Neutrophil Extracellular Traps Structures Research Articles

LL37/FPR2 regulates neutrophil mPTP promoting the development of neutrophil extracellular traps in diabetic retinopathy.

Diabetic retinopathy (DR) is characterized by chronic, low-grade inflammation. This state may be related to the heightened production of neutrophil extracellular traps (NETs) induced by high glucose (HG). Human cathelicidin antimicrobial peptide (LL37) is an endogenous ligand of G protein-coupled chemoattractant receptor formyl peptide receptor 2 (FPR2), expressed on neutrophils and facilitating the formation and stabilization of the structure of NETs. In this study, we detected neutrophils cultured under different conditions, the retinal tissue of diabetic mice, and fibrovascular epiretinal membranes (FVM) samples of patients with proliferative diabetic retinopathy (PDR) to explore the regulating effect of LL37/FPR2 on neutrophil in the development of NETs during the process of DR. Specifically, HG or NG with LL37 upregulates the expression of FPR2 in neutrophils, induces the opening of mitochondrial permeability transition pore (mPTP), promotes the increase of reactive oxygen species and mitochondrial ROS, and then leads to the rise of NET production, which is mainly manifested by the release of DNA reticular structure and the increased expression of NETs-related markers. The PI3K/AKT signaling pathway was activated in neutrophils, and the phosphorylation level was enhanced by FPR2 agonists invitro. Invivo, increased expression of NETs markers was detected in the retina of diabetic mice and in FVM, vitreous fluid, and serum of PDR patients. Transgenic FPR2 deletion led to decreased NETs in the retina of diabetic mice. Furthermore, invitro, inhibition of the LL37/FPR2/mPTP axis and PI3K/AKT signaling pathway decreased NET production induced by high glucose. These results suggested that FPR2 plays an essential role in regulating the production of NETs induced by HG, thus may be considered as one of the potential therapeutic targets.

DNase I rescues goat sperm entrapped by neutrophil extracellular traps

Artificial insemination has been a predominant technique employed in goat husbandry for breeding purposes. Subsequent to artificial insemination, sperm can elicit inflammation in the reproductive tract, resulting in substantial the accumulation of neutrophils. Recognized as foreign entities, sperm may become entrapped within neutrophil extracellular traps (NETs) released by neutrophils, thereby exploiting their properties of pathogen elimination. Deoxyribonuclease I (DNase I), which is known for disintegrating NETs and causing loss of function, has been utilized to ameliorate liver and brain damage resulting from NETs, as well as to enhance sperm quality. This study investigated the mechanism of sperm-induced NETs and further explored the impact of DNase I on NETs. Sperm quality was evaluated using optical microscopy, while the structure of NETs was observed through immunofluorescence staining. The formation mechanism of NETs was examined using inhibitors and PicoGreen. The findings revealed that sperm induced the formation of NETs, a process regulated by glycolysis, NADPH oxidase, ERK1/2, and p38 signaling pathways. The composition of NETs encompassed DNA, citrullinated histone H3 (citH3), and elastase (NE). DNase I protects sperm by degrading NETs, thereby concurrently preserving the integrity of plasma membrane and motility of sperm. In summary, the release of sperm-induced NETs leads to its damage, but this detrimental effect is counteracted by DNase I through degradation of NETs. These observations provide novel insights into reproductive immunity in goats.

Unobvious Neutrophil Extracellular Traps Signification in the Course of Oral Squamous Cell Carcinoma: Current Understanding and Future Perspectives.

