Flow Cytometry Analysis of Human Neutrophil Extracellular Traps in vitro v1 (protocols.io.3yhgpt6)

protocols.io ◽  
2019 ◽  
Author(s):  
Haruchika Masuda
Keyword(s):  
Flow Cytometry ◽  
Human Neutrophil ◽  
Neutrophil Extracellular Traps ◽  
Flow Cytometry Analysis ◽  
Extracellular Traps

In Vitro Identification and Isolation of Human Neutrophil Extracellular Traps

2020 ◽  
pp. 97-117
Author(s):  
Guillermina Calo ◽  
Analia Silvina Trevani ◽  
Esteban Grasso ◽  
Irene Angelica Keitelman ◽  
Rosanna Ramhorst ◽  
...  
Keyword(s):  
Human Neutrophil ◽  
Neutrophil Extracellular Traps ◽  
Extracellular Traps

scholarly journals Synthesis of Human Neutrophil Extracellular Traps Contributes to Angiopoietin-Mediated In Vitro Proinflammatory and Proangiogenic Activities

2018 ◽  
Vol 200 (11) ◽  
pp. 3801-3813 ◽  
Author(s):  
Simon S. Lavoie ◽  
Elizabeth Dumas ◽  
Branka Vulesevic ◽  
Paul-Eduard Neagoe ◽  
Michel White ◽  
...  
Keyword(s):  
Human Neutrophil ◽  
Neutrophil Extracellular Traps ◽  
Extracellular Traps

scholarly journals Gastric Cancer-Induced Neutrophil Extracellular Traps: A Potent Mediator of Cancer Associated Thrombosis

2021 ◽  
Author(s):  
Jiacheng Li ◽  
Xiaoming Zou ◽  
Shifeng Yang ◽  
Jiaqi Jin ◽  
Lei Zhu ◽  
...  
Keyword(s):  
Gastric Cancer ◽  
Flow Cytometry ◽  
Activated Protein C ◽  
Neutrophil Extracellular Traps ◽  
Mice Model ◽  
Tissue Samples ◽  
Extracellular Traps ◽  
Cancer Associated Thrombosis

Abstract Background: Development of venous thromboembolism (VTE) is associated with high mortalities among gastric cancer (GC) patients. Neutrophil extracellular traps (NETs) have been reported to correlated to procoagulant and prothrombotic in some diseases. We aimed to clarify that NETs participates in the development of cancer-associated thrombosis in GC.Method: The level of NETs in blood and tissue samples of patients were analyzed by ELISA and flow cytometry. NETs generation in vitro were observed by immunofluorescence (IF). The NETs procoagulant activity (PCA) was performed by fibrin formation and thrombin-antithrombin complex (TAT) assays. Hypercoagulation of platelets and endothelial cells (ECs) stimulated by NETs were measured by IF and flow cytometry. Thrombosis in vivo was measured in an established mice model of VTE induced by flow stenosis in the inferior vena cave (IVC).Result: NETs are likely to form in blood and tissue samples of GC patients compared with healthy individuals. In vitro studies that GC cells and their conditioned medium (CM), but not gastric mucosal epithelial cell can stimulate NETs releasing from neutrophils. In addition, NETs induced hypercoagulation of platelets by up-regulating the expression of phosphatidylserine (PS) and P-selectin on the cells. Furhter, NETs stimulate adhesion of normal platelets on glass surfaces. Similarly, NETs trigger the conversion of ECs to hypercoagulable phenotypes by down-regulating the expression of their intercellular tight junctions but up-regulating that of tissue factor (TF). Treatment of normal platelets or ECs with NETs augmented the level of plasma fibrin generation and TAT complex. Meanwhile, in the models of IVC stenosis, tumor-bearing mice demonstrate stronger ability to form thrombi and NETs were abundantly accumulated in the thrombi compared with control mice. Notably, combination of DNase-1, activated protein C (APC) and Sivelestat markedly abolished the PCA of NETs.Conclusion: Our findings demonstrate that GC-induced NETs strongly increase the risks of VTE development both in vitro and in vivo. Given that inhibitors of NETs disrupt hypercoagulation, NETs are potential therapeutic target against VTE.

scholarly journals In vitro activation of coagulation by human neutrophil DNA and histone proteins but not neutrophil extracellular traps

Blood ◽  
2017 ◽  
Vol 129 (8) ◽  
pp. 1021-1029 ◽  
Author(s):  
Denis F. Noubouossie ◽  
Matthew F. Whelihan ◽  
Yuan-Bin Yu ◽  
Erica Sparkenbaugh ◽  
Rafal Pawlinski ◽  
...  
Keyword(s):  
Human Neutrophil ◽  
Neutrophil Extracellular Traps ◽  
Human Neutrophils ◽  
Coagulation Activation ◽  
Histone Proteins ◽  
Extracellular Traps ◽  
Key Points ◽  
In Vitro Activation ◽  
Normal Human

Key Points Individual histone proteins and DNA purified from normal human neutrophils promote coagulation activation. Neither intact NETs nor nucleosomes directly promote coagulation activation in plasma in vitro.

scholarly journals Neutrophil extracellular traps induce MCP-1 release from endothelial cells at the plaque rupture site in acute myocardial infarction

2020 ◽  
Vol 41 (Supplement_2) ◽  
Author(s):  
A Ondracek ◽  
T.M Hofbauer ◽  
A Mangold ◽  
T Scherz ◽  
V Seidl ◽  
...  
Keyword(s):  
Myocardial Infarction ◽  
Acute Myocardial Infarction ◽  
Endothelial Cells ◽  
Plaque Rupture ◽  
Ex Vivo ◽  
Coronary Intervention ◽  
Neutrophil Extracellular Traps ◽  
Funding Source ◽  
Extracellular Traps

