猪流行性腹泻病毒诱导的T细胞依赖性sIgA+ B细胞分化机制

The majority infections are initiated at mucosal surfaces. Porcine endemic diarrhea virus (PEDV) is a classical pathogen that initiates the infection on intestinal mucosa and causes severe morbidity or mortality in piglets. Lacking of active immune responses due to immature immune system for suckling piglets, maternal vaccines to induce lactogenic immunity, and their transmission to suckling neonates via colostrum and milk, are critical for early passive protection. Therefore, the issue that how to improve the secretory IgA level in breast milk has been becoming a popular topic for prevention of gastro-intestinal infectious diseases. It is known that the maternal sIgA level is regulated by immune responses within gut-mammary gland-sIgA axis, specifically by the differentiation of IgA+ B cells in gut-associated lymphoid tissues (GALT). This proposal is focusing on the key events of sIgA+ B cell differentiation within germinal center of Payer’s patches (PPs) after PEDV infection. The relationships and interactions between the follicular helper T cells (Tfh), follicular dendritic cells (FDC), interleukins within PP germinal center microenvironment, and the class switch recombination (CSR) of sIgA+ B cell will be analyzed and discussed. The studies in this proposal will lead to reveal of the molecular basis of T-dependent sIgA+ B cell CSR induced by PEDV infection and therefore facilitate the analysis of pathogenesis, dissecting host-pathogen interaction, optimizing strategies for prevention and control of PEDV, and research and development of novel vaccines and adjuvants.

黏膜是大多数病原体攻击机体致病并激发免疫反应的始发部位，猪流行性腹泻病毒（PEDV）是一种典型的消化道黏膜感染的病原体，对哺乳仔猪危害甚为严重，母源sIgA抗体水平对该病的发生发展具有决定性的影响，因此，如何提高母乳中特异性IgA水平是目前的研究热点。而母源sIgA抗体水平受肠道-乳腺-sIgA轴免疫反应的调控，尤其是肠道相关淋巴组织内sIgA+ B细胞的分化起关键作用。本项目以PEDV为研究对象，围绕发生于派伊尔集合淋巴结（PP）生发中心（GC）内分泌IgA的B细胞分化这一关键事件，分析淋巴滤泡辅助T细胞（Tfh）和淋巴滤泡树突状细胞（FDC）及微环境中的细胞因子在B细胞抗体类别转换（CSR）中的作用，阐明PEDV感染后T细胞依赖性sIgA+ B细胞分化的分子机制。为猪流行性腹泻的发病机理、病原-宿主相互作用关系的剖析、疾病防控策略的优化以及新型疫苗和佐剂等的研发提供理论依据。

猪流行性腹泻病毒（PEDV）是一种典型的消化道黏膜感染的病原体，对哺乳仔猪危害甚为严重，母源sIgA抗体水平对该病的发生发展具有决定性的影响，因此，如何提高母乳中特异性IgA水平是目前的研究热点。而母源sIgA抗体水平受肠道-乳腺-sIgA轴免疫反应的调控，尤其是肠道相关淋巴组织内sIgA+ B细胞的分化起关键作用。为了顺利开展本项目工作，首先建立了筛选猪肠道病毒的6重RT-PCR方法以及猪肠道冠状病毒4重荧光定量RT-PCR方法，并利用上述方法从田间样品分离获得一株生长性能良好的PEDV流行毒株，采用该PEDV毒株在实验室条件下感染仔猪，通过病原检测、病理解剖、生化中心标志物检测等分析，结果表明成功建立了动物感染及黏膜免疫应答模型，且PEDV感染诱导猪肠道PP结内IgA+ B细胞关键酶AID的表达。由于猪肠道黏膜免疫应答机制研究在国内外尚属空白，以致相关抗体、试剂缺乏，为深入研究PEDV诱导的肠道黏膜免疫应答机制，本项目首先成功制备Tfh细胞表面关键标志物CXCR5抗体；经反复尝试与优化，成功获得了猪B细胞发生IgA抗体类别转换过程中关键标志物PST、GLT和CT的基因并建立了其评价方法。本项目研究发现PEDV的感染显著上调猪肠道PP结内Tfh细胞的IL-10和IL-21表达水平，而上调的IL-10和IL-21显著促进了PP结中IgA的分泌水平，此外，进一步研究结果显示，IL-10和IL-21可抑制PP结中IgM+ B细胞凋亡并促进B细胞增殖与分化。为探明IL-10和IL-21促进B细胞增殖分化的分子机制，本研究筛选了相关信号通路，结果显示，IL-10和IL-21激活了PP结B细胞内JAK/STAT信号通路, 采用抑制剂阻断PI3K/AKT/ERK信号通路后，B细胞的IgA抗体分泌水平显著降低。由此可见，PEDV感染后肠道PP结内生发中心的Tfh细胞分泌IL-10和IL-21，后者与B细胞相互作用激活了PI3K/AKT/ERK信号通路，从而促进B细胞增殖与分化以及抗体类别转换，最终导致IgA分泌量上调。该研究结果将为猪流行性腹泻的发病机理、病原-宿主相互作用关系的剖析、疾病防控策略的优化以及新型疫苗和佐剂等的研发提供理论依据。