盐度通过应激轴影响香鱼免疫系统的调控研究

Salinity is one of the important stress factors in marine fish. However, it is still unclear what the mechanisms underlying the effect of salinity on the immune system in fish are. In previous work, we found that brackish water acclimation for one week decreased the survival of infected ayu. While the level of cortisol of stress axis was up-regulated after brackish water acclimation and cortisol reduced the phagocytosis of monocytes/macrophages (MO/MФ) in ayu. Both cortisol and adreno-cortico-tropic-hormone (ACTH) receptors are detected in ayu MO/MФ. Thus, we aimed to investigate the function and mechanisms of stress axis on immune system in ayu after salinity acclimation, especially coritisol and two kinds of ACTH. Firstly, we try to investigate the hormone levels and receptor expressions of stress axis (HPI axis) to demonstrate salinity dependent pathway. Then, we try to illustrate the role of salinity dependent hormones in the host defense of infected ayu by measuring survival, bacterial burden, and cytokine expression. Since the important role of MO/MФ in host defense of ayu, we will also investigate the role of salinity dependent hormones on migration, phagocytosis, bacterial killing, and cytokine expression in primary MO/MФ culture and also in MO/MФ like cell line to probe the cellular mechanisms underlying stress axis dependent alteration in immune system. The effects of salinity dependent transcriptional factors on MO/MФ chemotaxis and activation are further determined after treatment with salinity dependent hormones to demonstrate the transcriptional mechanisms of salinity in ayu. This work will reveal the effect of salinity dependent hormones of stress axis on host defense in ayu fish, which is the base to provide methods for marine fish protection.

盐度是海洋洄游鱼类面临的主要环境应激。申请人前期研究发现短期咸淡水应激降低香鱼免疫能力，并发现应激轴(HPI轴)的皮质醇抑制单核巨噬细胞(MO/MФ)的吞噬，而咸淡水引起的渗透压改变不影响MO/MФ的吞噬。由此假设盐度主要通过应激轴影响鱼类免疫系统，拟阐明HPI轴的皮质醇和两种ACTH对免疫系统的调控机制。拟检测香鱼盐度应激后应激轴激素水平和相应受体的表达变化，探明盐度特异性的应激轴途径；拟分析香鱼存活率等免疫指标变化，明确盐度特异性应激轴信号对整体动物免疫能力的影响；拟通过细胞培养、流式细胞术等技术测定应激轴信号对MO/MФ趋化和活性的影响，阐明应激轴影响免疫系统的细胞机制；拟通过Western blot和RNA干扰等技术检测盐度相关转录因子是否介导应激轴影响MO/MФ功能，探索应激轴作用于免疫系统的转录机制。该项目的实施有望揭示盐度对鱼类免疫系统的作用机制，将为海洋鱼类的保护提供指导。

应激可诱导内分泌系统释放多种激素从而影响多种免疫反应，然而在鱼类盐度条件下应激激素对免疫系统的调控功能未知。我们从香鱼（Plecoglossus altivelis）中克隆了尿皮质素（UCN）、阿黑皮素原（POMC）-A、POMC-B和糖皮质激素受体（GR）的cDNA序列。香鱼UCN（PaUCN）、PaPOMC-A、PaPOMC-B和PaGR转录本在所有检测组织中均有表达，并且在感染条件下，免疫组织和单核/巨噬细胞（MO/MФ）中上述基因表达发生改变。UCN调控MO/MΦ细胞因子的表达、促进MO/MΦ吞噬和杀菌能力、提高感染香鱼的存活率和降低细菌载量。PaPOMC-A来源的香鱼促肾上腺皮质激素（PaACTH）-A、PaPOMC-B来源的PaACTH-B调控MO/MФ炎性细胞因子的产生并促进鳗弧菌刺激的MO/MΦ的杀菌活性。PaACTH-A提高感染香鱼的存活率并降低细菌载量。PaGR调控MO/MΦ中炎性细胞因子的产生，抑制吞噬作用，促进细菌杀伤和细胞凋亡。此外，糖皮质激素上调免疫细胞中CXC基序趋化因子受体4（CXCR4）基因表达。与PaCXCR4b+ HSPCs相比，PaCXCR4a+ HSPCs优先分化为香鱼中的髓系细胞。该研究发现应激轴影响香鱼免疫系统的调控，有助于指导海洋鱼类的保护和利用，同时也将加深我们对脊椎动物免疫系统的理解。研究期限内共发表SCI论文12篇，培养9名研究生，参加3次会议，授权1项发明专利，荣获1项省部级科技进步奖二等奖（排名3/16）。入选二区SCI期刊Front Immunol编委，并主持两期专刊。