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

Alexander N R Weber,Long Chu,Karen Aymonnier,Clare M Waterman,Deya Cherpokova,Denisa D Wagner,Lai Shi,Patrick Münzer,Venkat Giri Magupalli,Sarah Gutch,Rüdiger E Scharf,Lucas Di Meglio,Nicoletta Sorvillo,Shoichi Fukui,Roberto Negro,Hao Wu

doi:10.3389/fimmu.2021.683803

Abstract

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.

Highlights

Described as part of the innate immune response to microbes [1], there is increasing evidence that neutrophil extracellular traps (NETs) are produced under sterile conditions
To investigate whether NLR family pyrin domain containing 3 (NLRP3) inflammasome assembles in neutrophils under sterile conditions, human peripheral neutrophils were activated with phorbol 12-myristate 13-acetate (PMA) or nigericin for 4 hours
Since ASC speck formation was observed in neutrophils forming NETs, we addressed the importance of the NLRP3 inflammasome assembly on NETosis

Summary

Introduction

Described as part of the innate immune response to microbes [1], there is increasing evidence that neutrophil extracellular traps (NETs) are produced under sterile conditions. They are implicated in a wide variety of inflammatory, (auto) immune, and thrombo-occlusive disorders. NET formation is stimulated by diseases, such as diabetes [7] and cancer [8], and contributes to cancer progression [9, 10]. NETs are decondensed chromatin meshworks ejected by neutrophils upon inflammatory stimulation or hypoxia. NET formation (NETosis) was determined to be a well-orchestrated sequence of cellular events, including disassembly of the cellular cytoskeletons, endomembrane fragmentation, nuclear rounding, plasma membrane permeabilization, and nuclear and plasma membrane rupture [12]

Methods

Results

Conclusion