Neutrophil Extracellular Traps Markers Research Articles

Dermatophagoides farinae induces conjunctival epithelial cell damage to promote neutrophil migration and neutrophil extracellular traps formation

To investigate the mechanisms underlying allergic conjunctivitis caused by conjunctival epithelial cell damage, neutrophil migration and neutrophil extracellular traps (NETs) formation induced by crude extracts of Dermatophagoides farinae mite (CDM). Human conjunctival epithelial cells were stimulated with 500, 1 000, 2 000, 4 000 ng/mL, and the expression levels of interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), interferon-γ (IFN-γ) and IL-8 were detected using quantitative real-time PCR (qPCR) assay and enzyme-linked immunosorbent assay (ELISA). The culture supernatant of human conjunctival epithelial cells was collected and co-cultured with neutrophils. Neutrophil migration was measured using Transwell migration assay, and the expression of NETs markers myeloperoxidase (MPO) and citrullinated histone H3 (CitH3) was quantified using immunofluorescence staining. Neutrophils were stimulated with phorbol 12-myristate 13-acetate (PMA), and then NETs were collected for treatment of human conjunctival epithelial cells. Cell apoptosis was detected using flow cytometry, and the levels of IL-6, TNF-α, IFN-γ and IL-8 were measured in the cell culture supernatant using ELISA. Treatment with CDM at concentrations of 2 000 ng/mL and 4 000 ng/mL up-regulated IL-6, TNF-α, IFN-γ and IL-8 expression in human conjunctival epithelial cells. Following treatment with CDM at concentrations of 2 000 ng/mL and 4 000 ng/mL, the culture supernatant of human conjunctival epithelial cells promoted neutrophil migration and induced increases in the staining intensity of MPO and CitH3. In addition, increased NETs triggered the apoptosis of human conjunctival epithelial cells and IL-6, TNF-α, IFN-γ and IL-8 secretion in the culture supernatant of human conjunctival epithelial cells. CDM induces human conjunctival epithelial cell damages, thereby promoting neutrophil migration and NETs formation, while the release of NETs further aggravates human conjunctival epithelial cell damages.

High-Throughput Analysis of Neutrophil Extracellular Trap Levels in Subtypes of People with Type 1 Diabetes.

Neutrophils might play an important role in the pathogenesis of autoimmune diseases, including type 1 diabetes (T1D), by contributing to immune dysregulation via a highly inflammatory program called neutrophil extracellular trap (NET) formation or NETosis, involving the extrusion of chromatin entangled with anti-microbial proteins. However, numerous studies reported contradictory data on NET formation in T1D. This might in part be due to the inherent heterogeneity of the disease and the influence of the disease developmental stage on neutrophil behavior. Moreover, there is a lack of a standardized method to measure NETosis in an unbiased and robust manner. In this study, we employed the Incucyte® ZOOM live-cell imaging platform to study NETosis levels in various subtypes of adult and pediatric T1D donors compared to healthy controls (HC) at baseline and in response to phorbol-myristate acetate (PMA) and ionomycin. Firstly, we determined that the technique allows for an operator-independent and automated quantification of NET formation across multiple time points, which showed that PMA and ionomycin induced NETosis with distinct kinetic characteristics, confirmed by high-resolution microscopy. NETosis levels also showed a clear dose-response curve to increasing concentrations of both stimuli. Overall, using Incucyte® ZOOM, no aberrant NET formation was observed over time in the different subtypes of T1D populations, irrespective of age, compared to HC. These data were corroborated by the levels of peripheral NET markers in all study participants. The current study showed that live-cell imaging allows for a robust and unbiased analysis and quantification of NET formation in real-time. Peripheral neutrophil measures should be complemented with dynamic quantification of NETing neutrophils to make robust conclusions on NET formation in health and disease.

Neutrophil extracellular trap burden correlates with the stenosis of coronary atherosclerosis.

Coronary atherosclerosis diseases (CADs) are associated with chronic inflammation. Neutrophil extracellular traps (NETs) are a type of novel proinflammatory cytokines whose levels are dramatically elevated in acute coronary syndrome. We conducted this study to further evaluate the association between circulating NET-associated markers and CAD in Chinese adults. A total of 174 patients with CAD and 55 healthy controls were screened using percutaneous coronary intervention or coronary computed tomography angiography. Blood lipid levels, blood glucose levels, and blood cell counts were determined using commercial kits. Serum levels of myeloperoxidase (MPO) and neutrophil elastase (NE) were measured using ELISA. Double-stranded DNA (dsDNA) in serum was quantified using the Quant-iT PicoGreen assay. We also compared the circulating NET levels with various parameters in the study subjects. The levels of serum NET markers, dsDNA, MPO, and NE, were significantly elevated in patients with CAD, particularly in the severe group, consistent with the increase in neutrophil counts. The levels of NET markers correlated with the risk factors of AS, increasing with the number of risk factors. NET markers were identified as independent risk factors for severe coronary stenosis and also as predictors of severe CAD. NETs may be related to AS and serve as indicators or predictors of stenosis in patients with severe CAD.

