Inhibition Of Neutrophil Extracellular Traps Formation Research Articles

Midkine drives cardiac inflammation by promoting neutrophil trafficking and NETosis in myocarditis.

Heart failure due to dilated cardiomyopathy is frequently caused by myocarditis. However, the pathogenesis of myocarditis remains incompletely understood. Here, we report the presence of neutrophil extracellular traps (NETs) in cardiac tissue of patients and mice with myocarditis. Inhibition of NET formation in experimental autoimmune myocarditis (EAM) of mice substantially reduces inflammation in the acute phase of the disease. Targeting the cytokine midkine (MK), which mediates NET formation in vitro, not only attenuates NET formation in vivo and the infiltration of polymorphonuclear neutrophils (PMNs) but also reduces fibrosis and preserves systolic function during EAM. Low-density lipoprotein receptor-related protein 1 (LRP1) acts as the functionally relevant receptor for MK-induced PMN recruitment as well as NET formation. In summary, NETosis substantially contributes to the pathogenesis of myocarditis and drives cardiac inflammation, probably via MK, which promotes PMN trafficking and NETosis. Thus, MK as well as NETs may represent novel therapeutic targets for the treatment of cardiac inflammation.

A Sampling of Highlights from the Literature

Tumor cells select for weak MHCII binding (by Wykis via Wikimedia Commons)Antitumor responses to neoantigens has largely focused on CD8+ T cells. However, CD4+ T-cell responses also contribute, and how this impacts the tumor mutational landscape has not been well documented. Marty and colleagues show that mutations with weak MHCII binding are strongly selected during tumor progression. Using TCGA datasets representing more than 5,000 tumor samples, a score linking MHCII genotype to presentation of specific mutations was generated and shown to reflect the mutated epitopes that are presented to immune cells. The analysis reveals not only that peptides binding poorly to MHCII are selected, but also that the ability to bind to MHCII has more influence on the selective pressure put on tumors than MHCI binding. These results highlight how CD4+ T cells may be playing a key role in shaping antitumor immunity.Marty R, …, Carter H. Cell 2018 Sep 20. doi: 10.1016/j.cell.2018.08.048.ALCAM-specific homing allows T cells to better infiltrate (by RRZEicons via Wikimedia Commons)T-cell infiltration into brain tumors remains a challenge. Samaha and colleagues demonstrate that tumor endothelium, as a mechanism of immune evasion, downregulates specific adhesion molecules commonly upregulated by inflammation and needed by immune cells for extravasation. However, the endothelium also upregulates the adhesion molecule, ALCAM, and this was used to develop a high-avidity ALCAM-specific homing system that allows circulating T cells to better adhere to cancer tissues, resulting in improved cytotoxic T-cell infiltration and potent antitumor responses.Samaha H, …, Ahmed N. Nature 2018 Sep 5;561:331–7.Autophagy in macrophages (green indicates LC3; by Kam23lesh via Wikimedia Commons)The role of autophagy proteins in tumors and the tumor microenvironment have not yet been fully elucidated. Cunha and colleagues show myeloid cell LC3-associated phagocytosis (LAP), which functions in engulfment of dying cells, contributes to immune suppression in the tumor microenvironment. Targeting LAP, which recruits components of the autophagy machinery, specifically in myeloid cells inhibits tumor progression. Tumor-associated macrophages deficient for LAP lose suppressive functions and gain a pro-inflammatory M1 phenotype, promoting T-cell antitumor activity. These data highlight that autophagy proteins can play a role in the suppression of antitumor responses in the tumor microenvironment.Cunha LD,…, Green DR. Cell 2018 Sep 20. doi: 10.1016/j.cell.2018.08.061.Disseminated cancer cells can enter into dormancy or proliferate causing recurrence (by AliMicJones via Wikimedia).Recurrence of many cancers occurs from activation of dormant tumor cells. The mechanisms behind awakening disseminated cancer from dormancy to cause metastasis are not fully known. Albrengues and colleagues show that chronic inflammatory conditions produce neutrophil extracellular traps (NETs) that result in remodeling laminin and integrin signaling that awakens dormant single disseminated cancer cells and metastases in both breast and prostate cancer models. By inhibiting NET formation, or by degrading the DNA scaffold in NETs, dormant cancer cells do not reactivate and metastasis is prevented. This highlights how targeting NETs could aid in the prevention of cancer recurrence in patients.Albrengues J, …, Egeblad M. Science 2018 Sep 28;361:eaao4227.Triple negative breast carcinoma (by Sarahkayb via Wikimedia Commons)Interactions occurring in the tumor microenvironment are complex. Using a multiplexed ion beam imaging system that detects 36 proteins with roles in cell phenotype, function, and regulation, Keren and colleagues analyzed the composition of the tumor microenvironment (TME) in triple-negative breast cancer samples. Coupling the imaging to machine learning allowed determination of the cellular composition, cell localization and spatial relationships to other cell types, and functionality across 41 patients. Immune composition could be classified as compartmentalized or mixed, with compartmentalization at the tumor border having a better prognoses. This analysis not only revealed a structured TME but also allows for characterizing the complex and dynamic interactions taking place in the TME, providing a deeper understanding of how tumor composition can affect prognosis.Keren L, …, Angelo M. Cell 2018 Sep 6;174:1373–87.e19.Gene expression affects what protein is expressed, as well as its function (by OldakQuill via Wikimedia Commons)CD19-negative acute lymphoblastic leukemia (ALL) relapse can occur with CAR T-cell therapy. Orlando and colleagues demonstrate that CAR-resistant tumor cells usually possess CD19 mutations that lead to a truncated protein leading to CD19 loss on the surface of ALL cells, rather than epitope loss as previously reported. This finding suggests concurrently targeting additional molecules such as CD22 should improve CD19 CAR T cell treatment.Orlando EJ, …, Winckler W. Nat Med 2018 Oct 1. DOI: 10.1038/s41591-018-0146-z.

