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

Abstract

Background and aims: 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.

Methods: 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.

Results: 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.

Conclusion: 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.