Induce Neutrophil Extracellular Traps Formation Research Articles

Flavonoids and 5-Aminosalicylic Acid Inhibit the Formation of Neutrophil Extracellular Traps

Neutrophil extracellular traps (NETs) have been suggested to play a pathophysiological role in several autoimmune diseases. Since NET-formation in response to several biological and chemical stimuli is mostly ROS dependent, in theory any substance that inhibits or scavenges ROS could prevent ROS-dependent NET release. Therefore, in the present comprehensive study, several antioxidative substances were assessed for their capacity to inhibit NET formation of primary human neutrophils in vitro. We could show that the flavonoids (−)-epicatechin, (+)-catechin hydrate, and rutin trihydrate as well as vitamin C and the pharmacological substances N-acetyl-L-cysteine and 5-aminosalicylic acid inhibited PMA induced ROS production and NET formation. Therefore, a broad spectrum of antioxidative substances that reduce ROS production of primary human neutrophils also inhibits ROS-dependent NET formation. It is tempting to speculate that such antioxidants can have beneficial therapeutic effects in diseases associated with ROS-dependent NET formation.

Circulating Histones Are Mediators of Trauma-associated Lung Injury

Acute lung injury is a common complication after severe trauma, which predisposes patients to multiple organ failure. This syndrome largely accounts for the late mortality that arises and despite many theories, the pathological mechanism is not fully understood. Discovery of histone-induced toxicity in mice presents a new dimension for elucidating the underlying pathophysiology. To investigate the pathological roles of circulating histones in trauma-induced lung injury. Circulating histone levels in patients with severe trauma were determined and correlated with respiratory failure and Sequential Organ Failure Assessment (SOFA) scores. Their cause-effect relationship was studied using cells and mouse models. In a cohort of 52 patients with severe nonthoracic blunt trauma, circulating histones surged immediately after trauma to levels that were toxic to cultured endothelial cells. The high levels were significantly associated with the incidence of acute lung injury and SOFA scores, as well as markers of endothelial damage and coagulation activation. In in vitro systems, histones damaged endothelial cells, stimulated cytokine release, and induced neutrophil extracellular trap formation and myeloperoxidase release. Cellular toxicity resulted from their direct membrane interaction and resultant calcium influx. In mouse models, cytokines and markers for endothelial damage and coagulation activation significantly increased immediately after trauma or histone infusion. Pathological examinations showed that lungs were the predominantly affected organ with edema, hemorrhage, microvascular thrombosis, and neutrophil congestion. An anti-histone antibody could reduce these changes and protect mice from histone-induced lethality. This study elucidates a new mechanism for acute lung injury after severe trauma and proposes that circulating histones are viable therapeutic targets for improving survival outcomes in patients.

Cytokines Induced Neutrophil Extracellular Traps Formation: Implication for the Inflammatory Disease Condition

Neutrophils (PMNs) and cytokines have a critical role to play in host defense and systemic inflammatory response syndrome (SIRS). Neutrophil extracellular traps (NETs) have been shown to extracellularly kill pathogens, and inflammatory potential of NETs has been shown. Microbial killing inside the phagosomes or by NETs is mediated by reactive oxygen and nitrogen species (ROS/RNS). The present study was undertaken to assess circulating NETs contents and frequency of NETs generation by isolated PMNs from SIRS patients. These patients displayed significant augmentation in the circulating myeloperoxidase (MPO) activity and DNA content, while PMA stimulated PMNs from these patients, generated more free radicals and NETs. Plasma obtained from SIRS patients, if added to the PMNs isolated from healthy subjects, enhanced NETs release and free radical formation. Expressions of inflammatory cytokines (IL-1β, TNFα and IL-8) in the PMNs as well as their circulating levels were significantly augmented in SIRS subjects. Treatment of neutrophils from healthy subjects with TNFα, IL-1β, or IL-8 enhanced free radicals generation and NETs formation, which was mediated through the activation of NADPH oxidase and MPO. Pre-incubation of plasma from SIRS with TNFα, IL-1β, or IL-8 antibodies reduced the NETs release. Role of IL-1β, TNFα and IL-8 thus seems to be involved in the enhanced release of NETs in SIRS subjects.

