| Object ives:To investigate the effect of 1,25 dihydroxy vitamin D3(1,25 dihydroxy vitamin D3,1,25(OH)2D3)combined with interleukin-6(Interleukin 6,IL-6)on the differentiation of myeloid-derived suppressor cells(MDSC)and its mechanism.Then explore the effect of 1,25(OH)2D3 on the surface molecules and functions of MDSC,to elucidate the immunomodulatory effect of 1,25(OH)2D3 on MDSC,and provides the new ideas of its application.Methods:1.Collect EDTA anticoagulant from healthy people and extract peripheral blood mononuclear cells(PBMCs)by density gradient centrifugation,separately/combined with interleukin 6(IL-6)and granulocyte mononuclear cell colony-stimulating factor(GM-CSF)and 1,25(OH)2D3 stimulated peripheral blood mononuclear cells of healthy people,and the expression frequency of HLA-DR-/lowCD33+CDllb+MDSC was detected by flow cytometry.The changes of MDSC in each stimulation group and untreated group were compared.2.The level of phosphorylation of STAT3 in MDSC cells of experimental group and untreated group was detected by flow cytometry.3.Collect 1,25(OH)2D3 and IL-6 stimulated PBMCs(experimental group)and untreated PBMCs,and analyze the CD80,CD86,PD-L1 molecules on the surface of two groups of MDSCs by flow cytometry.4.Flow-through sorting of normal human CD3+T cells and experimental group HLA-DR-/lowCD33+CDllb+MDSC.Normal human CD3+T cells were cultured separately as controls;normal human CD4+T cells were co-cultured with normal human HLA-DR-/lowCD33+CDllb+MDSC.CD4+T and CD8+T cell proliferation were detected by flow cytometry.5.Full-wavelength microplate reader was used to detect the activity of arginine-1 and nitric oxide in the supernatant of the co-cultured HLA-DR-/lowCD33+CD11b+MDSC and CD3+T cells in the experimental group.6.Flow cytometry was used to detect the percentage of MDSC phagocytosing fluorescent latex microspheres in experimental and untreated groups.7.Flow cytometry was used to detect the expression of reactive oxygen species(ROS)in MDSCs co-cultured with HLA-DR-/lowCD33+CD11b+MDSC and CD3+T cells and MDSC alone.Results:1.the proportion of MDSC in untreated group was(2.83±1.38)%,the proportion of MDSC in positive control group(GM-CSF+IL-6 group)was(10.41±1.12)%,the proportion of MDSC in 1,25(OH)2D3 group was(6.67±1.71)%,the proportion of MDSC in 1,25(OH)2D3+IL-6 group was(10.99±1.37)%,and the proportion of MDSC in GM-CSF+1,25(OH)2D3 group was(4.73±0.94)%.The proportion of MDSC cells in the 1,25(OH)2D3+IL-6 group increased,and the difference was significant(P=0.000).In addition,the MDSC cells induced by 1,25(OH)2D3+IL-6 group were all mononuclear MDSC(M-MDSC).2.Compared with the untreated group,the histogram of PBMC intracellular p-STAT3 stimulated by 1,25(OH)2D3 combined with IL-6 significantly shifted to the right,indicating that 1,25(OH)2D3 combined with IL-6 may induce PBMC differentiation to MDSC by increasing the intracellular STAT3 phosphorylation level of PBMC.3.CD3+T lymphocytes of normal subjects were cultured separately as the control group;1,25(OH)2D3+IL-6 induced CD3+T lymphocytes co-cultured with normal CD3+T lymphocytes were the experimental group.The basic conditions of proliferation of CD4+T lymphocytes and CD8+T lymphocytes were as follows:(87.57±3.59)%,(72.13±2.02)%and(57.33±8.46)%,(38.30±5.19)%.Among them,1,25(OH)2D3+IL-6 induced MDSC could not only inhibit the proliferation of CD4+T lymphocytes and CD8+T lymphocytes,the results were statistically significant(P=0.005)and(P=0.001).At the same time,comparison with normal MDSC showed that 1,25(OH)2D3 combined with IL-6 induced MDSC had stronger ability to inhibit the proliferation of CD8+T lymphocytes.4.The mean fluorescence intensity(MFI)of CD80,CD86 and pd-11 in the untreated group were(370.47±26.50),(863.97±65.93)and(666.80±16.63),respectively.The MFI values of CD80,CD86 and PD-L1 in 1,25(OH)2D3+IL-6 group were(381.35±29.78),(1690.20±32.17)and(1590.20±295.59),respectively.Compared with the untreated group,the expressions of CD86 and PD-L1 in MDSC in group 1,25(OH)2D3+IL-6 were significantly increased(P=0.000)and(P=0.032),and there was no statistical difference in the expression of CD80(P=0.661).5.The basic information of the arginase activity and NO concentration in the supernatant of the control group and the experimental group are as follows:(0.31±0.08)U/L,(62.00±6.21)umol/L and(0.42±0.05)U/L,(75.78±11.37)umol/L.Compared with the control group,the concentration of NO in the supernatant of the experimental group increased significantly,and the difference was statistically significant(P=0.045).It was concluded that 1,25(OH)2D3 combined with IL-6-induced MDSC inhibited the proliferation of T lymphocytes through the iNOS metabolic pathway,and there was no change in the arginine activity of the experimental group and the control group(P=0.268).6.The percentage of MDSC phagocytic fluorescent microspheres in the untreated group was(35.73±6.75)%,and that in the 1,25(OH)2D3+IL-6 group was(60.83±7.68)%.Compared with the untreated group,the phagocytic capacity of MDSC in the 1,25(OH)2D3+IL-6 group was significantly enhanced(P=0.049),and the difference was statistically significant.The phagocytic function of MDSC in the 1,25(OH)2D3+IL-6 group was not significantly different from that of mononuclear cells(P=0.000).7.The proportion of weakly positive and strongly positive ROS in the untreated group were(45.90±11.11)%,(54.10±11.11)%;The proportion of weakly positive ROS and strongly positive ROS in 1,25(OH)2D3+IL-6 group were(19.43±9.97)%and(80.57±9.97)%,respectively.Compared with the untreated group,the proportion expression of ROS strong positive of MDSC in group 1,25(OH)2D3+IL-6 increased(P=0.012),and the proportion expression of ROS weak positive decreased(P=0.012).Conclusions:1,25(OH)2D3 combined with IL-6 induces PBMC differentiation into monocyte-type MDSCs by stimulating STAT3 phosphorylation in PBMCs.At the same time,MDSCs significantly inhibit CD4+T and CD8 + T lymphocytes proliferate via inducible nitric oxide synthase(iNOS)metabolic pathways.Compared with normal MDSC,it was found that MDSC induced by 1,25(OH)2D3combined with IL-6 has a stronger ability to inhibit the proliferation of CD8+T lymphocytes,and the expression of CD86 and PD-L1 molecules on the surface of MDSC is stronger.In addition,1,25(OH)2D3 combined with IL-6 can promote the secretion of ROS in MDSC and enhance the phagocytosis of MDSC. |
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