Neutrophil accumulation and NET release contribute to thrombosis in HIT.

Heparin-induced thrombocytopenia (HIT) is an immune-mediated thrombocytopenic disorder associated with a severe prothrombotic state. We investigated whether neutrophils and neutrophil extracellular traps (NETs) contribute to the development of thrombosis in HIT. Using an endothelialized microfluidic system and a murine passive immunization model, we show that HIT induction leads to increased neutrophil adherence to venous endothelium. In HIT mice, endothelial adherence is enhanced immediately downstream of nascent venous thrombi, after which neutrophils undergo retrograde migration via a CXCR2-dependent mechanism to accumulate into the thrombi. Using a microfluidic system, we found that PF4 binds to NETs, leading them to become compact and DNase resistant. PF4-NET complexes selectively bind HIT antibodies, which further protect them from nuclease digestion. In HIT mice, inhibition of NET formation through Padi4 gene disruption or DNase treatment limited venous thrombus size. PAD4 inactivation did affect arterial thrombi or severity of thrombocytopenia in HIT. Thus, neutrophil activation contributes to the development of venous thrombosis in HIT by enhancing neutrophil-endothelial adhesion and neutrophil clot infiltration, where incorporated PF4-NET-HIT antibody complexes lead to thrombosis propagation. Inhibition of neutrophil endothelial adhesion, prevention of neutrophil chemokine-dependent recruitment of neutrophils to thrombi, or suppression of NET release should be explored as strategies to prevent venous thrombosis in HIT.

Hypoxia increases neutrophil-driven matrix destruction after exposure to Mycobacterium tuberculosis

The importance of neutrophils in the pathology of tuberculosis (TB) has been recently established. We demonstrated that TB lesions in man are hypoxic, but how neutrophils in hypoxia influence lung tissue damage is unknown. We investigated the effect of hypoxia on neutrophil-derived enzymes and tissue destruction in TB. Human neutrophils were stimulated with M. tuberculosis (M.tb) or conditioned media from M.tb-infected monocytes (CoMTB). Neutrophil matrix metalloproteinase-8/-9 and elastase secretion were analysed by luminex array and gelatin zymography, gene expression by qPCR and cell viability by flow cytometry. Matrix destruction was investigated by confocal microscopy and functional assays and neutrophil extracellular traps (NETs) by fluorescence assay. In hypoxia, neutrophil MMP-8 secretion and gene expression were up-regulated by CoMTB. MMP-9 activity and neutrophil elastase (NE) secretion were also increased in hypoxia. Hypoxia inhibited NET formation and both neutrophil apoptosis and necrosis after direct stimulation by M.tb. Hypoxia increased TB-dependent neutrophil-mediated matrix destruction of Type I collagen, gelatin and elastin, the main structural proteins of the human lung. Dimethyloxalylglycin (DMOG), which stabilizes hypoxia-inducible factor-1α, increased neutrophil MMP-8 and -9 secretion. Hypoxia in our cellular model of TB up-regulated pathways that increase neutrophil secretion of MMPs that are implicated in matrix destruction.

Control of the inflammatory response during pregnancy: potential role of VIP as a regulatory peptide.

