Mei-Rong Du

Thymic stromal lymphopoietin from trophoblasts induces dendritic cell-mediated regulatory TH2 bias in the decidua during early gestation in humans

Thymic stromal lymphopoietins (TSLPs) play critical roles in dendritic cell-mediated immune responses. In this study, we found that human trophoblasts and decidual epithelial cells in maternal-fetal interface of early placentas express TSLP mRNA and protein, but only trophoblast cells secret soluble TSLP. Human decidual CD1c(+) DCs (dDCs) highly express the functional TSLP receptor complex TSLP receptor and interleukin-7 receptor-α. Recombinant human TSLP activates CD1C(+) decidual DCs and peripheral monocyte-derived DCs with increased costimulatory molecules, major histocompatibility complex class II, and OX-40L. Human TSLP or supernatants from human trophoblasts specifically stimulate dDCs to highly produce interleukin-10 and T(H)2-attracting chemokine CCL-17. The TSLP-activated dDCs prime decidual CD4(+) T cells for T(H)2 cell differentiation, involved in maternal-fetal immunotolerance. Interestingly, the protein expression of TSLP in normal pregnancy with significant T(H)2 bias is much higher than that of miscarriage showing T(H)1 bias at the maternal-fetal interface. Therefore, human trophoblasts may contribute to maternal-fetal tolerance by instructing dDCs to induce regulatory T(H)2 bias in human early pregnancy via TSLP.

Chemokine CXCL12 promotes the cross-talk between trophoblasts and decidual stromal cells in human first-trimester pregnancy

BACKGROUND The precise mechanisms in the materno-fetal dialogue still remain unclear. The aim of this study was to investigate the role of the chemokine CXCL12 and its receptor CXCR4 in the interaction of trophoblasts and decidual stromal cells (DSCs). METHODS Expression of CXCL12/CXCR4 in trophoblasts and DSCs was detected by reverse transcription-polymerase chain reaction and immunochemical staining. The secretion of CXCL12 by trophoblasts was determined by enzyme-linked immunosorbent assay. The effects of CXCL12 on the biological functions of trophoblasts and DSCs were analyzed using a cell viability assay, matrigel invasion assay and zymography. Finally, a co-culture model was established to investigate the modulation of CXCL12/CXCR4 in the interaction of trophoblasts and DSCs. RESULTS CXCR4 was transcribed and translated by both human trophoblasts and DSCs. Human trophoblasts secreted CXCL12 spontaneously in vitro, but DSCs did not. CXCL12 induced an apparent increase in the invasiveness of trophoblasts (P < 0.01), and up-regulated matrix metalloproteinase (MMP) 9 and MMP2 activity of both trophoblasts and DSCs (both P < 0.01) in an autocrine and paracrine manner. The invasiveness and MMP9 and MMP2 activity of trophoblasts in co-culture with DSCs increased significantly (P < 0.01), and these could be inhibited by anti-CXCR4 neutralizing antibody. CONCLUSIONS CXCL12 secreted by human trophoblasts enhances the coordination between trophoblasts and DSCs, via the regulation of MMP9 and MMP2, which may improve the functional materno-fetal interface.

The integrative roles of chemokines at the maternal–fetal interface in early pregnancy

Embryos express paternal antigens that are foreign to the mother, but the mother provides a special immune milieu at the fetal–maternal interface to permit rather than reject the embryo growth in the uterus until parturition by establishing precise crosstalk between the mother and the fetus. There are unanswered questions in the maintenance of pregnancy, including the poorly understood phenomenon of maternal tolerance to the allogeneic conceptus, and the remarkable biological roles of placental trophoblasts that invade the uterine wall. Chemokines are multifunctional molecules initially described as having a role in leukocyte trafficking and later found to participate in developmental processes such as differentiation and directed migration. It is increasingly evident that the gestational uterine microenvironment is characterized, at least in part, by the differential expression and secretion of chemokines that induce selective trafficking of leukocyte subsets to the maternal–fetal interface and regulate multiple events that are closely associated with normal pregnancy. Here, we review the expression and function of chemokines and their receptors at the maternal–fetal interface, with a special focus on chemokine as a key component in trophoblast invasiveness and placental angiogenesis, recruitment and instruction of immune cells so as to form a fetus-supporting milieu during pregnancy. The chemokine network is also involved in pregnancy complications.

