Neutrophil Extracellular Traps Markers Research Articles

Plasma Markers of Neutrophil Extracellular Trap Are Linked to Survival but Not to Pulmonary Embolism in COVID-19-Related ARDS Patients.

IntroductionCoronavirus disease 2019 (COVID-19) can cause life-threatening acute respiratory distress syndrome (ARDS). Recent data suggest a role for neutrophil extracellular traps (NETs) in COVID-19-related lung damage partly due to microthrombus formation. Besides, pulmonary embolism (PE) is frequent in severe COVID-19 patients, suggesting that immunothrombosis could also be responsible for increased PE occurrence in these patients. Here, we evaluate whether plasma levels of NET markers measured shorty after admission of hospitalized COVID-19 patients are associated with clinical outcomes in terms of clinical worsening, survival, and PE occurrence.Patients and MethodsNinety-six hospitalized COVID-19 patients were included, 50 with ARDS (severe disease) and 46 with moderate disease. We collected plasma early after admission and measured 3 NET markers: total DNA, myeloperoxidase (MPO)–DNA complexes, and citrullinated histone H3. Comparisons between survivors and non-survivors and patients developing PE and those not developing PE were assessed by Mann–Whitney test.ResultsAnalysis in the whole population of hospitalized COVID-19 patients revealed increased circulating biomarkers of NETs in patients who will die from COVID-19 and in patients who will subsequently develop PE. Restriction of our analysis in the most severe patients, i.e., the ones who enter the hospital for COVID-19-related ARDS, confirmed the link between NET biomarker levels and survival but not PE occurrence.ConclusionOur results strongly reinforce the hypothesis that NETosis is an attractive therapeutic target to prevent COVID-19 progression but that it does not seem to be linked to PE occurrence in patients hospitalized with COVID-19.

Tissue accumulation of neutrophil extracellular traps mediates muscle hyperalgesia in a mouse model

Accumulation of uric acid (UA) during muscular trauma is a factor involved in the development of muscle hyperalgesia. Neutrophil extracellular traps (NETs), DNA-based reticular structures to capture UA, play a central role in the pain onset of gout attacks; however, the involvement of NETs via the elevation of local UA level in muscle hyperalgesia due to injuries from muscle overuse remains unknown. The triceps surae muscles (TSMs) in the unilateral hindlimb of mice were electrically stimulated to induce excessive muscle contraction. Mechanical withdrawal thresholds, tissue UA levels, neutrophil recruitment, and protein amount of citrullinated histone 3 (citH3), a major marker of NETs, were investigated. Furthermore, whether neutrophil depletion, extracellular DNA cleavage, and administration of the urate-lowering agent febuxostat improved muscle hyperalgesia caused by NET formation was examined. CitH3 expression upon neutrophil recruitment was significantly increased in the stimulated TSMs with increased tissue UA levels, whereas febuxostat administration improved muscle hyperalgesia with decreased citH3 and tissue UA levels, as observed in neutrophil depletion and extracellular DNA digestion. The underlying mechanism of muscle hyperalgesia associated with locally recruited neutrophils forming NETs due to increased tissue UA levels potentially plays a significant role in creating a vicious circle of muscle pain.

Neutrophil extracellular traps (NETs) in patients with STEMI. Association with percutaneous coronary intervention and antithrombotic treatments

BackgroundNeutrophil extracellular traps (NETs) are formed by DNA, histones and proteolytic enzymes, and are produced by activated neutrophils through different mechanisms. In turn, NETs can activate platelets and coagulation cascade favoring thrombotic processes. The aims of this study were to analyze levels and kinetics of NETs in ST-segment elevation myocardial infarction (STEMI) patients and correlate them with antithrombotic therapy and cardiovascular outcomes at follow-up. Methods150 consecutive STEMI patients referred to primary percutaneous coronary intervention (pPCI) were included. Citrate anticoagulated blood was extracted immediately before pPCI, 30 min and 24 h after the procedure. As markers of NETS cell free DNA (cfDNA), nucleosomes and citrullinated Histone 3 (citH3) were determined. 46 healthy subjects were included as controls. Patients were follow-up for 1.4 ± 0.56 years. ResultsBefore pPCI, NETs markers were elevated in STEMI patients compared to healthy controls (p < 0.05); these increased significantly 30 min post pPCI (p ≤ 0.001) and decreased at 24 h but remained elevated compared with the control group (p < 0.05). Patients treated with bivalirudin presented a lower increase of NETs 30 min post pPCI compared to patients treated with heparin (p < 0.05). Cardiovascular risk factors or type of stent implanted did not modify NETs levels. Cit3H (HR = 3.74; 95%CI 1.05–13.4; p = 0.042) and left ventricular ejection fraction ≤35% (HR = 6.84; 95%CI 2–23; p = 0.002) were independent predictors of composite endpoint of myocardial infarction, stroke, stent thrombosis and/or cardiovascular-cause death. ConclusionsNETs were elevated in STEMI patients, increased by pPCI and decreased thereafter. One of the most specific NETs markers was associated with cardiovascular outcomes.

