Inhibition Of Neutrophil Extracellular Traps Formation Research Articles

Colchicine Inhibits Neutrophil Extracellular Trap Formation in Patients With Acute Coronary Syndrome After Percutaneous Coronary Intervention.

BackgroundRelease of neutrophil extracellular traps (NETs) after percutaneous coronary intervention (PCI) in acute coronary syndrome (ACS) is associated with periprocedural myocardial infarction, as a result of microvascular obstruction via pro‐inflammatory and prothrombotic pathways. Colchicine is a well‐established anti‐inflammatory agent with growing evidence to support use in patients with coronary disease. However, its effects on post‐PCI NET formation in ACS have not been explored.Methods and ResultsSixty patients (40 ACS; 20 stable angina pectoris) were prospectively recruited and allocated to colchicine or no treatment. Within 24 hours of treatment, serial coronary sinus blood samples were collected during PCI. Isolated neutrophils from 10 patients with ACS post‐PCI and 4 healthy controls were treated in vitro with colchicine (25 nmol/L) and stimulated with either ionomycin (5 μmol/L) or phorbol 12‐myristate 13‐acetate (50 nmol/L). Extracellular DNA was quantified using Sytox Green and fixed cells were stained with Hoechst 3342 and anti‐alpha tubulin. Baseline characteristics were similar across both treatment and control arms. Patients with ACS had higher NET release versus patients with stable angina pectoris (P<0.001), which was reduced with colchicine treatment (area under the curve: 0.58 versus 4.29; P<0.001). In vitro, colchicine suppressed unstimulated (P<0.001), phorbol 12‐myristate 13‐acetate–induced (P=0.009) and ionomycin‐induced (P=0.002) NET formation in neutrophils isolated from patients with ACS post‐PCI, but not healthy controls. Tubulin organization was impaired in neutrophils from patients with ACS but was restored by colchicine treatment.ConclusionsColchicine suppresses NET formation in patients with ACS post‐PCI by restoring cytoskeletal dynamics. These findings warrant further investigation in randomized trials powered for clinical end points.RegistrationURL: https://anzctr.org.au; Unique identifier: ACTRN12619001231134.

Neutrophils From Patients With Invasive Candidiasis Are Inhibited by Candida albicans Biofilms.

Invasive candidiasis frequently involves medical device placement. On the surfaces of these devices, Candida can form biofilms and proliferate in adherent layers of fungal cells surrounded by a protective extracellular matrix. Due in part to this extracellular matrix, biofilms resist host defenses and antifungal drugs. Previous work (using neutrophils from healthy donors) found that one mechanism employed to resist host defenses involves the inhibition of neutrophil extracellular traps (NET) formation. NETs contain nuclear DNA, as well as antimicrobial proteins that can ensnare pathogens too large or aggregated to be effectively killed by phagocytosis. Given that these neutrophil structures are anticipated to have activity against the large aggregates of C. albicans biofilms, understanding the role of this inhibition in patients could provide insight into new treatment strategies. However, prior work has not included patients. Here, we examine NET formation by neutrophils collected from patients with invasive candidiasis. When compared to neutrophils from healthy participants, we show that patient neutrophils exhibit a heightened background level of NET release and respond to a positive stimulus by producing 100% more NETs. However, despite these physiologic differences, patient neutrophil responses to C. albicans were similar to healthy neutrophils. For both groups, planktonic cells induce strong NET release and biofilms inhibit NET formation. These results show that a mechanism of immune evasion for fungal biofilms translates to the clinical setting.

Colchicine inhibits neutrophil extracellular trap formation in acute coronary syndrome patients post percutaneous coronary intervention

Colchicine inhibits neutrophil extracellular trap formation in acute coronary syndrome patients post percutaneous coronary intervention

Особенности плазменного звена гемостаза у пациентов с Ph-негативными миелопролиферативными новообразованиями

