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First visualization of circulating neutrophil extracellular traps using cell fluorescence during human septic shock-induced disseminated intravascular coagulation
Université de Strasbourg (UNISTRA), Faculté de Médecine; Hôpitaux universitaires de Strasbourg, Nouvel Hôpital Civil, Service de Médecine Intensive Réanimation, Strasbourg, FranceUMR 1260, Regenerative Nano Medecine, INSERM, Fédération de Médecine Translationnelle de Strasbourg (FMTS), Université de Strasbourg, Strasbourg, France
Université de Strasbourg (UNISTRA), Faculté de Médecine; Hôpitaux universitaires de Strasbourg, Nouvel Hôpital Civil, Service de Médecine Intensive Réanimation, Strasbourg, FranceImmunoRhumatologie Moléculaire, INSERM UMR_S1109, LabEx TRANSPLANTEX, Centre de Recherche d'Immunologie et d'Hématologie, Faculté de Médecine, Fédération Hospitalo-Universitaire (FHU) OMICARE, Fédération de Médecine Translationnelle de Strasbourg (FMTS), Université de Strasbourg, Strasbourg, France
Université de Strasbourg (UNISTRA), Faculté de Médecine; Hôpitaux universitaires de Strasbourg, Nouvel Hôpital Civil, Service de Médecine Intensive Réanimation, Strasbourg, FranceUMR 1260, Regenerative Nano Medecine, INSERM, Fédération de Médecine Translationnelle de Strasbourg (FMTS), Université de Strasbourg, Strasbourg, France
Laboratoire d'hématologie cellulaire, Hôpitaux universitaires de Strasbourg, Strasbourg, FranceINSERM UMR 1113: Interface Recherche Fondamentale et Appliquée en Cancérologie (IRFAC), Strasbourg, France
Netosis features can be detected in blood PMNs using simple fluorescence microscopy.
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PMNs purified from patients with DIC-induced septic shock display NETosis features.
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Morphological analysis of PMNs can distinguish septic shock patients with DIC.
Abstract
Disseminated intravascular coagulation (DIC) is a severe complication of septic shock. Polymorphonuclear neutrophils (PMNs) may play a key role in septic shock-induced DIC via the release of neutrophils extracellular traps (NETs). NETs capture invading pathogens, but also act as a pro-coagulant surface at the interface between immunity and thrombosis. During septic shock-induced DIC, neutrophil activation may result in excessive NET formation. Herein, we originally report the presence of circulating NETs in human blood during septic shock-induced DIC.
To investigate NET formation during shock-induced DIC neutrophils were isolated from patients in septic shock associated with (n = 3) or without (n = 3) DIC. Neutrophils from healthy donors (n = 3) were stimulated in vitro with ionomycin as NET formation positive controls. PMNs smears were stained with mouse anti-human FITC anti-myeloperoxidase antibody and the blue-fluorescent DAPI nucleic acid stain. NETs were identified as elongated extracellular DNA fibers associated to myeloperoxidase detected by immunofluorescence.
NETs were unambiguously observed in PMNs from septic shock patients with DIC but not from patients without DIC. NETs features in DIC+ patients were undistinguishable from those observed in ionomycin-induced PMNs from healthy donors. Fluorescence images of NETs were associated to extracellular cytoplasmic expansions.
Our data report for the first time the direct visualization of circulating NETs in patients with septic shock-induced DIC. The in vivo relevance of previously reported indirect markers of NETosis (neutrophil side fluorescence) is confirmed.
Septic shock is the most severe form of infection, defined as a subset of sepsis in which circulatory, cellular and metabolic abnormalities are profound [
Singer M, Deutschman CS, Seymour CW, Shankar-Hari M, Annane D, Bauer M, et al. The third international consensus definitions for sepsis and septic shock (sepsis-3). JAMA. 2016 23;315(8):801–10.
