Neutrophil Extracellular Traps Components Research Articles

Cooperative Action of Oxidized Low-Density Lipoproteins and Neutrophils on Endothelial Inflammatory Responses Through Neutrophil Extracellular Trap Formation.

The function of oxidatively modified low-density lipoprotein (oxLDL) in the progression of cardiovascular diseases has been extensively investigated and well-characterized with regards to the activation of multiple cellular responses in macrophages and endothelial cells. Although accumulated evidence has revealed the presence of neutrophils in vascular lesions, the effect of oxLDL on neutrophil function has not been properly investigated. In the present decade, neutrophil extracellular traps (NETs) gained immense attention not only as a primary response against pathogenic bacteria but also due to their pathological roles in tissue damage in various diseases, such as atherosclerosis and thrombosis. In this study, we investigated if oxLDL affects NET formation and if it is a risk factor for inflammatory reactions in endothelial cells. HL-60-derived neutrophils were stimulated with phorbol 12-myristate 13-acetate (PMA) for 30 min to induce NET formation, followed by incubation with 20 μg/mL native or oxidized LDL for additional 2 h. Culture media of the stimulated cells containing released NETs components were collected to evaluate NET formation by fluorometric quantitation of released DNA and detection of myeloperoxidase (MPO) by western blot analysis. NET formation of HL-60-derived neutrophils induced by PMA was significantly enhanced by additional incubation with oxLDL but not with native LDL. Treatment of HL-60-derived neutrophils with oxLDL alone in the absence of PMA did not induce NET formation. Furthermore, the culture media of HL-60-derived neutrophils after NET formation were then transferred to human aortic endothelial cell (HAECs) culture. Treatment of HAECs with the culture media containing NETs formed by HL-60-derived neutrophils increased the expression of metalloproteinase-1 protein in HAECs when HL-60-derived neutrophils were incubated with native LDL, and the expression was accelerated in the case of oxLDL. In addition, the culture media from NETs formed by HL-60-derived neutrophils caused the elongation of HAECs, which was immensely enhanced by coincubation with native LDL or oxLDL. These data suggest that oxLDL may act synergistically with neutrophils to form NETs and promote vascular endothelial inflammation.

Neutrophil Extracellular Traps

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.

Platelet-Neutrophil Interplay: Insights Into Neutrophil Extracellular Trap (NET)-Driven Coagulation in Infection.

Well established for their central role in hemostasis, platelets have increasingly been appreciated as immune cells in recent years. This emerging role should not come as a surprise as the central immune cells of invertebrates, hemocytes, are able to phagocytose, secrete soluble mediators and promote coagulation of hemolymph, blurring the line between immunity and hemostasis. The undeniable evolutionary link between coagulation and immunity becomes even clearer as the role of platelets in inflammation is better understood. Platelets exert a range of immune-related functions, many of which involve an intimate interplay with leukocytes. Platelets promote leukocyte recruitment via endothelial activation and can serve as “landing pads” for leukocytes, facilitating cellular adhesion in vascular beds devoid of classic adhesion molecules. Moreover, platelets enhance leukocyte function both through direct interactions and through release of soluble mediators. Among neutrophil-platelets interactions, the modulation of neutrophil extracellular traps (NETs) is of great interest. Platelets have been shown to induce NET formation; and, in turn, NET components further regulate platelet and neutrophil function. While NETs have been shown to ensnare and kill pathogens, they also initiate coagulation via thrombin activation. In fact, increased NET formation has been associated with hypercoagulability in septic patients as well as in chronic vascular disorders. This review will delve into current knowledge of platelet-neutrophil interactions, with a focus on NET-driven coagulation, in the context of infectious diseases. A better understanding of these mechanisms will shed a light on the therapeutic potential of uncoupling immunity and coagulation through targeting of NETs.

