DNA-bound elastase of neutrophil extracellular traps degrades plasminogen, reduces plasmin formation, and decreases fibrinolysis: proof of concept in septic shock plasma.

Activation of platelets and neutrophils in septic shock results in the formation of microvascular clots containing an intricate scaffold of fibrin with neutrophil extracellular traps (NETs) DNA. NETs contain multiple components that might impact endogenous fibrinolysis, resulting in failure to lyse clots in the microcirculation and residual systemic microthrombosis. We propose herein that the reservoir of human neutrophil elastase (HNE) on NETs may directly interfere with the fibrinolytic mechanism via a plasminogen proteolytic pathway. To investigate this mechanism, we constructed fibrin-NETs matrices by seeding and activating neutrophils onto a fibrin surface and monitored plasminogen activation or degradation. We demonstrate that the elastase activity of HNE-DNA complexes is protected from inhibition by plasma antiproteases and sustains its ability to degrade plasminogen. Using mass spectrometry proteomic analysis, we identified plasminogen fragments composed of kringle (K) domains (K1+2+3, k1+2+3+4) and the serine protease (SP) region (K5-SP). We further demonstrate that patients with septic shock with disseminated intravascular coagulation have circulating HNE-DNA complexes, HNE-derived plasminogen fragments, a low plasminogen concentration, and a reduced capacity to generate plasmin onto fibrin. In conclusion, we show that NETs bearing active HNE-DNA complexes reduce plasminogen into fragments, thus impairing fibrinolysis by decreasing the local plasminogen concentration, plasminogen binding to fibrin, and localized plasmin formation.-Barbosa da Cruz, D., Helms, J., Aquino, L. R., Stiel, L., Cougourdan, L., Broussard, C., Chafey, P., Riès-Kautt, M., Meziani, F., Toti, F., Gaussem, P., Anglés-Cano, E. DNA-bound elastase of neutrophil extracellular traps degrades plasminogen, reduces plasmin formation, and decreases fibrinolysis: proof of concept in septic shock plasma.

Neutrophil Fluorescence: A New Indicator of Cell Activation During Septic Shock-Induced Disseminated Intravascular Coagulation.

OBJECTIVE: To investigate the contribution of neutrophil activation as innate immune cells during septic shock-induced disseminated intravascular coagulation. DESIGN: Prospective study. SETTING: One University Hospital ICU. PARTICIPANTS: Hundred patients with septic shock. Thirty-five patients had disseminated intravascular coagulation according to Japanese Association for Acute Medicine 2006 score. INTERVENTION: None. MEASUREMENTS AND MAIN RESULTS: Neutrophil chromatin decondensation was assessed by measuring neutrophil fluorescence (NEUT-side-fluorescence light) labeled by a fluorochrome-based polymethine reagent using a routine automated flow cytometer Sysmex XN20 (Sysmex, Kobe, Japan) and neutrophil-derived CD66b microparticles by prothrombinase assay. Measurements in disseminated intravascular coagulation and no disseminated intravascular coagulation patients showed that a mean value of NEUT-side-fluorescence light above 57.3 arbitrary units had a sensitivity of 90.91% and a specificity of 80.60% for disseminated intravascular coagulation diagnosis. NEUT-side-fluorescence light was correlated to the CD66b microparticles/neutrophil count, a surrogate of neutrophil activation associated with septic shock-induced disseminated intravascular coagulation. CONCLUSION: NEUT-side-fluorescence light, routinely available, could prove an accurate biomarker of neutrophil activation.

Insulin therapy and blood glucose management in critically ill patients: a 1-day cross-sectional observational study in 69 French intensive care units.

