NETosis, the process wherein neutrophils release highly decondensed chromatin called neutrophil extracellular traps (NETs), has gained much attention as an alternative means of killing bacteria. In vivo, NETs are induced by bacteria and pro-inflammatory cytokines. We have reported that peptidylarginine deiminase 4 (PAD4), an enzyme that converts Arg or monomethyl-Arg to citrulline in histones, is essential for NET formation. The areas of extensive chromatin decondensation along the NETs were rich in histone citrullination. Here, upon investigating the effect of global citrullination in cultured cells, we discovered that PAD4 overexpression in osteosarcoma U2OS cells induces extensive chromatin decondensation independent of apoptosis. The highly decondensed chromatin is released to the extracellular space and stained strongly by a histone citrulline-specific antibody. The structure of the decondensed chromatin is reminiscent of NETs but is unique in that it occurs without stimulation of cells with pro-inflammatory cytokines and bacteria. Furthermore, histone citrullination during chromatin decondensation can dissociate heterochromatin protein 1 beta (HP1β) thereby offering a new molecular mechanism for understanding how citrullination regulates chromatin function. Taken together, our study suggests that PAD4 mediated citrullination induces chromatin decondensation, implicating its essential role in NET formation under physiological conditions in neutrophils.
pad4; hypercitrullination; neutrophil extracellular traps; chromatin decondensation; heterochromatin protein 1; histone modifications
In response to inflammation, neutrophils deiminate histones and externalize chromatin. Neutrophil extracellular traps (NETs) are an innate immune defense mechanism, yet NETs also may aggravate chronic inflammatory and autoimmune disorders. Activation of peptidylarginine deiminase 4 (PAD4) is associated with NET release (NETosis) but the precise mechanisms of PAD4 regulation are unknown. We observed that, in human neutrophils, calcium ionophore induced histone deimination, whereas phorbol myristate acetate (PMA), an activator of protein kinase C (PKC), suppressed ionophore-induced deimination. Conversely, low doses of chelerythrine and sanguinarine, two inhibitors of PKC, reversed PMA inhibition and enhanced ionophore-stimulated deimination. In addition, a peptide inhibitor of PKCα superinduced ionophore activation of PAD4, thus identifying PKCα as the PMA-induced inhibitor of PAD4. At higher doses, chelerythrine, sanguinarine, and structurally unrelated PKC inhibitors blocked histone deimination, suggesting that a different PKC isoform activates histone deimination. We identify PKCζ as activator of PAD4 because a specific peptide inhibitor of this PKC isoform suppressed histone deimination. Confocal microscopy confirmed that, in the presence of PMA, NETosis proceeds without detectable histone deimination, and that ionophore cooperates with PMA to induce more extensive NET release. Broad inhibition of PKC by chelerythrine or specific inhibition of PKCζ suppressed NETosis. Our observations thus reveal an intricate antagonism between PKC isoforms in the regulation of histone deimination, identify a dominant role for PKCα in the repression of histone deimination, and assign essential functions to PKCζ in the activation of PAD4 and the execution of NETosis. The precise balance between opposing PKC isoforms in the regulation of NETosis affirms the idea that NET release underlies specific and vitally important evolutionary selection pressures.
NETosis; PAD4; protein kinase C; deimination; inflammation
Spectacular images of neutrophils ejecting nuclear chromatin and bactericidal proteins, in response to microbes, were first reported in 2004. As externalized chromatin could entangle bacteria, these structures were named neutrophil extracellular traps (NETs). Subsequent studies identified microorganisms and sterile conditions that stimulate NETs, and additional cell types that release extracellular chromatin. NETs’ release is the most dramatic stage in a cell death process called NETosis. Experimental evidence suggests that NETs participate in pathogenesis of autoimmune and inflammatory disorders, with proposed involvement in glomerulonephritis, chronic lung disease, sepsis and vascular disorders. Exaggerated NETosis or diminished NET clearance likely increases risk of autoreactivity to NET components. The biological significance of NETs is just beginning to be explored. A more complete integration of NETosis within immunology and pathophysiology will require better understanding of NET properties associated with specific disease states and microbial infections. This may lead to the identification of important therapeutic targets.