A network of cell-cell communications through contact and soluble factors supports the maternal-placental interaction and provides a suitable environment for fetal growth. Trophoblast cells take center stage at these loops: they interact with maternal leukocytes to sustain the varying demands of gestation, and they synthesize hormones, cytokines among other factors that contribute to the maintenance of immune homeostasis. Here, we discuss vasoactive intestinal peptide (VIP) and its potential as a regulatory neuropeptide in pregnancy. VIP is synthesized by trophoblast cells; it regulates trophoblast cell function and interaction with the major immune cell populations present in the pregnant uterus. VIP activity produces an anti-inflammatory microenvironment by modulating the functional profile of monocytes, macrophages, and regulatory T cells. Trophoblast VIP inhibits neutrophil extracellular trap formation and accelerates neutrophil apoptosis, enabling their silent clearance by phagocytic cells. The effects of VIP on the trophoblast-immune interaction are consistent with its regulatory role throughout pregnancy for immune homeostasis maintenance. These observations may provide new clues for pharmacological targeting of pregnancy complications associated with exacerbated inflammation.

TNFSF14 inhibits neutrophil extracellular trap formation in neutrophil‐like HL‐60 cells.

Tissue injury and infection induce a rapid inflammatory response, in which inflammatory cells, primarily neutrophils, leave the blood vessels and move into the affected tissue. Upon their arrival in this tissue, neutrophils use a variety of methods to kill potential pathogens, including phagocytosis, the generation of reactive oxygen species (ROS) that non‐specifically damage microbes, and formation of neutrophil extracellular traps (NETs) that trap microbes in an anti‐microbial environment. In addition to eliminating potential pathogens, ROS and NETs damage host tissues, promoting a positive feedback loop of tissue damage and inflammation that normally ends during the resolution of inflammation. The resolution of inflammation is associated with the inhibition of neutrophil recruitment and function, as well as induction of neutrophil apoptosis. Previous studies comparing wound healing in wild type and knockout mice found that the cytokine TNFSF14 is necessary for timely resolution of inflammation in mouse wounds, though the mechanism remains unclear. TNFSF14 could promote the resolution of inflammation by reducing cytokine production in the affected tissue, increasing neutrophil apoptosis, and/or reducing pro‐inflammatory neutrophil functions, such as ROS production and NET formation. In this study, we investigated whether TNFSF14 decreases HL‐60 neutrophil‐like cell production of ROS or NETs in response to multiple stimuli, using fluorimetry of DCF‐DA‐stained HL‐60 cells to quantify ROS production and fluorimetry of Sytox Green‐stained extracellular DNA to quantify NET formation. We found that, while TNFSF14 does not inhibit ROS production in these cells, it does decrease NET formation in response to PMA and LPS, suggesting that TNFSF14 may promote the resolution of inflammation through its inhibition of NET formation.Support or Funding InformationResearch start‐up and faculty development funding from DePauw UniversityThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Inhibition of Immune Complex Complement Activation and Neutrophil Extracellular Trap Formation by Peptide Inhibitor of Complement C1

Two major aspects of systemic lupus erythematosus (SLE) pathogenesis that have yet to be targeted therapeutically are immune complex-initiated complement activation and neutrophil extracellular trap (NET) formation by neutrophils. Here, we report in vitro testing of peptide inhibitor of complement C1 (PIC1) in assays of immune complex-mediated complement activation in human sera and assays for NET formation by human neutrophils. The lead PIC1 derivative, PA-dPEG24, was able to dose-dependently inhibit complement activation initiated by multiple types of immune complexes (IC), including C1-anti-C1q IC, limiting the generation of pro-inflammatory complement effectors, including C5a and membrane attack complex (sC5b-9). In several instances, PA-dPEG24 achieved complete inhibition with complement effector levels equivalent to background. PA-dPEG24 was also able to dose-dependently inhibit NET formation by human neutrophils stimulated by PMA, MPO, or immune complex activated human sera. In several instances PA-dPEG24 achieved complete inhibition with NETosis with quantitation equivalent to background levels. These results suggest that PA-dPEG24 inhibition of NETs occurs by blocking the MPO pathway of NET formation. Together these results demonstrate that PA-dPEG24 can inhibit immune complex activation of the complement system and NET formation. This provides proof of concept that peptides can potentially be developed to inhibit these two important contributors to rheumatologic pathology that are currently untargeted by available therapies.

