Levels Of Neutrophil Extracellular Traps Research Articles

Longitudinal plasma proteomic analysis of 1117 hospitalized patients with COVID-19 identifies features associated with severity and outcomes.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection is characterized by highly heterogeneous manifestations ranging from asymptomatic cases to death for still incompletely understood reasons. As part of the IMmunoPhenotyping Assessment in a COVID-19 Cohort study, we mapped the plasma proteomes of 1117 hospitalized patients with COVID-19 from 15 hospitals across the United States. Up to six samples were collected within ~28 days of hospitalization resulting in one of the largest COVID-19 plasma proteomics cohorts with 2934 samples. Using perchloric acid to deplete the most abundant plasma proteins allowed for detecting 2910 proteins. Our findings show that increased levels of neutrophil extracellular trap and heart damage markers are associated with fatal outcomes. Our analysis also identified prognostic biomarkers for worsening severity and death. Our comprehensive longitudinal plasma proteomics study, involving 1117 participants and 2934 samples, allowed for testing the generalizability of the findings of many previous COVID-19 plasma proteomics studies using much smaller cohorts.

Enhancement of NETosis by ACE2-cross-reactive anti-SARS-CoV-2 RBD antibodies in patients with COVID-19.

High levels of neutrophil extracellular trap (NET) formation or NETosis and autoantibodies are related to poor prognosis and disease severity of COVID-19 patients. Human angiotensin-converting enzyme 2 (ACE2) cross-reactive anti-severe acute respiratory syndrome coronavirus 2 spike protein receptor-binding domain (SARS-CoV-2 RBD) antibodies (CR Abs) have been reported as one of the sources of anti-ACE2 autoantibodies. However, the pathological implications of CR Abs in NET formation remain unknown. In this study, we first assessed the presence of CR Abs in the sera of COVID-19 patients with different severity by serological analysis. Sera and purified IgG from CR Abs positive COVID-19 patients as well as a mouse monoclonal Ab (mAb 127) that can recognize both ACE2 and the RBD were tested for their influence on NETosis and the possible mechanisms involved were studied. An association between CR Abs levels and the severity of COVID-19 in 120 patients was found. The CR Abs-positive sera and IgG from severe COVID-19 patients and mAb 127 significantly activated human leukocytes and triggered NETosis, in the presence of RBD. This NETosis, triggered by the coexistence of CR Abs and RBD, activated thrombus-related cells but was abolished when the interaction between CR Abs and ACE2 or Fc receptors was disrupted. We also revealed that CR Abs-induced NETosis was suppressed in the presence of recombinant ACE2 or the Src family kinase inhibitor, dasatinib. Furthermore, we found that COVID-19 vaccination not only reduced COVID-19 severity but also prevented the production of CR Abs after SARS-CoV-2 infection. Our findings provide possible pathogenic effects of CR Abs in exacerbating COVID-19 by enhancing NETosis, highlighting ACE2 and dasatinib as potential treatments, and supporting the benefit of vaccination in reducing disease severity and CR Abs production in COVID-19 patients.

Neutrophil Extracellular Trap Formation and Deoxyribonuclease I Activity in Patients with Otitis Media with Antineutrophil Cytoplasmic Antibody-Associated Vasculitis

Introduction: No previous studies have evaluated the levels of neutrophil extracellular trap (NET) remnants or the importance of deoxyribonuclease (DNase) I activity based on the disease activity of otitis media with antineutrophil cytoplasmic antibody-associated vasculitis (OMAAV). The aim of this study was to explore the formation of NETs in the middle ear of patients with OMAAV during the onset and remission phases of the disease, with a particular focus on the relationships between the quantifiable levels of NET remnants and DNase I activity. Methods: OMAAV patients were eligible for inclusion. Patients with otitis media with effusion (OME) were examined as controls. The levels of cell-free deoxyribonucleic acid (DNA), citrullinated-histone H3 (cit-H3)-DNA complex, and myeloperoxidase (MPO)-DNA complex were quantified using an enzyme-linked immunosorbent assay. DNase I activity was measured using a fluorometric method. Results: The quantifiable levels of cell-free DNA, cit-H3-DNA complex, and MPO-DNA complex in the middle ear lavage of patients with OMAAV at onset were significantly higher than those in patients with OMAAV at remission and in patients with OME. DNase I activity in the patients with OMAAV at onset was significantly lower than those in patients with OMAAV at remission and OME and was negatively correlated with the level of MPO-DNA complex. Conclusions: This study suggests that NET remnants and DNase I activity may be potentially useful biomarkers for the diagnosis and disease activity of OMAAV.