Background: The current standards of treatment for oral squamous cell carcinoma (OSCC) include surgery, radiotherapy, and chemotherapy. In recent years, research on the effectiveness of immunotherapy in the treatment of OSCC has also been conducted.Purpose: Studies indicate that nonspecific immune mechanisms involved in the course of the anticancer response also need to be taken into account.Research Design: This review summarizes the results of our research on the active participation of neutrophils, which are previously underestimated, in the antitumor response in the course of OSCC, taking into account the ability of these cells to generate neutrophil extracellular traps (NETs).Results: We proved that the formation of NETs accompanies not only inflammatory changes but also the neoplastic process and that lipopolysaccharide (LPS) or interleukin 17 (IL-17) plays a critical role in inducing the formation of NETs during the OSCC. The greatest achievement of our published findings was the demonstration of the formation and release of NETs from neutrophils cocultured with tumor cells, as well as after stimulation with supernatant from the SCC culture with a PI3K-independent Akt kinase activation mechanism. Moreover, the pioneering achievement of our studies was the localization of NET structures in the tumor tissue, as well as the observation of high concentrations of NET markers in the serum of OSCC patients with low concentrations in the saliva, indicating the differences in the course of immune response between the periphery and the local reactions.Conclusions: The data presented here provide surprising but important information on the role of NETs in the course of OSCC, thus pointing to a promising new direction in the development of management strategies for early noninvasive diagnosis and monitoring of the disease course, and perhaps immunotherapy. Furthermore, this review raises further questions and elaborates on the process of NETosis in cancer.

Neutrophil extracellular traps in autoimmune diseases: Analysis of the knowledge map.

Recent studies have shown much progress in the research of exosomes in AIDs. However, there is no bibliometric analysis in this research field. This study aimed to provide a bibliometrics review of the knowledge structure and research hotspots of neutrophil extracellular traps (NETs) in autoimmune diseases (AIDs). Articles relevant to NETs in AIDs from 2010 to 2022 were retrieved through the Web of Science Core Collection (WoSCC) database. This bibliometric analysis was performed by VOSview, CiteSpace, and Scimago Graphica. A total of 289 papers analyzed in this research were from 493 organizations in 47 countries by 1537 authors. They were published in 133 journals and cited 20,180 citations from 2,465 journals. The number of annual publications in this field is growing steadily and rapidly, with the United States, China and Germany leading the research effort. Frontiers in Immunology and Journal of Immunology have significantly impacted research in this field. Kaplan, Mariana J, from the National Institutes of Health (The United States), has the most published articles, and Brinkmann, v, from Max Planck Institute for Infection Biology (Germany), is the most co-cited author. Systemic lupus erythematosus and rheumatoid arthritis are the leading topics in this field. The trend of clinical application in the future is the development of new therapies by controlling NETs in the progression of AIDs. Our study summarized the research trends and developments of NETs in AIDs in recent years and would provide a reference for scholars in this field.

Gasdermin-D activation by SARS-CoV-2 triggers NET and mediate COVID-19 immunopathology

BackgroundThe release of neutrophil extracellular traps (NETs) is associated with inflammation, coagulopathy, and organ damage found in severe cases of COVID-19. However, the molecular mechanisms underlying the release of NETs in COVID-19 remain unclear.ObjectivesWe aim to investigate the role of the Gasdermin-D (GSDMD) pathway on NETs release and the development of organ damage during COVID-19.MethodsWe performed a single-cell transcriptome analysis in public data of bronchoalveolar lavage. Then, we enrolled 63 hospitalized patients with moderate and severe COVID-19. We analyze in blood and lung tissue samples the expression of GSDMD, presence of NETs, and signaling pathways upstreaming. Furthermore, we analyzed the treatment with disulfiram in a mouse model of SARS-CoV-2 infection.ResultsWe found that the SARS-CoV-2 virus directly activates the pore-forming protein GSDMD that triggers NET production and organ damage in COVID-19. Single-cell transcriptome analysis revealed that the expression of GSDMD and inflammasome-related genes were increased in COVID-19 patients. High expression of active GSDMD associated with NETs structures was found in the lung tissue of COVID-19 patients. Furthermore, we showed that activation of GSDMD in neutrophils requires active caspase1/4 and live SARS-CoV-2, which infects neutrophils. In a mouse model of SARS-CoV-2 infection, the treatment with disulfiram inhibited NETs release and reduced organ damage.ConclusionThese results demonstrated that GSDMD-dependent NETosis plays a critical role in COVID-19 immunopathology and suggests GSDMD as a novel potential target for improving the COVID-19 therapeutic strategy.

