Indoleamine 2,3-dioxygenase expression is restricted to fetal trophoblast giant cells during murine gestation and is maternal genome specific

Pharmacologic inhibition of indoleamine 2,3 dioxygenase (IDO) activity during murine pregnancy results in rejection of allogeneic fetuses by the maternal immune system. Here, we show that IDO expression is restricted to perinuclear regions of primary trophoblast giant cells (TGCs) of fetal origin at mid-gestation (E10.5). After placentation (E14), no IDO expression was detected at the maternal-fetal interface. Matings involving IDO-deficient females revealed that paternally inherited IDO alleles were inactive in primary TGCs, presumably due to paternal genome-specific gene inactivation. Allogeneic matings in which both parents were genetically IDO-deficient produced litters of normal sizes at normal rates compared to IDO-sufficient parental mice, implying that compensatory or redundant immunosuppressive mechanisms protected allogeneic fetuses during gestation in IDO-deficient mice. Consistent with this notion, treatment with IDO inhibitor did not affect allogeneic pregnancy rates when both parents were IDO-deficient, confirming that IDO was the relevant pharmacologic target of the IDO inhibitor in matings involving IDO-sufficient mice. Hence, IDO is a key immunosuppressive mechanism in normal murine pregnancies, and it is regulated entirely through maternally inherited fetal genes.     

What is the role of regulatory T cells in the success of implantation and early pregnancy?

Problem The immune system is well controlled by the balance between immunostimulation and immunoregulation. CD4⁺CD25⁺ regulatory T (Treg) cells and an enzyme called indoleamine-2, 3-dioxygenase (IDO) mediate maternal tolerance of the allogeneic fetus. Treg cells, therefore, may prevent early pregnancy loss due to maternal ‘rejection.’ Methods The latest understanding of tolerance during pregnancy is reviewed. Results and conclusions Recent data show that CD4⁺CD25⁺ Treg cells play essential roles in the induction and maintenance of tolerance, and that they augment the IDO activity in dendritic cells and macrophages. Therefore, CD4⁺CD25⁺ Treg cells and IDO enzyme may cooperate in the induction of tolerance during pregnancy. Treg deficiency is associated with very early post-implantation loss and spontaneous abortion in animal models, and low Treg levels are associated with recurrent miscarriages in humans.     

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.     

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.     

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.     

Indoleamine 2,3-dioxygenase (IDO) expression in invasive extravillous trophoblast supports role of the enzyme for materno-fetal tolerance

It is still not understood how the fetus escapes from being attacked by the maternal immune system. Recent reports based on mouse and in vitro models have suggested that the enzyme indoleamine 2,3-dioxygenase (IDO) is important for materno-fetal tolerance. IDO activity in the human placenta is known to be high and might lead to inhibition of T-cell proliferation, thus preventing fetal tissue from rejection by the maternal immune system. In an attempt to elucidate the precise location of IDO at the feto-maternal junctional zone, we investigated human placental and decidual tissue of first and third trimester of pregnancy using an immunohistochemical approach. In placental tissues, only syncytiotrophoblast and endothelial cells showed moderate expression of IDO. This pattern was observed regardless of whether first or third trimester tissue was investigated. In early and term decidua, cells with the typical morphology of invasive extravillous trophoblast (EVT) were strongly positive for IDO. Blocking immunohistochemical experiments with cytokeratin and IDO antibodies identified invasive EVT as the location of predominant IDO expression. Since EVT are the fetal cells with the closest contact to the maternal immune system, our results suggest that it is EVT which protects the fetus from rejection by downregulating local maternal T-cell responses.     

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.     

Immunosuppression by placental indoleamine 2,3-dioxygenase: a role for mesenchymal stem cells

