中性粒细胞胞外诱捕网和血栓形成的研究进展

中性粒细胞胞外诱捕网是由DNA骨架、组蛋白、颗粒成分以及胞浆蛋白组成的网状物,它通过捕获致病微生物,抑制其扩散、灭活毒力因子以及清除病原体来发挥抗菌活性,中性粒细胞这种与其经典的趋化、吞噬作用不同的胞外杀菌方式发挥着重要的固有免疫应答作用。然而,中性粒细胞胞外诱捕网的产生是一把双刃剑。近年来有研究表明它可以导致凝血异常、刺激血栓形成并为其提供支架,但这一过程的机制并不十分清楚,可能与中性粒细胞胞外诱捕网的促凝活性以及凝血级联反应的激活有关。本文将就中性粒细胞胞外诱捕网及其与血栓形成之间关系的研究进展做一综述。     

Neutrophil Extracellular Traps Confine Pseudomonas aeruginosa Ocular Biofilms and Restrict Brain Invasion

1. Download high-res image (165KB)
2. Download full-size image

     

Characterization of Mucosal Candida albicans Biofilms

C. albicans triggers recurrent infections of the alimentary tract mucosa that result from biofilm growth. Although the ability of C. albicans to form a biofilm on abiotic surfaces has been well documented in recent years, no information exists on biofilms that form directly on mucosal surfaces. The objectives of this study were to characterize the structure and composition of Candida biofilms forming on the oral mucosa. We found that oral Candida biofilms consist of yeast, hyphae, and commensal bacteria, with keratin dispersed in the intercellular spaces. Neutrophils migrate through the oral mucosa and form nests within the biofilm mass. The cell wall polysaccharide β-glucan is exposed during mucosal biofilm growth and is more uniformly present on the surface of biofilm organisms invading the oral mucosa. We conclude that C. albicans forms complex mucosal biofilms consisting of both commensal bacterial flora and host components. These discoveries are important since they can prompt a shift of focus for current research in investigating the role of Candida-bacterial interactions in the pathogenesis of mucosal infections as well as the role of β-glucan mediated signaling in the host response.     

Cytokines Induced Neutrophil Extracellular Traps Formation: Implication for the Inflammatory Disease Condition

Neutrophils (PMNs) and cytokines have a critical role to play in host defense and systemic inflammatory response syndrome (SIRS). Neutrophil extracellular traps (NETs) have been shown to extracellularly kill pathogens, and inflammatory potential of NETs has been shown. Microbial killing inside the phagosomes or by NETs is mediated by reactive oxygen and nitrogen species (ROS/RNS). The present study was undertaken to assess circulating NETs contents and frequency of NETs generation by isolated PMNs from SIRS patients. These patients displayed significant augmentation in the circulating myeloperoxidase (MPO) activity and DNA content, while PMA stimulated PMNs from these patients, generated more free radicals and NETs. Plasma obtained from SIRS patients, if added to the PMNs isolated from healthy subjects, enhanced NETs release and free radical formation. Expressions of inflammatory cytokines (IL-1β, TNFα and IL-8) in the PMNs as well as their circulating levels were significantly augmented in SIRS subjects. Treatment of neutrophils from healthy subjects with TNFα, IL-1β, or IL-8 enhanced free radicals generation and NETs formation, which was mediated through the activation of NADPH oxidase and MPO. Pre-incubation of plasma from SIRS with TNFα, IL-1β, or IL-8 antibodies reduced the NETs release. Role of IL-1β, TNFα and IL-8 thus seems to be involved in the enhanced release of NETs in SIRS subjects.     

Effect of Tetrandrine against Candida albicans Biofilms

Candida albicans is the most common human fungal pathogen and has a high propensity to develop biofilms that are resistant to traditional antifungal agents. In this study, we investigated the effect of tetrandrine (TET) on growth, biofilm formation and yeast-to-hypha transition of C. albicans. We characterized the inhibitory effect of TET on hyphal growth and addressed its possible mechanism of action. Treatment of TET at a low concentration without affecting fungal growth inhibited hyphal growth in both liquid and solid Spider media. Real-time RT-PCR revealed that TET down-regulated the expression of hypha-specific genes ECE1, ALS3 and HWP1, and abrogated the induction of EFG1 and RAS1, regulators of hyphal growth. Addition of cAMP restored the normal phenotype of the SC5314 strain. These results indicate that TET may inhibit hyphal growth through the Ras1p-cAMP-PKA pathway. In vivo, at a range of concentrations from 4 mg/L to 32 mg/L, TET prolonged the survival of C. albicans-infected Caenorhabditis elegans significantly. This study provides useful information for the development of new strategies to reduce the incidence of C. albicans biofilm-associated infections.     