Neutrophil extracellular traps; autoimmunity; neutrophils; infections
Netosis is a recently described neutrophil function that leads to the release of neutrophil extracellular traps (NETs) in response to various stimuli. NETs are filaments of decondensed chromatin associated with granular proteins. In addition to their role against microorganisms, NETs have been implicated in autoimmunity, thrombosis, and tissue injury. Access to a standardized source of isolated NETs is needed to better analyze the roles of NETs. The aim of this study was to develop a procedure yielding soluble, well-characterized NET preparations from fresh human neutrophils. The calcium ionophore A23187 was chosen to induce netosis, and the restriction enzyme AluI was used to prepare large NET fragments. DNA and proteins were detected by electrophoresis and specific labeling. Some NET proteins [histone 3, lactoferrin (LF)] were quantified by western blotting, and double-stranded DNA (dsDNA) was quantified by immunofluorescence. Co-existence of dsDNA and neutrophil proteins confirmed the quality of the NET preparations. Their biological activity was checked by measuring elastase (ELA) activity and bacterial killing against various strains. Interindividual differences in histone 3, LF, ELA, and dsDNA relative contents were observed in isolated NETs. However, the reproducibility of NET preparation and characterization was validated, suggesting that this interindividual variability was rather related to donor variation than to technical bias. This standardized protocol is suitable for producing, isolating, and quantifying functional NETs that could serve as a tool for studying NET effects on immune cells and tissues.
neutrophil; netosis; neutrophil extracellular traps; isolation; quantification; characterization; microbicidal activity
Neutrophil extracellular traps (NETs) are chromatin structures loaded with antimicrobial molecules. They can trap and kill various bacterial, fungal and protozoal pathogens, and their release is one of the first lines of defense against pathogens. In vivo, NETs are released during a form of pathogen-induced cell death, which was recently named NETosis. Ex vivo, both dead and viable neutrophils can be stimulated to release NETs composed of either nuclear or mitochondrial chromatin, respectively. In certain pathological conditions, NETs are associated with severe tissue damage or certain auto-immune diseases. This review describes the recent progress made in the identification of the mechanisms involved in NETosis and discusses its interplay with autophagy and apoptosis.
NETosis; autophagy; superoxide; NADPH oxidase; cell death
Netosis is a recently described type of neutrophil death occurring with the release to the extracellular milieu of a lattice composed of DNA associated with histones and granular and cytoplasmic proteins. These webs, initially named neutrophil extracellular traps (NETs), ensnare and kill microorganisms. Similarly, other cell types, such as eosinophils, mast cells, and macrophages, can also dye by this mechanism; thus, it was renamed as ETosis, meaning death with release of extracellular traps (ETs). Here, we review the mechanism of NETosis/etosis, emphasizing its role in diseases caused by protozoan parasites, fungi, and viruses.
Neutrophil extracellular traps (NETs) are extracellular chromatin structures that can trap and degrade microbes. They arise from neutrophils that have activated a cell death program called NET cell death, or NETosis. Activation of NETosis has been shown to involve NADPH oxidase activity, disintegration of the nuclear envelope and most granule membranes, decondensation of nuclear chromatin and formation of NETs. We report that in phorbol myristate acetate (PMA)-stimulated neutrophils, intracellular chromatin decondensation and NET formation follow autophagy and superoxide production, both of which are required to mediate PMA-induced NETosis and occur independently of each other. Neutrophils from patients with chronic granulomatous disease, which lack NADPH oxidase activity, still exhibit PMA-induced autophagy. Conversely, PMA-induced NADPH oxidase activity is not affected by pharmacological inhibition of autophagy. Interestingly, inhibition of either autophagy or NADPH oxidase prevents intracellular chromatin decondensation, which is essential for NETosis and NET formation, and results in cell death characterized by hallmarks of apoptosis. These results indicate that apoptosis might function as a backup program for NETosis when autophagy or NADPH oxidase activity is prevented.
neutrophil extracellular trap; granulocyte; chronic granulomatous disease; superoxide; autophagy; live cell imaging
Neutrophils constitute a critical part of innate immunity and are well known for their ability to phagocytose and kill invading microorganisms. The microbicidal processes employed by neutrophils are highly effective at killing most ingested bacteria and fungi. However, an alternative non-phagocytic antimicrobial mechanism of neutrophils has been proposed whereby microorganisms are eliminated by neutrophil extracellular traps (NETs). NETs are comprised of DNA, histones, and antimicrobial proteins extruded by neutrophils during NETosis, a cell death pathway reported to be distinct from apoptosis, phagocytosis-induced cell death, and necrosis. Although multiple laboratories have reported NETs using various stimuli in vitro, the molecular mechanisms involved in this process have yet to be definitively elucidated, and many questions regarding the formation and putative role or function of NETs in innate host defense remain unanswered. It is with these questions in mind that we provide some reflection and perspective on NETs and NETosis.