Quantification of NETs formation in neutrophil and its correlation with the severity of sepsis and organ dysfunction

BackgroundPrevious study from this lab has discerned oxidative, nitrosative stress and their relationship with cytokines contributing to the severity of sepsis and organ dysfunction. Cytokines are known to induce neutrophil extracellular traps (NETs) formation via free radicals generation. Hyper-activation of neutrophil leads to the increased NETs formation or ineffective clearance of NETs would likely increase the risk of auto-antibody generation against NETs components and being partly responsible for the sepsis severity and organ dysfunction. The present study was undertaken to further assess the status of NETs formation and their correlation with severity of sepsis, with the cytokines and organ dysfunction. MethodsThe level of NETs formation, DNA release, elastase release, and inflammatory cytokines was determined in 80 sepsis patients and 45 healthy volunteers. Their linearity with organ parameters and associations with sepsis severity were also assessed. ResultsNETs formation experiment was carried out and it was significantly higher in sepsis (70%) compared to control (30%). NETs % were positively correlated with severity of sepsis and organ dysfunction. Pearson's correlation coefficient demonstrated a direct relation between NETs components and organ parameters with Sepsis severity scores. ConclusionNETs formation is significantly higher due to which it is contributing to the sepsis severity and organ failure.

Triggering NETosis via protease-activated receptor (PAR)-2 signaling as a mechanism of hijacking neutrophils function for pathogen benefits.

Neutrophil-derived networks of DNA-composed extracellular fibers covered with antimicrobial molecules, referred to as neutrophil extracellular traps (NETs), are recognized as a physiological microbicidal mechanism of innate immunity. The formation of NETs is also classified as a model of a cell death called NETosis. Despite intensive research on the NETs formation in response to pathogens, the role of specific bacteria-derived virulence factors in this process, although postulated, is still poorly understood. The aim of our study was to determine the role of gingipains, cysteine proteases responsible for the virulence of P. gingivalis, on the NETosis process induced by this major periodontopathogen. We showed that NETosis triggered by P. gingivalis is gingipain dependent since in the stark contrast to the wild-type strain (W83) the gingipain-null mutant strain only slightly induced the NETs formation. Furthermore, the direct effect of proteases on NETosis was documented using purified gingipains. Notably, the induction of NETosis was dependent on the catalytic activity of gingipains, since proteolytically inactive forms of enzymes showed reduced ability to trigger the NETs formation. Mechanistically, gingipain-induced NETosis was dependent on proteolytic activation of protease-activated receptor-2 (PAR-2). Intriguingly, both P. gingivalis and purified Arg-specific gingipains (Rgp) induced NETs that not only lacked bactericidal activity but instead stimulated the growth of bacteria species otherwise susceptible to killing in NETs. This protection was executed by proteolysis of bactericidal components of NETs. Taken together, gingipains play a dual role in NETosis: they are the potent direct inducers of NETs formation but in the same time, their activity prevents P. gingivalis entrapment and subsequent killing. This may explain a paradox that despite the massive accumulation of neutrophils and NETs formation in periodontal pockets periodontal pathogens and associated pathobionts thrive in this environment.

UVA and UVB radiation induce the formation of neutrophil extracellular traps by human polymorphonuclear cells

Prolonged exposure of the skin to ultraviolet radiation (UV) leads to its damage and loss of protective properties. This condition called photoaging of the skin is caused by a number of destructive factors, such as reactive oxygen species (ROS) and proteolytic enzymes that cause damage to the extracellular matrix, e.g. collagen fibers. Many cells of the immune system, including neutrophils, are involved in the photoaging process. The presence of neutrophils in the skin exposed to UV irradiation is known; however, the mechanism of neutrophil activity at these conditions remains unclear.In our study, we focused on the ability of neutrophils to release neutrophil extracellular traps (NETs) and the role of these structures in the photoaging process. NET release occurs in response to various stimuli; however, we hereby showed that the UVA and UVB radiation that reaches the Earth's surface could activate the mechanism of netosis. UV-induced netosis was much faster than that activated by chemical or biological factors; however, it also occurred due to the production of ROS, known signal mediators in netosis. In this work, we also identified the probable netosis signaling pathway involved in the neutrophil response to UV.The participation of NET components may explain the ongoing process of skin photoaging, but it is also important to indicate netosis as a potential target for skin protection therapy. Antioxidants tested in this work, such as N-acetylcysteine, ethamsylate, as well as vitamin B1 (thiamine), can successfully inhibit UV-induced netosis, and thus be used as protective components against the negative effects of solar radiation.

Extracellular DNA, Neutrophil Extracellular Traps, and Inflammasome Activation in Severe Asthma.