Mitochondrial DNA Released by Trauma Induces Neutrophil Extracellular Traps

Neutrophil extracellular traps (NETs) are critical for anti-bacterial activity of the innate immune system. We have previously shown that mitochondrial damage-associated molecular patterns (mtDAMPs), including mitochondrial DNA (mtDNA), are released into the circulation after injury. We therefore questioned whether mtDNA is involved in trauma-induced NET formation. Treatment of human polymorphoneutrophils (PMN) with mtDNA induced robust NET formation, though in contrast to phorbol myristate acetate (PMA) stimulation, no NADPH-oxidase involvement was required. Moreover, formation of mtDNA-induced NETs was completely blocked by TLR9 antagonist, ODN-TTAGGG. Knowing that infective outcomes of trauma in elderly people are more severe than in young people, we measured plasma mtDNA and NET formation in elderly and young trauma patients and control subjects. MtDNA levels were significantly higher in the plasma of elderly trauma patients than young patients, despite lower injury severity scores in the elderly group. NETs were not visible in circulating PMN isolated from either young or old control subjects. NETs were however, detected in PMN isolated from young trauma patients and to a lesser extent from elderly patients. Stimulation by PMA induced widespread NET formation in PMN from both young volunteers and young trauma patients. NET response to PMA was much less pronounced in both elderly volunteers’ PMN and in trauma patients’ PMN. We conclude that mtDNA is a potent inducer of NETs that activates PMN via TLR9 without NADPH-oxidase involvement. We suggest that decreased NET formation in the elderly regardless of higher mtDNA levels in their plasma may result from decreased levels of TLR9 and/or other molecules, such as neutrophil elastase and myeloperoxidase that are involved in NET generation. Further study of the links between circulating mtDNA and NET formation may elucidate the mechanisms of trauma-related organ failure as well as the greater susceptibility to secondary infection in elderly trauma patients.     

中性粒细胞胞外诱捕网与肺部炎症性疾病的关系

中性粒细胞募集/浸润是肺部炎症性疾病的特征性表现,是肺部抵抗病原微生物入侵的第一道防线,主要通过吞噬作用杀灭病原微生物.然而,新近的研究发现,中性粒细胞被刺激后可形成一种以DNA为骨架并镶嵌有大量活性蛋白质的网状物质——中性粒细胞胞外诱捕网(neutrophil extracellular traps,NETs),这种特殊形式的生物结构能捕获并杀灭病原微生物.尽管就NETs的生物学功能而言,其对肺部炎症性疾病应该是有益的,但是越来越多的研究表明,NETs对肺上皮细胞和内皮细胞均具有直接的细胞毒性作用,并可能促进肺部炎症性疾病的发生发展.为了系统地了解NETs与肺部相关炎症性疾病的关系,本综述首先简述了NETs的结构、功能和形成过程,然后分别叙述了NETs与哮喘、慢性阻塞性肺病、细菌性肺炎、肺结核、肺囊性纤维化、间质性肺疾病、流感病毒感染和急性肺损伤的关系.最后总结、展望了NETs在肺部炎症性疾病中的潜在研究方向和针对性治疗策略.     

