The Study On The Possible Pathway Of Microparticles-mediated Hepatitis B Virus Infecting Macrophages And Capability Of HBx To Induce Expression Of MHC-Ⅰand Grp78

Background and objectiveThe fact that immune cells can be infected by HBV has been proved in recent researches, but whether and how HBV can infect macrophages is unclear. Microparticles represent a heterogeneous population of vesicles, which are veritable vectors for the intercellular exchange of biological signals and information. The purpose of this study was to investigate whether the macrophages could be infected by HBV through the micropaticles isolated from blood of HBV positive patients. Furthermore, we try to investigate the whether the expression of MHC-â… and Grp78 molecules can be regulated by HBx.Materials and methods1. Blood samples of HBV negative group were obtained from laboratory staffs that have been confirmed as HBV non-infected adults.2. The MPs were isolated from blood samples, and then mixed cultured with macrophages (U937) for 8h and 24h.3. The micropaticles and macrophages were dyed by PKH-26, CFSE.4. The micropaticles in macrophages were detected by confocal microscope.5. The HBV particles in macrophages were detected by transmission electron microscope.6. The expression of HBcAg in macrophages was detected by immunocytochemistry; the expression of HBsAg and HBeAg were detected by ELISA, and the expression of HBx protein and HBcAg were detected by immunofluorescence.7. The level of HBV DNA and cccDNA in macrophages was measured by real-time PCR; the level of HBV DNA in the supernatant was also measured by the same method.8. The MPs released from different generations of HBV-infected macrophages were mixed cultured with next generation of macrophages, and the levels of HBV DNA in these macrophages were measured by real-time PCR.9. HBx plasmid was transfected into HepG2 cells, the MHC-â… , Grp78 and MICA/B moleculars expression on surface of HepG2 were detected by FCM.10. Confocal microscope was used to observe the localization of MHC-â… and Grp78 molecules on cell surface.11. The siGrp78 and HBx plasmid, the siHLA and HBx plasmid were co-transfected into HepG2 cells respectively, the MHC-â… , and Grp78 molecular expression on surface of HepG2 were detected by FCM.12. HBx plasmid was transfected into T2 cells and H22 cells, the MHC-â… and Grp78 molecular expression on surface of T2 and H22 were detected by FCM.13. NK cells were mixed cultured with H22 cells which had been transfected by HBx plasmid, the killing effect of NK cells was detected by FCM.Results:1. The HBV particles were found in MPs from blood samples in HBV positive group by confocal microscope, and micropaticles were observed in macrophages at 8h after mixed cultivation.2. HBV particles were observed in macrophages by transmission electron microscope at 24h after mixed cultivation.3. HBcAg staining was positive in macrophages by immunocytochemistry, and the expression of HBsAg and HBeAg were deteced by immunofluorescence.4. The expression of HBsAg and HBeAg were detected as positive by ELISA not only in the macrophages but also in supernatant from HBV positive group.5. There were high levels of intracellular HBV DNA and cccDNA in macrophage from HBV positive group after mixed cultivation for 24h, and the levels of HBV DNA in supernatant is higher than the control group. 6. After mixed cultured with the MPs released from different generations of HBV-infected macrophages, the HBV DNA in different generations of macrophages were positive.7. The expression of MHC-â… and Grp78 moleculars on surface of HepG2 were up-regulated after tranfected by HBx plasmid, while MICA/B molecular expression had no significant change.8. It was found that MHC-â… and Grp78 molecules were located in the same position by confocal microscope.9. The expression of MHC-â… and Grp78 moleculars in HepG2 cells showed positive correlation after co-transfected by the siGrp78 and HBx plasmid, the siHLA and HBx plasmid.10. The killing efficiency of NK cells to the H22 cells which were transfected by HBx plasmid was decreased.Conclusion:The macrophages can be infected by HBV, and micropaticles are the vector for HBV to infect macrophages. The HBV can replicate in macrophages, and then be packaged in the new generation of MPs and released from HBV-infected macrophages. The MPs released from HBV-infected macrophages have the capacity to infect more macrophages. HBx can up-regulate the expression of MHC-â… and Grp78 moleculars on surface of HBV infected cells, two molecules may form the complex on the cell surface. And the HBV infected cells can escape immune response, because HBx can up-regulate the expression of MHC-â… and Grp78 molecules, which lead to the decreasing of cytotoxic activity of NK cells.