Pathogenic Bacterium Acinetobacter baumannii Inhibits the Formation of Neutrophil Extracellular Traps by Suppressing Neutrophil Adhesion

Hospital-acquired infections caused by Acinetobacter baumannii have become problematic because of high rates of drug resistance. A. baumannii is usually harmless, but it may cause infectious diseases in an immunocompromised host. Although neutrophils are the key players of the initial immune response against bacterial infection, their interactions with A. baumannii remain largely unknown. A new biological defense mechanism, termed neutrophil extracellular traps (NETs), has been attracting attention. NETs play a critical role in bacterial killing by bacterial trapping and inactivation. Many pathogenic bacteria have been reported to induce NET formation, while an inhibitory effect on NET formation is rarely reported. In the present study, to assess the inhibition of NET formation by A. baumannii, bacteria and human neutrophils were cocultured in the presence of phorbol 12-myristate 13-acetate (PMA), and NET formation was evaluated. NETs were rarely observed during the coculture despite neutrophil PMA stimulation. Furthermore, A. baumannii prolonged the lifespan of neutrophils by inhibiting NET formation. The inhibition of NET formation by other bacteria was also investigated. The inhibitory effect was only apparent with live A. baumannii cells. Finally, to elucidate the mechanism of this inhibition, neutrophil adhesion was examined. A. baumannii suppressed the adhesion ability of neutrophils, thereby inhibiting PMA-induced NET formation. This suppression of cell adhesion was partly due to suppression of the surface expression of CD11a in neutrophils. The current study constitutes the first report on the inhibition of NET formation by a pathogenic bacterium, A. baumannii, and prolonging the neutrophil lifespan. This novel pathogenicity to inhibit NET formation, thereby escaping host immune responses might contribute to a development of new treatment strategies for A. baumannii infections.

Dimethyl fumarate modulates neutrophil extracellular trap formation in a glutathione- and superoxide-dependent manner.

Neutrophil (polymorphonuclear) granulocytes (PMN) have been shown to contribute to the pathogenesis of psoriasis by releasing interleukin-17 and LL37-DNA complexes via neutrophil extracellular traps (NETs), webs of chromatin strands decorated with antimicrobial peptides, in psoriatic skin. Fumaderm® , a fumaric acid ester (FAE) formulation consisting of different FAE salts, has been successfully used to treat psoriasis for decades. Most recently, FAE treatment was reported to inhibit NET formation in murine epidermolysis bullosa acquisita. To elucidate the effect of FAE treatment on human psoriasis and healthy donor NET formation. Among the compounds present in the FAE formulation, dimethyl fumarate (DMF) pretreatment of human psoriasis and healthy donor PMN resulted in a consistent inhibitory effect on NET formation in response to phorbol 12-myristate 13-acetate but not to platelet activating factor and ionomycin. This effect was l-glutathione (GSH) dependent and involved a decrease in reactive oxygen species (ROS) production, a key event in NET formation. In contrast, G-protein-coupled signalling and protein synthesis were not involved. Monomethyl fumarate (MMF) was found to slightly reduce ROS production without affecting NET formation. We report DMF as a potent, stimulus-specific, GSH- and ROS-dependent modulator of NET formation. Our results support the notion that modulation of NET formation contributes to the beneficial effects of FAEs in a variety of inflammatory conditions.

Neutrophil extracellular traps are downregulated by glucocorticosteroids in lungs in an equine model of asthma

BackgroundSevere neutrophilic asthma is poorly responsive to glucocorticosteroids (GC). Neutrophil extracellular traps (NETs) within the lungs have been associated with the severity of airway obstruction and inflammation in asthma, and were found to be unaffected by GC in vitro. As IL-17 is overexpressed in neutrophilic asthma and contributes to steroid insensitivity in different cell types, we hypothesized that NETs formation in asthmatic airways would be resistant to GC through an IL-17 mediated pathway.MethodsSix neutrophilic severe asthmatic horses and six healthy controls were studied while being treated with dexamethasone. Lung function, bronchoalveolar lavage fluid (BALF) cytology and NETs formation, as well as the expression of CD11b and CD13 by blood and airway neutrophils were evaluated. The expression of IL-17 and its role in NETs formation were also studied.ResultsAirway neutrophils from asthmatic horses, as opposed to blood neutrophils, enhanced NETs formation, which was then decreased by GC. GC also tended to decrease the expression of CD11b in blood neutrophils, but not in airway neutrophils. IL-17 mRNA was increased in BALF cells of asthmatic horses and was unaffected by GC. However, both GC and IL-17 inhibited NETs formation in vitro.ConclusionGC decreased NETs formation in vitro and also in vivo in the lungs of asthmatic horses. However, airway neutrophil activation during asthmatic inflammation was otherwise relatively insensitive to GC. The contribution of IL-17 to these responses requires further study.

