Expression Of IL-37Contributes To The Immunosuppressive Property Of Human CD4~+CD25~+Regulatory T Cells

OBJECTIVE1.To prove that interleukin-37is expressed in human peripheral blood CD4+CD25+regulatory T cells.2.To do Research in the relation between activative degree of CD4+CD25+Treg andexpression degree of IL-37.3.To prove that the expression of IL-37has relation to the immunosuppressive effectof CD4+CD25+Treg.METHODSPartⅠ: We isolated CD4+CD25+Tregs from health volunteers`peripheral bloodmononuclear cell(PBMC) with human CD4+CD25+Regulatory T Cell Isolation kit.Analyzing the purity of isolated CD4+CD25+Tregs by flow cytometry.1.Proliferation activity of CD4+CD25+Tregs was compared in expander group,non-expander group, and control group(only10%FCS-RPMI1640).Using CCK-8theresults were tested by24h,48h and72h.2.Under the stimulation by expander the expression of IL-37in CD4+CD25+Tregs wasanalyzed by Western blot.3.Under the stimulation by expander for72h, the location of IL-37expression inCD4+CD25+Tregs was analyzed by laser scanning confocal microscope.PartⅡ:We isolated CD4+CD25+Tregs and CD4+CD25-T cells from health volunteers`PBMC with human CD4+CD25+Regulatory T Cell Isolation kit.Using siRNAsilented IL-37expression in Treg cells.1.Under the stimulation by expander the expression of IL-37in CD4+CD25+Tregs wasanalyzed by Western blot.There were three groups of CD4+CD25+Regulatory T Cell:normal group, il-37siRNA group, and scramble group. 2.CD4+CD25+Tregs and CD4+CD25-T cells were mixed by1:1ratio. The mixed cellswere seted in cell culture plate, stimulated with Dynabeads Human Treg Expender.The three groups of T-cell proliferative activity were tested by CCK-8.3.Using Elisa technology, we tested different time of three CD4+CD25+Treg/CD4+CD25-T cells groups and blank control group. We tested the level changes in thesupernatant fluid of IL-2, IFN-γ/IL-4,IL-10and TGF-β.RESULTS1.The purity of CD4+CD25+Tregs was above93%after isolated by human CD4+CD25+Regulatory T Cell Isolation kit, and the activity of the cells was97%.2.CD4+CD25+Treg cells were stimulated by Dynabeads Human Treg Expender. Incomparison with non-expander and control groups, proliferation activity in expandergroup was significantly increased along with the prolongation of stimulation. However,the non-expander groups showed no significant proliferation.3.With the Western blot, it was identified the expression of IL-37in humanCD4+CD25+Tregs. Under the stimulation by expander for72h, the expression of IL-37in CD4+CD25+Tregs was markedly enhanced.4.With laser scanning confocal microscope, expression of IL-37was obvious in thecytoplasm and it showed high density near the cell membrane by laser scanningconfocal microscope.5.Human CD4+CD25+Tregs and CD4+CD25-T cells were mixed by1:1ratio under thestimulated of Dynabeads Human Treg Expender. The proliferation capacity haddecreased when cocultured with CD4+CD25+Tregs cells, however, the proliferationrates soared after the expression of IL-37silenced by siRNA. The results showed thatsiRNA group of CD4+CD25-T cells had a weak, no difference proliferation capacitywith the control group.6.We used flow cytometry to detect SiRNA interference-IL-37Treg group and normalTreg cells at24under the stimulated of Dynabeads Human Treg Expender to test Foxp3,CTLA-4express. The results showed that Foxp3and CTLA-4molecule expressionsdeclined significantly in SiRNA-IL-37interference group compared with normal group (P <0.05).We can conclude thatCD4+CD25+expression of IL-37in Treg cellscan significantly affect the the expression of CTLA4and Foxp3, and they are at thesame change trend in expression.7.Using Elisa technology, we testd IL-10and TGF-βbetween normalCD4+CD25+Treggroup and siRNA interference-IL-37CD4+CD25+Treg group in the cell culturesupernatant. The results showed that the two factor levels were significantly decreased(P <0.01).So we ensured that in siRNA group the expression of IL-37ofCD4+CD25+Treg cells may affects their inhibitory cytokines production.8. At72h to test SiRNA interference-IL-37Treg group, we detected IL-2concentrations in the supernatant. According to the results of IL-2concentration, afteradded CD4+CD25+Treg cells in CD4+CD25-T cells, the concentration decreased(P<0.01). But in the siRNA interference-IL-37group, IL-2levels significantly increased(P <0.01). Negative control Treg group and CD4+CD25+Treg cells group showed nodifference (P>0.05). We indicated that silence of IL-37expression maybe an importantreason to increase of IL-2and also may leads to an decreased suppression ofCD4+CD25+Treg-mediated.9.SiRNA silenced37expression in CD4+CD25+Treg decreasing the polarization to Th2and rising IFN-gamma/IL-4concentration ratio. Compared with normal control group,it showed statistically difference (P <0.01). Experimental results showed that silencedexpression of IL-37leaded a significant rise in IFN-gamma/IL-4. Therefore, IL-37proteins may partially mediated polarization of CD4+CD25+Treg teword Th2.CONCLUSIONS1.In healthy human peripheral blood, IL-37can positively express in CD4+CD25+Tregs.2.With the stimulation by expander, the expression of IL-37might further enhance inCD4+CD25+Tregs.3.The expression of IL-37has relation to the immunosuppressive effect ofCD4+CD25+Treg.Silencing experssion of IL-37in CD4+CD25+Treg lead a decreace inimmune inhibitory effect twords CD4+T cell.