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1.
Sci Rep ; 12(1): 7581, 2022 May 09.
Artigo em Inglês | MEDLINE | ID: mdl-35534522

RESUMO

The epidemiological association between exposure to particulate matter (PM10) and various respiratory and cardiovascular problems is well known, but the mechanisms driving these effects remain unclear. Neutrophils play an essential role in immune defense against foreign agents and also participate in the development of inflammatory responses. However, the role of these cells in the PM10 induced inflammatory response is not yet fully established. Thus, this study aims to evaluate the effect of PM10 on the neutrophil-mediated inflammatory response. For this, neutrophils from healthy adult human donors were in vitro exposed to different concentrations of PM10. The cell viability and cytotoxic activity were evaluated by MTT. LDH, propidium iodide and reactive oxygen species (ROS) were quantified by flow cytometry. Interleukin 8 (IL-8) expression, peptidyl arginine deiminase 4 (PAD4), myeloperoxidase (MPO), and neutrophil elastase (NE) expression were measured by RT-PCR. IL-8 was also quantified by ELISA. Fluorescence microscopy was used to evaluate neutrophil extracellular traps (NETs) release. The in vivo inflammatory responses were assessed in BALB/c mice exposed to PM10 by histopathology and RT-PCR. The analysis shows that PM10 exposure induced a cytotoxic effect on neutrophils, evidenced by necrosis and LDH release at high PM10 concentrations. ROS production, IL-8, MPO, NE expression, and NETs release were increased at all PM10 concentrations assessed. Neutrophil infiltration in bronchoalveolar lavage fluid (BALF), histopathological changes with inflammatory cell infiltration, and CXCL1 expression were observed in PM10-treated mice. The results suggest that lung inflammation in response to PM10 could be mediated by neutrophils activation. In this case, these cells migrate to the lungs and release pro-inflamatory mediators, including ROS, IL-8, and NETs. Thus, contributing to the exacerbation of respiratory pathologies, such as allergies, infectious and obstructive diseases.


Assuntos
Armadilhas Extracelulares , Neutrófilos , Animais , Modelos Animais de Doenças , Armadilhas Extracelulares/metabolismo , Humanos , Interleucina-8/metabolismo , Pulmão/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Neutrófilos/metabolismo , Material Particulado/metabolismo , Material Particulado/toxicidade , Espécies Reativas de Oxigênio/metabolismo
2.
Front Immunol ; 13: 823511, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35603144

RESUMO

Neutrophil extracellular traps (NETs) play important roles in hepatic ischemic reperfusion injury (IRI) and acute rejection (AR)-induced immune responses to inflammation. After liver transplantation, HMGB1, an inflammatory mediator, contributes to the development of AR. Even though studies have found that HMGB1 can promote NET formation, the correlation between NETs and HMGB1 in the development of AR following liver transplantation has not been elucidated. In this study, levels of serum NETs were significantly elevated in patients after liver transplantation. Moreover, we found that circulating levels of NETs were negatively correlated with liver function. In addition, liver transplantation and elevated extracellular HMGB1 promoted NET formation. The HMGB1/TLR-4/MAPK signaling pathway, which is initiated by HMGB1, participates in NET processes. Moreover, in the liver, Kupffer cells were found to be the main cells secreting HMGB1. NETs induced Kupffer cell M1 polarization and decreased the intracellular translocation of HMGB1 by inhibiting DNase-1. Additionally, co-treatment with TAK-242 (a TLR-4 inhibitor) and rapamycin more effectively alleviated the damaging effects of AR following liver transplantation than either drug alone.


Assuntos
Armadilhas Extracelulares , Proteína HMGB1 , Armadilhas Extracelulares/metabolismo , Rejeição de Enxerto , Proteína HMGB1/metabolismo , Humanos , Macrófagos do Fígado/metabolismo , Fígado/metabolismo , Neutrófilos , Receptor 4 Toll-Like/metabolismo
3.
Thromb Res ; 214: 132-137, 2022 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-35561448

RESUMO

BACKGROUND: The release of neutrophil extracellular traps (NETs) is the basis of immune-mediated thrombosis. Data on the clinical relevance of NETs in antiphospholipid syndrome-related thrombosis are scarce. OBJECTIVE: The aim of this study was to evaluate whether the NET regulator proteins PADI4, ELANE, and MPO are associated with thrombosis in APS. METHODS: A total of 152 thrombotic APS (t-APS) patients and 123 individuals without thrombosis (controls) were included. The following markers of NETs were evaluated: PADI4, ELANE, and MPO gene expression by qPCR and circulating levels of citrullinated histone H3 (H3cit) and myeloperoxidase-DNA complexes (MPO-DNA) by ELISA. RESULTS: The levels of circulating MPO-DNA and MPO mRNA expression and PADI4 mRNA expression were higher in t-APS patients than in controls. The mean differences were 0.05 OD (95% CI 0.01 to 0.09) in MPO-DNA levels, 1.07 AU (95% CI 0.20 to 1.93) for MPO mRNA and 0.20 AU (95% CI 0.03 to 0.36) in PADI4 mRNA fold-change. These differences were more pronounced in triple-positive patients, who had 56% increased levels of MPO-DNA, 44% increased MPO mRNA expression and 69% increased PADI4 mRNA expression compared to controls. Additionally, circulating MPO-DNA levels and MPO mRNA expression were higher in patients with recurrent thrombosis than in patients with incident thrombosis and controls. In recurrent thrombosis, levels of MPO-DNA were 43.8% higher and MPO mRNA expression was 2-fold higher than in controls. Levels of circulating MPO-DNA and PADI4 mRNA expression did not differ substantially between primary and secondary APS. CONCLUSION: Thrombotic APS was associated with increased NET formation, which was more pronounced among patients with poorer prognosis, such as those with triple antiphospholipid positivity and recurrent thrombosis. Our results provide evidence on the association of NETs and the severity of APS-related thrombosis.


