scholarly journals How Neutrophil Extracellular Traps Become Visible

2016 ◽  
Vol 2016 ◽  
pp. 1-13 ◽  
Author(s):  
Nicole de Buhr ◽  
Maren von Köckritz-Blickwede
Keyword(s):  
Electron Microscopy ◽  
Nuclear Dna ◽  
Defense Mechanism ◽  
Neutrophil Extracellular Traps ◽  
Immune Defense ◽  
Extracellular Traps ◽  
Activated Neutrophils ◽  
Microscopy Techniques

Neutrophil extracellular traps (NETs) have been identified as a fundamental innate immune defense mechanism against different pathogens. NETs are characterized as released nuclear DNA associated with histones and granule proteins, which form an extracellular web-like structure that is able to entrap and occasionally kill certain microbes. Furthermore, NETs have been shown to contribute to several noninfectious disease conditions when released by activated neutrophils during inflammation. The identification of NETs has mainly been succeeded by various microscopy techniques, for example, immunofluorescence microscopy, transmission electron microscopy (TEM), and scanning electron microscopy (SEM). Since the last years the development and improvement of new immunofluorescence-based techniques enabled optimized visualization and quantification of NETs. On the one handin vitrolive-cell imaging led to profound new ideas about the mechanisms involved in the formation and functionality of NETs. On the other hand different intravital,in vivo, andin situmicroscopy techniques led to deeper insights into the role of NET formation during health and disease. This paper presents an overview of the main used microscopy techniques to visualize NETs and describes their advantages as well as disadvantages.

scholarly journals To Trap a Pathogen: Neutrophil Extracellular Traps and Their Role in Mucosal Epithelial and Skin Diseases

Cells ◽  
2021 ◽  
Vol 10 (6) ◽  
pp. 1469
Author(s):  
Carolina Domínguez-Díaz ◽  
Gael Urait Varela-Trinidad ◽  
Germán Muñoz-Sánchez ◽  
Karla Solórzano-Castanedo ◽  
Karina Elizabeth Avila-Arrezola ◽  
...  
Keyword(s):  
Skin Diseases ◽  
Inflammatory Diseases ◽  
Neutrophil Extracellular Traps ◽  
Immune Defense ◽  
Cathepsin G ◽  
Acid Hydrolases ◽  
Extracellular Traps ◽  
Health And Disease

Neutrophils are the most abundant circulating innate immune cells and comprise the first immune defense line, as they are the most rapidly recruited cells at sites of infection or inflammation. Their main microbicidal mechanisms are degranulation, phagocytosis, cytokine secretion and the formation of extracellular traps. Neutrophil extracellular traps (NETs) are a microbicidal mechanism that involves neutrophil death. Since their discovery, in vitro and in vivo neutrophils have been challenged with a range of stimuli capable of inducing or inhibiting NET formation, with the objective to understand its function and regulation in health and disease. These networks composed of DNA and granular components are capable of immobilizing and killing pathogens. They comprise enzymes such as myeloperoxidase, elastase, cathepsin G, acid hydrolases and cationic peptides, all with antimicrobial and antifungal activity. Therefore, the excessive formation of NETs can also lead to tissue damage and promote local and systemic inflammation. Based on this concept, in this review, we focus on the role of NETs in different infectious and inflammatory diseases of the mucosal epithelia and skin.

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

2021 ◽  
Vol 19 ◽  
pp. 205873922110623
Author(s):  
Hisatake Mori ◽  
Muhammad Aminul Huq ◽  
Md. Monirul Islam ◽  
Naoshi Takeyama
Keyword(s):  
Endothelial Cell ◽  
Pulmonary Artery ◽  
Barrier Function ◽  
Neutrophil Extracellular Traps ◽  
Endothelial Barrier ◽  
Endothelial Barrier Function ◽  
Extracellular Traps ◽  
Activated Neutrophils ◽  
Human Pulmonary Artery

