The Effects And Mechanisms Of Mifepristone In Uterine Macrophage Activation

Drug contraception is a milestone in the history of women contraception.Since 1960s,steroidal hormone oral contraceptives(OCs),have been widely used in the world.However,OCs containing estrogen are prone to thrombotic diseases,hormone-dependent tumors and weight gain and other side effects,limiting its application.And progesterone only contraceptives due to irregular vaginal bleeding and poor control of menstrual cycle and other shortcomings,are unfavorable in its clinical application.Therefore,the development of a new generation of OCs,which is capable of avoiding and reducing the above mentioned adverse effects is imminent.Mifepristone is a synthetic 19-norsteroid compound with potent anti-glucocorticoid and anti-progesterone characteristics.Since its discovery it has had a wide range clinical applications including obstetrics and gynecology,oncology,immunology and endocrinology.Currently,mifepristone is also a highly effective emergency contraception(EC)method after an unprotected intercourse,and has been shown to have the potential of being a novel regular contraception with few side effects by achieving "endometrial contraception" via the mechanism of preventing implantation without compromising ovulation.However,the precise mechanism of action underlying the contraceptive effects of mifepristone remains to be elucidated.Macrophages,the most plastic immune cells,are found in all tissues and can change their functions in response to microenvironments where they reside.Due to the near absence of dendritic cells in the human uterus,macrophages represent the major subset of antigen-presenting cells(APCs).Uterine macrophages may play a critical role in several processes required for a successful implantation,including remodeling of the endometrium,immunoregulation of neighboring immune cells,regulation of trophoblast invasion and fetal-maternal immune tolerance.Deeper understanding of the precise contraceptive mechanisms of mifepristone may provide theory support for the development of new methods for fertility control Part ? Uterine macrophages are alternative activated under basal conditions.Objective:To determine the macrophage activation state of uterine macrophages under basal conditions.Methods:Mononuclear cells isolated from peripheral blood of early pregnant women were induced to differentiate into classically activated(M1)macrophages and alternatively activated(M2)macrophages.At the same time uterine macrophages of high purity isolated from early pregnancy decidual tissue were cultured.The morphology of uterine macrophages was observed under microscope.CD14+ surface markers of uterine macrophages were detected by flow cytometry.The production of IL-12p70,IL-23,IL-p40 and IL-10 in the supernatant of the culture of uterine macrophages was detected by ELISA and was compared with the corresponding cytokines secreted by Ml macrophages and M2 macrophages,respectively.Results:The mean purity of the isolated uterine macrophages purified using immunomagnetic cell sorting was 90%or higher.In contrast with M1 macrophages,uterine macrophages produced significantly higher levels of IL-10 and significantly lower levels of IL-12p70,IL-23,and IL-p40,whereas uterine macrophages showed a similar cytokine profile with M2 macrophages.Conclusion:Uterine macrophages are alternative activated under basal conditions,which may facilitate embryo implantation and pregnancy.Part II Mifepristone switches uterine macrophages from an M2 phenotype toward an Ml phenotype via GR antagonismObjective:To explore the effects of mifepristone on uterine macrophage activation.Methods:Uterine macrophages isolated from human decidua were exposed to the indicated concentrations(65nmol,200nmol/L)of mifepristone in the absence or presence of progesterone(2umol/L,6umol/L)alone or dexamethasone(2umol/L,6umol/L)alone in vitro.After 24h exposure,the supernatants of cultured uterine macrophages were collected to detect IL-12 p70,IL-23,IL-12p40 and IL-10 via enzyme-linked immunosorbent assay(ELISA).Results:Low-dose mifepristone-treated uterine macrophages significantly increased the production of IL-12p70,IL-23,and IL-12p40 while significantly decreased the production of IL-10 compared with untreated uterine macrophages,and 200 nmol/L mifepristone-treated uterine macrophages enhanced more IL-12p70,IL-23,and IL-12p40 production and inhibited more IL-10 production than 65 nmol/L mifepristone-treated uterine macrophages.We found that there was no significant difference of cell viability among different treated macrophages.Compared with 200nmol/L mifepristone-treated macrophages,progesterone co-treatment did not reverse the production of IL-12p70,IL-23,IL-p40 and IL-10.Compared with 200nmol/L mifepristone-treated macrophages,dexamethasone co-treatment significantly inhibited the production of IL-12p70,IL-23 and IL-12p40 while significantly increased the production of IL-10.Conclusion:Mifepristone promotes classical macrophage activation via glucocorticoid receptor(GR)antagonism,which may enhance Thl immune response,inhibit Th2 immune response,and be one of its contraceptive mechansims.Part? Mifepristone promotes classical macrophage activation via TAK1 activationObjective:To explore the molecular mechanisms of the effects of mifepristone on uterine macrophage activation.Methods:Uterine macrophages of high purity isolated in vitro were treated with 200 nmol/L mifepristone in the absence or presence of 6umol/L dexamethasone.The phosphorylation of NF??,p38 MAPK,ERK,JNK and TAK1 was detected by Western blotting.The effects of NF??,p3 8 MAPK and TAK1 specific inhibitors JSH-23,SB203580 and 5Z-7-oxozeaenol on the mifepristone-mediated classical macrophage activation were detected,respectively.Results:Mifepristone treatment significantly increased phosphorylation of I??? in uterine macrophages,which was significantly inhibited by dexamethasone.In accordance with the inhibition of I??? phosphorylation,mifepristone-mediated I??? degradation in uterine macrophages was also inhibited by dexamethasone.In addition,mifepristone-induced translocation of phospho-p65 to the nucleus was impaired by dexamethasone.We found that mifepristone treatment resulted in induction of p38 MAPK phosphorylation in uterine macrophages,which was significantly suppressed by dexamethasone.JSH-23 significantly suppressed mifepristone induced production of IL-12p70,IL-23 and IL-12p40,whereas JSH-23 did not increase IL-10 production.We found SB203580 treatment had no effect on mifepristone-induced classical macrophage activation.Treatment with 5z-7-oxozeaenol significantly suppressed mifepristone-induced NF?? activation and significantly inhibited the production of IL-12p70,IL-23 and IL-12p40 while significantly increased IL-10 production.Conclusion:Mifepristone switches uterine macrophages from an M2 phenotype toward an M1 phenotype via TAK1 activation,which may result in implantation failure and achieve contraception.