CXCL12/CXCR4 signal involved in the regulation of trophoblasts on peripheral NK cells leading to Th2 bias at the maternal-fetal interface.

OBJECTIVE: In the early pregnancy, large number of decidual natural killer (dNK) cells are present in decidua, which exhibit distinctive phenotype and functions from peripheral blood NK cells (pNK)1. Unlike the cytotoxic pNK cells, dNK cells display more pronounced characteristics of immune tolerance, which contribute the Th2 bias at the maternal-fetal interface and ensure successful pregnancy2. However, the origin and the differentiation program of dNK still remain unknown. MATERIALS AND METHODS: Our previous study has demonstrated that the CXCL12/CXCR4 signal axis is involved in the shaping of Th2 bias at the maternal-fetal interface3,4. RESULTS: In this study, we demonstrated the first-trimester human trophoblast cells secrete chemokine CXCL12 that can recruit pNK cells to the decidua. We've also found that the pNK cells differentiate locally under the influence of trophablast cells. After co-culture with trophoblast cells, pNK cells could acquire dNK characteristics phenotypically while compared to the dNK cells; however, the blocking of CXCL12/CXCR4 signal of pNK cells has abrogated the modulation of trophoblast cells on the pNK cells. We've also found that JNK1/2/MAPK and ERK/MAPK signal pathways were required for the modulation of trophoblast cells on the pNK cells. MAPK signal pathway is involved in the functional modulation of human first-trimester trophoblast cells and decidual stromal cells on pNK and dNK cells. CONCLUSIONS: Our study has elucidated that CXCL12/CXCR4 can recruit pNK cells to the decidua, then positively modulate pNK cells differentiation into the dNK cells, which thus results in Th2 bias and maternal-fetal immune tolerance.

PD-1 and Tim-3 pathways are associated with regulatory CD8+ T-cell function in decidua and maintenance of normal pregnancy.

CD8+ T cells are critical in the balance between fetal tolerance and antiviral immunity. T-cell immunoglobulin mucin-3 (Tim-3) and programmed cell death-1 (PD-1) are important negative immune regulatory molecules involved in viral persistence and tumor metastasis. Here, we demonstrate that Tim-3+PD-1+CD8+ T cells from decidua greatly outnumbered those from peripheral blood during human early pregnancy. Co-culture of trophoblasts with CD8+ T cells upregulated PD-1+ and/or Tim-3+ immune cells. Furthermore, the population of CD8+ T cells co-expressing PD-1 and Tim-3 was enriched within the intermediate memory subset in decidua. This population exhibited high proliferative activity and Th2-type cytokine producing capacity. Blockade of Tim-3 and PD-1 resulted in decreased in vitro proliferation and Th2-type cytokine production while increased trophoblast killing and IFN-γ producing capacities of CD8+ T cells. Pregnant CBA/J females challenged with Tim-3 and/or PD-1 blocking antibodies were more susceptible to fetal loss, which was associated with CD8+ T-cell dysfunction. Importantly, the number and function of Tim-3+PD-1+CD8+ T cells in decidua were significantly impaired in miscarriage. These findings underline the important roles of Tim-3 and PD-1 pathways in regulating decidual CD8+ T-cell function and maintaining normal pregnancy.

Hyaluronan up-regulates growth and invasion of trophoblasts in an autocrine manner via PI3K/AKT and MAPK/ERK1/2 pathways in early human pregnancy.

INTRODUCTION: As one of the key molecules in the extracellular matrix in human conceptus, hyaluronan (HA) has been receiving particular attention. Here, we have investigated the expression and regulation of different molecular weight HA on the biological behaviors of primary human trophoblasts during the first trimester of pregnancy. METHODS: The expression of HA and HA synthetase (HAS) by human first trimester trophoblasts was analyzed in placentae from normal pregnancy or miscarriage by immunochemistry and real-time RT-PCR, respectively. ELISA was used to measure the secretion of HA by primary trophoblasts. The effects of HA on the proliferation, apoptosis and invasiveness of trophoblasts were examined. We also investigated the signaling pathways involved in HA activation in human trophoblasts. RESULTS: The higher HAS2 expression and HA secretion were observed in normal villi than that of miscarriage, and the primary trophoblasts secreted HA continuously. High molecular weight HA (HMW-HA) and medium molecular weight HA (MMW-HA) promoted proliferation and invasiveness while inhibited apoptosis of trophoblasts. However, low molecular weight HA (LMW-HA) had no obvious effect on the growth or invasiveness of human trophoblasts. In addition, HMW-HA showed more efficiently than MMW-HA on the growth while MMW-HA displayed a more obvious effect on the invasiveness of trophoblasts than HMW-HA. HMW-HA activated PI3K/AKT and MAPK/ERK1/2 signaling pathways in trophoblasts. Blocking PI3K/AKT or MAPK/ERK1/2 signaling inhibited the HA-upregulated growth and invasiveness of human trophoblasts. CONCLUSION: Our results suggest that higher level and greater molecular mass of HA can promote trophoblast growth and invasion in an autocrine manner, which was beneficial to placentation and maintenance of human early pregnancy.

