NLRP3 Inflammasome Assembly in Neutrophils Is Supported by PAD4 and Promotes NETosis Under Sterile Conditions

Front Immunol. 2021 May 28;12:683803. doi: 10.3389/fimmu.2021.683803. eCollection 2021.


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.

Keywords: MCC950; NETs; NLRP3 inflammasome; Neutrophils; PAD4; deep vein thrombosis.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, N.I.H., Intramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Caspase 1 / pharmacology
  • Disease Models, Animal
  • Extracellular Traps / metabolism*
  • Humans
  • Inflammasomes / metabolism*
  • Male
  • Mice
  • Mice, Inbred C57BL
  • Mice, Knockout
  • NLR Family, Pyrin Domain-Containing 3 Protein / deficiency*
  • NLR Family, Pyrin Domain-Containing 3 Protein / genetics
  • Neutrophils / drug effects
  • Neutrophils / enzymology*
  • Protein-Arginine Deiminase Type 4 / metabolism
  • Venous Thrombosis / blood
  • Venous Thrombosis / enzymology
  • Venous Thrombosis / genetics


  • Inflammasomes
  • NLR Family, Pyrin Domain-Containing 3 Protein
  • NLRP3 protein, human
  • Nlrp3 protein, mouse
  • Caspase 1
  • Protein-Arginine Deiminase Type 4
  • peptidylarginine deiminase 4, mouse