胞外ATP信号在褐牙鲆先天免疫应答中的作用研究

Japanese flounder (Paralichthys olivaceus) is an economical important species in marine culture industry in China. However, the frequent outbreak of infectious disease caused by bacterial and viral pathogens has caused serious economic loss in Japanese flounder culture industry. As the first barrier to defense infections, innate immunity plays important roles in fish immune system. Many lines of increasing evidence have indicate that the danger-associated molecular pattern molecule, extracellular ATP (eATP), released by host cells during the infections by different pathogens, is a potent immunostimulator in induction of host innate immune response, defense against pathogen invasion and repairing of infection-caused damages. Although the role of eATP in innate immunity has been intensively investigated in mammals, little is understood in lower vertebrates such as in fish. Our previous study have revealed that eATP was induced and released in the head kidney primary cells of Japanese flounder by immune challenges with pathogen-associated molecular patterns including LPS and Poly(I:C). We have also demonstrated that eATP has important immune significance in triggering Japanese flounder innate immune response through modulation of pro-inflammatory cytokines expression via activation of ATP-gated purinergic receptor P2X7. These results addressed the importance of eATP in induction of fish innate immune response. However, more details are still needed to better understand the broad-spectrum actions of eATP in fish innate immunity. Using P. olivaceus as an animal model with combined biochemistry, cell biology and molecular biology approaches, in this project we are setting out to extend our previous study by investigation the roles of eATP in defense against Edwardsiella tarda and lymphocystis disease virus infections, and dissect the molecular mechanisms of eATP-mediated innate immune response through elucidating the functions of ectonucleoside triphosphate diphosphohydrolase1 (ecto-NTPDase1，also termed CD39) and eATP-activated nucleotide-binding domain leucine-rich repeat family pyrin domain-containing receptor 3 (NLRP3), two critical components in modulating eATP-dependent innate immune response in fish. This study will expand our understanding of the complexity and diversity of immune regulation in fish innate immunity, benefit further investigations of the immune significances of eATP in other aquatic animals, and may provide novel insights in control of infectious diseases in fish.

褐牙鲆是我国北方主要的海水养殖鱼类之一，但近年来病害的频繁爆发已严重制约了其工厂化养殖的健康可持续发展。先天免疫是机体抵御病原侵染的第一道防线，在鱼类免疫防御系统中具有举足轻重的地位。最近的研究发现，细胞外ATP是宿主细胞受到病原刺激等条件下产生的具有免疫刺激功能的重要内源性信号分子，在高等脊椎动物先天免疫防御中起着重要作用，但在鱼类中的相关研究还基本是空白。本项目以褐牙鲆为研究对象，拟在我们对胞外ATP参与调节鱼类先天免疫的研究基础上，深入分析病原诱导的牙鲆胞外ATP信号分子释放的规律及其调控的分子机理，解析胞外ATP信号在褐牙鲆应答爱德华氏菌和淋巴囊肿病毒侵染中的先天免疫功能及其相关机理，研究胞外ATP活化的免疫炎症小体组成蛋白并阐明其在鱼类细胞因子IL-1beta成熟加工中的作用，预期研究成果将有助于揭示鱼类先天免疫调控的分子机制，并为鱼类传染性病害的免疫防治提供新的思路和理论支持。

细胞外ATP是机体在胁迫或病原侵染条件下产生的内源性信号分子，其在高等脊椎动物天然免疫防御中起着重要作用，但在鱼类等低等脊椎动物中尚无相关研究报道。本课题以牙鲆为对象，研究了免疫和病原刺激条件下鱼类胞外ATP信号分子释放的特征和规律，证明pannexin1和connexin43通道参与了免疫和病原刺激诱导的鱼类胞外ATP释放。证明胞外ATP可以迅速、显著提高牙鲆不同类型免疫细胞促炎性细胞因子、趋化因子和免疫相关基因表达，诱导一氧化氮和活性氧等炎性因子生成，促进鱼类产生强烈天然免疫应答。而且，该效应具有明显的细胞类型和浓度依赖特性，提示对胞外ATP具有不同亲和力的P2XRs可能共同参与了鱼类胞外ATP介导的天然免疫应答。分离鉴定了多个表达于细胞膜上的牙鲆胞外ATP受体P2XRs，发现虽然不同P2XRs在牙鲆免疫细胞中共表达，但优势表达的P2XRs具有明显的细胞类型依赖特征，证明P2X4R可以与pannexin1通道互作，形成嘌呤免疫信号通路，进而活化鱼类天然免疫。从牙鲆中分离鉴定了NLR免疫炎症体信号通路中包含NLRC3、ASC和Caspases等功能基因，证明鱼类免疫炎症体NLRC3信号通路和胞外ATP介导的天然免疫信号通路存在对话交流；证实牙鲆Caspase1蛋白可以与接头蛋白ASC相互作用，胞外ATP信号通过调节不同Caspase凋亡酶活性参与了鱼类免疫细胞凋亡，而胞外核苷酸酶CD39可以将胞外ATP转化为AMP，通过降低胞外ATP浓度，对鱼类胞外ATP介导的天然免疫应答起负调控作用。综上，本课题首次研究报道了免疫和病原刺激条件下鱼类胞外ATP的释放及其分子机制，证明在鱼类中胞外ATP是一种强效促炎性信号分子，可以促进鱼类天然免疫应答和细胞凋亡，并参与了对牙鲆炎症体NLRC3免疫功能的调节。本研究还进一步揭示了胞外核苷酸酶CD39对鱼类胞外ATP信号介导的天然免疫应答的负调控作用。