Indoleamine-dioxygenase is expressed in human decidua at the time maternal tolerance is established

The semi-allogeneic fetus has to be tolerated by the maternal immune system. In mice, it has been shown that inhibiting indoleamine-dioxygenase (IDO) leads to fetal rejection, suggesting a central significance for IDO in establishing maternal tolerance. Consequently, we have analyzed IDO expression in human endometrium and decidua to determine whether it may be of significance in human reproduction. Endometrial (n=60) and decidual (n=68; first and second trimester) tissue samples and isolated cells were analyzed for IDO mRNA and protein expression by real-time PCR, Western blot and immunohistochemistry. IDO expression in the decidua of proven fertile women (n=34) was compared to women presenting with their first pregnancy (n=22) and women with a history of miscarriages (n=12). Expression of IDO was localized in glandular epithelial cells and scattered stromal leukocytes. Expression started at the mid-luteal phase in the menstrual cycle and was high until the second trimester of pregnancy. However, glandular expression of IDO decreased during the second trimester, whereas expression in villous trophoblast started at this time. There were no significant differences in decidual IDO expression between proven fertile women and women presenting with their first pregnancy or women with a history of miscarriages. From the expression pattern we conclude that IDO may play a central role in human pregnancies for the establishment of maternal tolerance of fetal antigens. Thereby, IDO expression may be needed in each pregnancy independently from prior pregnancies, and a history of miscarriage may not reflect a general deficiency in IDO expression.     

Evolution of mammalian pregnancy in the presence of the maternal immune system

Eutherian mammals have inherited a typical vertebrate immune system, which protects the body against infectious organisms by detecting and destroying foreign biological material. However, with the evolution of longer gestation periods, this protective mechanism became a potential threat to the 'semi-foreign' fetus and so eutherians have developed systems to prevent immune rejection of their developing fetuses. In many species, this is achieved by reducing placental expression of major histocompatibility complex (MHC) genes, the products of which are responsible for most transplantation rejection reactions. Unexpectedly, however, major histocompatibility complex expression is often re-established in the most invasive trophoblast cells. It is not known why transplantation antigen expression in the fetal cells most exposed to the maternal immune system is advantageous. It is possible that such expression aids the process of invasion or exerts an immunoprotective effect on the fetus. It may prove possible to identify the essential steps that all eutherian fetuses take to ensure their survival in the face of potential maternal immune attack by studying the common features of the placental immunology of different species.     

Mouse model for allogeneic immune reaction against fetus recapitulates human pre-eclampsia

AIM: We have previously demonstrated that mRNA expression and enzyme activity levels of placental indoleamine 2,3-dioxygenase (IDO), which degrades L-tryptophan and blocks the proliferation of T cells, are significantly low in patients with severe pre-eclampsia. From this observation, we hypothesized that induction of maternal allogeneic immune reaction by reduced IDO activity is one of the causes of pre-eclampsia. METHODS: To examine this hypothesis, we administered an IDO inhibitor to pregnant female mice carrying allogeneic concepti. Since administration of an IDO inhibitor to pregnant mice starting at E4.5 is already reported to cause allogeneic fetal rejection, we modified the regimen and started the administration at E6.5 when the fetus and placenta have already been established. RESULTS: Pregnant mice treated with an IDO inhibitor developed high blood pressure and proteinuria in addition to local circulation impairment in the placenta, which is analogous to the lesions that are characteristic of human pre-eclampsia. In contrast, pregnant mice carrying syngeneic concepti did not manifest such symptoms. CONCLUSIONS: Our findings reveal a pivotal role for IDO activity in the etiology of pre-eclampsia. These data also lend support to the current hypothesis that pre-eclampsia is one of the possible manifestations of a maternal immunological reaction against an allogeneic fetus.     

Differential expression of complement regulatory proteins on subpopulations of human trophoblast cells.

