IL-22 secreted by decidual stromal cells and NK cells promotes the survival of human trophoblasts

Abstract

Interleukin-22 (IL-22) has been implicated as an important immune regulator in many physiologic and pathological processes, but little is known about the IL-22 in the fetal-maternal interface. In this study, we demonstrated that co-culture of decidual stromal cells (DSCs) and decidual natural killer (dNK) cells resulted in increased secretion of IL-22, compared to culture of DSCs or dNK cells alone. The trophoblast cell line, HTR8/SVneo, expresses IL-22 receptor α1 (IL-22R1). Combinant human (rh) IL-22 significantly promoted the proliferation and viability, and inhibited the apoptosis of HTR8/SVneo cells. By Western blotting and immunohistochemistry, we confirmed that villi expressed IL-22R1, and the villi from unexplained spontaneous miscarriage patients expressed reduced levels of IL-22R1 than those from normal early pregnancy. These findings indicate that the IL-22 secreted by DSCs and dNK might promote the survival of trophoblasts and participate in the maintenance of pregnancy by binding to the IL-22R1. The reduced level of IL-22/IL-22R1 in villi might be involved in the occurrence of spontaneous miscarriage.

Introduction

Trophoblast cells of the human placenta proliferate, differentiate and invade the endometrium leading to successful implantation of the embryo. It involves extensive cross-talk between the embryo-derived trophoblast cells and mother-derived cells at the site of implantation. A maternal component at the maternal-fetal interface is composed mainly of decidual stromal cells (DSCs), and a large and specific population of leukocytes (decidual leukocytes cells, DLCs). Among the various types of DLCs, decidual natural killer (dNK) cells are major cellular component in human decidua. However, dysfunction between the interaction of trophoblasts and decidual cells has been demonstrated in association with not only fetal intrauterine growth restriction and pre-eclampsia, but also first-trimester and late-term miscarriage [1-4].

IL-22 is one of the IL-10-family cytokines, which also include IL-10, IL-19, IL-20, IL-24, and IL-26, as well as more distantly related IL-28 and IL-29. IL-22 was first identified as an IL-10-related T cell-derived inducible factor (IL-TIF) from a lymphoma cell line and mainly secreted by active T helper cells and NK cells [5]. The functional IL-22 receptor complex consists of two chains, IL-22R1 and IL-10R2, which are ubiquitously expressed in various organs and cell types [6]. IL-22 activates a signal transduction cascade that results in the rapid activation of several transcription factors including STAT proteins via binding the receptor complex [7]. In addition, there is another receptor of IL-22, IL-22BP, a soluble molecule which inhibits cellular IL-22 effects [8].

Data from in vitro studies with various primary cells and cell lines implicate the potential roles of IL-22 in host defense, inflammation, and tissue repair [9-13]. Comprehensive analyses have demonstrated that IL-22 played an important role in the autoimmune diseases such as psoriasis [9,12,14,15], inflammatory bowel disease [16,17], rheumatoid arthritis [18], systemic lupus erythematous [19], infection and tumor [20]. However, the study of IL-22 in the reproductive system has not been widely performed yet. Only in a very recently published article [21], expression of IL-22R1 mRNA was shown in equine pregnant endometrium. The aim of this study is to elucidate, the role of IL-22 in the fetal-maternal interface and its relevance to human pregnancy.

In this study, we showed co-culture of dNK and DSCs could secrete increased level of IL-22. This cytokine could promote the proliferation, enhance the cell viability and inhibit the apoptosis of trophoblast cell line HTR8/SVneo. Furthermore, expression of IL-22R1 was detected in the trophoblasts from both normal pregnant women and patients with spontaneous miscarriage and the level of IL-22R1 from the latter group was significantly lower than that from the former one.

