Song-Cun Wang

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.

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.

CD56(bright)CD25+ NK cells are preferentially recruited to the maternal/fetal interface in early human pregnancy.

Decidual natural killer (dNK) cells are believed to be critical for maintaining maternal/fetal tolerance and regulating placental vascular remodeling based upon their abundance and unique phenotype during early pregnancy. However, the mechanism for how the dNK cells play such important roles in successful pregnancy remains undefined. Here, we identified a subtype of dNK cells characterized as having a CD3−CD56brightCD25+ phenotype. We found that CD56brightCD25+ NK cells preferentially localize to the maternal/fetal interface during early human pregnancy. CD25+ dNK cells account for approximately 75% of CD25-expressing decidual immune cells (DICs). However, less than 5% of CD25-positive peripheral blood mononuclear cells are CD25+ NK cells. Furthermore, CD25+ and CD25− dNK cells exhibit distinct phenotypes: CD25+ dNK cells display a more activated phenotype and greater cytokine-secreting capacity. Interestingly, coculture of peripheral NK (pNK) cells with primary trophoblasts upregulates the percentage of CD25-expressing pNK cells, resulting in increased expression of activation markers and cytokine production by pNK cells. In addition, we demonstrated that the CXCL12/CXCR4 axis is crucial for the recruitment of CD25+ dNK cells and contributes to the accumulation of CD3−CD56brightCD25+ dNK cells at the maternal/fetal interface. Thus, our data reveal that the crosstalk between trophoblasts and pNK cells leads to the accumulation of CD3−CD56brightCD25+ dNK cells, which exert a regulating effect at the maternal/fetal interface.

Programmed cell death-1 (PD-1) and T-cell immunoglobulin mucin-3 (Tim-3) regulate CD4+ T cells to induce Type 2 helper T cell (Th2) bias at the maternal–fetal interface

STUDY QUESTION Are the immune regulatory molecules programmed cell death-1 (PD-1) and T-cell immunoglobulin mucin-3 (Tim-3) involved in regulating CD4+ T cell function during pregnancy? SUMMARY ANSWER PD-1 and Tim-3 promote Type 2 helper T cell (Th2) bias and pregnancy maintenance by regulating CD4+ T cell function at the maternal-fetal interface. WHAT IS KNOWN ALREADY The maternal CD4+ T cell response to fetal antigens is thought to be an important component of maternal-fetal tolerance during pregnancy. PD-1 and Tim-3 are important for limiting immunopathology. The co-expression of PD-1 and Tim-3 on T cells identifies a T cell subset with impaired proliferation and cytokine production. Combined blockade of Tim-3 and PD-1 could restore T cell function to the greatest degree. STUDY DESIGN, SIZE, DURATION The expression of PD-1 and Tim-3 on CD4+ T cells was analyzed by flow cytometry, and in vitro and in vivo analyses were used to investigate the role of PD-1/Tim-3 signal in the regulation of CD4+ T cells function and pregnancy outcome. PARTICIPANTS/ MATERIALS, SETTING, METHODS A total of 88 normal pregnant women, 37 women with recurrent spontaneous abortion, 36 normal pregnant mice and 45 abortion-prone mice were included. We measure the expression of PD-1 and Tim-3 on CD4+ T cells and their relationship to the function of CD4+ T cells and pregnancy outcome, as well as the effects of blocking PD-1 and Tim-3 pathways on decidual CD4+ T (dCD4+ T) cells during early pregnancy. MAIN RESULTS AND THE ROLE OF CHANCE PD-1 and Tim-3, by virtue of their up-regulation on dCD4+ T cells during pregnancy, define a specific effector/memory subset of CD4+ T cells and promote Th2 bias at the maternal-fetal interface. Using in vitro and in vivo experiments, we also found that combined targeting of PD-1 and Tim-3 pathways results in decreased production of Th2-type cytokines by dCD4+ T cells and increased fetal resorption of normal pregnant murine models. Moreover, decreased PD-1 and Tim-3 on dCD4+ T cells may be associated with miscarriage. LIMITATIONS AND LIMITS OF CAUTION Further study is required to examine the mechanism of PD-1 and Tim-3 effects on Th2 cytokine production by CD4+ T cells during pregnancy. WIDER IMPLICATIONS OF THE FINDINGS These results have important implications for understanding the physiological mechanisms that promote maternal-fetal tolerance. Our study also indicates that targeting Tim-3 and PD-1 pathways may represent novel therapeutic strategies to prevent pregnancy loss. STUDY FUNDING/COMPETING INTERESTS This study was supported by the National Basic Research Program of China (2015CB943300); National Nature Science Foundation of China (81490744, 91542116, 31570920, 81070537, 31171437, 81370770, 31270969, 31570920, 91542116); the Key Project of Shanghai Municipal Education Commission (14ZZ013) and the Key Project of Shanghai Basic Research from Shanghai Municipal Science and Technology Commission (12JC1401600). None of the authors have any conflict of interest to declare.

