2018, Vol. 20
系统性红斑狼疮(systemic lupus erythematosus, SLE)是一种以自身抗体产生和免疫复合物沉积为主要特点的自身免疫性疾病。儿童系统红斑狼疮(childhood-onset systemic lupus erythematosus, cSLE)发生率较成人低, 每10万名儿童中有6~30名cSLE患儿, 但严重程度更高, 脏器损伤和药物负担也更大[1]。因此, 了解SLE的发病机制对于cSLE的管理至关重要。近年来, 固有免疫细胞, 包括树突状细胞(dendritic cell, DC)、中性粒细胞(neutrophil, NEU)等在自身免疫性疾病中的作用越来越受到关注。中性粒细胞胞外诱捕网(neutrophil extracellular traps, NETs)是Brinkmann等[2]于2004年首次报道的一种中性粒细胞杀菌机制, 激活后的中性粒细胞凋亡或者坏死可释放出一种由DNA和组蛋白等构成的纤维网状结构, 它可捕获病原微生物并对其消化加工而起到杀菌作用, 而中性粒细胞因为NETs释放而导致的特殊形式细胞死亡的过程称为中性粒细胞胞外诱捕网过程(neutrophil extracellular traposis, NETosis)。NETosis伴有大量胞内和核内自身抗原暴露, 长期的NETosis形成与降解失衡可能是导致SLE患者系统性损伤的原因[3-4]。本综述介绍两种NETs致病机制假说, 并重点阐述其在SLE中作用。
1 NETs的致病机制假说 1.1 NETs的形成和活化异常NETosis的特征在于激活的中性粒细胞形成氧爆发和特殊形式的细胞死亡[4]。它分为两个连续的步骤:1)中性粒细胞对病原体趋化及吞噬形成早期NETosis; 2)细胞质、细胞膜破裂, 核酸和颗粒蛋白复合物释放到胞外并展开成纤维网状结构, 形成晚期自杀性的NETosis[5-6]。早期的NETosis至关重要, 通常由金黄色葡萄球菌感染介导的补体受体和Toll样受体2(Toll like receptor-2, TLR2)诱导激活[7-8], 以及由大肠杆菌感染直接介导的Toll样受体4(Toll like receptor-4, TLR4)或间接介导的TLR4激活血小板诱导激活[5, 9]。此外, 由胆固醇结晶诱导的晚期自杀性NETosis不仅可导致中性粒细胞释放NETs并活化巨噬细胞产生白介素-1β(interleukin-1β, IL-1β), 白介素-18(interleukin-18, IL-18), 而且在炎症小体激活后胆固醇结晶还会通过与巨噬细胞膜表面蛋白(如αvβ3、CD14、CD36)结合诱导动脉粥样硬化斑块形成[10-11]。而巨噬细胞分泌的IL-1β、IL-18可诱导激活固有免疫系统, 包括中性粒细胞NETosis的发生。类似现象在NETs活化浆细胞样树突状细胞(plasmacytoid dentritic cells, pDCs)介导的Ⅰ型干扰素α(interferon-α, IFN-α)分泌过程中也有发现[12-13]。肽酰基精氨酸脱亚氨酶-4(peptidylarginine deiminases-4, PAD-4)、微管聚合、肌动蛋白和自噬等介导的组蛋白瓜氨酸化途径也参与NETosis形成[14-15]。
活性氧(reactive oxygen species, ROS)是NETosis形成的关键因子。烟酰胺腺嘌呤二核苷磷酸(nicotinamide adenine dinucleotide phosphate, NADPH)氧化酶和线粒体呼吸链是ROS的两个不同来源途径[16]。NADPH的氧化酶抑制剂二联苯碘(diphenyleneiodonium, DPI)可以阻止NETs的发生[17]。此外, 慢性肉芽肿患者体内的中性粒细胞因具有NADPH氧化酶突变, 也不能形成NETs[18]。Lood等[19]发现, 线粒体可在免疫复合物介导下去极化而诱导ROS生成, 更重要的是可引发线粒体向细胞表面动员外排线粒体DNA(mitochondrial DNA, mtDNA), 并通过自身介导的ROS氧化mtDNA; 该研究还表明, mtDNA在NETosis过程中氧化, 而氧化的mtDNA在诱导编码促炎细胞因子mRNA转录的作用中更具活性。
1.2 NETs的降解途径异常NETs是由DNA骨架所构成的复合物, 因此脱氧核糖核酸酶(deoxyribonuclease, DNase)可能参与到NETs的降解途径。2010年, Hakkim等[20]发现SLE患者血清中的DNase Ⅰ负责降解NETs, 但部分血清中的NETs未得到显著降解; 进一步在血清中加入微球菌核酸酶后, 部分患者的NETs可降解。这可能与血清中存在DNase Ⅰ抑制剂或者DNA保护性抗体有关。Leffler等[21]研究发现补体C1q可与NETs大量结合而保护NETs免遭降解; 部分SLE患者抗双链DNA抗体水平升高, 并与NETs的DNA结合, 使其降解受损[13-22]。对于NETs的清除, 一些不具备吞噬作用的细胞也可能参与, 如纤维母细胞和内皮细胞的膜表面表达A型清道夫受体可直接识别DNA并使其内化降解[23]。
