. 2016 Apr 13;185(1):86–97. doi: 10.1111/cei.12790

17β‐Oestradiol enhances the expansion and activation of myeloid‐derived suppressor cells via signal transducer and activator of transcription (STAT)−3 signalling in human pregnancy

T Pan ^1,^2,^†, L Zhong ^2,^†, S Wu ^3,^†, Y Cao ², Q Yang ², Z Cai ³, X Cai ³, W Zhao ³, N Ma ⁴, W Zhang ⁴, H Zhang ^2,⁵, J Zhou ^1,^2,^5,^✉

PMCID: PMC4908292 PMID: 26969967

Summary

During a successful pregnancy, the maternal immune system plays a critical role in maintaining immunotolerance towards semi‐allogeneic fetal antigens. Recent studies have indicated that myeloid‐derived suppressor cells (MDSCs) are active players in establishing fetal–maternal tolerance; however, the underlying mechanism remains poorly understood. In this study, we observed a significant expansion of monocytic MDSCs (M‐MDSCs) in the peripheral blood of pregnant women, which suppressed T cell responses in a reactive oxygen species‐dependent manner and required cell–cell contact. The number of M‐MDSCs correlated positively with serum oestrogen and progesterone levels. Administration of 17β‐oestradiol, but not progesterone, enhanced both the expansion and suppressive activity of M‐MDSCs through signal transducer and activator of transcription (STAT)‐3. Pretreatment with STAT‐3 inhibitor JSI‐124 almost completely abrogated the effects of 17β‐oestradiol on MDSCs. Collectively, these results demonstrate that 17β‐oestradiol‐induced STAT‐3 signalling plays an important role in both the expansion and activation of MDSCs during human pregnancy, which may benefit the development of novel therapeutic strategies for prevention of immune‐related miscarriage.

Keywords: 17β‐oestradiol, human pregnancy, MDSC, STAT‐3

Introduction

Tolerance towards fetal alloantigens is the major challenge for maternal immune system during the course of pregnancy. The maternal–fetal interface between the uterine mucosa and the embryonic tissues comprises distinct compartments that undergo dramatic changes during pregnancy, including implantation, placentation, fetal growth and parturition 1, 2. In mammals, implantation of an embryo in the mother's womb involves a series of steps leading to effective cross‐talk between embryo and mother. Once the embryo is implanted, it suppresses the maternal immune response and prevents being rejected. Simultaneously, the mother's immune system will develop a reaction against graft‐versus‐host to favour immunological tolerance 3, 4, 5. Although some types of immune cells were demonstrated to participate in the establishment of fetomaternal tolerance, the detailed mechanisms still remain to be understood 6, 7, 8.

Human trophoblasts are kept in direct contact with maternal tissues. The expression of polymorphic major histocompatibility complex molecules of maternal and paternal origin on the surface of trophoblasts allows them to interact functionally with surrounding immune cells 9. Meanwhile, some types of immune cells such as natural killer cells, dendritic cells and regulatory T cells are either enriched in or excluded from the decidua, which ultimately affect the pregnancy 8, 10, 11, 12. Furthermore, when endometrial stromal cells differentiate, genes encoding the T cell‐attracting chemokines CXCL9–12 and CCL5 become silenced epigenetically in decidual stromal cells 13, which limits T cell trafficking to the decidua and attenuates the immune responses. Investigating the mechanisms underlying maternal–fetal tolerance will lead to the development of more effective therapies for the pregnancy‐related complications.

Myeloid‐derived suppressor cells (MDSCs) were described originally as immune‐suppressive cells of myeloid origin in cancer 14, 15. They are a heterogeneous population consisting of myeloid progenitor cells at different stages and immature myeloid cells that have, under some pathological conditions, been prevented from differentiating fully. Targeting MDSCs has become an attractive therapeutic strategy in cancer and other disorders 16, 17, 18, 19. Due to their heterogeneity, MDSCs can be classified into monocytic (M) and granulocytic (G) subsets based on specific surface markers, both in mice and humans 15, 20. These two subsets differ in various aspects, including origin, molecular mechanisms, immunological functions and tissue localization. They should therefore be considered separately 21, 22.

In 2011, Mauti et al. reported that the expanded MDSCs might explain the enhanced metastatic breast cancer in gestation mice 23. More recent studies have reported higher levels of G‐MDSCs present in the cord blood and the peripheral blood of healthy pregnant women, in which they actively suppress T cell responses 24, 25, 26. Patients with miscarriage displayed a reduced level of MDSCs in both the blood and endometrium 27. In a mouse study, infiltration of MDSCs into the uterus responded to the maternal immunological signalling and contribute to tolerance 28. Collectively, these studies indicate that MDSCs may represent a major player in fetal–maternal tolerance. The mechanisms dictating the pregnancy‐derived MDSCs remains poorly understood.

In this study, we aimed to investigate the signalling involved in the expansion and activation of MDSCs during pregnancy. Peripheral blood was sampled from pregnant and postpartum women and MDSCs were analysed. We report that M‐MDSCs are expanded in human pregnancy, probably via the oestrogen‐mediated signal transducer and activator of transcription 3 (STAT‐3) pathway. Our study revealed a complicated interaction between reproduction and the immune system during the course of pregnancy.

Methods

Ethics statement

This research was approved by the Ethics Review Board of Shenzhen No. 2 People's Hospital (the first affiliated hospital of Shenzhen University) and the Ethics Review Board of Sun Yat‐Sen University. Written informed consent was provided by study participants and/or their legal guardians.

