Number Of Neutrophil Extracellular Traps Research Articles

Fibroblast Growth Factor 21 Suppressed Neutrophil Extracellular Traps Induced by Myocardial Ischemia/Reperfusion Injury via Adenosine Monophosphate-Activated Protein Kinase.

Previous investigations have established the anti-inflammatory properties of fibroblast growth factor 21 (FGF21). However, the specific mechanism through which FGF21 mitigates myocardial ischemia/reperfusion (I/R) injury by inhibiting neutrophil extracellular traps (NETs) remains unclear. A mice model of myocardial I/R injury was induced, and myocardial tissue was stained with immunofluorescence to assess NETs. Serum NETs levels were quantified using a PicoGreen kit. In addition, the expression levels of adenosine monophosphate (AMP)-activated protein kinase (AMPK) and FGF21 were evaluated by Wes fully automated protein blotting quantitative analysis system. Moreover, a hypoxia/reoxygenation (H/R) model was established using AMPK inhibitor and agonist pretreated H9c2 cells to further explore the relationship between FGF21 and AMPK. Compared with the control group, serum NETs levels were significantly higher in I/R mice, and a large number of NETs were formed in myocardial tissues (97.63 ± 11.45 vs. 69.65 ± 3.33, P < 0.05). However, NETs levels were reversed in FGF21 pretreated mice (P < 0.05). Further studies showed that FGF21 enhanced AMPK expression, which was significantly increased after inhibition of AMPK and decreased after promotion of AMPK (P < 0.05). FGF21 may exert cardioprotective effects by inhibiting I/R injury-induced NETs via AMPK.

Neutrophil Extracellular Traps in Myocardial Tissue Drive Cardiac Dysfunction and Adverse Outcomes in Patients With Heart Failure With Dilated Cardiomyopathy.

The immune systems and chronic inflammation are implicated in the pathogenesis of dilated cardiomyopathy (DCM) and heart failure. However, the significance of neutrophil extracellular traps (NETs) in heart failure remains to be elucidated. We enrolled consecutive 62 patients with heart failure with idiopathic DCM who underwent endomyocardial biopsy. Biopsy specimens were subjected to fluorescent immunostaining to detect NETs, and clinical and outcome data were collected. Ex vivo and in vivo experiments were conducted. The numbers of NETs per myocardial tissue area and the proportion of NETs per neutrophil were significantly higher in patients with DCM compared with non-DCM control subjects without heart failure, and the numbers of NETs were negatively correlated with left ventricular ejection fraction. Patients with DCM with NETs (n=32) showed lower left ventricular ejection fraction and higher BNP (B-type natriuretic peptide) than those without NETs (n=30). In a multivariable Cox proportional hazard model, the presence of NETs was independently associated with an increased risk of adverse cardiac events in patients with DCM. To understand specific underlying mechanisms, extracellular flux analysis in ex vivo revealed that NETs-containing conditioned medium from wild-type neutrophils or purified NET components led to impaired mitochondrial oxygen consumption of cardiomyocytes, while these effects were abolished when PAD4 (peptidyl arginine deiminase 4) in neutrophils was genetically ablated. In a murine model of pressure overload, NETs in myocardial tissue were predominantly detected in the acute phase and persisted throughout the ongoing stress. Four weeks after transverse aortic constriction, left ventricular ejection fraction was reduced in wild-type mice, whereas PAD4-deficient mice displayed preserved left ventricular ejection fraction without inducing NET formation. NETs in myocardial tissue contribute to cardiac dysfunction and adverse outcomes in patients with heart failure with DCM, potentially through mitochondrial dysfunction of cardiomyocytes.

The protective mechanism of human umbilical cord mesenchymal stem cell-derived exosomes against neutrophil extracellular trap-induced placental damage

IntroductionPreeclampsia (PE) is a pregnancy-specific complication. Its etiology and pathogenesis remain unclear. Previous studies have shown that neutrophil extracellular traps (NETs) cause placental dysfunction and lead to PE. Human umbilical cord mesenchymal stem cell-derived exosomes (hUCMSC-EXOs) have been widely used to treat different diseases. We investigated whether hUCMSC-EXOs can protect against NET-induced placental damage. MethodsNETs were detected in the placenta by immunofluorescence. The impact of NETs on cellular function and the effect of hUCMSC-EXOs on NET-induced placental damage were evaluated by 5-ethynyl-20-deoxyuridine (EdU) cell proliferation, lactate dehydrogenase (LDH), reactive oxygen species (ROS), and cell migration, invasion and tube formation assays; flow cytometry; and Western blotting. ResultsThe number of placental NETs was increased in PE patients compared with control individuals. NETs impaired the function of endothelial cells and trophoblasts. These effects were partially reversed after N-acetyl-L-cysteine (NAC; ROS inhibitor) or DNase I (NET lysing agent) pretreatment. HUCMSC-EXOs ameliorated NET-induced functional impairment of endothelial cells and trophoblasts in vitro, partially reversed NET-induced inhibition of endothelial cell and trophoblast proliferation, and partially restored trophoblast migration and invasion and endothelial cell tube formation. Exosomes inhibited ROS production in these two cell types, suppressed p38 mitogen-activated protein kinase (p38 MAPK) signaling activation, activated extracellular signal-regulated kinase 1/2 (ERK1/2) signaling, and modulated the Bax, Bim, Bcl-2 and cleaved caspase-3 levels to inhibit apoptosis. DiscussionHUCMSC-EXOs can reverse NET-induced placental endothelial cell and trophoblast damage, possibly constituting a theoretical basis for the treatment of PE with exosomes.

