负性协同刺激分子参与调控危重症手足口病T细胞功能障碍的机制研究

Severe hand, foot and mouth disease (HFMD) caused by enterovirus 71(EV71) has become one of the most critical public health problems due to its high mortality. In July 2012, we published a clinicopathologic study of HFMD in children in NEJM, and this study has proved neurogenic pulmonary edema is the leading cause of death. The previous study further showed that there was T-cell exhaustion and impaired IFN-gamma secretion in severe HFMD, however the mechanism remains unknown. Recent evidence suggests that negative co-stimulatory molecule T cell immunoglobulin and mucin domain (Tim-3) and program death factor-1(PD-1) plays a crucial role in mediating T cell exhaustion or dysfunction both in viral infections. Data from our previous study (unpublish) have identified that high Tim-3 expression was associated with T-cell exhaustion and impaired IFN-gamma secretion in the severe case with pulmonary edema caused by EV71. Based on this finding, we will examine the associations among Tim-3, PD-1 gene polymorphism and the severity of HFMD caused by EV71, and examine the relevant mechanisms of Tim-3, PD-1 expression in T cell, IFN - gamma secretion in EV71 clearance and mixed antagonist response syndrome(MARS) for severe HFMD case with pulmonary edema caused by EV71. This project will reveal the mechanism of negative co-stimulatory molecule involved in regulation of T cell exhaustion or dysfunction in severe case with pulmonary edema caused by EV71, which may provide theoretical basis for developing new therapeutic strategies.

EV71相关危重症手足口病死亡率高,2012年项目组在NEJM发表的临床病理研究证实并发神经源性肺水肿是死亡最主要原因；其体内存在T细胞耗竭及IFN-γ分泌障碍,但机制不明。近来的研究表明负性协同刺激分子Tim-3，PD-1是一些病毒感染性疾病T细胞耗竭/功能障碍的重要机制。我们前期研究首次证实EV71相关肺水肿体内IFN-γ分泌障碍与其高表达Tim-3相关。本项目在此基础上,分析Tim-3,PD-1基因多态性与疾病严重度的相关性；采用临床标本、细胞模型，通过体内检测、体外共培养、细胞转染及功能阻断实验研究EV71能否直接诱导T细胞Tim-3、PD-1表达上调，Tim-3、PD-1及其共同高表达对T细胞凋亡、IFN-γ分泌的调节作用机制，及对EV71清除、混合拮抗反应的影响。本项目初步阐明负性协同刺激分子参与调控EV71相关肺水肿T细胞耗竭/功能障碍的机制,为制定新的治疗策略提供理论依据。

危重症手足口病患儿出现的肺水肿是致死性并发症。近年的研究提示机体T细胞功能状态及异常的炎症反应异常可能与肺水肿的发病有关。本研究前瞻性评估危重症手足口病患儿CD4+ T细胞及其功能状态。研究发现危重症患儿促炎因子IFN-γ、IL-1β、IL-2、IL-6及抑炎因子TGF-β、IL-10水平较S组、M组、NC组显著升高(P＜0.05)。危重症中死亡组IL-8、IL-18、IL-10水平较存活组显著升高(P＜0.05)。危重症组IL-10、GM-CSF在病程的第4-6天水平高于在病程1-3天水平。危重症组全血IFN-γ释放功能减低；危重症组干扰素释放指数与CD4+T细胞(%)呈正相关 (r=0.578, P=0.049)。IL-8、IL-10、IL-18水平升高是病情加重的预警信号。危重症HFMD患儿处于代偿性抗炎反应或混合性拮抗反应状态，可能与CD4+T细胞功能障碍/耗竭有关。我们进一步探讨负性协同刺激因子在危重症手足口病发病中的作用。HFMD患儿外周血CD4、CD8T细胞表面表达PD1、Tim-3比例显著上调，并且与疾病严重程度有关,危重症表达PD1、Tim-3比例最高；高表达的PD1、Tim-3支持危重症手足口病存在T细胞功能障碍/耗竭。Tim-3负性调节CD4、CD8T细胞分泌IFN-γ功能，体外单一阻断Tim-3信号或联合PD1、Tim-3阻断通路可以改善危重症患儿T细胞分泌IFN-γ功能。本课题为危重症手足口病发病机制的研究提供新的思路，也为危重症手足口病潜在的免疫调控治疗措施提供理论依据和数据支持。