Patients and healthy donors

The peripheral blood samples of healthy pregnant women (n = 118, pregnant weeks 6 −35) and healthy postpartum women (n = 23) were recruited at Shenzhen No. 2 People's Hospital. Healthy controls (n = 42) were a group of local volunteers who were not pregnant and had no reported history of chronic illness. The basic characteristics of pregnant/postpartum subjects and healthy donors are outlined in Supporting information, Table S1.

Peripheral blood mononuclear cell (PBMC) isolation and flow cytometric analysis

PBMCs were isolated from whole blood by Ficoll centrifugation and analysed immediately 29. The following anti‐human fluorescence‐conjugated antibodies and their corresponding isotype controls were purchased from eBioscience (San Diego, CA, USA): CD11b‐fluoresceion isothiocyanate (FITC), CD33‐phycoerythrin (PE), human leucocyte antigen D‐related (HLA‐DR)‐PE‐cyanin (Cy)5, CD14‐PE‐Cy7, CD15‐eFluor450, CD4‐PE, CD8‐PE‐Cy5 and CD3‐PE‐Cy7. The cell phenotype was analysed by flow cytometry on a flow cytometer (BD LSR fortessa; BD Biosciences, San Jose, CA, USA) and the data were analysed with FlowJo software package (Treestar Inc., Ashland, OR, USA). For analysis, 200 000 or more total cells were evaluated, considering that MDSCs are a rare population in PBMCs. For flow cytometric sorting, a fluorescence activated cell sorter (FACS)Aria II (BD, Mountain View, CA, USA) was used. The strategy for MDSC sorting was HLA‐DR^–/low CD11b⁺ CD33^+/high cells from live PBMCs. Depletion of MDSCs was performed by harvesting the remaining PBMCs after MDSC sorting. These experiments were performed as described previously 30.

T cell proliferation assay

T cell proliferation was evaluated by carboxyfluorescein succinimidyl ester (CFSE) (5, 6 carboxyfluorescein diacetate, succinimidyl ester) dilution 31. PBMCs or purified T cells (1 × 10^–6 cells/ml) were labelled with CFSE (3 μM) (Invitrogen, Carlsbad, CA, USA) and then stimulated with anti‐CD3/CD28 antibodies (5 μg/ml) (eBioscience). After co‐culture alone or with MDSCs (1 : 2, 1 : 4, 1 : 8) for 3 days, T cell proliferation was determined by a flow cytometer. No stimulation was represented as a negative control.

Arginase activity assay

The experiment was performed as described previously 30. Briefly, cells were lysed with 0·1% Triton X‐100 for 30 min, followed by the addition of 25 mM Tris‐HCl and 10 mM MnCl₂. The enzyme was activated by heating for 10 min at 56°C. Arginine hydrolysis was performed by incubating the lysate with 0·5 M L‐arginine at 37°C for 120 min. The urea concentration was measured at 540 nm after the addition of α‐isonitrosopropiophenone (dissolved in 100% ethanol), followed by heating at 95°C for 30 min.

Reactive oxygen species (ROS) production

The experiment was performed following the manufacturer's procedures. Cells (5 × 10^–5 cells) were incubated at 37°C in the presence of 1 μM CM‐H2DCFDA (Invitrogen) for 30 min, and then labelled with fluorescence‐conjugated antibodies against CD33 and CD11b. The ROS content in MDSCs was analysed by flow cytometry.

Enzyme‐linked immunosorbent assay (ELISA) assay

Interferon (IFN)‐γ quantification in culture supernatants was determined using ELISA, following the manufacturer's instructions (R&D Systems, Minneapolis, MN, USA).

Quantitative reverse transcription–polymerase chain reaction (qRT–PCR)

RNA was extracted with an RNase Minikit, and cDNA was synthesized using a SuperScript III reverse transcriptase kit (Qiagen, Valencia, CA, USA). PCR was performed as described previously 32. Briefly, reactions were performed in triplicate using SYBR Green (TaKaRa, Otsu, Japan) and normalized to endogenous actin and glyceraldehyde 3‐phosphate dehydrogenase (GAPDH) mRNA levels using gene‐specific primers for each target (see Supporting information, Table S2).

Transwell assays

The MDSC‐T cell co‐culture experiments were performed with Transwells to determine the cell contact dependency of the MDSC function 30. For these studies, MDSCs isolated by flow cytometric sorting were cultured in Transwell inserts (0·4 μm pore; Millipore, Billerica, MA, USA), and fresh autologous T cells (1 × 10^–6 cells/ml) were cultured in 96‐well plates. For T cell proliferation assays, MDSCs (5 × 10^–5 cells/ml) were co‐cultured with CFSE‐labelled T cells at different ratios in the presence of anti‐CD3/CD28 stimuli for 3 days. Proliferation of T cells was evaluated by flow cytometry.

Generation of human MDSCs in vitro

Human PBMCs were isolated from healthy volunteer donors. PBMCs were then cultured in 24‐well plates at 1 × 10⁶ cells/ml in complete RPMI‐1640 medium, supplemented with recombinant human cytokines granulocyte–macrophage colony‐stimulating factor (GM‐CSF) (20 ng/ml) and interleukin (IL)−6 (20 ng/ml) (Peprotech, Oak Park, CA, USA). Media was refreshed every 2 days. Cells were analysed by flow cytometry on day 6 33.

Intracellular staining

Intracellular staining of STAT‐3 phosphorylation (pSTAT‐3) (phospho Tyr705) was performed following the manufacturer's protocol (Cell Signalling Technology, Beverly, MA, USA). Cells (5 × 10^–5 cells) were fixed with formaldehyde to stabilize the cell membrane, and permeabilized using BD Fix&Perm Solution (BD Biosciences). After washing, the cells were then stained with Alexa Fluor 488‐conjugated pSTAT‐3 and analysed by flow cytometry.

