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Abstract

Objectives

To localize indoleamine 2,3-dioxygenase (IDO) mRNA and protein and to undertake a functional study at the first trimester fetal–maternal interface in order to determine whether the distribution and function of IDO are related to recurrent spontaneous abortion (RSA).

Methods

Women undergoing legal pregnancy termination and women with RSA participated in this prospective study. Immunohistochemistry and real-time reverse transcription–polymerase chain reaction were used to analyse levels of IDO protein and mRNA in placenta, decidua and HTR-8/SVneo cells. Culture medium collected from trophoblast villous explant or HTR-8/SVneo cell cultures was used to measure IDO activity in response to interferon (IFN)-γ treatment.

Results

A total of 40 healthy women and 26 women with RSA provided samples of placenta and decidua. For normal pregnancies, IDO protein and mRNA was identified in placental trophoblasts, invasive extravillous trophoblasts and decidual glandular epithelium. IFN-γ significantly increased IDO activity in trophoblast villous explants and HTR-8/SVneo cells. Levels of IDO protein and mRNA in the placenta and decidua from normal pregnancies were significantly higher than in those from RSA.

Conclusions

Decreased levels of IDO protein and mRNA in the placenta and decidua from RSA suggest an important role for IDO in the maintenance of normal pregnancy.

Keywords

Abortion decidua indoleamine 2 3-dioxygenase reproductive immunology trophoblasts

Introduction

Recurrent spontaneous abortion (RSA) is one of the most common complications of pregnancy and is defined as two or more clinical pregnancies lost before 20 weeks’ gestation. Up to 5% of women experience RSA around the world, primarily in the first trimester,^1–3 and affected women are at an increased risk of developing clinical depression and anxiety.⁴ Despite extensive investigation, the exact causes of RSA cannot be determined in almost 50% of cases.⁵ The process of implantation may include mechanisms preventing allograft rejection, and tolerance to the fetal allograft represents an important mechanism for maintaining a pregnancy.⁶ It has been postulated that a proportion of these recurrent pregnancy losses might be due to immunological dysfunction,¹ but the precise mechanisms for the disturbance of immune tolerance at the fetal–maternal interface are still poorly understood.

Indoleamine 2,3-dioxygenase (IDO) is the key metabolic enzyme responsible for tryptophan degradation via the kynurenine pathway.⁷ IDO is widely produced in human tissues and cell subsets, including at the fetal–maternal interface, where it modulates their function by increasing tolerogenic capacities in vivo.^8–11 Munn et al.⁸ showed that inhibition of IDO by exposure of pregnant mice to 1-methyl-tryptophan induced a T cell-mediated rejection of allogeneic concepti, whereas syngeneic concepti were not affected; this suggests that IDO expression at the fetal–maternal interface is necessary to prevent rejection of the fetal allograft. Accumulating evidence indicates that IDO production and normal function at the fetal–maternal interface may play a prominent role in pregnancy tolerance.^8,12 It has been shown that reduced activity or decreased levels of IDO protein may participate in the pathogenesis of the immune complications of pregnancy, such as pre-eclampsia and RSA,^13–16 but the relationship between levels of IDO protein and RSA at the fetal–maternal interface remain largely unknown.

Several research groups have reported the presence of IDO at the fetal–maternal interface, however, there have been some conflicting data in the literature concerning the distribution of IDO protein in human first trimester placenta and deciduas.^17–24 It has been reported that there was variable levels of IDO staining for the placental syncytiotrophoblast, invasive extravillous cytotrophoblast (evCT) cell columns and macrophages in the villous stroma.^17,18 Conversely, others have found that IDO was only present in subtrophoblastic capillaries of the placental villi of first trimester pregnancies.^19–21 Hönig et al.²² demonstrated that the invasive evCTs were strongly positive for IDO, but that syncytiotrophoblasts were negative in the first trimester of pregnancy. Moreover, IDO was localized to the decidual glandular epithelium, decidual stromal cells, monocytes or ‘lineage negative, human leucocyte antigen D-related positive dendritic cells’ according to numerous studies.^17,18,23,24

Taking into account the particular immunological situation of the uteroplacental unit, it was considered worthwhile to investigate levels of IDO mRNA and protein, and IDO activity, in first trimester pregnancies on both sides of the fetal–maternal interface (Figure 1). The current study aimed to localize IDO mRNA and protein, and to undertake a functional study at the first trimester fetal–maternal interface, in order to determine whether the distribution and function of IDO are related to the occurrence of RSA.