Neutrophil extracellular traps (NETs) reduce the diffusion of doxorubicin which may attenuate its ability to induce apoptosis of ovarian cancer cells

PurposeAlthough neutrophil extracellular traps (NETs) are present in various tumors, their roles in tumor biology have not been clarified yet. In this study, we examined how NETs affect the pharmacokinetics and effects of doxorubicin (DOX). MethodsNETs were generated by neutrophils stimulated with phorbol 12-myristate 13-acetate (PMA) or lipopolysaccharide (LPS). DOX was added to NETs and their distribution was observed under fluorescein microscopy, and the diffusion of DOX through 3 μM pores from lower to upper chambers was evaluated with a fluorescence-based assay. Ovarian cancer cells, KOC-2S and SKOV3, were embedded in collagen gel droplets and cultured in 3D way and their apoptosis was examined with flow cytometry. ResultsDOX was mostly co-localized with NETs. The transfer of DOX to upper chambers increased over time, which was significantly decreased by the presence of neutrophils stimulated with PMA or LPS in the lower chamber. DOX outside of the gel increased the rates of annexin V (+) apoptotic cells, which were significantly reduced by the addition of LPS-stimulated neutrophils in media both in KOC-2S and SKOV3. The reduced diffusion and apoptosis were mostly restored by the destruction of the NETs structure with 1000 u/ml DNAse I. ConclusionNETs efficiently trap and inhibit the diffusion of DOX which may attenuate its ability to induce apoptosis of ovarian cancer cells. Degradation of NETs with DNAse I may augment the response of ovarian cancer to DOX.

Tumor-Associated Neutrophil Extracellular Traps Regulating Nanocarrier-Enhanced Inhibition of Malignant Tumor Growth and Distant Metastasis.

Tumor-associated neutrophil extracellular traps (NETs) play a critical role in promoting tumor growth and assisting tumor metastasis. Herein, a smart nanocarrier (designated as mP-NPs-DNase/PTX) based on regulating tumor-associated NETs has been developed, which consists of a paclitaxel (PTX) prodrug nanoparticle core and a poly-l-lysine (PLL) conjugated with the matrix metalloproteinase 9 (MMP-9)-cleavable Tat-peptide-coupled deoxyribonuclease I (DNase I) shell. After accumulating at the site of the tumor tissue, the nanocarrier can release DNase I in response to MMP-9 to degrade the structure of NETs. Then, the remaining moiety can uptake the tumor cells via the mediation of exposed cell penetrating peptide, and the PTX prodrug nanoparticles will lyse in response to the high intracellular concentration of reduced glutathione to release PTX to exert a cytotoxic effect of tumor cells. Through in vitro and in vivo evaluations, it has been proven that mP-NPs-DNase/PTX could serve as potential NET-regulated nanocarrier for enhanced inhibition of malignant tumor growth and distant metastasis.

Coronavirus disease 2019 (COVID-19): NETosis-associated mechanisms of progression and prospects for therapy regulating the formation of neutrophil extracellular traps (NETs)

Infectious disease COVID-19 caused by the SARS-CoV-2 coronavirus is characterized by high contagiousness, complexity of pathogenesis and unpredictability of the clinical course. In severe cases, which are especially susceptible to men, the elderly and people with underlying medical conditions such as obesity, diabetes, hypertension, cardiovascular and chronic respiratory diseases, the infection leads to respiratory failure and death due to the development of an extensive inflammatory reaction. As a result of many studies, it has been established that one of the leading causes of the severe course and death of patients with COVID-19 is the development of coagulopathy, that is, increased thrombus formation in small vessels due to excessive activity of neutrophils, which form the so-called neutrophil extracellular traps (NETs). Although NETs play a useful role in protecting their host from pathogens, their overgrowth can trigger a cascade of adverse reactions including: the production of antibodies against the host’s DNA (autoimmunization); damage to surrounding tissue; or the occurrence of thromboembolic complications. Therefore, extracellular neutrophil traps and their markers have been identified as targets for new therapeutic strategies aimed at reducing the severity of COVID-19 disease and/or mortality. This article describes the structure of NETs, as well as analyzes the molecular mechanisms that contribute to their overgeneration. In addition, the prospects for COVID-19 therapy aimed at regulating the formation of extracellular traps by creating drugs both limiting the production of NET structures and dissolving their excess amounts in the body of patients are discussed.

