Neutrophils activate plasmacytoid dendritic cells by releasing self-DNA-peptide complexes in systemic lupus erythematosus

Systemic lupus erythematosus (SLE) is a severe and incurable autoimmune disease characterized by chronic activation of plasmacytoid dendritic cells (pDCs) and production of autoantibodies against nuclear self-antigens by hyperreactive B cells. Neutrophils are also implicated in disease pathogenesis; however, the mechanisms involved are unknown. Here, we identified in the sera of SLE patients immunogenic complexes composed of neutrophil-derived antimicrobial peptides and self-DNA. These complexes were produced by activated neutrophils in the form of web-like structures known as neutrophil extracellular traps (NETs) and efficiently triggered innate pDC activation via Toll-like receptor 9 (TLR9). SLE patients were found to develop autoantibodies to both the self-DNA and antimicrobial peptides in NETs, indicating that these complexes could also serve as autoantigens to trigger B cell activation. Circulating neutrophils from SLE patients released more NETs than those from healthy donors; this was further stimulated by the antimicrobial autoantibodies, suggesting a mechanism for the chronic release of immunogenic complexes in SLE. Our data establish a link between neutrophils, pDC activation, and autoimmunity in SLE, providing new potential targets for the treatment of this devastating disease.     

Neutrophil proteases degrade autoepitopes of NET-associated proteins

Neutrophils can form neutrophil extracellular traps (NETs) to capture microbes and facilitate their clearance. NETs consist of decondensed chromatin decorated with anti-microbial proteins. Here, we describe the effect of neutrophil proteases on the protein content of NETs. We show that the neutrophil serine proteases degrade several neutrophil proteins associated with NETs. Interestingly, the anti-bacterial proteins associated with NETs, such as myeloperoxidase, calgranulin B and neutrophil elastase (NE), seem to be less susceptible to proteolytic degradation than other NET proteins, such as actin and MNDA. NETs have been proposed to play a role in autoimmune reactions. Our data demonstrate that a large number of the autoepitopes of NET proteins that are recognized by autoantibodies produced by systemic lupus erythematosus (SLE) and rheumatoid arthritis (RA) patients are also removed by the proteases. In conclusion, neutrophil serine proteases have a major impact on the NET proteome and the proteolytic changes of NET-associated proteins may counteract autoimmune reactions to NET components.     

还原型谷胱甘肽对活性氧介导的系统性红斑狼疮患者中性粒细胞NETosis的影响

目的　探讨抗氧化剂还原型谷胱甘肽（GSH）能否通过调控中性粒细胞ROS水平从而抑制中性粒细胞发生NETosis,减少中性粒细胞胞外诱捕网（neutrophil extracellular traps, NETs）的形成,为临床开发新的治疗系统性红斑狼疮（SLE）的药物提供理论基础。 方法　采用梯度离心法分离SLE患者外周血中性粒细胞,利用佛波酯（PMA）诱导中性粒细胞发生NETosis后,检测各组中性粒细胞胞内ROS水平并观察其NETs产生情况。 结果　与PMA处理组比较,经过GSH预处理的SLE患者中性粒细胞体外诱导NETs产生明显减少。同时,GSH也能明显降低SLE患者中性粒细胞胞内ROS水平。 结论　GSH可能通过调控SLE患者中性粒细胞ROS的水平,从而抑制其发生NETosis,提示GSH可能在SLE治疗中发挥作用。     

中性粒细胞胞外诱捕网在感染性疾病中的研究进展

中性粒细胞在非特异性免疫防御系统中起重要作用.Brinkmann等首次发现中性粒细胞可以形成一种捕获并杀灭病原体的特殊网状结构,将其命名为中性粒细胞胞外诱捕网(neutrophil extracellular traps,NETs).后续研究表明,NETs的生成和降解会影响感染性疾病的病理过程.因此,揭示NETs的产生机制及其对病原体的影响为深入理解感染性疾病发生发展的免疫机制提供重要理论依据,并为疾病的干预提供新策略.现拟对NETs的构成、产生机制及其在感染性疾病中的病理生理作用等作一综述.     