RANKL promotes the growth of decidual stromal cells in an autocrine manner via CCL2/CCR2 interaction in human early pregnancy.

OBJECTIVE: Receptor-activator of NF-κB ligand (TNFSF11, also known as RANKL) and its receptor RANK are essential regulators on bone remodeling, mammary gland development and hormone-associated breast cancer development. However, the expression pattern and role of RANKL/RANK axis in decidual stromal cells (DSCs) are unclear in human early pregnancy. STUDY DESIGN: We analyzed RANKL/RANK expression in DSCs by real-time PCR, immunhistochemistry, enzyme-linked immunosorbent assay (ELISA) and flow cytometry, respectively. Then BrdU cell proliferation assay, flow cytometry assay and ELISA were performed to investigate the effect of recombinant human RANKL and DSCs-derived RANKL on the proliferation, apoptosis, chemokine (C-C motif) ligand 2 (CCL2) secretion, C-C chemokine receptor type 2 (CCR2) and other target proteins expression in DSCs in vitro, respectively. RESULTS: Here we show that DSCs co-express RANKL/RANK. Not only recombinant human (rh) RANKL but also the DSC-secreted RANKL stimulate proliferation and anti-apoptosis, and elevate CCL2 secretion and CCR2 expression of DSCs. Furthermore, the stimulatory effects on the proliferation, anti-apoptosis and the expression of Bcl-2 and Ki67 and inhibitory signaling on Fas ligand (FasL) in DSCs induced by RANKL can be partly reversed by the way of blocking CCL2 and or CCR2. CONCLUSIONS: Our results have revealed that RANKL/RANK signal promotes Bcl-2 and Ki67 and decreases FasL expression, and further as a positive regulator for stimulating the proliferation and growth of DSCs through up-regulating CCL2/CCR2 signal, which finally contributes to the establishment and maintenance of physiological pregnancy.

Cyclosporine A promotes in vitro migration of human first-trimester trophoblasts via MAPK/ERK1/2-mediated NF-κB and Ca2+/calcineurin/NFAT signaling.

INTRODUCTION: As a calcineurin inhibitor in T-cell activation, cyclosporine A (CsA) has provided the pharmacologic foundation for organ transplantation. We have previously demonstrated that CsA promotes trophoblast growth and invasion in vitro. Here, we further investigated the regulation of CsA on trophoblast migration and the intracellular signaling pathways involved. METHODS: We evaluated the migration of human primary trophoblasts by using transwell migration assay. CsA-mediated induction of nuclear factor-kappa B (NF-κB) was evaluated by cotransfection with luciferase reporter constructs and luciferase activity assays. RESULTS: Treatment with CsA-promoted migration of primary trophoblasts and the HTR8 cell line in a dose-dependent manner. CsA also increased NF-κB-transcriptional activity in trophoblasts in time- and dose-dependent manners. Pharmacologically inhibiting mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) 1/2 signaling with U0126 attenuated the CsA-induced cell migration and NF-κB activity in trophoblasts. Furthermore, pretreatment with PDTC, a specific NF-κB inhibitor, inhibited the CsA-induced migration of trophoblasts in dose-dependent manners. Although NFAT activation by ionomycin via calcineurin is accompanied by increased transactivation of NF-κB, pretreatment with the NFAT inhibitor did not affect NF-κB-transcriptional activity. Interestingly, ionomycin and CsA synergize to transactivate NF-κB. Ionomycin-inhibited basal migration of trophoblasts, and pretreatment with CsA reversed the ionomycin-inhibited trophoblast migration. However, the NFAT inhibitor increased basal migration, but not CsA-induced migration, of trophoblasts. CONCLUSION: These observations indicate that both the MAPK/ERK/NF-κB pathway and Ca(2+)/calcineurin/NFAT pathways are involved in the CsA-promoted trophoblast migration. Our findings may help expand the clinical applications of this drug in trophoblast disorder.