Extracellular Nucleic Acids Present in the Candida albicans Biofilm Trigger the Release of Neutrophil Extracellular Traps.

Neutrophils, the first line of the host’s defense, use a variety of antimicrobial mechanisms to fight invading pathogens. One of the most crucial is the production of neutrophil extracellular traps (NETs) in the process called NETosis. The unique structure of NETs effectively inhibits the spread of pathogens and ensures their exposure to a high concentration of NET-embedded antimicrobial compounds. NETosis strategy is often used by the host to defend against fungal infection caused by Candida albicans. In immunocompromised patients, this microorganism is responsible for developing systemic fungal infections (candidiasis). This is correlated with the use of a vast array of virulence factors, leading to the acquisition of specific resistance to host defense factors and available drug therapies. One of the most important features favoring the development of drug resistance is a C. albicans ability to form biofilms that protect fungal cells mainly through the production of an extracellular matrix (ECM). Among the main ECM-building macromolecules extracellular nucleic acids have been identified and their role is probably associated with the stbilization of the biofilm structure. The complex interactions of immune cells with the thick ECM layer, comprising the first line of contact between these cells and the biofilm structure, are still poorly understood. Therefore, the current studies aimed to assess the release of extracellular nucleic acids by C. albicans strains at different stages of biofilm formation, and to determine the role of these molecules in triggering the NETosis. We showed for the first time that fungal nucleic acids, purified directly from mature C. albicans biofilm structure or obtained from the whole fungal cells, have the potential to induce NET release in vitro. In this study, we considered the involvement of TLR8 and TLR9 in NETosis activation. We showed that DNA and RNA molecules initiated the production of reactive oxygen species (ROS) by activation of the NADPH oxidase complex, essential for ROS-dependent NETosis. Furthermore, analysis of the cell migration showed that the nucleic acids located in the extracellular space surrounding the biofilm may be also effective chemotactic factors, driving the dynamic migration of human neutrophils to the site of ongoing fungal infection.

Histamine triggers the formation of neutrophil extracellular traps via NADPH oxidase, ERK and p38 pathways

Histamine plays a central role in various allergic diseases, such as allergic asthma and allergic rhinitis. Neutrophil extracellular traps (NETs) formation is a novel effector mechanism of neutrophils to defend against various stimuli. In this present study, we aimed to investigate the role of histamine on bovine NET formation, and examined its preliminary molecular mechanisms. Cell Counting Kit-8 (CCK8) and Lactate dehydrogenase assays showed that histamine had no significant influence on PMNs (polymorphonuclear leukocytes) viability. Confocal microscopy analyses identified NET structures by co-localizing the main components of NETs, and NET quantification revealed that histamine-triggered NETs were released in a dose-dependent manner. Furthermore, we found reactive oxygen species (ROS) production, phosphorylated extracellular signal-regulated kinase (ERK) and p38 proteins were significantly elevated in histamine-challenged PMNs. By applying functional inhibitors of nicotinamide adenine dinucleotide phosphate-oxidase (NADPH oxidase), ERK and p38, histamine-triggered NETs were markedly reduced, indicating their importance in histamine-triggered NET formation. Our findings described histamine-triggered NET formation, and revealed its potential molecular mechanisms via NADPH oxidase, ERK and p38 pathways. This is the first study to depict histamine-triggered NET formation, which could provide a new insight into histamine-related diseases.

IL-17A expressed on neutrophil extracellular traps promotes mesenchymal stem cell differentiation toward bone-forming cells in ankylosing spondylitis.