Введение: Тромбозы являются одним из основных осложнений Ph-негативных миелопролиферативных новообразований (МПН), таких как истинная полицитемия (ИП), эссенциальная тромбоцитемия (ЭТ) и первичный миелофиброз (ПМФ). Имеются многочисленные свидетельства активации тромбоцитов, лейкоцитов, эндотелия и коагуляции при МПН; однако результаты определения показателей гемостаза, применимых в клинической практике для выявления протромботических состояний при МПН, неоднозначны. Цель исследования: оценить состояние плазменного звена гемостаза у пациентов с Ph-негативными МПН и выявить показатели, отражающие протромботическую направленность изменений в системе гемостаза при данных патологиях. Материалы и методы: В исследование было включено 116 пациентов с МПН (43 с ИП, 35 с ЭТ, 38 с ПМФ). Пациенты находились на антиагрегантной, циторедуктивной и таргетной терапии ингибиторами янус-киназ. Контрольная группа состояла из 43 практически здоровых лиц. В плазме крови всех обследованных определяли индекс активированного парциального тромбопластинового времени (АПТВ) — отношение АПТВ исследуемого образца к АПТВ нормальной плазмы, протромбиновый тест по Квику (ПТ), содержание фибриногена, активности фактора VIII (FVIII) и естественных антикоагулянтов антитромбина (АТ) и протеина С (PC), уровень свободного протеина S (PS), а также показатели теста генерации тромбина (ТГТ), в том числе с добавлением тромбомодулина (ТМ). Результаты: У пациентов с МПН по сравнению с контрольной группой наблюдали сдвиг АПТВ и ПТ в сторону гипокоагуляции, было удлинено время инициации генерации тромбина, снижена скорость и количественные показатели ТГТ без добавления ТМ. При проведении ТГТ с добавлением ТМ у больных МПН выявлено снижение чувствительности к ТМ, которое коррелировало с падением активности PC и уровня свободного PS. Содержание фибриногена и активность FVIII у обследованных пациентов были повышены. Заключение: Для пациентов с МПН характерны разнонаправленные изменения показателей плазменного звена гемостаза. Важной детерминантой развития протромботических состояний при МПН выступает недостаточная эффективность естественных антикоагулянтов системы протеина С, приводящая к выраженному дисбалансу в системе гемостаза. Background: Thromboses are one of the main complications of Ph-negative myeloproliferative neoplasms (MPN), such as polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis (PMF). There are abundant evidences of platelet, leukocyte, endothelial, and coagulation activation in MPN; however, studies of hemostatic parameters applicable in clinical practice for revealing of prothrombotic states in MPN are not identical. Objectives: to assess plasma hemostasis in patients with Ph-negative MPN and to identify parameters reflecting the prothrombotic tendency of hemostatic changes in these pathologies. Patients/Methods: The study included 116 patients with MPN (43 with PV, 35 with ET, 38 with PMF). Patients were treated with antiplatelet, cytoreductive and targeted therapy with Janus kinases inhibitors. The control group consisted of 43 healthy individuals. In blood plasma of all examined persons we determined the index of activated partial thromboplastin time (APTT) — the ratio of APTT of the test sample to APTT of normal plasma, Quick prothrombin test (PT), fi brinogen content, activities of factor VIII (FVIII) and natural anticoagulants — antithrombin (AT) and protein C (PC), free protein S (PS) level, as well as thrombin generation assay (TGA) parameters, including thrombomodulin (TM) addition. Results: As compared with control group, in patients with MPN a shift in APTT and PV to hypocoagulation was observed, the initiation time of thrombin generation was prolonged, the rate and quantitative parameters of TGA without TM addition were decreased. In patients with MPN TGA with TM addition showed reduced sensitivity to TM that correlated with decreased PC activity and reduction of free PS level. Fibrinogen content and FVIII activity in the examined patients were increased. Conclusions: Patients with MPN are characterized by multidirectional changes of plasma hemostasis parameters. The important determinant of prothrombotic states development in MPN is insufficient effectiveness of the natural anticoagulants of protein C system that leads to expressed hemostasis imbalance.

NETs in APS: Current Knowledge and Future Perspectives.

Antiphospholipid syndrome (APS) is a thrombo-inflammatory disease that is primarily treated with anticoagulation. Better understanding the inflammatory aspects of APS could lead to safer, more effective, and more personalized therapeutic options. To this end, we sought to understand recent literature related to the role of neutrophils and, in particular, neutrophil extracellular traps (NETs) in APS. Expression of genes associated with type I interferons, endothelial adhesion, and pregnancy regulation are increased in APS neutrophils. APS neutrophils have a reduced threshold for NET release, which likely potentiates thrombotic events and perhaps especially large-vein thrombosis. Neutrophil-derived reactive oxygen species also appear to play a role in APS pathogenesis. There are new approaches for preventing and disrupting NETs that could potentially be leveraged to reduce the risk of APS-associated thrombosis. Neutrophils and NETs contribute to APS pathophysiology. More precisely understanding their roles at a mechanistic level should help identify new therapeutic targets for inhibiting NET formation, enhancing NET dissolution, and altering neutrophil adhesion. Such approaches may ultimately lead to better clinical management of APS patients and thereby reduce the chronic burden of this disease.