Neutrophil Extracellular Traps: How to Generate and Visualize Them

Neutrophil granulocytes are the most abundant group of leukocytes in the peripheral blood. As professional phagocytes, they engulf bacteria and kill them intracellularly when their antimicrobial granules fuse with the phagosome. We found that neutrophils have an additional way of killing microorganisms: upon activation, they release granule proteins and chromatin that together form extracellular fibers that bind pathogens. These novel structures, or Neutrophil Extracellular Traps (NETs), degrade virulence factors and kill bacteria¹, fungi² and parasites³. The structural backbone of NETs is DNA, and they are quickly degraded in the presence of DNases. Thus, bacteria expressing DNases are more virulent⁴. Using correlative microscopy combining TEM, SEM, immunofluorescence and live cell imaging techniques, we could show that upon stimulation, the nuclei of neutrophils lose their shape and the eu- and heterochromatin homogenize. Later, the nuclear envelope and the granule membranes disintegrate allowing the mixing of NET components. Finally, the NETs are released as the cell membrane breaks. This cell death program (NETosis) is distinct from apoptosis and necrosis and depends on the generation of Reactive Oxygen Species by NADPH oxidase⁵. Neutrophil extracellular traps are abundant at sites of acute inflammation. NETs appear to be a form of innate immune response that bind microorganisms, prevent them from spreading, and ensure a high local concentration of antimicrobial agents to degrade virulence factors and kill pathogens thus allowing neutrophils to fulfill their antimicrobial function even beyond their life span. There is increasing evidence, however, that NETs are also involved in diseases that range from auto-immune syndromes to infertility⁶.We describe methods to isolate Neutrophil Granulocytes from peripheral human blood⁷ and stimulate them to form NETs. Also we include protocols to visualize the NETs in light and electron microscopy.     

病原体逃逸中性粒细胞胞外诱捕网机制

中性粒细胞胞外诱捕网（NETs）是新发现的中性粒细胞抗病原机制,是天然免疫系统的重要组成部分。但病原体在进化中形成了针对NETs的免疫逃逸机制。不同的病原体逃逸NET的机制不同,本文主要介绍3种机制：降解NETs-DNA、表面分子机制和NETosis调控。     

The Fungal Exopolysaccharide Galactosaminogalactan Mediates Virulence by Enhancing Resistance to Neutrophil Extracellular Traps

Of the over 250 Aspergillus species, Aspergillus fumigatus accounts for up to 80% of invasive human infections. A. fumigatus produces galactosaminogalactan (GAG), an exopolysaccharide composed of galactose and N-acetyl-galactosamine (GalNAc) that mediates adherence and is required for full virulence. Less pathogenic Aspergillus species were found to produce GAG with a lower GalNAc content than A. fumigatus and expressed minimal amounts of cell wall-bound GAG. Increasing the GalNAc content of GAG of the minimally pathogenic A. nidulans, either through overexpression of the A. nidulans epimerase UgeB or by heterologous expression of the A. fumigatus epimerase Uge3 increased the amount of cell wall bound GAG, augmented adherence in vitro and enhanced virulence in corticosteroid-treated mice to levels similar to A. fumigatus. The enhanced virulence of the overexpression strain of A. nidulans was associated with increased resistance to NADPH oxidase-dependent neutrophil extracellular traps (NETs) in vitro, and was not observed in neutropenic mice or mice deficient in NADPH-oxidase that are unable to form NETs. Collectively, these data suggest that cell wall-bound GAG enhances virulence through mediating resistance to NETs.     

Neutrophil Extracellular Traps Induce Organ Damage during Experimental and Clinical Sepsis

Organ dysfunction is a major concern in sepsis pathophysiology and contributes to its high mortality rate. Neutrophil extracellular traps (NETs) have been implicated in endothelial damage and take part in the pathogenesis of organ dysfunction in several conditions. NETs also have an important role in counteracting invading microorganisms during infection. The aim of this study was to evaluate systemic NETs formation, their participation in host bacterial clearance and their contribution to organ dysfunction in sepsis. C57Bl/6 mice were subjected to endotoxic shock or a polymicrobial sepsis model induced by cecal ligation and puncture (CLP). The involvement of cf-DNA/NETs in the physiopathology of sepsis was evaluated through NETs degradation by rhDNase. This treatment was also associated with a broad-spectrum antibiotic treatment (ertapenem) in mice after CLP. CLP or endotoxin administration induced a significant increase in the serum concentrations of NETs. The increase in CLP-induced NETs was sustained over a period of 3 to 24 h after surgery in mice and was not inhibited by the antibiotic treatment. Systemic rhDNase treatment reduced serum NETs and increased the bacterial load in non-antibiotic-treated septic mice. rhDNase plus antibiotics attenuated sepsis-induced organ damage and improved the survival rate. The correlation between the presence of NETs in peripheral blood and organ dysfunction was evaluated in 31 septic patients. Higher cf-DNA concentrations were detected in septic patients in comparison with healthy controls, and levels were correlated with sepsis severity and organ dysfunction. In conclusion, cf-DNA/NETs are formed during sepsis and are associated with sepsis severity. In the experimental setting, the degradation of NETs by rhDNase attenuates organ damage only when combined with antibiotics, confirming that NETs take part in sepsis pathogenesis. Altogether, our results suggest that NETs are important for host bacterial control and are relevant actors in the pathogenesis of sepsis.     