Activated protein C inhibits neutrophil extracellular trap formation in vitro and activation in vivo

Activated protein C (APC) is a multifunctional serine protease with anticoagulant, cytoprotective, and anti-inflammatory activities. In addition to the cytoprotective effects of APC on endothelial cells, podocytes, and neurons, APC cleaves and detoxifies extracellular histones, a major component of neutrophil extracellular traps (NETs). NETs promote pathogen clearance but also can lead to thrombosis; the pathways that negatively regulate NETosis are largely unknown. Thus, we studied whether APC is capable of directly inhibiting NETosis via receptor-mediated cell signaling mechanisms. Here, by quantifying extracellular DNA or myeloperoxidase, we demonstrate that APC binds human leukocytes and prevents activated platelet supernatant or phorbol 12-myristate 13-acetate (PMA) from inducing NETosis. Of note, APC proteolytic activity was required for inhibiting NETosis. Moreover, antibodies against the neutrophil receptors endothelial protein C receptor (EPCR), protease-activated receptor 3 (PAR3), and macrophage-1 antigen (Mac-1) blocked APC inhibition of NETosis. Select mutations in the Gla and protease domains of recombinant APC caused a loss of NETosis. Interestingly, pretreatment of neutrophils with APC prior to induction of NETosis inhibited platelet adhesion to NETs. Lastly, in a nonhuman primate model of Escherichia coli-induced sepsis, pretreatment of animals with APC abrogated release of myeloperoxidase from neutrophils, a marker of neutrophil activation. These findings suggest that the anti-inflammatory function of APC at therapeutic concentrations may include the inhibition of NETosis in an EPCR-, PAR3-, and Mac-1-dependent manner, providing additional mechanistic insight into the diverse functions of neutrophils and APC in disease states including sepsis.

NETosis in Alzheimer's Disease.

Alzheimer’s disease (AD) is a neurodegenerative disorder characterized by the progressive deterioration of cognitive functions. Its neuropathological features include amyloid-β (Aβ) accumulation, the formation of neurofibrillary tangles, and the loss of neurons and synapses. Neuroinflammation is a well-established feature of AD pathogenesis, and a better understanding of its mechanisms could facilitate the development of new therapeutic approaches. Recent studies in transgenic mouse models of AD have shown that neutrophils adhere to blood vessels and migrate inside the parenchyma. Moreover, studies in human AD subjects have also shown that neutrophils adhere and spread inside brain vessels and invade the parenchyma, suggesting these cells play a role in AD pathogenesis. Indeed, neutrophil depletion and the therapeutic inhibition of neutrophil trafficking, achieved by blocking LFA-1 integrin in AD mouse models, significantly reduced memory loss and the neuropathological features of AD. We observed that neutrophils release neutrophil extracellular traps (NETs) inside blood vessels and in the parenchyma of AD mice, potentially harming the blood–brain barrier and neural cells. Furthermore, confocal microscopy confirmed the presence of NETs inside the cortical vessels and parenchyma of subjects with AD, providing more evidence that neutrophils and NETs play a role in AD-related tissue destruction. The discovery of NETs inside the AD brain suggests that these formations may exacerbate neuro-inflammatory processes, promoting vascular and parenchymal damage during AD. The inhibition of NET formation has achieved therapeutic benefits in several models of chronic inflammatory diseases, including autoimmune diseases affecting the brain. Therefore, the targeting of NETs may delay AD pathogenesis and offer a novel approach for the treatment of this increasingly prevalent disease.

Mesenchymal stem cells improves survival in LPS-induced acute lung injury acting through inhibition of NETs formation.

Acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) are syndromes of acute hypoxemic respiratory failure resulting from a variety of direct and indirect injuries to the gas exchange parenchyma of the lungs. During the ALI, we have an increase release of proinflammatory cytokines and high reactive oxygen species (ROS) formation. These factors are responsible for the release and activation of neutrophil-derived proteases and the formation of neutrophil extracellular traps (NETs). The excessive increase in the release of NETs cause damage to lung tissue. Recent studies have studies involving the administration of mesenchymal stem cells (MSCs) for the treatment of experimental ALI has shown promising results. In this way, the objective of our study is to evaluate the ability of MSCs, in a lipopolysaccharide (LPS)-induced ALI model, to reduce inflammation, oxidative damage, and consequently decrease the release of NETs. Mice were submitted lung injury induced by intratracheal instillation of LPS and subsequently treated or not with MSCs. Treatment with MSCs was able to modulate pulmonary inflammation, decrease oxidative damage, and reduce the release of NETs. These benefits from treatment are evident when we observe a significant increase in the survival curve in the treated animals. Our results demonstrate that MSCs treatment is effective for the treatment of ALI. For the first time, it is described that MSCs can reduce the formation of NETs and an experimental model of ALI. This finding is directly related to these cells modulate the inflammatory response and oxidative damage in the course of the pathology.

Bordetella parapertussis Circumvents Neutrophil Extracellular Bactericidal Mechanisms.