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

scholarly journals ELISA detection of MPO-DNA complexes in human plasma is error-prone and yields limited information on neutrophil extracellular traps formed in vivo

PLoS ONE ◽  
2021 ◽  
Vol 16 (4) ◽  
pp. e0250265
Author(s):  
Hubert Hayden ◽  
Nahla Ibrahim ◽  
Johannes Klopf ◽  
Branislav Zagrapan ◽  
Lisa-Marie Mauracher ◽  
...  
Keyword(s):  
Human Plasma ◽  
Dynamic Range ◽  
Neutrophil Extracellular Traps ◽  
Plasma Samples ◽  
Dna Complexes ◽  
High Background ◽  
Isotype Control ◽  
Extracellular Traps

Over the past years, neutrophil extracellular traps (NETs) were shown to contribute to states of acute and chronic inflammatory disease. They are composed of expelled chromatin and decorated by neutrophil-derived proteins. Therefore, the analysis of DNA complexes with myeloperoxidase (MPO) by ELISA has become an attractive tool to measure NET formation in in vitro and in vivo samples. When we used a published MPO-DNA ELISA protocol and included an isotype control for the anti-MPO coating antibody, we observed high assay specificity for in vitro prepared NET samples, whereas the specificity for in vivo plasma samples was low. In addition, the assay failed to detect in vitro generated MPO-DNA complexes when spiked into plasma. Therefore, we set out to improve the specificity of the MPO-DNA ELISA for plasma samples. We found that the use of Fab fragments or immunoglobulins from different species or reversal of the antibody pair led to either a high background or a low dynamic range of detection that did not improve the specificity for plasma samples. Also, the use of higher plasma dilutions or pre-clearing of plasma immunoglobulins were ineffective. Finally, we found that a commercial reagent designed to block human anti-mouse antibodies and multivalent substances increased the detection window between the MPO antibody and isotype control for highly diluted plasma. We applied this modified ELISA protocol to analyze MPO-DNA complexes in human blood samples of acute and chronic inflammatory conditions. While markers of neutrophil activation and NET formation such as MPO, elastase and citrullinated histone H3 correlated significantly, we observed no correlation with the levels of MPO-DNA complexes. Therefore, we conclude that ELISA measurements of MPO-DNA complexes in human plasma are highly questionable regarding specificity of NET detection. In general, plasma analyses by ELISA should more frequently include isotype controls for antibodies to demonstrate target specificity.

scholarly journals The Emerging Role of Neutrophil Extracellular Traps in Arterial, Venous and Cancer-Associated Thrombosis

2021 ◽  
Vol 8 ◽  
Author(s):  
Yilu Zhou ◽  
Weimin Tao ◽  
Fuyi Shen ◽  
Weijia Du ◽  
Zhendong Xu ◽  
...  
Keyword(s):  
Current Knowledge ◽  
Neutrophil Extracellular Traps ◽  
Vital Role ◽  
Extracellular Traps ◽  
Protein Components ◽  
Cancer Associated Thrombosis ◽  
Coagulation Pathway

Neutrophils play a vital role in the formation of arterial, venous and cancer-related thrombosis. Recent studies have shown that in a process known as NETosis, neutrophils release proteins and enzymes complexed to DNA fibers, collectively called neutrophil extracellular traps (NETs). Although NETs were originally described as a way for the host to capture and kill bacteria, current knowledge indicates that NETs also play an important role in thrombosis. According to recent studies, the destruction of vascular microenvironmental homeostasis and excessive NET formation lead to pathological thrombosis. In vitro experiments have found that NETs provide skeletal support for platelets, red blood cells and procoagulant molecules to promote thrombosis. The protein components contained in NETs activate the endogenous coagulation pathway to promote thrombosis. Therefore, NETs play an important role in the formation of arterial thrombosis, venous thrombosis and cancer-related thrombosis. This review will systematically summarize and explain the study of NETs in thrombosis in animal models and in vivo experiments to provide new targets for thrombosis prevention and treatment.