Material and methods

Primary tissues

The decidual and villi tissues were obtained from clinically normal pregnancies, which were terminated for social reasons and unexplained spontaneous early abortions (USA) at the Obstetrics and Gynecology Hospital of Fudan University. Each patient completed a signed, written consent form. The Fudan University Human Investigation Committee approved this study and the use of the placental tissue.

Cell line

The human trophoblast cell line HTR8/SVneo was cultured in DMEM/F12 medium (GIBCO BRL Life Technologies, Grand Island, NY) supplemented with 10% fetal bovine serum (FBS; Hyclone Laboratories, Logan, UT, USA) and 1% penicillin and streptomycin in a humidified incubator at 37°C and 5% CO₂.

Isolation of DSC and decidual NK cells (dNK)

The detailed description of the procedures used to isolate these cells from first-trimester placentas (n=16) have been reported in the literatures [22,23]. DSC was cultured in DMEM/F12 medium supplemented with 10% FBS (Gibco) and incubated in 5% CO₂ at 37°C. Total decidual lymphocyte was incubated with microbeads of conjugated anti-human CD3 and anti-human CD56 mAb (Miltenyi Biotec), and then CD3^-CD56⁺dNK cells were purified. Separation was performed with the AutoMACS instrument (Miltenyi Biotec).

Flow cytometric analysis

The evaluation of cell surface expression of CD3/CD56 was performed on dNK cells by direct cell surface labeling after pre-saturation of cells at 4°C for 20 min with goat normal serum diluted 1/20 in PBS. The cells were then washed twice and incubated at 4°C for 20 min with Percp5.5-conjugated mouse anti-human CD3, Allophycocyanin (APC)-conjugated mouse anti-human CD56, or the relevant isotype control (all from Biolegend, San Diego, USA). Then their purity was detected by flow cytometry (FCM). Samples were analyzed in a FACS Calibur flow cytometer (Becton Dickinson, USA) using Cellquest software (Becton Dickinson). Three independent experiments were performed, each in triplicate.

Enzyme-linked immunosorbent assay (ELISA)

Primary cultured decidual stromal cells are seeded or not in the 24-well plate (1*10⁵ cells/well), after attachment, the fresh isolated dNK (1*10⁵ cells/well) were seeded in and co-cultured for another 24 hs. At last, the supernatants were collected and detected by IL-22 ELISA kit (R&D Systems, Abingdon, UK) according to the instructions of the manufacturer. The minimal detectable concentration for IL-22 was 2.7 pg/ml. 6 individual placental samples were tested independently.

Immunostaining

For immunocytochemical staining, HTR8/SVneo cells were fixed in 4% paraformaldehyde for 20 min at room temperature after cultured for 48 hours, washed in PBS and permeabilized for 20 min in 0.1% Triton X-100-PBS. The cells were then incubated with 10% FBS in PBS for 30 min to reduce non-specific binding. Rabbit Anti-human IL-22R1 polyclonal antibody (10 ug/ml, 5 ug/ml, 2.5 ug/ml; Abcam, USA) and isotype were administrated overnight at 4°C to detect whether HTR8/SVneo cells express IL-22R1. The cells were then incubated with a biotinylated secondary antibody for 30 min at room temperature. Streptavidin-HRP was applied for another 30 min at room temperature. The cells were stained with DAB (Zhongshan Cambridge Company, Beijing, China) and counterstained with haematoxylin. The experiments were repeated three times.

For immunohistochemistry (IHC), paraffin-embedded villi specimens (6 samples of normal pregnancies and unexplained miscarriages selectively) were sliced at a 5 um thickness. Tissue sections were de-waxed in xylene, rehydrated in alcohol, and antigen retrieval was performed by heating each section at 100°C for 30 min in 0.01 mol/L sodiumcitrate buffer (pH 6.0). Then the sections were immersed in 3% hydrogen peroxide for 15 min to suppress endogenous peroxidase activity. After blocking with 10% fetal bovine serum for 1 hour at room temperature, the sections were incubated with anti-IL22R1 polyclonal antibody (5 ug/ml, Abcam, USA) and isotype overnight at 4°C. They were then sequentially treated with a biotinylated anti-rabbit antibody and HRP-labeled streptoavidin. Development was performed by treating the sections with a Liquid DAB-Plus Substrate kit (Zhongshan Cambridge Company, Beijing, China). After counterstaining with hematoxylin, immuno-staining of IL22R1 on the tissue sections was detected by light microscopy.