Assuntos
Síndrome Antifosfolipídica , Armadilhas Extracelulares , Trombose , Síndrome Antifosfolipídica/complicações , Síndrome Antifosfolipídica/metabolismo , DNA , Armadilhas Extracelulares/metabolismo , Humanos , Neutrófilos/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Trombose/metabolismo
4.
Front Immunol ; 13: 841445, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35479063

RESUMO

The rate of complete remission of acute promyelocytic leukemia (APL) is currently over 90% because of the use of all-trans retinoic acid (ATRA) with arsenic trioxide (ATO). However, hemorrhagic mortality has emerged as the most significant barrier to APL-induced remission. Neutrophils extracellular traps (NETs/ETs) cause vascular leakage by damaging the integrity of endothelial cells. We have previously demonstrated that APL cells treated with ATRA/ATO undergo a cell death process, releasing extracellular chromatin, termed ETosis/NETosis. However, the mechanism underlying the involvement of ETs in endothelial injury in APL remain largely unknown. Here, we analysed the ability of mature and immature neutrophils to release ETs, and their interaction with platelets (PLTs) in APL. Importantly, the effect of ETs on vascular endothelium in APL was discussed. Our results showed that the ability of immature neutrophils to release ETs was impaired in APL, whereas mature neutrophils produced ETs, which were associated with activated PLTs. Moreover, ATRA+ATO induced immature neutrophil differentiation, as well as increased the release of ETs from mature neutrophils. The excessive ETs damaged endothelial cells, causing blood cell leakage. Removing ETs using DNase 1 alleviated endothelial damage and improved blood cells leakage. Our results indicate that vascular endothelial injury is at least partially associated with ETs in APL, and that targeting ETs production may be an effective approach for relieving vascular leakage and reducing the burden of bleeding in APL.


Assuntos
Armadilhas Extracelulares , Leucemia Promielocítica Aguda , Trióxido de Arsênio , Células Endoteliais/metabolismo , Armadilhas Extracelulares/metabolismo , Hemorragia , Humanos , Leucemia Promielocítica Aguda/metabolismo , Tretinoína/farmacologia
5.
Braz J Biol ; 84: e252936, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35475990

RESUMO

Neutrophil extracellular traps (NETs) were first reported as a microbicidal strategy for activated neutrophils. Through an immunologic response against several stimuli, neutrophils release their DNA together with proteins from granules, nucleus, and cytoplasm (e.g., elastase and myeloperoxidase). To date, NETs have been implicated in tissue damage during intense inflammatory processes, mainly when their release is dependent on oxygen radical generation. Flavonoids are antioxidant and anti-inflammatory agents; of these, quercetin is commonly found in our daily diet. Therefore, quercetin could exert some protective activity against tissue damage induced by NETs. In our in vitro assays, quercetin reduced NETs, myeloperoxidase (MPO), and elastase release from neutrophils stimulated with phorbol 12-myristate 13-acetate (PMA). The activity of these enzymes also decreased in the presence of quercetin. Quercetin also reduced the cytotoxic effect of NETs on alveolar cells (A549 cell line). Further, in silico assays indicated favorable interactions between quercetin and NET proteins (MPO and elastase). Overall, our results demonstrate that quercetin decreases deleterious cellular effects of NETs by reducing their release from activated neutrophils, and diminishing the enzymatic activity of MPO and elastase, possibly through direct interaction.


