Decidual stromal cells recruit Th 17 cells into decidua to promote proliferation and invasion of human trophoblast cells by secreting IL-17

T helper 17 （Th17） cells have both regulatory and protective roles in physiological conditions. The Th17 subset and the cytokine interleukin-17A （IL-17A） have been implicated in the pathogenesis of certain autoimmune diseases, several types of cancer and allograft rejection. However, the role of Th17 cells at the maternal/fetal interface remains unknown. Here, we demonstrate that Th17 cells are present in decidua and are increased in the peripheral blood of 10 clinically normal pregnancies based on intracellular cytokine analysis. Our results suggest a potential role of Th17 cells in sustaining pregnancy in humans. Furthermore, we demonstrate that decidual stromal cells （DSCs） but not trophoblast cells recruit peripheral Th17 cells into the decidua by secreting CCL2. The recruited Th17 cells promote proliferation and invasion and inhibit the apoptosis of human trophoblast cells by secreting IL-17 during the first trimester of pregnancy. These findings indicate a novel role for Th17 cells in controlling the maternal-fetal relationship and placenta development.

Galectin-9 Promotes Human Trophoblast Cell Invasion through Matrix Metalloproteinase-2 and p38 Signaling Pathway

Objective:Adequate extravillous trophoblast(EVT)invasion plays a crucial role in the establishment of successful pregnancy.Insufficient trophoblast migration and invasion can result in defective placentation,which is associated with a number of clinical pathological conditions of pregnancy including spontaneous abortion and preeclampsia.Galectin-9(Gal-9)has a wide variety of regulatory functions in innate and adaptive immunity during infection,tumor growth,and organ transplantation.Methods:We utilized immortalized human first-trimester EVT cells(HTR8/SVneo)for our functional study.We examined the effects of Gal-9 on viability,proliferation,and invasion of HTR8/SVneo cells,as well as on matrix metalloproteinase-2(MMP-2)production in HTR8/SVneo cells.Furthermore,we observed the effects of different MAPK-signaling pathway inhibitors on the stimulatory functions of Gal-9 on HTR8/SVneo cells’invasion.Results:We verified the secretion of Gal-9 by trophoblasts and detected a correlation between low levels of Gal-9 and spontaneous abortion.Gal-9 promoted the invasion of HTR8/SVneo cells through its interaction with Tim-3,not CD44,and subsequently increased MMP-2 production.Blockade of p38 signaling pathway inhibited Gal-9 activities in HTR8/SVneo cells.Conclusion:Gal-9 promotes human trophoblast cell invasion through MMP-2 and p38 signaling pathway in a Tim-3-dependent manner.

CCL2 Enhances the Viability of Human Chorionic Trophoblast Cell Line HTR-8/SVneo Cells by Inhibiting Interleukin-24 Expression

Background:To investigate the regulatory effect of interleukin-24(IL-24)on cell viability of human chorionic trophoblast cell line(HTR-8/SVneo cells).Methods:Immunohistochemical staining was used to detect the expression of IL-24 and its receptors IL-20R1,IL-20R2,and IL-22R1 in villus tissue at early normal pregnancy.The effect of thymic stromal lymphopoietin(TSLP)and chemokine CCL2 on the expression of IL-24 in human chorionic trophoblast cell line HTR-8/SVneo cells was analyzed by In-cell Western.In addition,the effect of recombinant human IL-24(rhIL-24)and CCL2 on the viability of HTR-8/SVneo cells was analyzed by MTT assay.Results:IL-24 and its receptors showed a strong positive staining in trophoblasts at early normal pregnancy.Compared with control group,expression of IL-24 in HTR-8/SVneo cells was significantly inhibited after in vitro stimulation of recombinant human CCL2 protein(rhCCL2)(P<0.001).The viability of HTR-8/SVneo cells was significantly decreased after treatment with rhIL-24(P<0.001).In contrast,anti-IL-24 neutralizing antibody significantly enhanced the viability of HTR-8/SVneo cells(P<0.01).In addition,rhCCL2(100μg/L);enhanced the viability of HTR-8/SVneo cells(P<0.01)in vitro,but this effect was inhibited by treatment with rhIL-24.Conclusions:CCL2 enhances the viability of human trophoblast cell line HTR-8/SVneo cells in vitro by inhibiting the secretion of IL-24,which may be beneficial to blastocyst implantation and placental development.

