Study On The Role Of SlanDC In HIV Infected Target Cells

Objective:The transmission of viruses between cells and cells is an important way to spread the human immunodeficiency virus?human immunodeficiency virus,HIV?in the human body.Dendritic cell?DC?is the most powerful antigen presenting cell in human body.It plays an important role in inducing T cells to proliferate and activate,and could trigger the initiate immune response.However,it also plays a bridge role in the process of HIV spreading to target cells.The traditional DC is mainly divided into Myeloid DC?Myeloid DC,MDC?and plasma like cell like DC?Plasmacytoid DC,PDC?.But then Knut Sch?kel found a new group of DC subgroup,6-disodoum N-acetyl galactosamine specific cell surface expression?6-sulfo LacNAc?,which is called slanDC,can be used to identify monoclonal antibody M-DC8.Unlike traditional DC,slanDC expresses CD16,which is more similar to monocytes than MDC and PDC.It has the characteristics of myeloid derived cells,and the number in Periperal blood momcyte cells is more than MDC and PDC,which accounts for about 0.6-2%of peripheral blood mononuclear cells?Peripheral blood mononuclear cell?.In vitro studies have shown that monocyte induced dendritic cells?monocyte-derived dendritic cell,MDDC?play an important role in trans-infection of HIV.It is suggested that slanDC may play an important role in the transmission of HIV to target cells.There are two major mechanisms for the spread of HIV virus in the cells.The The most widely known way is cis-infection,HIV-1 infects target cells,and its RNA is formed by reverse transcription,and it's DNA is integrated into DNA of target cells.Following the replication of host cells,the progeny virus is released to the target cell near the cell to infect new target cells.The second way is tran-infection.The virus does not complete reverse transcription,integration,replication in cells,but only the cells as a bridge,through a number of surface molecules to capture and carry the HIV virus on the cell surface,thereby forming the virological synapse spread the virus to target cells,because the cell itself is not infected with HIV,just carry the virus,so this way is called trans spread.This paper through the study of HIV infected poeple,highly active antiretroviral therapy people and healthy people's slanDC surface siglec1 and viral load,CD4 quantity;in vitro co-culture target cell witn slanDC after exposure to the HIV-1_NL4-3,to determine whether slanDC can play a role in the spread of HIV to the target cells,to verify whether this effect is related with siglec1,provide the basis for the transmission of slanDC revealed in the process of HIV infection among cells.Methods:1.14 cases of HIV infection without treatment,28 cases of highly active antiretroviral therapy?HAART?were effective,all from the First Affiliated Hospital of China Medical University red ribbon clinic.19 cases of healthy control group?NC?were randomly selected as a healthy person with negative HIV antibody and unexposed to HIV.2.Determination of CD4⁺T cell counts.CD4⁺T cell counts were measured by flow cytometry?FACS Calibur,Becton-Dickinson,USA?.A single platform lyses,but no wash,procedure was performed for CD4⁺T cell counts with Trucount tubes and TriTEST CD4FITC/CD8PE/CD3PerCP?Becton-Dickinson,USA?.3.HIVviralload measurement.HIV RNA extracted from plasma samples was amplified by a standardized reverse transcription PCR assay according to the manufacturer's recommendations?COBAS Amplicor,HIV-1 Monitor Test Version 1.5,Roche Diagnostics,USA?.4.Amplication of HIV isolation.PBMC from healthy donors were stimulated with PHA at37?,5%CO₂.After 72h,cells were infected with 5×10⁶for 2.5h at 37?,5%CO₂.After infection,cells were resuspended to the concentration of 1×10⁶/ml in R10 supplemented with 100 U of interleukin-2/ml.Cultures were fed with 1×10⁶/ml stimulated PBMC every week.Viral growth was monitored by p24 enzyme-linked immunosorbent assay?ELISA?twice a week.Virus was harvested when the concentration of p24 in the supernatant was at least 10⁴?pg/ml?and stored at-156?.5.Luciferase detection.Take out the 96-hole culture plate in the incubator,suck up the supernatant,and add 100ul PBS to each hole.50ul luciferase detection reagent was added to each hole for 2 minutes?avoid light?.Mix3-5 times,take 100ul mixture to the whiteboard.Put the whiteboard into instrument to detected.6.The detection of siglec1 on the surface of slanDC and monocyte subsets.The flow tubes were marked well,and the extracted PBMC was added to the flow tube in turn.Each tube was added with the corresponding fluorescent antibody combinations,slanDC:HLA-DR-APC-cy7andM-DC8-FITC,others:CD14-Percp-cy5.5,CD16-PE-Cy7 and siglec1-PE.After mixing,4 degrees Celsius dyeing 30min,two times with a PBS containing 2%FBS,and 200 ul containing 1%polyformaldehyde.7.SlanDC sorting.50ml EDTA