BACKGROUND/OBJECTIVES: Mesenchymal stem cells (MSC) can be isolated from human placenta and have the potential to contribute to the immunosuppressive properties of placental tissue. The objectives of this study were to investigate the phenotype and differentiation characteristics of MSC derived from human placenta and evaluate the role of the tryptophan degrading enzyme, indoleamine 2,3 dioxygenase (IDO), in mediating their immunosuppressive affect. METHODS: MSC obtained from placental tissue (pMSC) were characterised using flow cytometry and tested for multipotency by determining differentiation into all mesenchymal lineages. The immunosuppressive properties of pMSC were tested in allogeneic mixed lymphocyte reactions and IDO expression and activity were measured by semi-quantitative real-time PCR and HPLC respectively. RESULTS: Multipotent stem cells were isolated from placenta and displayed chondrogenic, osteogenic and limited adipogenic differentiation. Cell surface antigen expression of pMSC was similar to bone marrow MSC (bMSC) with lack of the haematopoietic and common leukocyte markers (CD34, CD45), and expression of adhesion (CD29, CD166, CD44) and stem cell (CD 90, CD105, CD73) markers. Placental MSC were suppressive of allogeneic T-cell proliferation, an effect which was intensified following IDO induction by IFN-gamma. Replenishment of tryptophan or treatment with the IDO-blocker, 1-methyl-tryptophan (1-MT), attenuated the immunosuppressive action of pMSC. CONCLUSIONS: These results suggest that placental tissue contains MSC, which are phenotypically and functionally similar to bMSC, and that IDO is a key mediator of their immunosuppressive effect. Further investigation is needed to determine if pMSC function effects pregnancy outcome.     

绒毛及合体滋养层细胞吲哚胺2,3-二氧化酶表达与流产的关系

目的:探讨人早孕绒毛组织吲哚胺2,3-二氧化酶(IDO)的表达与流产的关系。方法:RT-PCR测正常妊娠和难免流产绒毛组织及JAR细胞株IDOmRNA表达;免疫组化分析两组绒毛组织IDO蛋白质表达;Westernblot检测体外培养的合体滋养层细胞IDO蛋白质表达;高效液相色谱法检测细胞培养上清液中有无犬尿氨酸。结果:难免流产组绒毛组织IDOmRNA及蛋白质表达均低于正常组;JAR细胞株不表达IDOmRNA;合体滋养层细胞表达IDO蛋白质;合体滋养层细胞培养上清中有犬尿氨酸。结论:绒毛组织IDO正常表达是维持妊娠所必需;体外培养的人早孕绒毛合体滋养层细胞表达的IDO具有活性。     

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.     

Humoral immune responses in murine pregnancy. V. Relationship to the differential immunogenicity of placental and fetal tissues

The nature of the humoral immune response induced in virgin female mice by injections of F1 placental and fetal tissues has been examined and compared to that induced by immunization with F1 adult spleen cells and by multiple allogeneic pregnancy. In a 'responder' strain mouse, as defined by the ability of multiple allogeneic pregnancy to elicit an anti-paternal humoral immune response, both F1 placental and fetal tissues induced the formation of alloantibodies primarily of the IgG1 sub-class, similar to those induced by allogeneic pregnancy, but different from those elicited by adult spleen cells. However, only the placental tissues induced alloantibodies possessing all the characteristics of those appearing in multiparous allogeneic pregnancy. In contrast, the alloantibodies induced by the injected fetal tissue possessed complement-dependent cytotoxic activity, indicating that the inability of pregnancy-induced alloantibodies to mediate cytotoxicity may not be related to their restriction to the IgG1 sub-class. In a 'non-responder' mouse strain, where multiple allogeneic pregnancy does not lead to a maternal alloantibody response, F1 placental tissues, in contrast to fetal and adult tissues, failed to induce a humoral immune response. Injection of F1 placental tissue therefore elicits responses that mimic both the properties and the strain-dependent distribution of the alloantibodies identified in normal murine pregnancy. This implies that the immunogenic stimulus in pregnancy emanates from the placental rather than the fetal compartment of the allogeneic conceptus.     

Priming of peripheral monocytes with prolactin (PRL) sensitizes IFN-gamma-mediated indoleamine 2,3-dioxygenase (IDO) expression without affecting IFN-gamma signaling

Prolactin (PRL) was originally identified by its ability to stimulate mammary development and lactation, and its essential roles other than lactation have recently been implicated in female reproduction. However, little is known about PRL-mediated events in pregnancy. The tryptophan catabolism enzyme indoleamine 2,3-dioxygenase (IDO) is interferon-gamma (IFN-gamma)-inducible and has recently become a focus for maternal-fetal tolerance for successful pregnancy. Based on recognition that PRL is one of the up-regulated hormones in pregnancy, in a previous study we have shown that PRL induces IDO expression in monocytes in cooperation with a suboptimal concentration of IFN-gamma. Here, we demonstrate that PRL sensitizes monocytes to induce IDO expression in response to low doses of IFN-gamma without affecting the typical IFN-gamma signaling events, such as STAT1 phosphorylation and IRF-1 induction. In addition, IDO induction in these cell cultures was observed only after 24 h pre-exposure to PRL. These results indicate a priming effect of PRL on monocytes that occurs before IFN-gamma signaling and increases their sensitivity to IFN-gamma for IDO induction, rather than a synergistic effect of PRL and IFN-gamma on IDO induction. These results offer new insights into the roles of PRL in female reproduction, as well as provide a better understanding as to how IDO expression is regulated and achieved in pregnancy.     