Statistics

Clinical and immunological parameters were compared by non‐parametric Mann–Whitney U‐tests. For in‐vitro experiments, statistical analyses were performed using unpaired or paired t‐tests. Correlations between different parameters were analysed using a Spearman's rank test. GraphPad Prism version 5·0a and spss version 17·0 software were used. P‐values less than 0·05 were considered significant.

Results

M‐MDSCs expand during human pregnancy

To investigate the role of MDSCs in human pregnancy, we first analysed the concentration of MDSCs in the peripheral blood from healthy pregnant women (n = 118) and age‐matched healthy non‐pregnant female donors (n = 42) by flow cytometry based on HLA‐DR^–/lowCD11b⁺CD33⁺ expression. As expected, the HLA‐DR^–/lowCD11b^intCD33^int and HLA‐DR^–/lowCD11b^highCD33^high populations corresponded to the G‐MDSC and M‐MDSC subsets, respectively, based on the expression of CD14/CD15 and cell morphology (Fig. 1a). The proportion of M‐MDSCs was elevated significantly in pregnant women compared with healthy donors and postpartum women (n = 23) (Fig. 1b). By contrast, no clear difference in G‐MDSCs was observed (data not shown). Interestingly, the level of M‐MDSCs increased from the first to the second trimesters and declined from the third trimester (Fig. 1c), indicating dynamic changes of MDSCs during the course of pregnancy. These observations demonstrated collectively that the M‐MDSC subset is elevated significantly in human pregnancy in a trimester‐dependent manner.

Expansion of monocytic myeloid‐derived suppressor cells (M‐MDSCs) in the course of human pregnancy. (a) Gating strategy of M‐MDSCs by flow cytometric analysis. Human leucocyte antigen D‐related (HLA‐DR)^–/low cells were first gated for live peripheral blood mononuclear cells (PBMCs) from pregnant women, and the CD11b⁺ CD33^+/high population was gated further as M‐MDSCs. The expression of cell surface markers CD14 and CD15 on this population was evaluated subsequently. Grey shows the isotype control. (b) Representative flow cytometric data of M‐MDSCs from healthy donors, pregnant and postpartum women. The boxed area (HLA‐DR^–/lowCD11b⁺ CD33^+/high cells) represents the percentage of M‐MDSCs in PBMCs. (c) Statistical analysis of MDSCs frequencies (left) and absolute cell counts (right) in the peripheral blood from healthy donors (n = 42), pregnant (*n =* 118) and postpartum (n = 23) women. *P < 0·05; **P < 0·01; unpaired t‐tests. (d) Statistical analysis of M‐MDSCs frequencies in PBMCs from different courses of pregnant women. *P < 0·05; unpaired t‐tests.

M‐MDSCs suppress T cell responses in pregnancy

The most important property of MDSCs is their immunosuppressive activity 34. Therefore, we evaluated the effect of pregnancy‐derived M‐MDSCs on T cell responses. We used two strategies: first, M‐MDSCs were depleted from peripheral blood mononuclear cells (PBMCs) by flow cytometric sorting, after which the PBMCs were stimulated with anti‐CD3/CD28 antibodies. The results showed that the proliferation of both CD4 and CD8 T cells was enhanced significantly by M‐MDSC depletion (Fig. 2a). The suppressive activity of MDSCs was confirmed further by co‐culture of MDSCs with T cells. M‐MDSCs actively suppressed the autologous T cell responses, including cell proliferation and IFN‐γ production, in a dose‐dependent manner (Fig. 2b,c). Secondly, to determine whether M‐MDSCs function through direct contact with T cells, M‐MDSC/T cell co‐culture experiments were performed using Transwells. The separation of M‐MDSCs from T cells eliminated their suppressive activity (Fig. 2d), indicating that the function of M‐MDSCs is cell contact‐dependent.

M‐MDSCs suppress T cell responses in a ROS‐dependent manner

Next, we investigated the mechanism underlying the immunosuppressive function of MDSCs. Inhibition of L‐arginine is believed to mediate the immunosuppressive effect of MDSCs under certain pathological conditions 25, 35. Therefore, we measured the arginase activity, nitric oxide (NO) and ROS levels in MDSCs from pregnant women and healthy non‐pregnant controls. ROS production was significantly higher in pregnancy‐derived MDSCs than in those from controls (Fig. 3a). No noticeable changes were observed in the level of arginase activity (Fig. 3b) or NO content (data not shown). Accordingly, the expression of p47phox, a major component of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase which is responsible for ROS synthesis, was induced markedly in pregnancy‐derived MDSCs. The expression of Arg1 and iNOS, responsible for arginase activity and NO production, respectively, did not display any differences (Fig. 3c). Furthermore, administration of the ROS inhibitor N‐acetyl‐L‐cysteine (NAC) abrogated the suppressive effect of MDSCs almost completely, whereas inhibitors of arginase or inducible nitric oxide synthase (iNOS) or L‐arginine supplementation did not show any effects (Fig. 3d,e). These results demonstrated that pregnancy‐derived MDSCs suppress T cells in a ROS‐dependent manner.