anticoagulant blood from healthy person was extracted the Periperal blood momcyte cell in three 50ml centrifuge tubes and washed two times,transferred to the three flow type sterile tubes,each tube joined 20ul HLA-DR-APC-cy7?BIolegend?and 20ul M-DC8-FITC?Miltenyi?,4?to avoid light reaction for 30 minutes,wash two times per tube on 2ml R10 medium,flow cytometry sorting of slanDC?BD?.8.Transfer of HIV-1NL4-3 to target cells by slanDC.After sorting,the slanDC was laid on the 96 hole plate?U bottom?.The number of cells in each pore was adjusted to 2*10⁵.The IFN-alpha was added to each hole?the final concentration was 1000U/ml?and placed at 37?,95%humidity and 5%CO₂ for 24 hours.96 holes were taken out,100ul HIV-1_NL4-3L4-3 was added to each hole,and R10 culture solution was added to 200ul,at 37?,95%humidity and 4 hours for 5%CO₂ culture.Take out 96 holes,transfer the cells to the flow tube,wash and discard the supernatant.Wash again,and take 100ul supernatant in a48 well culture plate blank hole,discard the rest of the supernatant,cultured cells resuspended and transferred to 48 well plates a blank hole with 100ul R10,each hole is added to the TZM-BL cell 2*10⁵,R10 medium added to 400ul at 37?,95%humidity5%CO₂,48 hours of coculture.9.statistical analysis.SPSS20.0 software package was applied to carry out statistical analysis,data processing was in the median.The comparison of experimental indexes between the two groups was conducted by Mann Whitney U test.The probability of statistics was tested by bilateral test,and P<0.05 was statistically significant.Result:1.Expression of siglec1 on slanDC surface of HIV infected people.The expression of siglec1 on slanDC cells in the untreated group was higher than that in the HARRT treatment group and the healthy control group?P<0.001,P<0.001?.There was no significant difference between the HARRT treatment group and the healthy control group?P=0.474?.CD14++CD16+,CD14+CD16+and CD14+CD16-are three subgroups of mononuclear cells,and the same trend is found in the subgroup of monocyte.The expression of untreated CD14++CD16+cells on siglec1 was higher than HARRT treatment group and healthy control group?P<0.001,P<0.001?,there was no significant difference between HARRT group and healthy control group?P=0.126?;the expression of untreated CD14+CD16+cells on siglec1 was higher than HARRT treatment group and healthy control group?P<0.001,P<0.001?and there is no significant difference between HARRT group and healthy control group?P=0.987?;the expression of untreated CD14+CD16-cells on siglec1 was higher than HARRT treatment group and healthy control group?P<0.001,P<0.001?,there was no significant difference between HARRT group and healthy control group?P=0.373?.2.The relationship between the expression of siglec1 on the slanDC surface of HIV infected people and the number of CD4+T.There was no significant correlation between the percentage of slanDC expressed in slanDC and the number of CD4+T cells?P=0.727?in slanDC infected people?HARRT treatment and untreated?.There was no significant correlation between the expression of siglec1and the number of CD4+T on the surface of CD14+CD16+?CD14+CD16-?CD14++CD16+cells?P=0.449,P=0.996,P=0.876?.3.The relationship between the expression of siglec1 on the slanDC surface of HIV infected people and the viral load.There was a significant correlation between the percentage of Siglec1+slanDC and the viral load in the plasma?P=0.0059,r=0.655?.With all subsets of monocytes compared to CD14+CD16+?CD14+CD16-and CD14++CD16+cells surface expression level of both siglec1 and plasma viral load was significantly correlated?P=0.0255,P=0.0011,P=0.0015?.4.SlanDC transmission of virus to target cells after HIV infection.After sorting slanDC by flow cytometry,the cells were treated for 24 hours,then incubated with HIVNL4-3 virus at 37 degrees centigrade for 4 hours,then washed and cultured with TZM-Bl cells for 48 hours,and luciferase was detected.It can be seen that slanDC has the role of transmitting HIV to the target cells.The Washing blank hole is used as a reference,and the virus is not completely washed,but slanDC,especially the IFNa induced siglec1 expression,can transmit the virus to the target cells.It was found that the transmission of HIV to the target cells was inhibited by siglec1a antibody closure,and 55.78%-79.68%was suppressed.Conclusion:HIV infected slanDC increased siglec1 expression and viral load were significantly correlated,no significant correlation with the number of CD4+T cells;the target cell and the exposure of slanDC after the co culture can be infected with the virus,the blocking of the slanDC surface after siglec1,the infection efficiency of target cells decreased significantly.It is suggested that siglec1 is expressed on the surface of slanDC,and its function plays an important role in the intercellular transmission of HIV.