neutrophil; apoptosis; necrosis; phagocytosis; inflammation
Neutrophil Extracellular Traps (NETs) consist of decondensed chromatin studded with granular and some cytoplasmic proteins. They are formed by activated neutrophil granulocytes, also called polymorphonuclear leukocytes (PMN) as the result of an active cell death program, named NETosis. NETosis can be induced by a wide range of stimuli including coculture of neutrophils with pathogens (bacteria, fungi, parasites, virus particles), activated platelets, or pathogen components. The first step of the NETotic cascade is stimulation of one or several receptors followed by activation of the Raf/MEK/ERK pathway that culminates in the assembly of the multimeric NADPH oxidase complex and the production of reactive oxygen species (ROS). Later, intracellular membranes disintegrate, the granular protein Neutrophil Elastase enters the nucleus and processes core histones that also get hypercitrullinated. This leads to decondensation and mobilization of chromatin. The amount of NET formation varies with the degree of stimulation, and this is dependent on the type and concentration of the stimulus. NETs can be quantified using various methods including fluorescence microscopy or measuring DNA release. Each of these methods have specific drawbacks: analysis of fluorescence microscopy is prone to subjective variations, and DNA release does not differentiate between DNA that has been released by NETosis or by other forms of cell death. Here we present a protocol to semi-automatically quantify NET formation. It relies on the observation that anti-chromatin antibodies bind more readily to decondensed chromatin present in the nuclei of cells undergoing NETosis and in the NETs. Relating the fluorescence signals of the anti-chromatin antibody to the signals of a DNA-binding dye allows the automatic calculation of the percentage of netting neutrophils. This method does not require sophisticated microscopic equipment, and the images are quantified with a public-domain software package.
NEtosis; chromatin; immunofluorescence; segmentation; quantification
Neutrophils are the most abundant leukocytes in circulation and represent one of the first lines of defense against invading pathogens. Neutrophils possess a vast arsenal of antimicrobial proteins, which can be released from the cell by a death program termed NETosis. Neutrophil extracellular traps (NETs) are web-like structures consisting of decondensed chromatin decorated with granular and cytosolic proteins. Both exuberant NETosis and impaired clearance of NETs have been implicated in the organ damage of autoimmune diseases, such as systemic lupus erythematosus (SLE), small vessel vasculitis (SVV), and psoriasis. NETs may also represent an important source of modified autoantigens in SLE and SVV. Here, we review the autoimmune diseases linked to NETosis, with a focus on how modified proteins externalized on NETs may trigger loss of immune tolerance and promote organ damage.
neutrophil; NETs; autoimmunity; posttranslational modifications; systemic lupus erythematosus (SLE); psoriasis; vasculitis; citrullination
Neutrophil extracellular traps (NETs) are beneficial antimicrobial defense structures that can help fight against invading pathogens in the host. However, recent studies reveal that NETs exert adverse effects in a number of diseases including those of the lung. Many inflammatory lung diseases are characterized with a massive influx of neutrophils into the airways. Neutrophils contribute to the pathology of these diseases. To date, NETs have been identified in the lungs of cystic fibrosis (CF), acute lung injury (ALI), allergic asthma, and lungs infected with bacteria, virus, or fungi. These microbes and several host factors can stimulate NET formation, or NETosis. Different forms of NETosis have been identified and are dependent on varying types of stimuli. All of these pathways however appear to result in the formation of NETs that contain DNA, modified extracellular histones, proteases, and cytotoxic enzymes. Some of the NET components are immunogenic and damaging to host tissue. Innate immune collectins, such as pulmonary surfactant protein D (SP-D), bind NETs, and enhance the clearance of dying cells and DNA by alveolar macrophages. In many inflammatory lung diseases, bronchoalveolar SP-D levels are altered and its deficiency results in the accumulation of DNA in the lungs. Some of the other therapeutic molecules under consideration for treating NET-related diseases include DNases, antiproteases, myeloperoxidase (MPO) inhibitors, peptidylarginine deiminase-4 inhibitors, and anti-histone antibodies. NETs could provide important biological advantage for the host to fight against certain microbial infections. However, too much of a good thing can be a bad thing. Maintaining the right balance of NET formation and reducing the amount of NETs that accumulate in tissues are essential for harnessing the power of NETs with minimal damage to the hosts.