Monocytic myeloid‐derived suppressor cells (M‐MDSCs) suppress T cell responses in a reactive oxygen species (ROS)‐dependent manner. (a) The content of ROS (CM‐H2DCFDA⁺) in MDSCs from pregnant women (P‐MDSC) and healthy controls (H‐MDSC) was measured by flow cytometry. Left panel: representative flow cytometry data from one individual; right panel: statistical analysis of five individuals. **P < 0·01; unpaired t‐tests. (b) Arginase activity in M‐MDSCs from pregnant women and healthy controls. Means and standard deviations (s.d.) are shown (n = 8). (c) The expression of L‐arginine metabolizing enzymes was measured by quantitative reverse transcription–polymerase chain reaction (qRT–PCR) (n = 3). *P < 0·05; Mann–Whitney U‐test. (d) Effects of different inhibitors on the suppressive function of MDSCs from pregnant women were evaluated by allogeneic mixed lymphocytes reaction. T cells were labeled with carboxyfluorescein succinimidyl ester (CFSE) and stimulated with anti‐CD3/anti‐CD28 (5 ng/ml). These cells were then co‐cultured with MDSCs from the same donor at a 2 : 1 ratio with treatments as indicated for 3 days, and T cell proliferation was evaluated by a flow cytometry. N‐hydroxy‐L‐arginine (NOHA) (an arginase inhibitor, 100 μM); L‐arginine (1 mM); N‐methyl‐L‐arginine [L‐NMMA, an inducible nitric oxide synthase (iNOS) inhibitor, 100 μM]; N‐acetyl‐l‐cysteine (NAC, a ROS inhibitor, 1 mM). Means and s.d. are shown. *P < 0·05; **P < 0·01, one‐way analysis of variance (anova). (e) Production of interferon (IFN)‐γ by T cell supernatants from (d) was measured by enzyme‐linked immunosorbent assay (ELISA). (d and e) The data represent means and s.d. for four subjects. *P < 0·05; **P < 0·01; Mann–Whitney U‐test.

17β‐oestradiol promotes both the expansion and activation of M‐MDSCs

In addition to their essential role in the reproductive system, the impact of pregnancy‐related hormones [oestrogen, progesterone and human chorionic gonadotrophin (hCG)] on the immune responses during pregnancy is well documented 1, 5. The fluctuation in the level of MDSCs during the course of pregnancy indicated a potential association between pregnancy‐related hormones and MDSC expansion. Pearson's correlation analysis revealed a positive correlation between serum 17β‐oestradiol (E2) and progesterone (P4) levels with M‐MDSCs, while the hCG content in the circulation was correlated negatively with M‐MDSC level in pregnancy (Fig. 4a). To determine the effects of hormones on MDSCs, in‐vitro differentiation of MDSCs was performed. E2 promoted MDSC expansion in a dose‐dependent manner (Fig. 4b), whereas P4 failed to have any effect (Supporting information, Fig. S1). The expression of S100A8 and S100A9, genes associated with MDSC expansion 36, was clearly higher in MDSCs upon E2 treatment (Supporting information, Fig. S2). Oestrogen receptor (ER)‐alpha was expressed highly in the pregnancy‐derived MDSCs (Fig. 4c). Furthermore, E2‐treated MDSCs displayed stronger capability in suppressing T cell responses (Fig. 4d), accompanied by higher ROS production, as expected (Fig. 4e). These results indicated that E2 promotes both the expansion and activation of MDSCs.

17β‐oestradiol (E2) promotes the expansion and activation of myeloid‐derived suppressor cells (MDSC). (a) The correlations between monocytic MDSCs (M‐MDSCs) and different plasma hormones. Left: oestradiol, middle: progesterone, right: human chorionic gonadotrophin (hCG); n = 72. Correlations between different parameters were analysed using a Spearman's rank test. (b) Peripheral blood mononuclear cells (PBMCs) from healthy donors were cultured in 20 ng/ml granulocyte–macrophage colony‐stimulating factor (GM‐CSF) and interleukin (IL)−6 for 6 days with or without different concentrations of 17β‐oestradiol; the proportions of MDSCs were evaluated by flow cytometry analysis. The blank group without any treatment was showed as a negative control and the GM‐CSF/IL‐6‐treated group was used as a positive control. Left: representative flow cytometry data; right: statistical analysis of three individuals. *P < 0·05; Mann–Whitney U‐test. (c) Representative images for agarose gel electrophoresis of reverse transcription–polymerase chain reaction (qRT–PCR) products of oestradiol or progesterone receptors in MDSCs from pregnant women. The beta‐actin lane was showed as an experimental control. (d) The comparison of suppressive activity on CD4⁺ (top) and CD8⁺ (bottom) T cells between E2‐derived MDSCs and control MDSCs are shown. Peripheral blood mononuclear cells (PBMCs) were cultured in 20 ng/ml GM‐CSF and IL‐6 for 6 days with oestradiol or ethanol control. MDSCs were then purified by flow cytometric sorting. T cells from the same donors were labelled with carboxyfluorescein succinimidyl ester (CFSE) and stimulated with anti‐CD3/anti‐CD28 (5 ng/ml) and then co‐cultured with isolated MDSCs at different ratios for 3 days. Unstimulated T cells were used as a negative control and the T cells only are shown as a positive control. Left: representative flow cytometric data; right: statistical analysis of three individuals. *P < 0·05; **P < 0·01; Mann–Whitney U‐test. (e) The production of reactive oxygen species (ROS) in MDSCs was measured by flow cytometry. Human leucocyte antigen D‐related (HLA‐DR)^–/lowCD33^+/high cells were gated first and then the percentage of CM‐H2DCFDA⁺ cells are shown. Left: representative flow cytometry data; right: statistical analysis of three independent experiments. *P < 0·05; **P < 0·01; Mann–Whitney U‐test.