Assuntos
Armadilhas Extracelulares , Células A549 , Armadilhas Extracelulares/metabolismo , Humanos , Elastase Pancreática/metabolismo , Peroxidase , Quercetina/farmacologia , Acetato de Tetradecanoilforbol/metabolismo , Acetato de Tetradecanoilforbol/farmacologia
6.
Vet Microbiol ; 269: 109438, 2022 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-35468400

RESUMO

Interleukin 5 (IL-5) regulates the maturation, activation, proliferation and function of immune cells, and plays an important role in the inflammatory response induced by an allergy. However, its anti-pathogen effect is poorly understood currently, especially on pneumonia. Here, this study was designed to elucidate the immunological role of IL-5 in the infection of mice with Actinobacillus pleuropneumoniae (APP). We established an acute lung infection model of APP in IL-5 knockout mice (IL-5-/-) and wild-type mice (WT) through nasal infusion or intraperitoneal injection, compared the survival rate, clinical symptoms, lung bacterial load, proportion of various immune cells, immune molecular expression, and neutrophil germicidal ability through flow cytometry, RT-qPCR, ELISA and immunofluorescence. Compared to WT mice, the IL-5-/- mice had a lower survival rate, more severe clinical symptoms, significantly increased bacterial load, and inflammatory cell infiltration in the lung after APP infection. In an uninfected state, IL-5 deficiency decreased the number of M1 interstitial macrophages and CD14- monocytes, while after infection, IL-5 deficiency significantly reduced the M2 alveolar macrophages, and increased PMN-II cells in the lung. Furthermore, the expression of IL-10, IL-4, IL-33, TNF-α, iNOS in the lung was lower in IL-5-/- mice under an uninfected condition, and the secretion of IL-18 was significantly increased after infection. In addition, IL-5 deficiency decreased bactericidal ability by inhibiting the formation of neutrophil extracellular traps (NETs). Collectively, these results provide evidence that IL-5 can enhance the resistance of APP infection, and its anti-infection mechanism, implying new targets and ideas for APP or similar respiratory agents' prevention and treatment.


Assuntos
Infecções por Actinobacillus , Actinobacillus pleuropneumoniae , Actinobacillus , Armadilhas Extracelulares , Infecções por Mycoplasma , Mycoplasma , Pleuropneumonia , Doenças dos Roedores , Infecções por Actinobacillus/veterinária , Animais , Armadilhas Extracelulares/metabolismo , Interleucina-5/genética , Interleucina-5/metabolismo , Pulmão/microbiologia , Camundongos , Camundongos Knockout , Infecções por Mycoplasma/veterinária , Pleuropneumonia/microbiologia , Pleuropneumonia/veterinária
7.
Front Immunol ; 13: 844781, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35464399

RESUMO

Sepsis consists of life-threatening organ dysfunction resulting from a dysregulated response to infection. Recent studies have found that excessive neutrophil extracellular traps (NETs) contribute to the pathogenesis of sepsis, thereby increasing morbidity and mortality. Lysophosphatidic acid (LPA) is a small glycerophospholipid molecule that exerts multiple functions by binding to its receptors. Although LPA has been functionally identified to induce NETs, whether and how LPA receptors, especially lysophosphatidic acid receptor 3 (LPA3), play a role in the development of sepsis has never been explored. A comprehensive understanding of the impact of LPA3 on sepsis is essential for the development of medical therapy. After intraperitoneal injection of lipopolysaccharide (LPS), Lpar3 -/-mice showed a substantially higher mortality, more severe injury, and more fibrinogen content in the lungs than wild-type (WT) mice. The values of blood coagulation markers, plasma prothrombin time (PT) and fibrinogen (FIB), indicated that the Lpar3 -/- mice underwent a severe coagulation process, which resulted in increased thrombosis. The levels of NETs in Lpar3 -/- mice were higher than those in WT mice after LPS injection. The mortality rate and degree of lung damage in Lpar3 -/- mice with sepsis were significantly reduced after the destruction of NETs by DNaseI treatment. Furthermore, in vitro experiments with co-cultured monocytes and neutrophils demonstrated that monocytes from Lpar3 -/- mice promoted the formation of NETs, suggesting that LPA3 acting on monocytes inhibits the formation of NETs and plays a protective role in sepsis. Mechanistically, we found that the amount of CD14, an LPS co-receptor, expressed by monocytes in Lpar3 -/-mice was significantly elevated after LPS administration, and the MyD88-p65-NFκB signaling axis, downstream of toll-like receptor 4 signaling, in monocytes was overactivated. Finally, after an injection of the LPA3 agonist (2S)-1-oleoyl-2-methylglycero-3-phosphothionate (OMPT), the survival rate of mice with sepsis was improved, organ damage was reduced, and the production of NETs was decreased. This suggested the possible translational value and application prospects of (2S)-OMPT in the treatment of sepsis. Our study confirms an important protective role of LPA3 in curbing the development of sepsis by suppressing NETs production and thrombosis and provides new ideas for sepsis treatment strategies.