Figure 1.

Diagram of the fetal (placental)–maternal (uterine decidual) interface near the end of the first trimester of human pregnancy. Stem cytotrophoblasts of the anchoring villus and the floating villus differentiate directly into syncytiotrophoblasts on the villous surface. During the invasion process, cytotrophoblasts of the anchoring villous form the trophoblastic columns that attach to the uterine wall. Cytotrophoblasts then invade the uterine interstitium, decidua and maternal vasculature, thereby anchoring the placenta to the uterus and gaining access to the maternal circulation. AV, anchoring villus; evCT, extravillous cytotrophoblast; FV, floating villus; Mϕ, macrophage; ST, syncytiotrophoblast; vCT, villous cytotrophoblast. The colour version of this figure is available at: http://imr.sagepub.com.

Patients and methods

Patients and tissue samples

This prospective study obtained first-trimester placenta and decidua directly from consecutive healthy women undergoing legal termination of normal pregnancy (5–9 weeks; healthy control group) and women with RSA (5–9 weeks; RSA group) at the Department of Obstetrics and Gynaecology, Qilu Hospital of Shandong University, Ji’nan, Shandong Province, China and the Maternity and Child Care Hospital of Ji’nan, Ji’nan, Shandong Province, China, between January 2007 and November 2009. The inclusion criteria for the control group were: healthy women; normal pregnancy; at 5–9 weeks’ gestation. The inclusion criteria for the RSA group were: a history of two or more consecutive spontaneous abortions; currently at 5–9 weeks’ gestation. In addition, patients were excluded from the study if they had systemic disease or had received previous treatment for abortion (such as progesterone and mifepristone) or treatment with immunomodulatory drugs.Among the healthy control subjects, six placental tissues were immediately placed in ice-cold sterile Dulbecco’s Modified Eagle’s Medium/Nutrient Mixture F12 (DMEM/F12) and kept for ≤30 min before subsequent trophoblast villous explant isolation. Other placental and decidual tissues were cut into small blocks, washed several times with 0.1 M phosphate-buffered saline (PBS; pH 7.2) to remove excess blood, either frozen at −80℃ or fixed for 24 h in 4% paraformaldehyde and embedded in paraffin wax.

Written informed consent was obtained from all study participants and this investigation was approved by the Ethics Committee of Qilu Hospital of Shandong University.

Cell lines and culture

The HTR-8/SVneo cell line was a gift from Dr Charles H Graham (Queen’s University, Kingston, ON, Canada). This cell line was derived from an explant culture of human first trimester placenta and has the biomarkers of evCT cells in situ.²⁵ HTR8/SVneo cells (10⁴ cells/well) were seeded into 96-well plates (Costar®, Shanghai, China) in RPMI 1640 medium (GIBCO® Cell Culture, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (Hyclone Laboratories, Logan, UT, USA), and were allowed to adhere overnight. Cells were then treated with interferon (IFN-γ) at concentrations of 10, 100, 1000 and 10 000 IU/ml (PeproTech, Rocky Hill, NJ, USA) or vehicle (0.1 M PBS, pH 7.4), and were incubated in a CO₂ incubator (Heraeus, Hanau, Germany) for a further 48 h at 37℃ with 5% CO₂. The cells were then used for immunocytochemistry and total RNA extraction as described below. The conditioned medium was collected and used for high-performance liquid chromatography (HPLC) analysis.

