脊索动物文昌鱼LITAF在先天免疫应答中的功能机制研究

Innate immunity is important for animals fighting against pathogens. LITAF (lipopolysaccharide-induced TNF-α factor), a transcription factor, plays an important role in innate immunity. LITAF mutations are related to the CMT1C type of Charcot-Marie-Tooth disease, but the mechanism is unknown. Amphioxus at the transition from invertebrate to vertebrate is emerging as a valuable model organism for comparative study of innate immunity. In this project, we will investigate the function of LITAF in amphioxus (Branchiostoma belcheri) innate immunity. Based on our previous results, qRT-PCR, in situ hybridization and western blot will be used to detect the spatial and temporal expression patterns in response to LPS stimulation. In order to further investigate the role of amphioxus LITAF and the mechanisms of LITAF signaling pathway in amphioxus innate immune response, in this proposal, we will study the interaction between the p38α, STAT6B and LITAF using p38α inhibitors, GST-Pulldown and CO-IP followed by mass spectrometry. Meanwhile, we will investigate the downstream cytokine TNF-α regulated by LITAF upon LPS treatment in amphioxus using luciferases reporter assay. Taking advantage of transfection mammal cell HEK293 with amphioxus LITAF, the subcellular localization and transcriptional regulation of TNFα by LITAF will be studied. In addition, the LITAF signaling pathway in chordate at network-level will be deeply analyzed using comparative genomics methods. Our study will not only reveal the conserved function of LITAF in the innate immune responses in Branchiostoma belcheri but also provide insights into origin and evolution of innate immunity mediated by LITAF signaling pathway across invertebrate and vertebrate.

先天免疫是动物对抗外源病原的重要方式。脂多糖诱导的TNFα因子（lipopolysaccharide-induced TNFα factor, LITAF）是一类保守的转录因子，在动物先天免疫中发挥重要作用。LITAF突变和腓骨肌萎缩症CMT1C型相关，其功能机制未明。本项目以进化地位关键物种文昌鱼为对象，研究LITAF在动物先天免疫中的功能。基于前期结果，拟采用qRT-PCR、原位杂交及western blot检测LITAF基因应答LPS刺激的时空表达谱；利用p38α抑制剂、GST-Pulldown、Co-IP串联质谱分析LITAF与上游蛋白p38α及STAT6B的互作关系，验证LITAF在先天免疫应答中的保守机制；在HEK293细胞内转染文昌鱼LITAF研究其亚细胞定位及对TNFα的转录调控。此外，以比较基因组学方法分析脊索动物LITAF信号通路进化，探索脊索动物先天免疫的起源和进化。

先天免疫普遍存在于无脊椎动物与脊椎动物中，是动物对抗外源病原的重要方式，对于维持机体的免疫平衡起着关键的作用。脂多糖诱导的TNFα因子（lipopolysaccharide-induced TNFα factor, LITAF）是一类保守的转录因子，在动物先天免疫中发挥重要作用。同时LITAF还与一些疾病密切相关，包括腓骨肌萎缩症CMT1C亚型、炎症性关节炎、前列腺癌及胰腺癌等。我们围绕进化地位上介于无脊椎与脊椎动物之间的的“活化石”物种文昌鱼开展LITAF介导的先天免疫应答机制研究。在项目实施期间，我们通过qRT-PCR及western blot方法明确了文昌鱼LITAF参与先天免疫调控的功能及机制；通过同源搜索及克隆方法获得了LITAF上游激酶p38及ERK2基因的全长序列，并进一步通过western blot方法确定了其参与文昌鱼体内的先天免疫应答，并进行了系统的进化分析；将LITAF互作蛋白STAT6B在文昌鱼基因组数据库中进行了同源搜索，文昌鱼中只包含STAT家族蛋白AmphiSTAT5.0及AmphiSTAT5.1，获得其全长序列，并分别构建了真核表达载体，CoIP法分析了LITAF蛋白及AmphiSTAT蛋白在细胞内的表达及互作关系。通过以上研究我们已经完善了文昌鱼LITAF介导的免疫通路的关键分子，并对相关分子的免疫应答机制做了系统的研究。在此基础上我们还进一步探究miRNA是否及如何参与文昌鱼先天免疫调控，通过高通量测序及芯片方法筛选了14个免疫相关的miRNA，并进行了初步的功能分析。本项目的完成有助于加深对LITAF介导的先天免疫调控机制的认识，为更全面的调节文昌鱼先天免疫调控网络奠定基础。