Quercetin alleviates rheumatoid arthritis by inhibiting neutrophil inflammatory activities

Rheumatoid arthritis (RA) is a prototypical autoimmune disorder mainly characterized by joint inflammation and cartilage destruction. Neutrophils actively take part in the initiation and progression of RA. Neutrophils express inflammatory mediators, including cytokines and chemokines. Aberrant formation of neutrophil extracellular traps (NETs) has been demonstrated in the pathogenesis of RA. Thus, neutrophils are regarded as important therapeutic targets in RA treatment. Quercetin is one of the major flavonoids found in fruits and vegetables. Previous studies have demonstrated that quercetin is a potential agent for the treatment of RA. However, the underlying antiarthritic mechanism of quercetin has not been investigated clearly. In this study, we analyzed the therapeutic mechanism of quercetin for RA. Our results showed that quercetin ameliorates inflammation in RA mice by inhibiting neutrophil activities. Quercetin inhibited neutrophil infiltration and reduced the plasma levels of inflammatory cytokines. Quercetin promoted the apoptosis of activated neutrophils. In addition, quercetin inhibited NET formation by suppressing autophagy. These findings suggest that quercetin may be an alternative agent for the treatment of RA by inhibiting neutrophil activities.

1336-P: Trimethylamine N-Oxide Inhibits the Formation of Neutrophil Extracellular Traps to Regulate Placental Development in Gestational Diabetes Mellitus

Objective: Trimethylamine N-oxide (TMAO) is associated with risk of gestational diabetes mellitus (GDM), and TMAO levels in peripheral/cord blood of GDM mothers increased. Diabetes primes neutrophils to produce neutrophil extracellular traps (NETs), but it has not been reported in GDM. We determined the relationship between NETs and GDM, and explored potential role of TMAO. Methods: Maternal peripheral blood was collected during OGTT tests, and cord blood and placenta were collected during delivery. Plasma concentrations of cell-free double-stranded DNA (dsDNA) were compared between GDM and control mothers and association analysis was performed. Expression of myeloperoxidase (MPO) in placental tissues of GDM and control mothers was examined. Neutrophils from the blood of healthy donors were used to observe effects of TMAO on NETs formation. The effect of TMAO on human placental extravillous trophoblast cell line HTR-8/Svneo was examined. Results: Compared with controls, plasma concentrations of dsDNA in peripheral blood and cord blood were significantly increased in GDM mothers. Association analysis found that concentrations of dsDNA in peripheral and cord blood plasma were positively correlated with BMI and blood glucose levels, but not blood pressure. In addition, the placental thickness was positively correlated with dsDNA levels in cord blood plasma. MPO expression in the placental tissues of GDM mothers was also significantly higher than that of control mothers. TMAO-treated neutrophils could resist the formation of NETs stimulated by PMA or LPS, and TMAO promotes the proliferation, migration, invasion and angiogenesis of the cells. Conclusion: The formation of NETs was increased in GDM, and was positively correlated with BMI and blood glucose. TMAO could inhibit NETs formation and promote invasion and angiogenesis of human placental trophoblast cells, which may be involved in the development of the placenta. Disclosure X. Lin: None. X. He: None. X. Xiao: None.

Neutrophil extracellular traps released by neutrophils impair revascularization and vascular remodeling after stroke

Neovascularization and vascular remodeling are functionally important for brain repair after stroke. We show that neutrophils accumulate in the peri-infarct cortex during all stages of ischemic stroke. Neutrophils producing intravascular and intraparenchymal neutrophil extracellular traps (NETs) peak at 3–5 days. Neutrophil depletion reduces blood-brain barrier (BBB) breakdown and enhances neovascularization at 14 days. Peptidylarginine deiminase 4 (PAD4), an enzyme essential for NET formation, is upregulated in peri-ischemic brains. Overexpression of PAD4 induces an increase in NET formation that is accompanied by reduced neovascularization and increased BBB damage. Disruption of NETs by DNase 1 and inhibition of NET formation by genetic ablation or pharmacologic inhibition of PAD increases neovascularization and vascular repair and improves functional recovery. Furthermore, PAD inhibition reduces stroke-induced STING-mediated production of IFN-β, and STING knockdown and IFN receptor-neutralizing antibody treatment reduces BBB breakdown and increases vascular plasticity. Collectively, our results indicate that NET release impairs vascular remodeling during stroke recovery.