Trophoblast cells forming the reactive interface between the mother and her semiallogeneic fetus risk attack by cellular and humoral elements of the maternal immune system. Biochemical, molecular, and immunohistologic studies have identified membrane cofactor protein (MCP) and decay accelerating factor (DAF) on trophoblast cells, which could assist in preventing lysis of the cells by complement-activating maternal antibodies. In this immunocytochemical study, differential expression of these two members of the family of complement regulatory proteins on subpopulations of human trophoblast cells and other types of cells in first and third trimester placentas was demonstrated. Staining with anti-MCP was particularly strong on villous cytotrophoblast cells and giant cells in first trimester tissues in comparison with other types of cells. In contrast, staining with anti-DAF was strong on proliferating cytotrophoblast in first trimester tissues, and on basal plate cytotrophoblast and decidual cells in term tissues. Placental villous mesenchymal cells but not trophoblast cells expressed a third regulatory protein, complement receptor 1. These observations support the postulate that complement regulatory proteins are critical to protection of the fetal allograft, and suggest specific requirements for trophoblast cells according to stage of differentiation and anatomic location.     

Expression of indoleamine 2,3-dioxygenase in the rhesus monkey and common marmoset

Indoleamine 2,3-dioxygenase (IDO) catalyzes the initial and rate-limiting step of tryptophan degradation along the kynurenine pathway, and is hypothesized to limit tryptophan availability at embryo implantation and prevent maternal T cell activation at the maternal–fetal interface. To determine if nonhuman primates are suitable models for investigating the role of IDO during pregnancy, we defined the expression of IDO in the rhesus monkey and common marmoset with particular attention to the female reproductive tract and placenta. IDO mRNA was detected by RT-PCR in the rhesus monkey term placenta, lung, small intestine, spleen, lymph node and nonpregnant uterus, and also in the common marmoset placenta. Immunohistochemical analysis of rhesus monkey tissues localized IDO to glandular epithelium of nonpregnant endometrium and first trimester decidua, vessel endothelium of nonpregnant myometrium, first trimester decidua and term decidua, and villous vessel endothelium and syncytiotrophoblast of term placenta. Western blot analysis confirmed IDO in rhesus monkey term placenta. In the common marmoset, IDO was detected in glandular epithelium of the nonpregnant uterus and in the decidua at day 60 and day 128 of gestation. IDO activity was higher in rhesus monkey and common marmoset decidua and placentas than in other tissues. Confirmation of IDO expression in rhesus monkey and common marmoset uterine and placental tissues supports the hypothesis that this enzyme regulates immune activation at the maternal–fetal interface and demonstrates that nonhuman primates may provide models with distinct similarities to human placentation to study the role of IDO in maternal–fetal immune dialogue.     

The chrondroitin sulfate proteoglycan (CSPG4) regulates human trophoblast function

Introduction

Trophoblast growth and invasion of the uterine endometrium are critical events during placentation and are tightly regulated by locally produced factors. Abnormal placentation can result in early miscarriage or preeclampsia and intrauterine growth restriction, leading to impaired fetal and/or maternal health. Chondroitin sulfate proteoglycan 4 (CSPG4) is involved in cancer cell migration and invasion, processes which are critical during placentation but unlike in cancer, trophoblast invasion is highly regulated. CSPG4 expression and function in trophoblast is unknown. We determined CSPG4 expression in human first trimester placenta and implantation sites, and investigated whether CSPG4 influenced proliferation, migration and invasion of a human extravillous trophoblast (EVT) cell line (HTR8/SVneo cells) as a model for extravillous trophoblast (EVT).

Methods and results

Immunoreactive CSPG4 localized to EVT cells in the trophoblast shell, subpopulations of interstitial EVT cells within the decidua and cytotrophoblast cells in placental villi. In HTR8/SVneo cells, siRNA knockdown of CSPG4 stimulated proliferation and decreased migration/invasion. In primary first trimester placental villi explants two cytokines, interleukin 11 (IL11) and leukemia inhibitory factor (LIF) with known roles in trophoblast function, stimulated CSPG4 mRNA expression and immunoreactive protein in the cyotrophoblast.

Discussion and conclusion

This is the first demonstration of the production and function of CSPG4 in human placentation. These data suggest that locally produced CSPG4 stimulates human EVT migration and invasion and suggests that IL11 and LIF regulate villous cytotrophoblast differentiation towards the invasive phenotype at least in part via CSPG4.     

