Demannosylated Hepatitis B Virus Escapes The Immune Recognition Of DC-SIGN On Dendritic Cells

Objective:To investigate whether HBV infection would increase the activity of Golgi M I in HepG2 cell line. The effect of inhibiting HBV demannosylation mediated by Golgi M I on recognition of DC-SIGN and maturation and activation of DC was observed, to investigate the function of DC-SIGN in HBV infection. The mechanism of demannosylated HBV escaping the immune recognition of DC-SIGN on DC to inhibit the activation of DC was approached as well.Methods:1. To investigate whether HBV infection would increase the activity of Golgi M I(1) HepG2 cells and HepG2.2.15 cells were cultured, and Golgi complexes of these cells were purified by sucrose density gradient centrifugation.(2) The activity of Golgi M I in Golgi complexes was detected by colorimetric method.(3) The mRNA expression of MAN1A1,MAN1A2 and MAN1C1 was performed by RT-PCR method.(4) The protein expression of MAN1A1,MAN1A2 and MAN1C1 was performed by Western blot method.2. To investigate the function of DC-SIGN in recognition of HBV and activation of DC (1) HepG2.2.15 cells were treated by different concentration of Golgi M I inhibitor kifunesine, and the Golgi complexes were purified after 24 hours to detect the activity of Golgi M I by colorimetric method.(2) Demannosylated HBV was obtained from the culture medium of HepG2.2.15 cells which had been treated by kifunesine(5ug/ml) for 5 days, HBV DNA was detected by fluorescence quantitative PCR.(3) The peripheral blood mononuclear cells of healthy individual were separated and differentiated to DC by the induction of GM-CSF and IL-4. The morphological characteristics were observed by light microscope and transmission electron microscope.(4) Obtained demannosylated HBV particles (5x106copies/ml) were added into the medium of DC which had been cultured for 5 days. The morphological characteristics of DC were observed by transmission electron microscope, the expression rates of CDla,CD83,CD80,CD86 and HLA-DR were assayed by flow cytometry analysis, the capacity of stimulating lymphocyte proliferation was detected by MTT assay, and the levels of IL-12 released by DC were measured by ELISA on day 7. DC treated with PBS or HBV obtained from untreated HepG2.2.15 cells were investigated as control.(5) DC cultured on day 5 was blocked by DC-SIGN antibody for 2 hours, and then treated by intervention and detected as above mentioned.3. To investigate the mechanism of demannosylated HBV escaping the immune recognition of DC-SIGN on DC(1) The eukaryotic expression vectors of HBV S, LS, MS, HBe, HBc, HBx protein were constructed by molecular cloning method, and then they were indentifed by enzyme digestion and DNA sequencing.(2) pEGFP-N1 and above-mentioned constructed plasmids were transfeced into HepG2 cells by Lipofectamine2000, respectively. Transfected efficiency was calculated by fluorescence microscopy, and the activity of Golgi M I was detected by colorimetric method. (3) pEGFP-N1-HBe and pEGFP-N1 plasmids were tranfected into HepG2 cells. The mRNA expression of MAN1A1,MAN1A2 and MAN1C1 was performed by RT-PCR method, and the protein expression of them was performed by Western blot method.Results:1. To investigate whether HBV infection would increase the activity of Golgi M IIt was found that the activity of Golgi Mâ… increased, the mRNA and protein expression of MAN1A2 and MAN1A1 increased too in HepG2.2.15 cells compared with HepG2 cells, but there was no difference in the mRNA and protein expression of MANIC1 between two groups.2. To investigate the function of DC-SIGN in recognition of HBV and activation of DC(1) The activity of GolgiM I was inhibited by kifunesine in HepG2.2.15 cells,5ug/ml was the appropriate intervented concentration.(2) There was no difference in HBV secretion between HepG2.2.15 cells treated by kifunesine and untreated HepG2.2.15 cells.(3) The nuclear of DC stimulated by TNF-a on day 7 turned bigger, and there were large amounts of sentus-like ecphyma stretching out around. Typical mature DC form was present.(4) Compared with native HBV group, the expression of CDla,CD83,CD80,CD86 and HLA-DR of DC were significantly improved, levels of IL-12 secretion were increased, and capacity of stimulating lymphocyte proliferation was enhanced in highly mannosylated HBV group.(5) When DC were treated with highly mannosylated HBV, prior block by DC SIGN specific antibody could induce the decreasion of CDla. CD83, CD80, CD86 and HLA-DR expression, reduction of IL-12 secretion, and weaken ability of stimulating lymphocyte proliferation.3. To investigate the mechanism of demannosylated HBV escaping the immune recognition of DC-SIGN on DC (1) The results of constructed plasmids by enzyme cutting and DNA sequencing were consistent with expectations.(2) The green fluorescent expression was visible in transfected cells by fluorescence microscopy.(3) It was found that the activity of Golgi M I increased in cells tranfected with pEGFP-N1-HBe plasmid than other plasmids.(4) The mRNA and protein expression of MAN1A2 and MAN1A1 was both higher in pEGFP-N1-HBe group than in pEGFP-N1 group, but there was no difference in the mRNA and protein expression of MAN1C1 between two groups.Conclusion:1. HBV infection could increase the activity of Golgi M I by up-regulating the mRNA and protein expression of MAN1A2 and MAN1A1.2. DC-SIGN could recognize highly mannosylated HBV and then promote the maturation and activation of DC. HBV may exploit demannosylation mediated by Golgi M I as a way to escape immune recognition by DC-SIGN and thereby induce the dysfunction of DC.3. HBe protein may induce the increase of Golgi M I activity by up-regulating the mRNA and protein expression of MAN1A2 and MAN1A1 during HBV infection.