Recognition of Fungal β‐glucan by Human Neutrophil CR3 Results in Homotypic Aggregation and Neutrophil Extracellular Traps

The armament of neutrophil‐mediated host defense against fungal and bacterial pathogens includes the formation of a lattice of DNA and microbicidal enzymes known as Neutrophil Extracellular Traps (NETs). The receptor mediated interactions and intracellular signaling events responsible for the elaboration of NETs in response to fungi are not known and were determined in this study. Recognition of the fungal cell wall component β‐glucan by human neutrophils resulted in rapid formation of cell aggregates and NET release. This response required fibronectin + β‐glucan demonstrating a role for CD11b/CD18. CD11b/CD18 blockade obviated this response which did not require production of oxidative metabolites. Downstream signaling events were determined using a global phosphoproteomic approach. Upon fibronectin + β‐glucan treatment, 46 phosphopeptides were upregulated and 15 were downregulated as compared to fibronectin alone. Validation revealed a functional role for ERK (Y204). NET formation to Candida albicans hyphae was also shown to be dependent on CD11b/CD18 recognition of hyphal β‐glucan, occur in the absence of reactive oxidant species, and depend on ERK. This study successfully identified a key receptor ligand interaction that induces NET formation and elucidates the global tyrosine phosphoproteome of human neutrophils to gain a better understanding of innate immune responses to fungal pathogens.NIH GM066194

Neutrophil Extracellular Trap Formation Is Associated with IL-1β and Autophagy-Related Signaling in Gout

BackgroundGout is a prevalent inflammatory arthritis affecting 1–2% of adults characterized by activation of innate immune cells by monosodium urate (MSU) crystals resulting in the secretion of interleukin-1β (IL-1β). Since neutrophils play a major role in gout we sought to determine whether their activation may involve the formation of proinflammatory neutrophil extracellular traps (NETs) in relation to autophagy and IL-1β.Methodology/Principal FindingsSynovial fluid neutrophils from six patients with gout crisis and peripheral blood neutrophils from six patients with acute gout and six control subjects were isolated. MSU crystals, as well as synovial fluid or serum obtained from patients with acute gout, were used for the treatment of control neutrophils. NET formation was assessed using immunofluorescence microscopy. MSU crystals or synovial fluid or serum from patients induced NET formation in control neutrophils. Importantly, NET production was observed in neutrophils isolated from synovial fluid or peripheral blood from patients with acute gout. NETs contained the alarmin high mobility group box 1 (HMGB1) supporting their pro-inflammatory potential. Inhibition of phosphatidylinositol 3-kinase signaling or phagolysosomal fusion prevented NET formation, implicating autophagy in this process. NET formation was driven at least in part by IL-1β as demonstrated by experiments involving IL-1β and its inhibitor anakinra.Conclusions/SignificanceThese findings document for the first time that activation of neutrophils in gout is associated with the formation of proinflammatory NETs and links this process to both autophagy and IL-1β. Modulation of the autophagic machinery may represent an additional therapeutic study in crystalline arthritides.

Neutrophil Extracellular Trap (NET)-Mediated Killing of Pseudomonas aeruginosa: Evidence of Acquired Resistance within the CF Airway, Independent of CFTR

The inability of neutrophils to eradicate Pseudomonas aeruginosa within the cystic fibrosis (CF) airway eventually results in chronic infection by the bacteria in nearly 80 percent of patients. Phagocytic killing of P. aeruginosa by CF neutrophils is impaired due to decreased cystic fibrosis transmembrane conductance regulator (CFTR) function and virulence factors acquired by the bacteria. Recently, neutrophil extracellular traps (NETs), extracellular structures composed of neutrophil chromatin complexed with granule contents, were identified as an alternative mechanism of pathogen killing. The hypothesis that NET-mediated killing of P. aeruginosa is impaired in the context of the CF airway was tested. P. aeruginosa induced NET formation by neutrophils from healthy donors in a bacterial density dependent fashion. When maintained in suspension through continuous rotation, P. aeruginosa became physically associated with NETs. Under these conditions, NETs were the predominant mechanism of killing, across a wide range of bacterial densities. Peripheral blood neutrophils isolated from CF patients demonstrated no impairment in NET formation or function against P. aeruginosa. However, isogenic clinical isolates of P. aeruginosa obtained from CF patients early and later in the course of infection demonstrated an acquired capacity to withstand NET-mediated killing in 8 of 9 isolates tested. This resistance correlated with development of the mucoid phenotype, but was not a direct result of the excess alginate production that is characteristic of mucoidy. Together, these results demonstrate that neutrophils can kill P. aeruginosa via NETs, and in vitro this response is most effective under non-stationary conditions with a low ratio of bacteria to neutrophils. NET-mediated killing is independent of CFTR function or bacterial opsonization. Failure of this response in the context of the CF airway may occur, in part, due to an acquired resistance against NET-mediated killing by CF strains of P. aeruginosa.