Embryonic Trophoblasts Induce Decidual Regulatory T Cell Differentiation and Maternal–Fetal Tolerance through Thymic Stromal Lymphopoietin Instructing Dendritic Cells

Physiological pregnancy requires the maternal immune system to recognize and tolerate embryonic Ags. Although multiple mechanisms have been proposed, it is not yet clear how the fetus evades the maternal immune system. In this article, we demonstrate that trophoblast-derived thymic stromal lymphopoietin (TSLP) instructs decidual CD11c+ dendritic cells (dDCs)with increased costimulatory molecules; MHC class II; and Th2/3-type, but not Th1-type, cytokines. TSLP-activated dDCs induce proliferation and differentiation of decidual CD4+CD25− T cells into CD4+CD25+FOXP3+ regulatory T cells (Tregs) through TGF-β1. TSLP-activated dDC–induced Tregs display immunosuppressive features and express Th2-type cytokines. In addition, decidual CD4+CD25+FOXP3+ Tregs promote invasiveness and HLA-G expression of trophoblasts, resulting in preferential production of Th2 cytokines and reduced cytotoxicity in decidual CD56brightCD16− NK cells. Of interest, decreased TSLP expression and reduced numbers of Tregs were observed at the maternal–fetal interface during miscarriage. Our study identifies a novel feedback loop between embryo-derived trophoblasts and maternal decidual leukocytes, which induces a tolerogenic immune response to ensure a successful pregnancy.

Cyclosporin A Improves Pregnancy Outcome by Promoting Functions of Trophoblasts and Inducing Maternal Tolerance to the Allogeneic Fetus in Abortion-Prone Matings in the Mouse

Abstract The embryo expresses paternal antigens foreign to the mother, and therefore has been viewed as a natural allograft. Cyclosporin A (CsA) is an immunosuppressant for preventing allograft rejection. Little is known, however, about the modulating effect of CsA on the materno-fetal relationship. In this study, pregnant CBA/J female mice mated with DBA/2 or BALB/c male mice as abortion-prone and normal pregnancy matings were administered, respectively, with CsA at Day 4 of gestation. We demonstrated that the administration of CsA at the window of implantation resulted in maternal T-cell tolerance to paternal antigen, and it improved pregnancy outcome in the CBA/J ⊠ DBA/2 abortion-prone matings. CsA administration enhanced Th2 and reduced Th1 cytokine production at the materno-fetal interface, and it expanded peripheral CD4+CD25+ FOXP3+ regulatory T cells in abortion-prone matings, implying development of Th2 bias and regulatory T cells. On the other hand, we observed that treatment with CsA led to enhanced growth and invasiveness of trophoblasts in the abortion-prone matings. Together, these findings indicate that CsA in lower dosages can induce materno-fetal tolerance and improve the biologic functions of trophoblast cells in the abortion-prone matings, leading to a successful pregnancy, which is useful in clinical therapeutics for spontaneous pregnancy wastage and other pregnancy complications.

The CXCL12/CXCR4 axis is involved in the maintenance of Th2 bias at the maternal/fetal interface in early human pregnancy