Abstract Introduction Leukocyte-mediated inflammation is crucial in acute myocardial infarction (AMI). We recently observed that neutrophil extracellular traps (NETs) are increased at the culprit site, promoting activation and differentiation of fibrocytes, cells with mesenchymal and leukocytic properties. Fibrocyte migration is mediated by monocyte chemoattractant protein (MCP)-1 and C-C chemokine receptor type 2 (CCR2). We investigated the interplay between NETs, fibrocyte function, and MCP-1 in AMI. Methods Culprit site and femoral blood of AMI patients was drawn during percutaneous coronary intervention. We characterized CCR2 expression of fibrocytes by flow cytometry. MCP-1 and the NET marker citrullinated histone H3 (citH3) were measured by ELISA. Fibrocytes were treated in vitro with MCP-1. Human coronary arterial endothelial cells (hCAECs) were stimulated with isolated NETs, and MCP-1 was measured by ELISA and qPCR. The influence of MCP-1 on NET formation in vitro was assessed using isolated neutrophils. Results We have included 50 consecutive AMI patients into the study. NETs and concentrations of MCP-1 were increased at the CLS. NET stimulation of hCAECs induced MCP-1 on mRNA and protein level. Increasing MCP-1 gradient was associated with fibrocyte accumulation at the site of occlusion. In the presence of higher MCP-1 these fibrocytes expressed proportionally less CCR2 than peripheral fibrocytes. In vitro, MCP-1 dose-dependently decreased fibrocyte CCR2 and reduced ex vivo NET release of healthy donor neutrophils. Conclusions NETs induce endothelial MCP-1 release, presumably promoting a chemotactic gradient for leukocyte and fibrocyte migration. MCP-1 mediated inhibition of NET formation could point to a negative feedback loop. These data will shed light on vascular healing. Funding Acknowledgement Type of funding source: Public grant(s) – National budget only. Main funding source(s): Austrian Science Fund

755 CXCR1 and CXCR2 chemokine receptor agonists produced by tumors induce neutrophil extracellular traps that interfere with immune cytotoxicity

2020 ◽  
Vol 8 (Suppl 3) ◽  
pp. A803-A803 ◽  
Author(s):  
Alvaro Teijeira ◽  
Saray Garasa ◽  
Itziar Migueliz ◽  
Assunta Cirella ◽  
Ignacio Melero
Keyword(s):  
Cancer Patients ◽  
Tumor Cells ◽  
Mouse Models ◽  
Chemokine Receptor ◽  
Suppressor Cells ◽  
Neutrophil Extracellular Traps ◽  
Myeloid Derived Suppressor Cells ◽  
Extracellular Traps ◽  
Tumor Associated Neutrophils

BackgroundNeutrophils are expanded and abundant in an important fraction (up to 35% of patients) in cancer-bearing hosts. When neutrophils are expanded, they usually promote exert immunomodulatory functions promoting tumor progression and the generation of metastases. Neutrophils can undergo a specialized form of cell death called NETosis that is characterized by the extrusion of their DNA to contain infections. In cancer NETs have been described to promote metastases in mouse models. IL-8, a CXCR1/2 ligand clinically targeted by blocking antibodies, has been described to induce NETosis and is upregulated in many cancer patients. Our hypothesis is that chemokines secreted by cancer cells can mediate NETosis in tumor associated neutrophils and that NETs can be one of the immunomodulatory mechanisms provided by tumor associated neutrophils.MethodsNETosis induction of peripheral neutrophils and granulocytic myeloid derived suppressor cells by different chemotactic stimuli, tumor cell supernatants and cocultures upon CXCR1/2 blockade. NET immunodetection in mouse models and xenograft tumors upon CXCR1/2 blockade. In vitro tumor cytotoxicity assays in the presence/absence of NETs, and videomicroscopy studies in vitro and by intravital imaging to test NETs inhibition of immune cytotoxicity by immune-cell/target-cell inhibition. Tumor growth studies and metastases models in the presence of NETosis inhibitors and in combination with checkpoint blockade in mouse cancer models.ResultsUnder the influence of CXCR1 and CXCR2 chemokine receptor agonists and other chemotactic factors produced by tumors, neutrophils, and granulocytic myeloid-derived suppressor cells (MDSCs) from cancer patients extrude their neutrophil extracellular traps (NETs). In our hands, CXCR1 and CXCR2 agonists proved to be the major mediators of cancer-promoted NETosis. NETs wrap and coat tumor cells and shield them from cytotoxicity, as mediated by CD8+ T cells and natural killer (NK) cells, by obstructing contact between immune cells and the surrounding target cells. Tumor cells protected from cytotoxicity by NETs underlie successful cancer metastases in mice and the immunotherapeutic synergy of protein arginine deiminase 4 (PAD4) inhibitors, which curtail NETosis with immune checkpoint inhibitors. Intravital microscopy provides evidence of neutrophil NETs interfering cytolytic cytotoxic T lymphocytes (CTLs) and NK cell contacts with tumor cells.ConclusionsCXCR1 and 2 are the main receptors mediating NETosis of tumor associated neutrophils in our in-vitro and in vivo systems expressing high levels of CXCR1 and 2 ligands. NETs limit cancer cell cytotoxicity by impeding contacts with cancer cells.

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