Immunohistochemistry

Tissue specimens were embedded in paraffin wax and 5 -µm sections were mounted onto silane-coated glass slides. Sections were dewaxed in histolene (2 × 5 min incubations) and rehydrated. After section rehydration, an antigen retrieval procedure was performed by boiling in 0.01 M citrate buffer (pH 6.0) in a microwave (3 × 2 min). Slides were cooled for 20 min and endogenous peroxidase activity was blocked with 3% hydrogen peroxide (H₂O₂) in methanol for 10 min. A 3,3'-diaminobenzidine (DAB) kit was purchased from Zhongshan Golden Bridge Biotechnology (Beijing, China). A nonspecific protein-binding block (10% normal goat serum; Zhongshan Golden Bridge Biotechnology) was applied for 10 min. Washes between each step of the immunostaining process were undertaken using 0.1 M PBS (pH 7.4) (3 × 2 min per wash). Mouse antihuman IDO monoclonal antibody (Chemicon International, Temecula, CA, USA) was diluted to 1 : 150 in 0.1 M PBS (pH 7.4) containing 1% bovine serum albumin (Hyclone Laboratories), then applied to sections overnight at 4℃. A negative control was performed by substituting isotype matched immunoglobulin (Ig) G1 at the same concentration as the primary antibody. Sections were then incubated with secondary biotin-labelled goat antimouse IgG antibody from the DAB kit for 30 min at 37℃. After being washed three times with 0.1 M PBS (pH 7.4), sections were incubated with peroxidase-labelled streptavidin-biotin for 10 min at 37℃, followed by staining with DAB solution from the DAB kit to allow visualization of the immunolabelling. Thereafter, sections were counterstained with haematoxylin, dehydrated in graded concentrations of ethanol and mounted with coverslips. The extent of the immunolabelling was evaluated using an Olympus IX81® microscope (Olympus, Tokyo, Japan).

Slides were examined at × 400 magnification and the extent of immunolabelling was scored according to the following method described by Lu et al:²⁶ slides were defined as +, ++ or +++ if 10–24%, 25–50% or >50% of the cells were stained positive, respectively. If <10% of the cells were stained, the section was considered negative. IDO immunostaining was scored blindly by two independent observers (Y.C. and Yan Fang, Department of Obstetrics and Gynaecology, Qilu Hospital of Shandong University) without knowing the study group from which the section came; 10 fields per subject were examined in each section.

Immunocytochemistry

A total of 200 µl of HTR-8/SVneo cell (5 × 10⁵ cells/ml) culture was transferred to the wells of a chambered slide and the cells were allowed to grow to confluence with the addition of fresh tissue culture medium. Cells were washed thoroughly (5 × 2 min washes) in 0.1 M PBS (pH 7.4), then fixed with 95% ethanol and 5% glacial acetic acid for 5 min. Cells were again washed thoroughly (5 × 2 min washes) in 0.1 M PBS (pH 7.4) then incubated in 0.25–0.5% Triton™ X-100 (Sigma-Aldrich, St Louis, MO, USA) in 0.1 M PBS (pH 7.4) for 10 min to permeabilize the membranes. After being washed (3 × 5-min washes) in 0.1 M PBS (pH 7.4), the cells were incubated in 3% H₂O₂ in 0.1 M PBS (pH 7.4) for 10–30 min in order to block endogenous peroxidase activity. Subsequently, 10% normal goat serum (Zhongshan Golden Bridge Biotechnology) was applied for 10 min as a nonspecific protein-binding block. Thereafter, the cells were incubated with mouse antihuman IDO monoclonal antibody, and routine DAB immunocytochemistry procedures were undertaken as described above.