Neutrophil extracellular traps (NETs), transfusion requirements and clinical outcomes in orthotopic liver transplantation.

Inflammatory phenomena have a direct impact on the prognosis of orthotopic liver transplantation (OLT). Neutrophil extracellular traps (NETs) contribute to OLT inflammation and hemostasis imbalance in OLT. The association between NETosis, clinical outcomes and transfusion requirements is not determined. To evaluate NETs release during OLT and the effect of NETosis ontransfusion requirements and adverse outcomes in a prospective cohort of patients submitted to OLT. We quantified citrullinated histones (cit-H3) and circulating-free-DNA (cf-DNA) in ninety-three patients submitted to OLT in three periods: pre-transplant, after graft reperfusion and before discharge. NETs markers were compared between these periods using ANOVA test. The association of NETosis and adverse outcomes was evaluated using regression models adjusted for age, sex and corrected MELD. We observed a peak of circulating NETs following reperfusion, evidenced by a 2.4-fold increase in cit-H3 levels in the post-graft reperfusion period (median levels of cit-H3 pre transplant: 0.5ng/mL, after reperfusion: 1.2ng/mL and at discharge 0.5ng/mL, p < 0.0001). We observed an association between increased levels of cit-H3 and in-hospital death (OR = 1.168, 95% CI 1.021-1.336, p = 0.024). No association was found between NETs markers and transfusion requirements. There is a prompt release of NETs after reperfusion that is associated with poorer outcomes and death. Intraoperative NETs release seems to be independent of transfusion requirements. These findings highlight the relevance of inflammation promoted by NETS and its impact on OLT adverse clinical outcomes.

Bladder cancer patients have increased NETosis and impaired DNaseI-mediated NET degradation that can be therapeutically restored in vitro.

Neutrophils, key players of the immune system, also promote tumor development through the formation of neutrophil extracellular traps (NETs) in a process called NETosis. NETs are extracellular networks of DNA, histones and cytoplasmic and granular proteins (calprotectin, myeloperoxidase, elastase, etc.) released by neutrophils upon activation. NETs regulate tumor growth while promoting angiogenesis and invasiveness, and tumor cells also stimulate NETosis. Although NETosis seems to be increased in cancer patients, an increase of NETs in plasma may also be mediated by an impaired degradation by plasma DNaseI, as evidenced in several immunological disorders like lupus nephritis. However, this has never been evidenced in bladder cancer (BC) patients. Herein, we aimed to evaluate the occurrence of increased NETosis in plasma and tumor tissue of BC patients, to ascertain whether it is mediated by a reduced DNaseI activity and degradation, and to in vitro explore novel therapeutic interventions. We recruited 71 BC patients from whom we obtained a plasma sample before surgery and a formalin-fixed paraffin embedded tumor tissue sample, and 64 age- and sex-matched healthy controls from whom we obtained a plasma sample. We measured NETs markers (cell-free fDNA, calprotectin, nucleosomes and neutrophil elastase) and the DNaseI activity in plasma with specific assays. We also measured NETs markers in BC tissue by immunofluorescence. Finally, we evaluated the ability of BC and control plasma to degrade in vitro-generated NETs, and evaluated the performance of the approved recombinant human DNaseI (rhDNaseI, Dornase alfa, Pulmozyme®, Roche) to restore the NET-degradation ability of plasma. In vitro experiments were performed in triplicate. Statistical analysis was conducted with Graphpad (v.8.0.1). NETosis occurs in BC tissue, more profusely in the muscle-invasive subtype (P<0.01), that with the worst prognosis. Compared to controls, BC patients had increased NETosis and a reduced DNaseI activity in plasma (P<0.0001), which leads to an impairment to degrade NETs (P<0.0001). Remarkably, this can be therapeutically restored with rhDNaseI to the level of healthy controls. To the best of our knowledge, this is the first report demonstrating that BC patients have an increased NETosis systemically and in the tumor microenvironment, in part caused by an impaired DNaseI-mediated NET degradation. Remarkably, this defect can be therapeutically restored in vitro with the approved Dornase alfa, thus Pulmozyme® could become a potential therapeutic tool to locally reduce BC progression.

NET Proteome in Established Type 1 Diabetes Is Enriched in Metabolic Proteins.