]. Septic shock is characterized by a broad inflammatory response and coagulation activation that both contribute to bacterial containment, but also leads to microangiopathy through uncontrolled thrombin and fibrin generation, potentially evolving towards disseminated intravascular coagulation (DIC) [
]. During septic shock, DIC exacerbates multiple organ failure and increase the risk of death, underlining the relevance of blood coagulation processes as therapeutic target of interest [
]. Polymorphonuclear neutrophils (PMNs or neutrophils) have recently been identified as potential players in the response to infection by releasing their content, including DNA, histones and granules enzymes [
]. NETs are thus involved in host defense against the invading pathogens. NETs also form a large procoagulant surface and activate the contact phase of coagulation [
] and contribute to subsequent mortality. Furthermore, cytokines released by neutrophils contribute to alter the membrane profile of vascular cells, especially endothelial cells, thus participating to septic shock-induced endothelial dysfunction and coagulation activation [
]. Thus, neutrophils may play a major role in immunothrombosis, defined by interactions between innate immunity, inflammation and thrombosis leading to thrombin generation, especially in microvessels [
]. Nevertheless, the pathophysiological role of neutrophils as modulators of the interactions between coagulation and immunity during infectious and thrombotic diseases, and their interaction with other blood cells remain a matter of debate [
]. Herein, we report an original proof of concept, highlighting circulating NETs in human blood during septic shock-induced DIC.
2. Material and methods
2.1 Patients and blood collection
Patients in septic shock with DIC (n = 3) and without DIC (n = 3) were enrolled in this study, as well as 3 healthy donors. Septic shock was assessed according to the third international consensus definition [
Singer M, Deutschman CS, Seymour CW, Shankar-Hari M, Annane D, Bauer M, et al. The third international consensus definitions for sepsis and septic shock (sepsis-3). JAMA. 2016 23;315(8):801–10.
], and DIC was diagnosed if the JAAM (Japanese Association for Acute Medicine)-DIC 2016 score was at least equal to 4 during the first 24 h of septic shock [
Revision of the Japanese Association for Acute Medicine (JAAM) disseminated intravascular coagulation (DIC) diagnostic criteria using antithrombin activity.
]. At the admission, 5 mL of blood were drawn on EDTA containing tubes (BD Vacutainer Plus Blood Collection Tubes, Becton Dickinson, Le Pont de Claix, France) that contain K2EDTA ethylene-diamine-tetraacetic acid dipotassium dihydrate (EDTA-2K) anticoagulant which is spray-dried to the walls of the tube (final concentration 7.2 mg/5 mL). The Strasbourg University Hospital Ethics Committee approved this study. Informed consent was obtained from the patient or relatives at admission and confirmed by the patient as soon as possible.
2.2 Neutrophil isolation
Neutrophils were immediately purified after blood collection using negative immuno-magnetic sorting method (EasySep®, Stemcell Technologies, Canada) [
]. A total of 5 × 104 cells purified PMNS were subsequently spotted on Thermo Scientific™ SuperFrost™ Microscope Slides (Fisher Scientific, Illkirch, France) using cytocentrifugation at 35 g for 5 min, which preserved cell integrity and morphology.
2.3 Positive controls
NET formation was induced in vitro by stimulation of purified PNNs from healthy donors (n = 3) with 4 μM ionomycin (Calbio-chem, Merk Biodevelopment, France) for 4 h at 37 °C.
Quantification was performed by blinded assessment of extracellular DNA release using fluorescent microscopy (20× magnification). Degree of NETs was quantified as the percentage of neutrophils forming NETs divided by the total number of neutrophils X 100. Samples were categorized into 5 groups: absence of NETs (0% neutrophils forming NETs per microscopic field), scarce number of neutrophils forming NETs (1–25%), moderate NETs (26% to 50%), strong NETs (50% to 75%), and very strong NETs (76% to 100%).
PMNs spots were also stained by standard May-Grünwald-Giemsa for patients, normal PMNs and PMNs after induction of NETosis with ionomycin. Three independent operators reviewed blindly the morphology in at least one hundred neutrophils for each condition. A differential count was established between neutrophils harboring a normal morphology and those harboring morphology similar to PMNs after ionomycin induced NETosis. NETosis-associated morphology in these conditions consisted in nuclear decondensation, cytoplasmic vacuolization and the presence of cytoplasmic membrane expansions visible on >1/3 of the cell circumference or the presence of one or more membrane prolongation larger than 1/3 of the cell size. These modifications reflect neutrophil activation.
