The Research Progress Of The Effect Of Gal-9 On SlanDC Of HIV-1 Infection Patients

Objective:slanDC is a new group of dendritic cells(dendritic cells,DC)expressing of6-sulpho N-acetyl lactose amine(6-sulfo LacNAc,slan),having very strong antigen presented function,it also plays a role in start the T cell response,activated NK cells secreted cytokines and induce the ADCC effect.During hiv-1 infection,the number of slanDC increased and the activation level increased.Galactose lectin-9(Gal-9)is a beta galactose binding protein with eosinophil chemotactic activity,is one of the members of the galactose lectin family,it plays an impor role in viral infections,autoimmune diseases,cancer and other diseases.Tim-3 is the main receptor of Gal-9,and the Gal-9/Tim-3signaling pathway plays an important role in regulating natural immune response and adaptive immune response.Whether the Gal-9 and Tim-3 can express on the surface of slanDC during hiv-1 infection should be further studied.There studies found that during the acute HIV-1 infection,chronic HIV-1 infections,even thoug the people receive HIV-1 ARV treatment,the Gal-9 plasma levels was higher than uninfected,and may be have a relationshipwith the viral load,thedisease progression,and the treatment.In vitro studies have found that Gal-9 has an effect on DC mature function,influence the secretion of cytokines.Restructuring Gal-9 can upregulate the expression ofCD40,CD54,CD80,CD83,CD86 and HLA-DR,induce the maturation of people Monocyte derived dendritic cells(Monocyte-derived dendritic cells,moDC),increase the secretion of IL-12.Human recombinant Gal-9 can also induce mouse bone marrow derived dendritic cells(BMDC)maturation and promote the mouse heart transplantation rejection response.In addition to promoting the mature function,the recombinant gal-9 can also inhibits the maturation of BMDCs in BALB/c mouse,decrease the secretion of pro-inflammatory cytokines related to Th2.SlanDC is a major member of peripheral blood DC,the number of slanDC increase during the HIV-1,the existing pilot study on the surface of Gal-9can affect mature moDC,BMDC and cytokine secretion function,then if the Gal-9 can influence the expression of CD40,CD80 and CD83 slanDC surface affects the function of slanDC mature as well as the secretion of cytokines during the HIV-1 infection has not been reported.Besides slanDC,according to thephenotype and function,the DC can also be divided into the m DC(myeloid Dendritic Cells,mDC)and pDC(plasmacytoid Dendritic Cells,pDC).It has been found that there is a large number of Gal-9 in the m DC and pDC cells of HIV-1 infected patients,and the expression of Gal-9 of slanDC on the surface and intracellular is still to be further studied.Methods:1.The subject.A total of 25 HIV-1 infected patients were selected in this study,all of them were treatment.The 25 HIV-1 infected patients were from the Red Ribbon Outpatient Department of First Affiliated Hospital of China Medical University and confirmed as positive by Western blotting.The healthy control group were composed of39 cases of HIV-1 negative normal people.All patients included in this study signed informed consents,and approved the Ethics Committee of the First Affiliated Hospital of China Medical University.2.Peripheral blood mononuclear cells(PBMCs)were extracted by density gradient centrifugation.Taking 1×10~6 cells to detect the expression of Gal-9 and Tim-3 on the surface of mDC,pDC and slanDC and taking 4×10~6 cells to detect the expression of Gal-9 in the cytoplasm and endonuclear.6×10~6 cells were used to detect the effect of exogenous Gal-9 on the expression of Tim-3,CD40,CD80 and CD83 on slanDC surface and the secretion of cytokines IL-10,IL-12,TNF-?and IFN-?.3.Detect the expression of Gal-9 and Tim-3 on slanDC surface.The expression of Gal-9 and Tim-3 on the surface of slanDC was detected by LSR II flow cytometry according to the instructions of BD company's cell surface staining instruction.4.The detection