scholarly journals Neutrophil extracellular traps activate IL-8 and IL-1 expression in human bronchial epithelia

2020 ◽  
Vol 319 (1) ◽  
pp. L137-L147 ◽  
Author(s):  
Kristin M. Hudock ◽  
Margaret S. Collins ◽  
Michelle Imbrogno ◽  
John Snowball ◽  
Elizabeth L. Kramer ◽  
...  
Keyword(s):  
Neutrophil Extracellular Traps ◽  
Gm Csf ◽  
Extracellular Traps ◽  
Tnf Α ◽  
Multiple Donors ◽  
Novel Target ◽  
Induced Changes ◽  
Air Liquid Interface

Neutrophil extracellular traps (NETs) provide host defense but can contribute to the pathobiology of diverse human diseases. We sought to determine the extent and mechanism by which NETs contribute to human airway cell inflammation. Primary normal human bronchial epithelial cells (HBEs) grown at air-liquid interface and wild-type (wt)CFBE41o- cells (expressing wtCFTR) were exposed to cell-free NETs from unrelated healthy volunteers for 18 h in vitro. Cytokines were measured in the apical supernatant by Luminex, and the effect on the HBE transcriptome was assessed by RNA sequencing. NETs consistently stimulated IL-8, TNF-α, and IL-1α secretion by HBEs from multiple donors, with variable effects on other cytokines (IL-6, G-CSF, and GM-CSF). Expression of HBE RNAs encoding IL-1 family cytokines, particularly IL-36 subfamily members, was increased in response to NETs. NET exposure in the presence of anakinra [recombinant human IL-1 receptor antagonist (rhIL-1RA)] dampened NET-induced changes in IL-8 and TNF-α proteins as well as IL-36α RNA. rhIL-36RA limited the increase in expression of proinflammatory cytokine RNAs in HBEs exposed to NETs. NETs selectively upregulate an IL-1 family cytokine response in HBEs, which enhances IL-8 production and is limited by rhIL-1RA. The present findings describe a unique mechanism by which NETs may contribute to inflammation in human lung disease in vivo. NET-driven IL-1 signaling may represent a novel target for modulating inflammation in diseases characterized by a substantial NET burden.

scholarly journals Antibodies Against Pseudomonas aeruginosa Alkaline Protease Directly Enhance Disruption of Neutrophil Extracellular Traps Mediated by This Enzyme

2021 ◽  
Vol 12 ◽  
Author(s):  
Chendi Jing ◽  
Chenghua Liu ◽  
Yu Liu ◽  
Ruli Feng ◽  
Run Cao ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Alkaline Protease ◽  
Chronic Infection ◽  
Active Sites ◽  
Neutrophil Extracellular Traps ◽  
Lung Infection ◽  
Bacterial Burden ◽  
Extracellular Traps

Extracellular traps released by neutrophils (NETs) are essential for the clearance of Pseudomonas aeruginosa. Alkaline protease (AprA) secreted by P. aeruginosa negatively correlates with clinical improvement. Moreover, anti-AprA in patients with cystic fibrosis (CF) can help identify patients with aggressive forms of chronic infection. However, the mechanism underlying the clinical outcomes remains unclear. We demonstrated that aprA deficiency in P. aeruginosa decreased the bacterial burden and reduced lung infection. AprA degraded NET components in vitro and in vivo but did not affect NET formation. Importantly, antibodies induced by AprA acted as an agonist and directly enhanced the degrading activities of AprA. Moreover, antisera from patients with P. aeruginosa infection exhibited antibody-dependent enhancement (ADE) similar to that of the antibodies we prepared. Our further investigations showed that the interaction between AprA and the specific antibodies might make the enzyme active sites better exposed, and subsequently enhance the recognition of substrates and accelerate the degradation. Our findings revealed that AprA secreted by P. aeruginosa may aggravate infection by destroying formed NETs, an effect that was further enhanced by its antibodies.