Neutrophil Extracellular Traps Contain Calprotectin,a Cytosolic Protein Complex Involved in Host Defense against Candida albicans

Neutrophils are the first line of defense at the site of an infection. They encounter and kill microbes intracellularly upon phagocytosis or extracellularly by degranulation of antimicrobial proteins and the release of Neutrophil Extracellular Traps (NETs). NETs were shown to ensnare and kill microbes. However, their complete protein composition and the antimicrobial mechanism are not well understood. Using a proteomic approach, we identified 24 NET-associated proteins. Quantitative analysis of these proteins and high resolution electron microscopy showed that NETs consist of modified nucleosomes and a stringent selection of other proteins. In contrast to previous results, we found several NET proteins that are cytoplasmic in unstimulated neutrophils. We demonstrated that of those proteins, the antimicrobial heterodimer calprotectin is released in NETs as the major antifungal component. Absence of calprotectin in NETs resulted in complete loss of antifungal activity in vitro. Analysis of three different Candida albicans in vivo infection models indicated that NET formation is a hitherto unrecognized route of calprotectin release. By comparing wild-type and calprotectin-deficient animals we found that calprotectin is crucial for the clearance of infection. Taken together, the present investigations confirmed the antifungal activity of calprotectin in vitro and, moreover, demonstrated that it contributes to effective host defense against C. albicans in vivo. We showed for the first time that a proportion of calprotectin is bound to NETs in vitro and in vivo.     

Simplified Human Neutrophil Extracellular Traps (NETs) Isolation and Handling

Neutrophil Extracellular Traps (NETs) have been recently identified as part of the neutrophil’s antimicrobial armamentarium. Apart from their role in fighting infections, recent research has demonstrated that they may be involved in many other disease processes, including cancer progression. Isolating purified NETs is a crucial element to allow the study of these functions.In this video, we demonstrate a simplified method of cell free NET isolation from human whole blood using readily available reagents. Isolated NETs can then be used for immunofluorescence staining, blotting or various functional assays. This enables an assessment of their biologic properties in the absence of the potential confounding effects of neutrophils themselves.A density gradient separation technique is employed to isolate neutrophils from healthy donor whole blood. Isolated neutrophils are then stimulated by phorbol 12-myristate 13-acetate (PMA) to induce NETosis. Activated neutrophils are then discarded, and a cell-free NET stock is obtained.We then demonstrate how isolated NETs can be used in an adhesion assay with A549 human lung cancer cells. The NET stock is used to coat the wells of a 96 well cell culture plate O/N, and after ensuring an adequate NET monolayer formation on the bottom of the wells, CFSE labeled A549 cells are added. Adherent cells are quantified using a Nikon TE300 fluorescent microscope. In some wells, 1000U DNAse1 is added 10 min before counting to degrade NETs     