Assuntos
Armadilhas Extracelulares , Sepse , Trombose , Animais , Armadilhas Extracelulares/metabolismo , Fibrinogênio/metabolismo , Lipopolissacarídeos/metabolismo , Lipopolissacarídeos/toxicidade , Camundongos , Receptores de Ácidos Lisofosfatídicos/genética , Receptores de Ácidos Lisofosfatídicos/metabolismo , Sepse/metabolismo , Trombose/metabolismo
8.
Zhonghua Wei Zhong Bing Ji Jiu Yi Xue ; 34(2): 198-201, 2022 Feb.
Artigo em Chinês | MEDLINE | ID: mdl-35387730

RESUMO

Sepsis is defined as a life-threatening organ dysfunction caused by the dysregulated host response to infection, and is one of the main causes of death in intensive care unit (ICU) patients. Coagulation dysfunction runs through the pathophysiological progress of sepsis whose severity should be closely related to the prognosis of sepsis. Neutrophil extracellular traps (NETs) is a three-dimensional network structure with DNA as the skeleton and inlaid with various protein components. The excessive production of NETs can lead to sepsis-induced coagulopathy (SIC) by activating the coagulation system, inhibiting the anticoagulation system, resisting fibrinolysis, damaging vascular endothelial cells and the interaction of platelets. At present, the treatment of SIC is mainly symptomatic treatment, and there is no recognized effective anticoagulation strategy. Interventions for NETs and their components, and drugs for antiplatelets are expected to become new directions for disease treatment.


Assuntos
Transtornos da Coagulação Sanguínea , Armadilhas Extracelulares , Sepse , Anticoagulantes , Transtornos da Coagulação Sanguínea/etiologia , Células Endoteliais , Armadilhas Extracelulares/metabolismo , Humanos , Neutrófilos/metabolismo , Sepse/metabolismo
9.
Int J Mol Sci ; 23(7)2022 Mar 30.
Artigo em Inglês | MEDLINE | ID: mdl-35409152

RESUMO

The first description of a new form of neutrophil cell death distinct from that of apoptosis or necrosis was discovered in 2004 and coined neutrophil extracellular traps "(NETs)" or "NETosis". Different stimuli for NET formation, and pathways that drive neutrophils to commit to NETosis have been elucidated in the years that followed. Critical enzymes required for NET formation have been discovered and targeted therapeutically. NET formation is no longer restricted to neutrophils but has been discovered in other innate cells: macrophages/monocytes, mast Cells, basophils, dendritic cells, and eosinophils. Furthermore, extracellular DNA can also be extruded from both B and T cells. It has become clear that although this mechanism is thought to enhance host defense by ensnaring bacteria within large webs of DNA to increase bactericidal killing capacity, it is also injurious to innocent bystander tissue. Proteases and enzymes released from extracellular traps (ETs), injure epithelial and endothelial cells perpetuating inflammation. In the context of autoimmunity, ETs release over 70 well-known autoantigens. ETs are associated with pathology in multiple diseases: lung diseases, vasculitis, autoimmune kidney diseases, atherosclerosis, rheumatoid arthritis, cancer, and psoriasis. Defining these pathways that drive ET release will provide insight into mechanisms of pathological insult and provide potential therapeutic targets.


Assuntos
Doenças Autoimunes , Armadilhas Extracelulares , Autoimunidade , DNA/metabolismo , Células Endoteliais , Armadilhas Extracelulares/metabolismo , Humanos , Inflamação/patologia , Neutrófilos/metabolismo
10.
Front Immunol ; 13: 798022, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35432310

RESUMO

Background: Neutrophils form extracellular net-like structures called neutrophil extracellular traps (NETs). Emerging evidence has shown that cancer can induce NET formation; however, it is not fully understood how NETs influence cancer biology, and no consensus has been reached on their pro- or antitumor effects. A comprehensive analysis of the global NET-associated gene regulatory network is currently unavailable and is urgently needed. Methods: We systematically explored and discussed NET enrichment, NET-associated gene regulatory patterns, and the prognostic implications of NETs in approximately 8,000 patients across 22 major human cancer types. We identified NET-associated regulatory gene sets that we then screened for NET-associated regulatory patterns that might affect patient survival. We functionally annotated the NET-associated regulatory patterns to compare the biological differences between NET-related survival subgroups. Results: A gene set variation analysis (GSVA) based on 23 major component genes was used to calculate a metric called the NET score. We found that the NET score was closely associated with many important cancer hallmarks, particularly inflammatory responses and epithelial-to-mesenchymal transition (EMT)-induced metastasis. Higher NET scores were related to poor immunotherapy response. Survival analysis revealed that NETs had diverse prognostic impacts among various cancer types. The NET-associated regulatory patterns linked to shorter or longer cancer patient survival were distinct from each other. Functional analysis revealed that more of the NET-associated regulatory genes linked to poor cancer survival were associated with extracellular matrix (ECM) remodeling and pan-cancerous risk factors. SPP1 was found to be highly expressed and correlated with NET formation in cancers with poor survival. We also found that the co-upregulation of NET formation and SPP1 expression was closely linked to increased EMT and poor survival, that SPP1 influenced NET-induced malignant capacity, and that SPP1 overproduction induced a robust formation of metastatic-promoting NETs. Conclusion: NETs were common across cancers but displayed a diverse regulatory pattern and outcome readouts in different cancer types. SPP1 is potentially the key to NET-related poor outcomes.