Acetylated histones contribute to the immunostimulatory potential of neutrophil extracellular traps in systemic lupus erythematosus

In addition to disturbed apoptosis and insufficient clearance of apoptotic cells, there is recent evidence for a role of neutrophils in the aetiopathogenesis of systemic lupus erythematosus (SLE). In response to various stimuli, neutrophils can rapidly release DNA fibres decorated with citrullinated histones and anti-microbial peptides. These structures are referred to as neutrophil extracellular traps (NETs). In addition to apoptotic cell-derived microparticles, these NETs may comprise a further source of autoantigens, able to drive the autoimmune response in SLE. Our group recently identified specific histone modifications occurring during apoptosis that play an important role in the autoimmune response in SLE. In the current study, we evaluated the presence and immunostimulatory potential of these previously identified histone modifications in NETs. Compared to NETs from healthy donors, the histones present in NETs formed by SLE-derived neutrophils contain increased amounts of acetylated and methylated residues, which we previously observed to be associated with apoptosis and SLE. Treatment of neutrophils with histone deacetylase (HDAC) inhibitor Trichostatin A (TSA), prior to induction of NETosis, induced NETs containing hyperacetylated histones, endowed with an increased capacity to activate macrophages. This implies that specific histone modifications, in particular acetylation, might enhance the immunostimulatory potential of NETs in SLE.     

Neutrophil extracellular traps (NETs) in autoimmune diseases: A comprehensive review

Neutrophil extracellular traps (NETs) are fibrous networks which protrude from the membranes of activated neutrophils. NETs are found in a variety of conditions such as infection, malignancy, atherosclerosis, and autoimmune diseases including rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), anti-neutrophil cytoplasmic antibodies (ANCA)-associated vasculitis (AAV), psoriasis, and gout. Studies suggest that an imbalance between “NETosis,” which is a process by which NETs are formed, and NET degradation may be associated with autoimmune diseases. Neutrophils, interleukin-8, ANCA and other inflammatory molecules are considered to play a key role in NET formation. Prolonged exposure to NETs-related cascades is associated with autoimmunity and increases the chance of systemic organ damage. In this review, we discuss the roles of various inflammatory molecules in relation to NETs. We also describe the role of NETs in the pathogenesis of autoimmune diseases and discuss the possibility of using targeted therapies directed to NETs and associated molecules to treat autoimmune diseases.     

Neutrophil extracellular traps prevent HIV infection in the female genital tract

Women acquire human immunodeficiency virus (HIV) mainly through sexual intercourse. However, low transmission rates per sexual act indicate that local immune mechanisms contribute to HIV prevention. Neutrophils represent 10–20% of the genital immune cells in healthy women. Neutrophils mediate mucosal protection against bacterial and fungal pathogens through different mechanisms, including the release of neutrophil extracellular traps (NETs). NETs are DNA fragments associated with antimicrobial granular proteins. Despite neutrophil abundance and central contributions to innate immunity in the genital tract, their role in protection against HIV acquisition is unknown. We found that stimulation of human genital neutrophils with HIV viral-like particles (HIV-VLPs) induced NET release within minutes of viral exposure, through reactive oxygen species-independent mechanisms that resulted in immediate entrapment of HIV-VLPs. Incubation of infectious HIV with pre-formed genital NETs prevented infection of susceptible cells through irreversible viral inactivation. HIV inactivation by NETs from genital neutrophils could represent a previously unrecognized form of mucosal protection against HIV acquisition.     

IL-17A expressed on neutrophil extracellular traps promotes mesenchymal stem cell differentiation toward bone-forming cells in ankylosing spondylitis