The Galectin-9/Tim-3 pathway is involved in the regulation of NK cell function at the maternal-fetal interface in early pregnancy

Decidual natural killer （dNK） cells actively participate in the establishment and maintenance of maternal-fetal immune tolerance and act as local guardians against infection. However, how dNK cells maintain the immune balance between tolerance and anti-infection immune responses during pregnancy remains unknown. Here, we demonstrated that the inhibitory molecule T-cell immunoglobulin domain and mucin domain-containing molecule-3 （Tim-3） are expressed on over 60% of dNK cells. Tim-3^＋ dNK cells display higher interleukin （IL）-4 and lower tumor necrosis factor （TNF）-α and perforin production. Human trophoblast cells can induce the transformation of peripheral NK cells into a dNK-like phenotype via the secretion of galectin-9 （Gal-9） and the interaction between Gal-9 and Tim-3. In addition, trophoblasts inhibit lipopolysaccharide （LPS）-induced pro-inflammatory cytokine and perforin production by dNK cells, which can be attenuated by Tim-3 neutralizing antibodies. Interestingly, a decreased percentage of Tim-3-expressing dNK cells were observed in human miscarriages and murine abortion-prone models. Moreover, T helper （Th）2-type cytokines were decreased and Thl-type cytokines were increased in Tim-3^＋ but not Tim-3- dNK cells from human and mouse miscarriages. Therefore, our results suggest that the Gal-9/Tim-3 signal is important for the regulation of dNK cell function, which is beneficial for the maintenance of a normal pregnancy.

Trophoblast-derived CXCL16 induces M2 macrophage polarization that in turn inactivates NK cells at the maternal–fetal interface

Decidual macrophages (dMΦ) are distinct from the conventional macrophages present in other tissues and express M2macrophage markers, but the molecular mechanisms of formation and the roles of M2 MΦ during pregnancy have not beencompletely elucidated. The crosstalk between decidual natural killer cells (dNK) and dMΦ plays an important role in themaintenance of maternal–fetal immune tolerance. Here, CXCL16 derived from first-trimester trophoblast cells induces thepolarization of human M2 macrophages. The M2 MΦ polarized by CXCL16 exhibit decreased interleukin-15 production, whichfacilitates the inactivation of NK cells. The cytotoxicity of NK cells is attenuated by the CXCL16-polarized M2 MΦ. The data shown inthe present study provide evidence to support the hypothesis that CXCL16 secreted by trophoblast cells is a key molecule involvedin decidual M2 MΦ polarization, which in turn regulates the killing ability of NK cells, thereby contributing to the homeostatic andimmune-tolerant milieu required for successful fetal development.

The integrative roles of chemokines at the maternal-feta 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.

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 Th 1-type and Th2-type cytoki nes 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-IO production in trophoblasts, and IL-IO production in DSCs. However, the coculture of trophoblasts with DSCs and DICs significantly increased interferon （IFN）-7 expression in DSCs, and tumor-necrosis factor （TNF）-a expression in DICs. No change was seen in Thl-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 Thl-type cytokines IFN-y and TNF-a, and downregulated the production of the Th2-type cytokines IL-4 and IL-IO, in trophoblasts, DSCs, DICs or their cocultures. Interestingly, rhCXCL12 inhibited production of the Thl-type cytokine TNF-a and enhanced the expression of the Th2-type cytokines such as IL-4 and IL-IO 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 viathe CXCL12/CXCR4 signal at the maternal/fetal interface in early human pregnancy.

Gene Expression Pattern of Histone Acetylation Enzymes Changed in the Hypothalamus of Middle-Aged Female Rats:A Putative Mechanism for Female Reproductive Aging

Objective:Female reproductive aging is characterized by reduced responsiveness of the hypothalamus to E2-positive feedback,which can result in alterations of gene expression and luteinizing hormone(LH)surge dysfunction.We hypothesize that age-related changes in E2-responsive gene expression are due to altered histone acetylation by histone deacetylases(HDACs)or estrogen receptor-alpha(ERα)coactivators with histone acetyltransferase(HAT)activity.Methods:In the present study,young and middle-aged female rats were ovariectomized(OVX)and treated with E2 or oil once per day for 2 days.At the time of the expected LH surge,the anterior and posterior hypothalami were dissected,and gene expression of 11 HDACs and 4 ERαcoactivators with HAT activity was measured using real-time polymerase chain reaction.Results:In the anterior hypothalamus,age affected the gene expression of 3 HDACs(Hdac3,Hdac5,and Hdac11)and 2 ERαcoactivators(Src2 and Crebbp).E2 treatment significantly decreased mRNA levels of 4 HDACs(Hdac4,Hdac5,Hdac10,and Hdac11)and 2 ERαcoactivators(Src2 and Crebbp)in young females(3-4 months).However,none of the genes responded to E2 in the middle-aged females(9-11 months),except Hdac10.In the posterior hypothalamus,age influenced Hdac5 and Src1 mRNA expression.E2 treatment increased Hdac4 and Crebbp mRNA levels in the young but not middle-aged females.Conclusions:These data suggest that E2 regulates HDACs and ERαcoactivators with HAT activity in an age-and E2-dependent manner,which may contribute to the age-related gene expression changes on the day of LH surge in female reproductive aging.

