Effects And Mechanisms Of Mifepristone In The Regulation Of Dendritic Cell Differentiation And Function

Mifepristone(RU486)is a synthet:ic 19-norsteroid,which is a potent antagonist of Progesterone and glucocorticoid.When it was first found,mifepristone has been used an abortion drug.Accumulating evidences from the basic and clinical researches Demonstrated a variety of potential applications for mifepristone in the areas of gynecology,endocrinology,oncology,and immunology.Mifepristone is a progesterone receptor modulator and has been the first choice for the first trimester abortion in some regions.Recently,numerous studies showed that low-dose mifepristone can be used to achieve the purpose of contraception,the so called "endometrium contraception".However,the mechanism about endometrial contraception is still need to be eluciated.DCs are efficient antigen-presenting cells,Indoleamine 2,3-dioxygenase(IDO)expressed in DCs is the rate-limiting enzyme in the pathway that degrades the essential aminoacid L-tryptophan to generate several biologically active metabolites known as kynurenines.IDO is highly expressed in the DCs and exerts immmunoregulatory activity through its metabolite kynurenine.IDO has been implicated in contributing to immunotolerance in early pregnancy.IDO+ DC regulate the immune responses by inducing T cell anergy,inducing Tregs.Therefore,in order to investigate the effects and possible mechanisms of mifepristone in the regulation of dendritic cells(DCs),DCs from human will be used.In our study,in vitro study will be done to investigate the possible mechanism of maternal-fetal interface acquired immune,in the cell and molecular levels,regulated by mifepristone through the methods of western blot,ELISA,flow cytometer and so on.We hope that current study will provide something new to the development of ideal contraceptive method,the understanding and treatment of diseases in human reproduction.Part Ⅰ Effect of mifepristone on differentiation of dendritic cellsObjective:DCs are sorted out exposed to mifepristone(20,65,200 nM).To observe the effect of mifepristone on the maturation and biological function of DCs.And to explore the role and mechanism of mifepristone in the regulation of acquired immunity at the maternal-fetal interface in the process of embryo implantation.Methods:CD14+ Mononuclear cells isolated from peripheral blood were induced into immature DCs by GM-CSF、IL-4 for 6 days.Then,the immature DCs were exposed to mifepristone(20,65,200nM)for another 48 hours.At the same time,set the solvent control group(treated with dimethyl sulphoxide,DMSO).Surface molecules CD80、CD86 and ICAM-I were determined by flow cytometry.The IL-12p70 level in the supernatant were tested by enzyme linked immunosorbnent assay(ELISA).Results:1.Mifepristone upregulated the expression of CD80 and CD86 and ICAM-I.2.Compared with the control group,the level of IL-12p70 secretion of DCs in the mifeprsitone treatment group was significantly higher than that of the control group:The ability of mifepristone in promoting DCs secretion of IL-12p70 is proportional to the concentration of mifepristone within limits(65nM-200nM).Conclusion:1.Mifepristone:induced phenotype maturation of DCs and the IL-12 secreiion of the DCs.2.The ability of mifepristone in promoting DCs maturation revealed dose dependence in effective concentration range.3.It’s probably that mifepristone reached Semi allogeneic fetus rejection and contraceptive effection through inducing DCs maturation.PartⅡ The role of mifepristone in the regulation of DCs affecting TregsObjective:Test the expression of FOXP3 on Tregs and secretion of IL-10 from Tregs to investigate the role of mifepristone in the regulation of DCs affecting T cell mediated acquired immunity.Methods:After exposed to different dose of mifepristone,DCs were mixed cultured with Tregs.Western blot was used to test the expression of forkhead box p3 and the IL-10 was evaluated by ELISA.Results:Unmature DCs were treated with 20,65,200 nM mifepristone and DMSO respectively.Allogeneic Tregs were used as reaction cells.The level.of Foxp3 expressed in 200nM