HBsAg对干扰素基因刺激蛋白信号通路诱导外周血浆样树突状细胞生成干扰素α的影响

Effect of HBsAg on the production of interferon-αin peripheral blood plasmacytoid dendritic cells induced by the stimulator of interferon genes signaling pathway

目的探究HBsAg对外周血中浆样树突状细胞(pDC)环鸟甘酸-腺苷酸(cGAMP)激活IFN基因刺激蛋白(STING)通路表达IFNα的影响。方法收集2016年2月—12月于徐州医学院附属医院感染性疾病科门诊及住院的慢性HBV感染者及体检的健康成年人外周静脉全血,分离提取外周血单个核细胞(PBMC)。分别在PBMC中加STING激动剂cGAMP培养后,ELISA法检测培养上清IFNα、IFNβ和TNFα水平。健康成人PBMC和HBsAg预先孵育后加入cGAMP刺激,收集上清检测IFNα。磁珠分选法从PBMC中去除pDC,加cGAMP培养后ELISA检测上清IFNα水平。健康成人PBMC中加入HBsAg和/或cGAMP刺激后采用流式细胞术计数检测pDC频数。计量资料2组间比较采用独立样本t检验。结果cGAMP体外刺激慢性HBV感染者的PBMC产生的IFNα水平显著低于健康对照,差异有统计学意义(469.72±18.95 vs 599.90±84.06,t=4.868,P=0.001)。健康人PBMC与HBsAg共培养后以cGAMP刺激产生IFNα明显低于未加HBsAg组(448.5±52.0 vs 571.0±30.8,t=4.500,P=0.011)。与未去除pDC的PBMC相比,cGAMP刺激去除pDC的PBMC分泌IFNα水平明显降低(164.50±40.73 vs 339.50±35.33,t=6.482,P=0.001)。与cGAMP刺激后的健康成人PBMC相比,pDC在HBsAg预先孵育后再以cGAMP刺激的PBMC中的频数明显降低(0.12%±0.04%vs0.24%±0.04%,t=5.176,P=0.014)。结论HBsAg对cGAMP激活pDC中STING通路表达IFNα有抑制作用。

Objective To investigate the effect of HBsAg on the expression of interferon-α(IFN-α)in peripheral blood plasmacytoid dendritic cells(pDCs)induced by the stimulator of interferon genes(STING)signaling pathway activated by cyclic GMP-AMP(cGAMP).Method Peripheral venous blood was collected from healthy adults and the patients with chronic hepatitis B virus(HBV)infection who attended the outpatient service or were hospitalized in Department of Infectious Diseases,The Affiliated Hospital of Xuzhou Medical University,from February to December 2016,and peripheral blood mononuclear cells(PBMCs)were isolated and extracted.After the STING agonist cGAMP was added to PBMCs,ELISA was used to measure the levels of IFN-α,interferon-β,and tumor necrosis factor-αin supernatant.PBMCs from healthy adults were pre-incubated with HBsAg and then stimulated by cGAMP,and supernatant was collected to measure IFN-α.The magnetic-activated cell sorting method was used to remove pDCs from PBMCs,and after culture with cGAMP,ELISA was used to measure the level of IFN-αin supernatant.PBMCs from healthy adults were stimulated by HBsAg and/or cGAMP,and then flow cytometry was used to measure the frequency of pDCs.The independent samples t-test was used for comparison of continuous data between two groups.Results PBMCs from the patients with chronic HBV infection stimulated by cGAMP in vitro had a significantly lower level of IFN-αthan healthy controls(469.72±18.95 vs 599.90±84.06,t=4.868,P=0.001).PBMCs from healthy adults co-cultured with HBsAg and stimulated by cGAMP had a significantly lower level of IFN-αthan those in the non-HBsAg group(448.5±52.0 vs 571.0±30.8,t=4.500,P=0.011).Compared with PBMCs containing pDCs,PBMCs without pDCs stimulated by cGAMP had a significant reduction in the level of IFN-α(164.50±40.73 vs 339.50±35.33,t=6.482,P=0.001).Compared with PBMCs from healthy adults stimulated by cGAMP,PBMCs pre-incubated with HBsAg and then stimulated by cGAMP had a significant reduction in the frequency of pDCs(0.12%±0.04%vs 0.24%±0.04%,t=5.176,P=0.014).Conclusion HBsAg can inhibit the expression of IFN-αinduced by the STING pathway in pDCs activated by cGAMP.