Indoleamine 2,3-dioxygenase,immunosuppression and pregnancy

Pharmacologic inhibition of indoleamine 2,3-dioxygenase (IDO) activity during murine pregnancy results in maternal T-cell-mediated rejection of allogeneic but not syngeneic conceptuses. Increased risk of allogeneic pregnancy failure induced by exposure to IDO inhibitor is strongly correlated with maternal C3 deposition at the maternal-fetal interface. Here we review evidence that cells expressing IDO contribute to immunosuppression by inhibiting T-cell responses to tumor antigens and tissue allografts, as well as fetal tissues.     

妊娠过程中凋亡级联的作用和调节

细胞凋亡是一种级联事件,在妊娠过程中发挥了重要的作用,主要体现在胎盘形成过程中滋养细胞层分化为合体细胞滋养层,滋养层入侵确保了胎盘锚固于母体血管系统以保证胎儿氧气及营养的供应。滋养层细胞的凋亡限制了子宫壁侵入,维持母体完整性,凋亡产生的多肽是胎儿特异性耐受建立的诱导物。整个级联过程受母体免疫系统的监控,妊娠妇女的免疫应答并没有显著降低,母体免疫系统能够识别胎儿移植物这个外源性抗原并作出应答。母胎界面特定的免疫耐受诱导机制使绒毛外滋养细胞（EVT）限制和容忍相互联系。细胞因子主要由自然杀伤细胞、T细胞分泌,白细胞通过细胞因子发挥其免疫调节作用。凋亡过程受到基因调控、激素及氧化应激等因素的影响。     

Indoleamine 2,3-dioxygenase: distribution and function in the developing human placenta

Studies in mice have suggested that the placenta is protected from immune rejection by maternal T cells by means of localised indoleamine 2,3-dioxygenase dependent depletion of tryptophan. To determine whether such mechanisms might operate in the human placenta, we have studied the physiological importance of human placental indoleamine 2,3-dioxygenase immunohistochemically and functionally. Indoleamine 2,3-dioxygenase is detectable immunohistochemically from day 6 human blastocysts and thereafter throughout pregnancy in syncytiotrophoblasts, extravillous cytotrophoblasts and macrophages in the villous stroma and in the fetal membranes. Interferon-gamma added to villous explants markedly stimulates indoleamine 2,3-dioxygenase protein expression in macrophages. Indoleamine 2,3-dioxygenase-mediated tryptophan degradation in the first trimester villous and decidual tissue explants is stimulated by interferon-gamma and inhibited by 1-methyl-tryptophan (an inhibitor of indoleamine 2,3-dioxygenase). Peripheral blood mononuclear cell proliferation is controlled by indoleamine 2,3-dioxygenase-mediated tryptophan degradation. These results suggest the cellular basis of a mechanism present at the human maternal-fetal interface involved in regulating the maternal immune response to conceptus.     

Apoptosis of extravillous trophoblast cells limits the trophoblast invasion in uterine but not in tubal pregnancy during first trimester

During the first trimester of pregnancy extravillous trophoblast cells (EVT) invade the maternal decidua. Invasion normally is reduced from the second trimester onwards and stops in the inner third of the myometrium. By contrast, in extrauterine tubal pregnancy, trophoblast invasion may even penetrate the tubal wall, which ultimately leads to the rupture of the fallopian tube. Induction of apoptosis of EVT cells, by maternal immune competent cells, may be an important mechanism to limit EVT invasion in uterine pregnancy. Tissue specimens from first and second trimester uterine pregnancy and first trimester tubal pregnancy were analyzed for apoptosis by TUNEL- and M30-staining. By immunohistochemical double labelling, maternal leukocyte subtypes were co-localized to apoptotic cells and in this context, the number of CD56(+)NK cells was analyzed. Our data show that apoptosis is confined to the decidua basalis. Most apoptotic cells are single cytokeratin-positive epithelial cells residing in the stromal compartment. Consequently these cells can only be EVT cells. Maternal leukocytes are not apoptotic. They are located in close contact to apoptotic cells. The number of apoptotic cells in the second trimester (1.8+/-0.7 per cent) is reduced compared to first trimester (5.6+/-0.7 per cent) of uterine pregnancy. In parallel, the number of NK cells declines from first (24.4+/-2.9) to second (12.4+/-1.8) trimester. Furthermore, apoptosis is significantly reduced in ectopic (0.9+/-0.3 per cent) compared to eutopic first trimester pregnancies. Consequently, we suggest that in first trimester uterine pregnancy, induction of EVT cell apoptosis by the maternal immune system is one mechanism to limit EVT invasion. During the second trimester, in parallel to declining numbers of NK cells, the mechanism changes. However, in tubal pregnancy due to differing immunological microenvironments at the ectopic implantation site, apoptosis induction fails, which deleteriously may result in uncontrolled invasion and penetration of the tubal wall.     