中性粒细胞胞外诱捕网在矽肺中的作用研究

目的:通过构建二氧化硅诱导动物矽肺模型,探讨中性粒细胞胞外诱捕网(neutrophil nxtracellular traps,NETs)在矽肺中可能的作用。方法:将C57BL/6J雄性小鼠完全随机分为磷酸盐缓冲液(phosphate buffered solution,PBS)组、脱氧核糖核酸酶Ⅰ(deoxyribonuclease Ⅰ, DNase Ⅰ)组、二氧化硅+PBS组、二氧化硅+DNase Ⅰ组。通过气管内滴注二氧化硅(0.2 g/kg)混悬液构建小鼠矽肺模型,PBS组与DNase Ⅰ组注入等体积的PBS。在二氧化硅(silicon dioxide,SiO_2)混悬液注入后的第0小时、10小时小鼠气管内注入DNase Ⅰ(5 mg/kg),以后DNase Ⅰ持续给药:5 mg/kg/day,直到SiO_2混悬液注入后的28天。二氧化硅(SiO_2)干预28天后,取各组小鼠肺组织与肺泡灌洗液,通过PicoGreen荧光染料检测支气管肺泡灌洗液(bronchoalveolar lavage fluid,BALF)中NETs水平,酶联免疫吸附实验(enzyme-linked immunosorbent assay, ELISA)检测BALF中转化生长因子β1 (transforming growth factor-β1,TGF-β1)与炎症因子白细胞介素6(interleukin-6,IL-6)、白细胞介素1β(interleukin-1β,IL-1β)、肿瘤坏死因子α(tumor necrosis factor-α,TNF-α)水平,HE染色和Masson染色观察肺组织的病理学变化,Western Blot检测肺组织中NETs特异性组分瓜氨酸化组蛋白3(citrullinated-histone3,Cit-H3)表达。结果:SiO_2干预28天后,与PBS组相比,二氧化硅+PBS组小鼠肺组织炎症损伤加重,BALF中促炎介质IL-1β、IL-6、TNF-α水平上升;肺组织发生纤维化,大量硅结节形成;肺组织中Cit-H3蛋白表达量增加,BALF中NETs水平显著升高。予以NETs抑制剂DNase Ⅰ进行干预后,肺组织NETs水平显著下降,二氧化硅诱导的肺部炎症损伤、纤维化显著减轻。结论:NETs水平升高可能介导了二氧化硅诱导的小鼠矽肺模型肺部炎症损伤与纤维化。     

A 96 Well Microtiter Plate-based Method for Monitoring Formation and Antifungal Susceptibility Testing of Candida albicans Biofilms

Candida albicans remains the most frequent cause of fungal infections in an expanding population of compromised patients and candidiasis is now the third most common infection in US hospitals. Different manifestations of candidiasis are associated with biofilm formation, both on host tissues and/or medical devices (i.e. catheters). Biofilm formation carries negative clinical implications, as cells within the biofilms are protected from host immune responses and from the action of antifungals. We have developed a simple, fast and robust in vitro model for the formation of C. albicans biofilms using 96 well microtiter-plates, which can also be used for biofilm antifungal susceptibility testing. The readout of this assay is colorimetric, based on the reduction of XTT (a tetrazolium salt) by metabolically active fungal biofilm cells. A typical experiment takes approximately 24 h for biofilm formation, with an additional 24 h for antifungal susceptibility testing. Because of its simplicity and the use of commonly available laboratory materials and equipment, this technique democratizes biofilm research and represents an important step towards the standardization of antifungal susceptibility testing of fungal biofilms.Download video file.^{(44M, mov)}     

Vibrio cholerae Evades Neutrophil Extracellular Traps by the Activity of Two Extracellular Nucleases

The Gram negative bacterium Vibrio cholerae is the causative agent of the secretory diarrheal disease cholera, which has traditionally been classified as a noninflammatory disease. However, several recent reports suggest that a V. cholerae infection induces an inflammatory response in the gastrointestinal tract indicated by recruitment of innate immune cells and increase of inflammatory cytokines. In this study, we describe a colonization defect of a double extracellular nuclease V. cholerae mutant in immunocompetent mice, which is not evident in neutropenic mice. Intrigued by this observation, we investigated the impact of neutrophils, as a central part of the innate immune system, on the pathogen V. cholerae in more detail. Our results demonstrate that V. cholerae induces formation of neutrophil extracellular traps (NETs) upon contact with neutrophils, while V. cholerae in return induces the two extracellular nucleases upon presence of NETs. We show that the V. cholerae wild type rapidly degrades the DNA component of the NETs by the combined activity of the two extracellular nucleases Dns and Xds. In contrast, NETs exhibit prolonged stability in presence of the double nuclease mutant. Finally, we demonstrate that Dns and Xds mediate evasion of V. cholerae from NETs and lower the susceptibility for extracellular killing in the presence of NETs. This report provides a first comprehensive characterization of the interplay between neutrophils and V. cholerae along with new evidence that the innate immune response impacts the colonization of V. cholerae in vivo. A limitation of this study is an inability for technical and physiological reasons to visualize intact NETs in the intestinal lumen of infected mice, but we can hypothesize that extracellular nuclease production by V. cholerae may enhance survival fitness of the pathogen through NET degradation.     

Hookworms Evade Host Immunity by Secreting a Deoxyribonuclease to Degrade Neutrophil Extracellular Traps

1. Download : Download high-res image (273KB)
2. Download : Download full-size image