乙型肝炎病毒抑制M1巨噬细胞TLR4、NLRP3及下游因子促进免疫逃逸

Hepatitis B virus inhibits TLR4,NLRP3 and downstream factors of M1 macrophages to promote immune escape

目的:探讨乙型肝炎病毒(HBV)抑制M1巨噬细胞促进免疫逃逸的机制,为抗病毒治疗提供靶点和策略。方法:人单核细胞系THP-1以PMA+LPS+IFN-γ诱导为M1巨噬细胞。通过细胞形态,流式细胞术及RT-qPCR检测CD68、CD86、HLA-DR及M1巨噬细胞功能性分子IL-1β、IL-6、TNF-α表达,以鉴定M1巨噬细胞。HBV稳定复制细胞系HepG2.2.15与M1巨噬细胞共培养,qPCR检测HBV-DNA的表达;流式细胞术检测CD68、CD86与HLA-DR表达;RT-qPCR与Western blot测定M1巨噬细胞功能相关分子TLR4、NLRP3、Caspase-1、pro-caspase-1、caspase-1 p20、IL-1β、IL-18的表达;流式细胞术检测细胞凋亡率,Western blot测定凋亡相关蛋白cleaved-caspase-3的表达。结果:成功诱导THP-1分化为M1巨噬细胞;M1巨噬细胞抑制HBV复制(P<0.05);HBV抑制M1巨噬细胞的CD68、CD86与HLA-DR的表达(P<0.01);HBV抑制M1巨噬细胞的TLR4、NLRP3,Cas-pase-1,caspase-1 p20、IL-1β、IL-18的表达(P<0.01);HBV诱导M1巨噬细胞凋亡(P<0.05)。结论:HBV通过抑制M1巨噬细胞及其功能分子TLR4、NLRP3及下游因子,减少炎症因子合成及分泌,诱导凋亡,进而促进免疫逃逸,造成HBV在体内持续存在并复制。

Objective:To explore the mechanism of hepatitis B virus(HBV)inhibiting M1 macrophages to promote immune escape,and to provide targets and strategies for antiviral therapy.Methods:The human monocyte cell line THP-1 was induced into M1 macrophages with PMA+LPS+IFN-γ.Cell morphological changes and the expressions of CD68,CD86,HLA-DR and functional molecules IL-1β,IL-6,TNF-αin M1 macrophages were detected by flow cytometry and RT-qPCR to identify M1 macrophages.HBV stable replication cell line HepG2.2.15 were co-cultured with M1 macrophages,and the expression of HBV-DNA was detected by qPCR.The expression of CD68,CD86 and HLA-DR were detected by flow cytometry.The expressions of functional molecules TLR4,NLRP3,Caspase-1,pro-caspase-1,caspase-1 p20,IL-1βand IL-18 in M1 macrophages were determined by RT-qPCR and Western blot.Apoptosis rate was detected by flow cytometry,and the expression of apoptosis related protein cleaved-caspase-3 was determined by Western blot.Results:THP-1 was successfully induced to differentiate into M1 macrophages.M1 macrophages inhibited HBV replication(P<0.05).HBV inhibited the expressions of CD68,CD86 and HLA-DR in M1 macrophages(P<0.01).HBV inhibited the expressions of TLR4,NLRP3,Caspase-1,caspase-1 p20,IL-1βand IL-18 in M1 macrophages(P<0.01).HBV induced M1 macrophage apoptosis(P<0.05).Conclusion:HBV inhibits M1 macrophages and their functional molecules TLR4,NLRP3 and downstream factors,reduces the synthesis and secretion of inflammatory factors,induces apoptosis,and then promotes immune escape,resulting in the persistence and replication of HBV in the body.