妊娠期及妊高征滋养细胞E-钙粘附素的表达

目的 :研究妊娠各期胎盘床绒毛外细胞滋养细胞 (EVT)E 钙粘附素 (E cad herin)表达的变化 ,并与妊高征胎盘床中的表达进行比较。方法 :用免疫组化法 ,对 4 0例标本进行染色 (孕早期 9份 ,孕中期 4份 ,孕晚期 2 7份 ,其中妊高征组 8份 ,非妊高征组 19份 )。结果 :孕 10周前绒毛的细胞滋养细胞仅小部分E cadherin染色呈弱阳性。孕 10周后 ,绒毛的细胞滋养细胞染色均为强阳性 ,细胞柱区域从近端到远端染色逐渐减弱。孕中期EVT染色几乎为阴性 ,孕晚期又稍有增强。妊高征时EVT染色减弱不明显。妊高征组和非妊高征组染色阳性的细胞分别为 2 7.4 5 %和 12 .74 % (P <0 .0 1)。结论 :E cadherin表达减弱或消失有利于细胞滋养细胞的浸润。妊高征时 ,E cadherin表达减弱不明显 ,细胞滋养细胞浸润能力减弱 ,以至螺旋动脉缺乏妊娠期生理性变化 ,导致发生妊高征。     

Expression of HLA-E molecules in the placental tissue of women infected with HIV-1 and uninfected women

IntroductionExpression of HLA-E molecule in the placental extravillous trophoblast is associated with immune system cell inhibition, resulting in immune tolerance to fetus during pregnancy. HIV-1 can infect trophoblast cells and modify the expression of HLA-E, which may inhibit the cytotoxic activity of the immune system.AimThe aim of this study was to evaluate HLA-E expression in third trimester placental tissue of women infected with HIV-1 and uninfected women.MethodsWe performed an immunohistochemistry assay to evaluate HLA-E staining in the placental tissue of 99 HIV-1 infected and 85 uninfected women. A pathologist analyzed and classified the HLA-E expression in the placental cells.ResultsIrrespective of the HIV status, HLA-E staining was observed in the extravillous trophoblast cells, endothelial cells and Hofbauer cells, but not in the syncytiotrophoblast. HLA-E staining showed no significant difference between the placental tissue of women infected with HIV-1 and uninfected women (P = 0.76). Considering HIV-1 infected women, HLA-E staining was not influenced by HIV-1 viral load (P = 0.48), CD4⁺ T-cell count (P = 0.10) and antiretroviral therapy used during pregnancy (P = 0.54).DiscussionDespite the presence of HIV-1 infection, the expression of HLA-E molecules in the placental tissue was not modified when the infection was under antiretroviral therapy control.     

Tryptophan catabolism prevents maternal T cells from activating lethal anti-fetal immune responses

The murine conceptus is protected from maternal immunity by cells expressing indoleamine dioxygenase (IDO), which catabolizes tryptophan. Induction of lethal maternal anti-fetal immunity requires effective pharmacologic inhibition of IDO enzyme activity and the presence of maternal T cells, but not B cells and also depends on the degree of maternal-fetal tissue incompatibility. Based on these findings, we propose a model to explain the role of IDO in suppressing maternal immunity and the mechanism of fetal allograft rejection, when IDO activity is inhibited during gestation. This model incorporates observations that fetal allograft rejection is T cell dependent, antibody-independent and is accompanied by a novel type of inflammation involving extensive complement deposition at the maternal-fetal interface, when IDO activity is blocked during murine pregnancy.     