Tim-3 protects decidual stromal cells from toll-like receptor-mediated apoptosis and inflammatory reactions and promotes Th2 bias at the maternal-fetal interface

Toll-like receptors (TLRs) are important in mediating immune responses against various pathogens during pregnancy. However, uncontrolled TLR-triggered inflammation will endanger normal pregnancy, resulting in pregnancy loss. Therefore, maintenance of a moderate inflammatory response is crucial for successful pregnancy under conditions of infection. Here, we demonstrated significantly lowered expression of T-cell immunoglobulin and mucin domain 3 (Tim-3) in miscarried decidual stromal cells (DSCs), indicating that Tim-3 might play important roles in maintaining successful pregnancies. Activation of TLR signaling induced pro-inflammatory cytokine production and apoptosis of DSCs, which was accompanied by up-regulated Tim-3 expression. Tim-3, in turn, protected DSCs from TLR-mediated apoptosis in an ERK1/2 pathway-dependent manner. In addition, Tim-3 inhibited TLR signaling-induced inflammatory cytokine production by DSCs through suppressing NF-κB activation. Tim-3 increased production of T helper 2 (Th2)-type cytokines by DSCs and reversed the inhibitory effect of LPS on Th2 cytokine generation by up-regulation of interferon regulatory factor 4 expression. Tim-3 blockade abolished the effect of Tim-3 on the inflammatory response to LPS stimulation. Thus, Tim-3 signaling could represent a “self-control” mechanism in TLR-triggered inflammation during pregnancy. These findings identify Tim-3 as a key regulator of DSCs and suggest its potential as a target for the treatment of spontaneous abortion.

PD-1 and Tim-3 Pathways Regulate CD8+ T Cells Function in Atherosclerosis

T cell-mediated immunity plays a significant role in the development of atherosclerosis (AS). There is increasing evidence that CD8+ T cells are also involved in AS but their exact roles remain unclear. The inhibitory receptors programmed cell death-1 (PD-1) and T cell immunoglobulin and mucin domain 3 (Tim-3) are well known inhibitory molecules that play a crucial role in regulating CD8+ T cell activation or tolerance. Here, we demonstrate that the co-expression of PD-1 and Tim-3 on CD8+ T cells is up-regulated in AS patients. PD-1+ Tim-3+ CD8+ T cells are enriched for within the central T (TCM) cell subset, with high proliferative activity and CD127 expression. Co-expression of PD-1 and Tim-3 on CD8+ T cells is associated with increased anti-atherogenic cytokine production as well as decreased pro-atherogenic cytokine production. Blockade of PD-1 and Tim-3 results in a decrease of anti-atherogenic cytokine production by PD-1+ Tim-3+ CD8+ T cells and in an augmentation of TNF-α and IFN-γ production. These findings highlight the important role of the PD-1 and Tim-3 pathways in regulating CD8+ T cells function in human AS.

Chemokine CCL28 induces apoptosis of decidual stromal cells via binding CCR3/CCR10 in human spontaneous abortion.

Spontaneous abortion is the most common complication of pregnancy. Immune activation and the subsequent inflammation-induced tissue injury are often observed at the maternal-fetal interface as the final pathological assault in recurrent spontaneous abortion. However, the precise mechanisms responsible for spontaneous abortion involving inflammation are not fully understood. Chemokine CCL28 and its receptors CCR3 and CCR10 are important regulators in inflammatory process. Here, we examined the expression of CCL28 and its receptors in decidual stromal cells (DSCs) by immunochemistry and flow cytometry (FCM), and compared their expression level in DSCs from normal pregnancy versus spontaneous abortion, and their relationship to inflammatory cytokines production by DSCs. We further analyzed regulation of the pro-inflammatory cytokines on CCL28 expression in DSCs by real-time polymerase chain reaction, In-cell Western and FCM. The effects of CCL28-CCR3/CCR10 interaction on DSC apoptosis was investigated by Annexin V staining and FCM analysis or DAPI staining and nuclear morphology. Higher levels of the inflammatory cytokines interleukin (IL)-1β, IL-17A and tumor necrosis factor-α, and increased CCR3/CCR10 expression were observed in DSCs from spontaneous abortion compared with normal pregnancy. Treatment with inflammatory cytokines differently affected CCL28 and CCR3/CCR10 expression in DSCs. Human recombinant CCL28 promoted DSC apoptosis, which was eliminated by pretreatment with neutralizing antibodies against CCR3/CCR10 and CCL28. However, CCL28 did not affect DSC growth. These results suggest that the inflammation-promoted up-regulation of CCL28 and its receptors interaction in DSCs is involved in human spontaneous abortion via inducing DSC apoptosis.

