Abstract
During pregnancy, the maternal immune system must tolerate the persistence of semi-allogeneic fetus in the maternal tissue. Inadequate recognition of fetal antigens may lead to pregnancy complications, such as recurrent miscarriage (RM) and recurrent implantation failure (RIF). Dendritic cells (DCs) are key regulators of protective immune responses and the development and maintenance of tolerance. Regarding that DCs are important in the establishment of immune tolerance in human pregnancy, it would be important to study the microenvironment in which DCs reside or are activated may affect their functions toward tolerance rather than active immune response. IL-10 plays a critical role in the maintenance of normal pregnancy, and the increased production of IL-10 is associated with successful pregnancy. In this study, we provide an in-depth comparison of the phenotype and cytokine production by DC-10 and other DC subsets, such as iDC and mDC. CD14+ monocyte-derived DCs were differentiated in the presence of IL-10 (DC-10) in vitro from ten normal fertile controls, six RM women and seven RIF women, and characterized for relevant markers. DC-10 was characterized by relatively low expression of costimulatory molecule CD86, as well as MHC class II molecule HLA-DR, high expression of tolerance molecules HLA-G, ILT2, ILT4 and immunosuppressive cytokine IL-10, but produced little or no proinflammatory cytokines, such as TNF-α, IL-6 and IL-12p70. Our study provides a better understanding of the phenotypical properties of DC-10, which may participate in the complex orchestration that leads to maternal immune tolerance and homeostatic environment in human pregnancy.
Introduction
Pregnancy is a unique situation during which the semi-allogeneic fetus is well protected from the attack of maternal immune cells. Inadequate recognition of fetal antigens may lead to pregnancy complications, such as recurrent miscarriage (RM) and recurrent implantation failure (RIF). RM was defined as two or more clinical miscarriages occurred prior to 20 weeks of gestation, whereas RIF refers to failure to get pregnant following transfer for at least six good-quality blastocysts/embryos totally in three or more embryo transfer cycles (Practice Committee of the American Society for Reproductive Medicine 2012, Simon & Laufer 2012). These two diseases may have overlapping causes by which the embryo cannot implant into the uterus or fails to continue normal development to term. Previous studies suggested that RM and RIF may share some common immunological pathological changes, such as increased uterine natural killer (NK) cells and decreased Foxp3+ regulatory T (Treg) cells (Tuckerman et al. 2007, 2010, Galgani et al. 2015). Although a few biomarkers have been identified in RM and RIF, the underlying mechanism of altered immunological status in RM and RIF needs further appraisal.
Dendritic cells (DCs) are highly specialized antigen-presenting cells (APCs) and possess the capability to stimulate naïve T cells (Banchereau & Steinman 1998). They are widely distributed in all tissues and represent an essential link between innate and adaptive immunity. DC precursors first develop into immature DCs (iDCs), which express lower levels of MHC class I and II proteins. Upon appropriate stimulation, iDCs undergo several steps of maturation and migrate to secondary lymphoid organs. The mature DCs (mDCs) transport MHC–peptide complexes to the cell surface and express costimulatory molecules, such as CD80, CD86, CD40 as well as CD83, which finally lead to the activation of specific T cells (Mellman & Steinman 2001). DCs are not only involved in the induction of protective immunity, but also implicated in the maintenance of tolerance in a different states of activation or differentiation (Pulendran et al. 2010, Hubo et al. 2013).