Assuntos
Armadilhas Extracelulares , Neoplasias , Armadilhas Extracelulares/metabolismo , Humanos , Imunoterapia , Neoplasias/patologia , Neutrófilos/metabolismo , Prognóstico
11.
Elife ; 112022 Apr 26.
Artigo em Inglês | MEDLINE | ID: mdl-35471185

RESUMO

Neutrophil extracellular traps (NETs) are web-like chromatin structures composed by dsDNA and histones, decorated with antimicrobial proteins. Their interaction with dendritic cells (DCs) allows DC activation and maturation toward presentation of NET-associated antigens. Differently from other types of cell death that imply protein denaturation, NETosis preserves the proteins localized onto the DNA threads for proper enzymatic activity and conformational status, including immunogenic epitopes. Besides neutrophils, leukemic cells can release extracellular traps displaying leukemia-associated antigens, prototypically mutant nucleophosmin (NPMc+) that upon mutation translocates from nucleolus to the cytoplasm localizing onto NET threads. We tested NPMc+ immunogenicity through a NET/DC vaccine to treat NPMc-driven myeloproliferation in transgenic and transplantable models. Vaccination with DC loaded with NPMc+ NET (NPMc+ NET/DC) reduced myeloproliferation in transgenic mice, favoring the development of antibodies to mutant NPMc and the induction of a CD8+ T-cell response. The efficacy of this vaccine was also tested in mixed NPMc/WT bone marrow (BM) chimeras in a competitive BM transplantation setting, where the NPMc+ NET/DC vaccination impaired the expansion of NPMc+ in favor of WT myeloid compartment. NPMc+ NET/DC vaccination also achieved control of an aggressive leukemia transduced with mutant NPMc, effectively inducing an antileukemia CD8 T-cell memory response.


Assuntos
Armadilhas Extracelulares , Leucemia , Animais , Armadilhas Extracelulares/metabolismo , Camundongos , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Vacinação
12.
Ecotoxicol Environ Saf ; 237: 113558, 2022 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-35483150

RESUMO

Bisphenol A (BPA) is an endocrine disruptor, that can cause immune dysfunction. Cineole (CIN) has that effect of regulating immune function and resist oxidation. Neutrophil extracellular traps (NETs) are one of the ways to resist pathogen invasion. In order to explore the effects of BPA and CIN on the release of chicken NETs and the antagonistic effect of CIN, take chicken peripheral blood neutrophils as object of study, grouping as NC, CIN, BPA + CIN and BPA. SEM, flow cytometry, RT-PCR, Western-blot and other methods were used to detect related indicators. The results showed that BPA inhibited the activities of GPX, SOD and CAT, increased the contents of MDA and NO, increased the activity of iNOS. BPA exposure inhibited the expression of myeloperoxidase (MPO), neutrophil elastase (NE) and histone, and inhibited the release of NETs. BPA activated downstream apoptosis and necroptosis through the p38 mitogen-activated protein kinase (p38-MAPK) pathway, which increased the expression of cytochrome C (CytC), bcl-2 associated K protein gene (bak), cysteinyl aspartate specific proteinase 3 (caspase-3), cysteinyl aspartate specific proteinase 9 (caspase-9), receptor-interacting protein kinase 1 (RIPK1), receptor-interacting protein kinase 1 (RIPK3) and mixed lineage kinase domain-like protein (MLKL), decreased the expression of B-cell lymphoma-2 (bcl-2). However, the co-exposure of CIN and BPA partially recovered the release of NETs, alleviated BPA-induced oxidative stress, and inhibited the activation of p38-MAPK pathway, necroptosis, and mitochondrial apoptosis pathway. These results indicated that CIN modulated p38 pathway alleviated BPA-induced neutrophil necroptosis and apoptosis, and increased NETs formation.


Assuntos
Armadilhas Extracelulares , Apoptose/genética , Ácido Aspártico , Compostos Benzidrílicos , Eucaliptol/metabolismo , Armadilhas Extracelulares/metabolismo , Fenóis , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/genética , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo
13.
Vet Microbiol ; 268: 109427, 2022 May.
Artigo em Inglês | MEDLINE | ID: mdl-35405476

RESUMO

Streptococcus agalactiae (S. agalactiae) continues to be challenging for milk quality in some countries and leads to huge economic losses. A large number of neutrophils are recruited into inflammatory foci when S. agalactiae infection occurs, and most studies have focused on the interaction between neutrophil extracellular traps (NETs) and this bacterium in the context of human pathogenicity. However, there is little information on the NET formation mechanism induced by S. agalactiae in the context of bovine mastitis. Here, neutrophils isolated from BALB/c mice were infected with S. agalactiae SAG-FX17, and NET formation was evaluated. SAG-FX17 could induce NADPH oxidase-derived reactive oxygen species (NOX-ROS)-dependent NET formation, and 21.8% of bacteria could be eliminated by NETs via NET DNA and associated proteins. SAG-FX17 could induce the phosphorylation of p38 MAPK, ERK1/2 MAPK, and JNK/SAPK in neutrophils. However, only ERK1/2 MAPK was shown to play an important role in SAG-FX17-induced NET formation. Importantly, NOX-ROS production occurs upstream of ERK1/2 MAPK activation and then induces NET release. ERK1/2 MAPK phosphorylation can, in turn, enhance NOX-ROS generation, which further contributes to NET release and bacterial elimination. This study provides evidence of the molecular mechanism underlying serotype Ia S. agalactiae SAG-FX17-induced NET formation and the interaction between bacteria and NETs, and these findings will increase our knowledge about bacterial mastitis in dairy cattle and contribute to the prevention and clinical treatment of bovine mastitis.