STAT‐3 signalling mediates the effects of E2 on MDSCs

The importance of STAT‐3 signalling in MDSC expansion and activation under tumour conditions is well established 37, 38, 39. Thus, we investigated the role of the STAT‐3 pathway in pregnancy‐derived MDSCs. M‐MDSCs from pregnant women displayed significantly higher levels of pSTAT‐3 than those from controls (Fig. 5a). Administration of E2 caused a clear elevation of STAT‐3 phosphorylation in MDSCs in a time‐ and dose‐dependent manner. The levels of pSTAT‐3 peaked at 1 h after E2 stimulation, and then declined at 2 h (Fig. 5b). In agreement with these findings, pretreatment with cucurbitacin‐I (JSI‐124), a potent inhibitor of pSTAT‐3 40, abrogated the effect of E2 on MDSC expansion almost completely (Fig. 5c). Blocking of STAT‐3 signalling by JS1‐124 also counteracted the suppressive activity of MDSCs toward T cells, as well as the enhanced ROS production (Fig. 5d,e). These observations indicated that STAT‐3 signalling mediates the effects of E2 on MDSCs.

Signal transducer and activator of transcription‐3 (STAT‐3) signalling mediates the effects of 17β‐oestradiol (E2) on myeloid‐derived suppressor cells (MDSCs). (a) STAT‐3 phosphorylation (pSTAT‐3) level in MDSCs from pregnant women and healthy controls was measured by flow cytometry. Human leucocyte antigen D‐related (HLA‐DR)^–/lowCD33^+/high cells were gated first and the percentages of pSTAT‐3⁺ cells are shown. Top: representative flow cytometric data; bottom: statistical analysis of results from five individuals. **P < 0·01; unpaired t‐tests. (b) The effect of E2 on pSTAT‐3 level in MDSCs was measured by flow cytometry. Peripheral blood mononuclear cells (PBMCs) were cultured in 20 ng/ml GM‐CSF, 20 ng/ml IL‐6 and 10 μM E2 for different times or different concentration of E2 for 1 h, the percentage of pSTAT‐3⁺ cells in MDSCs (HLA‐DR^‐/lowCD33^+/high) was measured by flow cytometry. Untreated cells were used as a negative control. Top: representative flow cytometric data; bottom: statistical analysis of results from three independent experiments. (c–e) PBMCs were pretreated with cucurbitacin I (JSI‐124) (10 nM) for 12 h and then cultured in 20 ng/ml granulocyte–macrophage colony‐stimulating factor (GM‐CSF) and interleukin (IL)−6 media. These cells were followed by E2 (10 μM) treatment for 6 days. Dimethylsulphoxide (DMSO) was used as a control. Representative flow cytometry data from one experiment (left) and statistical analysis of results from three independent experiments (right) are shown. (c) The percentage of MDSCs was determined by flow cytometric analysis. (d) Reactive oxygen species (ROS) production in MDSCs was measured by flow cytometry, as described previously. (e) The suppressive function of MDSCs from different treatment was measured. (b–e) *P < 0·05; **P < 0·01; Mann–Whitney U‐test.

Discussion

MDSCs have a well‐established role in maintaining immune tolerance under a variety of pathological conditions, especially in the context of cancer and inflammatory disorders 30, 38. Their importance under some special physiological circumstances requiring immunotolerance, such as pregnancy, is beginning to be appreciated. Understanding their roles in physiological tolerance will facilitate the recognization of MDSCs’ importance in the immune system.

Recently, Köstlin et al. reported that G‐MDSCs were elevated in pregnant women 25. In this study, a significant expansion of M‐MDSCs was observed in human pregnancy; however, no changes were noted for G‐MDSC. The differences observed from these two studies may be caused by the participants’ genetic backgrounds, or cytokine milieus produced in the individuals, or for some unknown reasons. We would point out that the similar phenomenon concerning different subsets of MDSCs was also reported in HIV‐1‐infected individuals 30, 41. It is therefore plausible that MDSC subsets may vary in populations from different regions. Further investigation will be needed to determine the exact mechanisms.

Interactions between oestrogen and the immune system have been well demonstrated 42, 43, 44. We provide evidence that oestrogen facilitates the expansion and activation of M‐MDSCs in human pregnancy, shedding new light on the mechanism of the immunosuppressive effect of oestrogen. The high expression of ER‐alpha on MDSCs further supports the involvement of oestrogen in myeloid cells. Myeloid‐specific deletion of ER would help in delineating the exact role of oestrogen in myeloid cell development and MDSC biology. In addition to a potential therapeutic function of ER in pregnancy, the regulation of MDSCs by oestrogen may have broad clinical significance in a variety of inflammatory‐related disorders in females, such as arthritis, osteoporosis, atherosclerosis, diabetes and some metabolic diseases.

Cross‐talk between oestrogen and STAT‐3 signalling has been noted in various cell types. Previous studies have reported that oestrogen receptor signalling is associated with the Janus kinase (JAK)/STAT‐3 pathway in tumour cells 45. In addition, the E2–ERa complex induced STAT‐3 phosphorylation in different cell lineages 46, 47. In tumour‐bearing hosts, cytokines or growth factors released by tumour tissues could activate STAT‐3 signalling, subsequently promoting MDSC expansion 39. Our findings revealed that a regulatory function of STAT‐3 mediates the effect of oestrogen on MDSCs during pregnancy. It is therefore plausible that mechanistic regulation of STAT‐3 signalling in MDSCs is context‐dependent. In conclusion, our study demonstrated that E2‐induced STAT‐3 signalling represents a mechanism of M‐MDSC expansion in human pregnancy.