BACKGROUND: Hyperglycaemia is common in critically ill patients, but blood glucose and insulin management may differ widely among intensive care units (ICUs). We aimed to describe insulin use practices and the resulting glycaemic control in French ICUs. We conducted a multicentre 1-day observational study on November 23, 2021, in 69 French ICUs. Adult patients hospitalized for an acute organ failure, severe infection or post-operative care were included. Data were recorded from midnight to 11:59 p.m. the day of the study by 4-h periods. RESULTS: Two ICUs declared to have no insulin protocol. There was a wide disparity in blood glucose targets between ICUs with 35 different target ranges recorded. In 893 included patients we collected 4823 blood glucose values whose distribution varied significantly across ICUs (P < 0.0001). We observed 1135 hyperglycaemias (> 1.8 g/L) in 402 (45.0%) patients, 35 hypoglycaemias (≤ 0.7 g/L) in 26 (2.9%) patients, and one instance of severe hypoglycaemia (≤ 0.4 g/L). Four hundred eight (45.7%) patients received either IV insulin (255 [62.5%]), subcutaneous (SC) insulin (126 [30.9%]), or both (27 [6.6%]). Among patients under protocolized intravenous (IV) insulin, 767/1681 (45.6%) of glycaemias were above the target range. Among patients receiving insulin, short- and long-acting SC insulin use were associated with higher counts of hyperglycaemias as assessed by multivariable negative binomial regression adjusted for the propensity to receive SC insulin: incidence rate ratio of 3.45 (95% confidence interval [CI] 2.97-4.00) (P < 0.0001) and 3.58 (95% CI 2.84-4.52) (P < 0.0001), respectively. CONCLUSIONS: Practices regarding blood glucose management varied widely among French ICUs. Administration of short or long-acting SC insulin was not unusual and associated with more frequent hyperglycaemia. The protocolized insulin algorithms used failed to prevent hyperglycaemic events.

Chronic critical illness and post-intensive care syndrome: from pathophysiology to clinical challenges.

BACKGROUND: Post-intensive care syndrome (PICS) encompasses physical, cognition, and mental impairments persisting after intensive care unit (ICU) discharge. Ultimately it significantly impacts the long-term prognosis, both in functional outcomes and survival. Thus, survivors often develop permanent disabilities, consume a lot of healthcare resources, and may experience prolonged suffering. This review aims to present the multiple facets of the PICS, decipher its underlying mechanisms, and highlight future research directions. MAIN TEXT: This review abridges the translational data underlying the multiple facets of chronic critical illness (CCI) and PICS. We focus first on ICU-acquired weakness, a syndrome characterized by impaired contractility, muscle wasting, and persisting muscle atrophy during the recovery phase, which involves anabolic resistance, impaired capacity of regeneration, mitochondrial dysfunction, and abnormalities in calcium homeostasis. Second, we discuss the clinical relevance of post-ICU cognitive impairment and neuropsychological disability, its association with delirium during the ICU stay, and the putative role of low-grade long-lasting inflammation. Third, we describe the profound and persistent qualitative and quantitative alteration of the innate and adaptive response. Fourth, we discuss the biological mechanisms of the progression from acute to chronic kidney injury, opening the field for renoprotective strategies. Fifth, we report long-lasting pulmonary consequences of ARDS and prolonged mechanical ventilation. Finally, we discuss several specificities in children, including the influence of the child's pre-ICU condition, development, and maturation. CONCLUSIONS: Recent understandings of the biological substratum of the PICS' distinct features highlight the need to rethink our patient trajectories in the long term. A better knowledge of this syndrome and precipitating factors is necessary to develop protocols and strategies to alleviate the CCI and PICS and ultimately improve patient recovery.

Leukocyte cell population data in patients with cardiac surgery and cardiopulmonary bypass: A potential readily available tool to monitor immunity.

