Mohmad Alsabani1, Simon T Abrams2, Zhenxing Cheng3, Ben Morton4, Steven Lane5, Samar Alosaimi6, Weiping Yu7, Guozheng Wang8, Cheng-Hock Toh9. 1. Department of Clinical Infection, Microbiology and Immunology, University of Liverpool, Liverpool, UK; College of Applied Medical Sciences, King Saud bin Abdulaziz University for Health Sciences, Riyadh, Saudi Arabia. 2. Department of Clinical Infection, Microbiology and Immunology, University of Liverpool, Liverpool, UK; Coagulation, Liverpool University Hospitals NHS Foundation Trust, Liverpool, UK. 3. The Medical School, Southeast University, Nanjing, China; Department of Gastroenterology, The First Affiliated Hospital, Anhui Medical University, Hefei, China. 4. Department of Clinical Science, Liverpool School of Tropical Medicine, Liverpool, UK; Malawi-Liverpool Wellcome Trust Clinical Research Programme, Blantyre, Malawi. 5. Medical Statistics, Institute of Translational Medicine, University of Liverpool, Liverpool, UK. 6. Department of Clinical Infection, Microbiology and Immunology, University of Liverpool, Liverpool, UK. 7. The Medical School, Southeast University, Nanjing, China. 8. Department of Clinical Infection, Microbiology and Immunology, University of Liverpool, Liverpool, UK; Coagulation, Liverpool University Hospitals NHS Foundation Trust, Liverpool, UK. Electronic address: wangg@liverpool.ac.uk. 9. Department of Clinical Infection, Microbiology and Immunology, University of Liverpool, Liverpool, UK; Roald Dahl Haemostasis & Thrombosis Centre, Liverpool University Hospitals NHS Foundation Trust, Liverpool, UK. Electronic address: toh@liverpool.ac.uk.
Abstract
BACKGROUND: Neutrophil extracellular traps (NETs) facilitate bacterial clearance but also promote thrombosis and organ injury in sepsis. We quantified ex vivo NET induction in septic humans and murine models of sepsis to identify signalling pathways that may be modulated to improve outcome in human sepsis. METHODS: NET formation in human donor neutrophils was quantified after incubation with plasma obtained from patients with sepsis or systemic inflammation (double-blinded assessment of extracellular DNA using immunofluorescence microscopy). NET formation (% neutrophils forming NETs) was correlated with plasma cytokine levels (MultiPlex assay). Experimental sepsis (caecal ligation and puncture or intraperitoneal injection of Escherichia coli) was assessed in C57/BL6 male mice. The effect of pharmacological inhibition of CXCR1/2 signalling (reparixin) on NET formation, organ injury (hepatic, renal, and cardiac biomarkers), and survival in septic mice was examined. RESULTS: NET formation was higher after incubation with plasma from septic patients (median NETs=25% [10.5-46.5%]), compared with plasma obtained from patients with systemic inflammation (14% [4.0-23.3%]; P=0.02). Similar results were observed after incubation of plasma from mice with neutrophils from septic non-septic mice. Circulating CXCR1/2 ligands correlated with NETosis in patients (interleukin-8; r=0.643) and mice (macrophage inflammatory protein-2; r=0.902). In experimental sepsis, NETs were primarily observed in the lungs, correlating with fibrin deposition (r=0.702) and lung injury (r=0.692). Inhibition of CXCR1/2 using reparixin in septic mice reduced NET formation, multi-organ injury, and mortality, without impairing bacterial clearance. CONCLUSION: CXCR1/2 signalling-induced NET formation is a therapeutic target in sepsis, which may be guided by ex vivo NET assays.
BACKGROUND: Neutrophil extracellular traps (NETs) facilitate bacterial clearance but also promote thrombosis and organ injury in sepsis. We quantified ex vivo NET induction in septic humans and murine models of sepsis to identify signalling pathways that may be modulated to improve outcome in human sepsis. METHODS: NET formation in human donor neutrophils was quantified after incubation with plasma obtained from patients with sepsis or systemic inflammation (double-blinded assessment of extracellular DNA using immunofluorescence microscopy). NET formation (% neutrophils forming NETs) was correlated with plasma cytokine levels (MultiPlex assay). Experimental sepsis (caecal ligation and puncture or intraperitoneal injection of Escherichia coli) was assessed in C57/BL6 male mice. The effect of pharmacological inhibition of CXCR1/2 signalling (reparixin) on NET formation, organ injury (hepatic, renal, and cardiac biomarkers), and survival in septic mice was examined. RESULTS: NET formation was higher after incubation with plasma from septic patients (median NETs=25% [10.5-46.5%]), compared with plasma obtained from patients with systemic inflammation (14% [4.0-23.3%]; P=0.02). Similar results were observed after incubation of plasma from mice with neutrophils from septic non-septic mice. Circulating CXCR1/2 ligands correlated with NETosis in patients (interleukin-8; r=0.643) and mice (macrophage inflammatory protein-2; r=0.902). In experimental sepsis, NETs were primarily observed in the lungs, correlating with fibrin deposition (r=0.702) and lung injury (r=0.692). Inhibition of CXCR1/2 using reparixin in septic mice reduced NET formation, multi-organ injury, and mortality, without impairing bacterial clearance. CONCLUSION: CXCR1/2 signalling-induced NET formation is a therapeutic target in sepsis, which may be guided by ex vivo NET assays.
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