Assuntos
Doenças dos Bovinos , Armadilhas Extracelulares , Mastite Bovina , Doenças dos Roedores , Animais , Bovinos , Doenças dos Bovinos/metabolismo , Armadilhas Extracelulares/metabolismo , Feminino , Sistema de Sinalização das MAP Quinases/fisiologia , Camundongos , NADPH Oxidases/metabolismo , Neutrófilos , Espécies Reativas de Oxigênio/metabolismo , Streptococcus agalactiae/metabolismo
14.
Sci Rep ; 12(1): 5978, 2022 Apr 08.
Artigo em Inglês | MEDLINE | ID: mdl-35396366

RESUMO

Presepsin, a biomarker discovered in Japan, has been clinically applied as a diagnostic aid for sepsis. Recently, however, it has been reported that presepsin levels are elevated in patients with severe systemic lupus erythematosus without infection, suggesting the existence of a production mechanism that does not involve bacterial phagocytosis. In this study, we aimed to elucidate the mechanism of presepsin production without bacterial phagocytosis and explore the clinical significance of presepsin. Neutrophil extracellular traps (NETs) were induced by Escherichia coli and phorbol myristate acetate (PMA) in neutrophils isolated from the peripheral blood of healthy subjects. NET induction alone did not increase presepsin levels, but co-culturing with monocytes significantly increased them. The addition of a NET formation inhibitor also suppressed presepsin levels, suggesting that presepsin production is greatly influenced by monocyte phagocytosis of NETs. Phagocytosis of NETs by THP-1 and U937 cells, which was induced by CD14 expression, also increased presepsin levels. This study suggests that presepsin can be used to assess the severity of inflammatory diseases, such as autoimmune diseases, and monitor treatment effects.


Assuntos
Armadilhas Extracelulares , Receptores de Lipopolissacarídeos , Monócitos , Fragmentos de Peptídeos , Armadilhas Extracelulares/metabolismo , Humanos , Receptores de Lipopolissacarídeos/metabolismo , Macrófagos/metabolismo , Monócitos/metabolismo , Neutrófilos/metabolismo , Fragmentos de Peptídeos/metabolismo , Fagocitose
15.
Am J Physiol Lung Cell Mol Physiol ; 322(5): L662-L672, 2022 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-35272488

RESUMO

Neutrophil extracellular trap (NET) formation is a key feature in sepsis. The aim of the present study was to examine the role of the actin cytoskeleton in regulating the expulsion of NETs. Actin-related protein 2/3 (Arp 2/3) complex is an important regulator of F-actin polymerization. Coincubation with CK666, a specific Arp 2/3 inhibitor, decreased 12-phorbol 13-myristate acetate-induced NET formation in vitro. CK666 not only abolished F-actin polymerization but also caused intracellular retention of NETs. Inhibition of Arp 2/3 reduced NET formation on circulating neutrophils and in the bronchoalveolar space in mice undergoing cecal ligation and puncture (CLP). Notably, treatment with CK666 attenuated CLP-induced neutrophil recruitment, edema formation, and tissue damage in the lungs. Moreover, Arp 2/3 inhibition decreased levels of C-X-C motif chemokine ligand 1 (CXCL-1) and interleukin-6 in the lung and plasma of septic animals. Taken together, this study shows that expulsion of NETs is regulated by the actin cytoskeleton and that inhibition of Arp 2/3-dependent F-actin polymerization not only decreases NET formation but also protects against pathological inflammation and tissue damage in septic lung injury. Thus, we suggest that targeting NET release is a novel and useful way to ameliorate lung damage in abdominal sepsis.


Assuntos
Armadilhas Extracelulares , Sepse , Proteína 2 Relacionada a Actina/metabolismo , Complexo 2-3 de Proteínas Relacionadas à Actina/metabolismo , Actinas/metabolismo , Animais , Modelos Animais de Doenças , Armadilhas Extracelulares/metabolismo , Pulmão/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Infiltração de Neutrófilos , Neutrófilos/metabolismo , Sepse/metabolismo
16.
Cell Mol Life Sci ; 79(4): 206, 2022 Mar 25.
Artigo em Inglês | MEDLINE | ID: mdl-35333979