Western blot

100 mg fresh villi tissues from normal pregnancies (n=6) or unexplained miscarriages (n=6) were added 1 ml RIPA lysis and 10 μl PMSF, centrifuged at 12,000 g at 4°C for 30 min, and cell lysates were assayed for protein concentration using the Bradford protein assay (Beyotime Company, Shanghai, China). Proteins (50 ug) were separated on 10% SDS-polyacrylamide gels, then transferred to a nitrocellulose membrane. After being blocked in blocking buffer, the membrane was incubated with anti-IL22R1 polyclonal antibody (1:400, Abcam, USA) overnight at 4°C, washed and incubated with HRP labeled secondary antibody (1:5000, Santa Cruz, CA, USA). Blots were developed using Amersham ECL detection kit (Amersham Pharmacia Biotech, Freiburg, Germany). The experiments were repeated three times.

Cell viability/proliferation/apoptosis assays

Two different techniques, MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) (Sigma Chemicals, St. Louis, MO) assay and BrdU (5-Bromo-2-deoxyUridine) (Millipore, USA) assay, were applied to measure the effect of rhIL-22 (Peprotech, USA) on viability/proliferation of HTR8 cells. HTR8 cells were re-suspended in DMEM/F12 with 10% FBS and seeded at a density of 3000 cells/well in 96-well fl at-bottom microplates. After 24 h, we replaced the medium by DMEM/F12 containing 1% FBS and cultured the cells for another 12 h. Then the medium was removed again, and the cells were stimulated with rhIL-22 (0, 25, 50, 100, 200 ng/ml), at 37°C for 24 h.

For the MTT assay, 20 ul of the MTT reagent was added to each well of the 96-well microplates and incubated at 37°C for 4 h, and 150 ul of DMSO was added, absorbency was measured at a wavelength of 570 nm on an automatic microplate reader.

For BrdU assay, 20 ul of BrdU reagent was added to each well of the 96-well microplates and incubated at 37°C for 24 h. Then we decanted the medium and washed cells for 3 times with PBS, added 200 ul fixation dilution and incubated for 30 min, washed 3 times, added 100 ul 1×BrDU primary monoclonal antibody, incubated for 1 hour at room temperature, washed 3 times, added 100 ul goat anti-mouse HRP-IgG, incubated for 30 min at room temperature, washed 3 times, added 100 ul TMB substrate solution for 30 min at room temperature and added 100 ul stop solution, at last, absorbency was measured at a wavelength of 450 nm - 570 nm on an automatic microplate reader. The experiments were repeated three times.

For apoptosis assay, the cells were digested by 0.25% trypsin without EDTA, and then centrifuged 1000 g for 5 min, re-suspended with PBS, labeled the cells by Annexin V and PI according to the instruction manual, and detected the percentage of early apoptosis cells by flow cytometry. The apoptosis detection kit was obtained from Merk Company (Merk, Germany).

Statistical analysis

All values are shown as mean±SEM. Comparison between controls and various treatments was performed by one-way ANOVA, with application of the Dunnett test. Differences were accepted as signifi cant at P<0.05.