2.5 Blood cell count and neutrophil analysis on Sysmex XN
Complete blood cell count was measured on an automated Sysmex™ XN20® analyzer (Sysmex Corporation, Kobe, Japan) according to the manufacturer without intervention. Cell permeabilization was performed by treatment with proprietary lysis reagents allowing the polymethine fluorescent XN-20 basic dye to enter the cells, where it binds to nucleic acids in the cytoplasmic organelles and the nucleus. NEUT-SFL reflects leukocyte activation and is correlated to NETosis [
]. NEUT-SFL values were extracted from the research screen of the analyzer software as previously described.
3. Results
Baseline characteristics, outcome and infection features are summarized in Table 1. Despite the limited number of enrolled that patients, no obvious bias concerning gender, age, comorbidities or type of infection was observed between patients with or without DIC. As expected, patients with DIC had a lower platelet and neutrophil count than non-DIC patients.
Table 1Description of patient characteristics at baseline.
Gender
Female
Male
Male
Female
Male
Male
Age (years)
30
32
81
64
67
71
Comorbidities
DIC
+
+
+
−
−
−
DVT
−
−
+
−
−
−
Arterial thrombosis
−
−
−
+
−
−
Immunosuppression
+
−
−
−
−
−
Chronic heart failure
−
−
−
+
+
−
Cirrhosis
−
−
−
−
−
−
Chronic renal failure
−
−
−
+
−
−
Chronic respiratory failure
−
−
−
+
+
−
Organ failure
Norepinephrine support
+
+
+
+
+
+
Mechanical ventilation
+
+
+
+
+
+
Extrarenal epuration
−
−
−
+
−
−
SOFA score
13
7
9
9
12
14
Mortality
Death at day 7
+
−
−
−
−
−
Death at day 28
−
−
−
−
−
CBC
Leucocytes (G/L) [4.10–10.50]
2.7
21.47
14.77
28.26
30.93
25.2
Neutrophils (G/L) [1.80–7.70]
1.81
20.34
11.5
19.37
26.19
23.18
Platelets (G/L) [150–400]
16
39
169
371
138
306
Hemoglobin (g/dL) [13.0–18.0]
12.5
8.2
9.9
11.9
9.3
10.2
Infection
Community-acquired
+
−
+
+
−
+
Nococomial
−
+
−
−
+
−
Site of infection
Lung
Abdomen
Urinary system
Urinary system
Abdomen
Lung
Microorganism
Streptococcus pneumoniae
Polymicrobial
Escherichiacoli
Klebisiella pneumoniae
Enterococcus faecium
Streptococcus pneumoniae
Blood-culture
+
+
+
−
−
+
PMNS imaging
Elongated extracellular DAPI stained DNA fibers associated to myeloperoxidase
All patients suffered from septic shock. DIC: Disseminated intravascular coagulation; DVT: Deep Venous Thrombosis; CBC: Complete Blood Cell Count. Normal values of CBC parameters are enclosed in square brackets.
PMNs from healthy donors stimulated by ionomycin released NETs visualized using fluorescence microscopy as double-stained DAPI-positive chromatin structures decorated with antimicrobial granule MPO in the extracellular space (Fig. 1B , upper panel).
Fig. 1Representative images of immunofluorescence and May-Grünwald-Giemsa staining showing NETs in purified peripheral blood polymorphonuclear neutrophils (PMNs).
Observations were reproduced at least 3 times in each condition in different donors and patients.
Upper panel: PMNs stained with a FITC-conjugated anti-myeloperoxidase antibody (green) and counterstained using DAPI (blue), ×20 and ×100 original magnification. NETs were identified as elongated extracellular DNA fibers (blue), associated to myeloperoxidase (green).
Lower panel: neutrophils from the same samples stained with May-Grünwald-Giemsa (×60 original magnification), showing extracellular cytoplasmic extensions associated with NET images
A: unstimulated PMNs from healthy donors
B: NETs induced in healthy donors PMNs exposed to ionomycin for 4 h
C: PMNs isolated from patients with septic shock without disseminated intravascular coagulation (DIC)
D: PMNs isolated from patients with septic shock with DIC. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
NET structures similar to those observed after ionomycin stimulation were unambiguously visualized in PMNs from septic shock patients with DIC (Fig. 1D, upper panel), but neither in PMNs from septic shock patients without DIC (Fig. 1C, upper panel), nor in unstimulated PMNs from healthy donors (Fig. 1A, upper panel).
