Da-Jin Li

Human First-Trimester Trophoblast Cells Recruit CD56brightCD16− NK Cells into Decidua by Way of Expressing and Secreting of CXCL12/Stromal Cell-Derived Factor 1

More than 70% of decidual lymphocytes are NK cells characterized by CD56brightCD16− phenotype, but the mechanisms by which these NK cells are recruited in the decidua are still almost unrevealed. In this study, we first analyzed the transcription of 18 chemokine receptors in the first-trimester decidual CD56brightCD16− NK cells. Among these receptors, CXCR4 and CXCR3 were found highly transcribed, and the expression of CXCR4 was verified in most of the decidual CD56brightCD16− NK cells by flow cytometry. The first-trimester human trophoblasts were found expressing CXCL12/stromal cell-derived factor 1, the specific ligand of CXCR4, by way of in situ hybridization and immunohistochemistry. The primary cultured trophoblast cells were also found to secrete stromal cell-derived factor 1α spontaneously, and its concentration was 384.6 ± 90.7 pg/ml after the trophoblast cells had been cultured for 60 h. All of the ligands for CXCR3 were below the minimal detectable concentration when trophoblast cells were cultured for up to 48 h. Both recombinant human SDF-1α and supernatants of the cultured trophoblast cells exhibited chemotactic activity on decidual CD56brightCD16− NK cells. Our findings suggest that human first-trimester trophoblast cells produce CXCL12, which in turn chemoattracts decidual CD56brightCD16− NK cells. This activity could contribute to the recruitment mechanism of decidual lymphocytes, especially CD56brightCD16− NK cells, in decidua, and may be used at a local level to modulate the immune milieu at the materno-fetal interface.

Blockade of CD86 Signaling Facilitates a Th2 Bias at the Maternal-Fetal Interface and Expands Peripheral CD4+CD25+ Regulatory T Cells to Rescue Abortion-Prone Fetuses

Abstract Intervention in B7 (CD80/CD86)/B7-ligand (CD28/CTLA-4) pathways is an effective way of preventing unwanted immune responses, such as allograft rejection. Pregnancy maintenance represents maternal tolerance to the fetal allograft, which is accompanied by a type 2 helper cell (Th2) bias at the maternal-fetal interface. Here, the costimulatory signal of CD86 was selectively blocked, and that of CD80 was kept unimpaired by administration of anti-murine CD86 monoclonal antibody at the early gestational stage in abortion-prone CBA/J×DBA/2 matings and normal pregnant CBA/J×BALB/c matings. It was demonstrated that in vivo blockade of CD86 costimulation could suppress maternal immune attack to the fetus by shifting cytokines from Th1 predominance to Th2 bias at the maternal-fetal interface, and expanding peripheral CD4+CD25+ regulatory T cells, which play an important role in the development and maintenance of maternal-fetal tolerance. Furthermore, the expression of CD28 and its ligands CD80/CD86 on peripheral lymphocytes was down-regulated, whereas that of CTLA-4 was up-regulated, which might facilitate the suppressive effect of CD4+CD25+ regulatory T cells on the alloreactive T cells. The maternal-fetal immunotolerance induced by CD86 blockade decreased fetal resorption in CBA/J×DBA/2 matings, but did not affect normal pregnant CBA/J×BALB/c matings. These results suggest that selective blockade of CD86 costimulation leads to maternal immune tolerance to embryo antigen, and might contribute to a rational immunoregulatory regimen for recurrent spontaneous abortion.

The CD4+CD25bright regulatory T cells and CTLA-4 expression in peripheral and decidual lymphocytes are down-regulated in human miscarriage

The present study was undertaken to analyze the changes in the proportion of CD4(+)CD25(bright) regulatory T (Treg) cells and the expression of costimulatory molecules, CTLA-4 and CD28, in the peripheral blood and deciduas in the setting of non-pregnancy, normal early pregnancy and miscarriage. In this study, we showed that CD4(+)CD25(bright) T cells significantly increased in the peripheral of normal pregnancy compared to that of non-pregnancy. The proportions of CD4(+)CD25(bright) T cells in both peripheral blood and deciduas were significantly lower in miscarriage than that of normal pregnancy. CD4(+)CD25(bright) T cells were characterized by high-level FoxP3 expression and low-level CD69 expression. An increase in the CD28 mRNA expression was accompanied by a decrease in the CTLA-4 mRNA expression in decidual tissues from human miscarriage. The ratios of CTLA-4(+)/CD28(+) in miscarriage were significantly lower than that of the normal pregnancy both in the peripheral and in deciduas. The ratio of CTLA-4(+)/CD28(+) in CD4(+)CD25(bright) T cells was significantly higher than that of the CD4(+)CD25(dim) T cells both in normal pregnancy and in miscarriage. The decidual T cells in the miscarriage appeared higher in responsiveness and IL-2 and IFN-gamma production in comparison with the decidual T cells in the early pregnancy. These results above suggest that CD4(+)CD25(bright) Treg cells might play a role in the maintenance of pregnancy via up-regulation of CTLA-4 expression. The down-regulation of Treg cells and their functions, and the imbalance of positive and negative regulators of costimulatory signals might lead to an abnormal immune milieu, which confer susceptibility to pregnancy loss.

