C5a/C5aR信号对CD4+ T细胞防御隐孢子虫感染的调节作用及机制

Analyzing the immunological mechanism of host against Cryptosporidium infection is the fundamental way to control cryptosporidiosis. Previous studies have indicated that Th1, Th2 and Th17 cells were key subsets to resolve Cryptosporidium infection, but the immunological mechanism of anti-infection effect has not been clearly stated. The C5a/C5aR signal has been suggested to have important role to regulate stimulation and effect of CD4+ T cells and its subsets. Our previous study found significantly up-regulated expression of C5a and C5aR in spleen and small intestine during C. parvum infection and association between this change and dynamics of IL-17 and IFN-gamma. Therefore, we assume that the C5a/C5aR signal has regulative role to Cryptosporidium specific CD4+ T cell effect. To address this hypothesis, this project will investigate the effect of C5a/C5aR signal to pathogenicity of C. parvum, maturation of dendritic cells induced by C. parvum, expression of activation costimulatory molecules of CD4+ T cells, and differentiation and response of CD4+ T cell subsets, using C. parvum, models of mice and swine intestine epithelial cells, and cellular and molecular technologies. The findings of this project will systematically clarify the regulative role and its mechanism of C5a/C5aR signal to CD4+ T cells against Cryptosporidium infection in vivo and in vitro, and shed new light to screen new effective drug targets and vaccine candidate genes to control cryptosporidiosis.

解析宿主抗隐孢子虫免疫机制是防控其感染的根本途径。已明确CD4+ T细胞中Th1、Th2和Th17是宿主抗隐孢子虫感染关键细胞亚群，但其抗感染效应调控机制尚不清楚。C5a/C5aR信号对病原特异CD4+ T细胞及其亚群产生和效应有重要调节作用，我们前期研究发现微小隐孢子虫感染鼠脾脏和小肠C5a及受体C5aR显著上调表达，且与IL-17和IFN-γ消长相关联。据此我们认为C5a/C5aR信号对隐孢子虫特异CD4+ T细胞效应有调节作用。本项目拟以微小隐孢子虫为对象，以小鼠和猪肠上皮细胞为模型，采用细胞和分子手段，探讨该信号对隐孢子虫致病性的影响，研究其对隐孢子虫诱发树突状细胞成熟及对CD4+ T细胞活化共刺激分子表达、细胞亚群分化和效应的作用，最终从体内外系统阐明C5a/C5aR信号调节CD4+ T细胞抗隐孢子虫感染的作用机制，为防控隐孢子虫病药靶和疫苗候选基因筛选提供新思路。

CD4+ T细胞是宿主防御隐孢子虫感染的关键细胞，但其在隐孢子虫感染中分化和效应的调节机制尚不清楚。本项目分别以人兽共患微小隐孢子虫和呼吸道贝氏隐孢子虫为研究对象，以小鼠、HCT-8细胞和雏鸡为模型，在体内外水平研究了C5a/C5aR信号对宿主CD4+ T细胞抗隐孢子虫感染的调节作用机制，取得了如下结果：（1）在2种隐孢子虫感染过程中，C5a和C5aR均出现了上调表达，并呈现动态变化；（2）致病性研究发现，使用C5aR的抗体、抑制剂或敲除C5aR增加了成年小鼠微小隐孢子虫的排卵囊量，抑制剂处理也增加了雏鸡贝氏隐孢子虫的排卵囊量，但是抑制剂处理显著降低了乳鼠微小隐孢子虫感染后期（144h）的荷虫量；（3）抑制剂处理造成微小隐孢子虫感染乳鼠CD4+ T细胞活化共刺激分子CD28和CD40的显著上调表达，但显著降低了CD40L的表达；（4）抑制剂处理造成微小隐孢子虫感染乳鼠Th1细胞主效应因子IFN-γ和转录因子T-bet的显著下调表达，Th2细胞主效应因子IL-4和转录因子GATA-3的显著下调表达，Treg细胞主效应因子TGF-β和转录因子Foxp3的显著下调表达，但Th17细胞主效应因子IL-17的显著上调表达，Th1、Th2和Th17细胞的变化趋势与其相似；（5）抑制剂处理造成贝氏隐孢子虫感染雏鸡气管、脾脏、法氏囊、泄殖腔中Th17细胞主效应因子IL-17及转录因子STAT3的显著下调表达，Th1细胞主效应因子IFN-γ及转录因子STAT4的显著下调表达，Th2细胞主效应因子IL-4及转录因子STAT6显著下调表达，Th1、Th2和Th17细胞的数量也显著下降。这些研究结果表明，C5a/C5aR信号可通过影响宿主CD4+ T细胞的分化和效应调节宿主对隐孢子虫的免疫防御反应。项目研究成果进一步深化了宿主对隐孢子虫的免疫防御机制，为防控隐孢子虫病药靶和疫苗候选基因筛选提供了新思路。