Singlet oxygen is essential for neutrophil extracellular trap formation

Neutrophil extracellular traps (NETs) that bind invading microbes are pivotal for innate host defense. There is a growing body of evidence for the significance of NETs in the pathogenesis of infectious and inflammatory diseases, but the mechanism of NET formation remains unclear. Previous observation in neutrophils of chronic granulomatous disease (CGD) patients, which defect NADPH oxidase (Nox) and fail to produce reactive oxygen species (ROS), revealed that ROS contributed to the formation of NETs. However, the active species were not identified. In this study, we discovered that singlet oxygen, one of the ROS, mediated Nox-dependent NET formation upon stimulation with phorbol myristate acetate. We also revealed that singlet oxygen itself could induce NET formation by a distinct system generating singlet oxygen with porfimer sodium (Photofrin) in CGD neutrophils, as well as healthy neutrophils. This was independent of Nox activation. These results show that singlet oxygen is essential for NET formation, and provide novel insights into the pathogenesis of infectious and inflammatory diseases.

Role of p38 MAPK in PMA‐induced NETs release from human neutrophils

Neutrophil extracellular traps (NETs) are produced by the neutrophils to bind and kill the extracellular microorganisms. It is composed of chromatin decorated with granular proteins such as elastase, myeloperoxidase, cathepsin G, pentraxin, lactoferrin etc. In the present study we have investigated the role of p38 MAPK in PMA induced reactive oxygen species (ROS) generation and NETs formation. Human neutrophils treated with PMA induced free radical generation and NETs release in a time (1–3 hrs.) and concentration (10 – 50 nM) dependent manner as demonstrated by DCF response and elastase/DNA release respectively. Since NETs release is dependent on free radicals effect of SB 202190 (50 μM, p38MAPK inhibitor) was investigated on PMA induced free radical formation as assessed by DCF and NBT, which was not significantly reduced. NETs formation was however inhibited by SB 202190 or DPI (10 μM, NADPH oxidase inhibitor), suggesting that p38 MAPK play a role downstream to the NADPH oxidase in PMA induced NETs release. NETs are made up of DNA and elastase, their release was also inhibited by DPI and SB 202190. PMA treatment induced the phosphorylation of p38 MAPK, which was inhibited by DPI. The results of the present study suggest that PMA induced NETs formation was mediated through the activation of NADPH oxidase and p38 MAPK.

Staphylococcus epidermidis Antimicrobial δ-Toxin (Phenol-Soluble Modulin-γ) Cooperates with Host Antimicrobial Peptides to Kill Group A Streptococcus

Antimicrobial peptides play an important role in host defense against pathogens. Recently, phenol-soluble modulins (PSMs) from Staphylococcus epidermidis (S. epidermidis) were shown to interact with lipid membranes, form complexes, and exert antimicrobial activity. Based on the abundance and innocuity of the cutaneous resident S. epidermidis, we hypothesized that their PSMs contribute to host defense. Here we show that S. epidermidis δ-toxin (PSMγ) is normally present in the epidermis and sparsely in the dermis of human skin using immunohistochemistry. Synthetic δ-toxin interacted with neutrophil extracellular traps (NETs) and colocalized with cathelicidin while also inducing NET formation in human neutrophils. In antimicrobial assays against Group A Streptococcus (GAS), δ-toxin cooperated with CRAMP, hBD2, and hBD3. In whole blood, addition of δ-toxin exerted a bacteriostatic effect on GAS, and in NETs, δ-toxin increased their killing capacity against this pathogen. Coimmunoprecipitation and tryptophan spectroscopy demonstrated direct binding of δ-toxin to host antimicrobial peptides LL-37, CRAMP, hBD2, and hBD3. Finally, in a mouse wound model, GAS survival was reduced (along with Mip-2 cytokine levels) when the wounds were pretreated with δ-toxin. Thus, these data suggest that S. epidermidis–derived δ-toxin cooperates with the host-derived antimicrobial peptides in the innate immune system to reduce survival of an important human bacterial pathogen.

Neutrophil Extracellular Traps Induce Platelet Adhesion and Thrombus Formation.

