1 NETs及其形成过程（NETosis）
NETs是存在于胞外的一种纤维网状结构，其在基因组DNA的基础上还包含组蛋白、髓过氧化物酶（myeloperoxidase，MPO）、中性粒细胞弹性蛋白酶（neutrophil elastase，NE）和组织蛋白酶G等抗菌活性蛋白
质[3] . 电镜下观察NETs的结构发现主要为直径15~17 nm的（DNA骨架）平滑纤维和直径为25 nm的镶嵌在纤维上的球形（蛋白质）区域组成[2] . 应用DNA染料染色后可发现感染部位NETs的网状结构，而以DNase处理后，NETs结构的完整性则被破坏. 与之相反，应用蛋白裂解酶处理NETs，其DNA结构仍可保持完整性，说明DNA的确是NETs的主要结构[2] . NETs特有的三维网状结构可以发挥良好的捕获细菌、真菌和病毒等病原微生物的功能，可防止病原体扩散、促进抗菌因子聚集于感染部位而有效地发挥作用，并且NETs可依赖自身大量的抗菌蛋白质发挥病原体杀灭作用. 然而，NETs同时也是一把“双刃剑”，如在肺部组织中形成过多或不被机体及时降解，NETs中诸多的活性蛋白质对肺组织和细胞也会起到损伤/破坏作用，进而加重炎症反应甚至促进肺部疾病的发展.NETs的形成过程称之为NETosis，它是一种中性粒细胞凋亡和坏死之外的另一种死亡途径，其主要包括核膜的破裂、染色体的解聚、相关蛋白质的嵌入、胞膜的破裂以及NETs的排出，整个过程不需要半胱氨酸天冬氨酸蛋白酶（caspase）的参与且不伴随DNA的片段
化[4] . 诸多物质如病原微生物、LPS、PMA和活化的血小板等均可刺激中性粒细胞NETs的形成[5] . 随着研究的深入，目前认为中性粒细胞的NETosis可通过如下3种模式发生：a. 自杀式NETosis，是目前了解最多也是最主要的一种模式. 当中性粒细胞被刺激后，通过蛋白激酶C激活Raf/MERK/ERK信号通路，导致烟酰胺腺嘌呤二核苷酸磷酸（nicotinamide adenine dinucleotide phosphate，NADPH）氧化酶活化进而刺激活性氧簇（reactive oxygen species，ROS）的产生，促使肽酰基精氨酸脱亚胺酶4（peptidylarginine deiminase 4，PAD4）催化组蛋白瓜氨酸化，进而使得核染色体解聚[6] . 同时，胞内作为PAD4辅酶的钙离子浓度升高，加速核染色体解聚. 并且，胞内嗜天青颗粒释放MPO和NE进入细胞核，进一步促进核染色体解聚. 随后核膜破裂，解聚后的染色体进入胞质并被颗粒蛋白等加以修饰. 最终胞膜破裂，NETs以弹射方式释放至细胞外. 综上可知，ROS的产生和PAD4介导的核染色体解聚是发生自杀式NETosis的必要条件[7,8] . b. 细胞核DNA释放式NETosis，不依赖于Raf/MERK/ERK信号通路的激活和ROS的产生，且细胞核膜和胞膜的完整性得以保留，释放NETs后的中性粒细胞仍可吞噬病原微生物并且寿命不受影响，补体C3蛋白和Toll样受体可促发此种形式的NETosis[6] . c. 线粒体DNA释放式NETosis，依赖于ROS的产生，当中性粒细胞受到LPS或粒细胞-巨噬细胞集落刺激因子（granulocyte-macrophage colony stimulating factor，GM-CSF）刺激后可发生此种类型的NETosis[6] .2 NETs与哮喘
支气管哮喘是临床上常见的一种慢性气道炎症性疾病. 传统观点认为嗜酸性粒细胞在哮喘的发病过程中发挥关键作用，因此既往有大量的研究报道了嗜酸性粒细胞在哮喘中的作用和机制，并且在一定程度上证明了嗜酸性粒细胞确实是促使哮喘这种慢性气道炎症发生发展的重要效应细
胞[9] . 但随着对哮喘发病机制的逐步认识，近年来发现中性粒细胞同样在哮喘的发病过程中发挥着不容忽视的重要作用[10,11] . 因而当前的研究观点认为可以根据哮喘患者诱导痰中的炎症细胞类型将其分为4型：嗜酸性粒细胞性哮喘、中性粒细胞性哮喘、混合细胞性哮喘和寡细胞性哮喘[12] . 而中性粒细胞性哮喘（neutrophilic asthma，NA）在上述4种哮喘类型中占比超过了20%[13] ，且NA与临床上重症和难治性哮喘等关联密切[14,15] .研究发现，中性粒细胞可以在人过敏性哮喘气道中产生NET