All DIC patients and ionomycin-stimulated PMNs were stratified into “strong NETs formation” (>50% of PMNs displaying DNA release) and “very strong NET formation” (>75% of PMNs displaying DNA release) groups (Fig. 2A ). Conversely, no-DIC patients and unstimulated normal PMNs were stratified into “scarce” or “absence” NET categories.
Fig. 2Quantification of NETS and cytological modifications.
A. NETs quantification using immunofluorescence microscopy (% neutrophils forming NETs per microscopic field, ×20 magnification)
B. Representation of cells harboring cytoplasmic expansions, expressed in percentage of PMNs, determined blindly by 3 different cytologists in all samples.
Ionomycin-stimulated PMNs stained with May-Grünwald-Giemsa harbored chromatin decondensation and vacuolization of the cytoplasm (Fig. 1B, lower panel), features associated to neutrophil activation. NETosis is one of the phenomena that participates to this activation [
]. These features were absent in unstimulated PMNs (Fig. 1A, lower panel). In addition, villous surface projections (also called pseudopods) were detected on the circumference of ionomycin-stimulated PMNs. These modifications were characterized by cytoplasmic membrane expansions visible on >1/3 of the cell circumference or the presence of one or more membrane prolongation larger than 1/3 of the cell size (Fig. 1B, lower panel). The percentage of PMNs harboring a NETosis-associated morphology reviewed by three blinded-operators were strongly elevated in PMNs from DIC patients (Fig. 1D, lower panel and Fig. 2B) but not in no-DIC patients (Fig. 1C, lower panel and Fig. 2B).
Accordingly, the side-fluorescence of PMNs (NEUT-SFL), a Sysmex™ CBC analyzers parameter previously associated to NETosis modifications [
] was increased in normal PMNs stimulated with ionomycin and DIC patients compared to patients without DIC and normal unstimulated normal PMNs (Fig. 3A, B ).
Fig. 3Neutrophil Side Fluorescence Light (NEUT-SFL) measured on Sysmex XN20.
The present report highlights the first direct visualization of circulating NETs in the blood of patients diagnosed with DIC during a septic shock. In this pilot study, PMNs from DIC patients displayed typical NETs structures, that were not observed in PNs from no-DIC patients.
The degree of NETs formation observed in all DIC patients using immunofluorescence microscopy was strong, comparable to the one observed in ionomycin-stimulated PMNs from healthy volunteers.
In previous studies, NETosis was mainly correlated to DIC and thrombosis by serum assays or flow cytometry [
]. We have recently reported that indirect markers of NETosis (MPO-DNA complexes) are elevated in septic shock-induced DIC patients compared to no-DIC patients [
]. These apparently conflicting results may be explained by the use of different NET measurement methods, the characteristics of the patient studied or the anticoagulation therapy administrated for DIC.
Surrogate markers of NETosis such as cfDNA and DNase have been associated to sepsis. These factors reflect cell injury and are elevated in other conditions like thrombotic events, autoimmune diseases or cancer [
Vallés J, Lago A, Santos MT, Latorre AM, Tembl JI, Salom JB, et al. Neutrophil extracellular traps are increased in patients with acute ischemic stroke: prognostic significance. Thromb. Haemost. 2017 05;117(10):1919–29.
]. Although this method is more specific than serum markers, the gating of NETs or NETotic PMNs population using side scatter/forward scatter is difficult and may cause measurement variability and interpretation biases [
]. Taken together, flow cytometry may detect only the earliest stage of NETs release (before plasma membrane perforation and cell lysis) and serum markers the components released after NETosis [
The immunofluorescence methodology used in this study, limited to MPO-DNA complexes visualization, may overpass the limitations of indirect methodologies. It provides a “snapshot” of circulating PMNs at the time of sampling. The staining of citrullinated histones may represent an additional evidence of the occurrence of NETosis in PMNs, but the technical simplicity of the approach used here may facilitate repeated measures during the evolution of the patient in further studies.