neutrophil extracellular traps (NETs); lung inflammation; lung infection; surfactant protein D (SP-D); cystic fibrosis (CF); acute lung injury (ALI); neutrophil
The release by neutrophils of DNA-based extracellular traps (NETs) is a recently recognized innate immune phenomenon that contributes significantly to control of bacterial pathogens at tissue foci of infection. NETs have also been implicated in the pathogenesis of non-infectious diseases such as small vessel vasculitis, lupus and cystic fibrosis lung disease. Reactive oxygen species (ROS) are important mediators of NET generation (NETosis). Neutrophils with reduced ROS production, such as those from patients with chronic granulomatous disease or myeloperoxidase (MPO) deficiency, produce fewer NETs in response to inflammatory stimuli. To better understand the roles of various ROS in NETosis, we explore the role of MPO, its substrates chloride ion (Cl−) and hydrogen peroxide (H2O2), and its product hypochlorite (HOCl) in NETosis.
In human peripheral blood neutrophils, pharmacologic inhibition of MPO decreased NETosis. Absence of extracellular Cl−, a substrate for MPO, also reduced NETosis. While exogenous addition of H2O2 and HOCl stimulated NETosis, only exogenous HOCl could rescue NETosis in the setting of MPO inhibition. Neither pharmacological inhibition nor genetic deletion of MPO in murine neutrophils blocked NETosis, in contrast to findings in human neutrophils.
Our results pinpoint HOCl as the key ROS involved in human NETosis. This finding has implications for understanding innate immune function in diseases in which Cl− homeostasis is disturbed, such as cystic fibrosis. Our results also reveal an example of significant species-specific differences in NET phenotypes, and the need for caution in extrapolation to humans from studies of murine NETosis.
Peripheral blood neutrophils form highly decondensed chromatin structures, termed neutrophil extracellular traps (NETs), that have been implicated in innate immune response to bacterial infection. Neutrophils express high levels of peptidylarginine deiminase 4 (PAD4), which catalyzes histone citrullination. However, whether PAD4 or histone citrullination plays a role in chromatin structure in neutrophils is unclear. In this study, we show that the hypercitrullination of histones by PAD4 mediates chromatin decondensation. Histone hypercitrullination is detected on highly decondensed chromatin in HL-60 granulocytes and blood neutrophils. The inhibition of PAD4 decreases histone hypercitrullination and the formation of NET-like structures, whereas PAD4 treatment of HL-60 cells facilitates these processes. The loss of heterochromatin and multilobular nuclear structures is detected in HL-60 granulocytes after PAD4 activation. Importantly, citrullination of biochemically defined avian nucleosome arrays inhibits their compaction by the linker histone H5 to form higher order chromatin structures. Together, these results suggest that histone hypercitrullination has important functions in chromatin decondensation in granulocytes/neutrophils.
Systemic lupus erythematosus (SLE) is a complex autoimmune disease characterized by an overproduction of autoantibodies. The loss of self-tolerance in SLE is believed to be caused by the dysregulation of both innate and adaptive immune systems. Neutrophils, the most abundant effector cells of innate immunity, have long been shown to be associated with SLE. However, their role in the pathogenesis of SLE was not clear until recent studies discovered abnormal regulation of neutrophil extracellular traps (NETs) in SLE patients. NETs are web-like structures composed of chromatin backbones and granular molecules. They are released by activated neutrophils through a process called “NETosis”. Nets were first described in 2004 as a novel host defense mechanism to trap and kill foreign pathogens. Recent evidence shows that NETs also participate in the pathogenesis of a variety of inflammatory and autoimmune diseases, including SLE. An imbalance between NET formation and clearance in SLE patients may play a prominent role in the perpetuation of autoimmunity and the exacerbation of disease, as well as the induction of end-organ manifestations. This review summarizes the current findings regarding the contribution of NETs to the pathogenesis of SLE.
