鱼类特有的干扰素调节因子11介导的免疫应答的机制

Interferon regulatory factors (IRFs) family consists of ten members in mammals, and are known as transcription mediators involved in the transcriptional regulation of interferon expression, and in regulation of MHC class I and II antigen presentation and antibody class switching, orchestrating adaptive immune response, and in the induction of cell apoptosis and tumor proliferation to maintain homeostasis. To data, orthologues of mammalian IRFs have all been found in fish, and most serendipitously, a new member of this gene family, termed as IRF11, was recently identified in our previous study. However, the function of IRF11 as well as its role in fish antiviral immune response are still unknown. In this proposed research, the molecular mechanism of its transcription and activation will be investigated by luciferase reporter assay and immunoblotting. The signal transduction pathway mediated by IRF11 will be identified through yeast two-hybrid technique and co-immunoprecipitation. Gene-regulatory networks of IRF11 will be investigated using gene knockdown and high-throughput microarray. The obtained results will serve as probably the first in understanding the immune response mediated through fish specific IRF, being thus significantly important in understanding the resistant mechanism in fish, and at least to some extent, filling the substantial gaps in our knowledge of the antiviral immune responses in fish, with the evolutionary implication of immune system in fish as well as in other classes of vertebrates.

干扰素调节因子是一类多功能的转录因子，在哺乳类中共有10个成员。IRF可诱导干扰素表达，参与机体抗病毒防御；可调控抗原递呈和抗体类别转换，参与适应性免疫应答；可调节细胞增殖和凋亡，维持机体免疫以及其他功能的平衡。硬骨鱼类IRF家族除了具有哺乳类所有成员外，还具有鱼类特有的IRF基因，即IRF11。目前，有关IRF11功能方面的研究在国内外尚未见报道。本项目以斑马鱼为研究对象，利用荧光素酶报告系统和免疫印迹分析，研究鱼类IRF11转录调控、激活的分子机制，通过酵母双杂交与免疫共沉淀技术，解析其信号转导通路，并结合基因敲除和基因芯片分析，阐明IRF11的调控网络的分子机理，揭示其在鱼类免疫应答中的作用。本项目的完成将首次阐明硬骨鱼类特有的IRF11的作用机理和免疫功能，对于深入了解鱼类的抗病机制，拓展鱼类免疫学，认识脊椎动物免疫系统的组成和进化都具有重要意义。

干扰素调节因子（Interferon regulatory factor, IRF）是一类多功能转录因子，广泛参与宿主抗病毒免疫应答、抗原递呈和抗体类别转换，调控细胞增殖和凋亡，维持机体自稳平衡。哺乳动物IRF家族共有10个成员，IRF1-10。在前期的研究中，我们发现硬骨鱼类除了具有哺乳动物IRF所有成员外，还具有其特有的IRF，即IRF11。本项目比较分析了硬骨鱼类IRF11基因座的构成及脊椎动物IRF家族序列特性，发现IRF11和IRF1、IRF2由同一个祖先分化形成，该分化事件发生在头索动物与脊椎动物祖先分化之后，且IRF11在四足类中发生了基因丢失。三者虽具有相似的结构，但其表达模式不同。IRF11主要表达于鱼类胸腺组织，Poly I:C刺激和副溶血弧菌感染未能显著上调各组织中IRF11的表达水平。结合定点突变和荧光素酶报告系统，我们发现IRF11的DBD区域中第40位组氨酸，46位色氨酸在IRF11特异性识别靶基因启动子至关重要。利用激光共聚焦显微镜观测到IRF11在转染0.5小时后在胞内积累，并于3小时后转位入核。此外，缺乏核定位序列119RRRK122的IRF11的入核进程及其对靶基因调控作用被抑制。体外实验证实，IRF11虽能显著上调I型干扰素启动子报告基因活性，但无直接抗病毒作用。采用CRISPR/Cas9技术构建了斑马鱼IRF11敲除品系，比较分析了野生型与IRF11-/-品系在抗细菌免疫应答方面的差异。发现IRF11-/-斑马鱼感染迟缓爱德华菌48小时后，其体细菌数量显著多于野生型幼鱼。生存率曲线统计结果显示，迟缓爱德华菌感染7天后，IRF11-/-斑马鱼全部死亡，而野生型斑马鱼死亡率为76%。此外，过表达IRF11的斑马鱼胚胎中的菌落数量要显著低于空白对照组，进一步证实了IRF11参与了宿主的抗细菌免疫应答。本项目解析了IRF11介导的转录调控作用的结构基础及分子机制，阐释了其在抗细菌免疫反应中的作用。项目研究成果丰富和提升了对鱼类免疫应答调控机制的认识，为进一步认识脊椎动物免疫系统的发生和进化奠定了基础。