s[16] ，中性粒细胞性哮喘与非中性粒细胞性哮喘相比，诱导痰中的细胞外DNA和NETs组分的含量明显较高，且其含量与气道内中性粒细胞水平呈正相关，而与肺功能、呼吸系统症状以及疾病控制呈负相关[17] . 另有研究表明NETs可以损伤气道上皮并引发炎症反应，从而加重哮喘的严重程度[18] . 在小鼠动物实验模型中的研究发现，仙台病毒感染通过将中性粒细胞募集到肺泡形成NETs而诱发哮喘[19] . 且在马动物实验模型中的研究结果也发现，中性粒细胞性重症哮喘马的气道中NETs的含量明显升高[20] . 既往已有研究报道哮喘患者气道内LPS和IL-8的水平明显升高，并且目前已知LPS和IL-8可诱发中性粒细胞的NETosis，表明哮喘患者气道内NETs的形成可能与过量的LPS和IL-8存在有关[2,18,21] . 综上可知，NETs在气道内的积聚加剧了哮喘病的疾病进展.3 NETs与慢性阻塞性肺病
慢性阻塞性肺疾病（chronic obstructive pulmonary disease，COPD）是一种以气道内气流受限，不完全可逆并呈进行性发展为特征的疾
病[22] . COPD的主要病理改变为气道重塑，主要表现为气道上皮损伤、皮下组织纤维化、气道平滑肌增生、黏液腺增生和化生，以及血管损伤及重构等[23] ，被认为是气道以中性粒细胞为主的炎症细胞和结构细胞之间相互作用的结果. 研究发现，COPD的患者不仅其气道痰液中中性粒细胞的比例明显升高并与疾病严重程度正相关[24] ，而且其诱导痰、支气管肺泡灌洗液和气道上皮中也均发现中性粒细胞的水平升高，并在急性加重期升高尤为明显[25] . 此外，还有研究表明中性粒细胞浸润与COPD气道慢性炎症和重塑密切相关[26,27] . 因此，气道内中性粒细胞性炎症是COPD的一个重要病理特点.对于COPD患者而言，气道内聚集的中性粒细胞可通过释放NETs及其相关活性物质引起慢性气道炎症和肺实质破坏. 通过对COPD患者的痰液标本进行研究发现，与健康对照组人群比较，无论COPD患者是处于稳定期还是急性加重期，其痰液中NETs的水平均明显升高，且急性加重期COPD患者痰液中NETs的含量升高更为显
著[28,29,30] ，进一步分析表明NETs的水平还与COPD患者的气流受限严重程度相关[30] ，说明NETosis是COPD病理过程中的重要环节[31] . 此外，已知吸烟是导致COPD的最为常见的原因，有超过50%的COPD发病由吸烟导致[32] ，且90%的COPD患者死亡与吸烟直接相关[33] . 香烟烟雾刺激不仅可导致中性粒细胞聚集/浸润于气道，而且研究发现尼古丁作为香烟烟雾的一种重要成分，可以通过介导尼古丁乙酰胆碱受体并以剂量依赖性方式诱导中性粒细胞释放NETs[34] . 另有研究发现，香烟烟雾提取物（cigarette smoke extract, CSE）诱发中性粒细胞形成的NETs不仅能刺激支气管上皮细胞分泌IL-6和IL-8[35] ，而且还可诱导浆细胞样树突状细胞的成熟与活化，启动T细胞介导的免疫应答[36] . 提示NETs在吸烟所致的COPD病理的过程中同样具有重要的促进作用.我们课题组利用LPS诱导构建小鼠COPD模型的研究发现，LPS可诱导小鼠气道内NETs的形成，而通过雾化吸入DNaseⅠ可降解NETs以减轻LPS诱导的小鼠气道炎症和黏液分泌过多，且这种减少的机制可能经由TLR4/NF-κB信号通路介
导[37] . 但NETs导致COPD发生发展的分子机制较为复杂，有待进一步深入研究阐明.4 NETs与细菌性肺炎
临床上最常见的细菌性肺炎是社区获得性肺炎（community acquired pneumonia，CAP）. 肺炎链球菌是CAP最常见的致病因素，发病率和死亡率都很高，其次是流感嗜血杆
菌[38,39] . 在CAP发生发展期间，大量中性粒细胞被募集到感染部位而引发炎症反应，并且产生过量的NETs. 研究表明，有3种不同的肺炎链球菌菌株（血清型3、4和19F）能够诱导小鼠肺部NETs的形成，且与肺组织病理学病变的严重程度相关[40] . 进一步研究发现肺炎链球菌α-烯醇化酶可能介导肺炎链球菌诱导NETs形成的过程[41] . 但是，肺炎链球菌自身能够抵抗NETs的杀菌作用. 肺炎链球菌能表达一种膜定位内切酶EndA，它能够在体外降解NETs并促进细菌从上呼吸道扩散到肺部再进而扩散至血液[42,43] . 除了表达EndA，肺炎链球菌还能通过使菌体表面带正电荷来保护自身以免被NETs捕获和杀伤[44] . 因此，在肺炎链球菌感染过程中释放的NETs可能只起到了破坏肺组织的作用，而并不能发挥杀菌活性.不可分型流感嗜血杆菌（缺乏荚膜）也是肺炎的重要病因，主要发生于慢性支气管炎和慢性阻塞性肺病患
