Cyclosporin A Improves The Cross-talking At Materno-fetal Interface Of Human First-trimester Pregnancy

Research Background Embryo can be viewed as a natural allografl owing to theexpression of patemal antigens foreign to its mother. Thereby, induction of matemalimmune tolerance toward the fetus becomes an important event in the maintenance ofpregnancy. The excessive activation of maternal immuno-competent cells againstembryo antigen results in rejection to the allogeneic fetus and pregnancy wastage. Themechanisms of matemo-fetal immune regulation during pregnancy and improvementof the pregnant outcome are of great interest for researchers in reproductive medicine.Cyclosporin A, CsA), first found in 1970s, has made transplantation abreakthrough. Our previous research has found that a low concentration of CsA(10^-4～1.0μmol/L) can remarkably promote proliferation, migration and invasion invitro of human first-trimester cytotrophoblasts while inhibit apoptosis of these cells,and the study in vivo showed that administration with CsA at the early stage ofpregnancy can increase the secretion of IL-10, decreased secretion of IFN-γ, expandperipheral CD4⁺CD25⁺Foxp3⁺ regulatory T cell, and reduce fetal resorption rate inthe abortion-prone murine CBA/J×DBA/2 matings, which suggests CsA appears to afavorable effect on the materno-fetal relationship by promoting functions oftrophoblast cells and inducing a Th2 bias and maternal tolerance to the allogeneicfetus, leading to a new therapeutic approach for some pregnancy complications.Objective There is a complicated cross-talking between fetus and mother duringpregnancy. Besides the embryo-derived trophoblasts, the maternal derived cells,mainly comprising decidual stromal cells (DSC) and decidual immuno-competentcells (DIC) are also involved in the establishment and maintenance of the matemo-fetal interface, a unique immune milieu. Therefore, we still have a long wayto elucidate the pharmacological action of CsA on the materno-fetal relationship. Thepurpose of this study was to further investigate the mechanisms of CsA in regulationof the first-trimester human trophoblast cells, and explore the potential mechanisms ofCsA in the functional integration between the embryo-derived trophoblast andmaternal cells, DSC and DIC, in decidua.1. CsA improves viability and invasion of human first-trimester trophoblastsvia SDF-1/CXCR4 axis in an autoerine mannerMethods and Results After isolation, purification and characterization of thefirst-trimester human trophoblast cells, the effects of CsA on transcription andtranslation of chemokine SDF-1 and its receptor CXCR4 in trophoblasts wereanalyzed by RT-PCR, In-cell western and ELISA, respectively. The secretion level ofMMPg/MMP2 in the supernatant collected from the invasion assay was determinedby gelatin zymography. It was found that CsA significantly promoted the mRNA andprotein expression of SDF-1/CXCR4 of human first-trimester trophoblast cells viaMAPK/ERK1/2 signaling pathway. Thereafter, we investigated whether the enhancedSDF-1/CXCR4 interaction was involved in the up-regulation of CsA on viability byMTT and invasiveness of trophoblasts by Matfigel invasion assay. SDF-1 or CXCR4neutralizing antibody could significantly decrease the enhanced viability and invasionof trophoblasts induced by CsA. It was also found that SDF-1 or CXCR4 neutralizingantibody could significantly abolish the enhanced secretion of MMP9/MMP2 bytrophoblasts up-regulated by CsA.Conclusion These results suggest CsA improves the expression of SDF-1 andCXCR4 in human first-trimester trophoblasts via MAKP pathway, and theup-regulated SDF-1/CXCR4 interaction promotes the viability and invasiveness oftrophoblasts in an autocrine manner.2. DSC enhances the CsA-promoted invasiveness of the human first-trimestertrophobastsMethods and Results The first-trimester human DSC was isolated and identifiedfor purity. Then, the expression of SDF-1/CXCR4 was detected at both transcriptional and translational level by RT-PCR, immunostaining and ELISA, respectively. Theresults showed that the human first-trimester DSCs only transcribed and translatedCXCR4 at a moderate level, and did not produce SDF-1 at either mRNA or proteinlevel with