Real-time RT–PCR

Real-time reverse transcription–polymerase chain reaction (RT–PCR) was used to measure the amount of IDO mRNA in tissue specimens and HTR8/SVneo cells. Total RNA was extracted from 100 mg of placenta and decidua tissue that were stored at –80℃ following collection, and from 1 × 10⁷ HTR8/SVneo cells, using TRIzol® reagent according to the manufacturer's instructions (Invitrogen, Carlsbad, CA, USA). The cDNA was synthesized from 2.0 µg (10 µl) of total RNA using oligo-dT18 primer (0.1 µg; Invitrogen). Moloney murine leukaemia virus reverse transcriptase (200 U; Promega, Madison, WI, USA) and 25 U of RNAsin (ribonuclease inhibitor; Promega) were used in a total volume of 20 µl. Reverse transcription was performed using a Mastercycler® instrument(Eppendorf, Hamburg, Germany). Subsequently, real-time RT–PCR was run on a LightCycler® 2.0 instrument (Roche Diagnostic Systems, Branchburg, NJ, USA) based on general fluorescence detection with SYBR® Green-based detection chemistry (Toyobo, Osaka, Japan). The levels of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA were used to internally standardize the levels of mRNA. Primers for IDO were used as previously described:²⁷ IDO (forward) 5′-AGAGTCAAATCCCTCAGTCC-3′; IDO (reverse) 5′-AAATCAGTGC CTCCAGTTCC-3′ (Life Technologies, Shanghai, China). Primers for GAPDH were as follows: GAPDH (forward) 5′-GCACCGTCAAGGCTGAGAAC-3′; GAPDH (reverse) 5′-TGGTGAAGACG CCAGTGGA-3′ (Life Technologies). Real-time PCR was performed using SYBR® Green Real-time PCR Master Mix (Toyobo, Osaka, Japan). The cycling programme involved preliminary denaturation at 95℃ for 10 min, followed by 45 cycles of denaturation at 95℃ for 15 s, annealing at 56℃ for 20 s, and elongation at 72℃ for 10 s. Amplification and detection of contaminating genomic DNA was avoided by constructing one of the primers over an exon/intron boundary. The PCR products were separated on 1.5% agarose gel and visualized using ethidium bromide staining and ultraviolet light. In all cases, the PCR products were confined to a single band of the expected size (222 base pairs) for IDO. Sequencing of the different bands (Life Technologies) confirmed homology with the reported sequences for human IDO and GAPDH. The quantitative endpoint for real-time PCR, the threshold cycle (C_T), was defined as the cycle number at which fluorescence passed a fixed threshold, which was determined automatically by the LightCycler® system. The level of IDO relative to GAPDH was calculated using the equation 2⁻^ΔCT, where ΔC_T = (C_{T IDO} − C_{T GAPDH}) and the ΔC_T value was negatively correlated with the level of IDO.

Human trophoblast villous explant culture

Trophoblast villous explant cultures were established from first trimester human placentas (collected from healthy control women who were undergoing legal termination of normal pregnancy by dilatation and curettage), using a modification of the method of Genbacev et al.²⁸ and methods described elsewhere.^29,30 Placental tissue was placed in ice-cold DMEM/F12 and processed within 1 h of collection. The tissue was washed in sterile DMEM/F12 and aseptically dissected using a standard microscope to remove endometrial tissue and fetal membranes. Small fragments of placental villi (15–20 mg wet weight) were teased apart and placed in a 24-well culture dish that was precoated with 0.2 ml diluted Matrigel™ Basement Membrane Matrix (3–4 mg/ml) (Becton Dickinson, Franklin Lakes, NJ, USA), polymerized at 37℃ for 30 min. Explants were cultured in either regular DMEM/F12 (with 100 mg/ml streptomycin, 100 IU/ml penicillin and 0.25 mg/ml ascorbic acid, pH 7.4), or DMEM/F12 with 1000 U/ml IFN-γ. Villous explants were kept in culture in a CO₂ incubator≤48 h at 37℃ in 5% CO_2, as described above, and the conditioned medium was collected at 48 h for measurement of tryptophan and L-kynurenine concentrations. In all experiments, a single placenta was used. Triplicate explants were set up for each treatment.

Measurement of IDO activity by HPLC

The IDO activity was measured by HPLC as described previously.¹⁶ The medium that had been conditioned by culture with trophoblast villous explants or HTR-8/SVneo cells was deproteinized with 4% trichloroacetic acid. Total free tryptophan and L-kynurenine were assayed by HPLC (Waters 2695 Alliance System and Waters 2996 photodiode array detector; Waters, Milford, MA, USA) on a C-18 column (Zorbax C18, 250 × 4.6 mm, 5 µm; Agilent Technologies, Wilmington, DE, USA) eluted isocratically using a solvent of 0.1 M phosphate buffer (pH 4.0) with 1.0 M sodium hydroxide. The ratio of L-kynurenine/tryptophan was used to assess the IDO activity.

Statistical analyses

All statistical analyses were performed using the SPSS® statistical software package, version 11.5 (SPSS Inc., Chicago, IL, USA) for Windows®. Differences between the groups were analysed by the Mann–Whitney U-test or Student’s t-test, according to the data distribution. A P-value < 0·05 was considered to be statistically significant.

