miR-30a-5p介导PAM自噬在猪链球菌2型和猪圆环病毒2型协同感染免疫逃逸中的作用机制

Streptococcus suis serotype 2 (SS2) is a respiratory pathogen that causes pneumonia, meanwhile the lung is the target organ of PCV2. Porcine alveolar macrophage (PAM) is one of the main defense against micro-organisms. Our previous results indicated that miR-30a-5p could promote the replication of PCV2 and SS2 through enhancing PAM autophagy, which co-infected with PCV2 and SS2. Moreover, synergistic infection promotes the proliferation of SS2 and PCV2 both in PAM and mouse, and increases the mortality of mouse. Therefore, we assume that autophagy may regulate the function of PAM to benefit the immune escape of SS2 and PCV2 synergistic infection. In this study, we will research four aspects to elucidate this mechanism: ⑴ evaluate the collaborative interaction of synergistic infection with autophagy induced by miR-30a-5p, ⑵ study the effect of autophagy on the polarization and pathogen clearance function of PAM, ⑶ analyze the signaling pathway activated by miR-30a-5p in SS2 and PCV2 synergistic infection, ⑷ preform the animal experiments to verify the function of autophagy induced by miR-30a-5p. These results will deepen illuminate the molecular mechanism of immune escape through the autophagy induced by miR-30a-5p in SS2 and PCV2 synergistic infection.

S.suis 2（SS2）是呼吸道致病菌，可引起肺炎，而肺脏又是PCV2的靶器官。PAM是宿主抵御外来病原体的主要防线之一，参与调节宿主免疫。前期研究表明，miR-30a-5p能够增强SS2与PCV2协同感染诱导PAM自噬的能力，促进病原增殖；协同感染提高了病原在PAM和小鼠体内的增殖能力，提高了小鼠的死亡率。我们设想自噬可能影响PAM功能，帮助SS2和PCV2在协同感染中实现免疫逃逸。本项目拟从以下4个方面进行：⑴协同或单一感染与miR-30a-5p介导自噬的相互作用；⑵miR-30a-5p介导的自噬对PAM细胞分型、清除病原等功能的影响；⑶协同感染中miR-30a-5p调节自噬的信号通路以及靶向自噬信号通路关键分子和靶基因的分析；⑷体内实验验证miR-30a-5p介导的自噬在协同感染中的作用。最终阐释miR-30a-5p介导PAM自噬在SS2和PCV2协同感染免疫逃逸中的作用机制。

PCV2和SS2是猪的两种主要病原体且都是人畜共患病原体，两者共感染病例在临床检测中较为常见。猪肺泡巨噬细胞是宿主抵御外来病原体的主要防线之一，参与调节宿主免疫。前期研究表明，miR-30a-5p能够增强SS2与PCV2协同感染诱导猪肺泡巨噬细胞自噬的能力，促进病原增殖；协同感染提高了病原在猪肺泡巨噬细胞和小鼠体内的增殖能力，提高了小鼠的死亡率。我们设想自噬可能影响猪肺泡巨噬细胞功能，帮助SS2和PCV2在协同感染中实现免疫逃逸。本项目拟从以下4个方面进行：⑴协同或单一感染与miR-30a-5p介导自噬的相互作用；⑵miR-30a-5p介导的自噬对PAM细胞分型、清除病原等功能的影响；⑶协同感染中miR-30a-5p调节自噬的信号通路以及靶向自噬信号通路关键分子和靶基因的分析；⑷体内实验验证miR-30a-5p介导的自噬在协同感染中的作用；最终阐释miR-30a-5p介导猪肺泡巨噬细胞自噬在SS2和PCV2协同感染免疫逃逸中的作用机制。体外实验结果表明，miR-30a-5p过表达可能通过激活PI3K，调控PI3K/Akt/mTOR信号通路调节自噬，显著增强猪肺泡巨噬细胞的自噬能力，促进巨噬细胞极化为促炎的M1状态，提高SS2的黏附和侵袭能力，有利于PCV2的复制。体内实验结果表明，与其他组相比，miR-30a-5p agomir+ PCV2+SS2-1组的感染情况最严重，表现出更高的PCV2病毒滴度、小鼠体重下降率和小鼠死亡率；miR-30a-5p过表达组肺脏组织的载菌量显著高于SS2-1组和PCV2+SS2-1组；与单一的SS2-1感染组相比，PCV2感染能有力地提高具有抗病毒活性的IFN-β和IFN-γ的表达，且在miR-30a-5p agomir+PCV2组更为明显；感染引起的巨噬细胞M1极化显著促进了PCV2和SS2的协同感染。