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.     

Immune interactions at the maternal-fetal interface

Models of murine allogeneic pregnancy have established that maternal T cells recognize fetal alloantigens and are normally suppressed or deleted. While the precise cellular interactions and mechanisms involved in maternal lymphocyte tolerance are not yet clear, the identity of some of the critical factors are beginning to be uncovered. Signals that have been shown in mice to have an obligatory role in immunological survival of the semiallogeneic fetus include, but are probably not limited to, indoleamine-2,3-dioxygenase and the newly discovered B7 family protein, B7-H1. Whether these proteins have intersecting functions is unknown, but it is possible that both are involved in the control of maternal T regulatory cells, which are also strictly required for successful allogeneic pregnancy in mice. Additional factors that are involved include trophoblast and/or maternally derived FasL, and in humans, class Ib HLA molecules. The potency of these mechanisms in protecting the fetal allograft is underscored by the scarcity of knockout and transgenic models in which pregnancy is immunologically compromised. Here, the current understanding of mechanisms of specific suppression of maternal lymphocytes is reviewed.     

Immunology of pregnancy: towards a unifying hypothesis.

The variable findings of hormonal-immunoregulation and the variable cellular and humoral immune responses in pregnancy have been considered in relationship to the physiological response. From such considerations it appears that the peripheral blood lymphocyte/leukocyte response in pregnancy is not important, but rather the local uterine immune response at implantation and throughout pregnancy. It is proposed, and evidence is presented, that a normal allogeneic immune response is initiated at the time of implantation of the blastocyst. This immune response regulates the invasive nature of the trophoblast and initiates the first stage of parturition. The initiation and maintenance of this immune response is based on an interplay between maternal and paternal HLA and trophoblast antigens. In the case of HLA-incompatible donor-recipient blastocyst transplants, a more pivotal role for immunoregulation by trophoblast antigens is proposed. This is because it is considered that the local uterine immune response suppresses the expression of allogeneic HLA. This concept is further developed in terms of haploid HLA suppression on maternal and fetal lymphocytes that cross the placenta. This is considered to allow the interaction of these lymphocytes with each other and explains maternal transfer of cell-mediated immunity.     

Trophoblast debris modulates the expression of immune proteins in macrophages: a key to maternal tolerance of the fetal allograft?

Interactions between maternal immune cells and the placenta are of substantial interest since diseases of pregnancy, such as recurrent miscarriage, villitis of unknown etiology and preeclampsia may arise due to inadequate adaptation of the maternal immune system. During normal pregnancy trophoblast debris is shed from the placenta into the maternal blood in large quantities. This trophoblast debris is then rapidly cleared from the maternal circulation. In this study, we exposed trophoblast debris generated from an in vitro placental explant model to peripheral blood-derived macrophages and quantified a variety of molecules that are important in immune responses by ELISA or flow cytometry. Phagocytosis of trophoblast debris resulted in reduced cell-surface expression of MHC-II molecules, the costimulatory molecules (CD80, CD86, CD40 and B7H3), monocyte chemoattractant protein-1 (MCP-1), inter-cellular adhesion molecule 1 (ICAM-1) and IL-8 receptors in macrophages while the expression of programmed death-1 ligand 1 (PD-L1) was upregulated. In addition, phagocytosis of trophoblast debris induced the secretion of the anti-inflammatory cytokines IL-10, IL6 and IL1Ra and decreased the secretion of pro-inflammatory cytokines IL-1β, IL12p70 and IL-8 by macrophages. Phagocytosis of trophoblast debris also increased macrophage expression of the immunosuppressive enzyme indoleamine 2,3-dioxygenase (IDO). We have shown that phagocytosis of trophoblast debris from normal placentae alters the phenotype of macrophages such that they are likely to deviate maternal immune responses towards tolerance and away from inflammation. This may be one of the mechanisms that allow the human fetal allograft to survive in direct contact with the maternal immune system.     

