The Impact Of Hepatitis B Virus On Interferon-Beta Expression In Hepatocytes

Hepatitis B virus, which is strictly hepatotropic and non-cytopathic, can cause liver pathology mediated by the host immune response. Both the innate and adaptive immunity contribute greatly to the control of HBV replication and spread, while the former is crucial in virus inhibition and clearance in the early stage of infection.Through pattern-recognition receptors (PRRs), the host cells monitor the pathogen-associated molecular patterns (PAMPs) of invading pathogens, activate the consequent signal pathways, and thus stimulate the production and secretion of interferons. The antiviral activity is indirectly performed by interferon in the innate immune system, and directly fulfilled by the antiviral proteins inducd by interferons. Since the major form of interferons expressed in hepatocytes is interferon-β, its production could be viewed as a hallmark of the innate immune response against pathogens in liver cells.There remains much controversy about the role of the innate immunity in HBV infection. Some researchers label HBV as a"stealth virus", which can sneak through the innate immune system and replicate without stirring IFNs production; while other studies lay emphasis on the importance of the innate immunity in inhibiting HBV replication and spread. The innate immunity in human beings infected by HBV deserves intensive study.This study focused on the innate immune response in human hepatocytes against HBV in natural infection, which provided some evidence that would promote both the development of antiviral drugs and clinical prevention and therapy of HBV infection. The detailed contents and results of this study are listed as follows:1. Investigate whether IFN-βcan be produced in HepG2 and L-O2 cells, with poly(I:C) as the inducer. The recombinant plasmid pIFNB-luc, in which the firefly luciferase gene is driven under IFN-βpromoter, was constructed by PCR amplification of IFN-βpromoter fragment and inserted into pGL3-Basic vector upstream of the firefly luciferase gene, then verified by PCR, endonuclease cleavage, and sequencing. Dual-luciferase reporter assay system and RT-PCR were applied to detect the transcription of IFN-β. Extracellular poly(I:C) failed to induce IFN-βexpression, but when poly(I:C) was transfected into both cells by liposomes, IFN-βtranscription level was changed. We demonstrated that receptors for dsRNA lied intracellularly, and the IFN signal pathways were available in HepG2 and L-O2 cells.2. Study whether HBV elicits IFN-βexpression in hepatocytes. To imitate the natural infection of HBV in the liver, we incubated cells with HBV positive human serum. First, to decide whether serum-derived HBV attack could trigger IFN-βproduction in hepatic cell lines, we tested the expression of IFN-βin HepG2 and L-O2 cells at various time points after incubating with HBV positive human serum. No obvious change in IFN-βtranscription was detected using luciferase reporter gene and RT-PCR. However, nested-PCR proved that HepG2 and L-O2 cells initiated IFN-βexpression in the face of serum-derived HBV attack. Real-time PCR results showed that there was no significant change in IFN-βexpression if intra-cellular HBV copy number was raised by DMSO induction, indicating that the low level of IFN-βexpression was unlikely due to low copy of HBV. Then we isolated hepatocytes from both the liver tissues of healthy body and that of HBsAg positive patient by collagenase digestion and precipitation, investigated IFN-βresponse against HBV in primary human hepatocytes and liver cells of hepatitis B patient, which were more close to the response in vivo. HBV infection in the liver cells of patient was confirmed by immunohistochemistry and ELISA. The transcription of IFN-βwas detected by nested-PCR in both HBV infected primary human hepatocytes and hepatitis B patient liver cells, but not by one-circle PCR. We proved that IFN-βwas produced in natural infection of HBV, which indicated the occurrence of the innate immune response in hepatocytes.3. Investigate the counteraction of HBV on IFN-βexpression. After cells were incubated with HBV positive human serum and transfected with poly(I:C) to trigger IFN-βexpression, the change in IFN-βexpression was detected by luciferase reporter gene system, western blot and immunofluorescence. Serum-derived HBV significantly enhanced the transcription level of IFN-βinduced by poly(I:C), and transfected poly(I:C) tremendously elevated IFN-βproduction in HepG2.2.15 cells. The data provided another proof supporting that HBV could be sensed by the innate immune system, and HBV did not evade it through inhibiting IFN-βproduction; but in the opposite, HBV promoted IFN-βexpression.Summarizing all the data, we have represented strong evidences proving the existence of innate immune response in natural HBV infection. HBV could induce and enhance the expression of IFN-β, which inhibit HBV replication efficiently. The results are conducive to understanding the mechanisms of HBV innate immunity and the development of drugs to prevent and control HBV infection.