Ankylosing spondylitis (AS) is an inflammatory disease characterized by excessive bone formation. We investigated the presence of neutrophil extracellular traps (NETs) in AS and how they are involved in the osteogenic capacity of bone marrow mesenchymal stem cells (MSCs) through interleukin-17A (IL-17A). Peripheral neutrophils and sera were obtained from patients with active AS and healthy controls. NET formation and neutrophil/NET-associated proteins were studied using immunofluorescence, immunoblotting, qPCR, and ELISA. In vitro co-culture systems of AS NET structures and MSCs isolated from controls were deployed to examine the role of NETs in the differentiation of MSCs toward osteogenic cells. Analysis was performed using specific staining and qPCR. Neutrophils from patients with AS were characterized by enhanced formation of NETs carrying bioactive IL-17A and IL-1β. IL-17A-enriched AS NETs mediated the differentiation of MSCs toward bone-forming cells. The neutrophil expression of IL-17A was positively regulated by IL-1β. Blocking IL-1β signaling on neutrophils with anakinra or dismantling NETs using DNase-I disrupted osteogenesis driven by IL-17A-bearing NETs. These findings propose a novel role of neutrophils in AS-related inflammation, linking IL-17A-decorated NETs with the differentiation of MSCs toward bone-forming cells. Moreover, IL-1β triggers the expression of IL-17A on NETs offering an additional therapeutic target in AS.     

New Aspects of the Biology of Neutrophil Extracellular Traps

The formation and release of neutrophil extracellular traps (NETs) discovered in 2004 by Volker Brinkmann and Arturo Zychlinsky cast a new light on the role of neutrophils in the non‐specific immune response of the body. This discovery has resulted in the rapid development of neutrophil studies in different bacterial and autoimmune diseases as well as neoplasms. Research is also being performed on the role of different signalling pathways engaged in the induction of NETosis, a unique form of a programmed cell death leading to the creation of NETs. The literature provides information on the structure and composition of neutrophil extracellular traps. This review presents the latest data on NET formation and the role of their key components, as well as describing the intracellular signalling pathways leading to the generation of NETs that have been discovered.     

Burning controversies in NETs and autoimmunity: The mysteries of cell death and autoimmune disease

Abstract

The causes and mechanisms of autoimmune disease pose continuing challenges to the scientific community. Recent clues implicate a peculiar feature of neutrophils, their ability to release nuclear chromatin in the form of neutrophil extracellular traps (NETs), in the induction or progression of autoimmune disease. Efforts to define the beneficial versus detrimental effects of NET release have, as yet, only partially revealed mechanisms that guide this process. Evidence suggests that the process of NET release is highly regulated, but the details of regulation remain controversial and obscure. Without a better understanding of the factors that initiate and control NET formation, the judicious modification of neutrophil behaviour for medically useful purposes appears remote. We highlight gaps and inconsistencies in published work, which make NETs and their role in health and disease a puzzle that deserves more focused attention.     

Zebrafish (Danio rerio) whole kidney assays to measure neutrophil extracellular trap release and degranulation of primary granules

The zebrafish (Danio rerio) is an excellent model system for studies in developmental biology, genetics, and toxicology, and is increasingly gaining importance in the field of immunology. The use of whole zebrafish kidneys as source of neutrophils for degranulation assays and detection of neutrophil extracellular traps is described for the first time. Neutrophils from zebrafish kidneys released neutrophil extracellular traps (NETs) and myeloperoxidase (MPO) upon stimulation with calcium ionophore, phorbol myristate acetate, and beta-glucan. Immunocytochemical study of zebrafish kidney cells revealed that NETs are made of DNA fibers associated with neutrophil granular proteins, but not with cytoskeleton. Rapid, direct MPO and extracellular DNA detection assays were developed to quantify NET release and degranulation of neutrophil primary granules from whole zebrafish kidneys. The assays were used to measure the effects of acute crowding and handling stress on neutrophils, and to examine the potential for use of zebrafish whole kidney assays in evaluation of neutrophil function under different conditions in vivo. The whole kidney NET release and degranulation assays are quantitative, can rapidly measure a large number of samples, and are capable of detecting inhibition of neutrophil activity in stressed fish, overcoming the limitations that prevented use of zebrafish in the investigations of cellular innate immune function. The assays can be used as a new research model to study effects of stress, immunomodulators, toxicants, and diseases on fish neutrophil biology.     