The cytological modifications observed in PMNs from DIC patients also need to be further explored, and the different mechanisms of neutrophil activation that cause these changes have to be deciphered with additional markers. However, if the morphological modifications observed in such patients is not entirely explained by NETosis occurrence, chromatin decondensation associated to the modifications of plasma membrane may indicate that NET formation occurs by a “vital NETosis” mechanism, as depicted by de Buhr et al. [
Nevertheless, our data support the in vivo existence and relevance of NETs in humans. For the first time, evidence of the presence of circulating NETs in the blood of patients with septic shock-induced DIC was provided, in accordance with the observation of McDonald et al. who showed that NETs promote intravascular coagulation in mice [
McDonald B, Davis RP, Kim S-J, Tse M, Esmon CT, Kolaczkowska E, et al. Platelets and neutrophil extracellular traps collaborate to promote intravascular coagulation during sepsis in mice. Blood. 2017 09;129(10):1357–67.
Intravascular NETs may induce platelet trapping and microvascular occlusion leading to multi-organ failure. Accordingly, destruction of NETs via DNAse treatment was associated to the impairment of intravascular coagulation, suggesting that the integrity of NETs is critical for coagulation activation [
McDonald B, Davis RP, Kim S-J, Tse M, Esmon CT, Kolaczkowska E, et al. Platelets and neutrophil extracellular traps collaborate to promote intravascular coagulation during sepsis in mice. Blood. 2017 09;129(10):1357–67.
]. New treatments targeting NET formation such as DNAse or Peptidyl arginine deiminase-4 (PAD4) enzyme that mediates histone citrullination may represent an interesting therapeutic approach during septic shock [
McDonald B, Davis RP, Kim S-J, Tse M, Esmon CT, Kolaczkowska E, et al. Platelets and neutrophil extracellular traps collaborate to promote intravascular coagulation during sepsis in mice. Blood. 2017 09;129(10):1357–67.
Most of the treatments administered to DIC patients exhibits anti-coagulant activity that increase the risk of bleeding. Standardization of the evaluation of NETs may help identify patients at different levels of risk in future randomized controlled trials. Multispectral imaging flow cytometry proposed by Zhao et al. is a promising sophisticated method to identify NETs [
], but is not routinely available in clinical practice. If confirmed, observation of the morphology of PMNs after cytocentrifugation and evaluation of their chromatin decondensation level using neutrophil side fluorescence (e.g. NEUT-SFL, [
In this pilot study, we have shown 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 NET features observed following in-vitro ionomycin stimulation of normal PMNs. NET images were furthermore associated to modifications of PMNs morphology observed with May-Grünwald-Giemsa staining. These features may of great interest for the stratification of patients undergoing future treatment protocols targeting NETs.
Funding statement
Association D'Aide aux Insuffisants Respiratoires d'Alsace-Lorraine (ADIRAL, Strasbourg, France).
Declaration of competing interest
Pr. Meziani's institution received funding from Stago.
The remaining authors have disclosed that they do not have any potential conflicts of interest.
We wish to acknowledge Association D'Aide aux Insuffisants Respiratoires d'Alsace-Lorraine for financial support. We also acknowledge Dr. Carine Gervais for precious technical assistance and Delphine Rolland for her careful and critical reading of the manuscript.
References
Singer M, Deutschman CS, Seymour CW, Shankar-Hari M, Annane D, Bauer M, et al. The third international consensus definitions for sepsis and septic shock (sepsis-3). JAMA. 2016 23;315(8):801–10.
Revision of the Japanese Association for Acute Medicine (JAAM) disseminated intravascular coagulation (DIC) diagnostic criteria using antithrombin activity.
Vallés J, Lago A, Santos MT, Latorre AM, Tembl JI, Salom JB, et al. Neutrophil extracellular traps are increased in patients with acute ischemic stroke: prognostic significance. Thromb. Haemost. 2017 05;117(10):1919–29.
McDonald B, Davis RP, Kim S-J, Tse M, Esmon CT, Kolaczkowska E, et al. Platelets and neutrophil extracellular traps collaborate to promote intravascular coagulation during sepsis in mice. Blood. 2017 09;129(10):1357–67.
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