Rationale: Extracellular DNA (eDNA) and neutrophil extracellular traps (NETs) are implicated in multiple inflammatory diseases. NETs mediate inflammasome activation and IL-1β secretion from monocytes and cause airway epithelial cell injury, but the role of eDNA, NETs, and IL-1β in asthma is uncertain. Objectives: To characterize the role of activated neutrophils in severe asthma through measurement of NETs and inflammasome activation. Methods: We measured sputum eDNA in induced sputum from 399 patients with asthma in the Severe Asthma Research Program-3 and in 94 healthy control subjects. We subdivided subjects with asthma into eDNA-low and -high subgroups to compare outcomes of asthma severity and of neutrophil and inflammasome activation. We also examined if NETs cause airway epithelial cell damage that can be prevented by DNase. Measurements and Main Results: We found that 13% of the Severe Asthma Research Program-3 cohort is "eDNA-high," as defined by sputum eDNA concentrations above the upper 95th percentile value in health. Compared with eDNA-low patients with asthma, eDNA-high patients had lower Asthma Control Test scores, frequent history of chronic mucus hypersecretion, and frequent use of oral corticosteroids for maintenance of asthma control (all P values <0.05). Sputum eDNA in asthma was associated with airway neutrophilic inflammation, increases in soluble NET components, and increases in caspase 1 activity and IL-1β (all P values <0.001). In in vitro studies, NETs caused cytotoxicity in airway epithelial cells that was prevented by disruption of NETs with DNase. Conclusions: High extracellular DNA concentrations in sputum mark a subset of patients with more severe asthma who have NETs and markers of inflammasome activation in their airways.

013 NETs generate structured antimicrobial peptide-nucleosome immune complexes with inter-DNA spacings optimal for TLR9 activation

The molecular pathogenesis of psoriasis is characterized in part by breakdown of immune tolerance to self-DNA. Recent work demonstrates that the human antimicrobial peptide (AMP) LL37 overexpressed in psoriasis organizes naked DNA into periodic nanocrystals to potently hyperactivate Toll-like receptor 9 (TLR9) in plasmacytoid dendritic cells (pDCs). Interestingly, a subset of self-DNA in psoriatic lesions remain bound to histones as histone-DNA nucleosome core particles (NCPs) released from neutrophil extracellular traps (NETs). At present, it is unknown how NET components like AMPs interact with NCPs, and whether AMP-NCP complexes form structures compatible with TLR9 activation in psoriasis. Here, we combine synchrotron X-ray scattering, cryo-electron microscopy, and computer simulations to demonstrate that under a broad range of conditions, NCPs stack into columns that present periodically arranged dsDNA ligands like threads on a screw, allowing for optimal interdigitation with clusters of TLR9. Remarkably, simulations and electron microscopy indicate that the superhelical pitch of DNA wrapped around the NCP column relaxes to a value that is well-matched with the steric size of TLR9, which predicts strong immune activation. Taken together, our results suggest that AMPs can remodel the structural organization of nucleosomes from NETs into potent amplifiers of inflammation. Preliminary immune activation experiments will be presented.

79 Burn Injury Alters Pulmonary Neutrophil Extracellular Trap (NET) Formation

It is well known that burn patients suffer from serious lung complications such as acute respiratory distress syndrome, acute lung injury and pneumonia. However, the reasons for the development of these conditions after burn injury (BI) are not clearly understood. Several studies have been examined the importance of macrophages after burn injury. However, the involvement of neutrophils in lung complications after BI has not been studied in detail. Particularly, the importance of recently identified neutrophil extracellular traps (NETs) in the lungs after BI is unknown. NETs are considered to help trap infectious agents to protect the host; however, excess NETs could damage and destroy the airways and cause lung dysfunction. We established an in vivo model to study NETosis in the lungs after BI using an LPS model and investigated neutrophil behavior in burn injured individuals with/without LPS-mediated pneumonia. We have first created a 15% body burn, and instilled various amounts of LPS (0–50 µg/kg) into the airways of BALB/c mice. After various time points (30mins, 3 h, 16 h, 1-7day), we have collected bronchoalveolar lavage (BAL) fluid, and lung tissues. Immune cells present in the BAL fluid were deposited on slides by Cytospin preparations, stained and quantified by microscopy. DNA-protein complexes present in the BAL supernatant and lung tissues were analyzed by agarose gel electrophoresis and PicoGreen assays. Presence of a NET marker, citrullinated histone, and further quantification of neutrophils with MPO in the lung, were analyzed by Western blots and immunohistochemistry. The data obtained from these studies show that neutrophils are not detectable in the airways under baseline or after BI; however, different numbers of neutrophils and amounts of NETs were present under various experimental conditions and time points-post BI. These data show that we could measure NET components in the airways of mice instilled with LPS after BI. We have successfully established a mouse model to study pulmonary NETosis in BI, and optimized the range of LPS concentrations and time points necessary to observe differences in NETosis under various experimental conditions. BI reduces the LPS-mediated pulmonary innate immune response. A decrease in innate immune response may be responsible for the increase in pneumonia incidence in patients with BI. This model should help understand the roles of NETs in pulmonary dysfunction after BI, and for testing potential drugs for correcting NET-mediated lung complications.