Zixue Powder attenuates septic thrombosis via reducing neutrophil extracellular trap through blocking platelet STING activation

Ethnopharmacological relevanceMicrothrombosis is commonly seen in sepsis and COVID-19. Zixue Powder (ZXP) is a traditional Chinese herbal formula with the potential to treat microvascular and infectious diseases. However, the role and mechanism of ZXP in sepsis-associated thrombosis remain unclear. Aim of the studyInvestigating the therapeutic effectiveness and underlying mechanisms of ZXP in septic thrombosis. Materials and methodsZXP's compositions were examined with UPLC-QTOF-MS. The efficacy of ZXP on sepsis-induced thrombosis was assessed through various methods: liver tissue pathology was examined using hematoxylin-eosin staining, platelet count was determined by a blood cell analyzer, and an enzyme-linked immunosorbent assay (ELISA) was used to detect the levels of serum tissue factor (TF), thromboxane B2 (TXB2), D-Dimer, and plasminogen activator inhibitor-1 (PAI-1). Neutrophil extracellular traps (NETs) were localized and expressed in liver tissues by immunofluorescence, and the number of NETs in peripheral blood was evaluated by ELISA, which measured the quantity of cf-DNA and MPO-DNA in serum. Platelet P-selectin expression and platelet-neutrophil aggregation were measured by flow cytometry, and plasma P-selectin expression was measured by ELISA. Furthermore, the mechanism of the stimulator of interferon genes (STING) signaling pathway in ZXP's anti-sepsis thrombosis effect was investigated using the STING agonist, Western blot experiments, and immunoprecipitation experiments. ResultsUPLC-QTOF-MS identified 40 chemical compositions of ZXP. Administration of ZXP resulted in significant improvements in liver thrombosis, platelet counts, and levels of TXB2, TF, PAI-1, and D-Dimer in septic rats. Moreover, ZXP inhibited NETs formation in both liver tissue and peripheral blood. Additionally, ZXP decreased the levels of P-selectin in both platelets and plasma, as well as the formation of platelet-neutrophil aggregates, thereby suppressing P-selectin-mediated NETs release. Immunoprecipitation and immunofluorescence staining experiments revealed that ZXP attenuated P-selectin secretion by inhibiting STING-mediated assembly of platelet soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) complex, ultimately preventing inhibition of NETs formation. ConclusionOur study showed that ZXP effectively mitigates platelet granule secretion primarily through modulation of the STING pathway, consequently impeding NET-associated thrombosis in sepsis. These findings offer valuable insights for future research on the development and application of ZXP.

Neutrophil extracellular traps in the anti-infectious defense of human organism

Background. Neutrophil extracellular traps (NETs) are net-like structures that have been investigated in inflammatory diseases. However, the presence of NETs in infected persons without clinical symptoms has not been yet studied.Aim. To reveal NETs in healthy persons during and after the H1N1 influenza pandemic as well as to study the functional activity of NETs.Materials and methods. The study included two groups of volunteers (n = 10 in each group) aged 20–25 years. The first group of volunteers was examined in the absence of acute diseases during one month before the study and in the absence of chronic diseases in the medical history. Volunteers of the second group were in contact with patients with influenza, but did not get sick. The comparative study also included patients with acute inflammation in the abdominal cavity (appendicitis, cholecystitis, abscess; 12 patients) and 9 patients with non-specific ulcerative colitis. Neutrophils were isolated from the blood by the traditional method of Ficoll density centrifugation. The number, morphology, and functional activity of NETs were determined (by capture of Klebsiella pneumoniae). SYBR Green I-based fluorescence microscopy was used to visualize and quantify NETs.Results. In healthy volunteers who were not in contact with infected patients, spontaneous NETs formation did not occur. Neutrophils of persons who were in contact with infected patients spontaneously formed NETs. In this case the number of NETs reached 8.58 ± 0.51%, and the size of NETs amounted to 39.68 ± 3.52 µm. NETs effectively captured cells of the tested microorganism, which was accompanied by retraction of network fibers and transformation of the network structure into a cloud-like one, which retained 89.38 ± 5.86 microbial cells. For comparison, the NETs in patients with acute inflammation in the abdominal cavity captured and bound 20.2 ± 1 .67 microbial cells and with non-specific ulcerative colitis – 5.53 ± 0.34 cells.Conclusion. High binding capacity of NETs is a factor contributing to effective defense of the body against the development of an infectious disease with manifested clinical symptoms.