Neutrophils; Neutrophil extracellular traps; Lupus; Pathogenesis; Autoimmune; Interferon
Peptidylarginine deiminases, or PADs, convert arginine residues to the non-ribosomally encoded amino acid citrulline in a variety of protein substrates. PAD4 is expressed in granulocytes and is essential for the formation of neutrophil extracellular traps (NETs) via PAD4-mediated histone citrullination. Citrullination of histones is thought to promote NET formation by inducing chromatin decondensation and facilitating the expulsion of chromosomal DNA that is coated with antimicrobial molecules. Numerous stimuli have been reported to lead to PAD4 activation and NET formation. However, how this signaling process proceeds and how PAD4 becomes activated in cells is largely unknown. Herein, we describe the various stimuli and signaling pathways that have been implicated in PAD4 activation and NET formation, including the role of reactive oxygen species generation. To provide a foundation for the above discussion, we first describe PAD4 structure and function, and how these studies led to the development of PAD-specific inhibitors. A comprehensive survey of the receptors and signaling pathways that regulate PAD4 activation will be important for our understanding of innate immunity, and the identification of signaling intermediates in PAD4 activation may also lead to the generation of pharmaceuticals to target NET-related pathogenesis.
PAD4; citrullination; deimination; neutrophil; NET
Neutrophils trap and kill bacteria by forming highly decondensed chromatin structures, termed neutrophil extracellular traps (NETs). We previously reported that histone hypercitrullination catalyzed by peptidylarginine deiminase 4 (PAD4) correlates with chromatin decondensation during NET formation. However, the role of PAD4 in NET-mediated bacterial trapping and killing has not been tested. Here, we use PAD4 knockout mice to show that PAD4 is essential for NET-mediated antibacterial function. Unlike PAD4+/+ neutrophils, PAD4−/− neutrophils cannot form NETs after stimulation with chemokines or incubation with bacteria, and are deficient in bacterial killing by NETs. In a mouse infectious disease model of necrotizing fasciitis, PAD4−/− mice are more susceptible to bacterial infection than PAD4+/+ mice due to a lack of NET formation. Moreover, we found that citrullination decreased the bacterial killing activity of histones and nucleosomes, which suggests that PAD4 mainly plays a role in chromatin decondensation to form NETs instead of increasing histone-mediated bacterial killing. Our results define a role for histone hypercitrullination in innate immunity during bacterial infection.
Introduction: Neutrophil extracellular trap (NET) formation was recently identified as a novel mechanism to kill pathogens. However, excessive NET formation in sepsis can injure host tissues. We have recently shown that parenteral vitamin C (VitC) is protective in sepsis. Whether VitC alters NETosis is unknown. Methods: We used Gulo−/− mice as they lack the ability to synthesize VitC. Sepsis was induced by intraperitoneal infusion of a fecal stem solution (abdominal peritonitis, FIP). Some VitC deficient Gulo−/− mice received an infusion of ascorbic acid (AscA, 200 mg/kg) 30 min after induction of FIP. NETosis was assessed histologically and by quantification for circulating free DNA (cf-DNA) in serum. Autophagy, histone citrullination, endoplasmic reticulum (ER) stress, NFκB activation and apoptosis were investigated in peritoneal PMNs. Results: Sepsis produced significant NETs in the lungs of VitC deficient Gulo−/− mice and increased circulating cf-DNA. This was attenuated in the VitC sufficient Gulo−/− mice and in VitC deficient Gulo−/− mice infused with AscA. Polymorphonuclear neutrophils (PMNs) from VitC deficient Gulo−/− mice demonstrated increased activation of ER stress, autophagy, histone citrullination, and NFκB activation, while apoptosis was inhibited. VitC also significantly attenuated PMA induced NETosis in PMNs from healthy human volunteers. Conclusions: Our in vitro and in vivo findings identify VitC as a novel regulator of NET formation in sepsis. This study complements the notion that VitC is protective in sepsis settings.