Neutrophil Extracellular Traps as a Drug Target to Counteract Chronic and Acute Inflammation.

Neutrophil extracellular traps (NET), extruded decondensated chromatin entangled with neutrophil proteases, have been first identified in neutrophils stimulated with bacteria or phorbol myristate acetate (PMA) via activation of NADPH oxidase and the generation of reactive oxygen species. Although the first findings demonstrated the beneficial role of NET formation by trapping the bacteria and limiting their dissemination, numerous studies in the recent decade revealed the multifunctional aspects of NET formation which manifests itself not only in the context of anti-microbial effect but also as a pathological trigger. Uncontrolled and exaggerated NET formation or inability to digest and remove NET have been reported in thrombosis, auto-immune diseases, cancer or even in infertility. Studies are ongoing to disclose the role of NET in different pathological situations and most importantly, NET regulation via compounds that either interfere with NET formation or target NET components such as DNA or neutrophil proteases. Although the final product of NET formation seems to be quite common i.e. DNA entangled with proteases, stimuli that induce NET have a wide range of varieties and the involved pathways are diverse too. Therefore, in every pathological condition, it is necessary to consider carefully the type of stimulus and the signaling pathways in order to target the disease more specifically. Here we briefly summarize some (out of many) NET triggers/pathways and discuss the potential interventions in the pathological situations.

Neutrophil extracellular trap components increase the expression of coagulation factors.

Neutrophil extracellular traps (NETs) represent an important link between inflammation and thrombosis. Here, the present study aimed to investigate the influence of the NET components, DNA and histone H4, on hemostatic gene expression. A further aim was to confirm the influence of H4 on the expression of tissue factor (TF) and investigate a potential effect of DNA, and to test the involvement of miR-17/92 and its paralog miR-106b-25 in this regulation. In HepG2 cells, the mRNA levels of factor VII and factor XII, which are crucial in the activation of the coagulation cascade, and of serpin family F member 2 (encoding α2-antiplasmin) were significantly upregulated by DNA and H4; while the mRNA levels of factor V, which is essential for thrombin generation of protein S, a cofactor of protein C that also has the ability to inhibit the factor X activation pathway, and of serpin family C member 1 (encoding antithrombin, the main endogenous anticoagulant) were significantly upregulated only by H4. H4 and DNA also provoked an increase in hepatocyte nuclear factor 4α (HNF4A) mRNA expression that could be responsible for the increase in the expression of certain coagulant factors. In THP-1 cells, it was also demonstrated that H4 caused an increase in TF mRNA while decreasing several of the microRNAs (miRNA/miRs) of the cluster miR-17/92, which may in part explain the increase in the expression of TF. The present results suggest the ability of NET components to alter the hemostatic balance and a possible involvement of HNF4α and miRNAs in this regulation.

Neutrophil Activation and Early Features of NET Formation Are Associated With Dengue Virus Infection in Human.

