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 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 LuxS Promotes Biofilm Maturation and Persistence of Nontypeable Haemophilus influenzae In Vivo via Modulation of Lipooligosaccharides on the Bacterial Surface

2009 ◽  
Vol 77 (9) ◽  
pp. 4081-4091 ◽  
Author(s):  
Chelsie E. Armbruster ◽  
Wenzhou Hong ◽  
Bing Pang ◽  
Kristin E. Dew ◽  
Richard A. Juneau ◽  
...  
Keyword(s):  
Haemophilus Influenzae ◽  
Otitis Media ◽  
Opportunistic Infections ◽  
Bacterial Species ◽  
Enzyme Linked Immunosorbent Assay ◽  
Nontypeable Haemophilus Influenzae ◽  
Bacterial Persistence ◽  
Soluble Mediator

ABSTRACT Nontypeable Haemophilus influenzae (NTHI) is an extremely common airway commensal which can cause opportunistic infections that are usually localized to airway mucosal surfaces. During many of these infections, NTHI forms biofilm communities that promote persistence in vivo. For many bacterial species, density-dependent quorum-signaling networks can affect biofilm formation and/or maturation. Mutation of luxS, a determinant of the autoinducer 2 (AI-2) quorum signal pathway, increases NTHI virulence in the chinchilla model for otitis media infections. For example, bacterial counts in middle-ear fluids and the severity of the host inflammatory response were increased in luxS mutants compared with parental strains. As these phenotypes are consistent with those that we have observed for biofilm-defective NTHI mutants, we hypothesized that luxS may affect NTHI biofilms. A luxS mutant was generated using the well-characterized NTHI 86-028NP strain and tested to determine the effects of the mutation on biofilm phenotypes in vitro and bacterial persistence and disease severity during experimental otitis media. Quantitation of the biofilm structure by confocal microscopy and COMSTAT analysis revealed significantly reduced biomass for NTHI 86-028NP luxS biofilms, which was restored by a soluble mediator in NTHI 86-028NP supernatants. Analysis of lipooligosaccharide moieties using an enzyme-linked immunosorbent assay and immunoblotting showed decreased levels of biofilm-associated glycoforms in the NTHI 86-028NP luxS strain. Infection studies showed that NTHI 86-028NP luxS had a significant persistence defect in vivo during chronic otitis media infection. Based on these data, we concluded that a luxS-dependent soluble mediator modulates the composition of the NTHI lipooligosaccharides, resulting in effects on biofilm maturation and bacterial persistence in vivo.

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 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 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 Degraded neutrophil extracellular traps promote the growth of Actinobacillus pleuropneumoniae

2019 ◽  
Vol 10 (9) ◽  
Author(s):  
Nicole de Buhr ◽  
Marta C. Bonilla ◽  
Jessica Pfeiffer ◽  
Silke Akhdar ◽  
Cornelia Schwennen ◽  
...  
Keyword(s):  
Immune Cell ◽  
Bacterial Species ◽  
Severe Pneumonia ◽  
Lavage Fluid ◽  
Neutrophil Extracellular Traps ◽  
Actinobacillus Pleuropneumoniae ◽  
Degraded Dna ◽  
Human Neutrophils ◽  
Extracellular Traps

Abstract Actinobacillus pleuropneumoniae (A.pp) causes severe pneumonia associated with enormous economic loss in pigs. Peracute diseased pigs die in <24 h with pneumonia. Neutrophils are the prominent innate immune cell in this infection that massively infiltrate the infected lung. Here we show that neutrophils release neutrophil extracellular traps (NETs) as response to A.pp infection. Numerous NET-markers were identified in bronchoalveolar lavage fluid (BALF) of A.pp-infected piglets in vivo, however, most NET fibers are degraded. Importantly, A.pp is able to enhance its growth rate in the presence of NETs that have been degraded by nucleases efficiently. A.pp itself releases no nuclease, but we identified host nucleases as sources that degrade NETs after A.pp infection. Furthermore, the nucleases of co-infecting pathogens like Streptococcus suis increase growth of A.pp in presence of porcine NETs. Thus, A.pp is not only evading the antimicrobial activity of NETs, A.pp is rather additionally using parts of NETs as growth factor thereby taking advantage of host nucleases as DNase1 or nucleases of co-infecting bacteria, which degrade NETs. This effect can be diminished by inhibiting the bacterial adenosine synthase indicating that degraded NETs serve as a source for NAD, which is required by A.pp for its growth. A similar phenotype was found for the human pathogen Haemophilus (H.) influenzae and its growth in the presence of human neutrophils. H. influenzae benefits from host nucleases in the presence of neutrophils. These data shed light on the detrimental effects of NETs during host immune response against certain bacterial species that require and/or efficiently take advantage of degraded DNA material, which has been provided by host nuclease or nucleases of other co-infecting bacteria, as growth source.

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 Phosphorylcholine Decreases Early Inflammation and Promotes the Establishment of Stable Biofilm Communities of Nontypeable Haemophilus influenzae Strain 86-028NP in a Chinchilla Model of Otitis Media

2006 ◽  
Vol 75 (2) ◽  
pp. 958-965 ◽  
Author(s):  
Wenzhou Hong ◽  
Kevin Mason ◽  
Joseph Jurcisek ◽  
Laura Novotny ◽  
Lauren O. Bakaletz ◽  
...  
Keyword(s):  
Haemophilus Influenzae ◽  
Otitis Media ◽  
Fluorescent Antibody ◽  
Phase Variable ◽  
Nontypeable Haemophilus Influenzae ◽  
Bacterial Surface ◽  
Culture Cells ◽  
Antibody Staining

ABSTRACT Nontypeable Haemophilus influenzae (NTHi) is a leading causative agent of otitis media. Much of the inflammation occurring during NTHi disease is initiated by lipooligosaccharides (LOS) on the bacterial surface. Phosphorylcholine (PCho) is added to some LOS forms in a phase-variable manner, and these PCho+ variants predominate in vivo. Thus, we asked whether this modification confers some advantage during infection. Virulence of an otitis media isolate (NTHi strain 86-028NP) was compared with that of an isogenic PCho transferase (licD) mutant using a chinchilla (Chinchilla lanigera) model of otitis media. Animals infected with NTHi 86-028NP licD demonstrated increased early inflammation and a delayed increase in bacterial counts compared to animals infected with NTHi 86-028NP. LOS purified from chinchilla-passed NTHi 86-028NP had increased PCho content compared to LOS purified from the inoculum. Both strains were recovered from middle ear fluids as long as 14 days postinfection. Biofilms were macroscopically visible in the middle ears of euthanized animals infected with NTHi 86-028NP 7 days and 14 days postchallenge. Conversely, less dense biofilms were observed in animals infected with NTHi 86-028NP licD 7 days postinfection, and none of the animals infected with NTHi 86-028NP licD had a visible biofilm by 14 days. Fluorescent antibody staining revealed PCho+ variants within biofilms, similar to our prior results with tissue culture cells in vitro (S. L. West-Barnette, A. Rockel, and W. E. Swords, Infect. Immun. 74:1828-1836, 2006). Animals coinfected with equal proportions of both strains had equal persistence of each strain and somewhat greater severity of disease. We thus conclude that PCho promotes NTHi infection and persistence by reducing the host inflammatory response and by promoting formation of stable biofilm communities.

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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