Ankylosing spondylitis (AS) is an inflammatory disease characterized by excessive bone formation. We investigated the presence of neutrophil extracellular traps (NETs) in AS and how they are involved in the osteogenic capacity of bone marrow mesenchymal stem cells (MSCs) through interleukin-17A (IL-17A). Peripheral neutrophils and sera were obtained from patients with active AS and healthy controls. NET formation and neutrophil/NET-associated proteins were studied using immunofluorescence, immunoblotting, qPCR, and ELISA. In vitro co-culture systems of AS NET structures and MSCs isolated from controls were deployed to examine the role of NETs in the differentiation of MSCs toward osteogenic cells. Analysis was performed using specific staining and qPCR. Neutrophils from patients with AS were characterized by enhanced formation of NETs carrying bioactive IL-17A and IL-1β. IL-17A-enriched AS NETs mediated the differentiation of MSCs toward bone-forming cells. The neutrophil expression of IL-17A was positively regulated by IL-1β. Blocking IL-1β signaling on neutrophils with anakinra or dismantling NETs using DNase-I disrupted osteogenesis driven by IL-17A-bearing NETs. These findings propose a novel role of neutrophils in AS-related inflammation, linking IL-17A-decorated NETs with the differentiation of MSCs toward bone-forming cells. Moreover, IL-1β triggers the expression of IL-17A on NETs offering an additional therapeutic target in AS.

Influence of iron- and zinc-chelating agents on neutrophil extracellular trap formation.

Release of neutrophil extracellular traps (NETs) is one of the neutrophils’ mechanisms involved in the response to infection. NETs are released from the cell in response to a biological or synthetic stimulus to entrap, immobilize and kill pathogens. Metal ions and metal binding proteins were identified in the structure of NETs, but their role in NET release remains unclear. The aim of this study was to assess how lack of iron and zinc generated by ion sequestration using chelators affects NET release. Neutrophils were isolated from whole blood or buffy coats of healthy blood donors by density gradient centrifugation and incubated with zinc chelators: 20 µM N,N,N',N'-Tetrakis(2-pyridylmethyl)ethylenediamine (TPEN), 40 µM diethylenetriaminepentaacetic acid (DTPA) or iron chelators: 400 µM deferoxamine mesylate salt (DFO) and 50 µM iminodiacetic acid (IDA). Next, 100 nM phorbol 12-myristate 13-acetate (PMA) was added to stimulate release of NETs. The amount of released DNA was measured by fluorometry and NETs were visualized by immunofluorescence microscopy. This study demonstrates that iron and zinc chelators are able to modulate NET release. Here we show that preincubation of neutrophils with TPEN and IDA inhibits NET release in cells stimulated with PMA. On the other hand, DFO stimulates NET release. Incubation of cells with DTPA does not affect release of NETs.

Neutrophil extracellular traps are associated with altered human pulmonary artery endothelial barrier function

Introduction: Acute respiratory response syndrome (ARDS) leads to increased permeability of the endothelial-epithelial barrier, which in turn promotes edema formation and hypoxemic respiratory failure. Although activated neutrophils are thought to play a significant role in mediating ARDS, at present the contribution of neutrophil extracellular traps (NETs) to lung endothelial barrier function is unclear. Methods: To clarify their role, we co-cultured in vitro NETs induced by phorbol myristate acetate (PMA)–activated neutrophils with lung endothelial cell monolayers and examined the barrier function of lung endothelial cells by immunofluorescence microscopy and albumin permeability in a double-chamber culture method. Results: Co-culture with stimulated neutrophils increased the albumin permeability of the human pulmonary artery endothelial cell (HPAEC) monolayer and altered cytoskeleton F-actin and vascular endothelial-cadherin in cell-cell junctions. Hyperpermeability to albumin and histological alterations were prevented by inhibition of NET formation with peptidyl arginine deiminase inhibitor or a neutrophil elastase inhibitor and were also prevented by increased degradation of NET structure with DNase. Conclusion: This in vitro experiment shows that altered HPAEC barrier function and increased albumin permeability are caused by the direct effect of PMA-induced NETs and their components. NET formation may be involved in the increased vascular permeability of the lung, which is a common feature in ARDS of various etiologies. These insights may help generate novel approaches for medical interventions.

Early Enteral Nutrition Preserves Intestinal Barrier Function through Reducing the Formation of Neutrophil Extracellular Traps (NETs) in Critically Ill Surgical Patients.