The involvement of the immune system in the protection and pathology of natural dengue virus (DENV) has been extensively studied. However, despite studies that have referred to activation of neutrophils in DENV infections, the exact roles of neutrophils remain elusive. Here, we explored the phenotypic and functional responses of neutrophils in a cohort of adult dengue patients. Results indicated that during an acute DENV infection, neutrophils up-regulate CD66b expression, and produce a more robust respiratory response as compared with that in convalescent or healthy individuals; this confirmed in vivo neutrophil activation during DENV infection. Spontaneous decondensation of nuclei, an early event of neutrophil extracellular trap (NET) formation, was also markedly increased in cells isolated from DENV-infected patients during the acute phase of the infection. In vitro incubation of NETs with DENV-2 virus significantly decreased DENV infectivity. Interestingly, increased levels of NET components were found in the serum of patients with more severe disease form—dengue hemorrhagic fever (DHF), but not uncomplicated dengue fever, during the acute phase of the infection. Levels of pro-inflammatory cytokines IL-8 and TNFα were also increased in DHF patients as compared with those in healthy and DF subjects. This suggested that NETs may play dual roles during DENV infection. The increased ability for NET formation during acute DENV infection appeared to be independent of PAD4-mediated histone H3 hyper-citrullination. Our study suggests that neutrophils are involved in immunological responses to DENV infection.

Systemic levels of anti-PAD4 autoantibodies correlate with airway obstruction in cystic fibrosis

Cystic fibrosis (CF) airway disease is characterized by the long-term presence of neutrophil granulocytes. Formation of neutrophil extracellular traps (NETs) and/or autoantibodies directed against extracellular components of NETs are possible contributors to neutrophil-mediated lung damage in CF. The goal of this study was to measure their levels in CF adults compared to healthy controls and subjects with rheumatologic diseases known to develop NET-related autoantibodies and pathologies, rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE). Sera were analyzed from the following number of subjects: 37 CF, 23 healthy controls (HC), 20 RA, and 21 SLE. CF had elevated serum myeloperoxidase (MPO) concentrations (347.5±56.1 ng/ml, mean+/-S.E.M., p = .0132) compared to HC (144.5±14.6 ng/ml) but not of neutrophil elastase (NE) complexed with alpha-1-antitrypsin, cell-free DNA or NE-DNA complexes. The peptidylarginine deiminase 4 (PAD4) enzyme is required for NET formation and associated DNA release in neutrophils. Serum levels of anti-PAD4 antibodies (Ab) were elevated in CF (p = .0147) compared to HC and showed an inverse correlation with a measure of lung function, FEV1% predicted (r = −0.5020, p = .015), as did MPO levels (r = −0.4801, p = .0026). Anti-PAD4 Ab levels in CF sera associated with lung infection by P. aeruginosa, but not that by S. aureus, age, sex, CF-related diabetes or the presence of musculoskeletal pain. Serum levels of anti-citrullinated protein Abs (ACPAs) and anti-nucleosome Abs were not elevated in CF compared to HC (p = .7498, p = .0678). In summary, adult CF subjects develop an autoimmune response against NET components that correlates with worsening lung disease.

Antibody Stabilization of Neutrophil Extracellular Trap-Platelet Factor 4 Complexes Is Therapeutic in a Murine Model of Endotoxemia