Type 1 diabetes (T1D) is a chronic autoimmune disease characterized by a T-cell-mediated destruction of the pancreatic insulin-producing beta cells. A growing body of evidence suggests that abnormalities in neutrophils and neutrophil extracellular trap (NET) formation (NETosis) are associated with T1D pathophysiology. However, little information is available on whether these changes are primary neutrophil defects or related to the environmental signals encountered during active disease. In the present work, the NET proteome (NETome) of phorbol 12-myristate 13-acetate (PMA)- and ionomycin-stimulated neutrophils from people with established T1D compared to healthy controls (HC) was studied by proteomic analysis. Levels of NETosis, in addition to plasma levels of pro-inflammatory cytokines and NET markers, were comparable between T1D and HC subjects. However, the T1D NETome was distinct from that of HC in response to both stimuli. Quantitative analysis revealed that the T1D NETome was enriched in proteins belonging to metabolic pathways (i.e., phosphoglycerate kinase, glyceraldehyde-3-phosphate dehydrogenase, and UTP-glucose-1-phosphate uridylyltransferase). Complementary metabolic profiling revealed that the rate of extracellular acidification, an approximate measure for glycolysis, and mitochondrial respiration were similar between T1D and HC neutrophils in response to both stimuli. The NETome of people with established T1D was enriched in metabolic proteins without an apparent alteration in the bio-energetic profile or dysregulated NETosis. This may reflect an adaptation mechanism employed by activated T1D neutrophils to avoid impaired glycolysis and consequently excessive or suboptimal NETosis, pivotal in innate immune defence and the resolution of inflammation.

NETosis induction reflects COVID-19 severity and long COVID: insights from a 2-center patient cohort study in Israel

BackgroundCOVID-19 severity and its late complications continue to be poorly understood. Neutrophil extracellular traps (NETs) form in acute COVID-19, likely contributing to morbidity and mortality. ObjectivesThis study evaluated immunothrombosis markers in a comprehensive cohort of acute and recovered COVID-19 patients, including the association of NETs with long COVID. MethodsOne-hundred-seventy-seven patients were recruited from clinical cohorts at 2 Israeli centers: acute COVID-19 (mild/moderate, severe/critical), convalescent COVID-19 (recovered and long COVID), along with 54 non-COVID controls. Plasma was examined for markers of platelet activation, coagulation, and NETs. Ex vivo NETosis induction capability was evaluated after neutrophil incubation with patient plasma. ResultsSoluble P-selectin, factor VIII, von Willebrand factor, and platelet factor 4 were significantly elevated in patients with COVID-19 versus controls. Myeloperoxidase (MPO)-DNA complex levels were increased only in severe COVID-19 and did not differentiate between COVID-19 severities or correlate with thrombotic markers. NETosis induction levels strongly correlated with illness severity/duration, platelet activation markers, and coagulation factors, and were significantly reduced upon dexamethasone treatment and recovery. Patients with long COVID maintained higher NETosis induction, but not NET fragments, compared to recovered convalescent patients. ConclusionsIncreased NETosis induction can be detected in patients with long COVID. NETosis induction appears to be a more sensitive NET measurement than MPO-DNA levels in COVID-19, differentiating between disease severity and patients with long COVID. Ongoing NETosis induction capability in long COVID may provide insights into pathogenesis and serve as a surrogate marker for persistent pathology. This study emphasizes the need to explore neutrophil-targeted therapies in acute and chronic COVID-19.

Pancreatic melatonin enhances anti-tumor immunity in pancreatic adenocarcinoma through regulating tumor-associated neutrophils infiltration and NETosis.

Tumor microenvironment contributes to poor prognosis of pancreatic adenocarcinoma (PAAD) patients. Proper regulation could improve survival. Melatonin is an endogenous hormone that delivers multiple bioactivities. Here we showed that pancreatic melatonin level is associated with patients' survival. In PAAD mice models, melatonin supplementation suppressed tumor growth, while blockade of melatonin pathway exacerbated tumor progression. This anti-tumor effect was independent of cytotoxicity but associated with tumor-associated neutrophils (TANs), and TANs depletion reversed effects of melatonin. Melatonin induced TANs infiltration and activation, therefore induced cell apoptosis of PAAD cells. Cytokine arrays revealed that melatonin had minimal impact on neutrophils but induced secretion of Cxcl2 from tumor cells. Knockdown of Cxcl2 in tumor cells abolished neutrophil migration and activation. Melatonin-induced neutrophils presented an N1-like anti-tumor phenotype, with increased neutrophil extracellular traps (NETs) causing tumor cell apoptosis through cell-to-cell contact. Proteomics analysis revealed that this reactive oxygen species (ROS)-mediated inhibition was fueled by fatty acid oxidation (FAO) in neutrophils, while FAO inhibitor abolished the anti-tumor effect. Analysis of PAAD patient specimens revealed that CXCL2 expression was associated with neutrophil infiltration. CXCL2, or TANs, combined with NET marker, can better predict patients' prognosis. Collectively, we discovered an anti-tumor mechanism of melatonin through recruiting N1-neutrophils and beneficial NET formation.

