Role Of Mig In Inflammatory Cells Recruitment Induced By Hepatitis B Virus In Mice

Background & Aim:HBV infection is one of the most common health problems worldwide. There are more than 350 million chronic carriers, and a large number of people with HBV infection are at increased risk of developing cirrhosis and hepatocellular carcinoma. Previous studies have demonstrated that HBV is a non-cytopathic virus and favored the host immune response responsible for liver injury after HBV infection. The infiltration of antigen-specific and antigen-nonspecific inflammatory cells into the liver contribute to liver damage. The expression of cytokine is closely related to chronic hepatitis B and liver damage. However, which cytokine play a crucial role in inflammatory cells recruitment after HBV infection is unclear. In this study, we sought to explore how HBV induced chemokine Mig secretion, then to investigate the effects of HBV-induced Mig on inflammatory cells infiltration and liver injury in mice.Methods:Ad-1.3×HBV plasmid was constructed and identified. The three cell lines (Human fetal liver cell line L02, hepatocellular carcinoma cell lines HepG2 and SMMC-7721) were infected with Ad-HBV HBV DNA, HBsAg and HBeAg were assessed by real-Time PCR and electrochemiluminescence immunoassay (ELISA). The expression of HBx was detected by RT-PCR and western blotting.90 BALB/c mice were divided into three groups. In Ad-HBV group, Ad-HBV was injected into the tail veins of the mice. Other groups were injected with adenovirus empty vector or saline. The secretion of HBsAg, HBeAg and Mig were detected by ECLIA and ELISA. Dection of Mig RNA expression in mouse liver was performed by in situ hybridization. HBV DNA and cccDNA were measured by real-Time PCR and serum ALT activity was determined by ELISA. The change of p50 and p65 subunits of NF-κB was assessed by western blotting and immunofluorescene staining. The mice treated with NF-κB MG-132 inhibitor and HBV-induced Mig expression was analyzed in the Ad-HBV group. At the same time, the effect of Ad-HBV on PI3K and MAPK signaling pathway was detected by western blotting. In addition, H&E staining was used to analyze liver inflammation and flow cytometry was used to analyze the percentage of inflammatory cells. The siRNA expression vector targeting the mRNA of Mig and a control vector were constructed. Mice were subjected to tail vein hydrodynamic injection of the Mig-shRNA/in vivo-jetPEI-Gal system in Ad-HBV group. The change of these indicators was detected by the above methods.Results:Restriction endonuclease analyses showed that Ad-1.3×HBV was constructed successfully. After enrichment, the recombinant adenoviral stock was characterized by the End-Point Dilution Assay, and viral titer was 6.138×1012VP/ml or 2.5×1011 pfu/ml. HBV DNA, HBsAg, HBeAg were detected in the cell culture medium and the expression of HBx was detected in the infected cells. Human fetal liver cells and hepatocellular carcinoma cells was successfully transfected with Ad-HBV. Then we established a BALB/c mouse model by tail vein injection of Ad-HBV. The secreted HBsAg, HBeAg (p<0.05), HBV DNA and HBV cccDNA was detected and the activity of ALT was significantly elevated (p<0.01) in different phases of Ad-HBV infection. Interestingly, the phase of HBsAg, HBeAg clearance coincided with the development of anti-viral antigen antibodies. These antibodies were first detected on day 14 and gradually increased. In addition, serum level of chemokine Mig was elevated rapidly and Mig RNA expression was detected in mouse liver after Ad-HBV infection (p<0.05). We also demonstrated that mouse endothelial cells and hepatocytes were the main cell sources that secreted Mig. Futhermore, NF-κB signaling pathway was activated and the translocation of p50, p65 from cytosol to nucleus in Ad-HBV group liver. The Ad-HBV mice were treated with NF-κB inhibitor, MG-132, which inhibited secretion of HBV-induced Mig (p<0.01). At the same time, we found that the upstream signaling pathway of NF-κB, such as PI3K/Akt, JNK and ERK1/2 were activated by western blottting. The level of phosphorylated Akt, JNK and ERK1/2 were elevated as control. However, Ad-HBV did not result in a notable increase in the level of phosphorylated p38. Finally, H&E staining of mouse liver showed that Ad-HBV was associated with the formation of liver inflammation foci that mainly portal inflammation. The flow cytometry indicated that the number of lymphocytes significantly increased in the liver, which corresponded with an increase in the percentage:CD4+, CD8+, NK and NKT cells. After tail vein injection of Mig-shRNA/in vivo-jetPEI-Gal complex, the expression of Mig could be partially inhibited in the Ad-HBV group (p<0.01). At the same time, we found that the migration of lymphocytes were inhibited (p<0.05), the severity of liver inflammation and the activity of ALT (p<0.01) were decreased after Mig-shRNA treatment. However, there was no notable difference in HBsAg, HBeAg, HBV DNA and cccDNA level in the Ad-HBV group before and after injection with Mig-shRNA.Conclusion:Adenovirus infection is a useful experimental method for HBV genome carried into mouse hepatocytes. Through activation of NF-κB signaling pathway, HBV induced chemokine Mig expression, recruited inflammatory cells into the liver and lead to liver injury. These results provide a better understanding of pathogenesis and develop new experimental basis for the immune treatment after HBV infection.