The Pathological Roles of Neutrophil Extracellular Traps (NETs) in Sepsis

Abstract

Neutrophils are amongst the first immune cells to defend against microbial infection and neutrophil extracellular traps (NETs) formation is an efficient microbicidal mechanism to prevent pathogen dissemination. However, NETs can also cause harm by promoting intravascular thrombosis and organ injury in animal models. Although NETs are recognised as targets for developing new therapeutic strategies, there is currently lack of robust and specific methods to quantify NETs, particularly in clinical settings. To progress the translational relevance of NETs formation, this thesis establishes a novel assay for quantifying NETs formation and assesses its clinical relevance. To evaluate the pathogenic roles of NETs, complementary in vivo studies were carried out in two septic mouse models, without or with anti-NETs therapy. The NETs formation assay was initially developed using plasma and sera from septic patients (n=54) admitted to the intensive care units (ICU) of Aintree and Royal Liverpool University Hospitals, or healthy volunteers (n=20). Differentiated neutrophil-like cell line (PLB-985) and isolated healthy (n=10) or patient (n=10) neutrophils were used to investigate the convenient source of neutrophils. Fluorescent staining of extracellular DNA demonstrated that ex vivo NETs formation was induced by directly incubating heterologous neutrophils with plasma or sera from ICU patients, but not normal plasma, unless supplemented with phorbol 12-myristate 13-acetate (PMA). Using this ex vivo NETs assay, critically ill patients can be stratified into 4 groups, absent, mild, moderate and strong NETs formation. To determine the clinical potential of this assay, the NETs-forming capacity was measured in plasma from a consecutive cohort of prospectively recruited adult ICU patients (n= 341) admitted to the Royal Liverpool University hospital. Strong NETsforming capacity was predominantly associated with critically ill patients diagnosed with sepsis. In addition, moderate to strong NETs formation was associated with higher sequential organ failure assessment (SOFA) scores on ICU admission and throughout the study duration (three days following admission). Multivariate regression analysis showed after adjusting for Acute Physiology and Chronic Health Evaluation (APACHE) II that measuring the degree of NETs formation in ICU admission could independently predict disseminated intravascular coagulation (DIC) and mortality whereas known NETs degradation markers, could not. High interleukin (IL)-8 levels were strongly associated with NETs-forming capacity of plasma and blocking IL-8 using either an anti-IL-8 monoclonal antibody or inhibitors of CXCR1/2 signaling (using reparixin) significantly attenuated NETs formation. The pathogenic implications of NETs was determined in two mouse models of sepsis including cecal ligation and puncture (CLP) and intraperitoneal injection of Escherichia coli; sham operated animals or intraperitoneal injection of saline were used as controls. The roles of NETs in both models of sepsis were investigated by depleting neutrophils and treating with DNase I or reparixin. Both the ex vivo assay and in vivo quantification of NETs showed dramatic increases, particularly in the lungs of septic mice. The agreement of both assays demonstrated that the ex vivo assay reflects in vivo NETs formation. The extent of NETs formation was strongly associated with fibrin deposition and lung injury, both of which were 2 dramatically reduced by neutrophil depletion and DNase I treatment. Macrophage inflammatory protein-2 (MIP-2), the CXCR1/2 ligand in mice, was significantly elevated and correlated to levels of NETs release. Inhibition of CXCR1/2 using reparixin decreased NETs formation, fibrin deposition and multiple organ injury without impairing bacterial clearance, and improved surviv