Some issues remain to be resolved before MDSCs can be used as a therapeutic target for immune‐related miscarriage. First, what is the difference between MDSCs from pheripheral blood and those infiltrating into the placenta during pregnancy? Understanding the biology and function of endometrium‐derived MDSCs will provide important clues concerning the exact role of MDSCs in prevention of fetal rejection. The second issue regards what kind of patients will benefit from administration of MDSCs, considering that the clinical miscarriage may be caused by complicated reasons. The third, but not last, issue is: if transfer of MDSCs could be used as therapeutic strategy of pregnancy‐related complications, how to develop enough human MDSCs in vitro?

Investigation of these important issues will advance the understanding and clinical application of MDSCs in immune‐related miscarriage.

Author contributions

All listed authors contributed to the idea generation, design and completion of this work. T. P., L. M. Z., S. W. and J. Z. contributed to the experimental work and manuscript preparation. Y. J. C., Q. Y., Z. M. C. and X. Y. C. prepared the experiments for the pregnant samples and healthy samples. W. H. Z., N. M. and W. Q. Z. contributed to the experiments of cell culture and flow cytometry. H. Z. and J. Z. contributed to the idea generation, experimental design and manuscript preparation. All authors reviewed the manuscript.

Disclosure

The authors declare no financial or commercial disclosures.

Supporting information

Additional Supporting information may be found in the online version of this article at the publisher's web‐site:

Fig. S1. Progesterone (P4) has no effect on the expansion of myeloid‐derived suppressor cells (MDSCs). Peripheral blood mononuclear cells (PBMCs) from healthy donors were cultured in 20 ng/ml granulocyte–macrophage colony‐stimulating factor (GM‐CSF) and interleukin (IL)−6 for 6 days with or without different concentration of P4, the proportions of MDSCs were evaluated by flow cytometry analysis. (a) Representative flow cytometry data and (b) statistical analysis of three individuals were shown.

Fig. S2. The mRNA level of related genes with different‐derived myeloid‐derived suppressor cells (MDSCs). (a) MDSCs were sorting from pregnant donors and healthy donors, and then the related genes expression were detected by quantitative reverse transcription–polymerase chain reaction (qRT–PCR). (b) Peripheral blood mononuclear cells (PBMCs) were isolated from healthy donors and cultured in 20 ng/ml granulocyte–macrophage colony‐stimulating factor (GM‐CSF) and interleukin (IL)−6 for 6 days with oestradiol or ethanol control. Then the related gene expressions were detected by qRT–PCR; n = 3 and *P < 0·05; unpaired t‐test.

Table S1. The basic characteristics of pregnant/postpartum subjects and healthy donors.

Table S2. The primer lists of quantitative reverse transcription–polymerase chain reaction (qRT–PCR).

Click here for additional data file.^{(329.4KB, docx)}

Acknowledgements

This work was supported by the following grants: National Key Basic Research Program of China (no. 2012CB524900), Guangdong Innovative Research Team Program (no. 2009010058), Guangdong Province Universities and Colleges Pearl River Scholar Funded Scheme (GDUPS, 2014), Programme for the 12th Five‐year Plan (no. 2012ZX10001003), National Natural Science Foundation of China (no. 91542112; no. 81571520; No.31270921; no. 301500740), Natural Science Foundation of Guangdong (no. S2011020006072) and the Fundamental Research Funds for the Central Universities, the Provincial Talents Cultivated by ‘Thousand‐Hundred‐Ten’ programme of Guangdong Province, 111 Project (no. B12003).