RESUMO

Cerebral Cavernous Malformation (CCM) is a brain vascular disease with various neurological symptoms. In this study, we describe the inflammatory profile in CCM and show for the first time the formation of neutrophil extracellular traps (NETs) in rodents and humans with CCM. Through RNA-seq analysis of cerebellum endothelial cells from wild-type mice and mice with an endothelial cell-specific ablation of the Ccm3 gene (Ccm3iECKO), we show that endothelial cells from Ccm3iECKO mice have an increased expression of inflammation-related genes. These genes encode proinflammatory cytokines and chemokines, as well as adhesion molecules, which promote recruitment of inflammatory and immune cells. Similarly, immunoassays showed elevated levels of these cytokines and chemokines in the cerebellum of the Ccm3iECKO mice. Consistently, both flow cytometry and immunofluorescence analysis showed infiltration of different subsets of leukocytes into the CCM lesions. Neutrophils, which are known to fight against infection through different strategies, including the formation of NETs, represented the leukocyte subset within the most pronounced increase in CCM. Here, we detected elevated levels of NETs in the blood and the deposition of NETs in the cerebral cavernomas of Ccm3iECKO mice. Degradation of NETs by DNase I treatment improved the vascular barrier. The deposition of NETs in the cavernomas  of patients with CCM confirms the clinical relevance of NETs in CCM.


Assuntos
Armadilhas Extracelulares , Hemangioma Cavernoso do Sistema Nervoso Central , Animais , Proteínas Reguladoras de Apoptose/genética , Células Endoteliais/metabolismo , Armadilhas Extracelulares/metabolismo , Hemangioma Cavernoso do Sistema Nervoso Central/genética , Hemangioma Cavernoso do Sistema Nervoso Central/metabolismo , Hemangioma Cavernoso do Sistema Nervoso Central/patologia , Humanos , Inflamação/patologia , Proteínas de Membrana/metabolismo , Camundongos
17.
Exp Mol Pathol ; 125: 104754, 2022 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-35259405

RESUMO

Neutrophils stand sentinel over infection and possess diverse antimicrobial weapons, including neutrophil extracellular traps (NETs). NETs are composed of web-like extracellular DNA decorated with antimicrobial substances and can trap and eliminate invading microorganisms. Although phorbol 12-myristate 13-acetate (PMA) is a potent NET inducer, previous studies have demonstrated that not all neutrophils exhibit NET formation even if stimulated by PMA at high concentrations. This study first showed that some neutrophils stimulated by PMA displayed a swollen nucleus but not NET formation and that hypoxic environments suppressed the NET release. Next, characterization of PMA-stimulated neutrophils with a swollen nucleus was accomplished by differentiating between suicidal-type NETosis and apoptosis. Furthermore, the significance of the phenomenon was examined using formalin-fixed, paraffin-embedded human lung disease tissues with and without pneumonia. As a result, histone H3 citrullination, DNA outflow, propidium iodide labeling, resistance to DNase I, and suspended actin rearrangement were characteristics of PMA-stimulated neutrophils with a swollen nucleus distinct from neutrophils that underwent either suicidal-type NETosis or apoptosis. Neutrophils stimulated by PMA under hypoxic conditions secreted matrix metalloproteinase-9 cytotoxic to human lung-derived fibroblasts. Further, deposition of neutrophil-derived citrullinated histone H3+ chromatin substances in pulmonary lesions was greater in patients with pneumonia than in patients without pneumonia and positively correlated with hypoxia-inducible factor-1α expression. The collective findings suggested that neutrophils activated under hypoxic conditions could be putative modulators of hypoxia-related disease manifestations.


Assuntos
Armadilhas Extracelulares , Pneumopatias , Acetatos/metabolismo , DNA , Armadilhas Extracelulares/metabolismo , Histonas/metabolismo , Humanos , Hipóxia/metabolismo , Pneumopatias/metabolismo , Ácido Mirístico/metabolismo , Neutrófilos/metabolismo , Forbóis , Acetato de Tetradecanoilforbol/metabolismo , Acetato de Tetradecanoilforbol/farmacologia
18.
Acta Neuropathol Commun ; 10(1): 38, 2022 03 24.
Artigo em Inglês | MEDLINE | ID: mdl-35331340

RESUMO

INTRODUCTION: Neutrophil accumulation is a well-established feature of Alzheimer's disease (AD) and has been linked to cognitive impairment by modulating disease-relevant neuroinflammatory and vascular pathways. Neutrophils express high levels of the oxidant-generating enzyme myeloperoxidase (MPO), however there has been controversy regarding the cellular source and localisation of MPO in the AD brain. MATERIALS AND METHODS: We used immunostaining and immunoassays to quantify the accumulation of neutrophils in human AD tissue microarrays and in the brains of APP/PS1 mice. We also used multiplexed immunolabelling to define the presence of NETs in AD. RESULTS: There was an increase in neutrophils in AD brains as well as in the murine APP/PS1 model of AD. Indeed, MPO expression was almost exclusively confined to S100A8-positive neutrophils in both human AD and murine APP/PS1 brains. The vascular localisation of neutrophils in both human AD and mouse models of AD was striking and driven by enhanced neutrophil adhesion to small vessels. We also observed rare infiltrating neutrophils and deposits of MPO around plaques. Citrullinated histone H3, a marker of neutrophil extracellular traps (NETs), was also detected in human AD cases at these sites, indicating the presence of extracellular MPO in the vasculature. Finally, there was a reduction in the endothelial glycocalyx in AD that may be responsible for non-productive neutrophil adhesion to the vasculature. CONCLUSION: Our report indicates that vascular changes may drive neutrophil adhesion and NETosis, and that neutrophil-derived MPO may lead to vascular oxidative stress and be a relevant therapeutic target in AD.