The mechanism of intravenous immunoglobulin (IVIG) in vascular endothelial injury in kawasaki disease based on neutrophil extracellular traps

This study explores the role of Neutrophil extracellular traps (NETs) in kawasaki disease (KD)-induced vascular inflammatory injury and the protective effect and mechanism of IVIG on vascular endothelial damage. A total of 37 children diagnosed with KD and admitted to Dongguan maternal and Child Health Care Hospital between March 2020 and June 2022 were included in the study. The children were divided into different groups based on their treatment and the presence or absence of coronary artery damage: IVIG treatment group (KDIVIG group), subgroup with coronary artery damage (KDCAL group), and subgroup without coronary artery damage (KDNCAL group), and a Control group consisting of 9 children who underwent surgical treatment. Flow cytometry was used to detect the proportion of neutrophils and the number of NETs in peripheral blood. It was found that the proportion of neutrophils in the peripheral blood of the acute KD group significantly increased with the presence of NETs. RT-PCR and ELISA detection showed that the levels of inflammatory factors TNF-α, IL-6 and CitH3 were abnormally elevated in this acute KD group, and the CAL group exhibited higher proportions of neutrophils and NETs-related markers compared to the NCAL group, while the IVIG group had significantly decreased proportions of neutrophils. PMA culture of neutrophils induced an increase expression of NETs marker protein, the content of NETs cfDNA increased. NETs culture could promote the secretion of TNF-α, whereas IVIG cultured cells inhibited the secretion of TNF-α. Finally, HCAEC cells were cultured with different levels of TNF-α, and the function of HCAEC cells was assessed using CCK8, scratch assay and flow cytometry. The high expression of TNF-α in the NETs group inhibited the proliferation and migration of HUVEC cells and enhanced their apoptosis. In contrast, the IVIG culture group exhibited similar effects to the TNF-α monoclonal antibody, as it inhibited HUVEC cell apoptosis and improved their viability by reducing TNF-α expression. Total protein was extracted from the cells using nano-magnetic beads, and RT-PCR and western blot detection indicated that the increase of TNF-α expression could increase the phosphorylation of NF-κB and and the expression of MMP-9. However, when TNF-α was inhibited by IVIG and TNF-α monoclonal antibody culture, the activity of NF-κB/MMP-9 athway was decreased. Therefore, IVIG may inhibit the production of NETs in KD children, thereby reducing TNF-α/NF-NF-κB/MMP-9 mediated inflammatory response process and protecting the function of vascular endothelial cells.

Metformin may improve the outcome of patients with colorectal cancer and type 2 diabetes mellitus partly through effects on neutrophil extracellular traps

BackgroundAlthough metformin reduces the risk of cancer-related mortality in patents with type 2 diabetes, the mechanism of its anti-cancer effects has not been fully understood.MethodImpact of metformin on survival was examined in patients who underwent curative colectomy for colorectal cancer (CRC). The effects of metformin in neutrophil extracellular traps (NETs) were examined with in-vitro experiments and multiplex immunohistochemistry of surgically resected CRC specimens.ResultsPrior intake of metformin prolonged relapse-free (P = 0.036) and overall survival (P = 0.041) in 289 patients with T2DM to the comparable levels to those of 1576 non-diabetic patients. Metformin reduced the production of NETs stimulated with lipopolysaccharide or HT-29 colon cancer cells to 60% of control. Neutrophils markedly suppressed the chemotactic migration of activated T cells in an NET-dependent manner, which was reversed by metformin treatment up to approximately half of the migration without neutrophils. Immunohistochemical analysis revealed a significant association between metformin intake and a reduction in the numbers of tumor-associated neutrophils (TANs) and NETs. Simultaneously, metformin intake was found to increase the presence of CD3(+) and CD8(+) tumor-infiltrating T cells (TILs), particularly at the tumor-invasion front, especially in areas with fewer TANs and NETs.ConclusionMetformin suppresses the diabetes-associated enhancement of NET formation, which can augment the infiltration of TILs in CRC tissues. The anti-tumor effect of metformin in patients with T2DM may be, at least partly, attributable to the inhibition of NETs.

Abstract 12043: Neutrophil Extracellular Traps in Heart Tissue Drive Cardiac Dysfunction and Adverse Outcomes in Dilated Cardiomyopathy

Background: Neutrophil extracellular traps (NETs) have emerged as crucial contributors to cardiovascular diseases, but the significance of NETs in heart failure remains unclear. Aims: We aimed to clarify the relevance of NETs in myocardial tissue in heart failure. Methods and Results: We studied consecutive 62 patients with idiopathic dilated cardiomyopathy (DCM) who underwent endomyocardial biopsy. Using immunohistochemistry, NETs were identified in heart tissue by detecting the colocalization of citrullinated histone H3, neutrophil elastase, and DAPI. DCM patients had significantly higher numbers of NETs per tissue area and per neutrophil compared to control subjects without heart failure (n=11, Figure A ) (P &lt; 0.05 and P &lt; 0.05, respectively). When categorizing DCM patients based on the presence or absence of NETs, patients with NETs (n=32) had lower left ventricular ejection fraction and higher B-type natriuretic peptide levels than those without NETs (P &lt; 0.05 and P &lt; 0.05, respectively). Kaplan-Meier analysis revealed that NETs-positive DCM patients had lower event-free survival rates from the composite of cardiac events including cardiac deaths, decompensated heart failure, and left ventricular assist device implantation over a median 768-day follow-up ( Figure B ). In a multivariable Cox proportional hazard model, the presence of NETs independently associated with the risk of adverse cardiac events (hazard ratio, 5.96; P &lt; 0.05). Ex vivo analysis revealed that NETs-containing conditioned medium from wild-type neutrophils impaired mitochondrial oxygen consumption in mouse adult cardiomyocytes (P &lt; 0.05), while genetic ablation of peptidyl arginine deiminase 4, a molecule essential for NETs formation, abolished this effect. Conclusion: NETs in myocardial tissue contribute to cardiac dysfunction and adverse outcomes in DCM patients, potentially through mitochondrial dysfunction of cardiomyocytes.