Focal adhesion kinase signaling is necessary for the Cyclosporin A-enhanced migration and invasion of human trophoblast cells.

OBJECTIVES: Our previous studies have shown that Cyclosporin A (CsA) promotes human trophoblast invasion via mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) pathway. E-cadherin and matrix metalloproteinases (MMPs) are important mediators in trophoblast migration and invasion. Here, we further investigate the role of focal adhesion kinase (FAK) signaling in the CsA-induced trophoblast migration and invasion and ERK activation. STUDY DESIGN: The migration and invasion of human primary trophoblasts and JEG-3 cells were measured by transwell migration and matrigel invasion assays. The activation of FAK, Src and ERK induced by CsA were examined by western blot. The colocalization of FAK and Src was detected by dual immunofluorescence assay. The regulation of E-cadherin expression and matrix metalloproteinases (MMPs) activity was evaluated by western blot and gelatin zymography, respectively. RESULTS: CsA increased the phosphorylation of FAK and Src in human primary trophoblasts and JEG-3 cells. Meanwhile, the activated FAK and Src colocalized in the cytoplasm of JEG-3 cells. The FAK inhibitor Y15 or Src inhibitor PP2 could abrogate the phosphorylation of ERK, the enhanced migration and invasion and the activity of MMP2, 9 induced by CsA. In addition, these inhibitors also restored the expression of E-cadherin which is down-regulated by CsA. However, U0126, an inhibitor of ERK, had no significant effect on the CsA-induced activation of FAK and Src. CONCLUSIONS: FAK-Src signaling, the upstream signaling cascade of ERK activation, plays an important role in the CsA-induced migration and invasion via down-regulating expression of E-cadherin and up-regulating activity of MMP2, 9 in trophoblast cells. These results may help provide a rationale to develop a novel therapeutic strategy for pregnancy disorders from insufficient trophoblast invasion.

Cyclosporin A promotes crosstalk between human cytotrophoblast and decidual stromal cell through up-regulating CXCL12/CXCR4 interaction.

BACKGROUND: Our previous studies have demonstrated that cyclosporin A (CsA) can increase the cell number in and invasion by human first-trimester trophoblasts and induce maternal-fetal tolerance. C-X-C chemokine receptor type 4 (CXCR4) and C-X-C chemokine ligand 12 (CXCL12) are important mediators at the maternal-fetal interface during early pregnancy. In this study, we further investigate the molecular mechanisms underlying modulation by CsA of the crosstalk between human cytotrophoblast and decidual stromal cell (DSC). METHODS: Human first-trimester cytotropoblast and DSC were treated with CsA in the absence or presence of U0126 pretreatment, and then the mRNA and protein levels of CXCL12 and CXCR4 were measured by RT-PCR, qPCR, in-cell western blots and enzyme-linked immunosorbent assay (ELISA), respectively. 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide and Matrigel invasion assays were used to determine the invasiveness of cytotrophoblast, respectively. The activity of matrix metalloproteinase (MMP)-9 and MMP-2 was detected by gelatin zymography. A co-culture with direct contact between cytotrophoblast and DSC was established and used to investigate the interaction between these two cells. RESULTS: CsA up-regulated CXCL12 and CXCR4 expression in human first-trimester cytotrophoblast cells, but not in DSCs. Blocking the mitogen-activated proteinkinase/extracellular signal-regulated kinase (MAPK/ERK1/2) signaling by U0126 abrogated the CsA-induced increase in CXCL12 and CXCR4 expression and neutralizing antibodies to CXCL12 or CXCR4 completely inhibited the CsA-induced increase in cell number, invasion and MMP-9 and MMP-2 activity of cytotrophoblast. CsA also significantly promoted the activity of MMP-9 and MMP-2 in DSCs, but this was unaffected by CXCL12 or CXCR4 neutralizing antibody. Furthermore, the CsA-induced MMP-9 and MMP-2 activity and the invasiveness of cytotrophoblast in the cytotrophoblast and DSC co-culture were significantly increased compared with CsA-treated trophoblast cultured alone, and CXCR4 blocking antibody effectively abolished the increased MMP activity and invasion of cytotrophoblasts in the cytotrophoblast-DSC co-culture stimulated by CsA. CONCLUSIONS: CsA can promote the crosstalk between cytotrophoblast and DSC through up-regulating CXCL12/CXCR4 interaction via MAPK signaling, resulting in the increased numbers of and invasion by human cytotrophoblast cells.