Hydroxychloroquine inhibiting neutrophil extracellular trap formation alleviates hepatic ischemia/reperfusion injury by blocking TLR9 in mice

Hepatic ischemia/reperfusion (I/R) injury may arise after partial hepatectomy and liver transplantation. Neutrophil extracellular traps (NETs) were involved in hepatic I/R injury. This study tested the hypothesis that blocking NETs formation could be a potential therapeutic target against hepatic I/R injury. NETs were excessively formed within liver and in serum of I/R mice models and were testified to be an independent contributor to hepatic I/R injury. Hydroxychloroquine (HCQ) alleviated hepatic I/R injury by inhibiting NETs formation in SCID and c57BL/6 mice models. In vitro, HCQ inhibited neutrophils to form NETs at a concentration of 100 μg/ml. CpG-ODN reversed the effect of HCQ inhibiting NETs formation. HCQ inhibited PAD4 and Rac2 expressions by blocking TLR9. NETs are essential contributors to hepatic I/R injury. HCQ blocking TLR9 protects against hepatic I/R injury by inhibiting NETs formation, which may suggest utility of HCQ or other TLR9 agonists for preventing hepatic I/R injury in clinical practices.

Neutrophils undergo switch of apoptosis to NETosis during murine fatty liver injury via S1P receptor 2 signaling

Inappropriate neutrophil infiltration and subsequent neutrophil extracellular trap (NET) formation have been confirmed to be involved in chronic inflammatory conditions. Fatty liver disease is an increasingly severe health problem worldwide and currently considered the most common cause of chronic liver disease. Sphingosine 1-phosphate (S1P), a product of membrane sphingolipid metabolism, regulates vital physiological and pathological actions by inducing infiltration and activation of various cell types through S1P receptors (S1PRs). Here, we seek to determine the S1PR-mediated effects on neutrophil activation during chronic liver inflammation. In this study, NETs are detected in the early stage of methionine-choline-deficient and a high-fat (MCDHF) diet-induced liver injury. NET depletion by deoxyribonuclease I intraperitoneal injection significantly protects liver from MCDHF-induced liver injury in vivo. Meanwhile, we show that levels of myeloperoxidase-DNA complex (NET marker) in the serum present positive correlation with sphingosine kinase1 (S1P rate-limiting enzyme) messenger RNA expression or S1P levels in the injured liver of MCDHF-fed mice. In vitro, S1PR2 participates in the redirection of neutrophil apoptosis to NETosis via Gαi/o, extracellular signal-regulated kinase, p38 mitogen-activated protein kinase, and reactive oxygen species signaling pathways. Moreover, S1PR2 knockdown in MCDHF-fed mice by S1PR2-siRNA intravenous injection significantly inhibits NET formation in damaged liver tissue and then alleviates hepatic inflammation and fibrosis. Conclusion: In the early stage of fatty liver disease, S1PR2-mediated neutrophil activation plays an important role in the evolvement of liver injury.

Therapeutic ACPA inhibits NET formation: a potential therapy for neutrophil-mediated inflammatory diseases

Excessive release of neutrophil extracellular traps (NETs) is associated with disease severity and contributes to tissue injury, followed by severe organ damage. Pharmacological or genetic inhibition of NET release reduces pathology in multiple inflammatory disease models, indicating that NETs are potential therapeutic targets. Here, we demonstrate using a preclinical basket approach that our therapeutic anti-citrullinated protein antibody (tACPA) has broad therapeutic potential. Treatment with tACPA prevents disease symptoms in various mouse models with plausible NET-mediated pathology, including inflammatory arthritis (IA), pulmonary fibrosis, inflammatory bowel disease and sepsis. We show that citrulline residues in the N-termini of histones 2A and 4 are specific targets for therapeutic intervention, whereas antibodies against other N-terminal post-translational histone modifications have no therapeutic effects. Because citrullinated histones are generated during NET release, we investigated the ability of tACPA to inhibit NET formation. tACPA suppressed NET release from human neutrophils triggered with physiologically relevant human disease-related stimuli. Moreover, tACPA diminished NET release and potentially initiated NET uptake by macrophages in vivo, which was associated with reduced tissue damage in the joints of a chronic arthritis mouse model of IA. To our knowledge, we are the first to describe an antibody with NET-inhibiting properties and thereby propose tACPA as a drug candidate for NET-mediated inflammatory diseases, as it eliminates the noxious triggers that lead to continued inflammation and tissue damage in a multidimensional manner.

