斑马鱼胞内模式识别受体NLRC5在抗感染中的作用及其机制

Members of the nucleotide-binding oligomerization domain-like receptor (NLR) family are pivotal regulators of innate and adaptive immune responses. NLRC5, the largest NLR family member, was recently proposed to function as a positive and negative regulator of antiviral immune responses and MHC class I transcription. Thus, the precise role of NLRC5 in regulating immune signaling remains controversial and needed to further investigation. In our research on zebrafish PRRs, NLRC5 (namely zfNLRC5) and its five splicing isoforms (namely zfNLRC5a, zfNLRC5b, zfNLRC5c, zfNLRC5d and zfNLRC5e) were identified, and our preliminary results showed that zfNLRC5 and its splicing isoforms were located in both cytoplasmic and nuclear compartments, and that the overexpression of zfNLRC5 in zebrafish embryos induced the expression of those genes involved in defense response to bacterium and virus, biotic stimulus, inflammatory response and so on. However, the function of NLRC5 splicing isoforms has never been reported in any species of vertebrates. The proposed research is designed to illustrate whether NLRC5 splicing isoforms are involved in mediating signaling pathways, and the similarity and difference of NLRC5 splicing isoforms in regulating signaling networks. Then, the ligand recognition, the roles in antibacterial and antiviral immunity, and the regulation on the expression of molecules associated with antigen presentation, especially MHC class I and MHC class II molecules, are to be further investigated in order to understand the function of NLRC5 splicing isoforms in the innate and adaptive immunity. The mechanisms of action of NLRC5 splicing isoforms will be evaluated by investigating the interactions between splicing isoforms and adaptor proteins involved in signaling pathways, or other antibacterial and antiviral PRRs, and by examining the effects of interactions among them on the anti-infection immune responses. It is believed that this proposed research will interpret the new mechanisms of anti-infection in teleost fish, and establish the immunogenetic basis of disease resistance in fish.

在哺乳动物中，NLRC5属于胞浆型模式识别受体，在调控机体的天然免疫以及炎症反应中起着重要作用。我们发现斑马鱼的NLRC5存在5种剪接异构体；NLRC5剪接异构体的功能还没有报道于任何脊椎动物。本项目将通过研究斑马鱼NLRC5不同剪接异构体在信号传导通路中的作用，阐明NLRC5的剪接异构体是否具有介导信号传导通路的作用，同时也将阐明这些剪接异构体在调控信号网络中的功能相同与差异性；通过研究NLRC5剪接异构体的配体识别、在抗细菌以及抗病毒感染中的作用以及对抗原提呈相关分子表达的调控，揭示NLRC5剪接异构体在先天免疫以及适应性免疫中的功能；通过研究NLRC5不同剪接异构体与接头蛋白、抗细菌以及抗病毒相关的模式识别受体之间的分子互作及其分子互作对抗感染免疫反应的影响，揭示NLRC5不同剪接异构体发挥功能的作用机制。该项目的完成将诠释鱼类抗感染的新机制；同时将奠定鱼类抗病力的免疫遗传学基础。

核苷酸结合寡聚化结构域受体（Nucleotide-binding oligomerization domain-like receptors, NLRs）是一类在先天免疫中对病原体感染起着重要作用的胞内模式识别受体；而NLRC5是NLRs家族中分子量最大的成员。尽管哺乳动物NLRC5在MHC转录调控中的作用得到公认，但其在调控先天免疫中的作用还存在争议。NLRC5的选择性剪接只是在人中被报道，其剪接异构体的功能还不清楚；而关于硬骨鱼类NLRC5在调控先天免疫以及适应性免疫中的作用更是一无所知。本项目对斑马鱼NLRC5的选择性剪接以及剪接异构体的功能进行了研究：除了斑马鱼NLRC5的正常形式（zfNLRC5）外，克隆得到zfNLRC5的5个截短形式的剪接异构体，分别被命名为zfNLRC5a，zfNLRC5b, zfNLRC5c, zfNLRC5d和zfNLRC5e；研究了它们在斑马鱼发育阶段的组成型表达以及在病原感染后的诱导型表达特征；阐明了zfNLRC5及其剪接异构体的亚细胞定位；探索了zfNLRC5及其剪接异构体对干扰素启动子活性、MHC启动子活性、MHC相关基因的转录调控、在细菌以及病毒感染中的作用、以及在介导信号传导通路中的作用。我们的结果首次揭示了硬骨鱼类NLRC5与哺乳动物NLRC5功能的差异：斑马鱼zfNLRC5不仅具有不依赖于类I干扰素产生的抗病毒功能；而且还能转录调控MHC-II类分子的表达。此外，我们的研究还首次揭示了zfNLRC5的剪接异构体在细菌以及病毒感染中的差异功能、zfNLRC5与zfNLRC5的剪接异构体对TLRs信号以及NF-κB信号通路的差异调控。除此之外，我们还完成了其它的工作：解析了NLRs 家族的两个新成员NALPL1和NALPL2在病原感染中的保护作用；RLRs经典抗病毒信号通路上的关键接头蛋白MAVS、关键激酶TBK1的选择性剪接及其剪接异构体的功能。这些研究对于揭示NLRs和RLRs家族基因功能多样性产生的机制、选择性剪接对NLRs和RLRs信号通路的调控作用、以及剪接异构体在病原体感染中的作用具有重要意义。