中性粒细胞细胞外诱捕网的研究进展

中性粒细胞是机体先天性免疫系统中的重要组成成分,是外周血中数量最多的免疫细胞,在非特异性免疫中至关重要,是构成机体内抵御微生物入侵的第一道防线。近年来,人们发现中性粒细胞除了经典的通过胞吞作用吞噬病原微生物、释放嗜天青颗粒及产生并分泌抑菌杀菌物质外,还具有抵抗微生物入侵的新机制——中性粒细胞细胞外诱捕网(neutrophil extracellular traps,NETs)。该机制主要通过活性氧和肽酰基精氨酸脱亚氨酶4等影响因子刺激中性粒细胞形成以DNA为骨架的网状结构,内含有多种蛋白酶,能非特异性捕获杀伤病原体,还能在机体某些疾病的产生和发展中起到协同作用。作者就NETs的形成、影响因素及与病原、疾病的相互关系作一概述,为进一步研究中性粒细胞的功能提供依据。     

中性粒细胞细胞外诱捕网的研究进展

中性粒细胞是机体先天性免疫系统中的重要组成成分,是外周血中数量最多的免疫细胞,在非特异性免疫中至关重要,是构成机体内抵御微生物入侵的第一道防线。近年来,人们发现中性粒细胞除了经典的通过胞吞作用吞噬病原微生物、释放嗜天青颗粒及产生并分泌抑菌杀菌物质外,还具有抵抗微生物入侵的新机制--中性粒细胞细胞外诱捕网（neutrophil extracellular traps,NETs）。该机制主要通过活性氧和肽酰基精氨酸脱亚氨酶4等影响因子刺激中性粒细胞形成以DNA为骨架的网状结构,内含有多种蛋白酶,能非特异性捕获杀伤病原体,还能在机体某些疾病的产生和发展中起到协同作用。作者就NETs的形成、影响因素及与病原、疾病的相互关系作一概述,为进一步研究中性粒细胞的功能提供依据。     

中性粒细胞胞外捕获网防御机制的研究进展

中性粒细胞作为先天免疫的吞噬细胞,在免疫防御中起核心作用。中性粒细胞胞外捕获网（NETs）的发现使中性粒细胞再次成为研究热点。NETs通过其内含的抗菌肽和诸多的酶系统进行捕获、抑制和杀灭病原微生物。NETs参与炎症和多种自身免疫调节疾病的发生发展。文章就NETs的形成机制以及NETs的宿主防御作用展开综述,以期为相关研究及应用提供参考。     

孕酮诱导猪中性粒细胞胞外诱捕网形成及其机制

中性粒细胞(polymorphonuclear neutrophils,PMN)胞外诱捕网(neutrophil extracellular traps,NETs)是一种重要的先天性免疫反应机制。为了探究孕酮是否与猪PMN NETs形成相关,本研究通过分离母猪外周血获得PMN,与不同质量浓度孕酮(10,20,40μg/L)共孵育2 h后,通过激光共聚焦显微镜观察NETs的形成;运用picoGreen~?试剂盒定量分析NETs的释放水平;通过乳酸脱氢酶(lactate dehydrogenase,LDH)试剂盒分析细胞外LDH活性。结果表明,孕酮可以显著诱导以DNA为骨架,装饰有PMN弹性蛋白(neutrophil elastase,NE)和髓过氧化物酶(myeloperoxidase,MPO)等蛋白颗粒的NETs形成,NETs形成与孕酮诱导剂量以及孵育时间呈正相关,且孕酮诱导猪PMN释放NETs的过程中,细胞外LDH活性无改变。本研究为有关雌性生殖激素参与调节母猪免疫系统的相关研究提供了新思路。     

高乙酰乙酸诱导中性粒细胞胞外诱捕网的形成

中性粒细胞胞外诱捕网(NETs)是几年前才发现的一种重要的先天免疫防御机制,目前NETs的发生机制尚不清楚,但是当缺乏有效抗衡调节时,它又能损伤组织、引发自身免疫等疾病。本试验为了研究作为酮病奶牛高酮血症的主要成分之一的乙酰乙酸(ACAC)对NETs表达量的影响,体外添加不同浓度ACAC处理中性粒细胞,运用免疫荧光技术和激光共聚焦显微成像定性评价NETs的生成;采用PicoGreen~dsDNA染料定量检测细胞上清游离DNA和Western blot反映NETs生成量。结果显示,高ACAC组产生NETs的量显著高于对照组组(P0.05),表明高ACAC可以体外成功诱导中性粒细胞产生NETs。     