vitamin C; sepsis; neutrophils; NETosis; cell free DNA; nuclear factor κB
During an inflammatory response, neutrophils migrate to the site of infection where they can kill invading pathogens by phagocytosis, secretion of anti-microbicidal mediators or the release of neutrophil extracellular traps (NETs). NETs are specialized anti-microbial structures comprised of decondensed chromatin decorated with microbicidal agents. Increased amount of NETs have been found in patients suffering from the chronic lung inflammatory disease cystic fibrosis, correlating with increased severity of pulmonary obstruction. Furthermore, acute lung inflammation during influenza A infection is characterized by a massive influx of neutrophils into the lung. The role of NETs during virus-mediated lung inflammation is unknown. Peptidylarginine deiminase 4 (PAD4)-mediated deimination of histone H3 and H4 is required for NET formation. Therefore, we generated a PAD4-deficient mouse strain that has a striking inability to form NETs. These mice were infected with influenza A/WSN, and the disease was monitored at the level of leukocytic lung infiltration, lung pathology, viral replication, weight loss and mortality. PAD4 KO fared comparable to WT mice in all the parameters tested, but they displayed slight but statistically different weight loss kinetics during infection that was not reflected in enhanced survival. Overall, we conclude that PAD4-mediated NET formation is dispensable in a mouse model of influenza A infection.
Neutrophil extracellular traps (NETs) are made of processed chromatin bound to granular and selected cytoplasmic proteins. NETs are released by white blood cells called neutrophils, maybe as a last resort, to control microbial infections. This release of chromatin is the result of a unique form of cell death, dubbed “NETosis.” Here we review our understanding of how NETs are made, their function in infections and as danger signals, and their emerging importance in autoimmunity and coagulation.
Neutrophils are armed with both oxidant-dependent and -independent pathways for killing pathogens. Activation of the phagocyte nicotinamide adenine dinucleotide phosphate (NADPH) oxidase constitutes an emergency response to infectious threat and results in the generation of antimicrobial reactive oxidants. In addition, NADPH oxidase activation in neutrophils is linked to activation of granular proteases and generation of neutrophil extracellular traps (NETs). NETosis involves the release of nuclear and granular components that can target extracellular pathogens. NETosis is activated during microbial threat and in certain conditions mimicking sepsis, and can result in both augmented host defense and inflammatory injury. In contrast, apoptosis, the physiological form of neutrophil death, not only leads to non-inflammatory cell death but also contributes to alleviate inflammation. Although there are significant gaps in knowledge regarding the specific contribution of NETs to host defense, we speculate that the coordinated activation of NADPH oxidase and NETosis maximizes microbial killing. Work in engineered mice and limited patient experience point to varying susceptibility of bacterial and fungal pathogens to NADPH oxidase versus NET constituents. Since reactive oxidants and NET constituents can injure host tissue, it is important that these pathways be tightly regulated. Recent work supports a role for NETosis in both acute lung injury and in autoimmunity. Knowledge gained about mechanisms that modulate NETosis may lead to novel therapeutic approaches to limit inflammation-associated injury.
NETs; NADPH oxidase; neutrophils; inflammation; injury
Neutrophil extracellular traps (NETs) consist of antimicrobial molecules embedded in a web of extracellular DNA. Formation of NETs is considered to be a defense mechanism utilized by neutrophils to ensnare and kill invading pathogens, and has been recently termed NETosis. Neutrophils can be stimulated to undergo NETosis ex vivo, and are predicted to contain high levels of serine proteases, such as neutrophil elastase (NE), cathepsin G (CG) and proteinase 3 (PR3). Serine proteases are important effectors of neutrophil-mediated immunity, which function directly by degrading pathogenic virulent factors and indirectly via proteolytic activation or deactivation of cytokines, chemokines and receptors. In this study, we utilized a diverse and unbiased peptide library to detect and profile protease activity associated with NETs induced by phorbol-12-myristate-13-acetate (PMA). We obtained a “proteolytic signature” from NETs derived from healthy donor neutrophils and used proteomics to assist in the identification of the source of this proteolytic activity. In addition, we profiled each neutrophil serine protease and included the newly identified enzyme, neutrophil serine protease 4 (NSP4). Each enzyme had overlapping yet distinct endopeptidase activities and often cleaved at unique sites within the same peptide substrate. The dominant proteolytic activity in NETs was attributed to NE; however, cleavage sites corresponding to CG and PR3 activity were evident. When NE was immunodepleted, the remaining activity was attributed to CG and to a lesser extent PR3 and NSP4. Our results suggest that blocking NE activity would abrogate the major protease activity associated with NETs. In addition, the newly identified substrate specificity signatures will guide the design of more specific probes and inhibitors that target NET-associated proteases.