HMGB1-Promoted Neutrophil Extracellular Traps Contribute to Cardiac Diastolic Dysfunction in Mice.

BackgroundHeart failure with preserved ejection fraction (HFpEF) remains an increasing public health problem with substantial morbidity and mortality but with few effective treatments. A novel inflammatory mechanism has been proposed, but the inflammatory signals promoting the development of HFpEF remain greatly unknown.Methods and ResultsSerum of patients with HFpEF was collected for measurement of circulating neutrophils and markers of neutrophil extracellular traps (NETs). To induce HFpEF phenotype, male C57BL/6 mice underwent uninephrectomy, received a continuous infusion of d‐aldosterone for 4 weeks, and maintained on 1.0% sodium chloride drinking water. Heart tissues were harvested, immune cell types determined by flow cytometry, NETs formation by immunofluorescence, and western blotting. Differentiated neutrophils were cultured to investigate the effect of HMGB1 (high mobility group protein B1) and SGLT2 (sodium‐glucose cotransporter‐2) inhibitor on NETs formation in vitro. Circulating neutrophils and NETs markers are elevated in patients with HFpEF, as are cardiac neutrophils and NETs formation in HFpEF mice. NETs inhibition with deoxyribonuclease 1 in experimental HFpEF mice reduces heart macrophages infiltration and inflammation and ameliorates cardiac fibrosis and diastolic function. Damage‐associated molecular pattern HMGB1 expression is elevated in cardiac tissue of HFpEF mice, and HMGB1 inhibition reduces heart neutrophil infiltration and NETs formation and ameliorates diastolic function. Lastly, SGLT2 inhibitor empagliflozin down‐regulates heart HMGB1 expression, attenuates NETs formation and cardiac fibrosis, and improves diastolic function in HFpEF mice.ConclusionsNETs contribute to the pathogenesis of HFpEF, which can be ameliorated by HMGB1 inhibition and SGLT2 inhibitors. Thus, HMGB1 and NETs may represent novel therapeutic targets for the treatment of HFpEF.

Ex Vivo and In Vitro Analysis Identify a Detrimental Impact of Neutrophil Extracellular Traps on Eye Structures in Equine Recurrent Uveitis.

Equine recurrent uveitis (ERU) is a common ocular disease of horses and described as a model for human autoimmune uveitis. This immune-mediated, inflammatory condition progressively destroys the eye, ultimately leading to blindness. Genetic and autoimmune factors, next to infections with Leptospira, are discussed as key factors in the pathogenesis. Furthermore, a release of neutrophil extracellular traps (NETs) by activated neutrophils is involved. NETs are composed of decondensed chromatin and proteins that can immobilize invading pathogens. However, if NETs accumulate, they can contribute to detrimental autoimmune processes. Thus, we aimed to investigate the impact of NETs in ERU patients. Therefore, we quantified several NET-markers (cell-free DNA, nucleosomes, citrullinated histone H3, histone-myeloperoxidase complexes, interleukin-17, equine cathelicidin 1 and DNase I activity) and NET-autoantibodies in sera and vitreous body fluids (VBF) of ERU-diseased horses and correlated the data with the disease status (signalment, ERU scores and Leptospira infection status). NET markers were detected to varying degrees in VBF of diseased horses, and partially correlated to disease severity and the presence of Leptospira spp. Cell-free DNA and nucleosomes as NET markers correlate with ERU severity in total and VBF scores, despite the presence of active DNases. Additionally, a significant correlation between fundus affection in the eye and NET autoantibodies was detectable. Therefore, we further investigated the influence of VBF samples from equine patients and isolated NETs on the blood-retina barrier in a cell culture model. VBF of diseased horses significantly induced cytotoxicity in retinal pigment epithelial cells. Moreover, partially digested NETs also resulted in cytotoxic effects. In the presence of lipopolysaccharide (LPS), the main component of the leptospiral surface, both undigested and completely digested NETs were cytotoxic. Correlations between the ERU-scores and Leptospira were also calculated. Detection of leptospiral DNA, and antibody titers of the serovar Grippotyphosa correlated with disease severity. In addition, a correlation between Leptospira and several NET markers was observed in VBF. Altogether, our findings suggest a positive correlation between NET markers with disease severity and involvement of Leptospira in the VBF of ERU-diseased horses, as well as a cytotoxic effect of NETs in eyes.