旋毛虫成虫排泄分泌物对中性粒细胞功能的影响

利用激光共聚焦显微镜和实时荧光定量PCR等技术,探究旋毛虫成虫排泄分泌物(核酸酶抑制剂ATA阻断DNase酶活性条件下)对中性粒细胞胞外诱捕网形成、活性氧生成和细胞因子产生的影响。结果显示,旋毛虫成虫排泄分泌物能够抑制中性粒细胞胞外诱捕网的形成,减少活性氧生成,促进细胞因子IL-1β、IL-10和TNF-α的表达,降低IL-4的表达,对IFN-γ表达水平无明显影响。结果表明,旋毛虫排泄分泌物能够影响中性粒细胞的功能,为进一步研究旋毛虫免疫逃避机制等奠定良好基础。     

沉默中性粒细胞Orai1影响NETs内ROS的表达

旨在研究不同时间与浓度的佛波肉豆蔻酸乙酸酯(PMA)对奶牛中性粒细胞(PMN)形成中性粒细胞胞外诱捕网(NETs)效果,筛选出PMA刺激时间与浓度,通过流式细胞术检测NETs状态下沉默中性粒细胞Orai1,检测钙离子与ROS水平。尾静脉肝素抗凝采集血液分离中性粒细胞,分别在不同时间与不同浓度PMA刺激下,根据激光共聚焦镜下观察NETs的形态状态。结果显示,在PMA在100 nm状态下刺激3 h时NETs形成状态效果最好。在沉默Orai1状态下,通过流式细胞检测结果显示,NETs内钙离子与ROS水平极显著降低。     

食源性病原菌激发固有免疫细胞胞外诱捕网的研究进展

固有免疫细胞在外源物质刺激后向细胞胞外环境释放由染色质DNA和多种胞内颗粒蛋白组成的纤维样网状物质，称为胞外诱捕网（extracellular traps，ETs）。ETs是一种新型的宿主防御机制，能够捕获或杀灭病原菌，有效控制病原菌的扩散，从而使机体免受感染。大肠杆菌、沙门菌等食源性人兽共患病病原体引发的疾病是全球广泛关注的公共卫生问题。本文就食源性病原菌激发固有免疫细胞ETs的形成、ETs的生物学活性以及细菌部分蛋白对ETs形成的影响等研究进展进行综述，以期为食源性疾病的防控提供相关理论参考。     

分泌型核酸酶SsnA介导猪链球菌2型逃避中性粒细胞胞外陷阱捕杀的研究

为了研究猪链球菌分泌型核酸酶SsnA能否介导猪链球菌2型逃避中性粒细胞胞外陷阱(NETs)的捕杀,试验通过构建猪链球菌2型(SS2)菌株的ssn A基因缺失突变株和回复株,比较三者在体外降解DNA的效率、降解NETs的能力及对NETs杀菌效率的影响。结果表明:ssn A基因缺失后的SS2不能在体外降解DNA及经PMA刺激诱导形成的NETs,同时降低了NETs对于SS2的捕杀作用。说明SS2能够通过胞外核酸酶SsnA来降解NETs的DNA骨架,从而逃避NETs的捕获作用而存活。     

禽异嗜性粒细胞与机体免疫研究进展

禽异嗜性粒细胞是存在于禽类血液中的一种主要的颗粒性白细胞,类似于哺乳动物的中性粒细胞,是禽类抵御外界微生物病原体入侵的一道重要防线,也是机体非特异性免疫系统中的重要成员和主要调节者。近年来,对于该细胞在机体急性炎症反应和吞噬杀伤作用中的研究已经深入到了基因和蛋白水平,为从禽类异嗜性粒细胞方面入手治疗禽类疾病,提供了理论依据和研究方向。论文从异嗜性粒细胞与中性粒细胞在形态结构,急性炎症反应和吞噬杀伤病原体作用中的区别,以及近年来该细胞的其他研究热点等方面综述了禽类异嗜性粒细胞的研究进展。     

Neutrophil extracellular trap formation by bovine neutrophils is not inhibited by milk

Neutrophils are the first line of defense in a mammary gland infection. However, the process of neutrophil transmigration across a membrane and ingestion of fat and/or casein when incubated in milk have been shown to inhibit bacterial phagocytosis and oxidative burst functions. Recently, a killing mechanism has been described whereby stimulated neutrophils release nuclear and granule material in fibrous webs that physically trap and kill bacteria. We demonstrate that these neutrophil extracellular traps are also produced by bovine blood neutrophils stimulated with PMA/ionomycin. Importantly, neutrophil extracellular traps can be formed when neutrophils have been incubated for up to 6h in milk prior to stimulation. This contrasts milk's rapid inhibition of bacterial phagocytosis and oxidative burst functions in the neutrophil. Furthermore, stimulation of neutrophils with bacteria common to mammary gland infections leads to neutrophil extracellular traps being formed in milk. Some bacteria tested stimulated enhanced formation of neutrophil extracellular traps in milk compared to culture media. Therefore, being unaffected by incubation in milk may indicate an important role for neutrophil extracellular traps in defense against mastitis.     

