cGAS/STING信号通路参与猪圆环病毒2型诱导猪肺泡巨噬细胞产生Ⅰ型干扰素

cGAS/STING signaling pathways induces the secretion of type Ⅰ interferon in porcine alveolar macrophages infected with porcine circovirus type 2

摘要:

为研究猪圆环病毒2型 (Porcine circovirus type 2，PCV2) 感染的猪肺泡巨噬细胞 (Porcine alveolar macrophages，PAMs) 分泌Ⅰ型干扰素信号通路，以PCV2病毒感染PAMs为研究对象，采用酶联免疫吸附测定 (Enzyme-linked immunosorbent assay，ELISA)、实时荧光定量PCR和Western blotting，分析PCV2感染对PAMsⅠ型干扰素的诱导、cGAS/STING信号通路相关基因mRNA和蛋白表达的影响，并应用靶向cGAS和STING特异性siRNA、抑制剂BX795和BAY 11-7082，解析cGAS、STING、TBK1和NF-κB/P65在PAMs生成Ⅰ型干扰素中的作用。结果显示，PAMs感染PCV2病毒48 h后Ⅰ型干扰素的表达量显著升高 (P＜0.05)，cGAS mRNA的表达量在感染48 h和72 h后显著升高 (P＜0.01)，STING mRNA表达量在PCV2感染72 h后显著上升 (P＜0.01)，TBK1 mRNA、IRF3 mRNA感染48 h后显著升高 (P＜0.01)。PCV2能够显著升高PAMs胞浆STING、TBK1和IRF3蛋白含量，降低胞浆NF-κB/p65的含量，促进NF-κB/p65和IRF3入核。敲低PAMs中cGAS或STING表达水平后，PCV2感染PAMs 48 h后，Ⅰ型干扰素的表达水平显著下降 (P＜0.01)；BAX795抑制TBK1后，PCV2感染PAMs 48 hⅠ型干扰素的表达水平显著下降 (P＜0.01)，BAY 11-7082 抑制NF-κB/P65表达后，PCV2感染PAMs 48 h I型干扰素的表达量与PCV2组相比无显著性差异 (P＞0.05)。结果表明，PAMs感染PCV2后通过cGAS/STING/TBK1/IRF3信号通路诱导Ⅰ型干扰素分泌。

Abstract:

In order to study the signal pathway secreting type Ⅰ interferon in porcine alveolar macrophages (PAMs) infected with porcine circovirus type 2 (PCV2), the protein and the mRNA expression levels of cGAS/STING pathways were analyzed by ELISA, Western blotting and quantitative reverse transcriptase PCR in PAMs infected with PCV2. In addition, the roles of cGAS, STING, TBK1 and NF-κB/P65 in the generation of type I interferon (IFN-I) from PAMs were analyzed by using the cGAS and STING specific siRNA, inhibitors BX795 and BAY 11-7082. The results showed that the expression levels of IFN-I increased significantly at 48 h after infection with PCV2 (P<0.05), the mRNA expression levels of cGAS increased significantly at 48 h and 72 h after infection (P<0.01), the mRNA expression levels of STING increased significantly at 72 h after infection (P<0.01), and the mRNA expression levels of TBK1 and IRF3 increased at 48 h after infection (P<0.01). The protein expression levels of STING, TBK1 and IRF3 in PAMs infected with PCV2 were increased, the content of NF-κB/p65 was decreased, and the nuclear entry of NF-κB/p65 and IRF3 was promoted. After knocking down cGAS or STING expression by siRNA, the expression level of IFN-I was significantly decreased after PCV2 infection for 48 h (P<0.01). BX795 and BAY 11-7082 inhibitors were used to inhibit the expression of IRF3 and NF-κB, the concentration of IFN-I in BX795-treated group was significantly reduced than that of the PCV2 group (P<0.01), while no significant difference was observed between the BAY 11-7028 group and the PCV2 group. The results showed that PAMs infected with PCV2 induced IFN-I secretion through the cGAS/STING/TBK1/IRF3 signaling pathway.