Association of neutrophil extracellular traps with the production of circulating DNA in patients with colorectal cancer

SummaryWe postulate that a significant part of circulating DNA (cirDNA) originates in the degradation of neutrophil extracellular traps (NETs). In this study, we examined the plasma level of two markers of NETs (myeloperoxidase (MPO) and neutrophil elastase (NE)), as well as cirDNA levels in 219 patients with a metastatic colorectal cancer (mCRC), and in 114 healthy individuals (HI). We found that in patients with mCRC the content of these analytes was (i) highly correlated, and (ii) all statistically different (p < 0.0001) than in HI (N = 114). These three NETs markers may readily distinguish between patients with mCRC from HI, (0.88, 0.86, 0.84, and 0.95 AUC values for NE, MPO, cirDNA, and NE + MPO + cirDNA, respectively). Concomitant analysis of anti-phospholipid (anti-cardiolipin), NE, MPO, and cirDNA plasma concentrations in patients with mCRC might have value for thrombosis prevention, and suggested that NETosis may be a critical factor in the immunological response/phenomena linked to tumor progression.

Circulating Markers of Neutrophil Extracellular Traps (NETs) in Patients With Ovarian Tumors.

Inflammation is a hallmark of cancer, and the role of neutrophils and neutrophil extracellular traps (NETs) in cancer and cancer-associated thrombosis has attracted a lot of interest. The NET-specific marker H3Cit has been found to be elevated in the plasma of patients with malignancies, suggesting NETs markers as novel cancer biomarkers. This study aimed to determine the levels of NETs markers (H3Cit and dsDNA) in the plasma of women with adnexal masses. Peripheral blood samples were obtained from 199 patients admitted for primary surgery of adnexal masses. Patients were grouped according to tumor type and stage. Plasma levels of H3Cit-DNA, dsDNA, and CA125 were quantified. Plasma levels of H3Cit-DNA and dsDNA were not elevated in women with borderline or malignant ovarian tumors compared with those of the benign group. Increased levels of CA125 were found in the borderline and ovarian cancer group (ptrend<0.001). In Cox regression analysis, CA125 levels dichotomized at 326 IU/ml (median) were associated with worse overall survival (HR=1.9; 95%CI=1.03-3.36; p=0.038). No differences were found in the survival analyses of malignant ovarian tumors by analyzing the dsDNA and H3Cit-DNA levels. There is no association between NETs markers and ovarian tumors.

Impaired Degradation of Neutrophil Extracellular Traps: A Possible Severity Factor of Elderly Male COVID-19 Patients

Neutrophil extracellular traps (NETs) have been described as a potential trigger of severe COVID-19. NETs are known as extracellular DNA fibers released by neutrophils in response to infection. If the host is unable to balance efficient clearance of NETs by dornases (DNases), detrimental consequences occur. Elevated levels of NETs in COVID-19 patients are associated with higher risk of morbid thrombotic complications. Here, we studied the level of NET markers and DNase activity in a cohort of COVID-19 patients compared to healthy controls. Our data confirmed an increased level of NET markers in the plasma of COVID-19 patients, with a higher level in male compared to female patients. At the same time, there was an increased DNase activity detectable in COVID-19 patients compared to healthy controls. Importantly, there was a negative correlation of DNase activity with the age of male patients. The antimicrobial peptide LL-37, which is known to stabilize NETs against DNase degradation, is embedded in NETs upon severe acute respiratory syndrome coronavirus-2-infection. The LL-37 plasma level correlates with the NET-marker level in male COVID-19 patients, indicating a potential role of LL-37 in the risk of NET-associated thrombosis in male COVID-19 patients by stabilizing NETs against DNase degradation. In conclusion, our data identify two potential risk factors of elderly male patients which may lead to inefficient NET degradation and a subsequently higher risk of NET-associated thrombosis during COVID-19: reduced DNase activity and an increased LL-37 level.

Inhibition of Sphingosine-1-Phosphate Receptor 2 Prevents Thoracic Aortic Dissection and Rupture.

Background: Numerous pieces of evidence have indicated that thoracic aortic dissection (TAD) is an inflammatory disease. Sphingosine-1-phosphate receptor 2 (S1PR2) signaling is a driver in multiple inflammatory diseases. Here, we examined the S1PR2 expression in TAD lesions and explored the effect of interfering with S1PR2 on TAD formation and progression.Methods: Aorta specimens and blood samples were collected from patients with TAD and matched controls. The expression of S1PR1, S1PR2, and S1PR3 was examined. The effect of inhibiting S1PR2 on TAD was evaluated in a TAD mouse model induced by β-aminopropionitrile fumarate (BAPN) and AngII. The presence of sphingosine kinase 1 (SPHK1), S1P, and neutrophil extracellular traps (NETs) was investigated. Further, the possible association between S1PR2 signaling and NETs in TAD was analyzed.Results: In the aortic tissues of patients with TAD and a mouse model, the S1PR2 expression was significantly up-regulated. In the TAD mouse model, JTE013, a specific S1PR2 antagonist, not only blunted the TAD formation and aortic rupture, but also preserved the elastic fiber architecture, reduced the smooth muscle cells apoptosis level, and mitigated the aortic wall inflammation. Augmented tissue protein expression of SPHK1, citrullinated histone H3 (CitH3, a specific marker of NETs), and serum S1P, CitH3 were detected in TAD patients. Surgical repair normalized the serum S1P and CitH3 levels. Immunofluorescence staining revealed that S1PR2 colocalized with NETs. The protein expression levels of SPHK1 and serum S1P levels positively correlated with the protein expression and serum levels of CitH3, separately. Furthermore, JTE013 treatment reduced NETs accumulation.Conclusion: Inhibiting S1PR2 attenuates TAD formation and prevents aortic rupture. Targeting S1PR2 may provide a promising treatment strategy against TAD.

