The Regulation Of HBcAg And TBK1 On Production Of IFN-β Induced By DsRNA In Hepatocytes

Hepatitis B viruses frequently establish persistent infection and cause chronic hepatitis and its serious complications,including cirrhosis and hepatocellular carcinoma. The mechanism involved in the pathogenesis of the chronic hepatitis is not elucidated and one of it is down-regulation of the immune response by viral protein encoded by HBV.With the recently advances in the research of the innate immune,the innate immunity initiated by HBV infection and the down-regulation of HBV protein received increasing attention.The innate immunity agonist may be applicated in treatment of chronic HBV infection.The innate immune system plays an important role in viral infection.Pattern recognition receptor(PRR) molecule that expressed on immune cells senses invading viral pathogens by recognition of pathogen-associated molecular pattern(PAMP) and initiates a series of signaling pathways leading to the induction of protective cellular genes,including typeⅠinterferon(IFN-α/β) and proinflammatory cytokines,then plays the antiviral innate immunity effect and also help to shape subsequent adaptive immune responses.Toll-like receptors(TLRs),as the representative of the PRR,such as TLR3,TLR7,TLR8 and TLR9 have been shown to detect infection by viruses.TLR3 recognize viral double-stranded RNA analogue polyinosinic-poly cytosine nucleotide (poly I:C).After binding with the ligand specifically,TLRs signal through two pathways, MyD88-dependent and TRIF-dependent pathway activate NF-κB and IRF-3,and induce the expression of antiviral immune effectors.In addition,the cytoplasm of the PRR, such as RIG-I and MDA5 were also able to identify the viral PAMP,such as dsRNA, and then induce the expression of IFN-β.The innate immune response signaling pathways mediated by TLRs and RLRs both activate the TANK binding kinase 1 (TBK1),which plays an important role in the innate immunity response.After activation of TBK1,IRF3 is activated and transferred to the nucleus,thereby enhancing the expression of typeⅠinterferon to regulate the innate immune response,and influence the adaptive immune.TBK1 may acts as potential agent in therapeutic application in chronic viral infection such as HBV and HCV.Objective:To investigate the regulation of HBcAg and TBK1 on innate immunity of hepatocytes,the expression of IFN-βinduced by dsRNA in hepatoma cell line HepG2 and HBV replication cell line HepG2.2.15 was tested.To investigate the regulation of HBcAg,HepG2 cells were transfected with eukaryotic plasmid expressing HBcAg and treated with HBcAg recombinant protein.To investigate the regulation of TBK1,we transfect eukaryotic plasmid expressing TBK1 into HepG2 and HepG2.2.15 cells.The results would be helpful to clarify the antiviral immunity in hepatocytes,and unraveling the mechanism of chronic HBV infection,and might provide new ideas and strategies for the development of treatments for chronic hepatitis.Methods:1.The regulation of HBcAg in production of IFN-βinduced by dsRNA in hepatocytes.1.1 Construction of pcDNA3-HBc plasmid:the plasmid pCP10 containing the HBV ayw subtype full-length as template,the gene encoding HBcAg was obtained by PCR and was inserted into plasmid pcDNA3.1.2 The expression of IFN-βinduced by poly I:C in HepG2 cells:HepG2 cells were stimulated by poly I:C at 0μg/ml,25μg/ml,50μg/ml,100μg/ml for 12 hours,24 hours, then the expression of IFN-βmRNA was assayed by real time PCR.1.3 The expression of IFN-βinduced by poly I:C in HepG2 cells transfected with pcDNA3-HBc:HepG2 cells were transfected with 1.2μg pcDNA3-HBc for 24 hours, poly I:C was added to the medium at 100μg/ml for 24 hours,then the expression of IFN-βwas assayed by real time PCR and ELISA.1.4 The regulation of HBcAg recombinant protein in production of IFN-βinduced by poly I:C in hepatocytes:HepG2 cells were treated with HBcAg recombinant protein at 0μg/ml,10μg/ml for 48 hours and poly I:C was added at 100μg/ml for 24 hours,then the expression of IFN-βwas assayed by real time PCR and ELISA.2.The regulation of TBK1 in production of IFN-βinduced by dsRNA in hepatocytes.2.1 Construction of pcDNA3-hTBK1 plasmid:The hTBK1 DNA as template,the gene encoding the human TBK1 was amplified by PCR and was inserted into plasmid pcDNA3.2.2 The expression of IFN-βinduced by poly I:C in Hela cells transfected with pcDNA3-hTBK1:Hela cells were transfected with pcDNA3-hTBK at 10μg,0.6μg, 1.2μg,1.8μg for 24 hours,poly I:C was added at 100μg/ml for 24 hours,then the expression of IFN-βwas assayed by real time PCR and ELISA.2.3 The expression of IFN-βinduced by poly I:C in HepG2 cells transfected with pcDNA3-hTBK1:HepG2 cells were transfected with different concentrations of pcDNA3-hTBK1 for 24 hours,poly I:C was added at 100μg/ml for 24 hours,then the expression of IFN-βwas assayed by real time PCR and ELISA.2.4 The expression of IFN-βinduced by poly I:C in HepG2.2.15 cells:HepG2.2.15 cells were stimulated by poly I:C at 0μg/ml,25μg/ml,50μg,/ml,100μg/ml for 24 hours,and the expression of IFN-βwas assayed by real time PCR and ELISA,the production of HBsAg,HBeAg