Mitochondrial dysfunction is not involved in Calciprotein particle-induced NLRP3 inflammasome activation in human monocytes Caroline Schmidt, Manuela Rossol, Ulf Wagner Department of Internal Medicine, Division of Rheumatology, Leipzig University, Liebigstraße 19, 04103 Leipzig, Germany In rheumatoid arthritis increased extracellular calcium ions, phosphate ions and fetuin A concentrations at the sites of erosive lesions lead to the formation of so called calciprotein particles (CPPs) and trigger activation of the NLRP3 inflammasome in monocytes. The NLRP3 inflammasome can be activated by a broad range of stimuli and they all share common activation mechanisms. One of these mechanisms is believed to be mitochondrial dysfunction, resulting in the production of mitochondrial ROS (mtROS). However, the role of mitochondria during CPP-induced NLRP3 inflammasome activation remains unknown. Freshly isolated human blood monocytes were primed with LPS and stimulated with CPPs and calcium. Calcium-induced micropinocytosis led to CPP uptake and therefore activation of NLRP3 inflammasome. Here we show that mtROS is not involved in CPP-induced NLRP3 inflammasome activation and that mitochondrial membrane polarization is unaffected. We found that inhibition of mtROS using MitoTEMPO had no effect on the release of IL-1β and thus NLRP3 inflammasome activation. In addition, Imaging Flow Cytometry was used to stain and detect mtROS. No increase of mtROS was observed in comparison to unstimulated control cells. Furthermore, by using TMRM we found that mitochondrial membrane polarization was also not effected by CPPs. In conclusion, our results demonstrate that mitochondrial dysfunction and the production of mtROS and are not involved in CPP-induced NLRP3 inflammasome activation and therefore do not represent a common mechanism for all NLRP3 inflammasome stimuli.