Hydrogen sulfide upregulates miR-16-5p targeting PiK3R1 and RAF1 to inhibit neutrophil extracellular trap formation in chickens.

Hydrogen sulfide (H2S) is a toxic air pollutant that causes immune damage. Recent studies have found that neutrophil extracellular trap (NET) formation is one way in which neutrophils exert immune functions. In addition, the formation of NETs is also related to thrombosis and autoimmune diseases. Recent studies have shown that miRNAs are involved in the regulation of a variety of pathophysiological processes. Here, we investigated the role of H2S in regulating the formation of NETs by affecting miR-16-5p. Our study established an in vitro H2S exposure model for neutrophils using phorbol-myristate-acetate (PMA) to induce NET formation. We observed the morphological changes of cells with scanning electron microscopy and fluorescence microscopy. Then, the content of extracellular DNA and the expression of MPO and NE in each group were detected. The results showed that H2S inhibited the formation of NETs. The expression of miR-16-5p and its target genes PiK3R1 and RAF1 was then measured by qRT-PCR. H2S upregulated miR-16-5p and inhibited expression of the target genes PiK3R1 and RAF1, and it subsequently inhibited the Pi3K/AKT and ERK pathways and decreased respiratory burst levels. Furthermore, H2S attenuated inositol 1,4,5-trisphosphate receptor (IP3R)-mediated endoplasmic reticulum calcium outflow as well as autophagy caused by PMA. This study enriches H2S immunotoxicity research and provides a possible solution for the treatment of NET-related diseases.

Inhibition of neutrophil elastase prevents neutrophil extracellular trap formation and rescues mice from endotoxic shock

Neutrophil elastase (NE) is a serine protease stored in the azurophilic granules of neutrophils and released into the extracellular milieu during inflammatory response or formation of neutrophil extracellular traps (NETs). Neutrophils release NETs to entrap pathogens by externalizing their cellular contents in a DNA framework decorated with anti-microbials and proteases, including NE. Importantly, excess NETs in tissues are implicated in numerous pathologies, including sepsis, rheumatoid arthritis, vasculitis, and cancer. However, it remains unknown how to effectively prevent NET formation. Here, we show that NE plays a major role during NET formation and that inhibition of NE is a promising approach for decreasing NET-mediated tissue injury. NE promoted NET formation by human neutrophils. Whereas sivelestat, a small molecule inhibitor of NE, inhibited the formation of NETs in vitro , administration of free sivelestat did not have any efficacy in a murine model of lipopolysaccharide-induced endotoxic shock. To improve the efficacy of sivelestat in vivo, we have developed a nanoparticle system for delivering sivelestat. We demonstrate that nanoparticle-mediated delivery of sivelestat effectively inhibited NET formation, decreased the clinical signs of lung injury, reduced NE and other proinflammatory cytokines in serum, and rescued animals against endotoxic shock. Collectively, our data demonstrates that NE signaling can initiate NET formation and that nanoparticle-mediated inhibition of NE improves drug efficacy for preventing NET formation.

461 Colchicine Inhibits Neutrophil Extracellular Trap Formation in Acute Coronary Syndrome Patients Post Percutaneous Coronary Intervention

Release of neutrophil extracellular traps (NETs) after PCI in ACS patients is associated with peri-procedural myocardial infarction, as a result of microvascular obstruction via pro-inflammatory and pro-thrombotic pathways. Colchicine is a potent, well-established anti-inflammatory agent with growing evidence to support its use in patients with coronary disease. However, its effects on post-PCI NET formation in ACS patients has not been explored. 60 patients (40 ACS; 20 stable angina pectoris[SAP]) were prospectively recruited and allocated to colchicine (1.5 mg) or no treatment. Within 24 h of treatment, serial coronary sinus blood samples were collected during PCI. Isolated neutrophils from 10 ACS patients post-PCI and 4 healthy controls were treated with colchicine (25 nM) and stimulated with either ionomycin (5 μM) or phorbol 12-myristate 13-acetate (PMA, 50 nM) in vitro. Extracellular DNA was quantified using Sytox Green and fixed cells were stained with hoechst and anti-alpha tubulin. Baseline characteristics were similar across both treatment and control arms. ACS patients had higher NET release versus SAP patients (p<0.001), which was reduced with colchicine treatment (AUC: 0.58 vs 4.29; p<0.001). In vitro, colchicine suppressed spontaneous (p=0.004), PMA-induced (p=0.03) and ionomycin-induced (p=0.02) NET formation in neutrophils isolated from ACS patients post-PCI, but not healthy controls. Tubulin organisation was impaired in neutrophils from patients with ACS but was restored by colchicine treatment. Colchicine suppresses NET formation in ACS patients post-PCI by restoring cytoskeletal dynamics. These findings warrant further investigation in randomised trials powered for clinical endpoints.

