猪繁殖与呼吸综合征病毒再感染激活猪IL-4表达的分子机制

Porcine interleukin-4 (IL-4) was almost undetectable from pigs infected with porcine reproductive and respiratory syndrome virus (PRRSV) for the first time. However, the level of IL-4 production was remarkably elevated after PRRSV reinfection and was significantly higher than the first infection. Previous studies found that porcine IL-4 significantly increased the percentages of CD4+ (Peng et al., 2013) and CD4+CD8+ (Li et al., 2015) T lymphocyte subpopulation in porcine peripheral blood but decreased the percentage of CD8+ T lymphocyte subpopulation (Peng et al., 2013). Also, the titer of neutralizing antibodies specific to PRRSV was significantly elevated in pigs co-administered with high level of IL-4 (Peng et al., 2013; Tang et al., 2014). However, the mechanism of IL-4 production activated by PRRSV reinfection is not yet clear. In this project, the PRRSV protein(s) activating IL-4 production will be investigated. Afterwards, the signal pathway mediating the activation of IL-4 production will be explored. Then, by analyzing the regulatory effect of the PRRSV protein with different amino acid mutants on the IL-4 production, the protein interaction between the PRRSV protein and corresponding signaling molecule, and signal transduction pathway mediating IL-4 production, the molecular mechanism of the activation of IL-4 production triggered by PRRSV reinfection could be clarified. Thus, it will lay a foundation in theory for further studies about the reason why the level of IL-4 production was significantly different when PRRSV infection and reinfection, and about the mechanism of IL-4 acting on immune response against PRRSV.

猪繁殖与呼吸综合征病毒（PRRSV）首次感染猪体内白细胞介素4（IL-4）表达水平极低，但PRRSV再感染则IL-4表达水平显著提高。研究发现，IL-4显著提高CD4+（Peng等，2013）、CD4+CD8+T细胞含量（Li等，2015）及特异性中和抗体水平（Peng等；Tang等，2014），降低CD8+T细胞含量（Peng等）。但是，关于PRRSV再感染激活IL-4表达的机制尚不清楚。本项目拟以PRRSV首次感染猪PAM为材料，在鉴定PRRSV再感染激活IL-4表达的PRRSV蛋白、筛选介导IL-4表达的信号通路基础上，分析PRRSV蛋白特定位点突变对IL-4表达的影响、PRRSV蛋白与相关信号分子互作以及相应通路信号转导途径，阐明PRRSV再感染激活IL-4表达的分子机制，为深入研究PRRSV感染与再感染IL-4表达差异的产生机理及IL-4对PRRSV免疫的作用机制奠定理论基础。

猪繁殖与呼吸综合征病毒（PRRSV）首次感染猪体内白细胞介素4（IL-4）表达水平极低，PRRSV再感染猪则IL-4表达水平显著提高。迄今，诱导猪IL-4表达的PRRSV蛋白及其相关信号级联途径、PRRSV感染及再感染后IL-4在猪外周淋巴组织中的变化规律尚不清楚。本项目从体外与体内两个方面对PRRSV感染诱导IL-4表达进行了研究。体外培养PRRSV JXA1-R首次感染猪的肺泡巨噬细胞16h后，再次接种PRRSV JXA1，同时加入猪外周血单核细胞和CD28单克隆抗体共培养18h时，经ELISA检测发现IL-4表达水平显著升高（p < 0.05）。该IL-4体外诱导表达系统，为筛选介导IL-4表达的PRRSV蛋白及其相关通路途径提供了条件。PRRS双阴性猪经PRRSV JXA1-R疫苗弱毒感染后28d，再次分别接种该弱毒或JXA1强毒，冰冻切片经荧光染色显示，PRRSV弱毒或强毒再感染后猪淋巴结和脾脏中IL-4 mRNA水平、外周血中IL-4蛋白水平皆显著升高（p < 0.05）。PRRSV感染前CD4+ T淋巴细胞与IL-4极其少量的、稀疏地散在于淋巴小结周围的副皮质区、淋巴小结内未有显现；首次感染后该细胞与IL-4的分布位置及数量与感染前相同。PRRSV再感染后数量较多的CD4+ T细胞、大量的IL-4出现于淋巴小结内，并且T细胞、IL-4和B细胞三者发生共定位。该研究表明，PRRSV再感染诱导产生的大量IL-4由淋巴小结外的副皮质区“迁移”到淋巴小结内，与进入淋巴小结的CD4+ T细胞一起聚集于生发中心的B细胞、发生相互作用。本项目建立了猪IL-4的体外细胞诱导表达系统，初步探明了PRRSV再感染诱导IL-4表达、IL-4与CD4+ T细胞及B细胞在猪外周淋巴组织中的位移及互作关系，为进一步阐明PRRSV感染后IL-4与T、B淋巴细胞的作用机制奠定了基础。