mifepristone group is lower than that in the control group(0.25±0.03 vs.0.62 ± 0.01;P<.01);also lower than that in the 65nM mifepristone group(0.25 ± 0.03 vs.0.34 ±0.01;P<01).However,there was no difference between the 20nM mifepristone group and the control group.The expression of IL-10 decreased in 65nM and 200nM groups.Levels of the changes in IL-10 and in Foxp3 were consistent.The IL-10 expressed in the 20nmol/L mifepristone group,65nmol/L mifepristone group and 200nmol/L mifepristone group were 158.34 ±9.46,127.36 ± 6.37,109.2 ± 9.45,respectively,as compared with control group(171.25 ± 7.85).Conclusion:After treated with mifepristone,DCs inhibited the expression of FOXP3 and IL-10 on Tregs.This hinted mifepristone have effect on the DCs regulation of the T cell mediated acquired immunity.PartⅢ The effects of IDO on mifepristone regulation of DCs functionsObjective:1.To explore the effect of mifepristone on the expression and activity of indoleamine 2,3-dioxygenase(IDO).2.To investigate the role of IDO in the mifepristone affecting the function of DCs.Methods:1.CD14+ Mononuclear cells isolated from peripheral blood were induced into immature DCs by GM-CSF、IL-4 for 6 days.Then,the immature DCs were exposed to mifepristone(20,65,200 nM)for another 48 hours.At the same time,set the solvent control group(treated with dimethyl sulphoxide,DMSO).IDO mRNA expression was measured by RT-PCR.IDO expression was analyzed by western blot:Kynurenine and tryptophan levels were tested by HPLC.2.CD14+ Mononuclear cells isolated from peripheral blood were induced into immature DCs by GM-CSF、IL-4 for 6 days.The immature DCs were exposed to 200nM mifepristone for another 48 hours.The mature DCs were divided into two groups:1-MT(1 ng/ml)was added to the group two while the group one was absent of it.After ten hours,the DCs were mixed-cultured with Tregs(1.0×105/ml)in 6 well plates for five days.The expression of Foxp3 was determined by western blot.IL-10 in the supernatant was detected by ELISA.Results:1.The expression and activity of IDO in DCs after treated with mifepristoneIDO mRNA expression in DCs was determined using Real-time RT-PCR.The mRNA expression of IDO in 20nmol/L mifepristone group,65nmol/L mifepristone group and 200nmol/L mifepristone group were 1,0.86±0.12,0.69 ±0.08 and 0.52± 0.05,respectively.The expression of IDO mRNA in DCs in the 65 and 200 nmol/L mifepristone group was lower than the control group(P<0.01).Mifepristone caused a dose-dependent downregulation of IDO between the 65nM and 200nM groups.There was no significant difference between the control group and the 20 nmol/L mifepristone group(P>.05)IDO protein level measured by Western blot was determined by Real-time RT-PCR.The protein expression of IDO in the 20nmol/L mifepristone group,65nmol/L mifepristone group and 200nmol/L mifepristone group were 0.75±0.11,0.67 ± 0.07,0.42 ± 0.23,respectively,as compared with control group(0.94 ± 0.10).To evaluate IDO activity,the Kyn/Trp ratio was measured by HPLC in each group.The Kyn/Trp ratio levels were similar to the mRNA and protein levels of IDO.The IDO activity in the 20nmol/L mifepristone group,65nmol/L mifepristone group and 200nmol/L mifepristone group were 1.39 ± 0.03,0.62±0.01,0.12 ± 0.02,respectively,-as-compared with control group(1.42±0.03).2.The expression of FOXP3 and IL-10 after treated with 1-MT,a competitive inhibitor of IDOOnce treated by 1-MT(the group two),as compared with group one,the IDO activity was inhibited and the expression of FOXP3 increased(0.12±0.01 vs.2.21 ±0.16.P<0.01).Also,IL-10 secreted by Tregs increased(106.47±11.58 vs.327.01±15.95.P<0.01).Conclusion:Mifepristone could down regulate DCs expression of functional IDO and decrease the activity of IDO.Mifepristone may have a role of anti-implantation by decreasing IDO expression and IDO activity in DC.However;when IDO activity was blocked with 1-MT,the FOXP3 and IL-10 significantly increased.This suggested that low-dose mifepristone regulated the function of DCs by reducing the production of IDO.Our findings provided possible immunological mechanisms for mifepristone as an acyeterion.