II类反式激活因子通过NLR信号调控Th2应答的分子免疫学机制

Thanks to the supports of NSFC projects, an unexpected phenomenon that the Th1 response could be inverted to the Th2 response, in addition to the down-regulated MHC class II expression, has been reported in our previous study when C57BL/6 mice were transgenic with mCIITA, a mutated class II transactivator gene. Further study indicated that the CIITA mutant behaved as a member of the NOD-like receptor (NLR) family to activate IL-33 gene via the NLR-initiating signal pathways, which resulted in a skewing production of the Th2-related cytokines. Based on these observations, together on the progresses of the pattern-recognition receptors (PRRs) and the controls of their signaling, this project focuses on four aspects to explore why mCIITA is able to activate the Th2-dominant immune responses. (1) To dissect the structure basis for the differential expression of functions with mCIITA and wtCIITA molecules, and their roles in stimulating NLRs by the cytosolic DAMPs (damage associated molecular patterns). (2) To reveal the epigenetic mechanisms by which the expression of transcriptional factors T-bet and Gata3 is declined and increased respectively to promote the Th2 responses. (3) To explore the pathways by which mCIITA stimulates the production of IL-33 in a big amount via the NLRP3/inflammasome-mediated signal pathway. (4) To understand the functional significance of the mCIITA-controlled regulation of IL-33 that functions as an inner alarmin. In brief, this project aims at the elucidation of the control mechanisms to the Nod2-/inflammasome-mediated signal pathways by using mCIITA as a probe and on their relationship with adoptive immunity: MHC expression and the Th1/Th2 subset transformation. This study is therefore one of the frontiers in innate immunity and would be helpful to understand how DAMP is involved in the non-infectious inflammations, which would lay a basis for the development of approaches to control the relavant clinical diseases.

II类反式激活因子（CIITA）是MHC II表达的主导开关。我们之前的研究意外发现CIITA突变体（mCIITA）转基因小鼠II类分子表达下调的同时，Th1应答转换为Th2应答；并确认mCIITA作为NLR启动Nod2和炎症小体介导的信号途径上调IL33，继而诱导Th2细胞因子表达。据此，本项目将结合NLR的调控进展提出"CIITA可通过NLRP信号途径调控Th2亚群分化和免疫应答"的假说，拟用转基因动物、ChIPseq等手段探索:⑴mCIITA在胞浆DAMP激发NLR的机理及其激活Th2应答中T-bet/Gata3消长的表观遗传因素;⑵mCIITA通过NLRP3炎症小体调控产生IL-33的机制；作为固有免疫研究前沿，以mCIITA为探针分析Nod2和炎症小体的信号调控特点，有助于就MHC表达、Th1/Th2亚群转换、非感染性炎症、与适应性免疫的关系，及相关研究的临床意义作出探索和评价。

本课题组之前研究发现II类反式激活因子突变体（mCIITA）除可以显著下调MHC II类分子表达之外，同时发现C57BL/6J小鼠由Th1型免疫应答背景转换为Th2型，并首先报道mCIITA可作为NLR启动Nod2下游的NF-B信号途径，通过激活巨噬细胞分泌IL33表达，从而活化Th2相关细胞因子。.据此，结合NLR领域的新进展及其调控机制的进展，我们在本项目中研究了mCIITA是否也可以通过结合NLRP3，激活炎症小体， 产生IL-33促进Th1→Th2转化的可能机制； 分析mCIITA激活Th2应答中T-bet/Gata3消长的表观遗传学因素；考察了mCIITA在胞浆DAMP激发NLR中的意义并探讨mCIITA在非感染性疾病中作为内源性警报素参与免疫应答的一些特点和意义。.结果发现：（1）mCIITA可通过活化NF-κB信号通路，促进NF-κB入核，诱导增强NLRP3的转录表达；（2）mCIITA可诱导NLRP3在胞浆中大量表达，进一步，共聚焦、WB/Co-IP等方法检测发现mCIITA在胞浆中与NLRP3结合，激活炎症小体，正向调控IL-33等相关细胞因子的表达，使免疫应答由Th1向Th2转变；（3）利用mCIITA+/+NLRP3-/-小鼠，首次证明体内mCIITA促进Th2细胞分化的作用依赖于NLRP3 的表达; （4）表观遗传学实验发现mCIITA逆转Th1→Th2格局主要依赖于其通过活化炎症小体影响T细胞转录因子T-bet/Gata3表达水平，并通过IL-4启动子区的去甲基化及组蛋白H3乙酰化作用增加而促使IL-4的表达增高，其中涉及细胞因子下游JAK/STAT信号通路的激活改变；（5）在喉癌及心脏瓣膜硬化两种非感染性炎症疾病中，发现了CIITA与NLRP3在巨噬细胞细胞质中的共表达/定位，而且荧光共定位强度与疾病进展和分期相关联，初步提示突变型的CIITA可作为以上病理状态下一种DAMP活化NLRP3的胞内感受器， 参与疾病发生，发展进程，以上疾病中CIITA分子的天然突变模式正在深入研究中。本项目的研究成果丰富了CIITA的生物学功能，具有重要的基础及临床意义。在该项目的资助下已申请新专利3项、 标注基金号发表19篇研究论文，其中12篇发表于 SCI 收录杂志，7篇发表在中文核心期刊。培养 2名博士，3名硕士。

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