The NLRP3 inflammasome is a pivotal sensor in innate immune cells that initiates the inflammatory cascade. Upon exposure to stimuli, it follows a two-stage activation mechanism involving an initial priming phase and a subsequent activation phase. Under pathological conditions, its activation leads to undesirable inflammation that drives or contributes to the pathogenesis of various complex diseases, such as colitis and metabolic diseases. Therefore, curbing NLRP3 inflammasome activity is highly advisable, but no specific NLRP3-targeted medications are available for clinical use. We previously found that shiitake mushroom-derived vesicle-like nanoparticles (S-VLNs) robustly inhibit the NLRP3 inflammasome. In this study, we aimed to identify the active components and found that ergosterol in S-VLNs potently suppresses both the priming and activating steps of the NLRP3 inflammasome. Among the upstream cellular events of NLRP3 inflammasome activation, ergosterol mitigates mitochondrial damage, trans-Golgi disassembly, and inflammasome complex formation without interfering with potassium efflux. At the molecular level, ergosterol promotes the transcriptional activity of Bmal1, a key component of the circadian clock. Knockdown of the Bmal1 gene abolishes the inhibitory effects of ergosterol on both the priming and activating steps of the NLRP3 inflammasome. Conversely, Bmal1 overexpression suppresses NLRP3 inflammasome activity. In vivo, ergosterol mitigates NLRP3 inflammasome signaling and inflammation in mouse disease models of peritonitis and colitis. Our study identifies ergosterol as an effective inhibitor of NLRP3 inflammasome signaling, with therapeutic potential in managing NLRP3 inflammasome-driven diseases. Our mechanistic investigation uncovers Bmal1 as a new regulator of NLRP3 inflammasome activity, mediating the anti-NLRP3 action of ergosterol.
