PAD4-Dependent Assembly of NLRP3 Inflammasome Promotes NET Formation Resulting in Venous Thrombus Progression

Neutrophil granulocytes are instrumental in innate immunity by defending the host against invading pathogens through phagocytosis, the formation of reactive oxygen species, degranulation, and the generation of neutrophil extracellular traps (NETs), a process known as NETosis. NETosis and the NLR family pyrin domain containing 3 (NLRP3) inflammasome assembly are associated with a similar spectrum of human disorders including thrombosis. Mainly described in macrophages, the NLRP3 inflammasome represents a multiprotein signaling platform that mediates pivotal responses of innate immunity after activation. Products of NLRP3 inflammasome activation, however, are suspected to play a role in NETosis, but limited information is known. This was the aim of Patrick Münzer, Ph.D., of Boston Children's Hospital in Boston, MA, and others. They sought to examine whether or not the NLRP3 inflammasome is implicated in NETosis, contributing to a stenosis model of deep vein thrombosis (DVT).

Via the use of microscopy and stenosis-induced DVT, Münzer demonstrated neutrophil stimulation with NLRP3 activators, resulting in assembly of the NLRP3 inflammasome, as observed by ASC speck formation. The ASC speck formation was significantly impaired in PAD4-deficient neutrophils, which showed diminished ASC and NLRP3 protein levels when compared with wild-type neutrophils. In an in vitro model of NETosis, genetic deficiency of NLRP3 or pharmacological inhibition with MCC950 resulted in significantly decreased NET formation after induction with NLRP3 activators. The time-lapse microscopy of ionomycin-stimulated NLRP3-deficient neutrophils revealed a role for NLRP3 in the breakage of the nuclear envelope during NETosis. Specific to the stenosis-induced DVT, he revealed that within tested mice, the role of NLRP3 played a significant role in the progression and expansion of venous thrombus size. Münzer concluded that NLRP3 signaling is a strong contributor to NETosis and is implicated in thrombus progression in stenosis-induced DVT, suggesting that NLRP3 inhibitors attenuating NETosis could be beneficial to curtail NET-mediated inflammatory and thrombotic diseases.

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