Tackling Severe Neutrophilic Inflammation in Airway Disorders with Functionalized Nanosheets.

Severe airway inflammatory disorders impose a significant societal burden, and the available treatments are unsatisfactory. High levels of neutrophil extracellular trap (NET) and cell-free DNA (cfDNA) were detected in the inflammatory microenvironment of these diseases, which are closely associated with persistent uncontrolled neutrophilic inflammation. Although DNase has proven to be effective in mitigating neutrophilic airway inflammation in mice by reducing cfDNA and NET levels, its clinical use is hindered by severe side effects. Here, we synthesized polyglycerol-amine (PGA) with a series of hydroxyl/amine ratios and covered them with black phosphorus (BP) nanosheets. The BP nanosheets functionalized with polyglycerol-50% amine (BP-PGA50) efficiently lowered cfDNA levels, suppressed toll-like receptor 9 (TLR9) activation and inhibited NET formation in vitro. Importantly, BP-PGA50 nanosheets demonstrated substantial accumulation in inflamed airway tissues, excellent biocompatibility, and potent inflammation modulation ability in model mice. The 2D sheet-like structure of BP-PGA50 was identified as a crucial factor for the therapeutic efficacy, and the hydroxyl/amine ratio was revealed as a significant parameter to regulate the protein resistance, cfDNA-binding efficacy, and cytotoxicity. This study shows the promise of the BP-PGA50 nanosheet for tackling uncontrolled airway inflammation, which is also significant for the treatment of other neutrophilic inflammatory diseases. In addition, our work also highlights the importance of proper surface functionalization, such as hydroxyl/amine ratio, in therapeutic nanoplatform construction for inflammation modulation.

Circulating neutrophil extracellular trap remnants as a biomarker to predict outcomes in lupus nephritis

ObjectiveTo determine if the serum levels of neutrophil extracellular trap (NET) remnants (Elastase-DNA and HMGB1-DNA complexes) at the time of a lupus nephritis (LN) flare predict renal outcomes in the...

DNA binding protein YB-1 is a part of the neutrophil extracellular trap mediation of kidney damage and cross-organ effects

Open-heart surgery is associated with high morbidity, with acute kidney injury (AKI) being one of the most commonly observed postoperative complications. Following open-heart surgery, in an observational study we found significantly higher numbers of blood neutrophils in a group of 13 patients with AKI compared to 25 patients without AKI (AKI: 12.9±5.4×109 cells/L; non-AKI: 10.1±2. 9×109 cells/L). Elevated serum levels of neutrophil extracellular trap (NETs) components, such as dsDNA, histone 3, and DNA binding protein Y-box protein (YB)-1, were found within the first 24 hours in patients who later developed AKI. We could demonstrate that NET formation and hypoxia triggered the release of YB-1, which was subsequently shown to act as a mediator of kidney tubular damage. Experimentally, in two models of AKI mimicking kidney hypoperfusion during cardiac surgery (bilateral ischemia/reperfusion (I/R) and systemic lipopolysaccharide (LPS) administration), a neutralizing YB-1 antibody was administered to mice. In both models, prophylactic YB-1 antibody administration significantly reduced the tubular damage (damage score range 1-4, the LPS model: non-specific IgG control, 0.92±0.23; anti-YB-1 0.65±0.18; and in the I/R model: non-specific IgG control 2.42±0.23; anti-YB-1 1.86±0.44). Even in a therapeutic, delayed treatment model, antagonism of YB-1 ameliorated AKI (damage score, non-specific IgG control 3.03±0.31; anti-YB-1 2.58±0.18). Thus, blocking extracellular YB-1 reduced the effects induced by hypoxia and NET formation in the kidney and significantly limited AKI, suggesting that YB-1 is part of the NET formation process and an integral mediator of cross-organ effects.

