2019, Vol. 46
白塞病(Behcet’s disease, BD)是以“眼-口-生殖器”三联征为特征、累及多系统的血管炎性疾病。BD基本临床特点为复发性口腔、生殖器溃疡和眼葡萄膜炎, 还可以累及皮肤、肠道、心血管、中枢神经和血液系统[1-3]。当BD合并眼部损害时诊断为眼BD, 占BD 60%~80%, 以双侧全葡萄膜炎和视网膜血管炎最多见, 病程反复, 重者失明[1]。近来多项研究认为, 辅助性T细胞17(T help cell 17, Th17)/调节性T淋巴细胞(T regular cell, Treg)失衡在BD葡萄膜炎发病中发挥重要作用[2-4]。
雷帕霉素靶蛋白(mammalian target of rapamycin, mTOR)是一种在进化上高度保守的丝氨酸/苏氨酸激酶, 可在糖皮质激素、营养物质(葡萄糖、脂肪酸等)和各种应激原作用下被激活并参与调控T细胞生长、增殖和分化[5]。在哺乳动物体内, mTOR存在两种蛋白复合体即mTORC1和mTORC2, 二者具有不同的特异功能蛋白即Raptor和Rictor [5-6]。mTORC1主要参与Th1和Th17细胞分化, 而mTORC2则与Th2细胞分化有关[5-8]。越来越多研究发现, mTOR通过打破Th17/Treg平衡在多种自身免疫疾病的发生发展中起重要作用, 如系统性红斑狼疮(systemic lupus erythematosus, SLE)及类风湿关节炎(rheumatoid arthritis, RA)[6-8]。本文就mTOR通过介导Th17/Treg失衡参与BD葡萄膜炎发病机制进行综述, 旨在为BD葡萄膜炎临床治疗提供新思路。
Th17/Treg失衡与BD葡萄膜炎
Th17细胞与BD葡萄膜炎 Th17细胞是一类新型的CD4+T细胞亚群, 主要分泌白细胞介素(interleukin, IL)-17、IL-17A、IL-17F、IL-21、IL-22和肿瘤坏死因子(tumor necrosis factor, TNF)-α来促进炎症过程[2, 9-11]。初始T细胞在IL-6和转化生长因子β(transforming growth factor β, TGF-β)共同作用下, 协同特异性转录因子维甲酸相关孤儿核受体(retinoid acid-related orphan nuclear receptor γt, RORγt)分化成Th17细胞[2-4]。此外, IL-1β和IL-23、IL-6因子对于Th17细胞分化和发育也不可或缺[1]。BD葡萄膜炎中, Th17细胞被抗原提呈细胞(antigen presenting cell, APC)激活并通过分泌效应因子(IL-17A、IL-17F、IL-21、IL-22、IL-6、IL-23)趋化单核/巨噬细胞、中性粒细胞、NK细胞等来共同促进眼内炎症的发生, 打破免疫赦免, 继而迁移至眼内组织[2]。在实验性自身免疫性葡萄膜炎(experimental autoimmune uveitis, EAU)小鼠模型中, 高水平IL-1β能打破血-视网膜屏障, 也可趋化中性粒细胞和单核细胞进入眼内组织。此外, IL-17单独或联合TNF-α通过增加趋化因子配体CXCL1/2/5/8水平来诱导上皮细胞和成纤维细胞高表达IL-8和粒细胞集落刺激因子(granulocyte colony-stimulating factor, G-CSF), 从而活化和募集中性粒细胞重建眼内微环境, TGF-β生成增多[9, 12]。有研究发现, 在BD葡萄膜炎患者外周血中IL-22过度表达, 通过降低机体整体抵抗力和诱导细胞凋亡来破坏人体视网膜色素上皮细胞层, 造成视力损害[3]。多项独立研究发现[2, 12-13], 在BD葡萄膜炎活动期患者血浆和房水中, Th17细胞及其细胞因子, 如IL-6、IL-23、干扰素γ(interferon-γ, IFN-γ)、IL-17较非活动期显著升高, 且与葡萄膜炎活动度呈正相关。EAU小鼠外周血Th17细胞和IL-17、IL-6水平升高, 治疗后则降低; 而加入抗体中和IL-17后, 葡萄膜炎明显减轻[1-2, 9-10, 12]。Nanke等[2]在BD葡萄膜炎小鼠模型中发现, 下调IL-6和TNF-α水平后, Th17细胞分化减少, 葡萄膜炎明显减轻, 且IL-23R基因位点rs17375018与BD葡萄膜炎中有较强的相关性。
