CXCL10/CXCR3轴对香鱼单核巨噬细胞迁移和活性的调控研究

Monocytes/macrophages play an important role in initiation of immune response and pathogen killing. The migration and activation of monocytes/macrophages are regulated by chemokine axes including CXCL9-11/CXCR3, CXCL12/CXCR4, CCL2/CCR2 and so on. In fish, several genes of CXCL10 and CXCR3 have been cloned and their tissue expression patterns are also measured. However, the precise function of CXCL10/CXCR3 axis is still unknown in fish. Here, we clone CXCL10 and CXCR3 genes from ayu, Plecoglossus altivelis, which is a popular and highly valued edible fish in Asia. CXCR3 is highly expressed in peritoneal monocytes/macrophages of infected ayu but lowly expressed in blood monocytes/macrophages. Moreover, CXCL10 protein is highly expressed in peritoneum after infection. Together, we hypothesize that CXCL10/CXCR3 axis regulates the migration and activation of ayu monocytes/macrophages to provide protective immunity. Firstly, we try to investigate the cellular origin of CXCL10 and determine whether reactive oxygen species (ROS) mediates infection-induced CXCR3 upregulation by RT-qPCR, ELISA, and flow cytometry. Then, we try to illustrate the role of CXCL10/CXCR3 in the migration and activation of monocytes/macrophages by primary cell culture and recombinant protein. The signaling implicated in migration and activation is investigated in monocytes/macrophages by microarray and antibody blockage. The effects of CXCL10/CXCR3 axis on bacterial infected ayu are further determined to confirm the cellular function of CXCL10/CXCR3 in vivo. This work will illustrate the CXCL10/CXCR3 dependent mechanisms underlying migration and activation of ayu monocytes/macrophages, which is the base to develop the immunotherapy of bacterial infection in aquaculture.

CXCL10/CXCR3轴调控哺乳动物单核巨噬细胞(MO/MФ)的体内迁移和活性，启动免疫反应和杀灭病原，但是鱼类中该轴的作用及机制尚不清楚。申请人前期发现香鱼CXCL10/CXCR3的表达与病原感染及MO/MФ的迁移现象相关。本项目旨在研究CXCL10/CXCR3轴在香鱼MO/MФ的迁移和活性调控中的作用及机制，拟通过分析感染位点CXCL10的免疫细胞来源、测定活性氧与MO/MФ中CXCR3表达的相关性，明确感染条件下CXCL10/CXCR3表达调控的机制；通过流式细胞术和基因表达谱芯片等技术研究CXCL10/CXCR3对MO/MФ迁移、活性和基因表达的影响，阐明调控机制及相关信号途径；通过整体动物存活率测定等试验研究CXCR3是否介导CXCL10增强香鱼保护性免疫。通过该项目实施，有望揭示CXCL10/CXCR3轴在鱼类免疫细胞调控中的作用机制，丰富对鱼类免疫应答机制的认识。

在多种硬骨鱼中研究趋化因子受体基因的多拷贝一直吸引研究者去探索免疫系统进化。CXCR3基因有两个亚基，CXCR3.1和CXCR3.2，这两个亚基在巨噬细胞中均有表达。以前的研究中硬骨鱼CXCR3两个亚基的作用没有被发现。我们发现CXCR3.1和CXCR3.2作用于硬骨鱼巨噬细胞的极化，这种现象在香鱼(Plecoglossus altivelis)等鱼类中均存在。在香鱼巨噬细胞中，香鱼的CXCR3.1（PaCXCR3.1）基因是组成型表达的，而CXCR3.2（PaCXCR3.2）基因是由大肠杆菌诱导表达的。当大肠杆菌感染后，PaCXCR3.1+ 和PaCXCR3.2+巨噬细胞分别显示出M1和M2表型。CXCL9-11样蛋白分别通过和巨噬细胞的PaCXCR3.1和 PaCXCR3.2作用来调节M1和M2型极化。香鱼STAT1和香鱼STAT3转录因子分别在PaCXCR3.1+的巨噬细胞和PaCXCR3.2+的巨噬细胞中被激活。另外，对患有脓毒血症的香鱼过继转移PaCXCR3.2+的巨噬细胞，脓毒血症的症状会减轻。我们的工作揭示了一个巨噬细胞极化的新机制，表明了硬骨鱼基因组复制产生的冗余基因在免疫系统中可能有重要的调节功能。