Direct Visualization of Circulating Neutrophil Extracellular Traps Using Cell Fluorescence during Human Septic Shock-Induced Disseminated Intravascular Coagulation
Authors: Stiel Laure , Mayeur-Rousse Caroline , Helms Julie , Meziani Fehrat , Mauvieux Laurent 
Published: November 2019

Septic shock is the most severe form of infection, defined as a subset of sepsis in which circulatory, cellular and metabolic abnormalities are profound and responsible for multiple organ failure and a high mortality-rate. Septic shock is characterized by a broad coagulation activation that can lead to uncontrolled thrombin and fibrin generation, which may evolve to disseminated intravascular coagulation (DIC). DIC increases the risk of death, thus representing a therapeutic target of interest. However, the pathophysiological mechanisms of DIC are not fully understood. Polymorphonuclear neutrophils (PMNs) have recently been identified as potential players of the response to infection by releasing their content, including DNA, histones and granules enzymes. These structures, called neutrophils extracellular traps (NETs), form a large net-like structure in which pathogens get trapped. NETs capture invading pathogens, but also represent a pro-coagulant surface at the interface between immunity and thrombosis. During septic shock-induced DIC, neutrophil activation may result in excessive NET formation. In this study, we originally report the presence of circulating NETs in human blood during septic shock-induced DIC using a simple and robust method.

Blood samples were obtained from healthy human volunteers (n=3), patients with septic shock without DIC (n=3) and with DIC (n=3). PMNs were immediately purified immediately after sampling using negative immune-magnetic sorting method. 5x104 purified PMNs were subsequently spotted on microscope slides using cytocentrifugation at 35g for 5 minutes, thus preserving cell integrity and morphology.

Positive controls for NET formation were obtained using PMNs isolated from healthy volunteers that were stimulated in-vitro with 4 μM ionomycin. PMNs were stained with mouse anti-human FITC anti-myeloperoxidase (MPO) antibody and the blue-fluorescent DAPI nucleic acid stain. NETs were identified as elongated extracellular DAPI stained DNA fibers associated to MPO detected by immunofluorescence microscopy.

The degree of NETs formation was blindly quantified into 5 categories according to the proportion of neutrophils forming NETs per microscopic field: absence (0% of neutrophils forming NETs), scarce (1-25%), moderate (26% to 50%), high (50% to 75%) or very high degree of NETS formation (76% to 100%).

PMNs from healthy donors stimulated by ionomycin released NETs, evidenced as double-stained DAPI-positive chromatin structures decorated with MPO in the extracellular space using fluorescence microscopy.

NET structures similar to those observed after ionomycin stimulation were unambiguously visualized in PMNs from septic shock patients with DIC, but neither in PMNs from septic shock patients without DIC, nor in unstimulated PMNs from healthy donors.

DIC patients were stratified into "high" or "very high" degree of NETs formation groups (&gt;75 % of PMNs displaying DNA release), not different from ionomycin-stimulated PMNs (50 to 75%). Conversely, PMNs from patients devoid of DIC as well as unstimulated normal PMNs from healthy volunteers were stratified into "scarce" or "absence" of PMNs forming NET categories.

Blind review of May-Giemsa-Grunwald (MGG) stained slides showed the correlation of the detection of NETs with nuclear decondensation and vacuolation of the cytoplasm of PMNs, features that are associated with neutrophil activation.

Accordingly, neutrophils' side-fluorescence (NEUT-SFL), a Sysmex™ CBC analyzers parameter previously associated to NETosis (Stiel et al., 2016, Delabranche et al., 2017) was increased during septic shock induced DIC as well as following in-vitro ionomycin stimulation of normal PMNs.

In this study, we showed for the first time direct evidence of circulating NETs in peripheral blood of patients with septic shock-induced DIC using immunofluorescence. These NETs are undistinguishable from NETs features observed following in-vitro ionomycin stimulation of normal PMNs. NETs images were furthermore associated with modifications of PMN morphology that could be detected using MGG staining. This observation may be of great interest in order to stratify patients eligible for future treatment protocols with molecules targeting NETs.

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Disclosures

Meziani: STAGO: Research Funding.