Results

Co-culture of DSCs and dNK cells produces increased level of IL-22

DSC and decidual NK cells (dNK) were isolated from human first-trimester placentas (n=16) as described before [22,23]. Using immunofl uo-rescent labeling for the CD56, CD3 and subsequent fl ow cytometric analysis, it was confi rmed that the purity of the CD3^-CD56⁺dNK cells isolated in our study was more than 95 percent (Figure 1A). And then, we mimicked the microenvironment of fetal-maternal interface by co-culturing dNK with DSCs, and detected the level of IL-22 in the cell culture supernatant. The results showed that both dNK cells and DSCs could secrete low level of IL-22. The co-culture system significantly elevated the secretion of IL-22 in the supernatant (P<0.01) (Figure 1B). These results indicated that the interaction between dNK and DSCs might play some roles at the fetal-maternal interface by secreting IL-22.

Figure 1.

Co-culture of DSCs and dNK cells produces increased level of IL-22. A: dNK cells were isolated and purified by CD56 and CD3 MACS beads from human first-trimester decidual tissues. The cells were then stained with CD56-APC and CD3-Percp5.5 and analyzed by FCM. Three independent experiments were performed, each in triplicate. Representative figures are shown. B: Primary dNK cells or DSCs were cultured alone or co-cultured for 48 h, and the supernatants were harvested and the level of IL-22 was measured by ELISA. **P<0.01 was compared to dNK cells alone or DSCs alone.

Human extravillous trophoblast cell line HTR8/SVneo expresses IL-22R1

To further study the functions of IL-22 at the fetal-maternal interface, we used the cell line HTR8/SVneo. The source of HTR8/SVneo is primary cultured trophoblast, so it has the most similar characterization to that of the primary cells. Firstly we identified the expression of IL-22 receptor IL-22R1 on this cell line by immunocytochemistry. We stained the cells with anti-human IL-22R1 antibody at different concentrations (1:50, 1:100, and 1:200) and demonstrated IL-22R1 specific expression. We repeated the experiment for 3 times and the results were accordant (Figure 2).

Figure 2.

Human extravillous trophoblast cell line HTR8/SVneo expresses IL-22R1. HTR-8/SVneo cells were stained with anti-IL-22RA1 polyclonal antibody in different concentration (1:50, 1:100, and 1:200) and the immunostain signal weakened followed by the dilution of antibody. Original magnification: ×100. These pictures are representatives of three individual experiments.

Recombinant human IL-22 promotes viability and proliferation of HTR-8/SVneo cells

The effect of recombinant human IL-22 (rhIL-22) on the viability and proliferation of HTR-8/SVneo cells was studied. The results of MTT assay showed that rhIL-22 could enhance the viability of HTR8 (P<0.01) (Figure 3A). Moreover, rhIL-22 promoted the proliferation of HTR8/SVneo in a dosage dependent manner (P<0.05 or P<0.01) (Figure 3B).

Figure 3.

Recombinant human IL-22 promotes viability and proliferation of HTR-8/SVneo cells. A: HTR-8/SVneo cell line was treated with different concentrations of rhIL-22 (0, 50, 100 and 200 ng/ml) for 48 h and then the cell viability and proliferation were detected by MTT and BrdU proliferation assays, respectively. The OD value of the control group which was not treated by the rhIL-22 was standardized as 1. The values of the treated cells were compared with the values generated from the untreated control cells and reported as the percentage viability and proliferation. *P<0.05 or **P<0.01 was compared to the vehicle control. Error bars represent the standard error of the mean.

rhIL-22 restricts the apoptosis of HTR-8/SVneo cells

Result in Figure 4 showed that the apoptosis percentage of HTR-8/SVneo cells pretreated with 50 ng/ml IL-22 decreased from 2.1%± 0.13% to 1.17%±0.13% (P<0.01) (Figure 4A and 4B). These data suggest that rhIL-22 significantly stimulated the viability and proliferation, inhibited the apoptosis of HTR-8/SVneo cells, which might promote the growth and survival of trophoblasts in early pregnancy.

Figure 4.

rhIL-22 restricts the apoptosis of HTR-8/SVneo cells. A, B: HTR-8/SVneo was treated with or without 50 ng/ml rhIL-22 for 48 h and then the apoptosis rate was analyzed by apoptosis assay. **P<0.01 was compared to the vehicle control. Error bars represent the standard error of the mean. Three independent experiments were performed.