Mifepristone (RU486) Inducing Abortion in a Mouse Model by Regulating Innate and Adaptive Immune Responses

Objective:Mifepristone(RU486),one of the most common medications for artificial abortion,attenuates the immunoregulatory effects of progesterone.However,the specific immune regulatory mechanism of RU486 in abortion remains unknown.We intended to investigate the immunomodulatory effects of RU486 on abortion.Methods:Sixty female mice were divided into the control group(0 mg RU486)and RU486 group(2 mg/kg RU486).The uterus,peripheral blood,and spleen were obtained for isolation of specific cell types.The population and phenotype of immune cells in the decidua,peripheral blood,and spleen were analyzed using flow cytometry.Statistical differences between groups were determined using two-tailed t-test.For all statistical tests,P<0.05 was considered statistically significant.Results:RU486 effectively induced abortion in pregnant mice,with a significantly higher number of decidual macrophages(dMφ)(control group=25.55%±2.467%,RU486 group=19.41%±1.423%;P<0.05),especially the major histocompatibility complex II high subset.No difference in Mφnumber was observed in the spleen or peripheral blood.Moreover,the dMφfrom mice with RU486-induced abortion displayed a remarkable activated phenotype,with increased expressions of inducible nitric oxide synthase,tumor necrosis factor-α,and interleukin(IL)-12 but decreased expressions of arginase-1 and IL-10.We also found elevated levels of decidual CD4+T-cells in the RU486 group that exhibited a higher level of the proinflammatory cytokine interferon-γand a lower level of the anti-inflammatory cytokines,IL-4 and IL-10.Conclusions:We report a new mechanism of RU486-induced abortion via the regulation of innate cell Mφactivation and the adaptive response of CD4+T-cells present in the decidua but not the periphery.

Correction: Trophoblast-derived CXCL16 induces M2 macrophage polarization that in turn inactivates NK cells at the maternal–fetal interface

In this article,published online 27 March 2018,in the“ACKNOWLEDGEMENTS”,it should be supplemented as follows:“This study was funded by a grant from the National Natural Science Foundation of China,number 81490744,awarded to Da-Jin Li.”The authors regret the omission.

Immune Regulation at Maternal-fetal Interface in Early Pregnancy

Decidual immune cells(DICs),including T-cells,regulatory T-cells,macrophages/dendritic cells,natural killer cells,and neutrophils,are resident at the maternal-fetal interface,and play vital roles in regulating trophoblast migration,decidual angiogenesis,immune tolerance,placentation,and decidualization during the early pregnancy.Extensive researches have revealed that these maternal DICs cooperated with each other,or with maternal decidual stromal cells,or with fetal-derived trophoblasts,and further formed a special maternal-fetal cross talk at the maternal-fetal interface,which was essential for the construction and maintenance of physiological pregnancy.Once aberrant cross talk and immune regulation arise,many pregnancy complications will inevitably occur,such as spontaneous abortion,intrauterine growth restriction(IUGR),preeclampsia(PE),and preterm birth.Here,we reviewed how critical immune cells are either enriched or excluded from the decidua,how their function is regulated within the decidua,and how they variously contribute to pregnancy success or failure.

Roles of Regulatory T Cells in Pathogenesis of Endometriosis

Numerous studies have shown aberrant immune cell function in endometriosis,including T cells,B cells,natural killer cells,and macrophages(MΦ).These alterations are thought to be induced by various mechanisms that promote the disease.Regulatory T cells(Tregs)may account for a decreased ability of newly recruited leukocytes to initiate effective immune responses against viable endometrial fragments,permitting their survival.Tregs differentiate during the development of endometriosis,which confer immunosuppression or play other roles in disease progression.In this review,we provide an overview of the regulation and roles of Tregs in endometriosis.These data provide further scientific evidence for the altered immune response in endometriosis,which could be a potential target in the treatment of endometriosis.This review could create new diagnostic strategies and effective immune-targeted therapies for this highly prevalent disease.Recent progress in the field indicates that these goals may be achieved in the future.

Role of Decidual Natural Killer Cells at the Maternal-Fetal Interface during Pregnancy

Pregnancy is a complicated process with intricate cell-to-cell crosstalk and immune regulation.Decidual natural killer(NK)cells account for 50%-70% of decidual immune cells in early pregnancy,suggesting that they play important roles in various events,such as embryo implantation and vascular remodeling.Many studies have shown that decidual NK cells interact with other cells either through direct contact or the secretion factors such as cytokines and chemokines.Hence,this review aimed to present the phenotypic characteristics,classification,and functions of decidual NK cells at the maternal-fetal interface during pregnancy.

Executive Editor-in-Chief’s introduction for This Special Issue

Prof.Da-Jin Li is a chief physician and senior scientist at Shanghai Medical College of Fudan University.Prof.Li is elected chairman of the Obstetrics and Gynecology Division of the Chinese Association of Integrative Medicine and vice president of the American Society for Reproductive Immunology.