Purpose: Cardiac surgery with cardiopulmonary bypass triggers sterile inflammation that is responsible for post-operative morbidity. Automated flow cytometry devices used for leucocyte count provide cell population data (CPD) regarding fluorescence intensity, size and granularity of leukocytes that have never been studied in the context of sterile inflammation. Our objective was to explore leukocyte cell population data in patients undergoing cardiac surgery with cardiopulmonary bypass in order to determine whether CPD could be used to monitor immune cell activation. Methods: This is an ancillary study of a cohort of patients undergoing cardiac surgery with cardiopulmonary bypass. Cell population data (CPD) extracted from a routine automated flow cytometer were analyzed (Fluorescence targeted to nucleic acids). The time points of interest were: pre-operative, postoperative and 5 days after surgery. The variations in those parameters were studied. Data were then compared between patients according to the occurrence of a composite criteria (supra-ventricular arrythmia, stroke, acute renal failure, and/or death). Results: Data from 1453 patients were analyzed. The neutrophil count, fluorescence granularity (NE-SCC), intensity (NE-SFL) and size (NE-FSC) increased with surgery. Heterogeneity of neutrophils decreased in terms of fluorescence granularity (NE-WX) and size (NE-WZ) but increased in terms of intensity (NE-WY). The lymphocyte count decreased with surgery. While fluorescence granularity (LY-X) and size increased (LY-Z), Lymphocyte intensity decreased (LY-Y). Lymphocytes were less heterogeneous in terms of their granularity, size and intensity after surgery (LY-WX, LY-WY, LY-WZ). Patients who developed the composite complication criteria had a higher pre-operative neutrophil count (5.08 [3.89;6.95] vs 4.76 [3.60;6.13], p = 0.02; AUC = 0.56 [0.51;0.60]), and more heterogeneous neutrophils in terms of fluorescence granularity (NE-WX, AUC = 0.57 [0.52;0.62]) and intensity (NE-WY, AUC 0.61 [0.56;0.65]). Those patients also had lower pre-operative lymphocyte count (1.49 [1.10;1.14] vs 1.81 [1.39;2.39], p<0.01, AUC = 0.61 [0.57;0.66]) and fluorescence granularity (LY-X, AUC = 0.57 [0.53;0.62]). NE-WX, NE-WY and LY-X were associated with post-operative complications after adjustment on the EuroSCORE 2 (adjusted odd ratio of 1.01 [1.00;1.02]; 1.01 [1.00;1.01] and 1.08 [1.02;1.15] respectively). Conclusion: Cardiac surgery with cardiopulmonary bypass was associated with substantial alterations of CPD probably reflecting leukocytes activation in sterile inflammation. Pre-operative NE-WX, NE-WY and LY-X biomarkers levels were associated with post-operative complications, independently of the EuroSCORE 2. Such routine, unexploited and low cost parameters might represent useful tools likely to monitor immune function and predict outcomes for patients undergoing cardiac surgery. Our findings requires validation on a larger external cohort.

NEUT-SFL in Patients with COVID-ARDS: A Novel Biomarker for Thrombotic Events?

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is an enveloped RNA virus first identified in December 2019 in Wuhan, China, and responsible for coronavirus disease 2019 (COVID-19). The ongoing COVID-19 pandemic is impacting healthcare worldwide. Patients who develop coagulopathy have worse outcomes. The pathophysiology of COVID-19 suggests a strong interplay between hemostasis and immune cells, especially neutrophils. Our purpose was to assess neutrophil fluorescence as a potential biomarker of deep vein thrombosis (DVT) in patients with COVID-acute respiratory distress syndrome (COVID-ARDS). Sixty-one patients with COVID-ARDS admitted to the four intensive care units (ICUs) of a French general hospital were included in this prospective study. Neutrophil activation was assessed by measuring neutrophil fluorescence (NEUT-Side Fluorescence Light, NEUT-SFL) with a specific fluorescent dye staining analyzed by a routine automated flow cytometer Sysmex XN-3000™ (Sysmex, Kobe, Japan). DVT was diagnosed by complete duplex ultrasound (CDU). We found that NEUT-SFL was elevated on admission in patients with COVID-ARDS (49.76 AU, reference value 46.40 AU, p < 0.001), but did not differ between patients with DVT (49.99 AU) and those without (49.52 AU, p = 0.555). NEUT-SFL is elevated in patients with COVID-ARDS, reflecting neutrophil activation, but cannot be used as a marker of thrombosis. Because neutrophils are at interface between immune response and hemostasis through release of neutrophil extracellular traps, monitoring their activation could be an interesting approach to improve our management of coagulopathy during COVID-ARDS. Further research is needed to better understand the pathophysiology of COVID-19 and identify high-performance biomarkers.