MP14-09 INCREASED NETOSIS AND IMPAIRED DNASEI-MEDIATED NET DEGRADATION IN BLADDER CANCER PATIENTS: APPROACH FOR AN IN VITRO THERAPEUTICAL RESTORATION

You have accessJournal of UrologyCME1 Apr 2023MP14-09 INCREASED NETOSIS AND IMPAIRED DNASEI-MEDIATED NET DEGRADATION IN BLADDER CANCER PATIENTS: APPROACH FOR AN IN VITRO THERAPEUTICAL RESTORATION Raquel Herranz, Julia Oto, Marta Hueso, María Castaño, Emma Plana, Fernando Cana, Isabel Aleman, Santiago Bonamad, Javier Perez-Ardavin, César David Vera Donoso, Manuel Martinez-Sarmiento, and Pilar Medina Raquel HerranzRaquel Herranz More articles by this author , Julia OtoJulia Oto More articles by this author , Marta HuesoMarta Hueso More articles by this author , María CastañoMaría Castaño More articles by this author , Emma PlanaEmma Plana More articles by this author , Fernando CanaFernando Cana More articles by this author , Isabel AlemanIsabel Aleman More articles by this author , Santiago BonamadSantiago Bonamad More articles by this author , Javier Perez-ArdavinJavier Perez-Ardavin More articles by this author , César David Vera DonosoCésar David Vera Donoso More articles by this author , Manuel Martinez-SarmientoManuel Martinez-Sarmiento More articles by this author , and Pilar MedinaPilar Medina More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000003234.09AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: An increased release of neutrophil extracellular traps (NETs) through NETosis, occurs in cancer. This enhances tumor growth and metastasis, which may be mediated by impaired NET degradation. However, this has never been evidenced in bladder cancer (BC). To evaluate the increase in NETosis in BC patients, to ascertain whether it is mediated by plasma DNaseI alterations and to in vitro explore therapeutic interventions. METHODS: NETs markers were measured in plasma samples of 73 BC patients and 64 controls. Plasma DNaseI activity was measured with the SRED assay. To evaluate the ability of plasma to degrade NETs, neutrophils were isolated from a healthy donor, seeded, and activated for 4 h with phorbol myristate acetate (PMA) to prompt NETosis. NETs were incubated for 6 h with 5 µl plasma from each participant and fluorescence was measured after staining with SytoxGreen. In parallel, BC plasma was supplemented with the recombinant human DNaseI Dornase Alfa (Pulmozyme, Roche), or vehicle (PBS 1X+0.1% BSA) to restore their DNaseI deficiency. We evaluated the presence of NETosis in tumor tissue by immunofluorescence (DNA, citrullinatedH3 and elastase). Statistical analysis was conducted with Graphpad (v.8.0.1). RESULTS: BC patients had significantly increased NETs markers (p<0.0001) and lower DNaseI activity (p<0.0001) in plasma compared to controls (Figure 1). In vitro NET degradation was impaired when incubated with BC plasma compared to control plasma (p<0.0001) (Figure 2). Addition of DNaseI restored NET degradation up to the level of healthy controls (p<0.0001) (Figure 2). NETosis occurs in BC tissue, more profusely in muscle-invasive BC. CONCLUSIONS: BC patients have increased NETosis in the tumor microenvironment and plasma, in part mediated by a reduced DNaseI activity. In vitro addition of Dornase Alfa, an approved treatment for cystic fibrosis, restores NET degradation in BC patients, thus becoming a potential therapeutic tool to reduce metastasis. Source of Funding: ISCIII-FEDER (PI17/00495, PI20/00075, FI21/00171), GVA (CIACIF/2021/192) and SETH © 2023 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 209Issue Supplement 4April 2023Page: e185 Advertisement Copyright & Permissions© 2023 by American Urological Association Education and Research, Inc.MetricsAuthor Information Raquel Herranz More articles by this author Julia Oto More articles by this author Marta Hueso More articles by this author María Castaño More articles by this author Emma Plana More articles by this author Fernando Cana More articles by this author Isabel Aleman More articles by this author Santiago Bonamad More articles by this author Javier Perez-Ardavin More articles by this author César David Vera Donoso More articles by this author Manuel Martinez-Sarmiento More articles by this author Pilar Medina More articles by this author Expand All Advertisement PDF downloadLoading ...

