OC 46.2 - Neutrophils protect against Staphylococcus aureus sepsis through neutrophil extracellular trap formation only in neutrophilic mice

Monday, June 24, 2024

15:00 – 15:15 ICT

Room: 111 A-C

Co-Author(s)

Caroline Martens

PhD Researcher
Center for Molecular and Vascular Biology, Department of Cardiovascular Sciences, KU Leuven, Leuven, Belgium
Leuven, Vlaams-Brabant, Belgium

Co-Author(s)

MS

Mathias Stroobants

Lab Technician
Center for Molecular and Vascular Biology, Department of Cardiovascular Sciences, KU Leuven, Leuven, Belgium
Leuven, Vlaams-Brabant, Belgium
Sara Zalghout, PhD

Postdoctoral fellow
Center for Molecular and Vascular Biology, Department of Cardiovascular Sciences, KU Leuven, Leuven, Belgium
Leuven, Vlaams-Brabant, Belgium
ML

Marleen Lox

Animal Technician
Center for Molecular and Vascular Biology, Department of Cardiovascular Sciences, KU Leuven, Leuven, Belgium
Leuven, Vlaams-Brabant, Belgium
Cécile V. Denis

Researcher
INSERM U1176, Université Paris Saclay, Le Kremlin-Bicêtre, France
Le Kremlin-Bicêtre, Ile-de-France, France
MP

Marijke Peetermans

Professor
Laboratory for Clinical Infectious and Inflammatory Diseases, Department of Microbiology, Immunology, and Transplantation, KU Leuven, Leuven, Belgium
Leuven, Vlaams-Brabant, Belgium
Thomas Vanassche

Professor
Department of Cardiovascular Diseases, University Hospitals Leuven, Leuven, Belgium
Leuven, Vlaams-Brabant, Belgium
Peter Verhamme, MD PhD

Professor
Department of Cardiovascular Diseases, University Hospitals Leuven, Leuven, Belgium
Leuven, Vlaams-Brabant, Belgium

Presenting Author(s)

Kimberly Martinod

Assistant Professor
Center for Molecular and Vascular Biology, Department of Cardiovascular Sciences - KU Leuven (Belgium)
Leuven, Vlaams-Brabant, Belgium

Background: Neutrophil extracellular traps (NETs) and von Willebrand factor (VWF) are key drivers of immunothrombosis and thromboinflammation in Staphylococcus aureus sepsis. Peptidylarginine deiminase 4 (PAD4) contributes to NET formation and citrullinates coagulation proteins. NETs interact with VWF and platelets, thus providing a scaffold for the formation of microthrombi that cause microvascular perfusion deficits.

Aims: To study the relationship between NETs, VWF, and PAD4 in murine S. aureus sepsis.

Methods: Murine sepsis was induced by injecting methicillin-resistant S. aureus (MRSA, USA300 strain) intravenously in C57BL/6J, MRP8Cre+PAD4(fl/fl) (NET-defective) or PAD4(fl/fl) (littermates), and VWF-deficient mice. Treatment with granulocyte colony stimulating factor (G-CSF) was used to induce neutrophilia in wild-type and NET-defective mice prior to administration of USA300. Cell counts and plasma levels of citrullinated histone H3-DNA (H3Cit-DNA) complexes, VWF antigen, and PAD4 antigen were measured.

Results: Wild-type mice injected with USA300 developed thrombocytopenia and had higher plasma levels of NETs, VWF, and PAD4 as compared to vehicle-injected mice. MRP8Cre+PAD4(fl/fl) mice had significantly reduced H3Cit-DNA and lower levels of VWF antigen 1 day after USA300 injection as compared to littermate controls, with comparable VWF levels in the later disease phase. Surprisingly, there was no difference in levels of cell-free PAD4 in plasma. VWF-deficient mice also developed similar clinical signs of sepsis, with thrombocytopenia and more substantial neutropenia as compared to wild-type mice. G-CSF-induced neutrophilic mice (prior to sepsis induction) had lower sepsis disease scores and significantly improved survival rates as compared to mice receiving saline control. This protection was absent in the NET-defective mice.

Conclusion(s): Individually eliminating NETs or VWF had little impact in MRSA sepsis in mice, and circulating PAD4 likely does not originate from neutrophils. Inducing neutrophilia protected mice against S. aureus sepsis, an effect which was abrogated in the absence of NET formation. Further interplay between NETs and VWF remains to be determined.