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Neutrophil extracellular trap formation (NETosis) and the NLR family pyrin domain containing 3 (NLRP3) inflammasome assembly are associated with a similar spectrum of human disorders. While NETosis is known to be regulated by peptidylarginine deiminase 4 (PAD4), the role of the NLRP3 inflammasome in NETosis was not addressed. Here, we establish that under sterile conditions the cannonical NLRP3 inflammasome participates in NETosis. We show apoptosis-associated speck-like protein containing a CARD (ASC) speck assembly and caspase-1 cleavage in stimulated mouse neutrophils without LPS priming. PAD4 was needed for optimal NLRP3 inflammasome assembly by regulating NLRP3 and ASC protein levels post-transcriptionally. Genetic ablation of NLRP3 signaling resulted in impaired NET formation, because NLRP3 supported both nuclear envelope and plasma membrane rupture. Pharmacological inhibition of NLRP3 in either mouse or human neutrophils also diminished NETosis. Finally, NLRP3 deficiency resulted in a lower density of NETs in thrombi produced by a stenosis-induced mouse model of deep vein thrombosis. Altogether, our results indicate a PAD4-dependent formation of the NLRP3 inflammasome in neutrophils and implicate NLRP3 in NETosis under noninfectious conditions in vitro and in vivo .

Peptidylarginine deiminase 4 (PAD4) catalyzes posttranslational modification of many target proteins through converting protein arginine or mono-methylarginine to citrulline. Neutrophil extracellular trap (NET) formation is the most dramatic manifestation of PAD4-mediated hypercitrullination reaction in neutrophils, which is characterized by the release of nuclear chromatin to form a chromatin network in the extracellular space. Histones H4, one of the major protein components of chromatin, is released into the extracellular space during sepsis, trauma, and ischemia-reperfusion injury and can also be released during the process of NET formation, along with its citrullinated form. The present study showed that histone H4 can induce NET formation in a calcium and PAD4 dependent manner. Histone H4 caused permeabilization of the neutrophil membrane and sustained rise in intracellular calcium that is necessary for activation of PAD4. In comparison, citrullinated histone H4 induced less calcium influx compared with its native form, leading to reduced NET formation. These studies suggest that citrullinated histone H4 could serve as a brake in the pathology of NETs, slowing down the vicious circle between histone H4 and NETs.

Key components of innate immune responses, the NLRP3 sensor, ASC adaptor and Caspase-1 effector form the NLRP3 inflammasome, whose assembly leads to both inflammatory cytokines secretion and cell death. Its priming and activation are regulated by numerous proteins including the serine/threonine kinase NEK7. PCNA is a nuclear scaffolding protein essential for DNA replication. In neutrophils, which are differentiated cells deprived of proliferative capacity, PCNA is exclusively cytoplasmic and regulates neutrophil functions through interactions with different protein partners. Among them, pro-Caspases-3/8/9/10 interact with PCNA to regulate apoptosis. We therefore hypothesize that cytoplasmic PCNA could serve as a regulatory platform for the NLRP3 inflammasome via its interaction with its components. In PMA-differentiated THP-1 macrophages, PCNA relocalized mainly in the cytoplasm. Furthermore, PCNA scaffold inhibitors inhibited IL-1β secretion, pro-Caspase-1 cleavage and pyroptosis-activating gasdermin D cleavage, induced by either nigericin, monosodium urate crystals or Escherichia coli bacteria. Nigericin-stimulation resulted in cytoplasmic PCNA binding to NEK7 and potentially NLRP3, and the p21 peptide, the highest competitive inhibitor of PCNA partner binding, disrupted PCNA-NEK7 interaction. Finally, use of bone marrow-derived macrophages from p21 knockout mice suggested that the cyclin-dependent kinase inhibitor p21 might be a natural repressor of the NLRP3 inflammasome. This work provides a proof-of-principle for the potential of disrupting PCNA interactions to regulate both canonical and non-canonical NLRP3 inflammasomes.