GSDMD- and PAD4-Independent NET Formation Mediates Vascular Occlusions in Septicemia

H. Englert; C. Rangaswamy; M. Divivier; J. Göbel; I. Hermans-Borgmeyer; U. Hermans-Borgmeyer; K. A. Mowen; T. Fuchs; T. Renné

doi:10.1055/s-0044-1779074

Hämostaseologie, Table of Contents

Hamostaseologie 2024; 44(S 01): S13
DOI: 10.1055/s-0044-1779074

Abstracts

Topics

T-03. Pathomechanisms of thrombosis

GSDMD- and PAD4-Independent NET Formation Mediates Vascular Occlusions in Septicemia

H. Englert

¹University Medical Center Hamburg-Eppendorf, Institute of Clinical Chemistry and Laboratory Medicine, Hamburg, Germany

,

C. Rangaswamy

¹University Medical Center Hamburg-Eppendorf, Institute of Clinical Chemistry and Laboratory Medicine, Hamburg, Germany

,

M. Divivier

¹University Medical Center Hamburg-Eppendorf, Institute of Clinical Chemistry and Laboratory Medicine, Hamburg, Germany

,

J. Göbel

¹University Medical Center Hamburg-Eppendorf, Institute of Clinical Chemistry and Laboratory Medicine, Hamburg, Germany

,

I. Hermans-Borgmeyer

²University Medical Center Hamburg-Eppendorf, Transgenic Mouse Unit, Center for Molecular Neurobiology Hamburg, Hamburg, Germany

,

U. Hermans-Borgmeyer

²University Medical Center Hamburg-Eppendorf, Transgenic Mouse Unit, Center for Molecular Neurobiology Hamburg, Hamburg, Germany

,

K. A. Mowen

³The Scripps Research Institute, Department of Chemical Physiology, La Jolla, USA

,

T. Fuchs

¹University Medical Center Hamburg-Eppendorf, Institute of Clinical Chemistry and Laboratory Medicine, Hamburg, Germany

,

T. Renné

¹University Medical Center Hamburg-Eppendorf, Institute of Clinical Chemistry and Laboratory Medicine, Hamburg, Germany

⁴Royal College of Surgeons in Ireland, Irish Centre for Vascular Biology, School of Pharmacy and Biomolecular Sciences, Dublin, Ireland

› Author Affiliations

Abstract

Full Text

Introduction Generation of Neutrophil Extracellular Traps (NETs), also termed NETosis, contribute to host defense; however, excessive NETosis leads to hyperinflammatory, autoimmune and thrombotic disease states. Histone citrullination by peptidyl arginine deiminase 4 (PAD4), and pore formation in the plasma membrane by gasdermin D (GSDMD) are believed to be involved in the formation and release of NETs [1] [2]. In this study, we aimed to study the role of GSDMD and PAD4 in NET formation in experimental models of septicemia and sterile neutrophilia.

Method We generated Gsdmd ^-/- and Pad4 ^-/-gene deficient mice on a Dnase1 ^-/- Dnase1l3 ^-/- deficient background to amplify intravascular NET occlusions in these mice. Animals were challenged in two models of NET formation, sterile neutrophilia, and septicemia, triggered by hepatic transgene expression of granulocyte colony stimulating factor (G-CSF) and injection of LPS and heat-killed E. coli, respectively [3]. The survival rate, body weight, and peripheral body temperature of the mice were measured. The murine lungs were stained with H&E and the NET markers chromatin, myeloperoxidase and citrullinated histone 3 to detect intravascular NET occlusions. We also assessed ex vivo NET formation from isolated murine neutrophils using phorbol-12-myristate-13-acetate (PMA) and calcium ionophore in the presence or absence of GSDMD and PAD4.

Results Here we tested the importance of PAD4, and GSDMD for NETosis in vivo. Gsdmd/Dnase1/Dnase1l3 ^-/- and Pad4/Dnase1/Dnase1l3 ^-/- mice were not protected from death in sterile neutrophilia, and septicemia. Histological analysis of lung sections showed that NET-derived clots occluded blood vessels in both mouse strains. Furthermore, we found that the genetic or pharmacological inhibition of GSDMD blocks ex vivo NETosis in PMA, but not calcium ionophore-activated murine neutrophils. The inhibition of PAD4 did not block ex vivo NETosis, independent of the respective stimulus.

Conclusion Taken together, we challenge the current role of GSDMD by newly reporting that intravascular NETosis is independent of GSDMD in experimental models of sterile neutrophilia and septicemia.

References

References
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