Parapneumonic Effusions Are Characterized by Elevated Levels of Neutrophil Extracellular Traps

Neutrophil extracellular traps (NETs) increasingly are implicated in acute and chronic conditions involving multiple organ systems. Are NET concentrations higher in parapneumonic effusions compared with effusions of other origin and does this reflect the inflammatory nature of these effusions? Patients (N= 101) seeking hospital treatment for undifferentiated pleural effusion underwent pleural fluid classification based on cytologic analysis results, biochemical findings, microbiological characteristics, and clinical judgement. Concentrations of NET markers (extracellular DNA [eDNA], citrullinated histone H3 [citH3]), neutrophils (α-defensins), and inflammation (IL-1β)-related proteins were quantified by enzyme-linked immunosorbent assay. Differences between groups were analyzed using the Kruskal-Wallis one-way analysis of variance. Correlations used Spearman coefficient. Receiver operating characteristic (ROC) curves were calculated. Effusions were classified into four groups: parapneumonic (n= 18), malignant (n= 35), transudative (n= 22), and unclassifiable (n= 26). Concentrations of NETs markers were significantly higher in the parapneumonic group compared with malignant, transudative, and unclassifiable groups (median eDNA, 12.8ng/mL vs0.77ng/mL, 0.44ng/mL, and 0.86ng/mL [P< .001]; and median citH3, 127.1ng/mL vs0.44ng/mL, 0.34ng/mL, and 0.49ng/mL [P< .001]). citH3 and eDNA were correlated highly with lactate dehydrogenase (LDH; Spearman r= 0.66 and r= 0.73, respectively; P< .001) and moderately negatively correlated with pH (r= -0.55 and r= -0.62, respectively; P< .001). α-Defensins and IL-1β were higher in the parapneumonic group than in other groups (median α-defensins, 124.4ng/mL vs4.7ng/mL,7ng/mL, and 6.9ng/mL [P< .001]; and median IL-1β, 145 pg/mL vs1.87 pg/mL, 1.39 pg/mL, and 2.6 pg/mL [P< .001]) and moderately correlated with LDH (r= 0.60 and r= 0.57; P< .001). ROC curves showed high sensitivity and specificity for NET markers for prediction of parapneumonic effusion. High levels of some NET-related mediators in parapneumonic effusions correlate with inflammation. Effusions of other causes do not show high levels of NETs. These results may have treatment implications because NETs may be an important contributor to the inflammation and viscosity of parapneumonic effusions and may help us to understand the therapeutic benefit of deoxyribonuclease in empyema.

High Levels of Neutrophil Extracellular Traps Persist in the Lower Respiratory Tract of Critically Ill Patients With Coronavirus Disease 2019.

Lower respiratory tract (LRT) disease induced by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) can deteriorate to acute respiratory distress syndrome (ARDS). Because the release of neutrophil extracellular traps (NETs) is implicated in ARDS pathogenesis, we investigated the presence of NETs and correlates of pathogenesis in blood and LRT samples of critically ill patients with COVID-19. Plasma NET levels peaked early after intensive care unit admission and were correlated with the SARS-CoV-2 RNA load in sputum and levels of neutrophil-recruiting chemokines and inflammatory markers in plasma samples. The baseline plasma NET quantity was correlated with disease severity but was not associated with soluble markers of thrombosis or with development of thrombosis. High NET levels were present in LRT samples and persisted during the course of COVID-19, consistent with the detection of NETs in bronchi and alveolar spaces in lung tissue from deceased patient with COVID-19. Thus, NETs are produced and retained in the LRT of critically ill patients with COVID-19 and could contribute to SARS-CoV-2-induced ARDS disease.