473 Role of Neutrophils and Neutrophil Extracellular Traps in Subarachnoid Hemorrhage in Mice

INTRODUCTION: Aneurysmal subarachnoid hemorrhage (aSAH) continues to be a devastating disease. The role of neutrophils and neutrophil extracellular traps (NETs) have been investigated in other cerebrovascular disorders. Recent evidence suggest that neutrophils might play a role in outcomes after aSAH. METHODS: Subarachnoid hemorrhage model: The endovascular perforation model was utilized to induce SAH in mice. Flow cytometry: Flow cytometry technique was utilized to quantify neutrophils. Neutrophils were identified as CD45+Cd11b+Ly6G+ cells. NETosis assay: Neutrophils were assayed for their NETosis potential using a previously described method. The ratio of PMA (SYTOX+) to DMSO (SYTOX+) defined the NETosis induction. RNAseq: RNA sequencing was performed on neutrophils isolated from skull and femur bone marrow. Immunohistochemistry: NETs were assessed using classical IHC techniques and in vivo microscopy. Neurobehavior score: Mice were assessed daily for their behavior score using previously reported techniques. RESULTS: Neutrophils infiltrate brain within 24 hours and normalize by day 5. RNAseq demonstrates segregation of skull and femur bone marrow neutrophils following SAH. SAH induces a NETosis phenotype in skull neutrophils. NETs form starting day 1 after injury and persist until at least day 7. Neutrophil depletion and NETs inhibition attenuates early brain injury after SAH. Patients developing DCI have significantly higher blood markers of NETs. CONCLUSIONS: Neutrophils infiltrate the brain within the first 24 hours with NETs forming as early as day 1. Depletion of neutrophils and NET inhibition mitigated early brain injury following SAH. Blood markers of NETs are elevated in DCI patients and may be used as predictors of DCI. This highlights NETs as potential therapeutic target for patients with SAH and paves the way for future studies.

Neutrophil extracellular traps contribute to coagulopathy after traumatic brain injury.

Coagulopathy contributes to the majority of deaths and disabilities associated with traumatic brain injury (TBI). Whether neutrophil extracellular traps (NETs) contribute to an abnormal coagulation state in the acute process of TBI remains unknown. Our objectives were to demonstrate the definitive role of NETs in coagulopathy in TBI. We detected NET markers in 128 TBI patients and 34 healthy subjects. Neutrophil-platelet (PLT) aggregates were detected in blood samples from TBI patients and healthy subjects using flow cytometry and staining with CD41 and CD66b. Endothelial cells (ECs) were incubated with isolate NETs and detected the expression of vascular endothelial (VE)-cadherin, syncanden-1, thrombomodulin, von Willebrand factor (VWF), phosphatidylserine (PS) and and tissue factor (TF). In addition, we established a TBI mouse model to detect the potential role of NETs in TBI associated coagulopathy. NETs generation was mediated by High Mobility Group Box 1(HMGB1) from activated platelets and contribute to procoagulant activity in TBI. Furthermore, coculture experiments indicated that NETs damaged the endothelial barrier and caused these cells to assume a procoagulant phenotype. Moreover, the administration of DNase I before or after brain trauma markedly reduced coagulopathy and improved the survival and clinical outcome of mice with TBI.

Neutrophil extracellular traps in urinary tract infection.

Urinary tract infections (UTI) are common types of bacterial infection in children. UTI treatment is aimed to prevent complications including hypertension, proteinuria, and progression to chronic kidney disease. Activated neutrophils release chromatin-based structures associated with antimicrobial proteins called neutrophil extracellular traps (NETs). We aimed to describe the role of NET-associated markers in children with UTI as well as the role of NETs formation in a mouse model of UTI. Markers of NETs including extracellular DNA (ecDNA), myeloperoxidase (MPO) and cathelicidin were analyzed in children with febrile UTI caused by E. coli (n = 98, aged 0.3-1.3 years) and in healthy controls (n = 50, 0.5-5.2 years). Moreover, an acute experimental model of UTI was performed on PAD4 knock-out mice with diminished NETs formation (n = 18), and on wild-type mice (n = 15). Children with UTI had significantly higher urinary NETs markers including total ecDNA, nuclear DNA and mitochondrial DNA, altogether with MPO and cathelicidin. The concentrations of MPO and cathelicidin positively correlated with ecDNA (r = 0.53, p ≤ 0.001; r = 0.56, p ≤ 0.001, respectively) and the number of leukocytes in the urine (r = 0.29, p ≤ 0.05; r = 0.27, p ≤ 0.05, respectively). Moreover, urinary MPO was positively associated with cathelicidin (r = 0.61, p ≤ 0.001). In the experimental model, bacterial load in the bladder (20-fold) and kidneys (300-fold) was significantly higher in PAD4 knock-out mice than in wild-type mice. Higher urinary NETs makers-ecDNA, MPO and cathelicidin and their correlation with leukocyturia in children with UTI confirmed our hypothesis about the association between NETs and UTI in children. Higher bacterial load in mice with diminished NETs formation suggests that NETs are not only a simple consequence of UTI, but might play a direct role in the prevention of pyelonephritis and other UTI complications.

Homeostatic, Non-Canonical Role of Macrophage Elastase in Vascular Integrity.

