role of glutathione in the activation of NLRP3 inflammasome

Abstract

NLRP3 inflammasome is an intracellular protein complex, which is responsible for the maturation of precursor forms of interleukin (IL)-1β and IL-18 into active proinflammatory cytokines and initiation of pyroptotic cell death. Although generation of reactive oxygen species (ROS) has been implicated in the activation of NLRP3 inflammasome, detail mechanism how it is regulated remains unclear. Here we describe that glutathione (GSH), an antioxidant peptide abundantly present in cells, plays an important regulatory role in ATP-dependent inflammasome activation. In LPS-primed J774.1 cells, treatment of cells with ATP induced rapid GSH decline, which led to cellular redox imbalance with concomitant generation of ROS. Similar level of GSH were detected in the culture supernatants suggesting that ATP stimulated GSH efflux. Moreover, ATP treatment also induced efflux of polysulfurated derivatives of GSH such as GSSH and GSSSH. P2X7 receptor antagonist A-804598 treatment remarkably inhibited both IL-1β production and GSH efflux. Importantly, the activation of NLRP3 inflammasome in J774.1 cells was significantly suppressed by adding GSH or its oxidized form GSSG extracellularly, as assessed by IL-1β production. Interestingly, LC-MS/MS data showed that addition of GSH attenuated ATP-induced GSH efflux and induced remarkable increase in persulfide levels of the cell. Immunoprecipitation data showed that addition of GSH or GSSG extracellularly was negatively regulates the NLRP3 inflammasome complex formation. Furthermore, GSH reduced IL-1β production induced by LPS and ATP in mice. Our findings represent a novel model for NLRP3 inflammasome activation by GSH efflux-mediated ROS generation.