Proteome Profiling of Recombinant DNase Therapy in Reducing NETs and Aiding Recovery in COVID-19 Patients

Severe coronavirus disease 2019 (COVID-19) can result in pneumonia and acute respiratory failure. Accumulation of mucus in the airways is a hallmark of the disease and can result in hypoxemia. Here, we show that quantitative proteome analysis of the sputum from severe patients with COVID-19 reveal high levels of neutrophil extracellular trap (NET) components, which was confirmed by microscopy. Extracellular DNA from excessive NET formation can increase sputum viscosity and lead to acute respiratory distress syndrome. Recombinant human DNase (Pulmozyme; Roche) has been shown to be beneficial in reducing sputum viscosity and improve lung function. We treated five patients pwith COVID-19 resenting acute symptoms with clinically approved aerosolized Pulmozyme. No adverse reactions to the drug were seen, and improved oxygen saturation and recovery in all severely ill patients with COVID-19 was observed after therapy. Immunofluorescence and proteome analysis of sputum and blood plasma samples after treatment revealed a marked reduction of NETs and a set of statistically significant proteome changes that indicate reduction of hemorrhage, plasma leakage and inflammation in the airways, and reduced systemic inflammatory state in the blood plasma of patients. Taken together, the results indicate that NETs contribute to acute respiratory failure in COVID-19 and that degrading NETs may reduce dependency on external high-flow oxygen therapy in patients. Targeting NETs using recombinant human DNase may have significant therapeutic implications in COVID-19 disease and warrants further studies.

Anti-citrullinated protein antibodies are associated with neutrophil extracellular trap formation in rheumatoid arthritis.

ObjectivesWe evaluated the associations of anti‐citrullinated protein antibodies (ACPAs) and serological and cytological levels of neutrophil extracellular trap (NET) formation in rheumatoid arthritis (RA) patients.MethodsSerum levels of myeloperoxidase‐DNA and elastase‐DNA complexes (NET remnants) were examined in 51 patients with RA and 40 healthy controls using a modified enzyme‐linked immunosorbent assay. Neutrophils were isolated by density gradient centrifugation. IgG antibodies were purified by affinity chromatography. NET formation in RA and control neutrophils was assessed by microscopy in vitro. NETs were purified and co‐incubated with fibroblast‐like synoviocyte (FLS) cells. Interleukin (IL)‐6 and IL‐8 mRNA expression and protein levels in FLS cells were determined by real‐time polymerase chain reaction and enzyme‐linked immunosorbent assay, respectively.ResultsIn RA patients, NET remnants in the peripheral circulation were higher in extremely high ACPA titers when compared to in moderate ACPA titers. And IgG antibodies containing ACPA can stimulate neutrophils to form NETs in a concentration‐dependent manner. Furthermore, significantly higher expression of the pro‐inflammatory cytokines IL‐6 and IL‐8 is detected after FLS cells interacted with NETs which derived from neutrophils stimulated with ACPA‐containing IgG antibodies.ConclusionsAnti‐citrullinated protein antibodies may enhance NET formation and contribute to inflammation development in RA by stimulating NET formation, such as by subsequent activation of FLS cells by NETs.

Increased Circulating Levels of Neutrophil Extracellular Traps During Cardiopulmonary Bypass

BackgroundThe intensity of inflammatory response triggered by cardiopulmonary bypass (CPB) during cardiac surgery has been associated with adverse outcomes. Neutrophils might contribute to organ injury through the liberation of DNA histone-based structures named “neutrophil extracellular traps” (NETs). Our objective was to assess circulating NETs levels before and after cardiac surgery in low-risk and high-risk patients. MethodsThis prospective cohort study included 2 groups of patients undergoing elective cardiac surgery with the use of CPB. The first group consisted of low-risk patients (European System for Cardiac Operative Risk Evaluation II ≤ 1%), and the second group included high-risk patients (European System for Cardiac Operative Risk Evaluation II ≥ 5%). Blood samples were drawn pre-CPB and 3 hours post-CPB separation. Measurements of circulating NETs, interleukin-6, C-reactive protein, myeloperoxidase, citrullinated histone 3, and pentraxin-related protein 3 levels were performed at each time point. ResultsTwenty-four patients, 12 high-risk and 12 low-risk patients, were included. Circulating NETs measurements changed from a median of 0.054 before CPB to 0.084 at 3 hours post-CPB separation, with a median increase of 0.037 (P < 0.001) per patient. No difference was noted between the high-risk and low-risk groups. A linear relationship was found between the circulating NETs measurements 3 hours post-CPB and CPB duration (ß = 0.047; confidence interval, 0.012-0.081; P = 0.01 R2 = 0.27). A correlation was found between the change in NETs with citrullinated histone 3 and myeloperoxidase levels, but not between NETs and other inflammatory biomarkers. ConclusionsCirculating NETs measurements increases during cardiac surgery with postsurgical levels proportional to CPB duration. The clinical significance of NETs production during cardiac surgery should be further investigated.