It is now well established that immunological recognition of pregnancy is crucial for the maintenance of gestation. As the first encounter with fetal antigens, uterine DCs appear to be important players in the maternal–fetal immune adjustment during pregnancy. In early human pregnancy, decidual DCs produce lower levels of Th1-skewing cytokine interleukin-12p70 (IL-12p70) compared to their peripheral counterparts, suggesting that decidual DCs might regulate Th1/Th2 balance to maintain a Th2-dominate state and lead to the maintenance of normal pregnancy (Miyazaki et al. 2003). Immunohistochemical studies showed an increase in the number of mDCs and decrease in the quantity of iDCs in the decidua of patients with RM, suggesting that decidual DCs may play an important role in the pathogenesis of RM (Qian et al. 2015). Immunoglobulin-like transcript 4 (ILT4) is a tolerance molecule which could interact with its ligand human leukocyte antigen (HLA)-G on T cells and induce T cell anergy and Treg cells enrichment (Lynge Nilsson et al. 2014). Our previous studies showed that there was an expansion of ILT4+ DCs in both peripheral blood and endometrium in normal fertile women compared to patients with RM and RIF, strongly suggesting that they may accumulate at the maternal–fetal interface to promote immune tolerance (Liu et al. 2018). Therefore, DCs appear to be one of the key regulators of immunological shifts necessary for implantation and the progression of pregnancy, involving both innate and adaptive mechanisms.
The immune polarizations of different DC subsets, which interestingly may switch their phenotypes, are ultimately decided by cytokines or mediators in the surrounding micromilieu. Regarding that DCs are important in the establishment of immune tolerance in human pregnancy, it would be critical to study the microenvironment in which DCs reside or are activated may affect their functions toward tolerance rather than active immune response. Several molecules that modulate DCs function toward tolerance have been identified during last decades. Tolerogenic DCs can be generated by culturing monocyte-derived DCs with several immunosuppressive drugs such as tacrolimus (Morelli et al. 2000), rapamycin (Turnquist et al. 2007, Naranjo-Gomez et al. 2011, Boks et al. 2012) or pharmacological mediators, including 1,25-dihudrocyvitamin D3 (Piemonti et al. 2000, Naranjo-Gomez et al. 2011, Boks et al. 2012), glucocorticoids (Piemonti et al. 1999), prostaglandin E2 (Kalinski et al. 1997, Steinbrink et al. 2000, Obermajer et al. 2011). IL-10 is an immunomodulatory cytokine that plays an important role in downregulating immune responses and inducing immune tolerance (Mosser & Zhang 2008). There have been numerous studies showing that IL-10 is essential in the maintenance of normal pregnancy, and the increased production of IL-10 is associated with successful pregnancy (Jenkins et al. 2000, Nagaeva et al. 2002). Several studies have showed that monocyte-derived DCs modulated with IL-10 in vitro (DC-10) from human peripheral blood acquire the ability to induce T regulatory type 1 (Tr1) cells, which are anergic in vitro and suppress immune responses mainly via IL-10 and TGF-β (Steinbrink et al. 1997, Naranjo-Gomez et al. 2011, Boks et al. 2012). DC-10-induced Treg cells could strongly suppress T cell reactivity (Boks et al. 2012). Additionally, there was a significantly high percentage of DC-10 in the human decidua than in the peripheral blood of pregnant women, demonstrating its important role in establishing and maintaining maternal tolerance in the first trimester (Amodio et al. 2013). In this scenario, DC-10 is a good candidate for cell therapy since they are phenotypically stable cells with potent suppressive functions.
In this study, we investigated the phenotypes and cytokine profiles of different DC subsets including iDCs, mDCs and DC-10 in normal fertile subjects, patients with RM and RIF. We are the first to compare the tolerogenic properties of different DC subsets from patients with RM and RIF. Our data showed that DC-10 exhibited immunological suppressive phenotype characterized by relatively low expression of costimulatory molecule CD86, as well as MHC class II molecule HLA-DR, high expression of tolerance molecules HLA-G, ILT2, ILT4 and immunosuppressive cytokine IL-10 and DC-10 from normal fertile controls may display a relatively more tolerogenic property. Therefore, we speculate that DC-10 is of great interest for inducing or re-establishing immunological tolerance during pregnancy.