Generation of neutrophil extracellular traps in patients with acute liver failure is associated with poor outcome.

Background and AimsAcute liver failure (ALF) is characterized by significant changes in the hemostatic system and by systemic inflammation. The formation of neutrophil extracellular traps (NETs), in which an activated neutrophil expels its DNA, histones, and granular enzymes, such as myeloperoxidase (MPO), has been associated with immune‐mediated and thrombotic diseases. We hypothesized that formation of NETs in patients with ALF contributes to progression of disease.Approach and ResultsA total of 676 patients with ALF (international normalized ratio [INR], ≥1.5) or severe acute liver injury (ALI; INR, ≥2.0) were recruited from the U.S. ALF Study Group Registry between 2011 and 2018, of whom 308 patients (45.6%) had acetaminophen‐induced ALF. Up to 21 days after admission, 483 patients (71.5%) survived without liver transplantation (LT). Levels of cell‐free DNA (cfDNA) and the specific NET marker MPO‐DNA complexes were measured in plasma samples obtained on admission and compared to levels in healthy controls. In addition, liver tissue obtained at transplantation of 20 ALF patients was stained for NETs. Levels of cfDNA were 7.1‐fold, and MPO‐DNA complexes 2.5‐fold, higher in patients with ALF compared to healthy controls. cfDNA levels were not associated with 21‐day transplant‐free survival, but were higher in those patients with more‐severe disease on admission, as reflected by various laboratory and clinical parameters. MPO‐DNA levels were 30% higher in patients with ALF who died or required urgent LT. Liver tissue of ALF patients stained positive for NETs in 12 of 18 evaluable patients.ConclusionsHere, we provide evidence for NET formation in patients with ALF. Elevated plasma levels of MPO‐DNA complexes in patients with ALF were associated with poor outcome, which suggests that NET formation contributes to disease progression.

Correlation of Neutrophil to Lymphocyte Ratio with Expression of Neutrophil Extracellular Traps Within Stroke Emboli.

It has been hypothesized that circulating neutrophils have a direct correlation with the composition of emboli in acute ischemic stroke (AIS). The aim of this study is to evaluate the association between neutrophil-lymphocyte ratio (NLR) in peripheral blood and the expression of neutrophil extracellular traps (NETs) within stroke emboli. Consecutive patients with acute ischemic stroke (AIS) due to large vessel occlusion (LVO) that underwent mechanical thrombectomy (MT) were included. Patients were divided into two groups based on NLR median value. Retrieved thrombi were histologically analyzed using Martius Scarlett Blue (MSB) for main thrombus components including red blood cells (RBCs), white blood cells (WBCs), fibrin and platelet. Immunohistochemistry staining for von Willebrand Factor (vWF) and anti-citrullinated H3 (H3Cit; NETs marker) was also performed. Samples from a total of 84 patients were included. The average percentage of RBCs, WBCs, fibrin, platelet, H3Cit, and vWF components in thrombi were 45.1%, 3.5%, 21.8%, 29.6%, 19.7% and 14.8% respectively. When stratifying by NLR group [low (≤3.94) versus high (>3.95)], high NLR group had significantly more WBCs (4.5%), fibrin (24.2%), H3Cit (22.7%) and vWF (17.1%) thrombus fractions compared to low NLR group. Additionally, RBC content (38.8%) was lower in the high NLR group. NLR is correlated with the amounts of WBCs, fibrin, NETs and vWF within the thrombi retrieved from AIS patients due to LVO.

Neutrophil Extracellular Traps Promote T Cell Exhaustion in the Tumor Microenvironment.