Abstract 1268: The effect of metformin for neutrophil extracellular traps (NETs) on tumor immune microenvironment of colorectal cancer with type 2 diabetes mellitus

Abstract Background: Although metformin (Met) is well known to suppress tumor growth, the detailed mechanisms of its anti-tumor effect remain unknown. In recent years, it has been pointed out that tumor-associated neutrophils (TAN) can produce neutrophil extracellular traps (NETs) in the tumor microenvironment (TME) which can tumor invasion and metastasis. Here, in this study, we investigated the effect of Met on tumor-associated NETs and its effect on the prognosis of cancer patients. Method: We retrospectively examined the outcome of colorectal cancer (CRC) patients with type 2 diabetes mellitus (T2DM) who received curative surgery in Jichi Medical University Hospital and asked the impact of Met intake on their outcome. In addition, we performed multicolor immunohistochemistry to examine the phenotypes of tumor-infiltrating lymphocytes (TIL), TAN and NETs in surgically removed tissues specimens in 40 (colorectal cancer) CRC patients who had taken Met before surgery and propensity score-matched 40 patients who had not. Results: A total of 1858 patients underwent curative colectomy from April 2010 to March 2021. Among them, 283 patients had T2DM at the time of surgery, and 62 patients had been treated with drugs including Met. The postoperative disease-free survival rate (DFS) was significantly improved in the Met intake group compared with non-intake group (HR=0.21, p&amp;lt;0.01). The number of CD66b(+) TANs significantly decreased in Met-intake group (Median(M)=169 (36-553)/mm2 vs 99 (34-322)/mm2, p&amp;lt;0.01). The number of tumor-associated NETs defined as CD66b(+) cit-H3(+) cells significantly decreased in Met-intake group (M=36 (8-272)/mm2 vs 20 (0-104)/mm2, p&amp;lt;0.01), and tumor-associated NETs/TANs ratio significantly decreased in Met-intake group (29.9% vs 19.8%, p&amp;lt;0.01). The number of CD3(+) TILs significantly increased in Met intake group (M=122 (28-257)/mm2 vs 142 (48-386)/mm2, p&amp;lt;0.05). The number of CD8(+) TILs significantly increased in Met-intake group (M=60 (14-180)/mm2 vs 99 (52-318)/mm2, p&amp;lt;0.01), and CD8/CD3 ratio significantly increased in Met-intake group (51.2% vs 72.1%, p&amp;lt;0.01). In addition, there was a negative correlation between TANs or tumor-associated NETs and CD8 positive TILs in all 80 patients (r=-0.26, p&amp;lt;0.05). Conclusion: Met can inhibit neutrophil infiltration and NETs formation while promote the infiltration of cytotoxic T cells in TME, which may lead to the improvement of the outcome of the patients with CRC. Citation Format: Akira Saito, Hideyuki Ohzawa, Yuki Kaneko, Kohei Tamura, Yurie Futoh, Kazuya Takahashi, Yuki Kimura, Rie Kawashima, Hideyo Miyato, Joji Kitayama, Naohiro Sata. The effect of metformin for neutrophil extracellular traps (NETs) on tumor immune microenvironment of colorectal cancer with type 2 diabetes mellitus [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1268.

Post-COVID syndrome is associated with increased extracellular purine bases and neutrophil extracellular traps in the blood plasma

