Bilirubin stabilizes the mitochondrial membranes during NLRP3 inflammasome activation

Mitochondria sense both intracellular and extracellular stress, with the subsequence released mt-ROS resulted from the interruption of its membrane integrity being a direct activator for NLRP3 inflammasome activation. Regulating the morphology and function of mitochondria could be a strategy against uncontrolled inflammation. We have previously reported that physiological concentrations of bilirubin exhibit anti-inflammatory effect by inhibiting both NF-κB and inflammasome activation. In the current study, we investigated its anti-NLRP3 inflammasome effect per se by means of detecting releasing of IL-1β and TNF-α, the formation of ASC oligomers and ASC-specks, as well as pro-caspase-1 recruitment. Mechanistically, with respect to the antioxidant nature of bilirubin, we evaluated the effect of bilirubin on the releasing of mt-ROS from mitochondria. In addition, mitochondrial morphofunction mainly including morphology and membrane potential in contact living macrophages was analyzed by applying a newly developed multiplexed high-content mitochondrial imaging analysis system using live-cell microscopy. We revealed that bilirubin targets and stabilizes mitochondrial membrane during NLRP3 inflammasome activation; defined doses of bilirubin could be considered as a mitochondria targeted medication against inflammasome-related diseases.