Background The gut was suggested as the driver of critical illness and organ injury. Recently, excessive formation of neutrophil extracellular traps (NETs) was associated with mucosal inflammation. Direct investigation of intestinal mucosa is essential to illuminate the potential mechanism of gut barrier in critically ill patients. We hypothesized that early enteral nutrition (EN) could decrease intestinal NETs and maintain the gut barrier. Methods Intestinal biopsies were obtained using biopsy forceps from critically ill surgical patients complicated with enterocutaneous fistula. Expressions of tight junction (TJ) proteins, mucosal inflammation, and apoptosis were evaluated. Moreover, NET-associated proteins were evaluated in intestinal specimens of patients by Western blot and immunofluorescence analysis. Results The intestinal barrier was significantly impaired in critically ill patients receiving early total parenteral nutrition (TPN), evidenced by intestinal villi atrophy, inflammatory infiltration, increased enterocyte apoptosis, and abnormal TJ expressions. Early EN significantly alleviated these intestinal injuries. In addition, we observed increased formation of the NET structure and elevated expressions of NET-associated proteins in intestines of critically ill surgical patients. Early EN was associated with the diminished presence of NETs and reduced expression of NET-associated proteins. Mechanically, analysis of the TLR4 pathway showed a significant increase in TLR4, NFκB, and MAPK signaling in patients receiving TPN when compared to those receiving early EN. Conclusion The intestinal barrier is disrupted in the human gut during critical illness. Our data suggests that an increased NET structure was showed in the gut of critically ill surgical patients, and early EN treatment was associated with the reduction of NET formation and the preservation of mucosal immunity.

Bioinspired DNase-I-Coated Melanin-Like Nanospheres for Modulation of Infection-Associated NETosis Dysregulation.

The current outbreak of the beta‐coronavirus (beta‐Cov) severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) began in December 2019. No specific antiviral treatments or vaccines are currently available. A recent study has reported that coronavirus disease 2019 (COVID‐19), the disease caused by SARS‐CoV‐2 infection, is associated with neutrophil‐specific plasma membrane rupture, and release excessive neutrophil extracellular traps (NETs) and extracellular DNAs (eDNAs). This mechanism involves the activation of NETosis, a neutrophil‐specific programmed cell death, which is believed to play a crucial role in COVID‐19 pathogenesis. Further progression of the disease can cause uncontrolled inflammation, leading to the initiation of cytokine storms, acute respiratory distress syndrome (ARDS), and sepsis. Herein, it is reported that DNase‐I‐coated melanin‐like nanospheres (DNase‐I pMNSs) mitigate sepsis‐associated NETosis dysregulation, thereby preventing further progression of the disease. Recombinant DNase‐I and poly(ethylene glycol) (PEG) are used as coatings to promote the lengthy circulation and dissolution of NET structure. The data indicate that the application of bioinspired DNase‐I pMNSs reduce neutrophil counts and NETosis‐related factors in the plasma of SARS‐CoV‐2 sepsis patients, alleviates systemic inflammation, and attenuates mortality in a septic mouse model. Altogether, the findings suggest that these nanoparticles have potential applications in the treatment of SARS‐CoV‐2‐related illnesses and other beta‐CoV‐related diseases.

Neutrophil Extracellular Traps: Signaling Properties and Disease Relevance.

Neutrophil extracellular traps (NETs) are characterized as extracellular DNA fibers comprised of histone and cytoplasmic granule proteins. NETs were first described as a form of innate response against pathogen invasion, which can capture pathogens, degrade bacterial toxic factors, and kill bacteria. Additionally, NETs also provide a scaffold for protein and cell binding. Protein binding to NETs further activate the coagulation system which participates in thrombosis. In addition, NETs also can damage the tissues due to the proteins they carry. Many studies have suggested that the excessive formation of NETs may contribute to a range of diseases, including thrombosis, atherosclerosis, autoimmune diseases, and sepsis. In this review, we describe the structure and components of NETs, models of NET formation, and detection methods. We also discuss the molecular mechanism of NET formation and their disease relevance. Modulation of NET formation may provide a new route for the prevention and treatment of releated human diseases.

