MBL Inhibits HCMV Capture And Transmission By Human MD-DC

Background:Human cytomegalovirus (HCMV) is a member of the herpes-viridae family with a lipid envelope,229kb double-stranded DNA, spreading through close or intimate contact. HCMV infection is common in utero, infants, organ transplantation, bone marrow transplantation, immune suppression or defects and AIDS patients, in the immunocompetent host, lethal infections are rarely reported. Because HCMV-borne virokines help HCMV escape host immune, it can cause productive, latent, cells transformation and abortive infections. Anti-cytomegalovirus therapy works after HCMV invading into the target cells, and it can not completely get rid of the virus. At present, we lack the anti-cytomegalovirus medicine which works before the virus getting into the target cells, there is, it blocks the virus adhesion, combine or fuse to the target cells membrane. In this study, natural MBL (hMBL) was purified, recombinant human wild-type MBL (rhMBL) was prepared and purified, DC were human peripheral blood monocyte-derived DC(MD-DC); the research was a study on hMBL/ rhMBL inhibiting MD-DC capturing HCMV and preventing transmission to T cells, and exploring its mechanism.Materials and methods1.The establishment of the Chinese hamster ovary (CHO) cell line secreting rhMBL To build PIRES2-AcGFP expression of rhMBL according to the method reported by Ohtani and improved. The above vector was transfected into CHO cell line, according to the instructions. Stable resistant cells were selected with G418, analyzed by RT-PCR and measured the MBL protein by ELISA.2. The preparation, purification, identification and quantification of hMBL/rhMBL the CHO/MBL cell line was cultivated in MDEM medium supplemented with 10% fetal bovine serum,37℃,5% CO2 and saturated humidity conditions. MBL was purified with mannan-sepharose 4B affinity chromatography from supernatant and human fresh plasma. Purity MBL was identified with SDS-PAGE, protein quantification was measured with Coomassie blue method.3. MD-DC were obtained from the blood of healthy seronegative donors by separation over a Ficoll-Hypaque gradient as previously reported. Briefly, peripheral blood mononuclear cells (PBMC) were seeded in culture flask at 1.8×10 cells/ml in 10 ml of RPMI 1640 medium containing 10% heat-inactivated endotoxin and mycoplasma-free fetal bovine serum,4mM 1-glutamine,50U/ml penicillin and 50μg/ml streptomycin. Two hours after plating and culturing the PBMC at 37℃in a humidified atmosphere enriched with 5% CO2, non-adherent cells were carefully removed by repeated washings with warm RPMI-1640 medium, leaving a monolayer of adherent cells which were eventually incubated in complete medium with 30ng/ml GM-CSF and 10ng/ml IL-4. Cells cultures treated under these experimental conditions have been shown to consist of> 85% MD-DC, as determined by cytofluorimetric analyses. 4. Observing the inhibitory of MBL on the ability of MD-DC to capture HCMV particlesHCMV were marked with PKH26 (red fluorescence) according to the instructions. Briefly,1×108 (DNA copy number) CMV re-hanging in lml diluent, quickly adding the final concentration of 5×10-6 M of the PKH-26, in dark incubation at 37℃after 30min by adding a large number of the volume of ice-cold containing 10% fetal calf serum PBS to stop reaction; wash four times with PBS to remove unbound PKH26 by 10KD filter. DC-SIGN on MD-DC surface were stained with CD209-FITC. MD-DC in control group didn't incubate with MBL. Centrifugal sedimentation was made into sheets to observation the inhibitory of MBL on the ability of MD-DC to capture HCMV particles under Immunofluorescence confocal microscopy.5. Effect of short exposure of MD-DC to the hMBL/rhMBL on HCMV-capture by MD-DCMD-DC were pre-exposed to several dilutions of the hMBL/rhMBL for 30 min at 37℃. Then HCMV suspensions (2×107 DNA copy number) were added to MD-DC for 2 h at 37℃. After the incubation period, the virus was removed and the cells were carefully washed four times with RPMI-1640 (10% FBS), to remove the unbound virus in the wells. To confirm the efficient removal of unbound virus, we collected 100μl from the last wash to be analyzed by RT-PCR. Then the cells were detached from the wells and collected to analyze the HCMV-capture by RT-PCR. This experiment was created with seven groups, namely control group "DC cells+HCMV", the test group "MD-DC+hMBL (1μg)+HCMV" group, "MD-DC+hMBL (5μg)+HCMV" group, "MD-DC+hMBL (10μg)+HCMV" group, "MD-DC+rhMBL (1μg/ml)+HCMV" group, "MD-DC+rhMBL (5μg/ml)+HCMV" group and "MD-DC+rhMBL (10μg/ml)+HCMV" group.6. Ability of MD-DC transmitting HCMV to T cellsMD-DC (2.5×105) were seeded in 48 well-plate, incubated with HCMV (2× 107 DNA copies) for 30min, four times wash with PBS to remove unbound HCMV, then co-cultured with 2.5×105 T cells stimulated with PHA in complete medium at 37℃,5% CO2 incubator. At the first 3 days and 4 days, HCMV were determined in the supernatant by RT-PCR. Each experiment was run in quintuple.7. Effect of short exposure of MD-DC to hMBL/rhMBL on virus transmission from MD-DC to T cellsHuman T cells (2.5×105 cells) were added to hMBL/rhMBL-exposed HCMV-captured MD-DC (prepared as described above). We then determined the HCMV transmission from virus-captured MD-DC to T cells by analyzing the CMV DNA by RT-PCR in the supernatants from the co-cultures at 3 and 4 days post co-cultivation. Each experiment was run in quintuple.8. Isolating CD4+T cells with CD4+ positive magnetic separation method according to kit instructions.9. Effects of anti-DC-SIGN and-MR antibodies on HCMV capture by MD-DC and subsequent transmission to T lymphocytesMD-DC were pre-incubated with antibodies (anti-dendritic cell mannose receptor (MR) antibody CD206,10μg/ml; anti-DC-SIGN antibody, 10μg/ml) for 15 min at 37℃.HCMV (2×107 DNA copies) was then added and incubated for 2 h at 37℃. Cells were washed four times with RPMI-1640 (10% FBS) to remove unbound virus. Human T cells (2.5×105 cells) were added to antibodies-exposed HCMV-captured MD-DC. We then determined the HCMV transmission from virus-captured MD-DC to T cells by analyzing the CMV DNA by RT-PCR in the supernatants from the co-cultures at 3 and 4 days post co-cultivation. Each experiment was run in quintuple.10. Determining PP65+GAM-FITC in cells by flow cytometry according to kit instructions.11. Statistical analysisThe experiments were carried out in quintuple, with five positive controls (untreated MD-DC) for each experiment, and the results are presented as mean values with standard deviation. All data processing with SPSS 13.0 statistical software, one-way analysis of variance, P≤0.05 is statistically significant.Results1. Detection of target gene expression in stably transferred cells by RT-Q-PCRGene expression F=2 to the (-△△ct)th power△△ct=(the average ct of target gene of testing samples-the average ct of housekeeping gene of the testing samples)-(the average ct of target gene of control samples-the average ct of housekeeping gene of the control samples), Note:2△△ct value is greater, indicating higher expression of the gene;3～5 PrimerF (bp514):5'-GGAGCCATTCAGAATCTCATCA-3' R (bp652C):5'-AACCAGCATTGTTGGGTTCA-3'Screening to get two kinds of stable cell lines, target gene expression in the wild-type is 103 times more than that in the empty vector.2. Obtaining large quantities of high purity hMBL/rhMBL320mg high purity MBL was purified from 200ml human plasma, and 510mg purity rhMBL was purified from 1000ml CHO/MBL supernatant, the purity was identified by SDS-PAGE, MBL monomer molecular weight after SDS reduction was 26KD, no obvious hybrid band was carried out by the SDS-PAGE, suggesting the MBL was high purity.3. Cells culture of DC and activated T cellsPeripheral blood mononuclear cells were stimulated with GM-CSF and IL-4, at the first 6 days cells were observed under inverted phase contrast microscope morphology:colony-like growth, strong refractive index, irregular cells, showing pseudopodia. the percentage of MD-DC (expression of CD209-FITC) was more than 85% with single-color flow cytometry.4. Observing MBL blocked the capture of MCV by DC-cells under Immunofluorescence confocal microscopyAfter MD-DC captured the HCMV labeled with PKH26 (red fluorescence), they were stained through CD209-FITC (green) combining to DC-SIGN molecule, and then used immunofluorescence confocal microscopy to observe the capture of HCMV by DC-SIGN;which was effected by MBL pre-incubated. MD-DC captured less HCMV if they were pre-incubated with 10μg/ml concentration hMBL or rhMBL; which showed 10μg/ml concentration of hMBL or rhMBL could efficiently inhibit the capture of HCMV.5.Inhibitory effect of HMBL/rhMBL on the ability of MD-DC to capture HCMVTo explore the inhibitory effect of MBL on the ability of MD-DC to capture HCMV, MD-DC were exposed to different