Post-COVID syndrome is characterized by fatigue, reduced exercise tolerance, muscle and joint pain, and psychoemotional disorders. In the development of a generalized body response in a viral infection, abnormal defense responses are of great importance. We studied neutrophils, neutrophil extracellular traps (NETs), DNA degradation products (purine nitrogenous bases, PNBs), and traditional biochemical parameters.Aim. To determine biochemical parameters and the number of NETs and PNBs in the peripheral blood of patients with post-COVID syndrome.Materials and methods. The study included outpatients (n = 21) aged 18–59 years (36 [27 ÷ 50]). The control group consisted of 20 individuals aged 18–59 years (38.5 [29 ÷ 51.5]) without a past medical history of the coronavirus infection. All patients underwent a physical examination, their medical history was assessed, and the level of NETs and PNBs in the venous blood was determined.Results. 11 patients had a mild form of the disease in their past medical history, 7 – moderate, and 3 – severe. The most common symptoms in the patients were fatigue, headache, epigastric pain, dizziness, and joint pain. Hair loss and dyspnea were less common. The concentration of NETs and PNBs was higher in the patients with post-COVID syndrome than in the control group (p &lt; 0.05). We detected NETs in the patients with post-COVID syndrome only in the form of filamentous structures. The concentration of extracellular purine bases in the blood of the patients with post-COVID syndrome was the highest in patients with moderate and severe acute periods. In patients with a mild acute period, the concentration of PNBs was 7.38 [0.0 ÷ 60.7] mg / ml, and in patients with moderate and severe acute periods – 19.15 [0.0 ÷ 33.5] and 34.19 [3.35 ÷ 70.0] mg / ml, respectively.Conclusion. Extracellular purine bases in concentrations capable of causing secondary alteration of cells are found in the peripheral blood of patients with post-COVID syndrome. Post-COVID syndrome is accompanied by the formation of filamentous NETs in the blood of patients.

Association of Neutrophil Extracellular Traps with Plaque Instability in Patient with Carotid Artery Stenosis

Vulnerable carotid plaques are related to cerebral thromboembolic and ischemic events. Neutrophil extracellular traps (NETs) can induce endothelial dysfunction and induce inflammation and coagulation. The aim of the present study was to investigate NETs in patients with carotid artery plaques. Carotid plaques were collected by carotid endarterectomy (CEA) from 26 symptomatic and 8 asymptomatic patients between August 2017 and January 2021. The specimens were stained with hematoxylin-eosin and Elastica-van Gieson. Immunohistochemistry was performed staining by CD31 for identifying endothelial cells. NETs were detected by digoxigenin-labeled antihistone H3 (HH3) (citrulline R2+R8+R17). The relationships between the presence of NETs and patient profile and histopathological findings were assessed. HH3-positive cells were detected in 17 (asymptomatic=2 symptomatic=15) of 34 carotid plaques (median=9.7/mm). The number of NETs was correlated with the number of diffusion-weighted imaging high-intensity lesions [P=0.01], plaque rupture [P=0.001], intraplaque hemorrhage [P=0.02], intra luminal thrombus [P=0.001], and thin fibrous cap [P=0.001]. The presence of NETs was associated with the instability of carotid plaques, intraluminal thrombus, which may lead to subsequent cerebral infarction. Clarifying the roles of NETs in carotid plaques may improve the treatment of carotid artery disease.

Formation of Neutrophil Extracellular Traps when Modeling Plague Infection in Mice Immunized with Yersinia pestis EV NIIEG

The purpose of the study was to determine the effect of Yersinia pestis EV NIIEG on the process of neutrophil extracellular traps formation in vivo when modeling plague infection and assess their contribution to antiplague protection.Materials and methods. BALB/c mice, which were immunized subcutaneously with the Y. pestis EV NIIEG vaccine strain, were used in the study. Animals were infected with a virulent strain Y. pestis 231 at a dose of 20 LD50 (103 CFU). To evaluate the contribution of neutrophil extracellular traps (NETs) to antibacterial protection, an experimental model was used based on fermenting NETs in the abdominal cavity of mice with nuclease. To calculate the number of NETs in peritoneal exudate (PE) fluorescent microscopy was applied. Phagocytic activity of PE cells was determined by flow cytometry. Bactericidal effect of NETs was recorded using bacteriological method.Results and discussion. In pre-immunized mice, the process of NETs formation in response to the reintroduction of plague microbe living cells was 5 times more intense than in intact animals and was accompanied by a significant increase in the killing of Y. pestis cells in PE. The use of micrococcus nuclease in the experiment for fermentation of the NETs, produced in the body of immunized animals, provided evidence of NET participation in conferring anti-infective protection against plague infection. Thus, the established fact of the NET formation in case of Y. pestis infection of mice immunized with Y. pestis EV NIIEG vaccine strain and the influence of this process on the effectiveness of protection against plague is the basis for further clarifying the immunopathogenetic role of neutrophil granulocytes in plague.

Neutrophil extracellular traps and echogenicity of carotid atherosclerotic plaques

Aim.To study the association between the number of neutrophil extracellular traps (NETs) formed by circulating neutrophils and echogenicity of carotid atherosclerotic plaques.Materials and Methods.We enrolled 72 patients (40-64 years of age) without the clinical signs of atherosclerotic vascular disease, who then underwent ultrasound duplex scanning of carotid arteries. Grayscale median analysis of the images was performed using Adobe Photoshop CS6 software. Neutrophils were stained and examined by means of light microscopy to count NETs.Results.Carotid atherosclerotic plaques were found in 69.4% of asymptomatic patients. Echogenicity of atherosclerotic plaques inversely correlated with the number of NETs (r = - 0.310; p = 0.029). The number of NETs in patients with echolucent plaques (GSM &lt; 60) was significantly higher in comparison with echogenic plaques (GSM ≥ 60, p = 0.042).