The regulatory mechanism of Th2 bias at the maternal/fetal interface remains unclear. In this study, we characterized cytokine production in decidual stromal cells (DSCs), decidual immune cells (DICs) and embryo-derived trophoblast cells, and investigated the regulation of CXCL12/CXCR4 interaction on Th2 bias at the maternal/fetal interface in early human pregnancy. We found differential production of Th1-type and Th2-type cytokines by trophoblasts, DSCs and DICs. The secretion of these cytokines varied in different cell cocultures, conduced to Th2 bias. Flow cytometry showed that coculture of trophoblasts with DSCs and DICs significantly increased IL-4 and IL-10 production in trophoblasts, and IL-10 production in DSCs. However, the coculture of trophoblasts with DSCs and DICs significantly increased interferon (IFN)-γ expression in DSCs, and tumor-necrosis factor (TNF)-α expression in DICs. No change was seen in Th1-type cytokine production in trophoblasts, and in Th2-type cytokine production in DICs in all cocultures. Furthermore, pre-treatment with anti-CXCR4 neutralizing antibody upregulated the production of the Th1-type cytokines IFN-γ and TNF-α, and downregulated the production of the Th2-type cytokines IL-4 and IL-10, in trophoblasts, DSCs, DICs or their cocultures. Interestingly, rhCXCL12 inhibited production of the Th1-type cytokine TNF-α and enhanced the expression of the Th2-type cytokines such as IL-4 and IL-10 in DICs; this effect was abrogated by anti-CXCR4 antibody. Our present study has elucidated the individual contributions of component cells to the shaping of Th2 bias, and uncovered a complicated cross-talk via the CXCL12/CXCR4 signal at the maternal/fetal interface in early human pregnancy.

Cyclosporin A Promotes Growth and Invasiveness In Vitro of Human First-Trimester Trophoblast Cells Via MAPK3/MAPK1-Mediated AP1 and Ca2+/Calcineurin/NFAT Signaling Pathways

Cyclosporin A (CsA) has provided the pharmacologic foundation for organ transplantation as a calcineurin inhibitor blocking T-cell activation. We have demonstrated that CsA promoted trophoblast viability/proliferation and invasion in vitro. In the present study, we further investigated the intracellular signalling pathways involved in enhancing cell viability/proliferation and invasiveness of the human trophoblast induced by CsA. We showed that blocking mitogen-activated protein kinase 3 (MAPK3)/MAPK1 signaling by U0126 attenuated CsA-increased cell viability and invasiveness of trophoblasts. Cyclosprin A inhibited ionomycin-stimulated nuclear factor of activated T-cells (NFAT) transactivation in JAR cells and reversed the ionomycin-inhibited trophoblast invasiveness. However, either activating calcineurin by ionomycin, resulting in NFAT transactivation, or inhibiting NFAT using an NFAT inhibitor had no effect on trophoblast cell viability/proliferation and apoptosis in vitro. Hence, the CsA-induced promotion of trophoblast growth and invasion occurred by overlapping but independent pathways. The MAPK3/MAPK1 pathway was essential for both trophoblast growth and invasion, whereas the Ca(2+)/calcineurin/NFAT pathway was only involved in the CsA-promoted trophoblast invasiveness. Finally, potential cross-talk between MAPK3/MAPK1 and Ca(2+)/calcineurin/NFAT and its relationship to activator protein 1 activation was investigated. Our findings explored possible signal transduction pathways modulated by CsA, which may lead to the expansion of the clinical applications of this drug.

CXCL12/CXCR4 Axis Triggers the Activation of EGF Receptor and ERK Signaling Pathway in CsA-Induced Proliferation of Human Trophoblast Cells