Methods
Subjects
Peripheral blood mononuclear cells (PBMCs) were collected from three groups of patients, including control, RM and RIF groups. The RM group includes ten women who had clinically spontaneous abortion for two or more times occurred prior to 20 weeks of gestation, confirmed by ultrasound. The RIF group includes seven women who had recurrent failure to get pregnant following transferred for at least six good-quality blastocysts/embryos totally in three or more embryo transfer cycles. The control group includes six women with proven fertility. All patients who currently or previously had autoimmune- or thyroid-related disease, abnormal karyotypes, uterine malformation, ultrasonographic evidence of hydrosalpinx and positive infectious disease tests including hepatitis B virus, hepatitis C virus, human immunodeficiency virus, Treponema pallidum particle assay, rapid plasma regain, TORCH and herpes virus were excluded from the study groups. The study was approved by the Ethics Committee of Shenzhen Zhongshan Urology Hospital upon informed consent with guarantees of confidentiality was obtained from all women.
DC differentiation
Fifteen milliliters heparinized blood samples were collected from control cases (n = 6; age 30.83 ± 2.93 years), RM cases (n = 10; age 33.30 ± 6.20 years) and RIF cases (n = 7; age 31.14 ± 4.41 years) during the mid-luteal phase (LH days 7–9) of the menstrual cycle. PBMCs were isolated by density gradient centrifugation using Ficoll–Paque (Amersham Bioscience), and the cells were washed with PBS twice. CD14+ monocytes were isolated by positive selection magnetic sorting using 20 μL of CD14 beads/1 × 107 PBMCs (Miltenyi Biotec, Bergisch Gladbach, Germany). Isolated CD14+ monocytes had an average purity of 96%, as analyzed by forward/side scatter and CD14 staining on flow cytometry. The cells were cultured as 5 × 105 cells/600 μL in 24-well flat-bottomed plates for 7 days at 37°C in RPMI 1640 (Gibco-BRL) supplemented with 1% antibiotics/antimycotics (Gibco-BRL), 10% FBS (Hyclone) and containing 10 ng/mL rhIL-4 (R&D Systems) and 50 ng/mL rhGM-CSF (R&D Systems) for iDC or in the presence of 10 ng/mL rhIL-10 for DC-10. After 5 days, iDCs were matured for another 2 days in this medium, but to which was added 1 μg/mL LPS. On day 7, DCs were collected and tested for levels of expression of CD14, CD83, HLA-DR, CD86, CD1a, ILT2, ILT4 and HLA-G. Supernatants were collected for testing the expression of cytokines TNF-α, IL-6, IL-10 and IL-12p70.
DC characterization
The cells were separated into two tubes for flow cytometric analysis, and one tube of cells were stained with the following anti-human antibodies: CD14-FITC, CD83-APC, HLA-G-PE, CD86-PE-Cy7 and HLA-DR-PerCP, while the other tube of cells were labeled with the following anti-human antibodies: CD1a-PE-Cy7, ILT2-FITC, ILT3-APC and ILT4-PE. The cell concentration of each tube was approximately 1 × 106 cells/mL. Control cells were stained with isotype-matched specificity antibodies. The cells were washed in PBS containing 3% NCS twice. The cell suspension was incubated with the appropriate monoclonal antibody for 15 min in the dark. The cells were then washed, suspended in 200 μL PBS and immediately analyzed in a flow cytometer (DxFLEX, Beckman Coulter). Details of all antibodies used in the study were listed in Table 1.
Antibodies table. Details of sources and concentrations of antibodies used for flow cytometric analysis.
