Abstract 120: Neutrophilic Mice Are Protected Against Staphylococcus Aureus Sepsis And Immunothrombosis Through Peptidylarginine Deiminase 4-mediated Neutrophil Extracellular Trap Formation

Background: Neutrophil extracellular traps (NETs) and von Willebrand factor (VWF) have been proposed as 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 PAD4, NETs, and VWF 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 flfl (NET-defective) or PAD4 fl/fl littermates. In separate experiments, granulocyte colony stimulating factor (G-CSF) was used to induce neutrophilia prior to administration of USA300. Mice were followed for up to 8 days for clinical scoring and reaching humane endpoints as a surrogate readout for survival. Cell counts and plasma levels of citrullinated histone H3-DNA (H3Cit-DNA) complexes, VWF antigen, and PAD4 were measured at days 1, 4, and 8. Results: WT mice injected with USA300 developed thrombocytopenia and had higher plasma levels of NETs, VWF, and PAD4 as compared to vehicle (saline)-injected mice. MRP8Cre + PAD4 flfl mice had significantly reduced H3Cit-DNA and lower levels of VWF antigen 1d after USA300 injection as compared to littermate controls, with comparable VWF levels in the later disease phase. There was no difference in levels of cell-free PAD4 in plasma indicating a non-neutrophil source of PAD4, but the substantial reduction of plasma NET levels supporting that neutrophil PAD4 was essential for NET release. 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, who surprisingly retained higher VWF levels. Conclusion: 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.