Early intravenous administration of tranexamic acid ameliorates intestinal barrier injury induced by neutrophil extracellular traps in a rat model of trauma/hemorrhagic shock

BackgroundEarly intravenous administration of tranexamic acid has been shown to protect the intestinal barrier after a model of trauma-hemorrhagic shock in the rat, but the potential mechanism remains unclear. Our previous studies have demonstrated that neutrophil extracellular traps contribute to the intestinal barrier dysfunction during sepsis and other critical conditions. Meanwhile, there are high levels of neutrophil infiltration in the intestine during trauma-hemorrhagic shock. Here, we hypothesized that neutrophil extracellular trap formation played a vital role during trauma-hemorrhagic shock-induced intestinal injury and that tranexamic acid, a serine protease inhibitor, may inhibit neutrophil extracellular trap formation and protect intestinal barrier function in trauma-hemorrhagic shock. MethodsA model of trauma-hemorrhagic shock in male rats was established. The rats were divided into 6 groups: (1) sham group; (2) trauma-hemorrhagic shock group; (3) trauma-hemorrhagic shock + DNase I group; (4) trauma-hemorrhagic shock + tranexamic acid group; (5) trauma-hemorrhagic shock + tranexamic acid (different time) group; and (6) trauma-hemorrhagic shock + tranexamic acid (different doses) group. The DNase I solution was injected intravenously to disrupt neutrophil extracellular traps immediately after the trauma-hemorrhagic shock model was completed. After 24 hours, the small intestine and blood were collected for analysis. Human neutrophils were harvested and incubated with phorbol-12-myristate-13-acetate or tranexamic acid, generation of reactive oxygen species, and key proteins expression were detected. ResultsTrauma-hemorrhagic shock induced the formation of intestinal neutrophil extracellular traps and disrupted the intestinal tight junction proteins. Clearing of neutrophil extracellular traps by DNase I resulted in increased expression of tight junction proteins and alleviated the intestinal injury. Early intravenous tranexamic acid administration (1 hour after trauma-hemorrhagic shock) decreased neutrophil extracellular trap formation and prevented tight junction protein disruption compared to the non-tranexamic acid group; however, after delayed administration of tranexamic acid (6 hours), there were no changes in neutrophil extracellular trap formation and intestinal injuries compared to the non-tranexamic acid group. Furthermore, tranexamic acid inhibited neutrophil extracellular trap formation and protected the intestinal barrier in a dose-dependent manner and high-dose (20 mg/kg) treatment of tranexamic acid showed a better effect compared with the therapeutic dose (10 mg/kg). The results of thromboelastography demonstrated that the R and K values in the high-dose group decreased (R, 1.85 ± 0.14 vs 3.87 ± 0.16 minutes, P < .001; K, 0.95 ± 0.04 vs 1.48 ± 0.07 minutes, P < .001), accompanied by a decrease in LY30, indicating that treatment with a high dose of tranexamic acid may cause hypercoagulability and shutdown of fibrinolysis. In addition, less neutrophil extracellular trap formation was detected in neutrophils incubated with neutrophils via an reactive oxygen species-dependent pathway. ConclusionWe first demonstrated a novel role of neutrophil extracellular traps in the pathophysiology of intestinal barrier dysfunction during trauma-hemorrhagic shock. Notably, early but not delayed intravenous administration of tranexamic acid effectively inhibits neutrophil extracellular trap formation and protects intestinal barrier function. Therefore, these results suggested a potential theoretic intervention for the protection of the intestinal barrier during trauma-hemorrhagic shock. In such a process, tranexamic acid appears to regulate neutrophil extracellular trap formation via the classic reactive oxygen species/mitogen-activated protein kinase pathway.