Bactericidal activity of porcine neutrophil secretions

Antimicrobial proteins in neutrophil granules exert their bactericidal activity both within the neutrophil phagolysosome and as components of neutrophil extracellular traps. This study evaluated the bactericidal activity of porcine neutrophil secretions against four bacterial pathogens of swine. Porcine neutrophils were treated with or without phorbol myristate acetate (PMA), then the resulting supernatants were incubated with Escherichia coli K-12, Streptococcus suis, Actinobacillus suis, or Pasteurella multocida, and the surviving colony forming units were enumerated. Supernatants of PMA-activated neutrophils killed an average of 95% of E. coli K-12 cells, relative to supernatants from untreated neutrophils. Inhibition of elastase activity using chloromethylketone (CMK) prior to PMA stimulation significantly reduced the bactericidal activity of the neutrophil supernatants; 57% of the PMA-induced bactericidal activity against E. coli K-12 was estimated to be elastase-dependent. The same neutrophil supernatants had lower bactericidal activity against S. suis, A. suis, and P. multocida, with 30%, 36% and 13% reduction in bacterial numbers, respectively. The cathelicidin porcine myeloid antimicrobial peptide (PMAP)-36 and lactotransferrin were among the proteins identified in the supernatants of PMA-stimulated neutrophils by mass spectrometry. These findings imply that elastase-activated proteins, such as cathelicidins, are partially responsible for the bactericidal effect of porcine neutrophil secretions, but non-elastase-dependent proteins such as lactoferrin may also contribute. Further, the secretions of activated neutrophils were effective in killing the avirulent E. coli K-12 but were less effective against the other bacteria tested, suggesting that these pathogens may have evolved mechanisms to resist neutrophil-mediated killing.     

Formation of NET,phagocytic activity,surface architecture,apoptosis and expression of toll like receptors 2 and 4 (TLR2 and TLR4) in neutrophils of mastitic cows

Neutrophils employ both oxidative and non oxidative mechanisms to destroy pathogens. Function of neutrophils coming in milk during mammary invasion is not clearly understood in dairy animals. Therefore, the present study was designed in 36 Sahiwal cows to see the changes occurring in the neutrophil activity of cows suffering from subclinical (SCM) and clinical mastitis (CM). Cows were divided into three groups as healthy (n?=?12), SCM (n?=?12) and CM (n?=?12) groups on the basis of CMT scoring, gross morphological changes in milk, bacteriological examination of milk and by counting milk SCC. Significantly (P?P?P?Toll like receptors 2 and 4 in blood and milk neutrophils were found to be significantly (P?P?TLR4 with the formation of NETs and change in surface architecture. Formation of NET like structures seemed to be an effective mode of defense employed by neutrophils of cows suffering from clinical mastitis.     

Interleukin 17 (IL-17) manipulates mouse bone marrow- derived neutrophils in response to acute lung inflammation

Interleukin 17 (IL-17) mediates neutrophil migration to the lungs during acute inflammation, potentially leading to lung tissue damage. In the present study, we evaluated whether IL-17 could facilitate certain neutrophil functions in a mouse model. Mice were divided into four groups and intranasally challenged with PBS (1 = Control), Influenza A (H1N1) and Klebsiella pneumoniae (2 = Mix), Influenza A alone (3 = Flu), or K. pneumoniae (4 = KP) alone. Bone marrow, BAL cells, and lung specimens were collected seven days post-challenge for analysis. Mice in the Flu group showed the highest mortality rate. Neutrophils were the prominent cell type in BAL from Mix and KP, whereas lymphocytes were most numerous in Flu. Lesions in the lungs revealed considerably damage in the Mix, Flu, and KP groups. Isolated bone marrow-derived neutrophils were in vitro primed with mouse recombinant IL-17A protein (rIL-17A) followed by various functional assays. The reactive oxygen species (ROS) levels in rIL-17A primed cells showed significant elevations in all groups. Phagocytosis and bacterial destruction showed no significant difference between (+) or (−) rIL-17A groups. The formation of neutrophil extracellular traps (NETs) in rIL-17A-primed neutrophils showed elevated NET production. We next monitored expressions of genes in neutrophils. IL-17A mRNA expression was significantly increased in Mix and Flu; IL-1β mRNA only significantly increased in Flu, and IL-17RA showed constitutive expressions in all groups. In summary, neutrophils may cause tissue damage during lung inflammation through specific functions influenced by IL-17.     