difference from the human first-trimester trophoblasts that coexpressSDF-1 and its receptor CXCR4. The viability of DSC in vitro could not be improvedby a different concentration of exogenous SDF-1. Simultaneously, though trophoblastconditioned medium (TCM) could remarkably increase the viability of DSC in vitro,SDF-1 or CXCR4 neutralizing antibody could not abolish the enhanced viability ofDSC induced by the TCM. But rhSDF-1 promoted the secretion of MMP9/MMP2 bythese cells in a dose-dependent manner. It was speculated that SDF/CXCR4 signalingpathway was involved in the cross-talk between trophoblats and DSCs throughregulating the production of MMP which is responsible for degradation of ECM.The effects of CsA on viability, expression of SDF-1/CXCR4 and secretion ofMMP9/MMP2 of DSC were investigated by MTT, in-cell western, ELISA and gelatinzymography, respectively. It was showed that there was no detectable change in theviability and expression of SDF-1/CXCR4 of DSC between the CsA-treated groupand the vehicle group. But CsA could significantly promote the secretion ofMMP9/MMP2 by DSC.A trophoblast/DSC coculture model including direct and indirect contact wasestablished to evaluate the effect of CsA on the invasion of trophoblasts. Compared tothe corresponding untreated coculture model, CsA of 1.0μmol/L could remarkablyimprove the invasion of trophoblasts. After having been treated with CsA, theinvasiveness of trophoblasts in both direct and indirect contact was much higher thanthat of the CsA-treated single trophoblast culture. The gelatin zymography showed thesame results. The secretion of MMP9/MMP2 of the CsA treatment in differentcoculture model was also obviously higher than that of the corresponding cuturewithout CsA. Compared to the CsA-treated single trophoblast culture, theMMP9/MMP2 secretion of the CsA treatment in both direct and indirect contact alsoincreased significantly.Conclusion The results above suggest CsA promotes the secretion of MMP9/MMP2by both trophoblats and DSC, and improves invasiveness of trophoblasts in coculturemodel, which contributes to the cross-talking at the matemo-fetal interface.3. Modulation of CsA on the secretion of IL-10/IFN-γat human first-trimester materno-fetal interface.Methods and Results After isolation and culture of human first-trimestertrophoblast cells, DSC and DIC, the secretion of IL-10 and IFN-γby these cells wasdetermined by ELISA. Then coculture in vitro in different array containing these cellswas established, and modulation of CsA on the production of IL-10/IFN-γby thecoculture system was investigated by ELISA. It was found that IL-10 might beproduced by the in vitro cultured human first-trimester trophoblast cells, DSC andDIC, respectively. There was no detectable difference of secretion level of IL-10between the CsA-treatment and the control in the single-cultured trophoblast cells,DSC or DIC, respectively. But after combination of two of the three cells,respectively, CsA significantly increased the secretion of IL-10 in the coculturesystem. Furthermore, when the three cells were all in direct contact coculture, theproduction of IL-10 further increased after treated with CsA. IFN-γcould be producedby DIC in vitro, and CsA did not change the secretion of IFN-γby DIC alone. Butwhen DIC was combined with trophoblast cells and/or DSC, the secretion level ofIFN-γin the three-cell-coculture model decreased after treatment with CsA.Conclusion It could be concluded that CsA promotes the secretion of IL-10 andinhibited that of IFN-γin the three-cell-coculture system, which contributes to a shiftfrom Th1 toward Th2 at the matemo-fetal interface.In conclusion, a low concentration of CsA has multiple modulating effects on thematemo-fetal interface: (1) up-regulation of the viability and invasiveness of humanfirst-trimester trophoblasts via SDF-1/CXCR4 autocrine manner; (2) improvement ofthe interaction between trophoblasts and DSC; (3) induction of Th2 bias. Thus thepharmacological mechanisms of CsA are far from being completely understood, andour results suggest CsA might be used as a unique immunotherapy for somepregnancy complications, such as recurrent spontaneous abortion, fetal growthrestriction and so on.