B. parapertussis is a whooping cough etiological agent with the ability to evade the immune response induced by pertussis vaccines. We previously demonstrated that in the absence of opsonic antibodies B. parapertussis hampers phagocytosis by neutrophils and macrophages and, when phagocytosed, blocks intracellular killing by interfering with phagolysosomal fusion. But neutrophils can kill and/or immobilize extracellular bacteria through non-phagocytic mechanisms such as degranulation and neutrophil extracellular traps (NETs). In this study we demonstrated that B. parapertussis also has the ability to circumvent these two neutrophil extracellular bactericidal activities. The lack of neutrophil degranulation was found dependent on the O antigen that targets the bacteria to cell lipid rafts, eventually avoiding the fusion of nascent phagosomes with specific and azurophilic granules. IgG opsonization overcame this inhibition of neutrophil degranulation. We further observed that B. parapertussis did not induce NETs release in resting neutrophils and inhibited NETs formation in response to phorbol myristate acetate (PMA) stimulation by a mechanism dependent on adenylate cyclase toxin (CyaA)-mediated inhibition of reactive oxygen species (ROS) generation. Thus, B. parapertussis modulates neutrophil bactericidal activity through two different mechanisms, one related to the lack of proper NETs-inducer stimuli and the other one related to an active inhibitory mechanism. Together with previous results these data suggest that B. parapertussis has the ability to subvert the main neutrophil bactericidal functions, inhibiting efficient clearance in non-immune hosts.

Disentangling the role of neutrophil extracellular traps in rheumatic diseases

A breakdown of immune tolerance to self-antigens in a genetically predisposing background, precipitated by environmental triggers, contributes to the development of systemic autoimmune diseases. Renewed interest in the immunomodulatory capabilities of neutrophils in systemic autoimmunity has identified neutrophil extracellular trap (NET) formation as a distinguishing action of neutrophils in afflicted hosts. Oxidation of nucleic acids and posttranslational modifications of proteins distinctly occur during NET formation and may promote enhanced immunogenicity. Various autoantibodies, immune complexes, and other inflammatory stimuli have been recently reported to promote NET formation in individuals with autoimmune diseases. Associations between level of NETosis and adverse outcomes in systemic autoimmune diseases, including thrombosis, adverse pregnancy outcomes, and renal disease, continue to be investigated. Understanding the putative pathogenic role and sequelae of NETosis in rheumatic diseases is a major focus of ongoing research efforts. Mechanisms elucidated by these discoveries may provide novel therapeutic targets to inhibit NET formation and/or promote the clearance of immunogenic NET material.

Trophoblast cells inhibit neutrophil extracellular trap formation and enhance apoptosis through vasoactive intestinal peptide-mediated pathways.

Do human trophoblast cells modulate neutrophil extracellular trap (NET) formation, reactive oxygen species (ROS) synthesis and neutrophil apoptosis through mechanisms involving vasoactive intestinal peptide (VIP)? Trophoblast cells inhibited NET formation and ROS synthesis and enhanced neutrophil apoptosis through VIP-mediated pathways in a model of maternal-placental interaction. Immune homeostasis maintenance at the maternal-placental interface is mostly coordinated by trophoblast cells. Neutrophil activation and NET formation increases in pregnancies complicated by exacerbated pro-inflammatory responses. VIP has anti-inflammatory and immunosuppressant effects and is synthesized by trophoblast cells. This is a laboratory-based observational study that sampled circulating neutrophils from 50 healthy volunteers to explore their response in vitro to factors derived from human trophoblast cells. Peripheral blood neutrophils were isolated from healthy volunteers and tested in vitro with first trimester trophoblast cell line (Swan-71 and HTR8) conditioned media (CM) or with VIP. The effect of VIP and trophoblast CM on NET formation was assessed by co-localization of elastase and DNA by confocal microscopy, DNA release and elastase activity measurement. Neutrophil apoptosis was determined by flow cytometry or fluorescence microscopy. ROS formation was assessed by flow cytometry with a fluorescent probe. VIP silencing was performed by siRNA transfection. For phagocytosis of apoptotic neutrophils, autologous monocytes were sampled, and engulfment and cytokines were assessed by flow cytometry and ELISA. Trophoblast CM and 10 nM VIP promoted neutrophil deactivation by preventing phorbol myristate acetate-induced NET formation and ROS synthesis while they increased neutrophil spontaneous apoptosis and reversed the anti-apoptotic effect of lipopolysaccharide (all P < 0.05 versus control). The effects of trophoblast CM were prevented by a VIP antagonist or when VIP knocked-down trophoblast cells were used (P < 0.05 versus control). Neutrophils driven to apoptosis by trophoblast CM could be rapidly engulfed by monocytes without increasing IL-12 production. Not applicable. The mechanisms of neutrophil deactivation by trophoblast VIP are based on the results obtained with neutrophils drawn from peripheral blood of healthy individuals interacting with trophoblast cell lines in vitro. These studies were designed to investigate biological processes at the cellular and molecular level; therefore, they have the limitations of studies in vitro and it is not possible to ascertain if these mechanisms operate similarly in vivo. We tested 50 neutrophil samples from healthy volunteers that have a normal variability in their responses. Cell lines derived from human trophoblast were used, and we cannot rule out a differential behavior of trophoblast cells in contact with neutrophils in vivo. Results presented here are consistent with an active mechanism through which neutrophils in contact with trophoblast cells would be deactivated and silently cleared by decidual macrophages throughout pregnancy. They support a novel immunomodulatory role of trophoblast VIP on neutrophils at the placenta, providing new clues for pharmacological targeting of immune and trophoblast cells in pregnancy complications associated with exacerbated inflammation. This work was funded by the National Agency of Sciences and Technology (PICT 2011-0144, 2014-0657 and 2013-2177) and University of Buenos Aires (UBACyT 20020130100040BA, 20020150100161BA and 20020130100744BA). The authors declare no competing interests.

