Aggregated NETs Sequester and Detoxify Extracellular Histones.
ABSTRACT: In response to various infectious and sterile stimuli neutrophils release chromatin decorated with bactericidal proteins, referred to as NETs. Their scaffolds are formed from chromatin fibers which display an apparent diameter of 15-17 nm and mainly consist from DNA (2 nm) and DNA-associated histones (11 nm). The NET-forming strands are thus not naked DNA but higher ordered chromatin structures. The histones may be released from the NET, especially if their tail arginines have been citrullinated. Several studies indicate that extracellular histones are toxic for mammalian epithelia and endothelia and contribute to the microvascular dysfunction observed e.g., in patients suffering from autoimmune diseases or sepsis. NETs formed at sites of very high neutrophil densities tend to clump and form fairly stable enzymatically active aggregates, referred to as aggNETs. The latter are endowed with a bunch of enzymes that cleave, bind, and/or modify autologous as well as foreign macromolecules. The tight binding of the serine proteases to the matrix precludes the spread of these toxic enzymes into the tissue but still allows the access of soluble inflammatory mediators to the enzymatic active internal surfaces of the NETs where they are degraded. Here, we describe that externally added histones are removed from culture supernatants of aggNETs. We will address the fate of the histones and discuss the feature on the background of neutrophil-driven diseases and the resolution of inflammation.
Project description:Neutrophil granulocytes, also called polymorphonuclear leukocytes (PMNs), extrude molecular lattices of decondensed chromatin studded with histones, granule enzymes, and antimicrobial peptides that are referred to as neutrophil extracellular traps (NETs). NETs capture and contain bacteria, viruses, and other pathogens. Nevertheless, experimental evidence indicates that NETs also cause inflammatory vascular and tissue damage, suggesting that identifying pathways that inhibit NET formation may have therapeutic implications. Here, we determined that neonatal NET-inhibitory factor (nNIF) is an inhibitor of NET formation in umbilical cord blood. In human neonatal and adult neutrophils, nNIF inhibits key terminal events in NET formation, including peptidyl arginine deiminase 4 (PAD4) activity, neutrophil nuclear histone citrullination, and nuclear decondensation. We also identified additional nNIF-related peptides (NRPs) that inhibit NET formation. nNIFs and NRPs blocked NET formation induced by pathogens, microbial toxins, and pharmacologic agonists in vitro and in mouse models of infection and systemic inflammation, and they improved mortality in murine models of systemic inflammation, which are associated with NET-induced collateral tissue injury. The identification of NRPs as neutrophil modulators that selectively interrupt NET generation at critical steps suggests their potential as therapeutic agents. Furthermore, our results indicate that nNIF may be an important regulator of NET formation in fetal and neonatal inflammation.
Project description:Silver nanoparticles (AgNPs) are widely used in various fields because of their antimicrobial properties. However, many studies have reported that AgNPs can be harmful to both microorganisms and humans. Reactive oxygen species (ROS) are a key factor of cytotoxicity of AgNPs in mammalian cells and an important factor in the immune reaction of neutrophils. The immune reactions of neutrophils include the expulsion of webs of DNA surrounded by histones and granular proteins. These webs of DNA are termed neutrophil extracellular traps (NETs). NETs allow neutrophils to catch and destroy pathogens in extracellular spaces. In this study, we investigated how AgNPs stimulate neutrophils, specifically focusing on NETs. Freshly isolated human neutrophils were treated with 5 or 100 nm AgNPs. The 5 nm AgNPs induced NET formation, but the 100 nm AgNPs did not. Subsequently, we investigated the mechanism of AgNP-induced NETs using known inhibitors related to NET formation. AgNP-induced NETs were dependent on ROS, peptidyl arginine deiminase, and neutrophil elastase. The result in this study indicates that treatment of 5 nm AgNPs induce NET formation through histone citrullination by peptidyl arginine deiminase and histone cleavage by neutrophil elastase.
Project description:In response to antineutrophil cytoplasmic antibody (ANCA), activated neutrophils release decondensed chromatin associated with cytoplasmic granular proteins composed of proteolytic enzymes and myeloperoxidase (MPO); these complexes are named neutrophil extracellular traps (NETs). NET formation requires peptidylarginine deiminase 4 (PAD4) to citrullinate chromatin histones and also MPO and neutrophil elastase to aid in decondensation and release of chromatin. Immunostaining of renal biopsy revealed that NET components of citrullinated histone, MPO and PAD4 were concurrently deposited both around fibrinoid necrosis in necrotizing glomerulonephritis in the early focal phase of ANCA-associated polyangiitis and along an interlobular arterial wall.