scholarly journals NontypeableHaemophilus influenzaeInitiates Formation of Neutrophil Extracellular Traps

2010 ◽  
Vol 79 (1) ◽  
pp. 431-438 ◽  
Author(s):  
Richard A. Juneau ◽  
Bing Pang ◽  
Kristin E. D. Weimer ◽  
Chelsie E. Armbruster ◽  
W. Edward Swords
Keyword(s):  
Otitis Media ◽  
Bacterial Species ◽  
Outer Membrane Proteins ◽  
Neutrophil Extracellular Traps ◽  
Bacterial Surface ◽  
Persistent Infections ◽  
Extracellular Traps ◽  
Molecular Patterns

ABSTRACTNontypeableHaemophilus influenzae(NTHI) is a leading cause of otitis media infections, which are often chronic and/or recurrent in nature. NTHI and other bacterial species persistin vivowithin biofilms during otitis media and other persistent infections. These biofilms have a significant host component that includes neutrophil extracellular traps (NETs). These NETs do not mediate clearance of NTHI, which survives within NET structures by means of specific subpopulations of lipooligosaccharides on the bacterial surface that are determinants of biofilm formationin vitro. In this study, the ability of NTHI and NTHI components to initiate NET formation was examined using anin vitromodel system. Both viable and nonviable NTHI strains were shown to promote NET formation, as did preparations of bacterial DNA, outer membrane proteins, and lipooligosaccharide (endotoxin). However, only endotoxin from a parental strain of NTHI exhibited equivalent potency in NET formation to that of NTHI. Additional studies showed that NTHI entrapped within NET structures is resistant to both extracellular killing within NETs and phagocytic killing by incoming neutrophils, due to oligosaccharide moieties within the lipooligosaccharides. Thus, we concluded that NTHI elicits NET formation by means of multiple pathogen-associated molecular patterns (most notably endotoxin) and is highly resistant to killing within NET structures. These data support the conclusion that, for NTHI, formation of NET structures may be a persistence determinant by providing a niche within the middle-ear chamber.

scholarly journals Erythromycin suppresses neutrophil extracellular traps in smoking-related chronic pulmonary inflammation

2019 ◽  
Vol 10 (9) ◽  
Author(s):  
Hui Zhang ◽  
Shi-Lin Qiu ◽  
Qi-Ya Tang ◽  
Xiu Zhou ◽  
Jian-Quan Zhang ◽  
...  
Keyword(s):  
Adaptive Immunity ◽  
Cigarette Smoke ◽  
Critical Role ◽  
Neutrophil Extracellular Traps ◽  
Airflow Limitation ◽  
Chronic Obstructive ◽  
Myeloid Dendritic Cells ◽  
Extracellular Traps

Abstract Neutrophil extracellular traps (NETs) may play a critical role in smoking-related chronic airway inflammation. However, the mechanism by which NETs induced by cigarette smoke initiate the adaptive immunity in chronic obstructive pulmonary disease (COPD) is not fully understood. In this study, we explored the effects of NETs induced by cigarette smoke on the myeloid dendritic cells (mDCs) and Th1 and Th17 cells. Additionally, we observed the inhibitory effect of erythromycin on NETs induced by cigarette smoke. We found that elevated NET levels in the sputum of COPD patients were correlated with the circulating Th1 response, mDC activation and airflow limitation. NETs induced by cigarette smoke extract (CSE) could activate monocyte-derived mDCs and promote Th1 and Th17 differentiation in vitro. Erythromycin effectively inhibited NET formation induced by CSE. In vivo, erythromycin decreased NETs in the airway and ameliorated emphysema with Th1 and Th17 cell down-regulation and CD40+ and CD86+ mDCs suppression in mice chronically exposed to cigarette smoke. These findings provide direct evidence that NETs promote the differentiation of Th1 and Th17 and play a role in the adaptive immunity of smoking-related chronic lung inflammation. Erythromycin is a potential therapeutic strategy for NETs inhibition in COPD.

