4-Hydroxynonenal Inhibits Pyroptosis by Preventing Inflammasome Activation in Macrophages Chia George Hsu#, Camila Lage Chavez*, Mark Sowden#, Bradford C. Berk# #Department of Medicine, Aab Cardiovascular Research Institute, University of Rochester School of Medicine and Dentistry, Rochester, NY, USA *Department of Biology, University of Rochester, Rochester, NY, USA Recent studies indicate that cysteine modification by itaconate on NLR Family Pyrin Domain Containing 3 (NLRP3) plays a critical role in pyroptosis, yet the precise role of this cysteine modification by lipid peroxidation products in pyroptosis remains unclear. 4-hydroxynonenal (HNE) is a major endogenous product of lipid peroxidation that covalently modifies cysteine-containing proteins. Here, we found that HNE at physiological concentration (3uM) blocked nigericin and ATP-induced cell death, as well as secretion of IL-1β and IL-18 in peritoneal macrophages, THP-1 differentiated macrophages and peripheral blood mononuclear cells (PBMC). HNE also prevented IL-1β and IL-18 release during lipopolysaccharide (LPS)-induced sepsis in mice. The antioxidant N-acetylcysteine (NAC) had no inhibitory effect on inflammasome activation, but eliminated the ability of HNE to protect peritoneal macrophages from nigericin-mediated pyroptosis. Mechanistically, HNE (3uM ) did not alter TNFa secretion, NLRP3 protein expression, or nuclear translocation of p65, suggesting specificity for inflammasome activation. Apoptosis-associated speck-like protein containing a CARD (ASC) assembles into a large protein complex (call speck formation) after inflammasome activation. HNE prevented ASC speck formation and oligomerization in response to nigericin treatment in LPS primed macrophages. To identify HNE-targeted proteins, we employed click chemistry by treating macrophages with HNE-Alkyne. We identified NLRP3 as a target protein of HNE. Our findings suggest that the generation of endogenous HNE during oxidative stress is likely to act as a signaling molecule to block inflammasome activation.