Neutrophils play an important role in innate immunity by defending the host organism against invading microorganisms. Antimicrobial activity of neutrophils is mediated by release of antimicrobial peptides, phagocytosis as well as formation of neutrophil extracellular traps (NET). These structures are composed of DNA, histones and granular proteins such as neutrophil elastase and myeloperoxidase. This study focused on the influence of NET on the host cell functions, particularly on human alveolar epithelial cells as the major cells responsible for gas exchange in the lung. Upon direct interaction with epithelial and endothelial cells, NET induced cytotoxic effects in a dose-dependent manner, and digestion of DNA in NET did not change NET-mediated cytotoxicity. Pre-incubation of NET with antibodies against histones, with polysialic acid or with myeloperoxidase inhibitor but not with elastase inhibitor reduced NET-mediated cytotoxicity, suggesting that histones and myeloperoxidase are responsible for NET-mediated cytotoxicity. Although activated protein C (APC) did decrease the histone-induced cytotoxicity in a purified system, it did not change NET-induced cytotoxicity, indicating that histone-dependent cytotoxicity of NET is protected against APC degradation. Moreover, in LPS-induced acute lung injury mouse model, NET formation was documented in the lung tissue as well as in the bronchoalveolar lavage fluid. These data reveal the important role of protein components in NET, particularly histones, which may lead to host cell cytotoxicity and may be involved in lung tissue destruction.
Recent evidence suggests that enhanced neutrophil extracellular trap (NET) formation activates plasmacytoid dendritic cells and serves as a source of autoantigens in SLE. We propose that aberrant NET formation is also linked to organ damage and to the premature vascular disease characteristic of human SLE. Here, we demonstrate enhanced NET formation in the New Zealand mixed 2328 (NZM) model of murine lupus. NZM mice also developed autoantibodies to NETs as well as the ortholog of human cathelicidin/LL37 (CRAMP), a molecule externalized in the NETs. NZM mice were treated with Cl-amidine, an inhibitor of peptidylarginine deiminases (PAD), to block NET formation and were evaluated for lupus-like disease activity, endothelial function, and prothrombotic phenotype. Cl-amidine treatment inhibited NZM NET formation in vivo and significantly altered circulating autoantibody profiles and complement levels while reducing glomerular IgG deposition. Further, Cl-amidine increased the differentiation capacity of bone marrow endothelial progenitor cells, improved endothelium-dependent vasorelaxation, and markedly delayed time to arterial thrombosis induced by photochemical injury. Overall, these findings suggest that PAD inhibition can modulate phenotypes crucial for lupus pathogenesis and disease activity and may represent an important strategy for mitigating cardiovascular risk in lupus patients.
There is emerging evidence that platelets are major contributors to inflammatory processes through intimate associations with innate immune cells. Here, we report that activated platelets induce the formation of neutrophil extracellular traps (NETs) in transfusion-related acute lung injury (TRALI), which is the leading cause of death after transfusion therapy. NETs are composed of decondensed chromatin decorated with granular proteins that function to trap extracellular pathogens; their formation requires the activation of neutrophils and release of their DNA in a process that may or may not result in neutrophil death. In a mouse model of TRALI that is neutrophil and platelet dependent, NETs appeared in the lung microvasculature and NET components increased in the plasma. We detected NETs in the lungs and plasma of human TRALI and in the plasma of patients with acute lung injury. In the experimental TRALI model, targeting platelet activation with either aspirin or a glycoprotein IIb/IIIa inhibitor decreased NET formation and lung injury. We then directly targeted NET components with a histone blocking antibody and DNase1, both of which protected mice from TRALI. These data suggest that NETs contribute to lung endothelial injury and that targeting NET formation may be a promising new direction for the treatment of acute lung injury.
The post translational modification of histones has significant effects on overall chromatin function. One such modification is citrullination, which is catalyzed by the protein arginine deiminases (PADs), a unique family of enzymes that catalyzes the hydrolysis of peptidyl-arginine to form peptidyl-citrulline on histones, fibrinogen, and other biologically relevant proteins. Overexpression and/or increased PAD activity is observed in several diseases, including rheumatoid arthritis, Alzheimer’s disease, multiple sclerosis, lupus, Parkinson’s disease, and cancer. This review discusses the important structural and mechanistic characteristics of the PADs, as well as recent investigations into the role of the PADs in increasing disease severity in RA and colitis and the importance of PAD activity in mediating neutrophil extracellular trap (NET) formation through chromatin decondensation. Lastly, efforts to develop PAD inhibitors with excellent potency, selectivity and in vivo efficacy are discussed, highlighting the most promising inhibitors.