While neutrophil extracellular traps (NETs) are important for directly promoting cancer growth, little is known about their impact on immune cells within the tumor microenvironment (TME). We hypothesize that NETs can directly interact with infiltrating T cells to promote an immunosuppressive TME. Herein, to induce a NET-rich TME, we performed liver Ischemia/Reperfusion (I/R) in an established cancer metastasis model or directly injected NETs in subcutaneous tumors. In this NET-rich TME, the majority of CD4+ and CD8+ tumor infiltrating lymphocytes expressed multiple inhibitory receptors, in addition these cells showed a functional and metabolic exhausted phenotype. Targeting of NETs in vivo by treating mice with DNAse lead to decreased tumor growth, decreased NET formation and higher levels of functioning T cells. In vitro, NETs contained the immunosuppressive ligand PD-L1 responsible for T cell exhaustion and dysfunction; an effect abrogated by using PD-L1 KO NETs or culturing NETs with PD-1 KO T cells. Furthermore, we found elevated levels of sPDL-1 and MPO-DNA, a NET marker, in the serum of patients undergoing surgery for colorectal liver metastases resection. Neutrophils isolated from patients after surgery were primed to form NETs and induced exhaustion and dysfunction of human CD4+ and CD8+ T cells. We next targeted PD-L1 in vivo by injecting a blocking antibody during liver I/R. A single dose of anti-PD-L1 during surgery lead to diminished tumors at 3 weeks and functional T cells in the TME. Our data thus reveal that NETs have the capability of suppressing T cell responses through metabolic and functional exhaustion and thereby promote tumor growth. Furthermore, targeting of PD-L1 containing NETs at time of surgery with DNAse or anti-PD-L1 lead to diminished tumor growth, which represents a novel and viable strategy for sustaining immune competence within the TME.

Inhibition of PAD4-mediated NET formation by cl-amidine prevents diabetes development in nonobese diabetic mice

Many evidences indicated that neutrophil extracellular traps (NETs) play pathogenic roles in type 1 diabetes (T1D). Peptidylarginine deiminases 4 (PAD4) has been proved to be indispensable for generation of NETs. In the current study, we investigated whether oral administration of cl-amidine, an effective inhibitor of PAD4, protects non-obese diabetic (NOD) mice from T1D development. Female NOD mice were orally administrated with cl-amidine (5 μg/g body weight) from the age of 8 weeks up to 16 weeks. It showed that cl-amidine inhibit NET formation in vitro and in vivo. The onset of T1D was delayed nearly 8 weeks and the incidence of disease was significantly decreased in cl-amidine treated mice compared with the control group. Moreover, cl-amidine decreased the serum levels of anti-citrullinated peptide antibody (ACPA) and anti-neutrophil cytoplasmic antibodies (ANCA) in NOD mice. Also, it decreased generation of T1D autoantibodies such as glutamic acid decarboxylase antibody (GADA), tyrosine phosphatase-related islet antigen-2 antibody (IA2A) and zinc transporter 8 antibody (ZnT8A), which were strongly correlated with the reduced serum PAD4 and MPO-DNA levels. Furthermore, cl-amidine administration inhibited pancreatic inflammation and increased frequency of regulatory T cells in pancreatic lymph nodes (PLNs). In addition, cl-amidine improved gut barrier dysfunction and decreased the serum level of lipopolysaccharide (LPS), which was positively correlated with the NETs markers (PAD4 and MPO-DNA) and T1D autoantibody IA2A. In conclusion, our data showed that orally delivery of cl-amidine effectively prevent T1D development and suggested inhibition of PAD4-dependent NET formation as a potential way of clinical treatment in T1D.

Assessment of Macrophage Inflammatory Biomarkers and Neutrophil Extracellular Traps Associated Proteins in COVID-19 Patients

