疟疾感染诱导Tregs细胞Foxp3表达的表观调控机制研究

The study on immune regulatory mechanism is important to development, treatment and prevention of malaria disease.Malaria infections display variation patterns of clinical course and outcome. Although CD4+CD25+Foxp3+ regulatory T cells (Tregs) play an essential role in immune homeostasis, the immune regulatory roles involved in malaria infection remains to be elucidated. In our previous studies, we found that the susceptibility to malaria was associated with Tregs-mediated immune regulation function abnormalities. Thus, the project intends to use rodents malaria models to systematic assessment of the role of epigenetic modification on Foxp3 expression and the effects on the functional differentiation of Treg subsets. Firstly, we will compare the difference of Treg cells phenotype and function, and assessment of malaria susceptibility and participate in the expression of Foxp3 regulated epigenetic interactions in the different rodent malaria models.Secondly, we will proceed a comparative analysis on CpG DNA methylation，histone acetylation and microRNA expression in Tregs cell.In addition, we aim to screening exogenous and endogenous signals which participate in the regulation of Foxp3 expression, differentiation characteristics of subsets of Tregs.Furthermore, we will reveal the critical possible mechanism of the regulation factor on Foxp3 expression. We aim to find new targets for intervention Tregs in vivo function, as well as the Tregs functional subsets, to provide a better Tregs classification markers. We wish to provide some useful informations to malaria immune regulatory mechanism theory.

免疫调控机制研究对疟疾疾病发生、发展、治疗和预防具有重要意义。疟疾感染显示了不同的临床表现和结局。尽管CD4+CD25+Foxp3+细胞发挥重要的免疫调控效应，但在疟疾感染中的免疫调控机制有待深入研究。本项目前期研究发现疟疾易感性与Tregs细胞介导的免疫调控功能异常有关。为此，本项目拟通过建立鼠疟模型，比较不同鼠疟模型Tregs细胞表型和功能差异，评估疟疾易感性与参与Foxp3表达调表观修饰相互关系，系统评估表观修饰作用对Foxp3表达的调控效应和对Tregs细胞亚群功能分化的影响，比较分析Foxp3座位的DNA甲基化、组蛋白乙酰化水平和microRNA表达谱系，筛选参与调控Foxp3表达和Tregs细胞亚群分化的关键性信号，揭示关键性调控因子对Foxp3表达的可能作用机制。旨在为体内干预Tregs细胞功能寻找新的靶点，同时为Tregs细胞功能亚群划分提供更好的分类标记，进而为疟疾免疫调控机制理论提供新的理论依据。

调节性T细胞的免疫调控作用在疟疾等感染性疾病的防治中具有重要作用。研究证实Foxp3(+)Treg细胞通过分化不同的亚群调节免疫应答的多种类型。表观调控作用在Foxp3(+)细胞发育和功能中起关键作用，其中miRNA介导的基因调控对Treg功能发挥至关重要。本研究利用鼠疟P.y17XL易感和抵抗模型对脾脏Treg细胞miRNA表达谱进行分析。检测到21个上调miRNA和5个下调miRNA（易感鼠vs正常鼠），26个上调miRNA和14个下调miRNA（易感鼠vs抵抗鼠）。qRT-PCR结果证实，P.y17XL感染导致脾脏Treg细胞miR-182-5p表达上调和miR-10a-5p表达下调。miRNA靶基因筛选和验证实验显示，miR-182-5p和miR-10a-5p分别调控靶基因Creb1和Bcl6。P.y17XL感染诱导肝脏产生趋化因子CXCL10，募集脾脏CD4(+)CXCR3(+)Foxp3(+)Th1-Treg细胞向肝脏趋化，脾脏Th1-Treg细胞在原虫血症达峰值时数量明显降低，CXCR3(+)Treg细胞Foxp3，Creb1和helios表达降低。由此提示，P.y17XL感染中脾脏Treg细胞miR-182-5p下调Creb1基因表达，调控胸腺来源Treg细胞（tTreg）的Foxp3表达。进一步，脾脏CXCR3(-)Treg细胞Bcl6和SOCS3表达增加，Treg相关抑制分子S1P1、Ezh2和CTLA-4表达上调。由此提示，P.y17XL感染中脾脏Treg细胞miR-10a-5p表达下调促进了Bcl6基因表达，增强CD4(+)Bcl6(+)Tfh-Treg细胞的免疫抑制作用，进而对脾脏Tfh介导的体液免疫应答产生调控。本研究结果表明，miR-182-5p和miR-10a-5p通过靶向Creb1和Bcl6基因，参与调控Th1-Treg和Tfh-Treg细胞功能进而影响疟疾感染结局。