Niflumic acid suppresses NLRP3 inflammasome activation by limiting chloride efflux and mitochondrial ROS production

Aberrant activation of the NLRP3 inflammasome contributes to the development of multiple inflammatory disorders, but clinically approved inhibitors targeting this pathway are still lacking. We investigated whether niflumic acid (NFA), a clinically used nonsteroidal anti-inflammatory drug, regulates NLRP3 inflammasome activation and explored the underlying mechanism.

NLRP3 inflammasome activation was induced in murine bone marrow-derived macrophages and PMA-differentiated THP-1 cells using nigericin, ATP, and monosodium urate crystals. Inflammasome activation, chloride efflux, mitochondrial injury, and mitochondrial ROS production were evaluated by immunoblotting, ELISA, flow cytometry, confocal microscopy, co-immunoprecipitation, and ASC speck analysis. TMEM16F was examined by molecular docking, cellular thermal shift assay, siRNA-mediated silencing, and pharmacological inhibition. The anti-inflammatory effects of NFA were further assessed in mouse models of LPS-induced endotoxemia, MSU-induced peritonitis, and acetaminophen-induced liver injury.

NFA preferentially inhibited NLRP3 inflammasome activation, as evidenced by reduced caspase-1 cleavage and decreased IL-1β and IL-18 maturation and secretion, without significantly affecting TNF-α production or the activation of the AIM2 and NLRC4 inflammasomes. Mechanistically, NFA suppressed ASC speck formation and disrupted NLRP3-ASC interaction, indicating impaired inflammasome assembly. NFA also blocked chloride efflux, alleviated mitochondrial damage, and reduced mitochondrial reactive oxygen species (mtROS) generation, while having minimal effects on K⁺ efflux and Ca²⁺ influx. Moreover, TMEM16F was found to contribute to NFA-mediated inhibition of NLRP3 inflammasome activation, and genetic depletion of TMEM16F or pharmacological inhibition of Ca²⁺-activated chloride channel activity mimicked the effects of NFA on chloride efflux, mtROS production, and NLRP3 inflammasome activation. In vivo, NFA reduced inflammatory injury and inflammasome-associated readouts in multiple inflammatory models.

NFA suppresses NLRP3 inflammasome activation through a mechanism associated, at least in part, with TMEM16F-related chloride efflux and mtROS production. These findings suggest an anti-inflammatory mechanism of NFA that is not fully explained by canonical COX inhibition and support further investigation of NFA as a potential modulator of inflammasome-associated inflammation.