and HBV DNA was detected by time-resolved fluoroimmunoassay (TRFIA) and real time PCR respectively.2.5 The regulation of antiviral effect induced by poly I:C in HepG2.2.15 cells transfected with pcDNA3-hTBK1:HepG2.2.15 cells were transfected with 1.8μg pcDNA3-hTBK1 for 24 hours,and was stimulated by poly I:C at 0μg/ml,25μg/ml, 50μg/ml,100μg/ml for 24 hours,the expression of IFN-βwas assayed by real time PCR and ELISA,the production of HBsAg,HBeAg and HBV DNA was detected by TRFIA and real time PCR respectively.Result:1.The regulation of HBcAg in production of IFN-βinduced by dsRNA in hepatocytes.1.1 Plasmid expressing HBcAg pcDNA3-HBc was identified by Agarose gel electrophoresis of the plasmid after enzyme EcoRⅠand BamHⅠdigested,and sequencing.1.2 Compared with the control,the expression of IFN-βinduced by poly I:C in HepG2 cells was significantly increased by poly I:C stimulation for 12 hours.With the increased concentration,the expression increased and there are significantly difference among different groups(P＜0.05).The expression of IFN-βinducted by poly I:C for 24 hours was significantly higher than 12 hours(P＜0.05).1.3 Compared with the control,there is no significantly difference in the expression of IFN-βinduced by poly I:C in HepG2 cells transfected with 1.2μg pcDNA3-HBc(P＞0.05),while the concentration of IFN-βwere significantly decreased(P＜0.05).1.4 Compared with the control,the expression and concentration of IFN-βtreated with HBcAg recombinant protein were significantly decreased(P＜0.05).2.The regulation of TBK1 in production of IFN-βinduced by dsRNA in hepatocytes.2.1 Plasmid expressing HBcAg pcDNA3-hTBK1was identified by Agarose gel electrophoresis of the plasmid after enzyme XbaⅠand BamHⅠdigested,and sequencing.2.2 Compared with the control and other groups,the expression of IFN-βinduced by poly I:C in Hela cells transfected with 1.8μg pcDNA3-hTBK1 increased significantly. Compared with the control and cells transfected with 0.6μg pcDNA3-hTBK1,the expression also increased significantly in cells transfected with 1.2μg pcDNA3-hTBK1 (P＜0.05).There is no significantly difference in the concentration of IFN-βin the different group(P＞0.05).2.3 The expression of IFN-βinduced by poly I:C in HepG2 cells transfected with pcDNA3-hTBK1 was significantly increased with the increased transfection except the control and low concentration(0.6μg).Compared with the control,there is no significantly difference among different groups in the concentration of IFN-βin supernatant(P＞0.05).2.4 The expression of IFN-βinduced by poly I:C in HepG2.2.15 cells,compared with the control,there was no significantly difference among different groups(P＞0.05) while the concentration of IFN-βin supernatant was significantly increased among different groups(P＜0.05).Compared with the control,there is no significantly difference among different groups in the production of HBsAg and HBeAg,and HBV DNA in supertanant and cells(P＞0.05).2.5 Compared with the control and 25μg/ml stimulation group,the expression of IFN-βincreased significantly by poly I:C stimulated as 100μg/ml(P＜0.05);compared with the control,there are no significantly difference among different groups in the concentration of IFN-β(P＞0.05).Compared with the control,the production of HBsAg significantly decreased in supernatant of cells stimulated by polyI:C at 50μg/ml and 100μg/ml(P＜0.05) while there are no significantly difference among different groups in the expression of HBeAg(P＞0.05).Compared with the control,HBV DNA in supernatant decreased significantly with poly I:C stimulation(P＜0.05),while there are no significantly difference among different groups(P＞0.05);and there is no significantly difference of HBV DNA in cells among different groups(P＞0.05).Conclusion:Poly I:C induces the expression of IFN-βin HepG2,HepG2.2.15 cells,suggesting that hepatocytes may play an important role in antiviral innate immune response. HepG2 cells transfected with eukaryotic plasmid expressing HBcAg,and directly treated with HBcAg recombinant protein,the expression of IFN-βinduced by poly I:C is down-regulated,suggesting that HBcAg is an antagonist antiviral innate immune response.HepG2 and HepG2.2.15 cells transfected with eukaryotic plasmid expressing hTBK1,the expression of IFN-βinduced by poly I:C is increased in HepG2 and HepG2.2.15 cells,at the same time,a certain concentration of hTBK1 can effectively inhibit the expression of virus protein HBsAg and the viral loads of HBV DNA in HepG2.2.15 cells,suggesting that TBK1 may inhibit the HBV replication by increasing the expression cytokines such as typeⅠinterferon(IFN-β) and other effectors.The results show that hepatocytes play a role in innate immune response during viral infection,while HBV can anti-host innate immune response by expressing viral protein HBcAg.Innate immunity effector signaling molecule such as TBK1 may enhance innate immune response.The result is helpful in unraveling the role of hepatocytes in innate immune response against HBV infection and the pathogenesis of persistent HBV infection,also might lead to design of novel therapeutic interventions in future.