Project description:The majority of patients with cancer undergo at least one surgical procedure as part of their treatment. Severe postsurgical infection is associated with adverse oncologic outcomes; however, the mechanisms underlying this phenomenon are unclear. Emerging evidence suggests that neutrophils, which function as the first line of defense during infections, facilitate cancer progression. Neutrophil extracellular traps (NETs) are extracellular neutrophil-derived DNA webs released in response to inflammatory cues that trap and kill invading pathogens. The role of NETs in cancer progression is entirely unknown. We report that circulating tumor cells become trapped within NETs in vitro under static and dynamic conditions. In a murine model of infection using cecal ligation and puncture, we demonstrated microvascular NET deposition and consequent trapping of circulating lung carcinoma cells within DNA webs. NET trapping was associated with increased formation of hepatic micrometastases at 48 hours and gross metastatic disease burden at 2 weeks following tumor cell injection. These effects were abrogated by NET inhibition with DNAse or a neutrophil elastase inhibitor. These findings implicate NETs in the process of cancer metastasis in the context of systemic infection and identify NETs as potential therapeutic targets.
Project description:The release of neutrophil extracellular traps (NETs), a mesh of DNA, histones and neutrophil proteases from neutrophils, was first demonstrated as a host defence against pathogens. Recently it became clear that NETs are also released in pathological conditions. NETs released in the blood can activate thrombosis and initiate a cascade of platelet responses. However, it is not well understood if these responses are mediated through direct or indirect interactions. We investigated whether cell-free NETs can induce aggregation of washed human platelets in vitro and the contribution of NET-derived extracellular DNA and histones to platelet activation response.Isolated human neutrophils were stimulated with PMA to produce robust and consistent NETs. Cell-free NETs were isolated and characterised by examining DNA-histone complexes and quantification of neutrophil elastase with ELISA. NETs were incubated with washed human platelets to assess several platelet activation responses. Using pharmacological inhibitors, we explored the role of different NET components, as well as main platelet receptors, and downstream signalling pathways involved in NET-induced platelet aggregation.Cell-free NETs directly induced dose-dependent platelet aggregation, dense granule secretion and procoagulant phosphatidyl serine exposure on platelets. Surprisingly, we found that inhibition of NET-derived DNA and histones did not affect NET-induced platelet aggregation or activation. We further identified the molecular pathways involved in NET-activated platelets. The most potent single modulator of NET-induced platelet responses included NET-bound cathepsin G, platelet Syk kinase, and P2Y12 and ?IIb?3 receptors.In vitro-generated NETs can directly induce marked aggregation of washed human platelets. Pre-treatment of NETs with DNase or heparin did not reduce NET-induced activation or aggregation of human washed platelets. We further identified the molecular pathways activated in platelets in response to NETs. Taken together, we conclude that targeting certain platelet activation pathways, rather than the NET scaffold, has a more profound reduction on NET-induced platelet aggregation.
Project description:Neutrophils are short-lived leukocytes that die by apoptosis, necrosis, and NETosis. Upon death by NETosis, neutrophils release fibrous traps of DNA, histones, and granule proteins named neutrophil extracellular traps (NETs), which can kill bacteria and fungi. Inoculation of the protozoan Leishmania into the mammalian skin causes local inflammation with neutrophil recruitment. Here, we investigated the release of NETs by human neutrophils upon their interaction with Leishmania parasites and NETs' ability to kill this protozoan. The NET constituents DNA, elastase, and histones were detected in traps associated to promastigotes by immunofluorescence. Electron microscopy revealed that Leishmania was ensnared by NETs released by neutrophils. Moreover, Leishmania and its surface lipophosphoglycan induced NET release by neutrophils in a parasite number- and dose-dependent manner. Disruption of NETs by DNase treatment during Leishmania-neutrophil interaction increased parasite survival, evidencing NETs' leishmanicidal effect. Leishmania killing was also elicited by NET-rich supernatants from phorbol 12-myristate 13-acetate-activated neutrophils. Immunoneutralization of histone during Leishmania-neutrophil interaction partially reverted Leishmania killing, and purified histone killed the parasites. Meshes composed of DNA and elastase were evidenced in biopsies of human cutaneous leishmaniasis. NET is an innate response that might contribute to diminish parasite burden in the Leishmania inoculation site.