A review of the interaction of Bacillus anthracis with cells of the innate immune response

Bacillus anthracis spores cause natural infections and can be used as biological weapons.The innate immune response is the first line of defence against invading pathogens and is likely to play a central role in this acute infection.To date the immune evasion mechanisms of B. anthracis are not well understood. Infection by inhalation with B. anthracis, the etiological agent of anthrax, is almost always lethal, yet cutaneous infections usually remain localized and resolve spontaneously in the majority of cases. Neutrophils are typically recruited to cutaneous but seldom to other forms of anthrax infections, raising the possibility that neutrophils kill B. anthracis. This is an overview of the interaction of B. anthracis with innate immune cells which also focuses on recent data on the role neutrophils play in anthrax pathogenesis. These data suggest that the timely recruitment of neutrophils can control the cutaneous and possibly other forms of B. anthracis infections and that the neutrophil granule proteins, a-defensins, play an important role in the potent anti-B. anthracis activity of neutrophils.     

Differential expression analysis of proteins from neutrophils in the periparturient period and neutrophils from dexamethasone-treated dairy cows

Neutrophils play an important role in the host immune system's defense against pathogens. It has been established that neutrophil functionality is suppressed in dairy cows at parturition. The periparturient immunosuppression seen in dairy cattle is associated with an increase in the incidence of mastitis. Using amine-reactive isobaric tagging reagents we have measured relative protein expression from normal prepartum neutrophils and neutrophils isolated during immunosuppression at parturition. We found over 40 proteins that are differentially expressed at parturition compared to prepartum. In addition, we measured relative protein expression from normal neutrophils and neutrophils obtained from cows treated with an immunosuppressive dose of dexamethasone. We found over 70 proteins are differentially expressed during dexamethasone treatment. We then compared protein expression changes in dexamethasone-induced immunosuppression to periparturient immunosuppression. A number of proteins underwent similar expression changes in both dexamethasone and periparturient immunosuppressed neutrophils. Most significantly, we found a significant number of proteins whose relative expression was not the same for these two different conditions that cause neutrophil dysfunction. The data demonstrates that there are both similarities and differences in neutrophil protein expression in the naturally occurring immunosuppression observed at parturition compared to dexamethasone-induced immunosuppression in the bovine neutrophil.     

Neutrophil extracellular traps in sheep mastitis

Neutrophil extracellular traps (NETs) are structures composed of DNA, histones, and antimicrobial proteins that are released extracellularly by neutrophils and other immune cells as a means for trapping and killing invading pathogens. Here, we describe NET formation in milk and in mammary alveoli of mastitic sheep, and provide a dataset of proteins found in association to these structures. Nucleic acid staining, immunomicroscopy and fluorescent in-situ hybridization of mastitic mammary tissue from sheep infected with Streptococcus uberis demonstrated the presence of extranuclear DNA colocalizing with antimicrobial proteins, histones, and bacteria. Then, proteomic analysis by LTQ-Orbitrap Velos mass spectrometry provided detailed information on protein abundance changes occurring in milk upon infection. As a result, 1095 unique proteins were identified, of which 287 being significantly more abundant in mastitic milk. Upon protein ontology classification, the most represented localization classes for upregulated proteins were the cytoplasmic granule, the nucleus, and the mitochondrion, while function classes were mostly related to immune defence and inflammation pathways. All known NET markers were massively increased, including histones, granule proteases, and antimicrobial proteins. Of note was the detection of protein arginine deiminases (PAD3 and PAD4). These enzymes are responsible for citrullination, the post-translational modification that is known to trigger NET formation by inducing chromatin decondensation and extracellular release of NETs. As a further observation, citrullinated residues were detected by tandem mass spectrometry in histones of samples from mastitic animals. In conclusion, this work provides novel microscopic and proteomic information on NETs formed in vivo in the mammary gland, and reports the most complete database of proteins increased in milk upon bacterial mastitis.

Electronic supplementary material

The online version of this article (doi:10.1186/s13567-015-0196-x) contains supplementary material, which is available to authorized users.     

热休克蛋白在冷应激引起的炎症反应和氧化应激中的调节作用

冬季持续低温引起的冷应激是降低北方畜牧业经济效益和动物福利的重要因素,可诱导动物产生炎症反应和氧化应激危害机体健康。热休克蛋白作为体内重要的分子伴侣蛋白,在维持机体内环境稳态、帮助动物抵抗应激方面有重要作用,低温环境可激活热休克蛋白快速产生,以此在细胞内外对免疫和抗氧化系统发挥重要调节作用：在胞外,具有保护细胞、参与调节免疫细胞功能、激发免疫反应和提高抗氧化酶活性等作用;在胞内,可抑制NF-κB信号通路保护机体免受炎症损伤,上调Nrf2信号通路提高机体抗氧化功能,缓解冷应激对机体造成的负面影响。本文总结了国内外关于在冷应激状态下热休克蛋白对机体免疫和抗氧化功能的调节作用及其机制,并给出部分提高热休克蛋白表达水平的方法,以期为后续畜禽的冷应激理论研究提供参考。