Fetal growth restriction is associated with reduced FasL expression by decidual cells

The Fas-Fas ligand (FasL) system contributes to immune tolerance at the feto-maternal site and has been ascribed a role in implantation and placental development by regulating trophoblast invasion and spiral artery remodelling. In the present study, we have examined FasL expression in decidual tissue from pregnancies with impaired placental development. Women with pre-eclampsia (PE) and/or fetal growth restriction (FGR) were enrolled as cases (n=33), and women with normal pregnancies were used as controls (n=27). Decidua basalis tissue was obtained by vacuum suction of the placental bed after delivery. FasL expression by extravillous trophoblasts (EVTs) and decidual cells (DeCs), together with EVT apoptosis, were assessed by immunohistochemistry. Levels of soluble FasL in maternal serum and apoptosis-related gene expression in decidual tissue were determined. The proportion of FasL-expressing DeCs was high in controls (72.0+/-10.2%), with a significant reduction among cases (58.1+/-19.7%; p=0.002), especially in those with FGR (54.3+/-19.9%; p<0.001). EVTs had a lower proportion of FasL expression than DeCs, with a less pronounced reduction in cases compared to controls (10.9+/-3.9 and 8.3+/-4.0%, respectively; p=0.02). Decidual FasL expression correlated with placental growth. The EVT apoptosis rate did not differ between cases and controls (1.1+/-1.9 and 1.1+/-1.3%, respectively). These findings indicate a reduction of immune privilege in decidua of PE/FGR pregnancies by reduced FasL expression and that DeCs may have a central role in the Fas-FasL-based feto-maternal immune balance.     

Differential expression of human placental PAPP-A2 over gestation and in preeclampsia

IntroductionPregnancy Associated Plasma Protein A2 (PAPP-A2) is a pregnancy related insulin-like growth factor binding protein-5 (IGFBP-5) protease, known to be elevated in preeclampsia. As the insulin-like growth factors are important in human implantation and placentation, we sought to determine the expression pattern of PAPP-A2 over human gestation in normal and preeclamptic pregnancies to evaluate its role in placental development and the pathogenesis of preeclampsia.MethodsPlacental basal plate and chorionic villi samples, maternal and fetal cord blood sera were obtained from preeclamptic and control pregnancies. Formalin-fixed tissue sections from across gestation were stained for cytokeratin-7, HLA-G, and PAPP-A2. PAPP-A2 immunoblot analysis was also performed on protein lysates and sera.ResultsPAPP-A2 expression is predominately expressed by differentiated trophoblasts and fetal endothelium. Chorionic villi show strong expression in the first trimester, followed by a progressive decrease in the second trimester, which returns in the third trimester. PAPP-A2 expression is not impacted by labor. PAPP-A2 is increased in the basal plate, chorionic villi and maternal sera in preeclampsia compared to controls, but is not detectable in cord blood.DiscussionPAPP-A2 is differentially expressed in different trophoblast populations and shows strong down regulation in the mid second trimester in chorionic villous samples. Both maternal sera and placental tissue from pregnancies complicated by preeclampsia show increased levels of PAPP-A2. PAPP-A2 levels are not altered by labor. Additionally, PAPP-A2 cannot be detected in cord blood demonstrating that the alterations in maternal and placental PAPP-A2 are not recapitulated in the fetal circulation.     