Влияние имплантационных материалов, созданных на основе внеклеточного матрикса ксенокости лошади и быка, на формирование экстрацеллюлярных нейтрофильных ловушек (экспериментальное исследование)

Purpose To study the effect of osteoplastic materials based on the extracellular xenomatrix of bovine and equine bone tissue on the formation of neutrophil extracellular traps (NETs) in the peripheral blood of rabbits in the early post-operative period after implantation. Materials and methods The study was carried out on 18 male rabbits of the Soviet Chinchilla breed, aged from 8 months to 1.2 years, weighing from 3.0 to 4.5 kg. A perforated bone defect of a cylindrical shape measuring 2 x 6 mm in the distal metaphysis of the right and left femurs was modeled in the animals. The rabbits were divided into three groups, six animals each. In group I, the bone defect was left unfilled; in group II, the defect was filled with a bovine bone tissue xenomatrix, and an equine bone tissue xenomatrix was implanted in group III animals. The implantation material had the appearance of a yellowish crumb with a particle size of 0.5– 1 mm. Blood smears stained according to Romanovsky-Giemsa were used for counting extracellular neutrophil traps (NETs). The percentage of neutrophils that passed the stages of nuclear transformation and emitted free chromatin into the extracellular space in the form of network-like structures was calculated. Results On days 3–7 of the experiment, the number of NETs increased in the early stages of NETosis in all groups. There were no significant differences between the groups. In group I, on days 7 and 14, the number of early forms of NETs (stages 1a and 1b) returned to the values of the preoperative period. In groups II and III, normalization of NETs (stage 1a) did not occur, and the content of NETs (stage 1b) returned to the initial level only by day 30 of the experiment. On days 3, 7, 14, the number of mature NETs increased in all groups. The highest values were noted in group II, where the bovine xenogeneic matrix was implanted. Conclusion Implantation materials based on the extracellular matrix of equine and bovine xenogeneic bone stimulate excessive formation of early NETs on days 14–30 of the experimental period in response to xenotransplantation. Xenomaterials of bovine bone tissue, in comparison with xenomaterials of equine bone tissue, induce a more pronounced inflammatory reaction in the nearest time after defect filling, which is manifested by higher production of mature NETs on days 3–14 of the experiment.

Quantitative Characteristics of Neutrophil Extracellular Traps in Tumor Tissues in Colorectal Cancer

BACKGROUND: Recently, the role of neutrophil extracellular traps (NETs) in oncopathology, including colorectal cancer (CRC), has become a subject of discussion. Literature review showed that in CRC, there are separate experimental and clinical studies devoted to assess the ability of neutrophils to form NET. A methodological approach, common to these studies, was that the ability of blood neutrophils in patients with CRC to form NET was evaluated in vitro.&#x0D; AIM: We undertook a study of the NETs presence in the tumor tissues, on the periphery of the tumor, and in healthy tissues with CRC at different stages and different types of therapy.&#x0D; METHODS: The study was carried out in the framework of the scientific and technical program “Personalized approach to the management of number of significant diseases” developed by the Non-commercial Joint-Stock Company “Karaganda Medical University,” funded by the Ministry of Education and Science of the Republic of Kazakhstan.&#x0D; RESULTS: Patients with CRC II and III stages are more likely to have NET in the center, on the periphery of the tumor and in healthy tissues adjacent to the tumor than in patients with CRC of Stage I. It was also found that radiation therapy as a pre-operative preparation contributed to an increase in the number of NET in the center of the tumor in patients with Stage I rectal cancer.&#x0D; CONCLUSION: Thus, our data revealed an increase in NET in the center and periphery of CRC, and an increase in their number after pre-operative radiation therapy was shown. The next stage of our study will be devoted to study the structural features of NET and their interaction with other effector cells and tumor cells.

Flow Cytometry-Based Quantification of Neutrophil Extracellular Traps Shows an Association with Hypercoagulation in Septic Shock and Hypocoagulation in Postsurgical Systemic Inflammation—A Proof-of-Concept Study

This proof-of-concept study aimed to evaluate a novel method of flow cytometry-based quantification of neutrophil extracellular traps (NETs) in septic shock patients and to identify possible interactions between the number of free-circulating NETs and alterations of the coagulatory system. Patients suffering from septic shock, a matched control group (CTRL), and patients suffering from systemic inflammation after cardiac (CABG) or major abdominal surgery (MAS) were enrolled in this prospective proof-of-concept study. Compared to the matched controls, free-circulating NETs were significantly elevated in septic shock and postsurgical patients (data are presented in median (IQR)); septic shock: (2.7 (1.9–3.9); CABG: 2.7 (2.1–3.7); MAS: 2.7 (2.1–3.9); CTRL: 1.6 (1–2); CTRL vs. septic shock: p = 0.001; CTRL vs. CABG: p < 0.001; CTRL vs. MAS: p < 0.001). NETs correlated positively with FIBTEM mean clot firmness (MCF) in septic shock patients (r = 0.37, p < 0.01) while they correlated negatively in surgical patients (CABG: r = −0.28, p < 0.01; MAS: r = −0.25, p = 0.03). Flow-cytometric quantification of NETs showed a significant increase in free-circulating NETs under inflammatory conditions. Furthermore, this study hints to an association of the number of NETs with hypercoagulation in septic shock patients and hypocoagulation in surgery-induced inflammation.