A Novel Assay of Neutrophil Extracellular Traps (NETs) Formation Independently Predicts DIC and Identifies the Rationale for Anti-IL-8 Therapies

Background Neutrophils are the first line of defence against bacterial infection and formation of neutrophil extracellular traps (NETs) is an important protective mechanism. However, NETs can also cause harm by promoting intravascular coagulation and multi-organ failure (MOF) in animal models. Although increasingly considered as important therapeutic targets, there is currently no robust and specific measure of NETs formation to inform clinical care and enable precision medicine in patients on the intensive care unit (ICU). The aim of this study is to establish a novel assay for measuring NETs and assess its clinical significance. Methods A prospective cohort of 341 consecutive adult ICU patients was recruited, following written informed consent. The NETs-forming capacity of ICU admission blood samples was semi-quantified by directly incubating patient plasma with isolated healthy neutrophils ex vivo. The association of NETs-forming capacity with sequential organ failure assessment (SOFA) scores, disseminated intravascular coagulation (DIC) and 28-day mortality were analysed and compared with available NETs assays. Cytokine analysis together with inhibitor studies was performed to determine the driving factors of NETs patients. To determine the pathological relevance of NETs, complementary in vivo studies were performed in mice models of sepsis (caecal ligation and puncture (CLP) or intraperitoneal injection of Escherichia coli), without or with anti-NETs therapy. Results We observed that NETs were directly induced by heterologous healthy neutrophils incubated with plasma taken from ICU patients, but not from healthy donors (unless incubated with 100 nM PMA). Using the novel assay, we could stratify ICU patients into 4 groups, those with absent (22.0%), mild (49.9%), moderate (14.4%) and strong (13.8%) NETs formation, respectively. Strong NETs formation was predominantly found in sepsis (P &amp;lt;0.0001) and was associated with higher SOFA scores on admission and throughout the study duration (72 hours post-admission). Adjusted by APACHE II, multivariate regression showed that measuring the degree of NETs formation in ICU admission could independently predict DIC and mortality whereas other NETs assays, e.g. cell-free DNA, myeloperoxidase and myeloperoxidase-DNA complexes, could not. Interleukin (IL)-8 levels were found to be strongly associated with NETs formation and inhibiting IL-8 significantly attenuated NETosis. MAPK activation by IL-8 has been identified as a major pathway of NETs formation in patients. Using mice models of sepsis, we specifically observed NETs positive staining (cit-H3) in the lung tissue. This was associated with increases in lung injury scores, along with circulating markers of liver (BUN [CLP: P=0.005, E.coli: P&amp;lt;0.001]), kidney (ALT [CLP: P=0.01, E.coli: P=0.002]) and cardiac injury (cTnI [(CLP: P&amp;lt;0.001, E.coli: P&amp;lt;0.001]). By targeting IL-8 we were able to significantly inhibit NETs formation, organ injury and also improved survival times in septic mice (P=0.004). Conclusions Our new NETs assay directly measures the NETs-forming capacity in patient plasma. This could guide clinical management and enable identification of NETs-inducing factors in individual patients for targeted treatment and personalised ICU medicine. We identify IL-8 as a major driving factor in sepsis, with anti-IL-8 therapy in septic mice significantly reducing NETs-induced organ damage and mortality. Disclosures No relevant conflicts of interest to declare.

Inhibition of NET formation by polydatin protects against collagen-induced arthritis

BackgroundRheumatoid arthritis (RA) is a systematic, inflammatory, autoimmune disease, associated with a high number of disabilities. Increasing evidence has demonstrated that neutrophil extracellular trap (NET) formation plays a significant role in the pathogenesis and progression of RA. In this study, we have aimed to investigate the effects of polydatin (PD) on NET formation and its effects on disease activity in a collagen-induced arthritis (CIA) mouse model. MethodsIn the presence of PD or vehicle, neutrophils isolated from RA patients and mice were treated with phorbol 12-myristate 13-acetate (PMA) for 4 h, and NET formation investigated. For in vivo experiments, PD was administered intraperitoneally (45 mg/kg per day) to collagen-induced arthritis (CIA) mice. The incidence and severity of collagen-induced arthritis were assessed and NET deposition tested. ResultsIn vitro, PD significantly suppressed NET formation of neutrophils from RA patients. Consistently, decreased NETs were observed in PD treated bone marrow-derived neutrophils. In CIA mouse model, PD treatment delayed the onset of arthritis and attenuated arthritis severity. Compared with vehicle-treated CIA mice, the deposition of NETs in ankle joints was also reduced in PD-treated CIA mice. ConclusionIn this study, we found that PD treatment markedly inhibited NET formation and protected CIA mice from the development of arthritis. These findings suggest that inhibition of NET formation by PD may serve as a novel mechanism for the treatment of RA.