| Antibody | Manufacturer | Catalog number | Isotype | Dilution or concentration (μg/mL) |
|---|---|---|---|---|
| CD14-FITC | eBioscience | 11-0149-42 | Mouse IgG1, κ | 10 |
| CD83-APC | eBioscience | 17-0839-42 | Mouse IgG1, κ | 5 |
| HLA-G-PE | eBioscience | 12-9957-42 | Mouse IgG2a, κ | 2.5 |
| CD86-PE-Cy7 | Biolegend | 305422 | Mouse IgG2b, κ | 1/40 |
| HLA-DR-PerCP | Biolegend | 307628 | Mouse IgG2a, κ | 1/40 |
| CD1a-PE-Cy7 | eBioscience | 25-0019-42 | Mouse IgG1, κ | 5 |
| ILT2-FITC | BD Biosciences | 555942 | Mouse IgG2b, κ | 1/10 |
| ILT3-APC | eBioscience | 17-5139-42 | Mouse IgG1 | 1.25 |
| ILT4-PE | eBioscience | 12-5149-42 | Mouse IgG2a, κ | 1/40 |
Cytokine determination
Following centrifugation for 5 min at 300 g to remove the cells and debris, the supernatant samples were stored at −80°C until further analysis. Multiplexed bead-based immunoassays were performed to assess the cytokine levels in each DC subpopulation. LEGENDplex™ Human immune response 4-plex panel (Biolegend) was used to assess the levels of TNF-α, IL-6, IL-10 and IL-12p70. The minimum detectable concentration of each cytokine is given as 2.4 pg/mL by the manufacturer. Samples were treated following the manufacturer’s instructions and measured with a Beckman device (DxFLEX; Beckman Coulter). Analysis was done using Legendplex software (Biolegend).
Statistical analysis
Statistical analyses were performed with SPSS, version 23.0 (SPSS). The data were presented as median with quartiles, and their comparisons were made by Kruskal–Wallis test. If a difference was found between groups, then a pairwise multiple comparison procedure was performed. The differences between comparison groups were considered to be statistically significant when P < 0.05.
Results
DC-10 display phenotypic characteristics of tolerogenic DC
Monocyte-derived DC-10 is generated in vitro by addition of exogenous IL-10, which is different from iDC and mDC. In RIF cases, in contrast to inflammatory mDCs, DC-10 showed a tolerogenic phenotype as demonstrated by decreased expression of the costimulatory molecule CD86 (MFI 129,951 (109,845–141,396) versus 212,434 (168,008–293,735); p = 0.037) (Fig. 1 and Supplementary Table 1, see section on supplementary data given at the end of this article). MHC class II molecule HLA-DR expression of DC-10 with IL-10 treatment was significantly lower compared with that in mDCs in RIF patients (MFI 32,100 (27,163–40,227) vs 53,004 (46,283–55,010); P = 0.048). IL-10 also induced an increase in the monocyte/macrophage-specific marker CD14 in DC-10 compared with iDC in all study groups, including control (MFI 38,140 (25,620–56,160) vs 3589 (1688–5714); P = 0.001), RM (MFI 30,863 (26,534–44,799) vs 5631 (3298–6708); P = 0.000) and RIF groups (MFI 38,070 (37,683–39,274) vs 3257 (2309-3812); P = 0.000). Analysis of the phenotype also illustrated that the DC-10 subset expressed immature molecule CD1a to a similar degree as iDCs. However, DC-10 exhibited a significantly higher expression of CD1a compared with mDCs in RM group (MFI 32,726 (14,430–54,849) vs 8423 (5191–10,061); P = 0.009) and RIF group (MFI 22,557 (17,345–32,999) vs 5827 (4839–6360); P = 0.033) (Fig. 1 and Supplementary Table 1).
Phenotype of DC-10. (A) Histograms of the different markers on different DC subpopulations are shown as open graphs, with matched isotype controls in gray. Three representative donors of six independent controls, ten independent patients with RM and seven independent patients with RIF analyzed are presented, respectively. (B) The mean fluorescence intensity (MFI; mean ± s.e.m.) is corrected for the MFI of matched isotype controls, and corrected MFI is shown. *P < 0.05, **P < 0.01, ***P < 0.001 when the data were analyzed by Kruskal–Wallis test followed by a pairwise multiple comparison procedure.