Results

First trimester placenta and decidua were obtained directly from healthy women undergoing legal termination of normal pregnancy (n = 40; mean ± SD age, 27.9 ± 4.1 years) and women with RSA (n = 26; mean ± SD age, 28.5 ± 4.7 years). There was no significant difference between the two groups in terms of mean age.In general, IDO protein was restricted to the cytosol and could not be detected in the nucleus of cells. For the samples from normal pregnancies, there was strong IDO staining for trophoblasts. In 22/40 samples, all types of placental trophoblast cells (including syncytiotrophoblast, cytotrophoblast and invasive evCT cell columns) were stained strongly for IDO (Figure 2A and 2B). In the remaining 18/40 samples there was strong staining for cytotrophoblast and invasive evCT cell columns, but no staining for syncytiotrophoblast (Figure 2C and 2D). In addition, some IDO-positive cells were seen in the villous stroma; this was probably localized to macrophages (Figure 2D). For the first trimester decidua, there was strong staining for the glandular epithelium (Figure 2G and 2H), with moderate staining on invasive evCT (Figure 2I).

Figure 2.

Representative photomicrographs of human first trimester placental and decidual tissues with immunohistochemical staining for indoleamine 2,3-dioxygenase (IDO). IDO staining was localized to syncytiotrophoblast, cytotrophoblast and invasive extravillous trophoblast (evCT) cell columns (A, B) or cytotrophoblast and evCT cell columns (C, D) of placental villi from normal pregnancies. IDO was present in syncytiotrophoblasts and evCT cell columns of placental villi from recurrent spontaneous abortion (E). IDO staining was localized to the endometrial glands (G, H) and invasive evCTs (I) of the decidua from normal pregnancies. IDO was present in the endometrial glands of decidua from recurrent spontaneous abortion (J, K). A negative control was performed by substituting isotype matched immunoglobulin G1 when immunostaining the placental villi (F) and decidua (L). The colour version of this figure is available at: http://imr.sagepub.com.

Using a semiquantitative scoring system, RSA placenta had a significantly lower level of immunostaining compared with normal pregnancy placenta (P < 0.01) (Table 1; Figure 2E). A significantly lower level of IDO immunostaining was observed in RSA decidua compared with normal pregnancy decidua (P < 0.01) (Table 2; Figure 2J and 2K).

Table 1.

Immunohistochemical staining for indoleamine 2,3-dioxygenase (IDO) in the human first trimester placental villi from normal pregnancies and from recurrent spontaneous abortions (RSA).

Groups	n	Intensity of IDO immunostaining^a				Statistical significance^b
Groups	n	−	+	+ +	+ + +	Statistical significance^b
Normal pregnancy	40	0	4	9	27
RSA	26	4	16	6	0	P < 0.01

Data presented as n study participants.

Semiquantitative scoring system: −, no staining (<10% of cells stained); + , mild staining (10–24% of cells stained); + + , moderate staining (25–50% of cells stained); + + + , strong staining (>50% of cells stained).

Mann–Whitney U-test.

Table 2.

Immunohistochemical staining for indoleamine 2,3-dioxygenase (IDO) in the human first trimester decidua from normal pregnancies and from recurrent spontaneous abortions (RSA).

Groups	n	Intensity of IDO immunostaining^a				Statistical significance^b
Groups	n	−	+	+ +	+ + +	Statistical significance^b
Normal pregnancy	40	0	6	20	14
RSA	26	6	16	3	1	P < 0.01

Data presented as n study participants.

Mann–Whitney U-test.

Immunocytochemical staining demonstrated that IDO protein was found in HTR-8/SVneo cells; the staining was restricted to the cytosol (Figure 3A and 3B).

Figure 3.

Representative photomicrographs showing the immunocytochemical localization of indoleamine 2,3-dioxygenase (IDO) in HTR-8/SVneo cells and the effect of interferon (IFN)-γ. HTR-8/SVneo cells cultured for 48 h in the absence (A) and presence of 1000 U/ml IFN-γ (B). A negative control was performed by substituting isotype matched immunoglobulin G1 when immunostaining the HTR-8/SVneo cells (C). The colour version of this figure is available at: http://imr.sagepub.com.

Human trophoblast villous explants cultured for 48 h were used to determine IDO activity by HPLC, following treatment with 1000 IU/ml IFN-γ. There was L-kynurenine in the cell culture medium of trophoblast villous explants. The presence of 1000 IU/ml IFN-γ in the culture medium of human trophoblast villous explants significantly increased IDO activity (P < 0.05) (Figure 4A). For HTR-8/SVneo cells, IFN-γ increased the IDO activity and mRNA levels in a concentration-dependent manner; the maximal increase was observed at 1000 IU/ml IFN-γ (Figure 4B and 4C).

