Golgi α-mannosidase Ⅰ Induce Hepatitis B Virus Demannosylation To Escape The Immunological Recognition Of DC-SIGN On Dendritic Cells

Objective:To investigate whether Golgi a-mannosidase I (Golgi M I) induced hepatitis B virus demannosylation could help the virus to escape immunological recognition of DC-SIGN on dendritic cells (DCs) and its mechanism. Silencing the DC-SIGN gene expression, observe the immunologic function of DCs that stimulated by high-mannose type HBV or wild type HBV, and research the influence, of HBV of the two type, to the maturation and immune activation of DCs, as well as study the function of DC-SIGN in the process of HBV infection.Base on our laboratory Previous studies, finding the possible core segment of HBe function protein gene squence which could promote the Golgi M I activity of host cells, to preliminary discussion the mechanism, of demannosylation related to Golgi M I, of HBV escape the immunological recognition of DC-SIGN, and the impedence of DCs immunoactivation.Method:1. The gene slicnce of DC-SING on DCs and its impace on DCs immunological function.(1) Cultured Peripheral blood mononuclear cells(PBMC) isolated from heparinized blood, inducted PBMC differentiation to DCs with GM-CSF, IL-4and TNF-alpha;(2) Designed and synthesized siRNAs for DC-SIGN and transferred them into DCs respectively;(3) Tested the expression of DC-SIGNmRNA in the cell by RT-PCR method, and detected the surface protein of DC-SIGN expression with flow cytometry, then Selected the siRNA sequences with stable gene silencing effect to carry out follow-up experiment;(4) In the groups of blank control, DC-SIGN gene slience and negative control siRNA geng slience, detected the surface protein of CD80、CD83、CD86. CDla. HLA-DR expression with flow cytometry;(5) In the3groups mentioned in (4), tested IL-10and IL-12p70secretion level of DCs supernant via ELIS A;(6) In the3groups mentioned in (4), tested DCs stimulate allogeneic lymphocyte proliferation by MTT method;(7) In the3groups mentioned in (4), detected DCs protein NF-icBp65and p38expression by westernblot.2. The influnence of high-mannose type or wild type HBV to DCs with DC-SIGN not gene slience or gene slience(1) Culture HepG2.2.15cells in vitro(2) Added kifunensine mother liquid to HepG2.2.15cell supernant with the final concentration of5ug/ml, and treated the cells for days;(3) Obtained the high mannose type HBV particles form kifunensine treated HepG2.2.15cell and obtained the wild type HBV form ordinary HepG2.2.15cells with Ultracentrifugation in low temperature, and then test the HBVDNA titer with fluorogenic quantitative PCR(4) Divided DCs that differentiation from PBMC in vitro to2groups, DC-SIGN gene slience group and or DC-SIGN non-slience group, each group contained of3subgroups: control, add wild type HBV, and add high mannose type HBV. On the5th day of DC culture, add HBV particles, with titer of5×106copies/ml, to the corresponding subgroups. continue culturing the DCs to7th day, harvested and tested DCs in the6subgroups severally as follow;(5) In the aforementioned6subgroups, detected the surface protein of CD80、CD83、 CD86、CDla、HLA-DR expression with flow cytometry; (6) In the aforementioned6subgroups, tested IL-10and IL-12p70secretion level of DCs supernant via ELISA;(7) In the aforementioned6subgroups, tested DCs stimulate allogeneic lymphocyte proliferation by MTT method;(8) In the aforementioned6subgroups, detected DCs protein NF-κBp65and p38expression by westernblot.3. To find the possible core segment in HBe gene segment which could promote the Golgi M I activity(1) Analyzed structure domain of HBe protein which express from eukaryote expression vector(plasmid) of HBe functional protein, The plasmid was constructed and identified by our laboratory and have been preserving to nowadays.