宿主microRNAs在Treg细胞介导的华支睾吸虫感染免疫逃避中作用及其机制研究

Clonorchiasis is an important food-born parasitic disease, which affects humans’ health severely. Regulatory T (Treg) cells prevent inflammatory diseases and play an important role in the immune evasion of parasites. In our previous study, we found that CD4+CD25+FoxP3+Treg cells were significantly increased during the middle and late infection of clonorchiasis. However, the mechanism of this remains unclear and recent studies demonstrate that microRNAs play a critical role in the development and differentiation of Tregs as well as suppressive function of Treg cells. Besides, miRNAs containing-exosomes excreted by Tregs can be contributed to the suppression of Tregs. Therefore, the aims of the present study are going to explore the detailed mechanisms by which the miRNAs regulate the differentiation, development and functions of Treg cells induced by Clonorchis sinensis. Using different mouse models of clonorchiasis including miRNA-/-FoxP3 KO mice, Rab27a/b-/- mice and wild type mice, the roles of miRNAs and exosome sourced from Tregs in regulation of Treg-induced suppression are to be analyzed in detail by Flow cytometry, next-general sequencing techniques and real-time PCR both in vivo and in vitro. Our foundlings will dramatically advance our understandings of the roles of miRNAs in regulation of Treg which contributes to the immune evasion of C. sinensis, as well as provide basic information and novel therapeutic targets to treat this disease.

华支睾吸虫病是一种严重影响人类健康的食源性寄生虫病。华支睾吸虫感染存在严重的免疫逃避现象。本课题组前期研究显示，华支睾吸虫感染能激活宿主的Treg细胞，但其机制尚不清楚。研究已表明，miRNAs可参与调控Treg细胞的活化，并可通过其外泌小体中miRNAs实现对效应性T细胞的免疫调控。本研究拟采用华支睾吸虫感染小鼠模型，分离小鼠Treg细胞，通过高通量测序等技术筛选感染小鼠Treg细胞差异表达miRNAs，并利用体外细胞模型干预实验和体内骨髓移植构建miRNA-/- FoxP3KO嵌合体小鼠感染模型，鉴定对Treg细胞活化具有调控功能的miRNAs。在此基础上，进一步分离活化Treg细胞的外泌小体，鉴定其中的miRNAs，并分析这些miRNAs对效应性T细胞的免疫调控作用。本研究以期阐明miRNAs调控Treg细胞在华支睾吸虫免疫逃避中的作用及分子机制，为创新华支睾吸虫病防治手段提供基础。

华支睾吸虫病是一种严重危害人类健康的寄生虫病，也是我国重点防治的寄生虫病之一。华支睾吸虫感染存在严重的免疫逃避现象，但目前有关于关华支睾吸虫产生免疫逃避分子机制的认识非常有限。本项目在前期研究的基础上，运用生物信息学技术、miRNA芯片技术、基因敲除/过表达、Pull-down等技术，较为详细探讨了华支睾吸虫产生免疫逃避的分子基础及调控机制，本项目研究发现：①CD4+CD25+Foxp3+Treg细胞诱导的虫体免疫逃避与华支睾吸虫对不同品系小鼠易感性密切相关，即易感型小鼠相对于抵抗型小鼠，其Treg水平明显升高，这种免疫逃避促使华支睾吸虫感染所致肝纤维化加重。②首次筛选鉴定出CsHscB蛋白，该蛋白作为TLR2的配基，可通过TLR2/ERK1信号通路促使巨噬细胞分泌高水平IL-10，促使虫体产生免疫逃避。③通过miRNA芯片技术鉴定、筛选出若干调控TGF/Smads信号通路的miRNAs，其中首次对miR-497进行了深入研究，发现其可通过靶向smad7促进TGFβ/Smads信号通路转导，鉴于TGFβ/Smads在Treg细胞分化、增殖中起着重要的作用，提示miR-497也可能调控Treg的分化、增殖。④分离并鉴定出华支睾吸虫来源的胞外囊泡，并证实其对巨噬细胞具有免疫调控功能，可分泌大量促炎因子，并抑制免疫负调控分子的产生，进而促进肝脏的损伤。上述研究丰富和发展了华支睾吸虫免疫逃避机制及其致病机制，将为创新华支睾吸虫病防治手段提供新的基础，也将为理解寄生虫感染免疫逃避这一重要科学问题提供新的理论和实验依据。