of Gal-9 in intracellular and nuclear of slanDC.The surface of Gal-9 in slanDC was stained with Gal-9-PE,and the expression of Gal-9 in the nucleus and nucleus of slanDC was detected by LSR?flow cytometry.5.The effect of human recombinant Gal-9 on maturation of slanDC,and expression of Tim-3 were studied in three groups,which were blank control group,positive control group and Gal-9 group.Each tube was added R10 medium 500ul,LPS 500ug/ml,Gal-9 10ug/ml,after mixing,into 37?,5%CO2 incubator for 40 hours.After culturing,the surface of slanDC were stained.At the end of the experiment,the expression of Tim-3?CD40?CD80?CD83 was detected by LSR?flow cytometry.6.The effect of human recombinant Gal-9 on the cytokine secretion of slanDC,there are the blank control group,the positive control group,the Gal-9 group.Each tube was added R10 medium 500ul,LPS 500ug/ml,Gal-9 10ug/ml,into 37?,5%CO2 incubator for 16 hours.After culturing,the surface of slanDC was stained at 4?for 30min,centrifuged,and protected from light for 30min at 4?.After the end,1ml/tube rupture membrane was centrifuged and repeated 2 times.Intracellular secretion of IL-12-Percp in slanDC was stained for 30min at 4?.At the end of the experiment,the effects of human recombinant Gal-9 on the secretion of cytokines by slanDC were detected by LSR?flow cytometry.7.The statistical analysis.Graphpad7.03package for statistical analysis and graphics were adopted,All data were expressed with t tests between different groups,P<0.05 was considered statistically significant.Result:1.The Gal-9 expression on the surface and the cytoplasm of slanDC,and the expression level of the cytoplasm were higher than expression level on the surface.The expression level of Gal-9 on the surface of slanDC in hiv-1 infected patients was higher than that in the healthy control group.The expression level of Gal-9 in the cytoplasm ofHIV-1patients was lower than that in the healthy control group.2.The Tim-3 can expression on the surface of slanDC in HIV-1 infected patients and healthy control group,the expression level of HIV-1 infected patients was lower than that in the healthy control group.Recombination Gal-9 and LPS can induce the expression of Tim-3 of slanDC inHIV-1 infected patients.3.The expression levels of CD40,CD80 and CD83 ofthe slanDCin HIV-1infected patients were lower than that in the healthy control group.4.The expression levels ofIL-12 secreted by slanDC in HIV-1 infected patients was lower than that in the healthy control group.5.Recombinant Gal-9 can reduced the expression level of CD40,CD80 and CD83 on slanDC surface in HIV-1 infected patients.6.The recombinant Gal-9 and LPS resulted in the decrease expression of the cytokines IL-12of slanDC in HIV-1 patients.7.The Gal-9 can expression both on the surface and the cytoplasm of mDC,pDC in healthy control group and the HIV-1 infected patients,and intracellular expression level was higher than that of surface.8.The expression level of Tim-3 of the mDC in HIV-1 infected patients was lower than that in the healthy control group.The Tim-3 expression on the surface of pDC in HIV-1infected patients and healthy control group,and the expression level of Tim-3 on pDC of HIV-1 infected patients was lower than that of healthy control group.In HIV-1infected patients and healthy control group,mDC express the highest level of Tim-3.Conclusion:The expression level of Gal-9 on the surface of slan DC was higher of HIV-1 infected patients,but the expression of intracellular Gal-9 was lower than that of healthy controls.The expression of Tim-3 on the surface of slanDC in HIV-1-infected was lower than that in healthy controls.Recombinant Gal-9 can increase the expression of Tim-3 on the surface of slanDC in HIV-1,infected patients,and it can also reduce the expression of CD40,CD80,and CD83 on the surface of slanDC and the level of IL-12secreted by slanDC in HIV-1 infected individuals,inhibition of the mature and secretory cytokine function of slan DC.