Assuntos
Doença de Alzheimer , Armadilhas Extracelulares , Doença de Alzheimer/metabolismo , Animais , Encéfalo/metabolismo , Armadilhas Extracelulares/metabolismo , Humanos , Camundongos , Neutrófilos/metabolismo , Peroxidase/metabolismo
19.
Thromb Res ; 213: 78-83, 2022 05.
Artigo em Inglês | MEDLINE | ID: mdl-35306431

RESUMO

BACKGROUND: Neutrophil extracellular traps (NETs) are formed by DNA, histones and proteolytic enzymes, and are produced by activated neutrophils through different mechanisms. In turn, NETs can activate platelets and coagulation cascade favoring thrombotic processes. The aims of this study were to analyze levels and kinetics of NETs in ST-segment elevation myocardial infarction (STEMI) patients and correlate them with antithrombotic therapy and cardiovascular outcomes at follow-up. METHODS: 150 consecutive STEMI patients referred to primary percutaneous coronary intervention (pPCI) were included. Citrate anticoagulated blood was extracted immediately before pPCI, 30 min and 24 h after the procedure. As markers of NETS cell free DNA (cfDNA), nucleosomes and citrullinated Histone 3 (citH3) were determined. 46 healthy subjects were included as controls. Patients were follow-up for 1.4 ± 0.56 years. RESULTS: Before pPCI, NETs markers were elevated in STEMI patients compared to healthy controls (p < 0.05); these increased significantly 30 min post pPCI (p ≤ 0.001) and decreased at 24 h but remained elevated compared with the control group (p < 0.05). Patients treated with bivalirudin presented a lower increase of NETs 30 min post pPCI compared to patients treated with heparin (p < 0.05). Cardiovascular risk factors or type of stent implanted did not modify NETs levels. Cit3H (HR = 3.74; 95%CI 1.05-13.4; p = 0.042) and left ventricular ejection fraction ≤35% (HR = 6.84; 95%CI 2-23; p = 0.002) were independent predictors of composite endpoint of myocardial infarction, stroke, stent thrombosis and/or cardiovascular-cause death. CONCLUSIONS: NETs were elevated in STEMI patients, increased by pPCI and decreased thereafter. One of the most specific NETs markers was associated with cardiovascular outcomes.


Assuntos
Armadilhas Extracelulares , Infarto do Miocárdio , Intervenção Coronária Percutânea , Infarto do Miocárdio com Supradesnível do Segmento ST , Armadilhas Extracelulares/metabolismo , Fibrinolíticos , Humanos , Infarto do Miocárdio/metabolismo , Infarto do Miocárdio com Supradesnível do Segmento ST/tratamento farmacológico , Infarto do Miocárdio com Supradesnível do Segmento ST/cirurgia , Volume Sistólico , Resultado do Tratamento , Função Ventricular Esquerda
20.
Sci Rep ; 12(1): 4136, 2022 03 09.
Artigo em Inglês | MEDLINE | ID: mdl-35264677

RESUMO

Accumulation of uric acid (UA) during muscular trauma is a factor involved in the development of muscle hyperalgesia. Neutrophil extracellular traps (NETs), DNA-based reticular structures to capture UA, play a central role in the pain onset of gout attacks; however, the involvement of NETs via the elevation of local UA level in muscle hyperalgesia due to injuries from muscle overuse remains unknown. The triceps surae muscles (TSMs) in the unilateral hindlimb of mice were electrically stimulated to induce excessive muscle contraction. Mechanical withdrawal thresholds, tissue UA levels, neutrophil recruitment, and protein amount of citrullinated histone 3 (citH3), a major marker of NETs, were investigated. Furthermore, whether neutrophil depletion, extracellular DNA cleavage, and administration of the urate-lowering agent febuxostat improved muscle hyperalgesia caused by NET formation was examined. CitH3 expression upon neutrophil recruitment was significantly increased in the stimulated TSMs with increased tissue UA levels, whereas febuxostat administration improved muscle hyperalgesia with decreased citH3 and tissue UA levels, as observed in neutrophil depletion and extracellular DNA digestion. The underlying mechanism of muscle hyperalgesia associated with locally recruited neutrophils forming NETs due to increased tissue UA levels potentially plays a significant role in creating a vicious circle of muscle pain.


Assuntos
Armadilhas Extracelulares , Animais , DNA/metabolismo , Modelos Animais de Doenças , Armadilhas Extracelulares/metabolismo , Febuxostat , Histonas/metabolismo , Hiperalgesia/tratamento farmacológico , Hiperalgesia/metabolismo , Camundongos , Músculos , Neutrófilos/metabolismo , Ácido Úrico/metabolismo
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