The Expression of CXCR4/CXCL12 in First-Trimester Human Trophoblast Cells

Abstract Chemokines and chemokine receptors have been implicated as pivotal players in many physiological and pathological situations, but little is known about the expression and function of chemokines and chemokine receptors at the materno-fetal interface. In this study, we first analyzed the transcription of 18 chemokine receptors in first-trimester human trophoblast cells. Among these receptors, CXCR4 was found highly transcribed. We demonstrated afterward that both CXCR4 and CXCL12 (stromal cell-derived factor-1; SDF-1) were expressed in trophoblast cells. Primary cultured trophoblast cells were also found secreting CXCL12 spontaneously. To identify the functional role of CXCR4/CXCL12 in these cells, we treated trophoblast cells with recombinant human (rh)SDF-1α and analyzed the cell viability and signaling pathway. The results showed that rhSDF-1α increased the viability of trophoblast cells and the activation of extracellular signal-regulated kinases signaling pathway in vitro. Our findings suggest that first-trimester trophoblast cells express functional CXCR4/CXCL12, which may play an important role in early pregnancy such as stimulating trophoblast cell proliferation or differentiation in an autocrine manner.

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.

Human Trophoblasts Recruited T Lymphocytes and Monocytes into Decidua by Secretion of Chemokine CXCL16 and Interaction with CXCR6 in the First-Trimester Pregnancy

During human early pregnancy, fetus-derived trophoblasts come into direct contact with maternal immune cells at the maternofetal interface. At sites of placental attachment, invasive extravillous trophoblasts encounter decidual leukocytes (DLC) that accumulate within the decidua. Because we first found chemokine CXCL16 was highly expressed in and secreted by the first-trimester human trophoblasts previously, in this study we tested the hypothesis of whether the fetal trophoblasts can direct migration of maternal T lymphocyte and monocytes into decidua by secreting CXCL16. We analyzed the transcription and translation of CXCL16 in the isolated first-trimester human trophoblast, and examined the kinetic secretion of CXCL16 in the supernatant of the primary-cultured trophoblasts. We demonstrated that the sole receptor of CXCL16, CXCR6, is preferentially expressed in T lymphocytes, NKT cells, and monocytes, hardly expressed in two subsets of NK cells from either the peripheral blood or decidua. We further demonstrated the chemotactic activity of CXCL16 in the supernatant of the primary trophoblast on the peripheral mononuclear cells and DLC. Moreover, the CXCL16/CXCR6 interaction is involved in the migration of the peripheral T lymphocytes, γδ T cells, and monocytes, but not NKT cells. In addition, the trophoblast-conditioned medium could enrich PBMC subsets selectively to constitute a leukocyte population with similar composition to that of DLC, which suggests that the fetus-derived trophoblasts can attract T cells, γδ T cells, and monocytes by producing CXCL16 and interaction with CXCR6 on these cells, leading to forming a specialized immune milieu at the maternofetal interface.

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.

CXCL8 enhances proliferation and growth and reduces apoptosis in endometrial stromal cells in an autocrine manner via a CXCR1-triggered PTEN/AKT signal pathway

BACKGROUND Chemokine CXCL8 (also known as IL-8) has been identified as a potential regulator of endometrial stromal cells (ESCs), but it is unclear how CXCL8 regulates the survival of ESCs in the pathogenesis of endometriosis. METHODS We assessed the secretion of CXCL8 by enzyme-linked immunosorbent assays and the expression of its receptors, CXCR1 and CXCR2, by in-cell Western assay and immunohistochemistry. The effects of CXCL8 on the activation or expression of various cell mediators were also investigated by in-cell Western assay. The effects of CXCL8 on the proliferation, growth and apoptosis of ESCs in vitro were assessed by BrdU assays, cell counts and annexin V labeling, respectively. RESULTS Secretion of CXCL8 and expression of CXCR1 in the eutopic ESCs from women with endometriosis were significantly higher than that in control ESCs, but the expression of CXCR2 showed no significant difference between these two cell types. CXCL8 stimulated proliferation and growth and reduced apoptosis of ESCs in an autocrine manner, and these effects were abolished by anti-human CXCL8 and CXCR1 neutralizing antibodies and by a PI3K/Akt inhibitor. Moreover, CXCL8 up-regulated the expression of the anti-apoptotic proteins, survivin and Bcl-2, inhibited the expression of the Phosphatase and tensin homolog (PTEN) and activated the phosphorylation of Akt. CONCLUSIONS This study suggests that CXCL8 and CXCR1 are involved in the pathogenesis of endometriosis by up-regulating proliferation and growth and restricting apoptosis in ESCs by activating the PTEN/Akt pathway and mediating the expression of survivin and Bcl-2.

The decidual gamma-delta T cells up-regulate the biological functions of trophoblasts via IL-10 secretion in early human pregnancy.

The functions of decidual γδT cells in early human pregnancy are not fully understood. The present study was undertaken to characterize the action of decidual γδT cells, and analyze their regulatory roles in proliferation and invasion of trophoblasts in early human pregnancy. It was revealed that decidual γδT cells were a CD4(-) and CD8(-) subset whose numbers increased significantly in either the decidua or peripheral blood. The main cytokines synthesized in decidual γδT cells in decreasing concentration were; IL-10>TGF-β>TNF-α≥IFN-γ, with little IL-2 or IL-4 being produced. Decidual γδT cells promoted trophoblast proliferation and invasion, and suppressed trophoblast apoptosis through IL-10 secretion in co-culture. These results suggest that γδT cells in human decidua might play an important role in the Th2 bias at maternal-fetal interface and in the development and progression of the placenta, which is beneficial to maternal immunotolerance toward the fetus in early human pregnancy.