Neutrophil Extracellular Traps (NETs) Are a Subset of Smudge Cells Identifiable By Peripheral Smear Autoanalyzers

Background. Neutrophil Extracellular Traps (NETs) are composed of extracellular decondensed chromatin networks that play an important role in immune and inflammatory response regulation. Simple and reliable identification of NETs has been challenging. Automated analyzers such as the CellaVision® Hematology Autoanalyzer identify a subset of cell-derived entities as smudge cells. We hypothesize that, in addition to the typical degenerated lymphocytes (DLs) forming smudge cells, a proportion of smudge cells as identified by the Autoanalyzer are actually NETs. Since patients with high numbers of NETs have increased and specific morbidities, accurate and rapid identification of NETs may be clinically useful. Methods. To test our hypothesis, we analyzed peripheral blood smears from samples processed by the CellaVision® Hematology Autoanalyzer. To differentiate NETs from DLs we used morphologic characteristics, immunohistochemistry, and flow cytometry. After immunohistochemistry and flow cytometry informed our morphologic classification of NETs and DLs, we relied solely on morphology. Smears with &gt;20% smudge cells were classified as the study group; the control group had &lt; 5% smudge cells. Medical chart review was performed by investigators blinded to sample group designation. Data obtained from chart review included patient demographics; presence of bacterial and/or viral infection occurring &lt;1 week of sample collection; myeloproliferative and lymphoproliferative disorders (LPD); solid organ malignancy and/or transplant; sickle cell disease; chronic kidney (CKD) and liver diseases; autoimmune disorders; HIV; thrombotic events. We reviewed the same sample laboratory data for hemoglobin, platelet counts, and white blood cell (WBC) counts. Statistical analyses were performed using two-sided t-test with α =0.05 for continuous variables, chi-square for categorical variables with samples size &gt;10 and Fisher's exact tests for categorical variables with sample size &lt;10. Results. 155 samples were used in the analysis: 96 in the study group, 59 in the control group. Cell-derived entities staining strongly with myeloperoxidase (MPO), neutrophil elastase (NE), and leukocyte alkaline phosphatase (LAP) were classified as NETs. On flow cytometry, these NETs are at least twice as large as WBCs, display extracellular DNA (as identified by Sytox dye) and stain positively for MPO [Figure 1]. On Wright-Giemsa stain, these appeared morphologically as cell remnants with decondensed nuclei, no intact cytoplasm, dispersed granules, and polarized chromatin projections that resemble spider webs [Figure 2]. Of 96 patient samples with &gt;20% smudge cells, morphologic analyses designated 88 as NETs and only 8 as DLs. Comparing patients with &gt;20% to &lt;5% total smudge cells, the former had a higher incidence of bacterial infections (p=0.0091), as well as higher WBC, lymphocyte, monocyte counts and hemoglobin (p=0.014, 0.009, 0.004, 0.01 respectively) [Table 1]. High percentage of smudge cells correlated with a higher percentage of bacterial infection when compared to those with fewer smudge cells despite a non-significant difference in total neutrophil counts. Only 3 of the 36 patients with documented bacterial infection in the &gt;20% smudge cell group had a neutrophils &gt;8.5 x109/L. Of 13 infants &lt;1yr of age with &gt;20%smudge cells, 5 had a bacterial infection with normal neutrophil counts. When the smudge cells were separated according to their category, NETs vs DLs, the DL group was older, had more LPD, higher total WBC, lymphocyte and monocyte counts (p=0.000044, 0.00036, 0.018, 0.022, 0.033) [Table 2]. Slides with mostly NETs had higher LAP scores than those classified as mostly DLs (172 +30.5 vs 103.3 +30.8, p=0.0087). Conclusions: NETosis is a relatively newly discovered tool in the neutrophil's arsenal. This study is the first to identify NETs on peripheral smear evaluations as performed by a routine Hematology Autoanalyzer and to differentiate them from DLs using a reliable set of morphologic characteristics, immunohistochemical stains, and flow cytometry probes. This study also supports data that associate NETs with bacterial infections. This could be clinically useful in diagnosis of infection in the absence of leukocytosis. Disclosures Billett: Albert Einstein College of Medicine: Patents &amp; Royalties: Patent application pending for NETs AI software. Kushnir:Janssen Pharmaceuticals: Research Funding. Reyes Gil:Albert Einstein College of Medicine: Patents &amp; Royalties: Patent application pending for NETs AI software.