Treg细胞与BD葡萄膜炎 Treg细胞又称作CD4+CD25+Foxp3+T细胞, 是一类通过产生抑制性细胞因子TGF-β和IL-10来维持免疫耐受、T细胞稳态的抑制性CD4+T细胞亚群[1, 9]。CD4+T细胞在高水平TGF-β单独作用下, 通过叉头状转录因子p3(forkhead transcription factor p3, Foxp3)促Treg细胞分化。在正常眼内微环境中, TGF-β还能结合视黄酸原位促进初始T细胞分化成Treg细胞[10]。Zhuang等[14]发现, 急性葡萄膜炎患者外周血和房水中CD4+CD25+Foxp3+Treg细胞及其mRNA表达水平显著下降, TGF-β减少。在EAU小鼠模型中, 治疗后的Treg细胞数量增多和功能增强[11]。
越来越多研究发现, Th17/Treg失衡在自身免疫疾病中起着不可或缺的作用, 如SLE、RA和多发性硬化症(multiple sclerosis, MS)等[6-8]。Ye等[15]观察到BD患者外周血和脑脊液中Th17细胞增多, Treg细胞减少, 且与疾病活动相关。Th17/Treg失衡与BD葡萄膜炎Th17细胞和Treg细胞在自身免疫反应中起着截然相反的作用, 二者相互拮抗制衡。Foxp3可以直接结合RORγt来抑制Th17细胞分化[14]。因此, Th17/Treg失衡对BD葡萄膜炎发病有重要作用。
mTOR通路介导Th17/Treg失衡
mTORC1促进Th17细胞分化 信号转导及转录激活因子3(signal transducer and activator of transcription, STAT3)和RORγt作为Th17细胞分化过程中主要的核转录因子, 在mTORC1活化后均表达上调。体内和体外EAU实验中, mTORC1缺乏时, 初始T细胞不能分化成Th17细胞, 其机制与STAT3磷酸化减少有关[1, 10, 15-16]。mTORC1抑制剂雷帕霉素治疗后小鼠RORγt表达降低, Th17细胞分化减少。Hu等[16]在小鼠结肠炎模型中观察到, 第二代mTOR抑制剂AZD8055能下调促炎细胞因子INF-γ、IL-17A、IL-1β、IL-6和TNF-α的表达, 同时使Th17细胞数量明显减少且功能减退。在实验性自身免疫性脑脊髓炎(experimental autoimmune encephalomyelitis, EAE)小鼠模型中, 雷帕霉素能通过阻断mTOR-STAT3信号通路来抑制Th17细胞分化[17]。
mTORC1抑制Treg细胞分化 mTOR-/-T细胞在IL-2和TGF-β缺乏的条件下能自发分化成Treg细胞, Foxp3表达增加[5, 18]。Zeng等[5]研究发现, 小鼠Treg细胞中Raptor蛋白缺失可增加多种自身免疫病的发生, mTORC1对于Treg细胞功能完整至关重要。对Treg细胞特异性敲除mTORC1上游抑制性因子TSC1后, mTORC1活化增强, 造成Foxp3表达降低和Treg细胞数量减少, 相反IL-17和IL-1β增多[5, 16]。在EAU小鼠模型中, mTORC1抑制剂雷帕霉素抑制IL-17和促进Foxp3表达, 进而选择性扩增Treg细胞, 并增强其稳定性和抗炎能力[11, 19]。