中性粒细胞胞外诱捕网与相关疾病的研究进展

中性粒细胞是机体防御系统的第一道防线,当机体遭受病原微生物的入侵,中性粒细胞即发挥强大的作用.研究发现,活化的中性粒细胞可以释放出非浓缩染色质形成网状支架,对病原体进行包围及限制,这一过程称为中性粒细胞胞外陷阱(neutrophil extracellular traps,NETs)形成(NETosis).NETs是由DNA骨架、组蛋白、颗粒成分以及胞浆蛋白组成的网状物,其能够网罗、杀伤病原体从而参与机体自身免疫应答,维护机体健康.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,它可以引发代谢性疾病,如糖尿病(diabetes mellitus,DM),引起心血管疾病动脉粥样硬化(atherosclerosis,AS),参与自身免疫性疾病,如系统性红斑狼疮(system lupus erythematosus systemic lupus erythematosus,SLE)、类风湿性关节炎(rheumatoid arthritis,RA)的产生,还可以使自身凝血异常,刺激血栓形成并为其提供支架,因而研究NETs及其与疾病之间的关系具有重要意义.     

Fish cast NETs: neutrophil extracellular traps are released from fish neutrophils

Neutrophil extracellular traps (NETs), which are extracellular DNA structures released from neutrophils, are described and characterized for the first time in fish using fluorescent confocal microscopy. Confocal images of fish neutrophil suspensions stained with 6'-diamino-2-phenylindole, dihydrochloride DNA fluorescent stain (DAPI) revealed the presence of NETs which appeared as fibrous structures connecting several cells. Co-localization of NETs with neutrophil granular proteins and actin was investigated using specific antibodies and probes. Double staining of neutrophils with SYTOX green and DAPI revealed that SYTOX stain applied to living cells stained extracellular DNA, but not nuclei. NETs are actively released from stimulated living cells, associated with granular proteins, but not with cytoskeleton, and are not a product of nuclear degradation seen in late apoptotic stages. Additionally, a fluorometric microtiter plate assay to quantify the release of NETs was adopted for use with fish neutrophils, and the effect of stress on NETs release was studied. This assay detected the inhibition of DNA release during stress conditions. In summary, NETs were released from living fish kidney neutrophils upon stimulation, characterized using fluorescence DNA-binding dyes, specific antibodies and probes, and quantified using a microtiter plate fluorometric assay that can rapidly measure a large number of samples. Detection of NETs can be used as an additional assay to an existing battery of functional tests, and as a new research model to study the effects of stress, immunomodulators, and diseases.     

Neutrophil proteinase 3 and dipeptidyl peptidase I (cathepsin C) as pharmacological targets in granulomatosis with polyangiitis (Wegener granulomatosis)

Neutrophils are among the first cells implicated in acute inflammation. Leaving the blood circulation, they quickly migrate through the interstitial space of tissues and liberate oxidants and other antimicrobial proteins together with serine proteinases. Neutrophil elastase, cathepsin G, proteinase 3 (PR3), and neutrophil serine protease 4 are four hematopoietic serine proteases activated by dipeptidyl peptidase I during neutrophil maturation and are mainly stored in cytoplasmic azurophilic granules. They regulate inflammatory and immune responses after their release from activated neutrophils at inflammatory sites. Membrane-bound PR3 (mbPR3) at the neutrophil surface is the prime antigenic target of antineutrophil cytoplasmic autoantibodies (ANCA) in granulomatosis with polyangiitis (GPA), a vasculitis of small blood vessels and granulomatous inflammation of the upper and/or lower respiratory tracts. The interaction of ANCA with mbPR3 results in excessive activation of neutrophils to produce reactive oxygen species and liberation of granular proteinases to the pericellular environment. In this review, we focus on PR3 and dipeptidyl peptidase I as attractive pharmacological targets whose inhibition is expected to attenuate autoimmune activation of neutrophils in GPA.     