Abstract 1345 IntroductionThrombus stability is provided by very large polymers adhering to platelets and anchoring the thrombus to the vessel wall. The best described polymers are fibrin and von Willebrand Factor (VWF). Activated neutrophils and other leukocytes can form an extracellular fibrous network which is composed of DNA, histones, and granular proteins. These neutrophil extracellular traps (NETs) are present in various inflammatory diseases. In deep vein thrombosis (DVT) inflammation closely cooperates with thrombosis. Here we examine whether NETs provide a new means to support the adhesion and recruitment of platelets and whether NETs are present in DVT. Methods and Results: To study the interaction of platelets with NETs, we isolated human neutrophils, induced NET formation and perfused over the NETs human platelets in plasma or whole blood anticoagulated with the thrombin inhibitor PPACK. Microscopic analysis revealed that under flow platelets adhere avidly to NETs. Perfusion of whole blood at physiological shear resulted in formation of thrombi on NETs in a time dependent manner. Addition of DNase1 degraded NETs and removed all platelets and thrombi demonstrating their adhesion to NETs. Thrombus formation on NETs was absent if blood was supplemented with EDTA indicating the requirement for divalent cations. Perfusion of NETs with heparinized blood dismantled NETs and prevented thrombus formation. Incubation of NETs with heparin alone released histones from NETs, indicating that heparin destroys the chromatin backbone of NETs. Furthermore, immunocytochemistry revealed that NETs were able to bind platelet adhesion molecules VWF and fibronectin from human plasma. Immunohistochemical analysis of a baboon deep vein thrombus showed abundant extracellular chromatin which co-localized with fibronectin and VWF. Conclusions: We show that extracellular traps are able to promote thrombosis in vitro and are abundant in vivo in DVT. We propose that extracellular chromatin provides a new type of scaffold that promotes platelet adhesion, activation, and aggregation and may be important for thrombus initiation or stability. DisclosuresNo relevant conflicts of interest to declare.

Neutrophil extracellular traps are induced by Mycobacterium tuberculosis

Due to the intracellular nature of mycobacterial infections, little attention has been paid to the possible extracellular role that neutrophils might play in tuberculosis. The recent discovery of neutrophil extracellular traps (NETs), composed of DNA and antimicrobial proteins,(1) introduces a new perspective to our understanding of the mechanism used by the innate immune system to contain and kill microorganisms. In this study, we tested in vitro whether Mycobacterium tuberculosis, an intracellular pathogen, can induce NETs formation and if this newly discovered mechanism is involved in a control response during mycobacterial infection. We found that two different genotypes of M. tuberculosis exerted, in vitro, a cytotoxic effect and induced subcellular changes on infected neutrophils, leading to NETs formation in a time dependent manner. NETs trapped mycobacteria but were unable to kill them. NETs formation induced by M. tuberculosis could help understand the early stages of mycobacterial pathogenesis.

IMPAIRED NEUTROPHIL EXTRACELLULAR TRAP FORMATION IN PLATELET-ACTIVATING FACTOR- AND LIPOPOLYSACCHARIDE-STIMULATED POLYMORPHONUCLEAR LEUKOCYTES ISOLATED FROM PRETERM INFANTS.

Background Activated human polymorphonuclear leukocytes (PMNs) participate in the innate immune response precipitated by tissue injury or bacterial invasion. As a recently described response to bacterial invasion, PMNs secrete lattices of DNA and antimicrobial proteins termed neutrophil extracellular traps (NETs) to effect extracellular killing of bacteria. However, PMNs isolated from infants born prematurely exhibit in vitro and in vivo functional deficits in bacterial killing. Whether these deficits result from impaired NET formation in preterm PMNs remains unknown. Hypothesis We hypothesize that preterm PMNs fail to robustly form NETs following activation in vitro. Methods We therefore stimulated human PMNs isolated from adults, term infants, and preterm infants with platelet-activating factor (PAF) (10−8M), lipopolysaccharide (LPS) (100 ng/mL), or live bacteria (Escherichia coli, 0.1-10 MOI) for 30 to 120 minutes. We visualized NET formation of live and fixed PMNs stained for extracellular DNA and myeloperoxidase expression via confocal microscopy, with and without time-lapse photography. We also obtained high-resolution images of NET formation in all three cell types via scanning electron microscopy of fixed PMNs following stimulation with PAF, LPS, or E. coli. Results We visualized limited or absent NET formation via confocal microscopy at 30 and 120 minutes in LPS- and E. coli-stimulated preterm PMNs compared with robust NET formation at 30 and 120 minutes in term and adult PMNs following the same stimuli. PAF failed to induce NET formation in preterm PMNs but did induce NET formation in term and adult PMNs at 30 minutes. Time-lapse confocal microscopy allowed visualization of NET formation in term and adult PMNs but failed to show NET formation in preterm PMN preparations. Furthermore, scanning electron microscopy confirmed robust NET formation at 30 minutes in term and adult PMNs following stimulation with PAF, LPS, and E. coli but noted minimal NET formation under those conditions in preterm PMNs. Conclusion We conclude that in vitro NET formation is impaired in preterm PMNs when compared with term and adult PMNs. Speculation We speculate that impaired NET formation may contribute to bacterial killing deficits documented in preterm infants, thus contributing to the morbidity and mortality in preterm infants associated with neonatal sepsis.