Matrix metalloproteinase (MMP)-12 is highly expressed in abdominal aortic aneurysms and its elastolytic function has been implicated in the pathogenesis. This concept is challenged, however, by conflicting data. Here, we sought to revisit the role of MMP-12 in abdominal aortic aneurysm. Apoe-/- and Mmp12-/-/Apoe-/- mice were infused with Ang II (angiotensin). Expression of neutrophil extracellular traps (NETs) markers and complement component 3 (C3) levels were evaluated by immunostaining in aortas of surviving animals. Plasma complement components were analyzed by immunoassay. The effects of a complement inhibitor, IgG-FH1-5 (factor H-immunoglobulin G), and macrophage-specific MMP-12 deficiency on adverse aortic remodeling and death from rupture in Ang II-infused mice were determined. Unexpectedly, death from aortic rupture was significantly higher in Mmp12-/-/Apoe-/- mice. This associated with more neutrophils, citrullinated histone H3 and neutrophil elastase, markers of NETs, and C3 levels in Mmp12-/- aortas. These findings were recapitulated in additional models of abdominal aortic aneurysm. MMP-12 deficiency also led to more pronounced elastic laminae degradation and reduced collagen integrity. Higher plasma C5a in Mmp12-/- mice pointed to complement overactivation. Treatment with IgG-FH1-5 decreased aortic wall NETosis and reduced adverse aortic remodeling and death from rupture in Ang II-infused Mmp12-/- mice. Finally, macrophage-specific MMP-12 deficiency recapitulated the effects of global MMP-12 deficiency on complement deposition and NETosis, as well as adverse aortic remodeling and death from rupture in Ang II-infused mice. An MMP-12 deficiency/complement activation/NETosis pathway compromises aortic integrity, which predisposes to adverse vascular remodeling and abdominal aortic aneurysm rupture. Considering these new findings, the role of macrophage MMP-12 in vascular homeostasis demands re-evaluation of MMP-12 function in diverse settings.

Worsening Thrombotic Complication of Atherosclerotic Plaques Due to Neutrophils Extracellular Traps: A Systematic Review.

Neutrophil extracellular traps (NETs) recently emerged as a newly recognized contributor to venous and arterial thrombosis. These strands of DNA, extruded by activated or dying neutrophils, decorated with various protein mediators, become solid-state reactors that can localize at the critical interface of blood with the intimal surface of diseased arteries alongside propagating and amplifying the regional injury. NETs thus furnish a previously unsuspected link between inflammation, innate immunity, thrombosis, oxidative stress, and cardiovascular diseases. In response to disease-relevant stimuli, neutrophils undergo a specialized series of reactions that culminate in NET formation. DNA derived from either nuclei or mitochondria can contribute to NET formation. The DNA liberated from neutrophils forms a reticular mesh that resembles morphologically a net, rendering the acronym NETs particularly appropriate. The DNA backbone of NETs not only presents intrinsic neutrophil proteins (e.g., MPO (myeloperoxidase) and various proteinases) but can congregate other proteins found in blood (e.g., tissue factor procoagulant). This systematic review discusses the current hypothesis of neutrophil biology, focusing on the triggers and mechanisms of NET formation. Furthermore, the contribution of NETs to atherosclerosis and thrombosis is extensively addressed. Again, the use of NET markers in clinical trials was considered. Ultimately, given the vast body of the published literature, we aim to integrate the experimental evidence with the growing body of clinical information relating to NET critically.

Elevated circulating levels of neutrophil extracellular traps after cardiopulmonary bypass surgery as risk factors of postoperative atrial fibrillation and mortality.

Cardiopulmonary bypass (CPB) can trigger a systemic inflammatory response during the perioperative period, which may lead to the consumption of the contact system and the production of neutrophil extracellular traps (NETs). This study attempted to determine whether the formation of NETs and contact activation are a vivid occurrence during CPB and whether they are related to post-operative atrial fibrillation (AF) and survival. A prospective observational study was conducted in 97 patients who underwent aortic valve and/or aorta replacement surgery with CPB. Circulating markers of NETs [histone-DNA complex, cell-free double stranded DNA (dsDNA), neutrophil elastase] and the contact system [prekallikrein, high molecular weight kininogen (HMWK), activated factor XII (FXIIa)] were measured at four-time points: before surgery (T0), immediately after surgery (T1), 1 day after surgery (T2), and 3 days after surgery (T3). Elevated levels of circulating NETs markers were observed across post-CPB time. Significantly elevated levels of histone-DNA complex and cell-free dsDNA measured T3 were detected in patients with post-operative AF compared to those without. In logistic regression analysis, levels of histone-DNA complex and cell-free dsDNA measured at T3 were significant markers of risk for occurrence of AF. The levels of cell-free dsDNA measured T2 were significantly higher in non-survivors than in survivors. The level of cell-free dsDNA showed significant prognostic value. NETs markers may be useful for the assessment of risk for post-operative AF and mortality. Conduct of additional research regarding the role of NETs as clinical markers and as a therapeutic target in CPB is anticipated.

IgD enhances the release of neutrophil extracellular traps (NETs) via FcδR in rheumatoid arthritis patients.