CIRCULATING NEUTROPHIL EXTRACELLULAR TRAPS IN TYPE 2 DIABETES MELLITUS PATIENTS WITH SUPPURATIVE AND NECROTIC COMPLICATIONS

Aim of the study. Neutrophil extracellular traps (NETs) have been reported to participate in many pathological processes, which points at NET concentration in blood as an important indicator of the body condition. When studying NETs, their blood concentration is usually evaluated indirectly, which has a number of shortcomings. We aimed to measure directly the amount of NETs as integral structures, using blood smears as a most suitable tool for clinical practice, and to carry out measurements of NET levels in blood of type 2 diabetes mellitus (T2DM) patients with necrotic suppurative complications to elucidate the usefulness of this assay. Materials and methods of the research. NETs were quantified by counting in standardized blood smears stained with Romanovsky dyes. Results. The mean level of circulating NETs in the blood of patients was higher than that in healthy subjects. Blood NET concentration in healthy persons and patients with high levels of NETs increased as glycated hemoglobin (HbA1c) concentration increased in the blood. In other patients, NET concentration decreased as HbAlc increased. Elevated NET levels and a direct correlation between NETs and HbAlc were observed in patients with phlegmon, large-area infected wound, lower leg gangrene, suppurative mediastinitis. In other patients, the prevailing lesions were infected wounds, abscesses, finger gangrene. Conclusion. This pilot study demonstrated the usefulness of measurement (using blood smear) of circulating NET concentration in the blood as a clinical parameter that would help to estimate patient’s condition, to predict the duration of post-surgical wound healing and the probability of complications in the form of thrombosis in T2DM.

High Level of Neutrophil Extracellular Traps Correlates With Poor Prognosis of Severe Influenza A Infection.

Most patients with severe infection with influenza A virus (IAV) progress to acute respiratory distress syndrome and even multiple organ dysfunction syndrome (MODS). Neutrophil extracellular traps (NETs) can be induced by pathogens and are responsible for immune tissue damage. We conducted a prospective study on the production and effects of NETs in H7N9 and H1N1 patients. We investigated NET production in plasma and supernatant of cultured neutrophils by measuring cell-free deoxyribonucleic acid (DNA) and myeloperoxidase (MPO)-DNA complexes with PicoGreen dye and enzyme-linked immunosorbent assay methods, respectively. We also observed NET structure by immunofluorescence staining. We found that patients with severe influenza showed elevated plasma NET level on the day of admission. Neutrophils from these patients showed higher capacity to release MPO-DNA complex in response to interleukin-8 or lipopolysaccharide stimulation. We also found that NETs from H7N9 and H1N1 patients increased the permeability of alveolar epithelial cells, and, consequently, NET production was positively correlated with acute physiology and chronic health evaluation (APACHE) II score and MODS. These data indicate that high level of NETs contributes to lung injury and is correlated with severity of disease. Thus, NETs might be a key factor to predict the poor prognosis in IAV patients.

GW28-e0962 Neutrophil extracellular traps(NETs) was considered to play a key role in disseminated intravascular coagulation(DIC).The chromatin filaments were released by activated polymorphonuclear neutrophil(PMNs) and decorated with granule proteins such as tissue factor(TF). Therefore, we investigated circulating levels of NETs in DIC caused by fulminant myocarditis to analyze coagulation complication severity and predict the outcome.

GW28-e0962 Neutrophil extracellular traps(NETs) was considered to play a key role in disseminated intravascular coagulation(DIC).The chromatin filaments were released by activated polymorphonuclear neutrophil(PMNs) and decorated with granule proteins such as tissue factor(TF). Therefore, we investigated circulating levels of NETs in DIC caused by fulminant myocarditis to analyze coagulation complication severity and predict the outcome.