者[45] ，NETs的持续存在同样可能加重这些患者的肺炎情况. 研究发现，不管是活性的或还是灭活的流感嗜血杆菌在体外均能诱导中性粒细胞释放NETs，其机制可能是由流感嗜血杆菌的脂寡糖与中性粒细胞表面的Toll样受体4结合而介导. 但是，流感嗜血杆菌不仅不会被刺激形成的NETs杀死而且还能在NETs中存活[46] . 进一步研究证明这些细菌可表达特定蛋白质分子如过氧化物氧还蛋白-谷氧还蛋白和过氧化氢酶，使其能抵抗宿主体内的氧化物质并可在NETs中存活[47] . 此外，研究还发现不可分型流感嗜血杆菌存在于呼吸道表面的生物膜群落，而NETs也是构成这些生物膜的一个组成部分[48] .5 NETs与结核
结核病目前仍然是人类的主要健康问题之一. 结核病是由结核分枝杆菌感染引起，主要影响肺部，其发病率和死亡率居高不
下[49] . 结核分枝杆菌通常通过感染者咳嗽或打喷嚏而呼出的微小液滴传播，一旦进入人体肺部，通常会被肺泡巨噬细胞吞噬并杀死. 然而，部分结核分枝杆菌可在巨噬细胞内存活，从而形成潜伏感染，并诱导肺实质形成肉芽肿. 虽然巨噬细胞一般被认为是携带结核分枝杆菌的主要细胞，但越来越多的证据表明被迅速募集到被感染肺组织的中性粒细胞同样可存储结核分枝杆菌. 研究发现，人中性粒细胞能够在体外吞噬结核分枝杆菌，但不能将其杀灭[50] ，而且中性粒细胞还是活动性肺结核患者痰和肺泡灌洗液中的主要免疫细胞类型[51] . 因此，中性粒细胞在活动性肺结核病中可能发挥保护结核分枝杆菌的作用从而促进肺结核的病理进程.研究发现，中性粒细胞被不同基因型的结核分枝杆菌（H37Rv和M. canettii）刺激时均可形成NETs，但其能够捕获结核分枝杆菌却发挥不了杀灭作
用[52] . 结核分枝杆菌诱导NETs形成的效应可能由其分泌的早期分泌抗原6介导，该抗原负责将结核分枝杆菌从吞噬体逃逸至细胞质中，其与NETs中的重要活性蛋白MPO存在共定位情况[53,54] . 在活动性肺结核患者的肉芽肿内，中性粒细胞被结核分枝杆菌刺激可释放大量的钙卫蛋白[55] ，这些蛋白质也是NETs的组成成分[56] . 另有研究发现，结核分枝杆菌在体外可诱导中性粒细胞释放含有基质金属蛋白酶8（matrix metalloproteinase，MMP-8）的NETs，且与健康受试者相比，来自肺结核病患者的诱导痰中含有更多的NETs，进一步分析表明肺结核患者中MMP-8的分泌水平与肺组织的破坏程度正相关[57] . 总而言之，对于肺结核患者而言，由结核分枝杆菌长期、反复刺激中性粒细胞而持续形成的NETs，为结核分枝杆菌对肺组织的破坏起到了协助作用.6 NETs与肺囊性纤维化
肺囊性纤维化是一种常染色体隐性遗传的先天性家族性疾病，常发病于北美洲白色人种，而在其他人种当中罕见. 肺囊性纤维化以外分泌腺病变为基础，由于支气管中的黏液分泌过多、黏稠且排出困难，导致支气管阻塞，使得细菌（如铜绿假单胞菌和金黄色葡萄球菌）易于生长繁殖，进而引起支气管和肺组织反复感染，最终导致肺囊性纤维化. 随着病情的进展，肺囊性纤维化的患者常出现右心肥大，继发心力衰竭. 肺囊性纤维化的炎症细胞浸润以中性粒细胞为主. 中性粒细胞能通过多种方式如吞噬作用、产生活性氧、脱粒作用和形成NETs等抗菌，但这些抗菌方式在肺囊性纤维化的疾病进程中均不能正常发挥作
用[58,59,60] .NETs在囊性肺纤维化中的抗菌作用非常有限. 尽管实验室培养的以及从临床囊性肺纤维化患者体内分离的铜绿假单胞菌均能强烈触发NETs释
放[61] ，但从晚期肺纤维化患者气道中分离获得的铜绿假单胞菌对NETs具有抗性[62,63] . 另有研究表明只有约25％的中性粒细胞形成的NETs在体外可杀死金黄色葡萄球菌[64] . 与健康对照相比，肺囊性纤维化患者的支气管肺泡灌洗液中的游离DNA含量明显升高[65] ，最初认为这些游离DNA来源于凋亡和坏死的细胞，但研究表明，其大多数来自于NETs，且NETs与肺囊性纤维化患者的较差肺功能有关[66,67] . 在临床上，DNase已被用于肺囊性纤维化患者的治疗，通过雾化吸入，可改善肺囊性纤维化儿童患者的肺功能，并对儿童和成人患者的疾病恶化率均有降低作用[68,69,70] . 由于DNase的主要作用是通过切割过量的细胞外DNA以降低痰液黏稠度而助于排痰，因此可以认为DNase在肺囊性纤维化患者治疗过程中发挥功效的主要靶标就是NETs.7 NETs与间质性肺疾病