Rheumatoid arthritis (RA) is a systemic autoimmune inflammatory disorder affecting primarily the joints. Neutrophils and the release of neutrophil extracellular traps (NETs) contribute to the pathogenesis of RA. However, IgD, which was abnormally higher in RA, has not been studied for its pathological role in neutrophil activation and NETs formation. To investigate the effects of IgD on neutrophil activation and NETs formation via IgD receptor (FcδR), we collect peripheral blood of RA patients and established adjuvant-induced arthritis (AA) rat model. We found that the expression of FcδR on neutrophils was significantly higher in RA patients compared with healthy controls. As a specific marker of NETs, the level of citrullinated histone H3 was positively correlated with sIgD and FcδR in RA patients. IgD enhances the release of NETs and promotes the proliferation of fibroblast-like synoviocytes (FLS) from RA patients by activating neutrophils. As a competitive FcδR blocker, IgD-Fc-Ig fusion protein could significantly reduce NETs formation and FcδR expression on neutrophils in vitro. In vivo, IgD-Fc-Ig could restrain IgD-induced neutrophil activation and NETs formation, thus inhibited FLS proliferation in AA rats. Data presented here demonstrate that neutrophils could be triggered by IgD to release NETs and take part in FLS proliferation in RA patients with excessive IgD. Blocking IgD-FcδR could inhibit neutrophil activation and NETs formation, and represent an additional attractive novel therapeutic strategy for the treatment of RA.

Unobvious Neutrophil Extracellular Traps Signification in the Course of Oral Squamous Cell Carcinoma: Current Understanding and Future Perspectives.

Background: The current standards of treatment for oral squamous cell carcinoma (OSCC) include surgery, radiotherapy, and chemotherapy. In recent years, research on the effectiveness of immunotherapy in the treatment of OSCC has also been conducted.Purpose: Studies indicate that nonspecific immune mechanisms involved in the course of the anticancer response also need to be taken into account.Research Design: This review summarizes the results of our research on the active participation of neutrophils, which are previously underestimated, in the antitumor response in the course of OSCC, taking into account the ability of these cells to generate neutrophil extracellular traps (NETs).Results: We proved that the formation of NETs accompanies not only inflammatory changes but also the neoplastic process and that lipopolysaccharide (LPS) or interleukin 17 (IL-17) plays a critical role in inducing the formation of NETs during the OSCC. The greatest achievement of our published findings was the demonstration of the formation and release of NETs from neutrophils cocultured with tumor cells, as well as after stimulation with supernatant from the SCC culture with a PI3K-independent Akt kinase activation mechanism. Moreover, the pioneering achievement of our studies was the localization of NET structures in the tumor tissue, as well as the observation of high concentrations of NET markers in the serum of OSCC patients with low concentrations in the saliva, indicating the differences in the course of immune response between the periphery and the local reactions.Conclusions: The data presented here provide surprising but important information on the role of NETs in the course of OSCC, thus pointing to a promising new direction in the development of management strategies for early noninvasive diagnosis and monitoring of the disease course, and perhaps immunotherapy. Furthermore, this review raises further questions and elaborates on the process of NETosis in cancer.

Diagnostic and prognostic role of circulating neutrophil extracellular trap markers and prekallikrein in patients with high-grade serous ovarian cancer.

Tumor-promoting inflammation is among the hallmarks of cancer. Prekallikrein is among the acute-phase reactants in the inflammatory response; moreover, neutrophils release nuclear contents into the extracellular space to create neutrophil extracellular traps (NET). We aimed to investigate the diagnostic and prognostic utilities of circulating plasma NET markers and prekallikrein for high-grade serous ovarian cancer (HGSOC). Circulating levels of three NET markers (histone-DNA complex, cell-free DNA, and neutrophil elastase) and prekallikrein were measured in 75 patients with HGSOC and 23 healthy controls. We used an area under the receiver operating characteristic curve (AUC) analysis to investigate their diagnostic and prognostic utilities for HGSOC. Compared with healthy controls, patients with HGSOC showed significantly higher levels of the three NET markers and prekallikrein. Patients with advanced-stage HGSOC showed significantly higher levels of the cell-free DNA (87.4 vs. 79.5 ng/ml; P = 0.013), compared with those with early-stage HGSOC. Further, the levels of histone-DNA complex, neutrophil elastase, and prekallikrein did not significantly differ according to the cancer stage. All markers showed significant diagnostic utility. Notably, a logistic regression-based model that comprised all four markers showed the strongest diagnostic power (AUC, 0.966; 95% confidence interval [CI], 0.933-1.000). Specifically, neutrophil elastase was identified as an independent poor prognostic factor for overall survival (adjusted hazard ratio [aHR], 10.17; 95% CI, 1.09-94.97; P = 0.042) and progression-free survival (aHR, 14.47; 95% CI, 1.52-137.35; P = 0.020) in patients with HGSOC. The levels of the three NET markers and prekallikrein might be novel diagnostic and prognostic markers for HGSOC.