▪Background : The SARS-CoV-2 infection activates both innate and adaptive immune responses and induces an exaggerated cytokine storm leading to causes septic shock, acute respiratory distress syndrome, and/or multiple organ failure in critically ill patients. The knowledge of this dysregulated immune response is not well explained. One hypothesis is that the response of macrophages and neutrophils to infection is disproportionate, resulting in overproduction of cytokines and activation of neutrophils leading to the most severe complications of infection, including the production of multiple microthrombi and endothelial damage. Aim of the study: To evaluate the activation levels of macrophage biomarkers as well as indicators of the development of neutrophil extracellular traps (NET) in the first phase of infection in hospitalized patients with COVID19. Patients and Methods: We selected dry blood spot from a total of 60 previously identified SARS-CoV-2 infected subjects. Plasma samples were provided by the Aragon Health System Biobank's collection and were extracted within 5 days of symptom onset. Plasma from 60 healthy controls were used to stablish the control range for NETosis determination. The study was authorized by the Ethics Committee of the Aragon Health System and complies with the European General Data Protection Regulation (GDPR 2016/679) and LO 3/2018. Demographic characteristics, clinical manifestations, follow-up during hospitalization, associated comorbidities, and thrombotic complications were obtained from the database. Chitotriosidase activity (ChT), YKL40 chitinase and CCL18/PARC cytokine were measured as markers of macrophage activation. Myeloperoxidase (MPO), Neutrophil Elastase (NE), and S100A8/S100A9 Heterodimer (MPR) were immuno-quantified, levels of circulating free DNA (cfDNA) were measured as well as the presence of DNAsases by fluorimetry as indicators of Netosis. For the comparative analysis, we stratified patients by age groups and disease severity and used the Mann-Whitney U test for statistical comparison, considering a p-value < 0.05 as statistically significant. Results : A statistically significant increase in ChT (p=0.032) and CCL18/PARC (p=0.0001) was observed in the patients´ group. A comparative study with clinical variables and other inflammation markers as ferritin, D-dimer will be shown upon acceptance. Concerning NET markers, we found a statistically significant increase in MPO, NE, and MRP in COVID-19 patients (p=0.0001; p=0.0290; p=0.0001 respectively), as well as a statistically significant decrease in DNAsa (p=0.0001). No differences in cfDNA levels were observed. The table shows the median (quartile1-quartile3) for each marker in the control group and in the patient group. Conclusions : In this study, biomarkers of macrophage activation do not appear to be more sensitive than the indicators of inflammation in routine clinical practice (ferritin, D-dimer). Clinical cases of severe COVID-19 disease show an excessive NET formation, which contributes to vascular damage and the development of thrombosis.This work was supported by a research grant from FEETEG [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Neutrophil Extracellular Trap Density Increases With Increasing Histopathological Severity of Crohn's Disease.

BackgroundIntestinal neutrophil recruitment is a characteristic feature of the earliest stages of inflammatory bowel disease (IBD). Neutrophil elastase (NE) and myeloperoxidase (MPO) mediate the formation of neutrophil extracellular traps (NETs); NETs produce the bactericidal oxidant hypochlorous acid (HOCl), causing host tissue damage when unregulated. The project aim was to investigate the relationship between NET formation and clinical IBD in humans.MethodsHuman intestinal biopsies were collected from Crohn’s disease (CD) patients, endoscopically categorized as unaffected, transitional, or diseased, and assigned a histopathological score.ResultsA significant linear correlation was identified between pathological score and cell viability (TUNEL+). Immunohistochemical analysis revealed the presence of NET markers NE, MPO, and citrullinated histone (CitH3) that increased significantly with increasing histopathological score. Diseased specimens showed greater MPO+-immunostaining than control (P < .0001) and unaffected CD (P < .0001), with transitional CD specimens also showing greater staining than controls (P < .05) and unaffected CD (P < .05). Similarly, NE+-immunostaining was elevated significantly in diseased CD than controls (P < .0001) and unaffected CD (P < .0001) and was significantly higher in transitional CD than in controls (P < .0001) and unaffected CD (P < .0001). The CitH3+-immunostaining of diseased CD was significantly higher than controls (P < .05), unaffected CD (P < .0001) and transitional CD (P < .05), with transitional CD specimens showing greater staining than unaffected CD (P < .01). Multiplex immunohistochemistry with z-stacking revealed colocalization of NE, MPO, CitH3, and DAPI (cell nuclei), confirming the NET assignment.ConclusionThese data indicate an association between increased NET formation and CD severity, potentially due to excessive MPO-mediated HOCl production in the extracellular domain, causing host tissue damage that exacerbates CD.

Complement activation is associated with neutrophil extracellular traps and all-cause mortality in ST-elevation myocardial infarction