References

1. Erlebacher A. Mechanisms of T cell tolerance towards the allogeneic fetus. Nat Rev Immunol 2013; 13:23–33. [DOI] [PubMed] [Google Scholar]
2. Red‐Horse K, Zhou Y, Genbacev O et al Trophoblast differentiation during embryo implantation and formation of the maternal–fetal interface. J Clin Invest 2004; 114:744–54. [DOI] [PMC free article] [PubMed] [Google Scholar]
3. Erlebacher A. Immunology of the maternal–fetal interface. Annu Rev Immunol 2013; 31:387–411. [DOI] [PubMed] [Google Scholar]
4. Makrigiannakis A, Karamouti M, Drakakis P, Loutradis D, Antsaklis A. Fetomaternal immunotolerance. Am J Reprod Immunol 2008; 60:482–96. [DOI] [PubMed] [Google Scholar]
5. Arck PC, Hecher K. Fetomaternal immune cross‐talk and its consequences for maternal and offspring's health. Nat Med 2013; 19:548–56. [DOI] [PubMed] [Google Scholar]
6. Tafuri A, Alferink J, Moller P, Hammerling GJ, Arnold B. T cell awareness of paternal alloantigens during pregnancy. Science 1995; 270:630–3. [DOI] [PubMed] [Google Scholar]
7. Lissauer D, Piper K, Goodyear O, Kilby MD, Moss PA. Fetal‐specific CD8+ cytotoxic T cell responses develop during normal human pregnancy and exhibit broad functional capacity. J Immunol 2012; 189:1072–80. [DOI] [PubMed] [Google Scholar]
8. Collins MK, Tay CS, Erlebacher A. Dendritic cell entrapment within the pregnant uterus inhibits immune surveillance of the maternal/fetal interface in mice. J Clin Invest 2009; 119:2062–73. [DOI] [PMC free article] [PubMed] [Google Scholar]
9. Madeja Z, Yadi H, Apps R et al Paternal MHC expression on mouse trophoblast affects uterine vascularization and fetal growth. Proc Natl Acad Sci USA 2011; 108:4012–7. [DOI] [PMC free article] [PubMed] [Google Scholar]
10. Rowe JH, Ertelt JM, Xin L, Way SS. Pregnancy imprints regulatory memory that sustains anergy to fetal antigen. Nature 2012; 490:102–6. [DOI] [PMC free article] [PubMed] [Google Scholar]
11. Hanna J, Goldman‐Wohl D, Hamani Y et al Decidual NK cells regulate key developmental processes at the human fetal–maternal interface. Nat Med 2006; 12:1065–74. [DOI] [PubMed] [Google Scholar]
12. Samstein RM, Josefowicz SZ, Arvey A, Treuting PM, Rudensky AY. Extrathymic generation of regulatory T cells in placental mammals mitigates maternal–fetal conflict. Cell 2012; 150:29–38. [DOI] [PMC free article] [PubMed] [Google Scholar]
13. Bromley SK, Mempel TR, Luster AD. Orchestrating the orchestrators: chemokines in control of T cell traffic. Nat Immunol 2008; 9:970–80. [DOI] [PubMed] [Google Scholar]
14. Movahedi K, Guilliams M, Van den Bossche J et al Identification of discrete tumor‐induced myeloid‐derived suppressor cell subpopulations with distinct T cell‐suppressive activity. Blood 2008; 111:4233–44. [DOI] [PubMed] [Google Scholar]
15. Youn JI, Nagaraj S, Collazo M, Gabrilovich DI. Subsets of myeloid‐derived suppressor cells in tumor‐bearing mice. J Immunol 2008; 181:5791–802. [DOI] [PMC free article] [PubMed] [Google Scholar]
16. Murdoch C, Muthana M, Coffelt SB, Lewis CE. The role of myeloid cells in the promotion of tumour angiogenesis. Nat Rev Cancer 2008; 8:618–31. [DOI] [PubMed] [Google Scholar]
17. Goedegebuure P, Mitchem JB, Porembka MR et al Myeloid‐derived suppressor cells: general characteristics and relevance to clinical management of pancreatic cancer. Curr Cancer Drug Targets 2011; 11:734–51. [DOI] [PMC free article] [PubMed] [Google Scholar]
18. De Veirman K, Van Valckenborgh E, Lahmar Q et al Myeloid‐derived suppressor cells as n hematological malignancies. Front Oncol 2014; 4:349. [DOI] [PMC free article] [PubMed] [Google Scholar]
19. Wesolowski R, Markowitz J, Carson WE III. Myeloid derived suppressor cells – a new therapeutic target in the treatment of cancer. J Immunother Cancer 2013; 1:10. [DOI] [PMC free article] [PubMed] [Google Scholar]
20. Talmadge JE, Gabrilovich DI. History of myeloid‐derived suppressor cells. Nat Rev Cancer 2013; 13:739–52. [DOI] [PMC free article] [PubMed] [Google Scholar]
21. Gabrilovich DI, Nagaraj S. Myeloid‐derived suppressor cells as regulators of the immune system. Nat Rev Immunol 2009; 9:162–74. [DOI] [PMC free article] [PubMed] [Google Scholar]
22. Ostrand‐Rosenberg S, Sinha P. Myeloid‐derived suppressor cells: linking inflammation and cancer. J Immunol 2009; 182:4499–506. [DOI] [PMC free article] [PubMed] [Google Scholar]
23. Mauti LA, Le Bitoux MA, Baumer K et al Myeloid‐derived suppressor cells are implicated in regulating permissiveness for tumor metastasis during mouse gestation. J Clin Invest 2011; 121:2794–807. [DOI] [PMC free article] [PubMed] [Google Scholar]
24. Rieber N, Gille C, Kostlin N et al Neutrophilic myeloid‐derived suppressor cells in cord blood modulate innate and adaptive immune responses. Clin Exp Immunol 2013; 174:45–52. [DOI] [PMC free article] [PubMed] [Google Scholar]
25. Kostlin N, Kugel H, Spring B et al Granulocytic myeloid derived suppressor cells expand in human pregnancy and modulate T‐cell responses. Eur J Immunol 2014; 44:2582–91. [DOI] [PubMed] [Google Scholar]
26. Gervassi A, Lejarcegui N, Dross S et al Myeloid derived suppressor cells are present at high frequency in neonates and suppress in vitro T cell responses. PLOS ONE 2014; 9:e107816. [DOI] [PMC free article] [PubMed] [Google Scholar]