CTLA4Ig gene transfer alleviates abortion in mice by expanding CD4+CD25+ regulatory T cells and inducing indoleamine 2,3-dioxygenase

Successful pregnancy requires a state of immunological tolerance since normally the maternal immune system does not reject the semi-allogeneic conceptus. Cytotoxic T lymphocyte-associated antigen-4 (CTLA-4), a ligand for B7, delivers negative signals to antigen presenting cells (APCs) to compete with CD28 for binding to B7 molecules and down-regulate proinflammatory responses, thus inhibiting T cell activation. Using CBA/J × DBA/2 matings as an abortion-prone model, we observed that adenovirus-mediated CTLA4Ig (Ad-CTLA4Ig) gene transfer improves pregnancy outcome. Ad-CTLA4Ig therapy skewed the ability of serum cytokine production toward a Th2 bias. Flow cytometry revealed that Ad-CTLA4Ig administration expanded peripheral CD4⁺CD25⁺ regulatory T cell populations in CBA/J × DBA/2 matings. Furthermore, Ad-CTLA4Ig administration induced indoleamine 2,3-dioxygenase (IDO) and Foxp3 mRNA expression at the materno-fetal interface. Our results demonstrate that adenovirus-mediated CTLA4Ig gene transfer improves pregnancy outcome in a murine model of abortion by expanding the CD4⁺CD25⁺ regulatory T cell population and inducing IDO mRNA expression.     

Elsevier Trophoblast Research Award Lecture: Unique Properties of Decidual T Cells and their Role in Immune Regulation during Human Pregnancy

Maternal lymphocytes at the fetal–maternal interface play a key role in the immune acceptance of the allogeneic fetus. Most studies focus on decidual NK cells and their interaction with fetal trophoblasts, whereas limited data are available on the mechanisms of fetus specific immune recognition and immune regulation by decidual T cells at the fetal–maternal interface. The aim of this review is to describe the phenotypic characteristics of decidual T cell subsets present at the fetal–maternal interface, their interaction with HLA-C expressed by fetal trophoblasts and their role in immune recognition and regulation at the fetal–maternal interface during human pregnancy.     

Indoleamine 2,3-dioxygenase is expressed in the endometrium of cycling mice throughout the oestrous cycle

Indoleamine 2,3-dioxygenase (IDO), an enzyme responsible for tryptophan catabolism, is thought to be required to prevent the rejection of the allogenic fetus by maternal T cells and to protect against intra- and extra-cellular pathogens. Consequently, we studied the expression of IDO in the endometrium of female Balb/c mice during the oestrous cycle. At each phase, the endometrium was peeled away and the relative expression of IDO mRNA was detected by semi-quantitative RT-PCR. The presence of IDO protein was confirmed in each phase by Western blotting and immunohistochemistry. Our results showed that IDO is expressed in the endometrium of cycling mice during all the phases of oestrous cycle. The expression of IDO was highest at the oestrus and lowest at the dioestrus. By means of Western blotting and immunohistochemistry, we obtained evidence that IDO protein is synthesised in the endometrium of cycling mice throughout the oestrous cycle. In accordance with RT-PCR results, IDO protein was predominant at the oestrus phase. IDO protein was mainly localised in the glandular and luminal epithelial cells. Our results support the concept of IDO providing a mechanism of innate immunity to protect from ascending infections of the female reproductive tract. In addition, considering the fact that mating only occurs during the oestrus phase, the high expression of IDO in this phase is likely to be a mechanism that induces immunological tolerance of the fetus.     

HLA-G transgenic mice.

We have generated a number of transgenic mice using DNA segments derived from the HLA-G gene. Using these mice we have examined the pattern of expression dictated by HLA-G promoter elements in mice and shown that HLA-G functions both as a restriction element and a transplantation antigen recognized by murine T cells. In addition, we have shown that trophoblast cells expressing H-2Kb under the control of HLA-G promoter elements affect maternal T cell phenotype and responsiveness during pregnancy. Using these same HLA-G/H-2Kb transgenic mice we have shown that trophoblast cells, expressing an inducible enzyme that degrades tryptophan, protects allogeneic conceptus expressing paternally-inherited transgenes from attack by maternal T cells that leads to fetal rejection.