间质性肺疾病的病理基础为弥漫性的肺实质和肺泡炎症以及间质纤维化，主要临床表现为进行性加重的活动性呼吸困难、限制性通气障碍、肺弥散功能降低和顽固性低氧血症. 同时，间质性肺疾病也是一种以中性粒细胞浸润为主的非感染性炎症性疾病. 研究发现，特发性肺纤维化（一种常见的间质性肺疾病）患者的支气管肺泡灌洗液中活化的中性粒细胞增加，且与患者的早期死亡率相
关[71] . 并发有间质性肺疾病的自身免疫性疾病患者血液中NETs的两种主要成分，游离DNA和抗菌肽LL-37的水平明显升高，且DNase的活性明显降低，提示NETs的慢性刺激导致了间质性肺疾病的发生[72] . 有间质性肺病并发症的皮肌炎患者中也可检测到升高的循环NETs水平，提示异常升高的NETs可能参与了皮肌炎患者间质性肺病的发病[73] .对间质性肺疾病患者的活检组织进行检测发现肌成纤维细胞附近有NETs聚集，而体外研究表明NETs可促进肺成纤维细胞的活化，并能促使其分化为肌成纤维细胞，用DNase降解NETs后，这种促纤维化作用明显下
降[74] . 另有研究发现NE抑制剂可通过抑制转化生长因子β1（transforming growth factor β1，TGF-β1）和肺部炎性细胞募集来减轻博来霉素诱导的肺纤维化[75] . NE在体外对肺成纤维细胞的增殖和肌成纤维细胞的分化都有促导作用，说明NETs导致间质性肺疾病的作用很可能是由其中的NE所介导[76] . 总之，这些研究均提示NETs在间质性肺疾病的发生发展中发挥着关键作用.8 NETs与流感病毒感染
流感病毒感染是呼吸系统最为常见的病症之一，其中以甲型流感病毒最为流行. 就临床抽血检验而言，单纯的流感病毒感染通常的表现是淋巴细胞升高而中性粒细胞比例无明显变化. 然而，在肺组织的局部，病毒感染同样可导致中性粒细胞的浸润. 研究表明流感病毒感染刺激的NETs形成依赖于PAD4对组蛋白的消
除[77] . 而流感病毒诱导的NETs形成过程可被抗菌肽LL-37加强[78,79] . 中性粒细胞被流感病毒感染刺激而形成的NETs对机体既可起到保护作用又能发挥损伤作用. NETs上所含的抗菌蛋白α防御素1能够通过抑制被流感病毒感染细胞中的蛋白激酶C而直接抑制流感病毒的复制[80] . NETs上的α防御素可以让被NETs隔离的病毒颗粒失活，从而阻止流感病毒感染肺组织中的靶细胞[81] . 然而，在流感病毒急性感染、病毒大量复制的过程中，肺组织内产生过量的NETs对机体而言却是有害的. 研究发现，被流感病毒感染后，NETs中所含的过量的组蛋白和MMP-9沉积于肺泡，可导致肺泡毛细血管损伤和小气道阻塞[82] ，使得损伤的肺组织易于继发细菌感染.9 NETs与急性肺损伤
急性肺损伤（acute lung injury，ALI）是由各种原因导致的肺毛细血管内皮和肺泡上皮的损伤，造成弥漫性肺泡和肺间质水肿，进而导致呼吸功能不全，主要临床表现为急性进行性低氧血症和呼吸窘迫，病情发展至严重阶段则被称为急性呼吸窘迫综合征（acute respiratory distress syndrome，ARDS）. 严重感染是导致急性肺损伤的最常见因素. 由于肺是全身炎症期间最敏感的靶器
官[83] ，所以急性肺损伤是严重脓毒症的一种常见并发症，其在临床上具有很高的发病率和死亡率[84] . 研究表明，与健康对照相比，发生严重急性呼吸窘迫综合征的脓毒症患者血清中游离NETs的含量明显升高[85] . 我们课题组通过收集临床标本进行研究发现，ARDS患者支气管吸出物中NETs的水平与病情的严重程度正相关，且发现在ALI小鼠模型的支气管肺泡灌洗液中NETs的含量明显高于对照组，靶向抑制NETs形成可显著减弱ARDS的强度[86] . 我们课题组的研究还发现，在LPS诱导的小鼠ALI的组织中也可检测到NETs的形成，而利用 DNaseⅠ进行治疗能显著降低NETs的水平，从而得以有效防止急性肺损伤[87] . 另有研究发现在脓毒症小鼠的肺泡腔和肺毛细血管中均能观察到NETs[88] ，且NETs的过度释放与心脏、肝脏和肺部的急性损伤直接相关，而联合rhDNase（人工重组DNA酶）和抗生素治疗脓毒症小鼠可显着降低这些器官的损伤[85] .10 总结与展望
