猪流行性腹泻病毒诱导B细胞活化的机制与特异抗体类型转换的关键调控因素

Porcine epidemic diarrhea virus (PEDV) is a major pathogen causing a severe diarrhea and death to suckling neonatal pigs and its damages will change with pig ages. Specific IgA antibody play a crucial role in host anti-PEDV infectious immunity, but we are not clear about the molecular mechanism of PEDV activating B cells and the regulation factors of specific antibody class switch. The proposed study will 1) explore in vitro the characterizations of key molecules expressed on the surface of matured dendritics and activated CD4+ T cells participating in B cell activation and on the surface of activated B cells, in which all cells come from suckling neonatal pigs, waned pigs and sows; 2) analyze the regulating effects of PI3K-AKT signaling pathway on PEDV-stimulated B cells by activating NF-κB; 3) reveal the crucial cytokines affecting PEDV-activated B cells and their regulating effects on specific antibody class switch; 4) together with in vivo experiment, evaluate the specific multiplication capacity of memory B cells derived from pigs of different ages, and the isotypes and levels of antibodies secreted by memory B. These results will illuminate the molecular mechanism of PEDV activating B cells and the regulating factors of specific antibody class switch, and will highlight the immune foundation on the correlation between B cell activation and pig ages. The study will enrich the theoretical basis of PEDV-infection immunity and guide PEDV prevention and control in pig farms.

猪流行性腹泻病毒（PEDV）是引起初生仔猪腹泻和死亡的主要病原，其对猪的危害具有年龄依赖性，特异IgA抗体在宿主抗感染免疫中发挥重要作用，但PEDV诱导B细胞活化的机制和特异抗体类别转换的关键调控因素尚不清楚。本项目拟在体外探究参与PEDV诱导不同年龄猪（初生仔猪、保育仔猪和成年母猪）B细胞活化的免疫细胞（成熟树突状细胞和活化CD4+ T细胞）以及活化B细胞的表面分子特征，解析PI3K-AKT信号通路激活NF-κB对PEDV活化B细胞的调节作用，揭示参与PEDV诱导B细胞活化并调控特异抗体类别转换的关键细胞因子。结合体内实验，分析不同年龄猪记忆性B细胞的特异增殖能力及其分泌的特异抗体类型与水平。综合上述结果，阐明PEDV活化B细胞的分子机制和特异抗体类别转换的关键调控因素，诠释PEDV诱导B细胞活化与猪年龄关系的免疫基础，丰富PEDV感染免疫机制理论基础，指导PEDV免疫防控实践。

猪流行性腹泻病毒（PEDV）是导致仔猪腹泻的主要病原，体液免疫在宿主抵抗该病毒感染过程中发挥重要作用，但PEDV诱导B细胞活化的机制尚不清楚。本项目结合体内和体外实验，应用流式细胞术、转录组学和siRNA等技术，探究了PEDV诱导B细胞活化分子机制，揭示了PEDV诱导B细胞活化及参与抗体类别转换的关键细胞因子，阐释了宿主抗PEDV感染体液免疫的记忆免疫反应特征，解析了B细胞应答与猪的生长阶段相关的主要免疫分子。结果发现① PEDV可诱导猪B细胞活化，引起B1（CD19+CD5+）细胞亚群数量增加，刺激产生趋化因子CXCL13，参与B细胞向组织趋化。但在获得性免疫应答过程中发挥主要抗感染作用的B2亚群和感染早期发挥抗感染作用的CD21+IgM+B细胞含量下降，B细胞受体信号通路的抑制性受体PIR-B、B细胞信号转导相关接头蛋白Bam32及NF-κ B信号通路中的B细胞活化因子（BAFF）的表达量降低，这些结果提示B细胞的进一步活化受到影响。② IL-4、IL-6、TGF-β和IFN-γ与PEDV特异性抗体类别转换有关，其中IL-4起主要作用；B细胞对PEDV刺激后的记忆免疫反应能力弱，当B细胞再次被PEDV刺激时仅有6.4%的CD21+B和3.7%的CD38+细胞亚群活化。③ PEDV刺激B细胞分泌的抗体与细胞因子与毒株毒力和猪的年龄相关。PEDV在体外不能刺激初生仔猪的B细胞产生特异性抗体；相对于保育猪，初生仔猪和母猪B细胞分泌的细胞因子种类少，尤其是初生仔猪B细胞没有分泌INF-和TGF-β，此结果可能与初生仔猪抗PEDV感染力低有关。上述研究结果，可以为深入揭示PEDV的感染免疫机制奠定重要基础，为新型PEDV疫苗的设计与研究提供科学指导。