mTOR与BD葡萄膜炎发病机制
mTOR-STAT3通路 在TGF-β存在的环境中, STAT3在mTOR活化后磷酸化, 刺激下游因子IL-6和IL-12、IL-23R的产生, 抑制Foxp3表达, 进而促进Th17细胞分化和抑制Treg细胞增殖[20]。活化的mTOR信号在STAT3磷酸化过程中起着至关重要的作用。小鼠胚胎成纤维细胞实验中, 雷帕霉素显著地降低P-Ser STAT3磷酸化水平, 而这一效应不受p38抑制剂的影响[21]。体外实验利用shRNA沉默mTOR基因后, 细胞内STAT3水平显著下降。进一步研究证明, 雷帕霉素通过抑制STAT3来促进Treg细胞分化、抑制Th17细胞增殖。Xu等[20]发现mTOR拮抗因子TSC1受抑可增加STAT3磷酸化水平, 提示mTOR-STAT3信号通路参与并调控Th17细胞分化。Chen等[22]认为STAT3通过上调细胞内ADP/ATP比值来激活AMPK α/mTOR通路, 从而抑制Th17细胞自噬; 此外, mTOR通过上调STAT3/p63/Notch信号来促进Th17细胞极化。
mTOR-HIF-1α通路 缺氧诱导因子-1α(hypoxia induced factor-1α, HIF-1α)广泛表达于T细胞, 尤其在Th17细胞中高表达, 是细胞内的缺氧感受器。低氧环境中, HIF-1α缺失可促进糖酵解过程和Foxp3表达, 从而抑制Th17细胞分化、加快Treg细胞增殖, 对于维持Th17/Treg平衡有重要作用[8, 10, 23]。活化的mTORC1通过MT1-MMP/Mint3通路在转录和翻译水平均能上调HIF-1α表达。反之, 雷帕霉素通过抑制mTOR可降低Mint3位于N末端氨基酸的磷酸化水平, 然后协同MT1-MMP进一步下调Mint3依赖的FIH-1表达水平, 最终抑制HIF-1α活化[23]。高水平HIF-1α主要作用于RORγt来促进Th17细胞分化:一方面HIF-1α通过招募形成HIF-1α/p300-RORγ t复合物, 活化转录因子RORγt和增强STAT3基因表达来促进Th17细胞因子的产生; 另一方面, HIF-1α还能诱导糖酵解酶类Glut1、HK2、PGK1、LDHA和PDK1表达来分别增加糖摄取和糖酵解以促进Th17细胞增殖[20, 23-25]。此外, HIF-1α还能协同Notch来增强Bcl-2表达促进Th17细胞分化[26]。体外实验中, 用miR-210靶向降解HIF-1α后能抑制Th17细胞分化[6]。同时HIF-1α水平升高还可以抑制Treg细胞分化, 其主要机制是直接结合Foxp3基因启动子区, 来降低Foxp3基因表达水平并加快其溶酶体降解速度[5, 10, 16]。
mTOR-Gfi1通路 独立生长因子1(growth factor independence1, Gfi1)是一种转录抑制因子。越来越多试验证实, mTORC1能通过抑制Gfi1上调STAT3和RORγt表达来介导Th17/Treg失衡[5, 15, 27]。Gfi1能阻断RORγt和增强STAT5表达进而抑制Th17细胞分化, 促进Treg细胞增殖[14, 28]。Suzuki等[28]研究发现, Gfi1能干扰RORγt募集与IL-17A启动子的结合来进一步下调IL-17A的表达。
结语 Th17/Treg失衡是BD葡萄膜炎发生发展中重要环节, 同时mTOR参与并通过多种途径调节Th17/Treg平衡, 提示mTOR介导Th17/Treg失衡可能是BD葡萄膜炎发病机制之一[1-3, 27, 29]。目前, mTOR抑制剂已经用于研究治疗RA和SLE等自身免疫病[6-7]。因此, 针对mTOR靶向治疗有望成为将来治疗BD葡萄膜炎的一种新方法。
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