Thrombosis in Myeloproliferative Neoplasms Is Linked to Increased Neutrophil Extracellular Trap (NET) Formation

▪Background: Philadelphia-negative myeloproliferative neoplasms (MPNs) are a group of clonal stem-cell disorders that are associated with an elevated risk for thrombosis. The reason for this association is incompletely understood. While elevated white blood cell (WBC) count is a risk factor for thrombosis in some models, the pathogenic pathways linking WBC abnormalities to thrombosis in MPNs are not clear.Neutrophil extracellular traps (NETs) are networks of extracellular neutrophil DNA, histones and serine proteases that contain and kill pathogens. There is a growing body of evidence linking NETs to thrombosis in various disease states, including cancer (Demers et al, PNAS 2012). We explored the contribution of NETs to the pro-thrombotic phenotype of MPNs.Methods: NET formation was assessed in neutrophils isolated from patients with MPNs and age-matched controls by using previously published immunofluorescence (IF) methods. c-KIT enriched marrow cells from Vav-Cre/Jak2V617F Knock-In (KI) mice were grafted into lethally irradiated wild-type (WT) recipients leading to constitutive heterozygous expression of the mutation in hematopoietic cells. Inferior vena cava (IVC) stenosis model was used to assess thrombotic tendency in mice. Mice were treated with ruxolitinib by oral gavage at a dose of 90 mg/kg BID for 3 days prior to thrombosis and NETosis experiments. NET formation was stimulated by ionomycin (4 µM).Results: Neutrophils from patients with MPNs (n=14) demonstrated significantly higher NET formation as compared to those from age-matched controls (n=10; p=0.003). This was true across specific MPN diagnoses and driver mutations (JAK2, CALR, MPL). Ex-vivo treatment of neutrophils with ruxolitinib (at 300nM for 2.5 hours) resulted in abrogation of NETosis as assessed by IF studies and was comparable to the effect achieved with the addition of compounds known to inhibit NET formation such as specific PAD4 and NADPH inhibitors; no difference was observed in Annexin V-mediated apoptosis between ruxolitinib treated and untreated neutrophils suggesting that ruxolitinib effect was not confounded by alternative apoptosis pathways.We next evaluated NETosis and its effect on thrombosis in MPN mouse models driven by Jak2 V617F. Neutrophils isolated from Vav-Cre/Jak2V617F demonstrated increased NET production ex-vivo as compared to WT littermate controls. Vav-Cre/Jak2V617F KI mice were grafted into lethally irradiated WT and assessed for their prothrombotic phenotype. VavCre/Jak2V617F KI demonstrated significantly higher thrombus formation at 2 hours using the IVC stenosis model (45% versus 0% for Jak2V617F KI (n=11) and Jak2WT (n=8), respectively; p=0.04 by Fisher's exact test). This was associated with significantly higher levels of free dsDNA in the plasma of KI stenosis mice as compared to WT stenosis controls (p=0.03 by Mann-Whitney). Furthermore, IHC and IF studies of lung tissue demonstrated that Vav-Cre/Jak2V617F KI have widespread thrombosis as compared to controls and IF studies demonstrated higher content of citrullinated histone H3 in the lung suggesting increased NETosis at that site. Treatment of Vav-Cre/Jak2V617F KI mice with ruxolitinib significantly abrogated thrombus formation (45% versus 0% for untreated (n=11) and treated (n=8) mice, respectively; p=0.04 by Fisher's exact test).We noted that expression of PAD4, an essential driver of NETosis, is elevated in MPN patients (Rampal Blood 2014). We found that PAD4 content was higher in protein lysates of neutrophils isolated from MPN patients as compared to controls. Moreover, abnormal nuclear localization was observed in patients. These observations may support a role for PAD4 in the excess NETosis observed in MPNs.Conclusion: We found that neutrophils from MPN patients and Jak2 V617F KI mice are sensitized to NET formation that may be mediated by increased PAD4 expression and nuclear localization. In mice, the increased ex-vivo NET formation is associated with an increased thrombosis rate and an abundance of NET related products in thrombus and tissue. Treatment with ruxolitinib abrogates NET formation in humans and significantly reduces thrombus formation in MPN mice models.These findings provide a pathogenic link between the genetic drivers of MPNs and thrombosis in these diseases. Pharmacologic inhibition of JAK-STAT pathway to reduce NETosis and thrombosis should be further investigated. DisclosuresDeAngelo:Novartis: Consultancy; Ariad: Consultancy; Celgene: Consultancy; Baxter: Consultancy; Amgen: Consultancy; Incyte: Consultancy; Pfizer: Consultancy.