Figure 4.

Effect of interferon (IFN)-γ on tryptophan catabolism by indoleamine 2,3-dioxygenase (IDO) in the supernatant above cultured human trophoblast villous explants from normal pregnancies in vitro or HTR-8/SVneo cells. Explants were cultured in either the absence (control) or presence of 1000 IUml IFN-γ for 48 h, and tryptophan catabolism by IDO in the supernatant was measured using high-performance liquid chromatography (HPLC) analysis (A). Tryptophan catabolism by IDO in the supernatant above HTR-8/SVneo cells was analysed using HPLC (B), and the relative amount of mRNA in each sample was calculated as the ratio between the IDO mRNA and the endogenous control glyceraldehyde-3-phosphate dehydrogenase (C). Data presented as mean ± SD (note: SD too small in B to be visible). *P < 0.05; Student’s t-test.

The level of IDO mRNA in RSA placenta was significantly lower than that in normal pregnancy placenta (P < 0.05) (Figure 5A). In addition, a significantly lower IDO mRNA level was observed in RSA decidua compared with normal pregnancy decidua (P < 0.05) (Figure 5B).

Figure 5.

Levels of indoleamine 2,3-dioxygenase (IDO) mRNA in the first trimester placenta and decidua tissue samples, as quantified using real-time reverse transcription–polymerase chain reaction and presented relative to the mRNA levels of the housekeeping protein glyceraldehyde-3-phosphate dehydrogenase. The level of IDO mRNA in the placenta from normal pregnancies was significantly higher than that from recurrent spontaneous abortion (RSA) samples (A); similar findings were observed for the decidua (B). Data presented as mean ± SD. *P < 0.05; Student’s t-test.

Discussion

Serving as an immunologically privileged tissue, the placenta (especially the syncytiotrophoblasts and evCTs) and the decidua play essential functions in pregnancy maintenance.^31–33 Increasing evidence suggests that IDO might induce immune tolerance by providing a low L-tryptophan environment.^8,12 Therefore, the detailed localization of IDO production and function in terms of mediating fetal–maternal tolerance at this site is worthy of further investigation. This current study demonstrated the precise distribution of IDO protein in placental trophoblasts from normal pregnancies. Interestingly, for some samples of normal pregnancy, it was the cytotrophoblasts rather than the syncytiotrophoblasts that demonstrated immunostaining for the IDO protein. To our knowledge, this is the first study to describe the characteristic localization of IDO in placental trophoblasts. For the other samples of placenta from normal pregnancies that were analysed in our study, the IDO protein was present in all types of placental trophoblast cells including syncytiotrophoblasts, cytotrophoblasts and invasive evCT cell columns, which was consistent with two other reports.^17,18 In contrast, another two studies reported that there was no IDO staining in placental villi in first trimester samples, and that it was first detected at around 14 weeks of gestation and increased rapidly thereafter.^19,20 The discrepancies may be due to individual differences, immune microenvironments and local cytokines such as IFN-γ, β-human chorionic gonadotropin, interleukin-4 and transforming growth factor-β,³⁴ suggesting the complexity of IDO distribution and function. Moreover, technical differences may also account for the discrepancies, since immunohistochemistry is a highly specialized procedure affected by many parameters such as antibody preparations, tissue processing and blocking steps. Sedlmayr et al.¹⁹ used the same monoclonal antibody to IDO as Takikawa et al.³⁵ at dilutions between 1 : 500 and 1 : 1000, whereas Hönig et al.²² used the same rabbit antiIDO polyclonal antibody as Munn et al.³⁶ diluted to 1 : 1000. In this current study, after titration, mouse antihuman monoclonal antibody (clone 10.1) was found to be optimal at a dilution of 1 : 150. Moreover, we used paraffin wax-embedded tissue sections and heat-mediated antigen retrieval in citrate buffer, which was consistent with the methods of Sedlmayer et al.;¹⁹ in contrast to Kudo et al.¹⁷ who used Tris-ethylenediaminetetra-acetic acid for antigen retrieval, whereas Hönig et al.²² used frozen sections. Certain antigens are very sensitive to the retrieval methods used and these methodological differences may explain the discrepant results obtained. Future work will need to establish which factors regulate IDO protein levels at the fetal–maternal interface. Moreover, this current study found that cultured trophoblast villous explants showed an increased IDO activity when stimulated by IFN-γ, suggesting an important role for the invasive evCTs in pregnancy tolerance.