Sepsis is characterized by life-threatening organ dysfunction caused by a dysregulated response to infection. Neutrophils play a crucial role in sepsis during which they release neutrophil extracellular traps (NETs), webs of negatively charged cell-free DNA (cfDNA) complexed with positively-charged histones that kill pathogens but also damage host tissue. While it has been proposed that NET digestion may be beneficial in the treatment of sepsis, it is also possible that this strategy leads to the release of harmful NET degradation products (NDPs), such as cfDNA, histones, and myeloperoxidase (MPO) that cause endothelial damage. An alternative approach would be treatment strategies that prevent NET lysis and the release of NDPs. We posited that NET-bound platelet factor 4 (PF4, CXCL4) may have this effect. PF4 is a highly-positively charged, platelet-specific chemokine released in high concentrations following platelet activation and aggregates polyanionic molecules like heparin, polyphosphates, and DNA. We have found that PF4 causes physical compaction of NETs without inducing histone release. We studied the effect of this compaction using an in vitro microfluidic assay in which NETs released from neutrophils stimulated by tissue-necrosis factor (TNF) α were infused over a layer of cultured human umbilical vein endothelial cells (HUVECs) ± PF4 (25 μg/ml) and incubated for 12 hours. Channels exposed to PF4 were “heathier” with significantly more residual attached endothelial cells (44±24 vs. 150±29 cells/hpf, n=6, p=0.0002). We have previously shown that mice that overexpress PF4 are protected from mortality in lipopolysaccharide (LPS) endotoxemia. Compared to wildtype animals, PF4 null mice have increased circulating levels of NET markers including cfDNA, citrullinated histones, and MPO. Treatment with exogenous PF4 leads to a decrease in plasma levels of NET components and a reduction in mortality. In a microfluidic assay in which channel-adherent NETs were treated with increasing concentrations of PF4 (0-25 µg/ml) and then infused with DNase I (100 U/ml), we found that compacted NET-PF4 complexes become resistant to DNase I digestion. Prior studies have revealed that when PF4 binds to polyanionic molecules, it exposes HIT-like antigenic sites. We confirmed that the NET-PF4 complexes similarly bind HIT IgG isolated from clinical samples and the HIT-like monoclonal antibody KKO. Interestingly, KKO binding further enhanced DNase I resistance, a phenomenon not seen with a polyclonal anti-PF4 antibody (abcam). We hypothesized that KKO which causes PF4 oligomerization unlike the polyclonal antibody, further stabilized the crosslinked NET-PF4 complexes, providing additional protection from DNase I digestion and preventing the release of NDPs. We then asked whether KKO may serve as a targeted therapy in the treatment of sepsis, enhancing NET-PF4 complex resistance to nuclease digestion and leading to the sequestration of NDPs. As KKO is an IgG2bk antibody, we were unable to use pepsin digestion to remove the Fc region that causes platelet activation. We therefore used an IgG-specific endoglycosidase to develop a deglycosylated version of KKO (DG-KKO) that retains the ability to bind to NET-PF4 complexes (data not shown), but has a reduced capacity to interact with hematopoietic cell Fc receptors. DG-KKO has a markedly decreased ability to activate human platelets compared to KKO in the presence of added PF4 as measured by P-selectin level (3710±140 control vs. 1580±300 treated MFI, n = 2). Compared to control, DG-KKO infusion in the murine endotoxemia model prevented thrombocytopenia (mean platelet count 286±16 control vs. 537±69 treated X103/µl, n=4-5, p=0.016), the release of cfDNA (8.5±0.3 vs. 3.7±0.7 µg/ml, n=4-5, p=0.016), and the emission of MPO-DNA complexes (234±14 vs. 99±5 % increase from non-LPS injected controls, n=4-5, p=0.016) As depicted in Figure 1, we propose that in sepsis, NETosis occurs (Step 1) causing the release of harmful NDPs (Step 2). PF4 expelled from activated platelets stabilizes PF4-NET complexes (Step 3). Infused DG-KKO enhances NET stability and decreases release of NDPs following DNase I digestion (Step 4). These studies provide mechanistic insights into the release of NDPs during endotoxemia and offer a targeted, novel therapeutic to prevent their contribution to inflammatory states such as sepsis. [Display omitted] DisclosuresPoncz:Incyte Corporation: Consultancy, Research Funding.

A Comparative Review of Neutrophil Extracellular Traps in Sepsis.

Sepsis is the leading cause of critical illness and mortality in human beings and animals. Neutrophils are the primary effector cells of innate immunity during sepsis. Besides degranulation and phagocytosis, neutrophils also release neutrophil extracellular traps (NETs), composed of cell-free DNA, histones, and antimicrobial proteins. Although NETs have protective roles in the initial stages of sepsis, excessive NET formation has been found to induce thrombosis and multiple organ failure in murine sepsis models. Since the discovery of NETs nearly a decade ago, many investigators have identified NETs in various species. However, many questions remain regarding the exact mechanisms and fate of neutrophils following NET formation. In humans and mice, platelet-neutrophil interactions via direct binding or soluble mediators seem to play an important role in mediating NET formation during sepsis. Preliminary data suggest that these interactions may be species dependent. Regardless of these differences, there is increasing evidence in human and veterinary medicine suggesting that NETs play a crucial role in the pathogenesis of intravascular thrombosis and multiple organ failure in sepsis. Because the outcome of sepsis is highly dependent on early recognition and intervention, detection of NETs or NET components can aid in the diagnosis of sepsis in humans and veterinary species. In addition, the use of novel therapies such as deoxyribonuclease and non-anticoagulant heparin to target NET components shows promising results in murine septic models. Much work is needed in translating these NET-targeting therapies to clinical practice.