目前已有许多证据表明NETs在各种肺部炎症性疾病当中均有存在，尽管在疾病的早期阶段NETs有捕获和杀灭病原微生物的作用，对防止病原微生物在肺部的扩散至关重要，但是越来越多的研究发现，呼吸道和肺组织中过量或持续存在的NETs会对肺造成不同程度的损伤，导致肺功能受损、加速疾病进展. NETs在肺部炎症性疾病发生发展的过程中，理论上来说应该存在一个“有害”与“有益”的临界点，如何发现和利用这个临界点是今后NETs在肺部炎症性疾病当中的研究重点. 目前重组人DNase是在少数肺部炎症性疾病中针对NETs的唯一治疗方法，但长期使用会产生严重的副作用. 因此，针对NETs进行靶向治疗的新药研发也是将来的重要研究方向.
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- 1
摘要
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.
Abstract
Neutrophil recruitment/infiltration is a characteristic manifestation of pulmonary inflammatory diseases and is the first line of defense against pathogenic microbial invasion in the lungs, killing pathogenic microorganisms mainly through phagocytosis. However, new research has found that neutrophils can form a network called neutrophil extracellular traps (NETs) that are DNA-based and inlaid with a large amount of active protein. This biological structure can capture and kill pathogenic microorganisms. Although it is beneficial for lung inflammatory diseases in terms of the biological function of NETs, more and more studies have shown that NETs have direct cytotoxic effects on lung epithelial cells and endothelial cells, and may promote the lung inflammatory diseases. In order to systematically understand the relationship between NETs and lung-related inflammatory diseases, this review first briefly describes the structure, function and formation process of NETs, and then describes the relationship of NETs and asthma, chronic obstructive pulmonary disease, bacterial pneumonia, tuberculosis, cystic fibrosis, interstitial lung disease, influenza virus infection and acute lung injury, finally summarizes the potential research directions and targeted treatment strategies of NETs in pulmonary inflammatory diseases.
呼吸系统是许多病原微生物入侵机体的主要部位，因此呼吸系统特别依赖于自身的先天性免疫监视，即以中性粒细胞为主的固有免疫细胞. 中性粒细胞募集到气道和肺组织是许多呼吸系统疾病的主要标志，一般情况下中性粒细胞利用其生物活性物质来抵抗细菌和真菌入侵肺部，但中性粒细胞的募集和活化也可见于病毒性呼吸系统感
NETs于2004年首次被Brinkmann