Glucocorticoids May Exacerbate Fungal Keratitis by Increasing Fungal Aggressivity and Inhibiting the Formation of Neutrophil Extracellular Traps

ABSTRACTPurpose: To evaluate whether glucocorticoids affect the prognosis of fungal keratitis by inhibiting the formation of neutrophil extracellular traps (NETs).Methods: A mouse model of Candida albicans (C.albicans) keratitis was established. Animals were randomly assigned to treatment with 0.1% dexamethasone (DXM) eye drops and normal saline (3 times each day for 3 days). The effects of DXM on fungal keratitis were assessed using clinical scores, immunofluorescence staining, histopathological examination, scanning electron microscopy (SEM), and pathogen burden assay. All the analyses were performed using SPSS software version 17.0 (Chicago, IL).Results: NETs formation was noteworthy in the cornea lesions of fungal keratitis. The clinical score of the DXM-treated group was significantly higher than that of the control group (P < .05). During the measured period, corneas from DXM-treated group contained more C.albicans than those from the control group by histology and pathogen burden assay. Compared with the control group, the DXM treatment group had a higher depth of infiltration of C.albicans. Histological and immunofluorescence staining showed that there were fewer neutrophils in the cornea focus of DXM-treated group (P < .05）, and the number of NETs formed in scrapings from control group was higher than that in the DXM treatment group on day 3 (P < .05, Z = −3.56)) and day 5 (P < .05, Z = −3.69). In a similar amount of cell scraping, the NETs of neutrophils formation from the DXM-treated group were also less than that from the control group.Conclusion: Our results indicated that NETs were involved in the immune response in C.albicans keratitis. Glucocorticoids may exacerbate fungal keratitis not only by increasing fungal aggressivity and reducing the infiltration of neutrophils but also by inhibiting the formation of NETs.

Propofol specifically reduces PMA-induced neutrophil extracellular trap formation through inhibition of p-ERK and HOCl

Neutrophil extracellular traps (NETs) are net-like chromatin fibers that can trap and kill microorganisms. Although several anti-inflammatory effects of intravenous anesthetics have been reported, it has not been investigated whether intravenous anesthetics influence NET formation. AimsTo compare the effects of four intravenous anesthetics (propofol, thiamylal sodium, midazolam, and ketamine) on phorbol myristate acetate (PMA)-induced NET formation and analyze the associated signaling pathways. Materials and methodsPMA-stimulated NETs formed in the absence or presence of intravenous anesthetics were stained with SYTOX Green and then quantified. Inhibitors were applied to investigate the related mechanism, which was confirmed by western blotting, and ROS were detected. Key findingsThe neutrophils incubated with propofol showed the lowest degree of NET formation compared with those incubated with the other intravenous anesthetics. Propofol significantly reduced the level of myeloperoxidase (MPO)-derived HOCl but not that of superoxide. Aminopyrine, an MPO inhibitor, markedly decreased the number of PMA-induced NETs, indicating the involvement of HOCl in the inhibitory effect of propofol on NET formation. According to western blotting results, the level of p-ERK was reduced by propofol during PMA-induced NET formation. The ERK inhibitor PD98059 decreased NET formation but did not inhibit PMA-induced HOCl generation, and aminopyrine did not reduce ERK phosphorylation. SignificanceThrough this study, we define a new anti-inflammatory effect of intravenous anesthetics. Of the four intravenous anesthetics tested, propofol was the most potent inhibitor of NET formation. Moreover, propofol resulted in a decrease in PMA-induced NET formation by two independent mechanisms: inhibition of HOCl and p-ERK.

Chlorpyrifos Suppresses Neutrophil Extracellular Traps in Carp by Promoting Necroptosis and Inhibiting Respiratory Burst Caused by the PKC/MAPK Pathway.

Neutrophil extracellular traps (NETs) are reticular structures formed by myeloperoxidase (MPO), histones, and neutrophil elastase (NE) that are released from neutrophils in response to pathogenic stimuli. Chlorpyrifos (CPF) is wildly used as an organophosphorus pesticide that causes a range of toxicological and environmental problems. Exposure to CPF can increase the production of neutrophils in carps, and this increase can be considered a biomarker of water pollution. To explore a relationship between NETs and CPF and its mechanism of influence, we treated neutrophils from the blood of carp with 1 μg/mL phorbol 12-myristate 13-acetate (PMA), 0.325 mg/L CPF, or 20 μM necrostatin-1 (Nec-1). The production of MPO and NETs was reduced in the CPF+PMA group compared with that in the PMA group. CPF can cause an increase in reactive oxygen species (ROS), while inhibiting respiratory burst caused by PMA stimulation. We found that the expression levels of protein-coupled receptor 84 (gpr84), dystroglycan (DAG), proto-oncogene serine/threonine kinase (RAF), protein kinase C (PKC), and mitogen-activated protein kinase 3 (MAPK3) in the CPF+PMA group were lower than those in the PMA group, indicating that the PKC-MAPK pathway was suppressed. The expression levels of cylindromatosis (CYLD), mixed lineage kinase domain-like pseudokinase (MLKL), receptor-interacting serine-threonine kinase 1 (RIP1), and receptor-interacting serine-threonine kinase 3 (RIP3) were increased, and the expression levels of caspase 8 were reduced by CPF, indicating that CPF may cause necroptosis. The addition of Nec-1 restored the number of NETs in the CPF+PMA group. The results indicate that CPF reduced the production of NETs by inhibiting respiratory burst and increasing necroptosis. The results contribute to the understanding of the immunotoxicological mechanism of CPF and provide a reference for comparative medical studies.