Citation: Reproduction 158, 3; 10.1530/REP-19-0172
In addition, we examined the expression of inhibitory molecules associated with tolerogenicity. IL-10 induced a significant increase in the expression of ILT2 compared with iDCs in the control group (MFI 17,184 (12,298–20,239) vs 7046 (2874–8088); P = 0.001) and RM group (MFI 14,062 (10,904–19,146) vs 9255 (4173–13,165); P = 0.018) (Fig. 2 and Supplementary Table 1). In RM group, the expression of HLA-G and ILT4 on DC-10 were significantly higher than those in iDCs (MFI 6022 (5303–8568) vs 4641 (2469–4961), P = 0.033; MFI 15,388 (7858–21,247) vs 6234 (4964–7611), P = 0.002, respectively) and mDCs (MFI 6022 (5303–8568) vs 3318 (1697–3550), P = 0.000; MFI 15,388 (7858–21,247) vs 7308 (6154–11,385), P = 0.031, respectively). Specifically, DC-10 generated from monocytes of healthy controls express significantly higher ILT4 compared with that from RIF patients (MFI 16,753 (10,549–26,364) vs 7248 (4192–8361), P = 0.019) (Fig. 2 and Supplementary Table 1).
Tolerance molecules expressed by DC-10. (A) Histograms of the different tolerogenic markers HLA-G, ILT2 and ILT4 on different DC subpopulations are shown as open graphs, with matched isotype controls in gray. Three representative donors of six independent controls, ten independent patients with RM and seven independent patients with RIF analyzed are presented, respectively. (B) The mean fluorescence intensity (MFI; mean ± s.e.m.) is corrected for the MFI of matched isotype controls, and corrected MFI is shown. *P < 0.05, **P < 0.01, ***P < 0.001 when the data were analyzed by Kruskal–Wallis test followed by a pairwise multiple comparison procedure.
Citation: Reproduction 158, 3; 10.1530/REP-19-0172
Cytokine production of iDCs, mDCs and DC-10
The production of inflammatory or regulatory cytokines by DCs during antigen presentation also plays a role in T cell differentiation. Thus, we investigated the influence of IL-10 on TNF-α, IL-6, IL-10 and IL-12p70 expressed by DCs. IL-12p70 production was similar among different DCs. DC-10 secretes significantly higher levels of IL-10 compared with iDC among control (19,897 (19,105–24,529) pg/mL vs 698 (433–1080) pg/mL; P = 0.007), RM (22,241 (13,435–27,180) pg/mL vs 184 (117–245) pg/mL; P = 0.007) and RIF women (14,074 (11,123–24,682) pg/mL vs 134 (46–386) pg/mL; P = 0.005) (Fig. 3 and Supplementary Table 2). However, DC-10 produced significantly lower amount of TNF-α compared with mDCs in all study groups (control: 2.06 (1.57–2.18) pg/mL vs 12.27 (4.49–64.57) pg/mL, P = 0.024; RM: 1.26 (0.98–1.80) pg/mL vs 23.69 (4.90–59.55) pg/mL, P = 0.007; RIF: 1.82 (1.46–1.98) pg/mL vs 31.04 (7.26–61.80) pg/mL, P = 0.013). Compared with mDCs, DC-10 secretes significantly lower amount of IL-6 in control group (3797 (2000–8624) pg/mL vs 29,387 (16,849–34,410) pg/mL; P = 0.032) and RM group (3907 (958–5274) pg/mL vs 30,339 (14,689–35,266) pg/mL; P = 0.013) (Fig. 3 and Supplementary Table 2). Overall, these findings demonstrate that DC-10, which are inducible in vitro from peripheral monocytes in the presence of exogenous IL-10, represent a DC subset with a unique cytokine production profile characterized by a high amount of IL-10 production, increased expression of tolerance molecules and reduced expression of activation markers.
Cytokine production by DC-10. Supernatants were collected and TNF-α, IL-6, IL-10 and IL-12p70 production by different DC subpopulations were determined by LEGENDplex. The mean ± s.e.m. levels of cytokines detected in four independent controls, four independent RM patients and four independent RIF patients are presented. *P < 0.05, **P < 0.01 when the data were analyzed by Kruskal–Wallis test followed by a pairwise multiple comparison procedure.