The expression of IL-22R1 is decreased in the villi from patients with miscarriage

Finally, we analyzed the expression level of IL-22R1, a subunit of IL-22 receptor complex in human villi tissues from 6 normal pregnant women and 6 unexplained miscarriage patients by IHC and Western blot. The results showed that the villi from both groups could express IL-22R1, while the villi from the normal group had significantly higher expression (P<0.05) (Figure 5A-D), indicating that villi tissue might be one of the target tissues of IL-22, and IL-22/IIL-22R1 signaling might participate in the maintenance of normal pregnancy.

Figure 5.

The expression of IL-22R1 is decreased in the villi from patients with miscarriage. The expression of IL-22R1 on human first-trimester villi of normal early pregnancy women (n=6) and unexplained miscarriage patients (n=6) were detected by Western blotting (A, B) and immunohistochemistry (C, D), respectively. Original magnification: ×100. *P<0.05 was compared to normal early pregnancy group. Error bars represent the standard error of the mean.

Discussion

During the first trimester of pregnancy, DSCs play an important role in regulating the growth and invasion of trophoblasts at the maternal-fetal interface [22]. The dNK cells account for more than 70 percent of total lymphoid population in the maternal deciduas [24]. This group of NK cells displays a unique functional profile and also plays important roles including promoting trophoblast cell invasion, vascular growth [25-27] and the induction of regulatory T cells that modulate the maternal physiologic immune response to prevent fetal rejection [28]. It is known that dNK cells may participate in the maintenance of successful pregnancy through secreting some Th2 type cytokines such as IL-4 and IL-10 [29]. In recent years, the Th-17 subset of T helper cells was confirmed in the fetal-maternal interface and was confirmed to induce the proliferation and invasion of trophoblast by secreting IL-17 (unpublished data). The concept of Th1/Th2/Th17 and regulatory T-cell paradigm in pregnancy is being established.

As a typical Th17 type cytokine, IL-22 is one of the IL-10-like family cytokines and first identified as an IL-10-related T cell-derived inducible factor (IL-TIF) from a lymphoma cell line [5]. Th17 cells are clearly the dominant IL-22 producer both at the mRNA and protein levels [30]. IL-22 could also be produced by innate immune cells, such as NK [14], gamma/delta T [31] and NKT cells. No expression was found in monocyte-derived macrophages or dendritic cells [32]. Victoria Male et al [33] found that uterine stage 3 NK cells, the CD34^-CD117+CD94^- NK cells, constitutively produced IL-22 by FCM. Similarly, we also found that dNK cells produce IL-22, and the co-culture with DSCs could increase the level of IL-22 in the co-culture unit.

In view of the biological effects, IL-22 increases the innate immunity of tissue cells, protects tissues from damage, and enhances their regeneration. Until recently, researchers have focused the function of this cytokine in a variety of immune diseases such as psoriasis. In fact, some unsuccessful pregnancies are the result of maternal immune rejection of fetus. Thus, we speculated whether there was relationship between IL-22 and unexplained miscarriage. IL-22 mediates its effects via a heterodimeric trans-membrane receptor complex consisting of IL-22R1 and IL-10R2 and subsequent Janus kinase–signal transducers and activators of transcription (JAK–STAT) signaling pathways including Jak1, Tyk2, and STAT3 [6,7]. In addition to this cell surface-associated IL-22 receptor complex, there is a secreted (“soluble”), single-chain, high affinity IL-22 binding receptor named IL-22 binding protein (IL-22BP) which could negatively regulate the activity of IL-22 [8]. Early studies [34] demonstrated that in contrast to other T and NK cell cytokines, no expression of IL-22R1 was detected in bone marrow, blood mononuclear cells, thymus, or spleen or in a variety of isolated resting or activated primary immune cells including monocytes, B cells, T cells, NK cells, macrophages, and immature and mature Dendritic cells. Some tissues and organs forming the body barriers and containing epithelial cells such as skin, kidney and those from the digestive (pancreas, small intestine, liver, colon) and the respiratory (lung, trachea) systems express the IL-22R1 and IL-10R2 complex. Recently, researchers [21] published the expression of IL-22R1 on the endometrium of D32 and D34 pregnancy and believed IL-22 facilitated re-epithelialization of the endometrium after trophoblast migration. In our previous studies [23], we have shown that trophoblasts derived from epithelial and were CK7 positive. Moreover, this study showed that the trophoblast cell line HTR-8/SVneo expressed the receptor IL-22R1. Therefore, trophoblast cells might be the target of IL-22.