Erythromycin suppresses neutrophil extracellular traps in smoking-related chronic pulmonary inflammation

Neutrophil extracellular traps (NETs) may play a critical role in smoking-related chronic airway inflammation. However, the mechanism by which NETs induced by cigarette smoke initiate the adaptive immunity in chronic obstructive pulmonary disease (COPD) is not fully understood. In this study, we explored the effects of NETs induced by cigarette smoke on the myeloid dendritic cells (mDCs) and Th1 and Th17 cells. Additionally, we observed the inhibitory effect of erythromycin on NETs induced by cigarette smoke. We found that elevated NET levels in the sputum of COPD patients were correlated with the circulating Th1 response, mDC activation and airflow limitation. NETs induced by cigarette smoke extract (CSE) could activate monocyte-derived mDCs and promote Th1 and Th17 differentiation in vitro. Erythromycin effectively inhibited NET formation induced by CSE. In vivo, erythromycin decreased NETs in the airway and ameliorated emphysema with Th1 and Th17 cell down-regulation and CD40+ and CD86+ mDCs suppression in mice chronically exposed to cigarette smoke. These findings provide direct evidence that NETs promote the differentiation of Th1 and Th17 and play a role in the adaptive immunity of smoking-related chronic lung inflammation. Erythromycin is a potential therapeutic strategy for NETs inhibition in COPD.

All trans-retinoic acid protects against acute ischemic stroke by modulating neutrophil functions through STAT1 signaling

Background and purposeRegulation of neural inflammation is considered as a vital therapeutic target in ischemic stroke. All-trans retinoic acid (atRA), a potent immune modulator, has raised interest in the field of stroke therapy. However, the immunological mechanisms for atRA-mediated neuroprotection remain elusive. The current study evaluated the impact of atRA on post-stroke neural inflammation and elucidated the mechanisms involved in the regulation of related neutrophil functions.MethodsatRA was prophylactically administered to mice 1 day before transient middle cerebral artery occlusion (tMCAO, 1 h) and repeated daily immediately after reperfusion for 3 days. Stroke outcomes, neutrophil polarization, and formation of neutrophil extracellular traps (NETs) in the stroke lesion were assessed. Neutrophil depletion was induced with anti-Ly6G antibodies. Primary neutrophil cultures were used to explore the mechanisms of atRA treatment.ResultsProphylactic atRA treatment reduced infarct volumes and neurological deficits at 1 day after tMCAO. Post-stroke neural inflammation was attenuated and neutrophil accumulation in lesion was downregulated. atRA treatment skewed neutrophil toward N2 phenotype which facilitated its clearance by macrophage and inhibited NETs formation. The functions of neutrophil were indispensable in the protective effects of atRA and were associated with suppression to STAT1 signaling by atRA. Administration of atRA after stroke still provided efficient protection to cerebral ischemia.ConclusionatRA displays potent therapeutic efficacy in ischemic stroke by attenuating neural inflammation. Treatment of atRA impeded neutrophil accumulation, favored N2 polarization, and forbade NETs formation in ischemic lesion. STAT1 signaling played a decisive role in the mechanisms of atRA-afforded regulation to neutrophil.

Protection against neutrophil extracellular trap (NET) toxicity by antioxidant monoHER

Background: Neutrophil extracellular traps (NET) are extracellular fibers produced by activated neutrophils to kill bacteria. NET was recently found to be associated with several diseases, such as autoimmune diseases. NET formation, called NETosis, is reactive oxygen species (ROS) dependent, thereby, prompting us to study its inhibition by potent antioxidant monoHER as well as to study monoHER protection against NET toxicity caused by NET constituent histone 3 on endothelial cells. Methods: Freshly isolated neutrophils from male donors were stimulated with PMA to induce NET formation. The effect of monoHER (50 µM) on oxidative burst (O2 ●− production) and NET formation was determined by fluorescence microscopy. Flow cytometry was used to determine the protective effect of monoHER against NET toxicity constituent histone 3 in EA.hy926 cells. Data was evaluated using ANOVA followed by the Bonferroni post-hoc test. Results: MonoHER significantly reduced (p < 0.01) O2 ●− production of PMA-stimulated neutrophils and consequently inhibited NET formation. MonoHER could also counteract histone 3 toxicity in EA.hy926 cells. Conclusions: MonoHER might inhibit ROS-dependent NETosis pathway and also protect the endothelial cells against NET toxicity.