Hydrogen sulfide primes diabetic wound to close through inhibition of NETosis

Diabetes-induced neutrophil NETosis impairs wound healing through neutrophil extracellular traps (NETs). Reactive oxygen species (ROS)-triggered activation of mitogen-activated protein kinase (MAPK) ERK1/2 and p38 is involved in NETosis. Hydrogen sulfide (H2S), an endogenous signaling molecule, accelerates diabetic wound healing (DWH), and inhibits ROS production, ERK1/2 and p38 activation, while its level is decreased in diabetes. However, it remains unknown whether H2S could accelerate DWH through inhibition of NETosis, and whether this inhibitory effect was associated with blockage of ROS-induced ERK1/2 and p38 activation. In order to solve these problems, serum NETs content was measured in diabetic foot patients and healthy individuals. Wound was created in dorsal skin of LepRdb/db and control mice and NETs content in wound tissues was tested. An in vitro NETosis model was induced by phorbol 12-myristate 13-acetate (PMA) in isolated neutrophils. Effects of H2S in form of Na2S on skin wound healing and NETosis were investigated both in vivo and in vitro. It was found that NETs level was highly increased in diabetic foot patients. Comparing with LepRm+/db mice, DWH was delayed in LepRdb/db mice, accompanied with high NETs level. In PMA-induced NETosis model, peptidylarginine deiminase (PAD)-4 and citrullinated histone H3, as well as NETs components dsDNA framework, myeloperoxidase and neutrophil elastase, were significantly increased. PMA-induced neutrophil NETosis and NETs formation were abolished by treatment with H2S. The delayed DWH of diabetic mice was partially restored by intraperitoneal injection of H2S, meanwhile, the highly expressed NETosis and NETs release were also down-regulated. The treatment with H2S not only attenuated ROS production but also abolished MAPK ERK1/2 and p38 activation. Like the effects of H2S, inhibition of MAPK ERK1/2 or p38 could decrease NETs release. These findings suggests that H2S attenuates NETosis and primes diabetic wound to heal through blockage of ROS-mediated MAPK ERK1/2 and p38 activation.

Elastase inhibition affects collagen transcription and prostaglandin secretion in mare endometrium during the estrous cycle.

We have shown that bacteria induce neutrophil extracellular traps (NETs) in mare endometrium. Besides killing pathogens, NETs may contribute for endometrosis (chronic endometrium fibrosis). Since elastase (ELA) is a NETs component that regulates fibrosis and prostaglandin (PG) output, the aim was to evaluate if inhibition of ELA would affect collagen 1 (COL1) transcription and PGs secretion by endometrium explants, in different estrous cycle phases. Follicular-FP (n=8) and mid luteal-MLP (n=7) phases explants were cultured for 24-48hr with medium alone (Control), ELA (0.5μg/ml,1μg/ml), sivelestat - ELA inhibitor (INH,10μg/ml), or ELA (0.5μg/ml,1μg/ml)+INH (10μg/ml). COL1 gene transcription was done by qRT-PCR and PGE2 and PGF2 α determination in culture medium by EIA. In FP, at 24hr, ELA0.5 increased COL1 transcription (p<0.001) but its inhibition (ELA0.5+INH10) decreased COL1 transcription (p<0.01) and PGF2 α production (p<0.05). Also, ELA0.5+INH10 or ELA1+INH10 raised PGE2 production (p<0.01). At 48hr, ELA1 increased COL1 transcription (p<0.01) and PGF2 α production (p<0.001), but its inhibition (ELA1+INH10) decreased these actions (p<0.01; p<0.05, respectively). Besides, ELA1+INH10 incubation increased PGE2 (p<0.05). PGF2 α also augmented with ELA0.5 (p<0.001), but lowered with ELA0.5+INH10 (p<0.01). In MLP, ELA0.5 up-regulated COL1 transcription (24hr, p<0.01; 48hr, p<0.001), but ELA0.5+INH10 decreased it (24hr, p<0.05; 48hr, p<0.001). At 48hr, incubation with ELA1 also increased COL1 transcription and PGF2 α production (p<0.05), but PGF2 α production decreased with ELA1+INH10 incubation (p<0.05). PGE2 production was higher in ELA1+INH10 incubation (p<0.05). Therefore, ELA inhibition may reduce the establishment of mare endometrial fibrosis by stimulating the production of anti-fibrotic PGE2 and inhibiting pro-fibrotic PGF2 α.