First visualization of circulating neutrophil extracellular traps using cell fluorescence during human septic shock-induced disseminated intravascular coagulation.

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.

Neutrophil Activation During Septic Shock.

In addition to their well-known role as the cellular mediators of immunity, key other roles have been identified for neutrophils during septic shock. Importantly, neutrophils indeed play a critical role in the recently described immunothrombosis concept and in septic shock-induced coagulopathy. Septic shock is one of the most severe forms of infection, characterized by an inadequate host response to the pathogenic organism. This host response involves numerous defense mechanisms with an intense cellular activation, including neutrophil activation. Neutrophils are key cells of innate immunity through complex interactions with vascular cells and their activation may participate in systemic tissue damages. Their activation also leads to the emission of neutrophil extracellular traps, which take part in both pathogen circumscription and phagocytosis, but also in coagulation activation. Neutrophils thus stand at the interface between hemostasis and immunity, called immunothrombosis.The present review will develop a cellular approach of septic shock pathophysiology focusing on neutrophils as key players of septic shock-induced vascular cell dysfunction and of the host response, associating immunity and hemostasis. We will therefore first develop the role of neutrophils in the interplay between innate and adaptive immunity, and will then highlight recent advances in our understanding of immunothrombosis septic shock-induced coagulopathy.

Evidence of Netosis in Septic Shock-Induced Disseminated Intravascular Coagulation.

INTRODUCTION: Neutrophils extracellular traps (NETs) have recently emerged as a new potential link between inflammation, immunity, and thrombosis and could play a key role in septic shock-induced disseminated intravascular coagulation (DIC) pathogenesis. The objective of our study was to investigate a potential link between NETosis and septic shock-induced DIC. METHODS: Twenty patients with septic shock (10 without and 10 with DIC according to JAAM 2006 score) were prospectively included in our study. Vascular cell activation was assessed by microparticle (MP) measurement. NETosis was investigated at days 1, 3, and 7 using two different approaches: probing and measurement of neutrophil DNA decompaction by neutrophil-side fluorescence light (NEUT-SFL) as recorded by an automated blood cell cytometer and the assessment of nucleosomes and NETs (DNA-bound myeloperoxidase, DNA-MPO). RESULTS: Endothelial-derived CD105-MPs, leucocyte-derived CD11a-MPs/leucocyte, and neutrophil-derived CD66b-MPs/neutrophil count ratios significantly increased in DIC compared with non-DIC patients, indicating on-going cell activation (P <0.05). NEUT-SFL, indicating DNA decompaction, was significantly higher in DIC patients. Circulating nucleosomes and DNA-MPO were increased in DIC patients (P <0.05). There were significant correlations between: nucleosomes and NETs (r = 0.397, P = 0.004), NEUT-SFL and nucleosomes (r = 0.243, P = 0.032), NEUT-SFL and DNA-MPO (r = 0.266, P = 0.024). CONCLUSION: NEUT-SFL, NETs, and elevated nucleosome concentrations were all correlated to DIC (P <0.05). We have shown that NETosis is significantly correlated to septic shock-induced DIC.

Extracorporeal life support in the treatment of colchicine poisoning.

