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Chromatin extracellular trap release in rainbow trout, Oncorhynchus mykiss (Walbaum, 1792).
Fish & Shellfish Immunology ( IF 4.1 ) Pub Date : 2020-01-25 , DOI: 10.1016/j.fsi.2020.01.040 Andre P Van 1 , Neila Álvarez de Haro 1 , James E Bron 1 , Andrew P Desbois 1
Fish & Shellfish Immunology ( IF 4.1 ) Pub Date : 2020-01-25 , DOI: 10.1016/j.fsi.2020.01.040 Andre P Van 1 , Neila Álvarez de Haro 1 , James E Bron 1 , Andrew P Desbois 1
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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.
中文翻译：
虹鳟Oncorhynchus mykiss（Walbaum，1792）释放出染色质细胞外陷阱。
中性粒细胞将装饰有抗菌蛋白的核染色质释放到细胞外环境中，作为抵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胞外陷阱（ETs），并通过称为NETosis的过程释放出来，已在哺乳动物，某些无脊椎动物和某些鱼类中发现，其中包括黑头fat鱼，斑马鱼，鲤鱼，大菱turbo，t和澳洲肺鱼。但是，鲑鱼科中没有关于ET的先前研究。ET是根据化学和生物刺激而释放的，但不同鱼类的观察结果不一致，尤其是在各种诱导剂和抑制剂的效力方面。因此，本研究旨在描述ET在鲑鱼（虹鳟，Oncorhynchus mykiss（Walbaum，1792）），并发现可以控制这种反应的诱导剂和抑制剂。通过三层Percoll梯度技术从头部肾脏组织中制备了高度浓缩的多形核细胞（PMN；主要是中性粒细胞）悬液。通过用核酸特异性染料染色质染色和预期修饰该结构的特征蛋白进行免疫细胞化学探测，可以在富含PMN的悬浮液中可视化ET结构。与已知会影响其他生物反应的诱导剂和抑制剂孵育后，对ET释放进行定量。类似于ET的结构被SYTOX Green（一种核酸特异的染料）阳性染色，而免疫细胞化学用于检测结构中的嗜中性粒细胞弹性蛋白酶，髓过氧化物酶和H2A组蛋白，它们是ET的诊断性蛋白质标记。与关于哺乳动物和某些鱼类的其他研究一致，钙离子载体和鞭毛蛋白是ET的有效诱导剂，而细胞松弛素D抑制NETosis。通常用于诱导ET的12肉豆蔻酸13肉豆蔻酸酯（PMA）仅具有弱的刺激活性，而热杀死的细菌和脂多糖则不诱导ET释放。出乎意料的是，ET抑制剂二苯二碘氯化铵充当了ET释放的诱导剂，这一发现在其他地方未见报道。综上所述，这些数据首次证实了鲑鱼PMN释放了ET，并鉴定了可用于控制NETosis的化合物，从而为进一步研究该机制在鱼类免疫中的作用提供了平台。这些新知识为翻译到服务器场设置奠定了基础，
更新日期：2020-01-26
中文翻译：
虹鳟Oncorhynchus mykiss（Walbaum，1792）释放出染色质细胞外陷阱。
中性粒细胞将装饰有抗菌蛋白的核染色质释放到细胞外环境中，作为抵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胞外陷阱（ETs），并通过称为NETosis的过程释放出来，已在哺乳动物，某些无脊椎动物和某些鱼类中发现，其中包括黑头fat鱼，斑马鱼，鲤鱼，大菱turbo，t和澳洲肺鱼。但是，鲑鱼科中没有关于ET的先前研究。ET是根据化学和生物刺激而释放的，但不同鱼类的观察结果不一致，尤其是在各种诱导剂和抑制剂的效力方面。因此，本研究旨在描述ET在鲑鱼（虹鳟，Oncorhynchus mykiss（Walbaum，1792）），并发现可以控制这种反应的诱导剂和抑制剂。通过三层Percoll梯度技术从头部肾脏组织中制备了高度浓缩的多形核细胞（PMN；主要是中性粒细胞）悬液。通过用核酸特异性染料染色质染色和预期修饰该结构的特征蛋白进行免疫细胞化学探测，可以在富含PMN的悬浮液中可视化ET结构。与已知会影响其他生物反应的诱导剂和抑制剂孵育后，对ET释放进行定量。类似于ET的结构被SYTOX Green（一种核酸特异的染料）阳性染色，而免疫细胞化学用于检测结构中的嗜中性粒细胞弹性蛋白酶，髓过氧化物酶和H2A组蛋白，它们是ET的诊断性蛋白质标记。与关于哺乳动物和某些鱼类的其他研究一致，钙离子载体和鞭毛蛋白是ET的有效诱导剂，而细胞松弛素D抑制NETosis。通常用于诱导ET的12肉豆蔻酸13肉豆蔻酸酯（PMA）仅具有弱的刺激活性，而热杀死的细菌和脂多糖则不诱导ET释放。出乎意料的是，ET抑制剂二苯二碘氯化铵充当了ET释放的诱导剂，这一发现在其他地方未见报道。综上所述，这些数据首次证实了鲑鱼PMN释放了ET，并鉴定了可用于控制NETosis的化合物，从而为进一步研究该机制在鱼类免疫中的作用提供了平台。这些新知识为翻译到服务器场设置奠定了基础，
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