The effects of the dental methacrylates TEGDMA, Bis-GMA, and UDMA on neutrophils in vitro.

ObjectivesThe prevalent usage of methacrylates in modern dentistry demands good knowledge of their biological impacts. While there have been several studies demonstrating the effects of different methacrylic monomers on mononuclear white blood cells, very little is known about the effects caused by these monomers on neutrophilic granulocytes. The objective of this study was to add novel knowledge about how neutrophils are affected by exposure to triethylene glycol dimethacrylate (TEGDMA), urethane dimethacrylate (UDMA), and bisphenol A glycol dimethacrylate (Bis‐GMA) alone or in combinations.Materials and MethodsIsolated neutrophils were cultured in the presence or absence of methacrylates. The IL‐8 release was measured using a DuoSet ELISA development kit. Apoptosis and necrosis were analyzed using flow cytometry. The formation of neutrophil extracellular traps (NETs) was investigated using Sytox green DNA staining combined with microscopically examination of released DNA and myeloperoxidase (MPO).ResultsThe release of IL‐8 was significantly increased after exposure to TEGDMA, Bis‐GMA, UDMA, or TEGDMA in combination with Bis‐GMA or UDMA compared to the unstimulated controls. Exposure to TEGDMA, UDMA, and Bis‐GMA for 24 hr separately or in combination did not affect apoptosis or necrosis of the exposed neutrophils. NET structures were formed by neutrophils after exposure to the different combinations of the methacrylates.ConclusionThe combination of TEGDMA and Bis‐GMA had a synergistic proinflammatory effect on neutrophils by increasing the release of IL‐8 and the formation of NET structures. The changes in the normal functions of neutrophils caused by methacrylate exposure may lead to altered inflammatory response and relate to previously reported adverse immune reactions caused by these substances.

Protocol for TRAUMADORNASE: a prospective, randomized, multicentre, double-blinded, placebo-controlled clinical trial of aerosolized dornase alfa to reduce the incidence of moderate-to-severe hypoxaemia in ventilated trauma patients

BackgroundAcute respiratory distress syndrome continues to drive significant morbidity and mortality after severe trauma. The incidence of trauma-induced, moderate-to-severe hypoxaemia, according to the Berlin definition, could be as high as 45%. Its pathophysiology includes the release of damage-associated molecular patterns (DAMPs), which propagate tissue injuries by triggering neutrophil extracellular traps (NETs). NETs include a DNA backbone coated with cytoplasmic proteins, which drive pulmonary cytotoxic effects. The structure of NETs and many DAMPs includes double-stranded DNA, which prevents their neutralization by plasma. Dornase alfa is a US Food and Drug Administration-approved recombinant DNase, which cleaves extracellular DNA and may therefore break up the backbone of NETs and DAMPs. Aerosolized dornase alfa was shown to reduce trauma-induced lung injury in experimental models and to improve arterial oxygenation in ventilated patients.MethodsTRAUMADORNASE will be an institution-led, multicentre, double-blinded, placebo-controlled randomized trial in ventilated trauma patients. The primary trial objective is to demonstrate a reduction in the incidence of moderate-to-severe hypoxaemia in severe trauma patients during the first 7 days from 45% to 30% by providing aerosolized dornase alfa as compared to placebo. The secondary objectives are to demonstrate an improvement in lung function and a reduction in morbidity and mortality. Randomization of 250 patients per treatment arm will be carried out through a secure, web-based system. Statistical analyses will include a descriptive step and an inferential step using fully Bayesian techniques. The study was approved by both the Agence Nationale de la Sécurité du Médicament et des Produits de Santé (ANSM, on 5 October 2018) and a National Institutional Review Board (CPP, on 6 November 2018). Participant recruitment began in March 2019. Results will be published in international peer-reviewed medical journals.DiscussionIf early administration of inhaled dornase alfa actually reduces the incidence of moderate-to-severe hypoxaemia in patients with severe trauma, this new therapeutic strategy may be easily implemented in many clinical trauma care settings. This treatment may facilitate ventilator weaning, reduce the burden of trauma-induced lung inflammation and facilitate recovery and rehabilitation in severe trauma patients.Trial registrationClinicalTrials.gov, NCT03368092. Registered on 11 December 2017.

