RNF20影响RNA病毒感染后的巨噬细胞极化

RNF20 affects macrophage polarization after RNA virus infection

目的 探究环指蛋白20(RNF20)在抗RNA病毒先天免疫中的作用。方法 在293T人胚肾上皮细胞中过表达RNF20,运用双荧光素酶报告基因实验,检测仙台病毒(SeV)感染诱导的干扰素a4(Ifna4)基因启动子活化。构建Rnf20基因髓系条件性敲除小鼠模型(Rnf20F/F;Lyz2-Cre),运用流式细胞术检测Rnf20F/F;Lyz2-Cre和对照Rnf20F/F小鼠骨髓、脾脏和外周血髓系细胞亚群的比例;利用建立的小鼠模型培养骨髓源巨噬细胞(BMDMs),在SeV和水疱性口炎病毒(VSV)感染后,运用免疫印迹检测转录因子干扰素调节因子3(IRF3)和核因子-κB (NF-κB)p65亚基的磷酸化,通过ELISA检测细胞因子IFN-β、TNF-α、IL-6的表达,通过高通量转录组测序分析转录组的改变,并通过LPS和IL-4分别诱导巨噬细胞M1和M2极化,证实RNF20对于转录组的影响。结果 293T细胞中RNF20过表达不影响SeV感染诱导的Ifna4基因启动子活化。Rnf20F/F;Lyz2-Cre和Rnf20F/F小鼠髓系细胞发育无明显差异,在RNA病毒感染2组小鼠的BMDMs后,IRF3、NF-κB p65的磷酸化和IFN-β、TNF-α、IL-6的表达相似,但是一些与巨噬细胞极化相关的基因表达有显著差异。RNF20的缺失有抑制巨噬细胞M1型极化、促进巨噬细胞M2型极化的趋势。结论RNF20不影响RNA病毒感染诱导的IRF3和NF-κB通路活化,但可通过影响巨噬细胞极化而参与感染后炎症的消退。

This study was designed to investigate the role of ring finger protein 20(RNF20) in host innate immunity against RNA viruses.Dual luciferase reporter assay was employed to examine the effects of RNF20overexpression on Sendai virus(SeV) infection-induced activation of interferon a4(Ifna4) gene promoter in 293T human embryonic kidney epithelial cells.Rnf20 myeloid conditional knockout mouse model was constructed(Rnf20F/F;Lyz2-Cre),and the frequency of myeloid subsets in the bone marrow,spleen,blood of Rnf20F/F;Lyz2-Cre mice and littermate Rnf20F/F mice were detected by flow cytometry.After bone marrow-derived macrophages(BMDMs) were cultured and subjected to the infection of SeV and vesicular stomatitis virus(VSV),Western blot was used to detect the phosphorylation of transcription factor interferon regulatory factor 3(IRF3) and nuclear factor-κB(NF-κB).ELISA was used to detect the production of IFN-β,TNF-α and IL-6,and Bulk RNA-sequencing was employed to explore transcriptional changes.Furthermore,M1 and M2 macrophage polarization was induced by LPS and IL-4,respectively,to confirm the changes revealed by transcriptome analysis.Data showed that RNF20 overexpression showed no significant effect on SeV infection-induced activation of Ifna4 gene promoter in 293T cells.There is almost no difference in the development of myeloid subsets between Rnf20F/F;Lyz2-Cre and Rnf20F/F mice.After RNA viral infection of BMDMs from Rnf20F/F;Lyz2-Cre and Rnf20F/F mice,the phosphorylation of IRF3 and NF-κB p65 and the expression of IFN-β,TNF-α and IL-6 were comparable between the two groups,while the expression levels of several genes related to macrophage polarization were significantly different.The loss of RNF20 showed the tendency of hindering M1 macrophage polarization while promoting M2 macrophage polarization.In conclusion,RNF20 does not affect RNA viral infection-induced activation of IRF3 or NF-κB pathways,but it might get involved in the resolution of inflammation afterwards.