Neonatal NET-inhibitory factor and related peptides inhibit neutrophil extracellular trap formation.

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.

Leukocytes coincubated with human sperm trigger classic neutrophil extracellular traps formation, reducing sperm motility

To determine whether the human spermatozoon is a sufficient stimulus to trigger the release of neutrophil extracellular traps (NETs) in a time- and dose-dependent manner. Experimental study. University-based laboratory. Semen samples from four men and blood samples from six healthy female donors. Polymorphonuclear neutrophils (PMN) isolated from peripheral blood were incubated with fresh human spermatozoa for 60, 90, 120, and 180minutes and at different PMN/sperm concentrations (1:1 [25 × 104], 1:3 [25 × 104:75 × 104], 1:6 [25 × 104:15 × 105], 1:18 [25 × 104:45 × 105]). During coincubation of PMN/sperm, the release of NETs was measured by PicoGreen. Immunofluorescence for histone H3, neutrophil elastase (NE), and myeloperoxidase (MPO) was performed. Different NETs inhibitors were tested: diphenylene iodonium, Suc-Ala- Ala-Pro-Val chloromethyl ketone (CMK), and 4-aminobenzoic acid hydrazide (ABAH) inhibitors of NADPH oxidase, NE, and MPO. Progressive mobility was assessed at increasing doses of neutrophils (1:18 [25 × 104:45 × 105], 6:18 [15 × 105:45 × 105], 9:18 [252 × 104:45 × 105]). The quantity of NETs increased at the ratio of 1:6 after 2hours and continued to increase subsequently. A ratio of 1:18 showed significant increases in NETs production at all times. Assessment of the inhibitors showed that CMK and ABAH inhibit NETs formation. Scanning and transmission electron microphotographs and immunofluorescence confirmed NETs formation induced by the spermatozoa. After 1hour, progressive motility diminished in the two groups with the highest proportion of neutrophils and after 2hours in all groups exposed to neutrophils. We show that the stimulus of the human spermatozoon triggers the release of NETs; this response is dose dependent and increases with exposure time. The motility of affected spermatozoa diminishes, suggesting that this interaction on a larger scale would decrease the probability of successful fertilization.

Neutrophil Extracellular Traps Promote the Development and Progression of Liver Metastases after Surgical Stress.

Risks of tumor recurrence after surgical resection have been known for decades, but the mechanisms underlying treatment failures remain poorly understood. Neutrophils, first-line responders after surgical stress, may play an important role in linking inflammation to cancer progression. In response to stress, neutrophils can expel their protein-studded chromatin to form local snares known as neutrophil extracellular traps (NET). In this study, we asked whether, as a result of its ability to ensnare moving cells, NET formation might promote metastasis after surgical stress. Consistent with this hypothesis, in a cohort of patients undergoing attempted curative liver resection for metastatic colorectal cancer, we observed that increased postoperative NET formation was associated with a >4-fold reduction in disease-free survival. In like manner, in a murine model of surgical stress employing liver ischemia-reperfusion, we observed an increase in NET formation that correlated with an accelerated development and progression of metastatic disease. These effects were abrogated by inhibiting NET formation in mice through either local treatment with DNAse or inhibition of the enzyme peptidylarginine deaminase, which is essential for NET formation. In growing metastatic tumors, we found that intratumoral hypoxia accentuated NET formation. Mechanistic investigations in vitro indicated that mouse neutrophil-derived NET triggered HMGB1 release and activated TLR9-dependent pathways in cancer cells to promote their adhesion, proliferation, migration, and invasion. Taken together, our findings implicate NET in the development of liver metastases after surgical stress, suggesting that their elimination may reduce risks of tumor relapse.