(2) Analysis the enzyme loci of the plasmid, and divided the HBe function protein gene sequence into3subsequence. connect enzyme loci Xho Ⅰ and BamH Ⅰ to upstream and downstream of each of the3sequence. Designed primers of the sequence and amplified them by PCR. Connect each sequence to carrier pEGP-N1separately to constructed3eukaryotic expression vectors that could express HBe subfragment,. Named them on HBe-1、HBe-2、HBe-3and identified the3new plasmids sequences.(3) The blank HepG2cell group, the HBe plasmid and the3new plasmid that could express HBe subfragment, were separately transfected in hepG2cells of4groups, with the help of Lipofectamine2000, obsevered the expression of green fluorescent protein(GFP)and estimated their transfection efficiency.(4) For MAN1A1and MAN1A2are two genotypes of Golgi M I, tested the expression of MAN1A1mRNA and MANlA2mRNA in the HepG2cells of the5groups as narrated above by RT-PCR method,(5) Detected the protein expression of MAN1A1and MAN1A2in the HepG2cells of the5groups as narrated above with flow cytometry. Result:1. The gene slicnce of DC-SING on DCs and its impace on DCs immunological function.(1) Successfully constructed the siRNA sequence that could silence DC-SIGN expression of DCs(2) Silence the DC-SIGN expression had no obvious effect to DCs surface protein of CD80、CD83、CD86、CDla、HLA-DR expression, or to IL-10and IL-12p70secretion level to DCs supernant; or to DCs stimulate allogeneic lymphocyte proliferation; or to protein NF-icBp65and p38expression of DCs.2. The influnence of high-mannose type or wild type HBV to DCs with DC-SIGN not gene slience or gene slience(1) In the3subgroups of DC-SIGN was not silenced, compared with high mannose HBV subgroup, the intervention of wild type HBV, promoted DCs, express more surface protein of CD80、CD83、CD86、CDla、HLA-DR, and created more IL-12p70but less IL-10to supernant; and have stronger ability to stimulate allogeneic lymphocyte proliferation; and express more NF-κBp65and p38protein in cells.(2) In the3subgroups of DC-SIGN gene slience, the intervention to DCs, compared between high mannose HBV subgroup and wild type HBV, have no statistical difference immune activation that judged by the observed index above-mentioned.(3) No matter whether DC-SIGN had been silenced, both wild type HBV and high mannose type HBV could statistical promote DCs immune activation that judged by the observed index above-mentioned.3. To find the possible core segment in HBe gene segment which could promote the Golgi M I activity(1) Successfully constructed constructed3eukaryotic expression vectors that contain partial HBe gene that could express HBe subfragment, and successfully transfected the plasmids to 5group HepG2cells;(2) HepG2cells in the groups that had transfected plasmid, compare with blank control group,express more MANlAl mRNA in cells each group, and express more more MAN1A2mRNA except HBe-1group;(3) HepG2cells in the groups that had transfected plasmid, compare with blank control group,express more MAN1Aland MAN1A2protein in cells each group;(4) HepG2cells mean expression level of mRNA and protein of MAN1A1and MAN1A2, in HBe-2group, was the most approach to that of HBe-full group; especially, the expression level of MAN1A2protein in HBe-2group, had no statistical difference with that of HBe-full group.(5) HepG2cells mean expression level of mRNA and protein of MAN1A1and MAN1A2, in HBe-1group and HBe-3group, were both statistical lower than that of HBe-full group.Conclusion:1. Silence DC-SIGN on DCs with siRNA have no obvious influence to DCs immunologic function;2. DC-SIGN promote DCs maturation and immune activation after recognizing HBV; compare to high mannose type HBV, the wild type HBV could escape the recognition of DC-SIGN to a certain extent, whose mechanism is related to HBV demannosylated mediated with Golgi M I. The escape mechanism may lead to functional defect of DCs and chronicity of HBV infection.3. The possible core segment of HBV which could promote the Golgi M I activity of host cells, may be in the gene segment of HBe-2plasmid, however, the core segment may need synergia from other segment to play its functional roles.