Citation: Reproduction 158, 3; 10.1530/REP-19-0172
Discussion
During pregnancy, specialized mechanisms have evolved to protect the semi-allogeneic fetus from maternal immune attack. Since the discovery of DCs by Steinman and Cohn (1973), DCs have been progressively established as central players in the regulation of immunity and tolerance. There have been numerous advances made in understanding how immunological tolerance is established and maintained during pregnancy, and DCs undoubtedly play a crucial role in these processes. This was demonstrated by the ablation of DCs led to spontaneous abortions in animal models (Krey et al. 2008, Plaks et al. 2008). Additional studies showed that decidual DCs could regulate the Th1/Th2 balance to maintain a Th2 dominant state, leading to the maintenance of normal pregnancy (Miyazaki et al. 2003). Present evidences clearly highlight the general importance of DCs in maintaining immunohomeostasis and is explained by their functional plasticity. The classification of DCs was not only defined by its immature and mature states, but also known that DCs can adopt distinct phenotype and function depending on their microenvironment. The exposure of DCs to immunosuppressive signals triggers the development of tolerogenic DCs, which are enriched with immunosuppressive characteristics. By understanding the mechanisms leading to DC immune tolerance, we will better understand the complex immune regulation during human pregnancy.
Activation of naïve T cells requires distinct signals delivered by DCs: signal I is mediated by MHC in complex with a peptide from captured antigens and is received by a specific T cell receptor; signal II is a costimulatory signal which is mandatory to T cell activation; signal III is in the form of several soluble factors such as IL-12, IL-15, IL-6 and TNF-α (Hubo et al. 2013). CD80 and CD86 expressed by DCs are probably one of the most important costimulatory pathway in T cell activation (Lenschow et al. 1996). Signaling through binding with CD28 on T cells confers production of IL-2, which is a factor that promotes expansion and survival of primary T cells (Linsley et al. 1991). Our results showed that DC-10 expressed significantly lower level of MHC class II molecule HLA-DR and costimulatory molecule CD86 in RIF patients, indicating that DC-10 may exhibit impaired capacity to activate naïve T cells and protect the allogeneic fetus from T cell activation.
Contrary to iDCs, whose tolerogenicity is mostly based on the absence of adequate costimulatory molecules, DC-10 possesses several specific elements of active tolerance. One of the major hallmarks of DC-10 is their extensive production of immunosuppressive cytokine IL-10, which directs the polarization of T cells into Tregs (Wakkach et al. 2003, Svajger & Rozman 2014). IL-10 also serves as a positive feedback loop, further strengthening the immunosuppressive biology of DCs and other immune cells in the microenvironment (Couper et al. 2008). IL-10 mediates immunosuppressive functions by binding to the IL-10 receptor complex and further leads to downstream activation and homodimerization of STAT3 through tyrosine phosphorylation of Tyk2/JAK1 (Moore et al. 2001). It has been well established that IL-10 plays an important role in the modulation of diseases such as RM and RIF (Kwak-Kim et al. 2003). In our study, no matter DC-10 was derived from peripheral blood of patients with RM, RIF or normal fertile controls, they all produced significantly higher IL-10 than iDC. Thus, we speculate that DC-10 expressing high amount of IL-10 may also contribute to the maternal immune tolerance to protect the fetal antigens from recognition by maternal immune system.