Cyclosporine A Enhances Th2 Bias at the Maternal-Fetal Interface in Early Human Pregnancy with Aid of the Interaction between Maternal and Fetal Cells

Our previous study has demonstrated that cyclosporine A (CsA) administration in vivo induces Th2 bias at the maternal-fetal interface, leading to improved murine pregnancy outcomes. Here, we investigated how CsA treatment in vitro induced Th2 bias at the human maternal-fetal interface in early pregnancy. The cell co-culture in vitro in different combination of component cells at the maternal-fetal interface was established to investigate the regulation of CsA on cytokine production from the interaction of these cells. It was found that interferon (IFN)-γ was produced only by decidual immune cells (DICs), and not by trophoblasts or decidual stromal cells (DSCs); all these cells secreted interleukin (IL)-4, IL-10, and tumor necrosis factor (TNF)-α. Treatment with CsA completely blocked IFN-γ production in DICs and inhibited TNF-α production in all examined cells. CsA increased IL-10 and IL-4 production in trophoblasts co-cultured with DSCs and DICs although CsA treatment did not affect IL-10 or IL-4 production in any of the cells when cultured alone. These results suggest that CsA promotes Th2 bias at the maternal-fetal interface by increasing Th2-type cytokine production in trophoblasts with the aid of DSCs and DICs, while inhibiting Th1-type cytokine production in DICs and TNF-α production in all investigated cells. Our study might be useful in clinical therapeutics for spontaneous pregnancy wastage and other pregnancy complications.

Tim-3 signaling in peripheral NK cells promotes maternal-fetal immune tolerance and alleviates pregnancy loss

A subset of natural killer cells suppresses the mother’s immune system to protect the fetus. Mediating maternal-fetal tolerance Many cases of recurrent miscarriage are due to a breakdown in immune tolerance between the mother and the fetus. Determining the underlying mechanism may aid in the identification of biomarkers for recurrent miscarriage and suggest possible therapies. Li et al. found that the abundance of the type I membrane receptor Tim-3 was increased on the surface of peripheral natural killer (pNK) cells in the first trimester of pregnancy. These cells produced anti-inflammatory cytokines and induced the generation of immunosuppressive cells. In contrast to pNK cells from donors with normal pregnancies, those from recurrent miscarriage patients had decreased Tim-3 abundance and were defective in immunosuppression. Experiments with abortion-prone mice showed that Tim-3+ pNK cells, but not Tim-3− pNK cells, protected against fetal loss, suggesting that Tim-3+ pNK cells promote maternal-fetal immune tolerance. Pregnancy loss occurs in about 15% of clinically recognized pregnancies, and defective maternal-fetal immune tolerance contributes to more than 50% of these events. We found that signaling by the type I membrane protein T cell immunoglobulin and mucin-containing protein 3 (Tim-3) in natural killer (NK) cells had an essential protective role during early pregnancy. Tim-3 on peripheral NK (pNK) cells was transiently increased in abundance during the first trimester of pregnancy, which depended on interleukin-4 (IL-4)–signal transducer and activator of transcription 6 (STAT6) and progesterone signaling. Tim-3+ pNK cells displayed immunosuppressive activities, including the production of anti-inflammatory cytokines and the induction of regulatory T cells (Tregs) in a transforming growth factor–β1 (TGF-β1)–dependent manner. Tim-3 on pNK cells was stimulated by its ligand galectin-9 (Gal-9), leading to signaling by the kinases c-Jun N-terminal kinase (JNK) and AKT. In recurrent miscarriage (RM) patients, Tim-3 abundance on pNK cells was reduced and the immunosuppressive activity of Tim-3+ pNK cells was impaired. Compared to Tim-3+ pNK cells from donors with normal pregnancies, RM patient Tim-3+ pNK cells exhibited changes in DNA accessibility in certain genetic loci, which were reversed by inhibiting accessible chromatin reader proteins. Furthermore, Tim-3+ pNK cells, but not Tim-3− pNK cells, reduced fetal loss in abortion-prone and NK cell–deficient mice. Together, our findings reveal a critical role for Tim-3–Gal-9 signaling–mediated immunoregulation by pNK cells in maternal-fetal immune tolerance and suggest that Tim-3 abundance on pNK cells is a potential biomarker for RM diagnosis.