Abstract Background The complement system and neutrophil extracellular traps (NETs) are both parts of the innate immune system, and have been implicated in the ischemia-reperfusion injury in patients with ST-elevation myocardial infarction (STEMI). There is experimental evidence of reciprocal activation between the complement system and NETs. Any such link in patients with STEMI has not been investigated. Purpose To investigate a potential association between complement activation and clinical outcomes after STEMI, and assess any interplay between complement activation and NETs in this situation. Methods Patients with ST-elevation myocardial infarction were included at a median of 18 hours after percutaneous coronary intervention (n=864). The terminal complement complex (TCC) was measured by ELISA as a marker of complement activation. As markers of NETs were myeloperoxidase-deoxynucleic acid (MPO-DNA) and citrullinated histone 3 (CitH3) measured by ELISAs, while double stranded DNA (dsDNA) was measured by a nucleic acid stain. Patients were followed for a median of 4.6 years. The primary endpoint was a composite of new myocardial infarction, unscheduled revascularization, stroke, hospitalization for heart failure and death, whichever occurred first. All-cause mortality was also recorded. Results The composite endpoint occurred in 184 (21.3%) patients, while 70 (8.1%) died during follow-up. When dichotomizing at median TCC, the group with above-median TCC levels did not have an increased risk of reaching the composite endpoint (hazard ratio (HR): 1.069, 95% CI: [0.801, 1.428], p=0.651). However, this group exhibited an increased risk of all-cause mortality (HR: 1.650, 95% CI: [1.020, 2.671], p=0.041). This risk persisted when adjusting for age, sex, hypertension and LDL-cholesterol (HR: 1.673, 95% CI: [1.014, 2.761], p=0.044), but the significance was lost when adjusting for NT-proBNP (HR: 1.492, 95% CI: [0.885, 2.515], p=0.133). TCC was correlated to dsDNA (r=0.127, p&amp;lt;0.001) and CitH3 (r=0.102, p=0.003), but not MPO-DNA. The group with both TCC and dsDNA in the highest quartile exhibited a significantly higher incidence of all-cause mortality than the remaining population (17.6% vs, 7.2%, p=0.002). When examining the predictive value of TCC and dsDNA on all-cause mortality in ROC curve analysis, the area under the curve (AUC) for TCC was 0.549 (95% CI: [0.472, 0.625]), while the AUC for dsDNA was 0.653 (95% CI: [0.584, 0.722]). When combining TCC and dsDNA the predictive value was marginally higher than for TCC alone (AUC: 0.649, 95% CI: [0.579, 0.720]) Conclusion In this STEMI population, complement activation measured by TCC was not associated with the primary composite endpoint, but was associated with increased risk of death. TCC was weakly correlated with markers of NETs. Despite a high mortality rate in patients with high levels of TCC and dsDNA, combining these variables did not increase the prognostic value compared to TCC alone. Funding Acknowledgement Type of funding sources: Foundation. Main funding source(s): Stein Erik Hagen's Foundation for Clinical Heart Research Survival according to cox regression

Thromboinflammatory profiling in atrial cardiopathy

Abstract Background Thrombotic cardioemboli originate largely from a diseased left atrium. Disturbed blood flow in atrial cardiopathy is proposed to create a thrombogenic environment and enhance clot formation, even independently of atrial fibrillation. The formation of neutrophil extracellular traps (NETs) has been identified as a crucial mechanism in thrombotic diseases, including cardioembolic thrombus formation. Moreover, emerging evidence suggests that von Willebrand factor (VWF) plays a vital role in NET-mediated pathologies, serving as a bridge between activated endothelium, platelets, and leukocytes. VWF and NETs have also both been shown to be involved in fibrotic remodeling of the heart. Purpose of the study The aim of this study is to identify thrombotic markers that correlate with structural abnormalities of the heart. This may provide insight into pathomechanisms that drive detrimental left atrial remodeling as well as clues regarding thromboembolic risks that are rooted in atrial cardiopathy Methods Paired blood samples were collected from patients undergoing catheterization with atrial transseptal puncture. We compared blood sampled locally from the left atrium to peripheral blood samples collected prior to the catheterization procedure from the same patients, analyzing them for a wide range of thrombotic markers, including: NETs biomarkers, VWF, ADAMTS13, D-dimers, fibrinogen, as well as indicators of fibrosis like BNP and C-terminal telopeptide of type I collagen (ICTP). In parallel, echocardiographic measurements including left atrium size and ejection fraction of the left ventricle were recorded in order to perform correlation analyses with the biochemical parameters in blood. Results A total of 71 patients (age 64,1±14, males 46,5%) were included in the study. Myeloperoxidase (MPO)-DNA complexes and citrullinated histone H3 (H3Cit) levels were significantly elevated in samples drawn from the left atrium compared to peripheral samples, and MPO-DNA levels in the left atrium correlated with left atrial size (Figure 1). H3Cit and PAD4 levels correlated with VWF activity and antigen, in central and peripheral samples. Reduced peripheral ADAMTS13 activity levels correlated with higher values throughout all NET markers, and PAD4, which has been shown to render ADAMTS13 inactive (Figure 2). NETs and PAD4 also correlated with BNP and ICTP, providing potential novel biomarkers for fibrosis development. Conclusion Blood drawn from the left atrium shows significantly higher levels of specific (not all) thrombotic markers as compared to blood drawn from peripheral veins, and a relationship between NETs and dysregulation of the VWF-ADAMTS13 axis. These elevations partly correlate with structural changes of the left atrium and, therefore, may point toward a disturbed local blood flow. This may help identify patients at risk for cardioembolic events, even independently of atrial fibrillation. Funding Acknowledgement Type of funding sources: Public grant(s) – EU funding. Main funding source(s): European Research Area Network - Cardio Vascular Disease (ERA-CVD)Fonds Wetenschapplijk Onderzoek Vlaanderen (FWO)

Neutrophil extracellular traps in patients with liver cirrhosis and hepatocellular carcinoma