Besides IL-10, inhibitory molecules expressed by DC-10 have also been associated with their tolerogenic functions. One of those is the family of receptors called ILTs, which have a long cytoplasmic tail that contains ITIMs. ILTs mediate negative intracellular signaling upon activation by recruiting tyrosine phosphatase Src homology-2-containing tyrosine phosphatase 1 (Munitz 2010). In our study, DC-10 expresses high levels of ILT2 and ILT4, which have been reported to be associated with their tolerogenicity (Manavalan et al. 2003, Gregori et al. 2010). They can be induced by IL-10, tryptophan deprivation, HLA-G and certain immunosuppressive pharmacological agents (Chang et al. 2002, Vlad et al. 2003, Ristich et al. 2005, Adorini & Penna 2009, Brenk et al. 2009). Among these stimulators, HLA-G is a nonclassical MHC class I inhibitory molecule and is shown to be the receptor for ILT2 and ILT4 (Menier et al. 2010). HLA-G is also expressed on DC-10 and is associated with immune regulatory mechanisms involved in pregnancy (Amodio et al. 2013). HLA-G mediates immunosuppressive function by activating signaling through ILT2 and ILT4. In our study, DC-10 expresses significantly higher level of HLA-G in RM patients. The HLA-G/ILT2/ILT4 pathway associated with DC-10 was also reported to play a role in the inhibition of NK and T cell function, as well as the suppression of DC maturation (Le Friec et al. 2003, Liang & Horuzsko 2003). Thus, the expression of these inhibitory molecules by DC-10 may contribute to the generation of a tolerogenic microenvironment and DC-10 may be regarded as suppressor cells capable of inhibiting other effector cells. Additionally, the high expression of ILT4, HLA-G and IL-10 by DC-10 are necessary for their tolerogenic activity and ability to prime T cells to become Tr1 cells, which are characterized by high secretion of IL-10 and TGF-β and predominantly induce tolerance by cytokine-mediated mechanisms. Addition of blocking antibodies against ILT4, HLA-G and IL-10R during co-culture of DC-10 and naïve T cells completely inhibited Tr1 cell induction (Gregori et al. 2010). Taken together, these tolerogenic DC-10 not only employ secreted mediators and inhibitory receptors to drive Tr1 cell generation but can also provide signals to inhibit other effector cells to maintain pregnancy.
Notably, our results showed that there were no significantly different expressions of maturation marker, costimulatory marker, MHC class II molecule, inhibitory molecules (except ILT4) and cytokine production of DC-10 among control, RM and RIF groups. This finding may be explained by the addition of exogenous IL-10 to DC cultures. DC-10 in our study was cultured from monocytes in vitro rather than identified from peripheral blood in vivo. IL-10 was added during the entire DC culture starting with monocytes from all patients included in the presence of IL-4 and GM-CSF. Our results also indicate that IL-10 signaling of monocytes from RM and RIF patients may not be impaired because of their similar phenotype with controls after IL-10 treatment. Additionally, considering that ILT4 plays an important role in the induction of Tr1 cells, our results showing that DC-10 from RIF patients expressed significantly lower ILT4 compared with normal controls may suggest that DC-10 from normal controls exert more tolerogenic functions. However, further in-depth investigations are required to elucidate the role of DC-10 in pregnancy complications.
To the best of our knowledge, this is the first comparison of different types of DCs from patients suffered from RM and RIF and normal fertile controls. DC-10 was characterized by relatively low expression of costimulatory molecule CD86, as well as MHC class II, high expression of tolerance molecules HLA-G, ILT2, ILT4 and immunosuppressive cytokine IL-10, but produced little or no proinflammatory cytokines, such as TNF-α, IL-6 and IL-12p70. A better understanding of the phenotypical properties, the precise molecular mechanisms and the immunological functions of DC-10 will provide important information for the maintenance of maternal immune tolerance and homeostatic environment in human pregnancy. In the long run, these findings will support the development of novel and innovative immunotherapeutic approaches for the control of pregnancy complications, such as RM and RIF.
Supplementary data
This is linked to the online version of the paper at https://doi.org/10.1530/REP-19-0172.
Declaration of interest
The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported.
Funding
This work was supported by Basic Research Program of Shenzhen (JCYJ20170307140647669), specific funding from Clinical Medical Research of Chinese Medical Association – Reproductive Medicine Clinical Research and Development Youth Programs (17020300699), Science and Technology Project of Health and Family Planning Commission of Shenzhen Municipality (SZBC2018003) and Sanming Project of Medicine in Shenzhen (SZSM201502035).
Acknowledgements
The authors would like to thank the staff at the Fertility Center at Shenzhen Zhongshan Urology Hospital for recruiting patients and helping with samples.
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