Chromatin extracellular trap release in rainbow trout, Oncorhynchus mykiss (Walbaum, 1792).
Chromatin extracellular trap release in rainbow trout, Oncorhynchus mykiss (Walbaum, 1792).
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DOI：
10.1016/j.fsi.2020.01.040
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发表时间：
2020-01
影响因子：
4.7
通讯作者：
A. P. Van;Neila Álvarez de Haro;J. Bron;Andrew P. Desbois
中科院分区：
文献类型：
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作者：
A. P. Van;Neila Álvarez de Haro;J. Bron;Andrew P. Desbois
中性粒细胞将修饰有抗菌蛋白的核染色质释放到细胞外环境中，作为对抗入侵微生物的先天免疫防御机制。这些染色质结构被称为细胞外陷阱（Extracellular traps，简称ETRs），通过一种名为NETosis的过程释放出来，已在哺乳动物、某些无脊椎动物和一些鱼类中检测到，包括黑头呆鱼、斑马鱼、鲤鱼、大菱鲆、比目鱼和肺鱼。然而，以前没有研究过鲑科中的寄生虫。鱼的释放是对化学和生物刺激的反应，但不同鱼类的观察结果不一致，特别是关于各种诱导剂和抑制剂的效力。因此，本研究旨在描述鲑鱼（虹鳟鱼，虹鳟鱼（Walbaum，1792））中的ET释放，并揭示可以控制这种反应的诱导剂和抑制剂。通过三层Percoll梯度技术从头肾组织制备高度富集的多形核细胞（PMNs;主要是中性粒细胞）悬浮液。通过用核酸特异性染料染色质并对预期装饰结构的特征蛋白进行免疫细胞化学探测，在富含中性粒细胞的悬浮液中观察ET结构。ET释放定量孵育后与诱导剂和抑制剂已知影响这种反应在其他生物体。用SYTOX绿色（一种核酸特异性染色剂）将类似于大肠杆菌的结构染色为阳性，同时使用免疫细胞化学检测结构中的中性粒细胞弹性蛋白酶、髓过氧化物酶和H2 A组蛋白，它们是大肠杆菌的诊断性蛋白质标记物。与哺乳动物和一些鱼类的其他研究一致，钙离子载体和鞭毛蛋白是有效的诱导剂，而细胞松弛素D抑制NETosis。佛波醇12-肉豆蔻酸酯13-乙酸酯（PMA），通常用于诱导内皮素，只产生弱的刺激活性，而热杀死的细菌和脂多糖不诱导ET释放。出乎意料的是，ET-抑制剂diphenyleneiodonium chloride作为ET释放的诱导剂，这一观察结果在其他地方没有报道。综上所述，这些数据首次证实了鲑鱼PMNs释放出的蛋白质，并鉴定了可用于操纵NETosis的化合物，从而为进一步研究探索这种机制在鱼类免疫中的作用提供了平台。这一新知识为转化为农场环境提供了基础，因为先天免疫反应的操纵提供了使用抗生素减轻微生物感染的潜在替代方案，特别是对于尚未实现疫苗接种保护的病原体。
Neutrophils release nuclear chromatin decorated with antimicrobial proteins into the extracellular milieu as an innate immune defence mechanism to counter invading microbes. These chromatin structures, called extracellular traps (ETs) and released by a process called NETosis, have been detected in mammals, certain invertebrates and some fish species, including fathead minnow, zebrafish, common carp, turbot, sole and barramundi. However, there have been no previous studies of ETs in the Salmonidae. ETs are released in response to chemical and biological stimuli, but observations from different fish species are inconsistent, particularly regarding the potency of various inducers and inhibitors. Thus, this present study aimed to describe ET release in a salmonid (rainbow trout,Oncorhynchus mykiss(Walbaum, 1792)) and uncover the inducers and inhibitors that can control this response. Highly enriched suspensions of polymorphonuclear cells (PMNs; mainly neutrophils) were prepared from head kidney tissues by a triple-layer Percoll gradient technique. ET structures were visualised in PMN-enriched suspensions through staining of the chromatin with nucleic acid-specific dyes and immunocytochemical probing of characteristic proteins expected to decorate the structure. ET release was quantified after incubation with inducers and inhibitors known to affect this response in other organisms. Structures resembling ETs stained positively with SYTOX Green (a stain specific for nucleic acid) while immunocytochemistry was used to detect neutrophil elastase, myeloperoxidase and H2A histone in the structures, which are diagnostic proteinaceous markers of ETs. Consistent with other studies on mammals and some fish species, calcium ionophore and flagellin were potent inducers of ETs, while cytochalasin D inhibited NETosis. Phorbol 12-myristate 13-acetate (PMA), used commonly to induce ETs, exerted only weak stimulatory activity, while heat-killed bacteria and lipopolysaccharide did not induce ET release. Unexpectedly, the ET-inhibitor diphenyleneiodonium chloride acted as an inducer of ET release, an observation not reported elsewhere. Taken together, these data confirm for the first time that ETs are released by salmonid PMNs and compounds useful for manipulating NETosis were identified, thus providing a platform for further studies to explore the role of this mechanism in fish immunity. This new knowledge provides a foundation for translation to farm settings, since manipulation of the innate immune response offers a potential alternative to the use of antibiotics to mitigate against microbial infections, particularly for pathogens where protection by vaccination has yet to be realised.