Neutrophil extracellular traps induce aggregation of washed human platelets independently of extracellular DNA and histones

BackgroundThe 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.MethodsIsolated 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.ResultsCell-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 αIIbβ3 receptors.ConclusionsIn 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.

399 Establishing an in Vivo Model to Study Pulmonary Neutrophil Extracellular Trap (NET) Formation After Burn Injury

It is well known that burn patients suffer from serious lung complications such as acute respiratory distress syndrome, acute lung injury and pneumonia. However, the reasons for the development of these conditions after burn injury (BI) are not clearly understood. Several studies examined the importance of macrophages after burn injury. However, the involvement of neutrophils in lung complications after BI has not been studied in detail. Particularly, the importance of recently identified neutrophil extracellular traps (NETs) in the lungs after BI is unknown. NETs are considered to help trap infectious agents to protect the host; however, excess NETs could damage and destroy the airways and cause lung dysfunction. There are no good animal models available to study the pulmonary NETs during BI. Therefore, we established an in vivo model to study NETosis in the lungs after BI using an LPS model. We have first created a 15% body burn, and instilled various amounts of LPS (0–50 mg/kg) into the airways of C57B/6 mice. After various time points (3 h, 16 h, 24 h, 48 h, 72 h) we have collected bronchoalveolar lavage (BAL) fluid and blood samples. Immune cells present in the BAL fluid were deposited on slides by Cytospin preparations, stained and quantified by microscopy. Cell and platelet counts in the blood samples were determined by an automated cell counter, and confirmed by blood smears, H and E staining and microscopy. DNA-protein complexes present in the BAL supernatant were analyzed by agarose gel electrophoresis and pocigreen assays. Presence of a NET marker, citrullinated histone, was analyzed by Western blots. The data obtained from these studies show that neutrophils are not detectable in the airways under baseline or after BI; however, different numbers of neutrophils and amounts of NETs were present under various experimental conditions and time points-post BI. These data show that we could measure NET components in the airways of mice instilled with LPS after BI. Blood analyses show that cells concentrations also differ among various experimental conditions, indicating the importance of neutrophil and NET-promoting components (e.g., platelets) in the blood after BI and/or LPS instillation. We have successfully established a mouse model to study pulmonary NETosis in BI, and optimized the range of LPS concentrations and time points necessary to observe differences in NETosis under various experimental conditions. This model should help to understand the roles of NETs in pulmonary dysfunction after BI, and for testing potential drugs for correcting NET-mediated lung complications.

The antidiabetic drug metformin blunts NETosis in vitro and reduces circulating NETosis biomarkers in vivo.

Diabetes is associated with an excess release of neutrophil extracellular traps (NETs) and an enhanced NETosis, a neutrophil cell death programme instrumental to anti-microbial defences, but also involved in tissue damage. We herein investigated whether the antidiabetic drug metformin protects against NETosis. We measured NET components in the plasma of patients with pre-diabetes who were randomized to receive metformin or placebo for 2months. To control for the effect on glucose, we also measured NET components in the plasma of patients with type 2 diabetes before and after treatment with insulin or dapagliflozin. In vitro, we used static and dynamic imaging with advanced live confocal two-photon microscopy to evaluate the effects of metformin on cellular events during NETosis. We examined putative molecular mechanisms by monitoring chromatin decondensation and DNA release in vitro. Metformin, as compared to placebo, significantly reduced the concentrations of NET components elastase, proteinase-3, histones and double strand DNA, whereas glucose control with insulin or dapagliflozin exerted no significant effect. In vitro, metformin prevented pathologic changes in nuclear dynamics and DNA release, resulting in a blunted NETosis in response to phorbol myristate acetate and calcium influx. Metformin prevented membrane translocation of PKC-βII and activation of NADPH oxidase in neutrophils, both of which diminished the NETosis response. Metformin treatment reduced the concentrations of NET components independently from glucose control. This effect was reproducible in vitro and was related to the inhibitory effect exerted by metformin on the PKC-NADPH oxidase pathway.