EV71诱导的pDC功能缺陷与CAP活化在危重症手足口病中的作用研究

Enterovirus 71（EV71）infection always induce the children with hand，foot and mouth disease (HFMD)worse. The epidemic rises fast as well as the number of patients with severe neurological disease and death increased evidently induced by Enterovirus 71（EV71）.As the first antiviral innate immune cells in peripheral blood, the antigen-presenting activity of pDCs directly determines the activation or in activation of T cells. Our previous study showed that EV71 infection could induce autophagy and apoptosis in pDCs. And our analyzed clinical data were concluded that the quantities of pDCs and T cells were decreased suddenly in the severe HFMD patients infected by EV71. Based upon these data, we speculate that pDCs in these patients might be utilized by EV71 leading to the defects in function, which also can trigger T cells to induce immune tolerance. Thus further to investigate the relationship between pDCs defects, pDCs autophgy and the degree of HFMD after EV71 infection, the mouse model infected by EV71 will be constructed to apply for studying the potential immnue escape strategy of EV71. Meanwhile, we will detect the activation of cholinergic anti-inflammatory pathway (CAP) that was proved associated with the regulation of pDCs in different patients samples. And the technique of High Throughout Screening will be adopted to discover the key mmune-neural signal molecules that can be considered as the target interfered molecules by gene interfere technique and applied for verifying the pathogenic mechanism induced by EV71 between the activation of CAP, the autophgy in pDCs and the defects of pDCs in function.

EV71是引发云南省儿童危重症手足口病的主要致病病毒，患者感染其后常出现严重的神经系统并发症。pDC作为外周血抗病毒天然免疫的第一道防线，其抗原递呈活性的高低直接决定了CD4+T细胞的活化程度。我们研究发现EV71感染可诱导pDC发生自噬及凋亡；临床数据显示在EV71感染的危重症手足口病患者体内pDC、CD4+T细胞数量极低，推测患者体内的pDC很可能被病毒利用产生失能、诱导T细胞出现免疫耐受。为了进一步证明EV71感染与pDC功能缺陷、pDC自噬以及病情危重程度之间的关系，本课题将通过构建EV71感染动物模型，对EV71产生免疫逃逸的相关免疫分子机制展开研究。同时，还将对能够调节DC活性的胆碱能抗炎通路的活化情况进行检测，通过高通量筛选技术发现与危重症手足口病发生密切相关的关键免疫-神经信号分子，通过基因敲除技术检测发现EV71感染后CAP活化与pDC自噬、pDC失能间存在的对话机制。

EV71是引发云南省儿童危重症手足口病的主要致病病毒。pDC作为外周血抗病毒天然免疫的第一道防线，其抗原递呈活性的高低直接决定了CD4+T细胞的活化程度。我们研究发现EV71感染可诱导pDC发生自噬及凋亡；临床数据显示在EV71感染的危重症手足口病患者体内pDCs、CD4+T细胞数量极低，推测患者体内的pDCs很可能被病毒利用产生失能、诱导T细胞出现免疫耐受。本课题通过构建EV71感染动物模型，对EV71产生免疫逃逸的相关免疫分子机制以及对能够调节DC活性的胆碱能抗炎通路的活化通路展开研究。研究发现：EV71感染早期，pDCs被快速激活并大量扩增、不断将病毒抗原递呈给CD4+T细胞，促进两者间细胞接触增多，释放大量干扰素和炎性细胞因子，与此同时，Treg细胞数迅速增多；CD4+T细胞相关的CAP通路活化明显；CD4+T细胞、pDCs内以NF-κB信号通路活化为主；Treg细胞内以JNK信号通路活化为主。随着EV71感染时间的延长，Treg细胞表达下调，进而影响其与pDCs间的相互作用，直接导致pDCs表面分子CD123不表达，促使pDCs不再扩增并发生功能缺陷；此后CD4+T、CD8+T细胞功能均受到pDCs的影响而表达受抑制，尤其是CD4+T细胞因承接病毒抗原过多而消耗过大、细胞死亡增多；部分CD4+T细胞表面集中有大量病毒抗原，导致其功能受损，免疫耐受产生；此时，干扰素和炎性细胞因子产生减少；CAP通路活化不明显；Treg细胞数缺失；同时NK细胞却因CD4+T、CD8+T以及Treg细胞表达受抑而异常高表达，NK细胞数异常增多，细胞内JNK及NF-κB信号通路均活化；推测高表达的NK细胞抗炎作用减弱，但细胞毒作用增强，可能参与杀伤未成熟的pDCs以及处于耐受状态的CD4+T细胞，导致两者数量急剧降低；但同时，已感染EV71的pDCs可能受病毒基因组重组及Treg细胞调节影响而使得pDCs表面受体出现重编辑，引起如pDCs表面CD123分子中CSF2R基因表达异常的情况，进而引发危重症发生。本研究为进一步阐明EV71的免疫病理致病机制提供了新的理论依据。