Neutrophil extracellular traps have auto-catabolic activity and produce mononucleosome-associated circulating DNA

BackgroundAs circulating DNA (cirDNA) is mainly detected as mononucleosome-associated circulating DNA (mono-N cirDNA) in blood, apoptosis has until now been considered as the main source of cirDNA. The mechanism of cirDNA release into the circulation, however, is still not fully understood. This work addresses that knowledge gap, working from the postulate that neutrophil extracellular traps (NET) may be a source of cirDNA, and by investigating whether NET may directly produce mono-N cirDNA.MethodsWe studied (1) the in vitro kinetics of cell derived genomic high molecular weight (gHMW) DNA degradation in serum; (2) the production of extracellular DNA and NET markers such as neutrophil elastase (NE) and myeloperoxidase (MPO) by ex vivo activated neutrophils; and (3) the in vitro NET degradation in serum; for this, we exploited the synergistic analytical information provided by specifically quantifying DNA by qPCR, and used shallow WGS and capillary electrophoresis to perform fragment size analysis. We also performed an in vivo study in knockout mice, and an in vitro study of gHMW DNA degradation, to elucidate the role of NE and MPO in effecting DNA degradation and fragmentation. We then compared the NET-associated markers and fragmentation size profiles of cirDNA in plasma obtained from patients with inflammatory diseases found to be associated with NET formation and high levels of cirDNA (COVID-19, N = 28; systemic lupus erythematosus, N = 10; metastatic colorectal cancer, N = 10; and from healthy individuals, N = 114).ResultsOur studies reveal that gHMW DNA degradation in serum results in the accumulation of mono-N DNA (81.3% of the remaining DNA following 24 h incubation in serum corresponded to mono-N DNA); “ex vivo” NET formation, as demonstrated by a concurrent 5-, 5-, and 35-fold increase of NE, MPO, and cell-free DNA (cfDNA) concentration in PMA-activated neutrophil culture supernatant, leads to the release of high molecular weight DNA that degrades down to mono-N in serum; NET mainly in the form of gHMW DNA generate mono-N cirDNA (2 and 41% of the remaining DNA after 2 h in serum corresponded to 1–10 kbp fragments and mono-N, respectively) independent of any cellular process when degraded in serum; NE and MPO may contribute synergistically to NET autocatabolism, resulting in a 25-fold decrease in total DNA concentration and a DNA fragment size profile similar to that observed from cirDNA following 8 h incubation with both NE and MPO; the cirDNA size profile of NE KO mice significantly differed from that of the WT, suggesting NE involvement in DNA degradation; and a significant increase in the levels of NE, MPO, and cirDNA was detected in plasma samples from lupus, COVID-19, and mCRC, showing a high correlation with these inflammatory diseases, while no correlation of NE and MPO with cirDNA was found in HI.ConclusionsOur work describes the mechanisms by which NET and cirDNA are linked. In doing so, we demonstrate that NET are a major source of mono-N cirDNA independent of apoptosis and establish a new paradigm of the mechanisms of cirDNA release in normal and pathological conditions. We also demonstrate a link between immune response and cirDNA.

Investigation of Neutrophil Extracellular Traps as Potential Mediators in the Pathogenesis of Non-Acute Subdural Hematomas: A Pilot Study.

Non-acute subdural hematomas (NASHs) are a cause of significant morbidity and mortality, particularly with recurrences. Although recurrence is believed to involve a disordered neuroinflammatory cascade involving vascular endothelial growth factor (VEGF), this pathway has yet to be completely elucidated. Neutrophil extracellular traps (NETs) are key factors that promote inflammation/apoptosis and can be induced by VEGF. We investigated whether NETs are present in NASH membranes, quantified NET concentrations, and examined whether NET and VEGF levels are correlated in NASHs. Samples from patients undergoing NASH evacuation were collected during surgery and postoperatively at 24 and 48 h. Fluid samples and NASH membranes were analyzed for levels of VEGF, NETs, and platelet activation. NASH samples contained numerous neutrophils positive for NET formation. Myeloperoxidase-DNA complexes (a marker of NETs) remained elevated 48 h postoperatively (1.06 ± 0.22 day 0, 0.72 ± 0.23 day 1, and 0.83 ± 0.33 day 2). VEGF was also elevated in NASHs (7.08 ± 0.98 ng/mL day 0, 3.40 ± 0.68 ng/mL day 1, and 6.05 ± 1.8 ng/mL day 2). VEGF levels were significantly correlated with myeloperoxidase-DNA levels. These results show that NETs are increasing in NASH, a finding that was previously unknown. The strong correlation between NET and VEGF levels indicates that VEGF may be an important mediator of NET-related inflammation in NASH.