In addition, our current study found, as far as is known for the first time, that HTR-8/SVneo cells produce IDO protein and mRNA, and that IDO activity was increased by culture with IFN-γ. Similar findings were reported for evCTs.²² Our current findings were compatible with the fact that the HTR-8/SVneo cell line was derived from an explant culture of human first trimester placenta and has the biomarkers of evCT cells in situ.²⁵ In contrast, others have reported that trophoblast cell lines such as JAR and BeWo cells do not constitutively produce IDO.^16,37 Therefore, the HTR-8/SVneo cell line might provide a valuable tool for the future investigation of the function and mechanism of IDO in the placenta.

The characteristic distribution of IDO in the trophoblasts and decidua has pointed to a critical role for IDO at the fetal–maternal interface. It has been shown that the placenta might be protected from immune rejection by maternal immune cells by means of IDO-dependent depletion of L-tryptophan.⁸ The levels of IDO protein at the fetal–maternal interface may generate a low L-tryptophan environment, which subsequently promotes immune pregnancy tolerance by inhibiting T cell and natural killer cell proliferation, or by inducing regulatory T cells and tolerogenic dendritic cells (DCs).^{7,8,12,38–45} It is possible that decreased activity or reduced levels of IDO protein may contribute to disorders of pregnancy such as RSA and pre-eclampsia.

Our findings that IDO protein immunostaining and levels of IDO mRNA in RSA were significantly lower than those observed in normal pregnancies were consistent with other work.^20,23 Levels of IDO protein on both peripheral blood DCs and decidual DCs and monocytes were upregulated during normal pregnancy. Conversely, levels of IDO on DCs and monocytes after IFN-γ or cytotoxic T lymphocyte-associated antigen-4 treatment were decreased in spontaneous abortion cases.²³ Munn et al.⁸ showed that 1-methyl-tryptophan (a pharmacological inhibitor of intestinal IDO administered to pregnant mice) could induce the loss of the fetus: rejection of the allogeneic fetus was accompanied by a unique form of inflammation that was characterized by T cell-dependent and antibody-independent activation of complement. However, a study reported that there was no association between IDO polymorphisms and susceptibility of Iranian women to RSA.⁴⁶ It is tempting to speculate that reduced IDO enzyme production or functional activity, rather than IDO polymorphisms, may contribute to RSA and the exact mechanism might be worthy of further investigation.

In conclusion, this current study demonstrated the characteristic distribution of IDO protein in the normal pregnancy placenta and deciduas. For the first time, IDO protein was found in cytotrophoblasts rather than syncytiotrophoblasts, in some of the samples and in HTR-8/SVneo cells. Significantly decreased levels of IDO protein and mRNA were observed in RSA samples, suggesting an important role for IDO in the maintenance of normal pregnancy. Therefore, the findings of our current study provide a basis for further research into the role of IDO in the immunoregulation of human pregnancy, and may open up an attractive drug target for the therapy of RSA.

Footnotes

Acknowledgements

We thank Dr Charles Graham, Department of Anatomy and Cell biology, Queen’s University, Kingston, Ontario, Canada for the gift of the HTR8/SVneo cell line.

Declaration of conflicting interest

The authors declare that there are no conflicts of interest.

Funding

This work was supported partly by the Excellent Youth and Middle Age Scientists Fund of Shandong Province (no. BS2012SW013) and the Science and Technology Research Found from Population and Family Planning Commission of Shandong Province.

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Indoleamine 2,3-dioxygenase levels at the normal and recurrent spontaneous abortion fetal–maternal interface

Abstract

Objectives

Methods

Results

Conclusions

Keywords

Introduction

Patients and methods

Patients and tissue samples

Cell lines and culture

Immunohistochemistry

Immunocytochemistry

Real-time RT–PCR

Human trophoblast villous explant culture

Measurement of IDO activity by HPLC

Statistical analyses

Results

Discussion

Footnotes

Acknowledgements

Declaration of conflicting interest

Funding

References