In-vivo concentrations of inhibins, activin A and follistatin in human early pregnancy

The aims of this study were to investigate the relationship between inhibins, activin A and follistatin in first trimester fetal fluids, maternal serum, placenta and decidua, and to investigate if these hormones are present in the circulation of the early second trimester human fetus. Amniotic and coelomic fluid, maternal serum, placental villi and decidual tissue were obtained from normal pregnancies at 8-12 weeks. Fetal blood by cardiocentesis and maternal blood were collected at 14-16 weeks gestation. Placental extracts had higher concentrations of inhibins, activin A and follistatin compared with decidual extracts. In the second trimester, inhibins and follistatin were detectable in fetal blood at 14-16 weeks gestation. Maternal serum concentrations of inhibin A (P < 0.001) and follistatin (P < 0.05) were significantly higher than fetal serum whereas inhibin B (P < 0.01) and pro-alpha C concentrations (P < 0.001) were higher in fetal serum. Inhibin B concentrations were also higher in male fetal serum samples that had higher concentrations of testosterone. The presence of all molecular forms of inhibins, activin A and follistatin in the first trimester fetal fluids, placental and decidual extracts in the first trimester confirms other reports. In the second trimester, high concentrations of inhibin B with testosterone in the fetal circulation indicate that these hormones may interact in the development of the male fetal gonads.     

The tumor suppressor gastrokine-1 is expressed in placenta and contributes to the regulation of trophoblast migration

Introduction

Gastrokine-1 (GKN1) is a secreted auto-/paracrine protein, described to be expressed in the gastric mucosa. In gastric cancers GKN1 expression is commonly down-regulated. While current research focusses on the exploration of tumor-suppressive properties of GKN1 with regard to its potential clinical use in the treatment of gastroenterologic tumor disease, nothing is known about GKN1 expression and function in other organ systems. We investigated GKN1 expression in placental tissue and cells.

Materials and methods

GKN1 was localized using immunohistochemistry in first and third trimester placental tissue, hydatidiform moles and various gestational trophoblastic neoplasias. We determined the expression of GKN1 in immunomagnetic bead-separated term placental cells and in choriocarcinoma cell lines. The role of GKN1 for JEG-3 migration was studied using live cell imaging. E-cadherin, MMP-2 and -9, TIMP-1 and -2, as well as urokinase (uPA) expression levels were determined.

Results

GKN1 is expressed in healthy third trimester placentas. Its expression is specifically limited to the extravillous trophoblast (EVT). GKN1 expression is significantly reduced in choriocarcinoma cell lines and gestational trophoblastic neoplasias. GKN1 attenuates the migration of JEG-3 choriocarcinoma cells in vitro, possibly via AKT-mediated induction of E-cadherin. GKN1 treatment reduced MMP-9 expression in JEG-3.

Discussion

Besides its role in gastric physiology our results clearly indicate regulatory functions of GKN1 in the EVT at the feto-maternal interface during pregnancy. Based on our findings in the JEG-3 choriocarcinoma cell line, an auto-/paracrine role of GKN1 for EVT motility and villous anchorage at the basal plate is conceivable. Thus, the tumor suppressor GKN1 is expressed in placental EVT and might contribute to the regulation of EVT migration/invasion.     

Novel model of placental tissue explants infected by cytomegalovirus reveals different permissiveness in early and term placentae and inhibition of indoleamine 2,3-dioxygenase activity

Human cytomegalovirus (HCMV) is the most common cause of viral intrauterine infection. Placental infection suggests hematogenous spread and permissiveness may vary according to the age of pregnancy. We set up and investigate permissivity of early and term placenta to HCMV with an ex vivo model of placental histocultures and evaluate the activity profile of IDO. Fourteen first trimester placentae were obtained following elective abortion and twelve term placentae after elective caesarean section. Fresh placental chorionic villi were isolated, washed and distributed on collagen sponge gels after overnight incubation with the virus. The culture medium was collected and fresh medium renewed regularly. Histology and immunohistochemistry showed preserved villous integrity in cultured placental histocultures. Infection could be seen in tissue sections of both early and term placentae, although early placentae were more permissive. Indoleamine 2,3-dioxygenase (IDO) is highly expressed in the placenta and is known to prevent maternal immune rejection. Constitutive IDO activity was higher in early, compared to term placentae and HCMV infection inhibited IDO activity in early placentae. IFN-γ-induced IDO activity was suppressed by HCMV in both early and term placentae. Our work shows a novel method of placenta organ culture. Our findings suggest that HCMV infects early placentae more strongly than term placentae. Early placental dysfunction through the inhibition of IDO activity may reveal a possible mechanism for miscarriages.