2 NETs在SLE发病机制中的作用通过肽酰精氨酸脱亚胺酶抑制剂和细胞外氧化线粒体DNA方式诱导狼疮易发小鼠, 最终导致NETs的形成[19]。然而, Campbell等[24]发现, Nox2(NADPH的亚单位)缺陷的MRLFaslpr小鼠的中性粒细胞不能进行NETosis, 而且狼疮易感症状加剧。这表明, Nox2依赖的NETosis并不是狼疮和NADPH氧化酶的驱动因素, 而是起到保护作用。类似的矛盾现象也在血管炎研究有所报道[25]。
Midgley等[26-28]研究显示, cSLE患者的中性粒细胞在体外经历了一个加速的自发凋亡过程, 可能与SLE患者的血清利于凋亡有关。而中性粒细胞凋亡将导致核ds-DNA、抗菌肽LL-37等抗原表达增加, 进而促进自身抗体产生并诱导NETs生成, 进一步激活pDCs产生IFN-α。反之, IFN-α又可诱导中性粒细胞更多的核抗原暴露及NETs生成。Garcia-Romo等[29]研究表明, cSLE患儿的抗核糖核蛋白(ribonucleoprotein, RNP)IgG也可导致中性粒细胞释放NETs, 而NETs则激活pDCs分泌IFN-α。
SLE患者单个核细胞中的低密度粒细胞(low density granuloctyes, LDGs)百分比增加[30]。LDGs吞噬能力缺陷却具更强的刺激pDCs分泌IFN-α的能力和对内皮细胞的损伤能力[31]。Carmona-Rivera等[32]研究表明, LDGs介导的内皮损伤与诱导基质金属蛋白酶-9(matrix metalloproteinase 9, MMP-9)外化激活MMP-2相关, 而且SLE患者血清MMP-9和抗MMP-9自身抗体的免疫复合物能诱导NETosis并增强MMP-9活性。
狼疮患者的免疫复合物沉积在皮肤、关节、小血管、肾小球等部位, 可被补体系统识别攻击, 导致局部组织坏死从而引发急慢性炎症表型, 或自身抗体直接与组织细胞抗原结合导致机体多系统受累。NETs作为一种宿主防御机制, 对狼疮患者所致的系统损伤以末端小血管内皮细胞损伤最常见, 并可促进血栓形成[25]。除小血管受累以外, 各种炎症的病理过程中均可观察到NETs沉积, 且NETs沉积与血浆和血清中的循环无细胞DNA(circulating cell-free DNA, cfDNA)水平相关[33-34]。有研究[35]证明中性粒细胞释放NETs会直接导致cfDNA水平提高, 并进一步形成DNA免疫复合物诱导pDCs等分泌IFN-α, 因此cfDNA相比IFN-α更适合用于监测SLE等自身免疫疾病活动程度和疗效评估。但Truszewska等[36]认为SLE患者cfDNA水平异常升高与各种抗体滴度有关, 与疾病活动程度无关。
3 调控NETs对SLE的影响调控NETs作为SLE的一种潜在治疗手段目前还处在临床前试验阶段, 比如基质金属蛋白酶抑制剂可以抑制LDGs介导的MMP-9对内皮细胞MMP-2的激活, 因此可用于SLE治疗[32]。Wang等[37]研究表明, 二甲双胍可下调mtDNA-pDCs-IFNα通路并抑制PMA诱导的NETs形成, 缩短激素疗程并降低SLE复发率。Pieterse等[38]发现, 组蛋白乙酰化可增强SLE患者NETs的免疫刺激潜能, 而组蛋白去乙酰化酶(histone deacetylases, HDACs)抑制剂如菌素A(trichostatin A, TSA)可以非竞争性地抑制HDACs而抑制组蛋白乙酰化, 可能成为SLE的一种治疗手段。Huang等[39]发现, 重组乳脂球表皮生长因子8能下调SLE患者中性粒细胞CXCR2表达, 减少中性粒胞迁移和NETs产生。此外, Handono等[40]发现, 维生素D可抑制SLE患者NETosis介导的中性粒细胞早期凋亡, 从而减少NETs所致的内皮损伤。
4 总结与展望NETs是一种不同于凋亡或坏死的细胞死亡形式, 展现出独特的宿主防御机制。然而, 在儿童自身免疫性疾病中, 如cSLE、类风湿性关节炎、ANCA相关血管炎等, 通常可观察到NETs的过度生成[41]。过度生成的NETs会通过暴露自身抗原诱导并释放大量自身抗体和干扰素, 激活补体级联反应, 从而参与到自身免疫性疾病的发病机制。而且cSLE的活动程度和肾脏受累与NETs关系密切。因此, NETs可能作为监测SLE疾病活动程度、预测IFN-α水平的指标[42]。但目前NETs的研究还处在起步阶段, 淋巴细胞刺激诱导DNA释放也与NETosis现象极其相似, 自身免疫性疾病的发病机制变得更为复杂[43]。最近有研究[44-45]指出, 通过遗传学方法敲除NAPDH氧化酶功能亚单位或者药理学抑制NAPDH氧化酶对于狼疮小鼠的发病没有影响, 对于肾小球肾炎模型小鼠的终末器官损伤也没有影响。这直接挑战了NETs促进SLE自身免疫和靶器官损伤的概念, 正如缺乏NAPDH氧化酶的慢性肉芽肿患者也很少发生SLE。因此, NETs在SLE等自身免疫性疾病发病机制的作用还需进一步研究, 以期为SLE的治疗提供新的治疗靶点。
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