NOD2调控PEDV免疫应答的分子机制研究

Porcine epidemic diarrhea (PED) has caused huge economic losses and greatly challenged the swine industry worldwide in recent years. However, poor understanding of the immune responses against PED limited its control and prevention. As we all know that the intestinal mucosa is the very first line of defense against porcine epidemic diarrhea virus (PEDV) invasion. To better understand the mechanism of initiation and development of immune responses induced by PEDV infection within the intestinal epithelium cells will definitely contribute to the effective prevention and control of PED. NOD2, one of the most important pattern recognition receptors in the intestine epithelium cells, recognizes the bacterial invasion into the intestines and regulates chronic inflammation diseases in the intestines. We have already proved that NOD2 can be activated by PEDV infection. In this project, we attempt to illuminate the molecular basis of immune responses regulated by NOD2 against PEDV infection. To do so, real-time PCR assay, western blot assay, knock-down and overexpression strategies will be employed to investigate the recognition of PEDV invasion and associated immune response signaling pathways regulated by NOD2. The ligands for NOD2 recognition on PEDV infection and the functional molecules responsible for PEDV replication and immune responses will also be explored. Finally, the in vitro results will be validated using PEDV infected piglets. All these experiments will come to a conclusion and reveal molecular basis of immune responses regulated by NOD2 against PEDV infection, facilitating invention of novel and rational strategies for PEDV prevention and control.

近年来，猪流行性腹泻（PED）在世界多国广泛流行，严重制约了养猪业的健康发展。然而，该病的有效防控仍存在理论瓶颈。肠道黏膜是机体产生免疫应答抵御猪流行性腹泻病毒（PEDV）入侵的第一道防线，全面了解肠上皮细胞感染PEDV后免疫发生及其调控路径将有助于解决这一瓶颈。NOD2是肠道细胞内的重要模式识别受体，在识别细菌入侵及调控肠道炎症等方面起重要作用，我们的前期研究已经证明PEDV感染可特异性激活NOD2。本项目以肠道细胞内重要模式识别受体NOD2为核心，将PEDV感染猪小肠上皮细胞，采用定量PCR、western blot、基因沉默和过表达等方法研究NOD2对下游免疫应答信号通路的调控，确定NOD2识别的配体；筛选不同信号通路的效应分子并探索其调控路径；并在本体动物验证体外实验结果。本项目对阐明NOD2调控PEDV感染的免疫应答机制具有重要意义，为探寻PEDV防控新策略提供理论基础。

近年来，猪流行性腹泻（PED）在世界多国广泛流行，严重制约了养猪业的健康发展。该病以小肠为主要靶标，全面了解肠上皮细胞感染后的免疫发生及其调控路径将有助于该病的防控。本项目发现，猪流行性腹泻病毒（PEDV）感染小肠后主要激活模式识别受体NOD2，并诱导多种细胞因子的表达，在肠道上皮细胞系IPEC-J2的感染中也出现了类似现象。进一步研究发现PEDV-S蛋白与NOD2存在特异性相互作用。NOD2的活化可激活下游NF-κB信号通路，促进p65的入核及磷酸化，从而激活多种细胞因子的表达，抑制PEDV复制；一旦沉默NOD2，即丧失该激活效应。同时，NOD2活化也可低水平诱导干扰素的产生。NOD2的下游结合蛋白RIPK2在对细胞因子和干扰素的诱导中也发挥重要作用，可单独激活细胞因子和干扰素的表达。沉默RIPK2，即便激活NOD2,也无法诱导下游信号通路的活化。此外，PEDV的感染也可增强宿主RNA的m6A修饰水平，从而启动NOD2信号通路的活化。我们也发现,在PEDV对大鼠肠道上皮细胞的感染中，NOD2同样具有重要的调控作用。综上，我们初步证明NOD2在调控PEDV诱导的肠道免疫中具有重要作用。