Newly excysted juveniles of Fasciola gigantica trigger the release of water buffalo neutrophil extracellular traps in vitro

Polymorphonuclear neutrophils (PMNs) are the most abundant leukocytes and are among the first line of immune system defense. PMNs can form neutrophil extracellular traps (NETs) in response to some pathogens. The release of NETs plays an important role in trapping and killing invading parasites. However, the effects of NETs on parasitic trematode infections remain unclear. In the present study, water buffalo NET formation, triggered by the newly excysted juveniles (NEJs) of Fasciola gigantica, was visualized by scanning electron microscopy. The major components of the structure of NETs were characterized by immunofluorescence. Viability of flukes incubated with water buffalo PMNs were examined under light microscopy. The results revealed that F. gigantic juveniles triggered PMN-mediated NETs. These NETs were confirmed to comprise the classic characteristics of NETs: DNA, histones, myeloperoxidase and neutrophil elastase. Although NETs were formed in response to viable larvae, the larvae were not killed in vitro. These results suggest that NET formation may serve as a mechanism to hamper the migration of large larvae to facilitate immune cells to kill them. This study demonstrates, for the first time, that parasitic trematode juveniles can trigger NET formation.

Inhibitors of nitric oxide synthase can reduce extracellular traps from neutrophils in asthmatic children in vitro.

This study aimed to explore the link between neutrophil extracellular traps (NETs) and childhood asthma, to investigate the ability of nitric oxide (NO) to induce NETs in asthmatic children and find inhibitors to reduce NETs in the NO synthesis pathway. A total of 49 children with mild persistent asthma were included in the study and 20 healthy children's blood samples were collected as healthy controls. Children with asthma were divided into symptomatic and asymptomatic groups according to the presence or absence of symptoms on the day of blood collection. Neutrophils in peripheral blood were isolated and plasma was preserved. NO donor (sodium nitroferricyanide(III) dehydrate [SNP]) could provide NO and proved by a fluorescent probe. A PicoGreen Kit was used to detect the NETs quantificationally. Fluorescence microscopy prepared to observe the main structures of NETs. We measured NETs components (extracellular free double-stranded DNA [dsDNA]) in healthy, symptomatic and asymptomatic groups' plasma samples, and we compared the ability of SNP with phosphate-buffered saline, lipopolysaccharides (LPS), and phorbol 12-myristate 13-acetate (PMA) to induce NETs. NO synthase (NOS) inhibitors were added to see the impact on NETs formation. Plasma was obtained from all blood samples of 69 children. The neutrophils of 40 asthmatic and 20 healthy children were successfully obtained, the recovery rate was over 95%, and the cell activity was over 80%. There was higher extracellular free dsDNA in the plasma of symptomatic group (n = 27) than asymptomatic group (n = 22) and healthy group (n = 20; P < .05). Studies on neutrophils from 40 children with asthma found that NO can be produced by adding SNP, PMA, and LPS. SNP could induce NETs with dose- and time-dependent. PMA (160 nM) had the strongest ability to induce NETs, LPS (200 ng/mL) followed, SNP (200 µM) was the weakest (P < .05), and the amount of NETs in the asthma group was significantly higher than that in the healthy group (P < .05). NOS inhibitors had the same blocking capacity for PMA- and LPS-induced NETs (P > .05), while NG-nitro- l-arginine methyl ester (500 µM) had the strongest inhibitory effect on SNP induction with time-dependent (P < .05). Inducible NOS was found in the NETs structure. Children with asthma had higher levels of NETs in peripheral blood, especially when they had asthma symptoms. We verified the ability of NO to induce NETs, and found neutrophils from asthmatic children can produce more NETs in vitro. NOS inhibitors blocked this process may provide new therapeutic targets for childhood asthma.