PAD4 Promotes NLRP3 Inflammasome Assembly Leading to NETosis

Abstract

Neutrophil extracellular trap formation (NETosis) and the NLR family pyrin domain containing 3 (NLRP3) inflammasome assembly are associated with a similar spectrum of disorders. We hypothesized that NLRP3 inflammasome may participate in NETosis. Using confocal microscopy, we observed the formation of ASC speck in stimulated neutrophils in the absence of infection, as well as the association of ASC speck with the released NETs. Peptidylarginine deiminase (PAD) 4, a mediator of NETosis, was needed for optimal NLRP3 inflammasome assembly in neutrophils and rendered bone marrow–derived macrophages more susceptible to inflammasome formation, possibly by increasing NLRP3 and ASC protein levels. NLRP3 deficiency or specific inhibition by MCC950 resulted in significantly impaired NETosis, and time-lapse visualization revealed defects in nuclear envelope and plasma membrane breakdown. Our results reveal PAD-dependent formation of NLRP3 inflammasome in neutrophils and macrophages and implicate the NLRP3 inflammasome in NETosis.