CONTEXT: Ingestions of Colchicum autumnale may lead to severe poisoning. It begins with gastrointestinal symptoms and leukocytosis, followed by multi-organ failure with shock and a possible late recovery phase. Mortality is highly dependent on the ingested dose. We report a case of accidental C. autumnale poisoning with refractory cardiogenic shock and eventual survival after extracorporeal life support (ECLS). CASE DETAILS: A 68-year-old woman was admitted to the intensive care unit (ICU) on day 3 after ingestion of C. autumnale in a meal. She first suffered from nausea and vomiting leading to severe dehydration. She then developed multi-organ failure and refractory cardiogenic shock, with a mean arterial pressure nadir of 50 mmHg despite high doses of catecholamines and a left ventricular ejection fraction at 5-10%. Venous-arterial ECLS was therefore started at an initial rate of 3.5 L/min and 3,800 rev/min. Her symptoms also included pancytopenia on day 4 with diffuse bleeding requiring iterative blood product transfusion. Platelet and leukocyte count nadirs were 13 × 10(9)/L (normal range: 150-400 × 10(9)/L) and 0.77 × 10(9)/L (normal range: 4.2-10.7 × 10(9)/L), respectively. ECLS allowed good cardiac contractility recovery within a few days, with complications including bleeding made controllable. Indeed, because of hemostasis disorders, the patient presented hemoptysis and hematuria. She was treated with tranexamic acid and transfused with blood products. She received 15 erythrocyte concentrates, 13 platelet concentrates, and 7 fresh frozen plasma. ECLS was removed by day 10, with subsequent weaning from mechanical ventilation as well as from hemodialysis in the following days. DISCUSSION: This patient survives after the use of ECLS in Colchicum poisoning, with controllable complications. Thus, ECLS might be indicated to overcome the potentially refractory cardiogenic shock phase.

Medium-chain triglyceride supplementation exacerbates peritonitis-induced septic shock in rats: role on cell membrane remodeling.

BACKGROUND AND AIMS: Lipid emulsions for parenteral nutrition interfere with immunity and may alter the cell plasma membrane and microparticle release, thus modulating their biological effects. Our aim was to evaluate the effect of two lipid emulsions for parenteral nutrition containing either a mixture of long- and medium-chain triglycerides (LCTs and MCTs) or LCTs only, to assess their role on microparticle release and acute inflammation during septic shock in rats. METHODS AND RESULTS: Septic rats (cecal ligation and puncture) and sham rats were infused with 5% dextrose or a lipid emulsion during 22 h. After 18 h, rats were resuscitated during 4 h and hemodynamic parameters monitored. Circulating microparticles and their phenotype were measured by prothrombinase assay; heart and aorta were collected for Western blotting and electron paramagnetic resonance measurements. No significant effect of lipid emulsions was observed in sham rats. In septic rats, norepinephrine requirements were increased in MCT/LCT-infused rats compared with 5% dextrose- or LCT-infused rats (2.7 ± 0.2 vs. 1.9 ± 0.8 and 1.2 ± 0.3 µg/kg per minute, respectively; P < 0.05) with increased procoagulant microparticle generation (38.6 ± 5.8 vs. 18.8 ± 3.1 and 19.2 ± 3.0 nM equivalent phosphatidylserine [Eq PhtdSer]; P < 0.05), leukocyte- (17.4 ± 3.5 vs. 7.7 ± 1.8 and 6.0 ± 1.1 nM Eq PhtdSer; P < 0.05), platelet- (13.9 ± 2.5 vs. 4.4 ± 0.7 and 5.4 ± 1.3 nM Eq PhtdSer; P < 0.05), and endothelial-derived microparticles (16.9 ± 3.6 vs. 6.4 ± 1.4 and 5.6 ± 0.8 nM Eq PhtdSer; P < 0.05). The mixture of MCTs/LCTs significantly increased cardiac and vascular nitric oxide and superoxide anion production, phosphorylated IκB, and cyclooxygenase 2 expression compared with the lipid emulsion containing only LCTs. CONCLUSIONS: Compared with 5% dextrose, MCT/LCT supplementation during septic shock in rats induced deleterious effects with increased inflammation and cell activation, associated to vascular hyporeactivity. During septic shock, LCT supplementation seemed to be neutral compared with 5% dextrose infusion.