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

P. Münzer^1,2,3, R. Negro^1,4, S. Fukui^1,2, L. di Meglio^1,5,6, D. Cherpokova^1,2, N. Sorvillo^1,2, L. Shi^1,2, V.G. Magupalli^1,4, S. Gutch^1,2, L. Chu^1,2, R.E. Scharf^1,2,7, C.M. Waterman^8,9, H. Wu^1,4, D.D. Wagner^1,2,8

¹Boston Children's Hospital, Program in Cellular and Molecular Medicine, Boston, United States, ²Harvard Medical School, Department of Pediatrics, Boston, United States, ³University of Tübingen, Department of Cardiology and Cardiovascular Medicine, Tübingen, Germany, ⁴Harvard Medical School, Department of Biological Chemistry and Molecular Pharmacology, Boston, United States, ⁵Marine Biological Laboratory, Whitman Center, Woods Hole, United States, ⁶INSERM University of Paris, Laboratory of Vascular Translational Science, Paris, France, ⁷Heinrich Heine University Medical Center, Division of Experimental and Clinical Hemostasis, Hemotherapy, and Transfusion Medicine, and Hemophilia Comprehensive Care Center, Düsseldorf, Germany, ⁸Whitman Center, Marine Biological Laboratory, Woods Hole, United States, ⁹National Heart, Lung, and Blood Institute of the National Institutes of Health, Cell Biology and Physiology Center, Bethesda, United States

Abstract Number: OC 14.2

Meeting: ISTH 2020 Congress

Theme: Vascular Biology » Innate and Adaptive Immunity

Background: 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 including thrombosis. Mainly described in macrophages, the NLRP3 inflammasome represents a multiprotein signaling platform that mediates pivotal responses of innate immunity after activation. However, products of NLRP3 inflammasome activation are also known to play a role in NETosis. Nevertheless, nothing is known about the role of NLRP3 in neutrophils or NETosis.

Aims: The present study examined whether the NLRP3 inflammasome is implicated in NETosis and thus contributes to a stenosis model of deep vein thrombosis.

Methods: The study employed confocal microscopy, time-lapse microscopy and stenosis-induced deep vein thrombosis.

Results: Neutrophil stimulation with the well-known NLRP3 activator nigericin or ionomycin resulted in assembly of the NLRP3 inflammasome as observed by ASC speck formation. Interestingly, ASC speck formation was significantly impaired in PAD4-deficient neutrophils, which showed diminished ASC and NLRP3 protein levels when compared to wild-type neutrophils. Moreover, in an in vitro model of NETosis, genetic deficiency of NLRP3 or pharmacological inhibition with the established inhibitor MCC950 resulted in significantly decreased NET formation after induction with inflammasome activators nigericin or monosodium urate crystals (MSU), as well as with ionomycin. Time-lapse microscopy of ionomycin-stimulated NLRP3-deficient neutrophils revealed a role for NLRP3 in the breakage of the nuclear envelope during NETosis, phenocopying PAD4 deficiency. Finally, a model of stenosis-induced deep vein thrombosis in mice unraveled a prominent role of NLRP3 in the progression and expansion of venous thrombus size.

Conclusions: The present observations uncover the importance of NLRP3 inflammasome/speck assembly in neutrophils. We show that NLRP3 signaling is a strong contributor to NETosis and is implicated in thrombus progression in stenosis-induced deep vein thrombosis. Thus, NLRP3 inhibitors attenuating NETosis could be beneficial to curtail NET-mediated inflammatory and thrombotic diseases.

To cite this abstract in AMA style:

Münzer P, Negro R, Fukui S, di Meglio L, Cherpokova D, Sorvillo N, Shi L, Magupalli VG, Gutch S, Chu L, Scharf RE, Waterman CM, Wu H, Wagner DD. PAD4-Dependent Assembly of NLRP3 Inflammasome Promotes NET Formation Resulting in Venous Thrombus Progression [abstract]. Res Pract Thromb Haemost. 2020; 4 (Suppl 1). https://abstracts.isth.org/abstract/pad4-dependent-assembly-of-nlrp3-inflammasome-promotes-net-formation-resulting-in-venous-thrombus-progression/. Accessed January 21, 2022.

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