gC1qR在PCV2诱导巨噬细胞M2型极化过程中的作用与调控机制

The immunosuppression caused by porcine circovirus type 2 (PCV2) infection is the major feature of PCV2-associated diseases. Recent studies have demonstrated that the M2 polarization of macrophages is associated with the immunosuppression caused by many viruses. Our previous studies have demonstrated that PCV2 infection could induce polarized M2 macrophages, whereas knockout of gC1qR gene could block the M2 polarization induced by PCV2, suggesting that gC1qR might play a pivotal role in the development of M2 polarization in PCV2-infected macrophages. However, it is still needed to further study the roles and regulatory mechanism of gC1qR in the M2 polarization of macrophage induced by PCV2. This project are firstly proposed to investigate that the changes of cell number, cell activity, polarized marker molecules, relative signaling pathways activities and miRNA expression profiles of porcine alveolar macrophages (PAMs), as well as the degree of tissue lesions, after PCV2 infect normal piglets or isolated primary PAMs, which will help us to determine the change pattern of polarized macrophage phonotype and correlation between polarization features of PCV2-infected macrophages and tissue lesions, and to identify the key signaling pathways and miRNAs that are involved in the regulation of M2 polarization induced by PCV2. Then, to develop gC1qR knockout porcine alveolar macrophages or piglet using CRISPR/cas9 genome editor tool, to investigate the changes of cell number, cell activity, polarized marker molecules, key signaling pathways activities, miRNA expression profiles, and to observe tissue lesions in either gC1qR knockout porcine alveolar macrophages or piglets, based on which to determine the roles and regulatory mechanism of gC1qR in the M2 polarization of macrophages induced by PCV2. The prospective results will help us to clarify the mechanisms of the macrophage M2 polarization during PCV2 infection , and provide a new insight into understanding the mechanism of PCV2 infection induction of immunosuppression.

PCV2感染诱导的免疫抑制是猪圆环病毒病的主要特征。近年来研究认为巨噬细胞M2型极化与多种病毒诱导的免疫抑制密切相关。我们前期研究发现，PCV2感染可诱导巨噬细胞向M2型分化，敲除gC1qR可阻止PCV2诱导的M2型极化，提示gC1qR可能在PCV2诱导巨噬细胞M2型极化中发挥关键作用。本项目拟首先在PCV2感染猪和猪肺泡巨噬细胞后检测细胞活性、极化标志分子、相关信号通路活性和miRNA表达谱的变化，观察组织病变，明确PCV2感染后猪肺泡巨噬细胞极化状态的变化特征及其与组织病变的相关性，确定调控M2型极化的关键通路和miRNA；然后通过Cas9技术敲除gC1qR基因，检测gC1qR敲除对PCV2诱导的M2型极化、相关通路、miRNA以及组织病变的影响，明确gC1qR及其调控的信号通路和miRNA的作用，阐明PCV2诱导巨噬细胞M2型极化的分子机制，为认识PCV2的免疫抑制机制提供理论数据。

PCV2感染诱导的免疫抑制是猪圆环病毒病的主要特征。近年来研究认为巨噬细胞M2型极化与多种病毒诱导的免疫抑制密切相关。本项目首先确定了PCV2体内或体外感染均诱导猪肺泡巨噬细胞向M2型极化，同时抑制其他病原诱导巨噬细胞向M1型极化；通过检测PCV2感染的gC1qR敲除的细胞和动物，明确PCV2感染后可通过其Cap与gC1qR结合进而激活PI3K/Akt和p38-MAPK通路诱导猪肺泡巨噬细胞M2型极化并抑制其它病原诱导M1型极化；同时筛选并鉴定出miR-23a、miR-29b、miR-93和miR-330是受gC1qR调控且参与调控巨噬细胞极化过程的关键miRNAs，PCV2感染后通过其Cap与gC1qR互作调控PI3K/Akt和p38-MAPK通路进而上调miR-23a和miR-29b表达、抑制巨噬细胞M1型极化和Th1免疫，通过上调miR-93和下调miR-330促进巨噬细胞M2型极化；PCV2感染后通过gC1qR募集PKCδ到核膜上，促进lamin A/C的磷酸化和迁移，使核膜结构变得疏松，同时募集病毒粒子到核膜上，促进PCV2病毒粒子的出核。在此基础上，筛选并确定PCV2的Cap蛋白N端‘24RRR26’是其与gC1qR结合的关键功能区域和关键氨基酸位点，突变获得了不与gC1qR结合的突变型PCV2毒株PCV2RmA，与野生毒株比较发现，PCV2RmA诱导巨噬细胞M2型极化的能力减弱，抑制M1型极化的能力减弱，复制能力减弱、致组织病变的能力减弱。该项研究阐明了PCV2感染诱导巨噬细胞M2极化和抑制M1型细胞因子产生的分子机制，以及gC1qR在调控PCV2感染的巨噬细胞极化中的作用及机制，筛选出不与gC1qR结合的突变型PCV2毒株PCV2RmA，并鉴定了该突变毒株诱导巨噬细胞极化的特征，为进一步从巨噬细胞极化调控方面认识PCV2的免疫抑制机制提供了新的理论数据。