concentrations and different types of MBL for 30 min, then with MBL-exposed MD-DC were cocultured with HCMV virus for 2 hours at 37℃, after which, MD-DC were thoroughly washed with PBS to remove free HCMV. MD-DC were detached and then analyzed for CMV DNA by fluorescence quantitative PCR. This result represents the amount of HCMV DNA captured by MD-DC. The CMV DNA content from the last wash supernatant was below detection limit. Data are expressed as a percent of the HCMV DNA value of initial HCMV DNA value or a percent of the HCMV DNA value of DC control cultures that were incubated with HCMV in the absence of the MBL.Compare to the control group, 1μg/ml hMBL and 1μg/ml rhMBL didn't show any inhibition on the ability of MD-DC to capture HCMV particles (P=0.207,95% CI:-0.0935-0.4135;P=0.117,95% CI:-0.0535-0.4535), while 5μg/ml and 10μg/ml of hMBL group,5μg/ml and 10μg/ml rhMBL group did (P=0.005,95% CI:0.1265-0.6335; P= 0.003,95% CI:0.1465-0.6535; P= 0.015,95% CI:0.0665-0.5735; P= 0.007,95% CI:0.1065-0.6135). The results show that the MBL dose-dependently inhibited capture of HCMV by MD-DC at an IC50 of hMBL and rhMBL was 4.5μg/ml and 7.4μg/ml respectively.6. The ability of MD-DC to transmit captured HCMV to T-cellsBecause MD-DC can capture HCMV particles and transmit HCMV to permissive cells, we want to investigate whether DC-SIGN expression enhances low-susceptible T cells sensitive to CMV infection.Data are expressed as a percent of the HCMV DNA value of initial HCMV DNA value. The results show that at the first 3 days and the first 4 days, the amount of HCMV DNA in the test group "MD-DC+HCMV+T" group culture supernatant was significantly higher than that in the control group "MD-DC+HCMV" group with statistical significance (F= 46.398, P= 0.000; F= 16.702, P=0.004), which indicates a large number of HCMV virus replication in T cells.7. inhibitory effect of hMBL/rhMBL on the ability of MD-DC to transmit captured HCMV particles to CD4+T cellsBecause MD-DC can capture HCMV particles and transmit HCMV to T cells, we want to investigate whether hMBL/rhMBL are able to prevent the transmission of the HCMV particles from MD-DC to cocultured CD4+T cells.The percentage of HCMV transmission was expressed as the percent of the HCMV DNA value of the positive control, in which MD-DC didn't exposure to MBL. Co-culture on day 3, the IC50 of hMBL/rhMBL to inhibit MD-DC in HCMV transmission were 4.7μg/ml/3.1μg/ml;co-culture on day 4, the IC50 of hMBL/ rhMBL were 4.6μg/ml/2.1μg/ml respectively.At the first 3 and 4 days of co-culture, there wasn't marked difference between the control group and 1μg/ml rhMBL group (P=0.220,95% CI:-5.2627-1.2627; P=0.199,95%CI:-1.5327-7.0527); 1μg/ml,5μg/ml and 10μg/ml of hMBL groups and 5μg/ml and 10μg/ml of rhMBL groups significantly inhibited the transmission comparing with the control group (P< 0.005), while there was neither statistically significant between 5μg/ml hMBL group and 10μg/ml hMBL group (P= 0.085,95% CI:-0.4227-6.1027; P=0.236,95%CI:-1.7527-6.8327), nor between 5μg/ml rhMBL group and 10μg/ml rhMBL group (P= 0.211,95% CI:-5.3027-1.2227;P=0.857, 95%CI:-3.9127-4.6727). hMBL and rhMBL has showed inhibitory effect on the ability of MD-DC to transmit captured HCMV particles to T cells with dose-dependent manner.At day 4 of the co-culture, MD-DC and T cells in the same group of 5 holes were collected together, totally for seven tubes, CD4+ T cells were selected by positive immune magnetic separation method, the percentage of T cells increased from 48%～55% before selection to 93%～99%, the percentage of HCMV PP65 protein in T cells were from 17.9%-35.4%analyzed by flow cytometry, and the amount of HCMV in T cells were from 28×103/ml～5.9×103/ml detected by fluorescence quantitative-PCR; which demonstrate that MD-DC transmit the captured HCMV particles to CD4+ T cells, and the HCMV can replicate in CD4+ T cells.Conclusions1. Successfully established secreting RhMBL cell lines (CHO/rhMBL), preparing high-purity rhMBL and hMBL;2. Successfully prepared human primary monocyte-derived dendritic cells (MD-DC) with high expression of DC-SIGN;3. MD-DC showed the ability of transmission captured HCMV to T cells;4. HMBL and rhMBL showed the inhibition of MD-DC to transmit captured HCMV particles to CD4+ T cells, but rhMBL inhibition effect slightly worse than hMBL;5. DC-SIGN molecule played an important role in MD-DC capturing and transmitting HCMV particles.