ANCA诱导中性粒细胞释放正五聚素蛋白3*

 目的：研究正五聚素蛋白3（PTX3）在中性粒细胞的表达及释放情况,为进一步探讨PTX3在抗中性粒细胞胞浆抗体（ANCA）相关性小血管炎（AAV）中的作用及机制奠定基础。方法：采用PolymorphprepTM进行密度梯度离心分离健康志愿者和AAV患者外周静脉血中性粒细胞,通过健康人血清、AAV患者血清、TNF-α和溶酶体膜蛋白2（LAMP-2）抗体分别刺激健康人外周血中性粒细胞,ELISA检测培养上清PTX3水平,免疫荧光观察中性粒细胞胞外捕网(NETs)的形成,激光共聚焦显微镜检测可溶性识别受体PTX3 的表达。结果：LAMP-2抗体及AAV患者血清均能促进中性粒细胞PTX3的释放。与正常中性粒细胞对照组相比,LAMP-2抗体刺激组中性粒细胞上清PTX3水平明显升高（P＜0.01）; AAV患者血清刺激组中性粒细胞中PTX3表达的荧光强度显著高于用健康人血清刺激的对照组（P＜0.01）,进一步通过激光共聚焦显微镜进行形态学观察,发现PTX3由中性粒细胞通过NETs形成释放。结论：PTX3可由ANCA诱导的NETs释放至胞外,参与血管炎的病理生理过程。     

Long pentraxin 3 (PTX3) expression and release by neutrophils in vitro and in ulcerative colitis

Pentraxin 3 (PTX3) is the first identified long pentraxin, and it is rapidly produced and released by several cell types in response to proinflammatory signals. The aim of this study was to investigate the behavior of neutrophils to produce PTX3 protein in response to proinflammatory cytokine IL-8 in vitro, as well as identify the expression pattern of PTX3 in human ulcerative colitis lesions. Pentraxin 3 protein was found to be present following release upon IL-8 stimulation in cultured neutrophils together with lactoferrin(+)-specific granules localized in neutrophil extracellular traps (NETs) formed by extruded DNA. Neutrophils in colonic mucosal tissue of patients with ulcerative colitis were the main cellular source of PTX3 protein, the expression of which is correlated well with the histological grades of inflammation. Immunofluorescence analysis against anti-lactoferrin antibody revealed the formation of NETs released from neutrophils within crypt abscess lesions, which were found to be activated through the expression of IL-8 receptor B (CXCR2). Of interest, neutrophils depleted of PTX3 protein were displayed, supporting the release of PTX3 from neutrophils in crypt abscess. We suspected that PTX3 protein may contribute to cell-mediated immune defense in inflamed colon tissue, and in particular in crypt abscess lesions, of patients with ulcerative colitis.     

Neutrophil extracellular traps in sterile inflammation: the story after dying?

Evidences accumulated that the death of neutrophils are not the end of their missions. The neutrophil extracellular traps (NETs), web-like structure, formed after neutrophils dying contribute greatly to immune defense, in both innate and adaptive immunity. Interestingly, previous studies revealed that the generation and activation of NETs do not only rely on bacteria induction, but also in patients with sterile inflammatory diseases, implying an undeniable correlation between NETs and these diseases. This review summarized the latest findings that the crucial roles of NETs in sterile inflammatory diseases, as well as novel targeted therapy based on these new discoveries.     

Toxoplasma gondii triggers release of human and mouse neutrophil extracellular traps

Neutrophils have recently been shown to release DNA-based extracellular traps that contribute to microbicidal killing and have also been implicated in autoimmunity. The role of neutrophil extracellular trap (NET) formation in the host response to nonbacterial pathogens has received much less attention. Here, we show that the protozoan pathogen Toxoplasma gondii elicits the production of NETs from human and mouse neutrophils. Tachyzoites of each of the three major parasite strain types were efficiently entrapped within NETs, resulting in decreased parasite viability. We also show that Toxoplasma activates a MEK-extracellular signal-regulated kinase (ERK) pathway in neutrophils and that the inhibition of this pathway leads to decreased NET formation. To determine if Toxoplasma induced NET formation in vivo, we employed a mouse intranasal infection model. We found that the administration of tachyzoites by this route induced a rapid tissue recruitment of neutrophils with evidence of extracellular DNA release. Taken together, these data indicate a role for NETs in the host innate response to protozoan infection. We propose that NET formation limits infection by direct microbicidal effects on Toxoplasma as well as by interfering with the ability of the parasite to invade target host cells.