Introduction Our previous study has demonstrated Cyclosporin A (CsA) promotes the proliferation of human trophoblast cells. Therefore, we further investigate the intracellular signaling pathway involved in the CsA-induced proliferation of human trophoblast cells. Methods Enzyme-linked immunosorbent assay (ELISA) was performed to evaluate the regulation of CsA on CXCL12 secretion in human trophoblast cells. Immunofluorescence analysis and western blotting analysis were used to investigate the role of CXCL12/CXCR4 axis in the CsA-induced epidermal growth factor receptor (EGFR) phosphorylation in human trophoblast cells. 5-bromo-2′-deoxyuridine (BrdU) cell proliferation assay was performed to analyze the involvement of EGFR and its downstream extracellular signal-regulated protein kinase (ERK) signaling pathway in the CsA-induced proliferation of human trophoblast cells. Results Low concentration of CsA promoted the secretion of CXCL12, and recombinant human CXCL12 promoted the phosphorylation of EGFR in primary human trophoblast cells and choriocarcinoma cell line JEG-3. The inhibition of CXCL12 or CXCR4 by either neutralizing antibodies or small interfering RNA (siRNA) could completely block the CsA-induced EGFR phosphorylation. The CsA-induced proliferation of human trophoblast cells was effectively abrogated by the EGFR inhibitor AG1478 as well as the ERK inhibitor U0126, but not by the PI3K/PKB inhibitor LY294002. CsA promoted the activation of ERK in JEG-3 cells, which was markedly abrogated in the presence of CXCL12 siRNA, or CXCR4 siRNA, or AG1478. Conclusions CsA may promote EGFR activation via CXCL12/CXCR4 axis, and EGFR downstream ERK signaling pathway may be involved in the CsA-induced proliferation of human trophoblast cells.

Cyclosporin A improves murine pregnancy outcome in abortion-prone matings: involvement of CD80/86 and CD28/CTLA-4

Immune regulation during pregnancy is complex, and thus an optimal therapy for pregnancy complications is always a big challenge to reproductive medicine. Cyclosporin A (CsA), a potent immunosuppressant, prevents rejection of allografts by hosts, but little is known about the modulating effect of CsA on the materno-fetal relationship. Here, pregnant CBA/J females mated with DBA/2 males as an abortion-prone model were administered with CsA on day 4.5 of gestation, and the pregnant CBA/J females mated with BALB/c males were established as successful pregnancy control. It was demonstrated that administration of CsA at the window of implantation significantly up-regulated the expression of CTLA-4, while down-regulating the levels of CD80, CD86, and CD28 at the materno-fetal interface in the CBA/J x DBA/2 abortion-prone matings, and the embryo resorption rate of the abortion-prone matings reduced significantly after CsA treatment, implying that modulation of costimulatory molecule expression by CsA might contribute to preventing the fetus from maternal immune attack. In addition, treatment with CsA induced enhanced growth and reduced cell apoptosis of the murine trophoblast cells. Together, these findings indicate that CsA has a beneficial effect on the materno-fetal interface in abortion-prone matings, leading to a pregnancy outcome improvement, which might provide new therapeutics for spontaneous pregnancy wastage.

E-Cadherin, as a Negative Regulator of Invasive Behavior of Human Trophoblast Cells, Is Down-Regulated by Cyclosporin A Via Epidermal Growth Factor/Extracellular Signal-Regulated Protein Kinase Signaling Pathway

Our previous study has demonstrated cyclosporin A (CsA) promotes the invasiveness of human first-trimester trophoblast cells. In the present study, we further investigated the intracellular signaling pathway responsible for the improvements in CsA-induced invasiveness of human trophoblast cells. We showed that CsA down-regulated E-cadherin transcription and translation in human primary cultured trophoblast cells and choriocarcinoma cell line JEG-3. U0126, an inhibitor of extracellular signal-regulated protein kinase (ERK), attenuated the CsA-induced transcriptional repressor SNAI2 (also called Slug) expression and restored E-cadherin expression inhibited by CsA in JEG-3 cells. We further demonstrated that CsA amplified epidermal growth factor (EGF)-stimulated EGF receptor (EGFR) tyrosine phosphorylation in JEG-3 cells, and inhibition of EGFR tyrosine phosphorylation by AG1478, an EGFR tyrosine kinase inhibitor, abolished the down-regulation of E-cadherin by CsA through ERK signaling pathway. Moreover, our data showed that E-cadherin expression was negatively correlated to the invasiveness of JEG-3 cells, and CsA could reverse the decreased invasiveness of JEG-3 cells that resulted from E-cadherin overexpression. In conclusion, these observations indicate that CsA may decrease E-cadherin expression via EGFR/ERK signaling pathway and, ultimately, contribute to the invasiveness improvement of human trophoblast cells.