27. Nair RR, Sinha P, Khanna A, Singh K. Reduced myeloid‐derived suppressor cells in the blood and endometrium is associated with early miscarriage. Am J Reprod Immunol 2015; 73:479–86. [DOI] [PubMed] [Google Scholar]
28. Zhao H, Kalish F, Schulz S, Yang Y, Wong RJ, Stevenson DK. Unique roles of infiltrating myeloid cells in the murine uterus during early to midpregnancy. J Immunol 2015; 194:3713–22. [DOI] [PubMed] [Google Scholar]
29. Grutzner E, Stirner R, Arenz L et al Kinetics of human myeloid‐derived suppressor cells after blood draw. J Transl Med 2016; 14:2. [DOI] [PMC free article] [PubMed] [Google Scholar]
30. Qin A, Cai W, Pan T et al Expansion of monocytic myeloid‐derived suppressor cells dampens T cell function in HIV‐1‐seropositive individuals. J Virol 2013; 87:1477–90. [DOI] [PMC free article] [PubMed] [Google Scholar]
31. Youn JI, Collazo M, Shalova IN, Biswas SK, Gabrilovich DI. Characterization of the nature of granulocytic myeloid‐derived suppressor cells in tumor‐bearing mice. J Leukoc Biol 2012; 91:167–81. [DOI] [PMC free article] [PubMed] [Google Scholar]
32. Zhou J, Cheng P, Youn JI, Cotter MJ, Gabrilovich DI. Notch and wingless signalling cooperate in regulation of dendritic cell differentiation. Immunity 2009; 30:845–59. [DOI] [PMC free article] [PubMed] [Google Scholar]
33. Lechner MG, Liebertz DJ, Epstein AL. Characterization of cytokine‐induced myeloid‐derived suppressor cells from normal human peripheral blood mononuclear cells. J Immunol 2010; 185:2273–84. [DOI] [PMC free article] [PubMed] [Google Scholar]
34. Srivastava MK, Sinha P, Clements VK, Rodriguez P, Ostrand‐Rosenberg S. Myeloid‐derived suppressor cells inhibit T‐cell activation by depleting cystine and cysteine. Cancer Res 2010; 70:68–77. [DOI] [PMC free article] [PubMed] [Google Scholar]
35. Rodriguez PC, Ochoa AC. Arginine regulation by myeloid derived suppressor cells and tolerance in cancer: mechanisms and therapeutic perspectives. Immunol Rev 2008; 222:180–91. [DOI] [PMC free article] [PubMed] [Google Scholar]
36. Sinha P, Okoro C, Foell D, Freeze HH, Ostrand‐Rosenberg S, Srikrishna G. Proinflammatory S100 proteins regulate the accumulation of myeloid‐derived suppressor cells. J Immunol 2008; 181:4666–75. [DOI] [PMC free article] [PubMed] [Google Scholar]
37. Kujawski M, Kortylewski M, Lee H, Herrmann A, Kay H, Yu H. Stat‐3 mediates myeloid cell‐dependent tumor angiogenesis in mice. J Clin Invest 2008; 118:3367–77. [DOI] [PMC free article] [PubMed] [Google Scholar]
38. Parker KH, Beury DW, Ostrand‐Rosenberg S. Myeloid‐derived suppressor cells: critical cells driving immune suppression in the tumor microenvironment. Adv Cancer Res 2015; 128:95–139. [DOI] [PMC free article] [PubMed] [Google Scholar]
39. Vasquez‐Dunddel D, Pan F, Zeng Q et al STAT‐3 regulates arginase‐I in myeloid‐derived suppressor cells from cancer patients. J Clin Invest 2013; 123:1580–9. [DOI] [PMC free article] [PubMed] [Google Scholar]
40. Ishdorj G, Johnston JB, Gibson SB. Inhibition of constitutive activation of STAT‐3 by curcurbitacin‐I (JSI‐124) sensitized human B‐leukemia cells to apoptosis. Mol Cancer Ther 2010; 9:3302–14. [DOI] [PubMed] [Google Scholar]
41. Vollbrecht T, Stirner R, Tufman A et al Chronic progressive HIV‐1 infection is associated with elevated levels of myeloid‐derived suppressor cells. AIDS 2012; 26:F31–37. [DOI] [PubMed] [Google Scholar]
42. Yuk JM, Kim TS, Kim SY et al Orphan nuclear receptor ERRalpha controls macrophage metabolic signalling and A20 expression to negatively regulate TLR‐induced inflammation. Immunity 2015; 43:80–91. [DOI] [PubMed] [Google Scholar]
43. Chen L, Li W, Qiu W et al RSRC1 SUMOylation enhances SUMOylation and inhibits transcriptional activity of estrogen receptor beta. FEBS Lett 2015; 589:1476–84. [DOI] [PubMed] [Google Scholar]
44. Maneix L, Antonson P, Humire P et al Estrogen receptor beta exon 3‐deleted mouse: the importance of non‐ERE pathways in ERbeta signalling. Proc Natl Acad Sci USA 2015; 112:5135–40. [DOI] [PMC free article] [PubMed] [Google Scholar]
45. Yamamoto T, Matsuda T, Junicho A, Kishi H, Saatcioglu F, Muraguchi A. Cross‐talk between signal transducer and activator of transcription 3 and estrogen receptor signalling. FEBS Lett 2000; 486:143–8. [DOI] [PubMed] [Google Scholar]
46. Villa A, Rizzi N, Vegeto E, Ciana P, Maggi A. Estrogen accelerates the resolution of inflammation in macrophagic cells. Sci Rep 2015; 5:15224. [DOI] [PMC free article] [PubMed] [Google Scholar]
47. Liang XH, Deng WB, Li M et al Egr1 protein acts downstream of estrogen–leukemia inhibitory factor (LIF)‐STAT‐3 pathway and plays a role during implantation through targeting Wnt4. J Biol Chem 2014; 289:23534–45. [DOI] [PMC free article] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Additional Supporting information may be found in the online version of this article at the publisher's web‐site:

Table S1. The basic characteristics of pregnant/postpartum subjects and healthy donors.

Table S2. The primer lists of quantitative reverse transcription–polymerase chain reaction (qRT–PCR).

Click here for additional data file.^{(329.4KB, docx)}

PERMALINK

17β‐Oestradiol enhances the expansion and activation of myeloid‐derived suppressor cells via signal transducer and activator of transcription (STAT)−3 signalling in human pregnancy

T Pan

L Zhong

S Wu

Y Cao

Q Yang

Z Cai

X Cai

W Zhao

N Ma

W Zhang

H Zhang

J Zhou