Project description:Neutrophils respond to various stimuli by decondensing and releasing nuclear chromatin characterized by citrullinated histones as neutrophil extracellular traps (NETs). This achieves pathogen immobilization or initiation of thrombosis, yet the molecular mechanisms of NET formation remain elusive. Peptidyl arginine deiminase-4 (PAD4) achieves protein citrullination and has been intricately linked to NET formation. Here we show that citrullination represents a major regulator of proteolysis in the course of NET formation. Elevated cytosolic calcium levels trigger both peptidylarginine deiminase-4 (PAD4) and calpain activity in neutrophils resulting in nuclear decondensation typical of NETs. Interestingly, PAD4 relies on proteolysis by calpain to achieve efficient nuclear lamina breakdown and chromatin decondensation. Pharmacological or genetic inhibition of PAD4 and calpain strongly inhibit chromatin decondensation of human and murine neutrophils in response to calcium ionophores as well as the proteolysis of nuclear proteins like lamin B1 and high mobility group box protein 1 (HMGB1). Taken together, the concerted action of PAD4 and calpain induces nuclear decondensation in the course of calcium-mediated NET formation.
Project description:Recent studies have demonstrated a role of neutrophils in both venous and arterial thrombosis. A key prothrombotic feature of neutrophils is their ability to release web-like structures composed of DNA filaments coated with histones and granule proteins referred to as neutrophil extracellular traps (NETs). NETs were discovered over a decade ago as part of our first line of host defense against invading microorganisms. Although NETs have a protective role against pathogens, recent data suggest that an uncontrolled and excessive NET formation within the vasculature may contribute to pathological thrombotic disorders. In vitro studies suggest that NETs promote vessel occlusion by providing a scaffold for platelets, red blood cells, extracellular vesicles, and procoagulant molecules, such as von Willebrand factor and tissue factor. In addition, NET components enhance coagulation by both activating the intrinsic pathway and degrading an inhibitor of the extrinsic pathway (tissue factor pathway inhibitor). NET formation has, therefore, been proposed to contribute to thrombus formation and propagation in arterial, venous, and cancer-associated thrombosis. This review will describe animal and human studies suggesting a role of NETs in the pathogenesis of various thrombotic disorders. Targeting NETs may be a novel approach to reduce thrombosis without affecting hemostasis.
Project description:Polymorphonuclear neutrophils have in recent years attracted new attention due to their ability to release neutrophil extracellular traps (NETs). These web-like extracellular structures deriving from nuclear chromatin have been depicted in ambiguous roles between antimicrobial defence and host tissue damage. NETs consist of DNA strands of varying thickness and are decorated with microbicidal and cytotoxic proteins. Their principal structure has in recent years been characterised at molecular and ultrastructural levels but many features that are of direct relevance to cytotoxicity are still incompletely understood. These include the extent of chromatin decondensation during NET formation and the relative amounts and spatial distribution of the microbicidal components within the NET. In the present work, we analyse the structure of NETs found in induced sputum of patients with acutely exacerbated chronic obstructive pulmonary disease (COPD) using confocal laser microscopy and electron microscopy. In vitro induced NETs from human neutrophils serve for purposes of comparison and extended analysis of NET structure. Results demonstrate that COPD sputa are characterised by the pronounced presence of NETs and NETotic neutrophils. We provide new evidence that chromatin decondensation during NETosis is most extensive and generates substantial amounts of double-helix DNA in 'beads-on-a-string' conformation. New information is also presented on the abundance and location of neutrophil elastase (NE) and citrullinated histone H3 (citH3). NE occurs in high densities in nearly all non-fibrous constituents of the NETs while citH3 is much less abundant. We conclude from the results that (i) NETosis is an integral part of COPD pathology; this is relevant to all future research on the etiology and therapy of the disease; and that (ii) release of 'beads-on-a-string' DNA studded with non-citrullinated histones is a common feature of in vivo NETosis; this is of relevance to both the antimicrobial and the cytotoxic effects of NETs.
Project description:Neutrophils are the most abundant innate immune cells and the first line of defense against many pathogenic microbes, including the human fungal pathogen Candida albicans. Among the neutrophils' arsenal of effector functions, neutrophil extracellular traps (NETs) are thought to be of particular importance for trapping and killing the large fungal filaments by means of their web-like structures that consist of chromatin fibers decorated with proteolytic enzymes and host defense proteins. Peptidylarginine deiminase 4 (PAD4)-mediated citrullination of histones in activated neutrophils correlates with chromatin decondensation and extrusion and is widely accepted to act as an integral process of NET induction (NETosis). However, the requirement of PAD4-mediated histone citrullination for NET release during C. albicans infection remains unclear. In this study, we show that although PAD4-dependent neutrophil histone citrullination is readily induced by C. albicans, PAD4 is dispensable for NETosis in response to the fungus and other common NET-inducing stimuli. Moreover, PAD4 is not required for antifungal immunity during mucosal and systemic C. albicans infection. Our results demonstrate that PAD4 is dispensable for C. albicans-induced NETosis, and they highlight the limitations of using histone citrullination as a marker for NETs and PAD4-/- mice as a model of NET-deficiency.