Neutrophil extracellular traps (NETs) are web-like structures consisting of DNA, histones and granule proteins, released from neutrophils in thrombus formation, inflammation, and cancer. We asked if plasma levels of the NET markers myeloperoxidase (MPO)-DNA and citrullinated histone H3 (H3Cit)-DNA, are elevated in liver cirrhosis and hepatocellular carcinoma (HCC) and if the levels correlate with clinical parameters. MPO-DNA, H3Cit-DNA, and thrombin–antithrombin (TAT) complex, as a marker of coagulation activity, were measured using ELISA in plasma from 82 patients with HCC, 95 patients with cirrhosis and 50 healthy controls. Correlations were made to clinical parameters and laboratory data and patients were followed for a median of 22.5 months regarding thrombosis development. H3Cit-DNA was significantly (p < 0.01) elevated in plasma from cirrhosis (66.4 ng/mL) and HCC (63.8 ng/mL) patients compared to healthy controls (31.8 ng/mL). TAT levels showed similar pattern (3.1, 3.7, and 0.0 µg/mL respectively, p < 0.01). MPO-DNA was significantly (p < 0.01) elevated in cirrhosis patients (0.53 O.D.) as compared to controls (0.33 O.D.). Levels of MPO-DNA and H3Cit-DNA correlated positively with Child–Pugh and MELD score. TAT was increased in all Child–Pugh and MELD groups. In multivariable logistic regression, Child B and C liver cirrhosis were independent predictors of elevated H3Cit-DNA in plasma. Levels of MPO-DNA and H3Cit-DNA were similar in patients with or without history of thrombosis, or thrombus formation during follow-up. In conclusion, plasma markers of NET formation are elevated in liver cirrhosis and correlate to the degree of liver dysfunction in patients with liver cirrhosis and/or HCC. The presence of HCC did not further increase the plasma levels of NET markers as compared to patients with cirrhosis only.

Auricular Vagus Nerve Stimulation Attenuates Lipopolysaccharide-Induced Acute Lung Injury by Inhibiting Neutrophil Infiltration and Neutrophil Extracellular Traps Formation.

Vagus nerve stimulation has been shown to exert anti-inflammation activities in sepsis. However, surgical implantation of stimulation devices is performed under general anesthesia, which limits its clinical application. Auricular vagus nerve stimulation (AVNS) is a minimal invasive technique that delivers electrical currents to the auricular branch of the vagus nerve. The purpose of this study was to determine the effects of AVNS on systemic inflammation, lung injury, neutrophil infiltration, and neutrophil extracellular traps (NETs) formation in the lung. In a LPS challenge lung-injury mice model, AVNS was applied to bilateral ears. Twelve hours after LPS administration, samples of blood, bronchoalveolar lavage fluid (BALF), and lung tissues were processed for investigations. We found that the treatment with AVNS significantly attenuated histopathological changes and neutrophil infiltration in the lung tissue, inhibited inflammatory cytokine elevations in serum and BALF, and decreased protein concentrations in BALF. Besides, AVNS decreased leukocyte and neutrophil accounts in BALF. Furthermore, colocalization of citrullination of histone H3 and myeloperoxidase expressions (highly specific marker of NETs) was reduced in AVNS mice. In conclusion, AVNS reduced systemic inflammation, attenuated lung edema, and inhibited neutrophil infiltration and NETs formation in the lung in LPS mice.

Involvement of Neutrophil Extracellular Traps in Cerebral Arteriovenous Malformations

Cerebral arteriovenous malformations (cAVMs) represent tangles of abnormal vasculature without intervening capillaries. High-pressure vascular channels due to abnormal arterial and venous shunts can lead to rupture. Multiple pathways are involved in the pathobiology of cAVMs including inflammation and genetic factors such as KRAS mutations. Neutrophil release of nuclear chromatin, known as neutrophil extracellular traps (NETs), plays a multifunctional role in infection, inflammation, thrombosis, intracranial aneurysms, and tumor progression. However, the relationship between NETs and the pathobiology of cAVMs remains unknown. We tested whether NETs play a role in the pathobiology of cAVMs. We analyzed samples from patients who had undergone surgery for cAVM and immunohistochemically investigated expression of citrullinated histone H3 (CitH3) as a marker of NETs. CitH3 expression was compared among samples from cAVM patients, epilepsy patients, and normal human brain tissue. Expressions of thrombotic and inflammatory markers were also examined immunohistochemically in samples from cAVM patients. Expression of CitH3 derived from neutrophils was observed intravascularly in all cAVM samples but not other samples. Nidi of AVMs showed migration of many Iba-I-positive cells adjacent to the endothelium and endothelial COX2 expression, accompanied by expression of IL-6 and IL-8 in the endothelium and intravascular neutrophils. Unexpectedly, expression of CitH3 was not necessarily localized to the vascular wall and thrombus. Our results offer the first evidence of intravascular expression of NETs, which might be associated with vascular inflammation in cAVMs.