To further study the detailed functions of IL-22 on trophoblasts, we used IL-22 to stimulate the HTR-8/SVneo cells and found that IL-22 markedly promoted the proliferation, enhanced the cell viability and reduced the apoptosis of HTR-8/SVneo cells. That is to say, IL-22 played an important role in modulating the functions of trophoblasts. The formulation of placenta represents an important biologic behavior in early pregnancy, in which process the proliferation, differentiation and invasion of trophoblasts are the critical events. The proliferation ability of trophoblasts was strong in early pregnancy and weakening alongside the gestation proceeding. The trophoblasts with high proliferation ability help to promote the embryo development and pregnancy maintenance while those with disturbance in the proliferation ability may lead to the occurrences of pregnancy-associated diseases such as recurrent spontaneous abortion (RSA), Fetal growth restriction (FGR) and pre-eclampsia. Other researchers [35] also found IL-22 could promote liver cell regeneration by increasing hepatic cell proliferation and hepatocyte migration through the activation of Akt and STAT signaling. Zhang W et al reported that IL-22 protected human lung cancer cells from starvation and chemotherapy drug-induced apoptosis via activation of STAT3 and its downstream anti-apoptotic proteins (Bcl-2 and Bcl-xL) and inactivation of extracellular signal regulated kinase (ERK1/2) [20]. To some extent, trophoblast cells have similar characterizations to tumor cells. Furthermore, both ERK1/2 [36] and STAT3 [37] signaling are involved in regulating the biological behavior of trophoblasts. So, we believed that IL-22 from dNK cells and DSCs might stimulate the growth and survival of trophoblasts at the fetal-maternal interface in a paracrine manner by binding the IL-22 receptor on the trophoblasts through regulating ERK1/2 and or STAT3 signaling.

Subsequently, we compared the expression of IL-22R1 in villi from normal early pregnant and unexplained miscarriage patients. The results showed that trophoblast from both normal early pregnant and miscarriage patients expressed IL-22R1. Our results also showed that trophoblast from miscarriage patients expressed significantly lower levels of IL-22R1, which might involve in the occurrence of disease by limiting the growth and proliferation of trophoblast.

Most recently, there was report [21] that IL-22 was expressed by a non-immune cell type, the invasive trophoblast cells of the equine chorionic girdle. This was the first study of non-lymphocytes derived IL-22 and expanded the resources of this cytokine and indicated the more widespread functions of it. Based on these results and our findings, IL-22 might be a beneficial cytokine for a successful pregnancy. The IL-22 secreted by the dNK cells, DSCs and trophoblasts at the maternal-fetal interface might promote the proliferation, enhance the cell variability and repress the apoptosis of trophoblasts in paracrine and autocrine manners through the cross-talking between dNK cells, DSCs and Trophoblasts. While the abnormal lower level of IL-22R1 in trophoblasts may be the reason for the dysfunction of trophoblasts growth and survival, which is not conducive to the formation and development of placenta and maintenance of normal pregnancy and may finally be involved in the occurrence of miscarriage.

Disclosure of conflict of interest

None.