Production Of IFN-β Induced By Viral PAMP In Hepatocytes

The innate immune system represents the first line of defense against viral infections in mammalian cells. Pattern recognition receptor (PRR) molecule that expressed on immune cells senses invading viral pathogens by recognition of conserved molecular structures known as pathogen-associated molecular patterns (PAMPs) and initiates signaling pathways leading to the induction of protective cellular genes, including type I interferons (IFN-α and IFN-β) and proinflammatory cytokines such as TNF-α that directly limit viral replication and also help to shape subsequent adaptive immune responses. The Toll-like receptors (TLRs) are a class of PRRs that have been shown to detect infection by many types of pathogens, including viruses. TLR3 is engaged specifically by double-stranded RNA that is present either in viral genomes or generated during viral replication. TLR7 and TLR8 detect GU-rich viral single-stranded RNA.TLR9 recognizes DNA containing unmethylated CpG motifs. The TLRs are not the only class of PAMP receptors thatcontribute to the recognition of virus infection. A cytoplasmic RNA helicase such as RIG-I and MDA5,also recognize dsRNA,and induce the expression of type I interferons (IFN-α and IFN-β) and proinflammatory cytokines.Hepatitis B and C viruses frequently establish persistent infection leading to chronic hepatitis, cirrhosis, and liver cancer. Type I IFN responses are important in the pathogenesis of chronic viral hepatitis. Both hepatitis B virus (HBV) and hepatitis C virus (HCV) cause persistent infections involving the hepatocytes, and both have evolved mechanisms to disrupt the induction of type I IFNs. The core protein of HBV inhibits the transcription of IFN-β. Similarly, the NS3/4A protease of HCV blocks Sendai virus (SenV)-induced activation of IFN regulatory factor 3 (IRF-3), a cellular transcription factor that plays a critical role in the expression of IFN-β. However, little is known of the mechanism how the liver produces IFN infected by HBV and HCV. Objective:To investigate the signaling pathways of hepatocytes defending against viral infections,and to better define the antiviral signaling pathways in hepatocytes lines,we investigated the expression of the innate immune receptor in various hepatocytes lines such as L02, SMMC7721 and HepG2 cells.by RT-PCR and the induction of IFN-β by different PAMPs such as polyI:C,polyU and CpG-ODN by the method of real timePCR.The result would be helpful in unraveling the pathogenesis of persistent HBV and HCV infections,and might possibly lead to design of novel therapeutic interventions in future..Methods:1. To investigate the expression of the innate immune pattern recognition receptor and the signal transduction protein in L02, SMMC7721 and HepG2 cell lines,specific primers of TLR3, TLR7, TLR8, TLR9, RIG-Ⅰ, MDA5, MyD88 and TRIF were synthesized and the expression of corresponding mRNA were detected by RT-PCR2. To investigate the innate immunorecognition of viral PAMPs analogs such as polyI:C,polyU ans CpG-ODN by hepatocyte cell lines. Cells were stimulated by ployI:C added directly to the medium at 0μg/ml, 25μg/ml, 50μg/ml,or at 0μg/ml,2.5μg/ml,5μg/ml complexed with lipofectamine for transfection,or ployU transfected with lipofectamine at 0μg/ml,1μg/ml,2μg/ml,or CpG-ODN was added directly to the medium at 0μg/ml,10μg/ml,20μg/ml and the expression of IFN-β by real time PCR.Cells were assayed after treated by 6h, 6h, 24h and 36h respectively.Result:1.TLR3, TLR7, TLR9, MDA5 and MyD88 and TRIF are expressed on L02 cell,TLR3, TLR7, TLR9, MDA5 and MyD88 are expressed on on SMMC7721 cell and HepG2 cell.2. The induction of IFN-β stimulated by PAMP in hepatocytes:(1)Compare with the control group (P>0.05),the induction of IFN-β in L02 cell, SMMC7721 cell, HepG2 cell was significantly increased by polyI:C stimulation (P<0.05),except the HepG2 cell stimulated by polyI:C as higher concentration (50 μg/ml). There is no significantly difference between the cells treated by polyI:C of 25 μg/ml and 50 μg/ml (P>0.05).(2) The expression of IFN-β in L02 cell was significantly increased by transfection of polyI:C,compare with the control group (P<0.05), there is no significantly difference between the different concentration (P>0.05).There is no significantly difference in SMMC7721 cell and HepG2 cell (P>0.05) after transfection of polyI:C.(3) The expression of IFN-β in L02 cell was no significantly increased by polyU transfection, compare with the control group (P>0.05).It is significantly increased in SMMC7721 cell(P<0.05),and it correlated with concentration(P<0.05). There is no significantly difference in HepG2 cell by polyU transfection (P>0.05).(4)The expression of IFN-β in L02 cell was significantly increased by CpG-ODN as higher concentration(20μg/ml), compare with the control group (P<0.05). There was no significantly difference in SMMC7721 cell and HepG2 cell (P>0.05).Conclusion:Innate immune receptor such as TLR3, TLR7, TLR9, MDA5 and signaling related protein TRIF, MyD88 can be detected in mRNA by RT-PCR. Induction of IFN-β in hepatocytes lines stimunlated by Viral PAMPs analogs polyI:C, polyU and CpG-ODN indicated that hepatocytes play a important role in innate antiviral response. To explore the role of hepatocytes in innate immune response against HBV/HCV infection and their interaction would be helpful in unraveling the pathogenesis of persistent HBV/HCV infection,and might lead to design of novel therapeutic interventions in future.