scholarly journals SARS-CoV-2–triggered neutrophil extracellular traps mediate COVID-19 pathology

2020 ◽  
Vol 217 (12) ◽  
Author(s):  
Flavio Protasio Veras ◽  
Marjorie Cornejo Pontelli ◽  
Camila Meirelles Silva ◽  
Juliana E. Toller-Kawahisa ◽  
Mikhael de Lima ◽  
...  
Keyword(s):  
Inflammatory Diseases ◽  
Tracheal Aspirate ◽  
Neutrophil Extracellular Traps ◽  
Angiotensin Converting Enzyme 2 ◽  
Extracellular Traps ◽  
Lung Epithelial ◽  
Activated Neutrophils ◽  
Epithelial Cell Death

Severe COVID-19 patients develop acute respiratory distress syndrome that may progress to cytokine storm syndrome, organ dysfunction, and death. Considering that neutrophil extracellular traps (NETs) have been described as important mediators of tissue damage in inflammatory diseases, we investigated whether NETs would be involved in COVID-19 pathophysiology. A cohort of 32 hospitalized patients with a confirmed diagnosis of COVID-19 and healthy controls were enrolled. The concentration of NETs was augmented in plasma, tracheal aspirate, and lung autopsies tissues from COVID-19 patients, and their neutrophils released higher levels of NETs. Notably, we found that viable SARS-CoV-2 can directly induce the release of NETs by healthy neutrophils. Mechanistically, NETs triggered by SARS-CoV-2 depend on angiotensin-converting enzyme 2, serine protease, virus replication, and PAD-4. Finally, NETs released by SARS-CoV-2–activated neutrophils promote lung epithelial cell death in vitro. These results unravel a possible detrimental role of NETs in the pathophysiology of COVID-19. Therefore, the inhibition of NETs represents a potential therapeutic target for COVID-19.

In vivo induction of neutrophil extracellular traps by Mycobacterium tuberculosis in a guinea pig model

2017 ◽  
Vol 23 (7) ◽  
pp. 625-637 ◽  
Author(s):  
Georgina Filio-Rodríguez ◽  
Iris Estrada-García ◽  
Patricia Arce-Paredes ◽  
María M Moreno-Altamirano ◽  
Sergio Islas-Trujillo ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Guinea Pig ◽  
Neutrophil Extracellular Traps ◽  
Post Inoculation ◽  
Extracellular Traps ◽  
Guinea Pig Model ◽  
In Vivo Induction ◽  
Proteins And Peptides

In 2004, a novel mechanism of cellular death, called ‘NETosis’, was described in neutrophils. This mechanism, different from necrosis and apoptosis, is characterized by the release of chromatin webs admixed with microbicidal granular proteins and peptides (NETs). NETs trap and kill a variety of microorganisms. Diverse microorganisms, including Mycobacterium tuberculosis, are NET inducers in vitro. The aim of this study was to examine whether M. tuberculosis can also induce NETs in vivo and if the NETs are bactericidal to the microorganism. Guinea pigs were intradermally inoculated with M. tuberculosis H37Rv, and the production of NETs was investigated at several time points thereafter. NETs were detected as early as 30 min post-inoculation and were clearly evident by 4 h post-inoculation. NETs produced in vivo contained DNA, myeloperoxidase, elastase, histones, ROS and acid-fast bacilli. Viable and heat-killed M. tuberculosis, as well as Mycobacterium bovis BCG were efficient NET inducers, as were unilamellar liposomes prepared with lipids from M. tuberculosis. In vitro, guinea pig neutrophils also produced NETs in response to M. tuberculosis. However, neither the in vivo nor the in vitro-produced NETs were able to kill M. tuberculosis. Nevertheless, in vivo, neutrophils might propitiate recruitment and activation of more efficient microbicidal cells.

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