补体抑制在泡型肝包虫病宿主免疫反应中的调控机制及其特异性作用靶点研究

Alveolar echinococcosis, a lethal zoonotic disease caused by Echinococcus multilocularis infection, generaly involves the hosts liver. This parasite could alter host immune responses and induce immune tolerance, which enables its long-term survival and direct damage to the host. The complement system, a crucial component of innate immune system, was recently found to be critical in regulating the adaptive immune system, particularly the T cells. In preliminary studies directly related to this project, we have found that levels of markers for T cells (CD3), B cells (CD19) and complement activation (C3b) were significantly decreased in hepatic tissues around the alveolar echinococcosis lesions. At the same time, levels of IFN-γ, IL-17A and sMAC were also markedly reduced, indicating a suppressed host T and B cell responses together with inhibited complement activation. We and others have previously shown that complement activation is required for robust T and B cell response development through complement receptor C3aR and C5aR signaling. All these insights, taken together, strongly suggest that alveolar echinococcosis could suppress the host adaptive immune system to induce immune tolerance by inhibiting complement activation. In this project, we will unveil the mechanism by which Echinococcus multilocularis actively inhibits the activation of complement and induces host immune tolerance using various modern techniques including flow cytometry, protein Pull-down and mass spectrometry. Furthermore, we will develop a novel immune therapy targeting the protein that alveolar echinococcusis depends to inhibit complement and evaluate its efficacy in treating parasite infection in a mouse model. The proposed studies, if successful, will not only provide new insights into the mechanism by which alveolar echinococcusis causes chronic liver inflammation, but also facilitate novel therapeutic approaches for treating this devastating disease.

泡型肝包虫病是泡球蚴寄生肝脏引起的严重致死性疾病，病原体通过主动干扰免疫应答、诱导免疫耐受在宿主体内长期存活。最新研究发现补体对特异性免疫系统，特别是T细胞具有重要的调节作用。我们研究发现：1）泡型肝包虫病人病灶旁肝组织CD3、CD19、C3b表达下降，提示补体活化被抑制，影响T和B细胞的增殖；2）泡型肝包虫病人病灶旁肝组织IFN-γ、IL-17A、sMAC表达下降，提示补体抑制阻碍Th0向Th1和Th17细胞分化。因此，我们推测：泡球蚴慢性感染宿主的过程中，补体抑制造成补体受体C3aR和C5aR信号通路表达下调，影响T细胞的增殖分化，从而诱导宿主免疫耐受形成。本研究将通过流式细胞术、蛋白pull down等技术，在细胞和蛋白质水平探索泡球蚴如何主动抑制补体系统活化、从而诱导宿主免疫耐受的机制，并利用制备的单克隆抗体和疾病动物模型，为以补体为靶点治疗泡型肝包虫病的免疫学疗法提供理论依据。

泡型肝包虫病是多房棘球蚴寄生肝脏引起的严重致死性疾病，是我国目前重点防治的寄生虫病之一。棘球蚴通过主动干扰免疫应答、诱导免疫耐受在宿主体内长期存活。既往研究表明，某些病原体在感染过程中可以通过抑制宿主补体活化逃避补体杀伤，同时调控下游免疫细胞功能诱导宿主免疫耐受形成。多房棘球蚴感染过程中可能存在类似过程。探索相关机制有助于深入理解泡球蚴诱导宿主免疫耐受的分子机理，同时对探索新的泡球蚴病药物治疗靶点具有重要意义。本项目中，我们首先分析了多房棘球蚴体内感染病灶局部免疫病理特征，包括多种补体及补体调控因子组分以及相关细胞因子的表达水平空间分布，发现尽管有大量补体活化片段沉积，但亦有大量补体负性调节因子围绕多房棘球蚴角皮层显著沉积，提示多房棘球蚴可能通过“劫持”宿主补体负性调节因子抑制补体在虫体表面黏附并以此逃避补体杀伤。通过生物信息学方法，我们首先筛选了多房棘球蚴所有可能的分泌性蛋白，功能富集分析提示这些蛋白的功能集中于能量代谢相关途径，其中，我们预测了一种潜在的分泌性的补体抑制蛋白，该蛋白由EmuJ_000439500基因编码，含有多个Sushi结构域，提示其可能具有C4-binding protein类似的功能—抑制补体通过经典途径激活。我们采用酵母重组蛋白体系制备了该蛋白，并在补体溶血实验中证实了其可以通过剂量依赖的方式抑制补体系统通过经典途径激活。我们进一步探究了多房棘球蚴抑制补体活化后对下游免疫细胞功能的影响，发现多房棘球蚴来源的蛋白可以显著抑制人体树突状细胞的成熟和分化，同时，多房棘球蚴感染小鼠体内髓源性抑制细胞比例明显升高，提示补体抑制诱导的免疫细胞功能变化可能是宿主形成免疫耐受的重要机制。本研究首次发现的多房棘球蚴来源的、具有补体抑制功能的分泌蛋白，可以作为潜在的免疫治疗干预靶点，也为棘球蚴-宿主免疫反应机制研究提供了新思路。