|Year : 2017 | Volume
| Issue : 4 | Page : 198-203
CCL2 enhances the viability of human chorionic trophoblast cell line HTR-8/SVneo Cells by inhibiting Interleukin-24 Expression
Jun Shao1, Yin-Yan He2, Da-Jin Li3, Ming-Qing Li3
1 Laboratory for Reproductive Immunology, Hospital and Institute of Obstetrics and Gynecology, Fudan University, Shanghai 200011, China
2 Department of Obstetrics and Gynecology, Shanghai General Hospital, Shanghai Jiao Tong University, Shanghai 200080, China
3 Laboratory for Reproductive Immunology, Hospital and Institute of Obstetrics and Gynecology, Fudan University, Shanghai 200011; Key Laboratory of Reproduction Regulation of NPFPC, Shanghai 200032, China
|Date of Submission||17-Nov-2017|
|Date of Web Publication||7-Feb-2018|
Laboratory for Reproductive Immunology, Hospital of Obstetrics and Gynecology, Fudan University Shanghai Medical College, Shanghai 200011
Source of Support: None, Conflict of Interest: None
Background: To investigate the regulatory effect of interleukin-24 (IL-24) on cell viability of human chorionic trophoblast cell line (HTR-8/SVneo cells).
Methods: Immunohistochemical staining was used to detect the expression of IL-24 and its receptors IL-20R1, IL-20R2, and IL-22R1 in villus tissue at early normal pregnancy. The effect of thymic stromal lymphopoietin (TSLP) and chemokine CCL2 on the expression of IL-24 in human chorionic trophoblast cell line HTR-8/SVneo cells was analyzed by In-cell Western. In addition, the effect of recombinant human IL-24 (rhIL-24) and CCL2 on the viability of HTR-8/SVneo cells was analyzed by MTT assay.
Results: IL-24 and its receptors showed a strong positive staining in trophoblasts at early normal pregnancy. Compared with control group, expression of IL-24 in HTR-8/SVneo cells was significantly inhibited after in vitro stimulation of recombinant human CCL2 protein (rhCCL2) (P < 0.001). The viability of HTR-8/SVneo cells was significantly decreased after treatment with rhIL-24 (P < 0.001). In contrast, anti-IL-24 neutralizing antibody significantly enhanced the viability of HTR-8/SVneo cells (P < 0.01). In addition, rhCCL2 (100 μg/L); enhanced the viability of HTR-8/SVneo cells (P < 0.01) in vitro, but this effect was inhibited by treatment with rhIL-24.
Conclusions: CCL2 enhances the viability of human trophoblast cell line HTR-8/SVneo cells in vitro by inhibiting the secretion of IL-24, which may be beneficial to blastocyst implantation and placental development.
Keywords: CCL2; Cell Viability; Chorionic Trophoblasts; HTR-8/SVneo Cells; Interleukin-24
|How to cite this article:|
Shao J, He YY, Li DJ, Li MQ. CCL2 enhances the viability of human chorionic trophoblast cell line HTR-8/SVneo Cells by inhibiting Interleukin-24 Expression. Reprod Dev Med 2017;1:198-203
|How to cite this URL:|
Shao J, He YY, Li DJ, Li MQ. CCL2 enhances the viability of human chorionic trophoblast cell line HTR-8/SVneo Cells by inhibiting Interleukin-24 Expression. Reprod Dev Med [serial online] 2017 [cited 2018 Aug 17];1:198-203. Available from: http://www.repdevmed.org/text.asp?2017/1/4/198/224918
| Introduction|| |
Normal pregnancy is similar to the natural allogeneic allograft, which not only requires the mother's specific immune tolerance to embryonic antigens but also depends on high viability, proliferation ability, and invasion ability of trophoblast cells, which is similar to malignant tumor cells. Cytotrophoblasts in inner layer of villi can differentiate into villous extravillous trophoblasts through division and proliferation. Extravillous trophoblasts can invade deeply into the decidual tissue of the mother to completely fix the placenta and fetus, and provide adequate nutrition for embryonic development. Extravillous trophoblasts can also replace uterine spiral artery endothelial cells, thereby recasting the maternal uterine vasculature. Viability, proliferation and invasion abilities of trophoblasts are crucial for blastocyst implantation, placental development, and establishment of appropriate maternal-fetal relationships. Abnormal viability, proliferation and invasion abilities of trophoblasts may be related to pregnancy-related diseases such as spontaneous abortion, intrauterine growth restriction, preeclampsia,,,,, and trophoblastic tumor. It is known that a variety of cytokines, chemokines, and proteases such as matrix metalloproteinases play important roles in the regulation of viability, proliferation, and invasion abilities of trophoblasts.,,, However, the regulatory mechanism of trophoblast viability in maternal-fetal interface under unique microenvironment remains unclear.
Interleukin-24 (IL-24), also known as melanoma differentiation associated gene 7 (MDA-7), was discovered in 1995 by Jiang et al. As a novel member of IL-10 superfamily cytokines, IL-24 can induce apoptosis of tumor cells. IL-24 has a wide range of antitumor biological effects, such as inducing apoptosis of various tumor cells and inhibiting tumor growth.,, Our previous study showed that human decidual stromal cells expressed IL-24 in early pregnancy, and estrogen could promote the proliferation of decidual stromal cells in vitro by inhibiting the expression of IL-24. However, expression of IL-24 in trophoblasts at early pregnancy and its effect on the biological behaviors of trophoblasts remains unclear.
Therefore, in our study, we first investigated the expression of IL-24 and its receptors in human chorionic trophoblast cells at normal early pregnancy stage. Then, we further explored the possible regulation of IL-24 expression in maternal-fetal interface microenvironment and its effects on cell viability of the trophoblast cell line HTR-8/SVneo in vitro to clarify the regulation mechanism of trophoblast viability and provide a new research direction for the clinical prevention and treatment of disorders related to trophoblast cell dysfunction.
| Methods|| |
This study was approved by the Ethics Committee of Obstetrics and Gynecology Hospital of Fudan University, and all patients signed informed consent. Villi tissues were obtained from 10 women (age: 23-36 years old) with clinically normal pregnancies, which were terminated for nonmedical reasons. No history of spontaneous miscarriage was found in those females. Females with infection, chromosome, and endocrine system disorders and other related diseases were excluded from the study.
Human trophoblast cell line HTR-8/SVneo was purchased from China Center for Type Culture Collection (Wuhan University Collection Center).
DMEM-F12 culture medium, fetal bovine serum (FBS) and 0.25% trypsin were from Gibco (USA). Triton X-100, rabbit anti-mouse ABC immunohistochemistry kit and goat anti-rabbit streptavidin staining kit were purchased from Beijing from Beijing Zhongshan Golden Bridge Biotechnology Co., Ltd. Rabbit anti-human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) monoclonal antibody, and mouse anti-human IL24, IL-20R1, and IL-22R1 monoclonal antibodies were purchased from the R and D Systems (USA). Mouse anti-human IL-20R2 monoclonal antibody isotype control antibody were purchased from Santa Cruz (USA). IRDye ™ 700DX goat anti-mouse and IRDye ™ 800DX goat anti-rabbit fluorescence secondary antibody was purchased from Rockland (USA). MTT was purchased from Sigma (USA).
Ten cases of villus tissue collected at early normal pregnancy stage were fixed, embedded in paraffin and sliced. Tissue sections were baked at 60°C for 1 h. After dewaxing, hydration and antigen retrieval, tissue sections were incubated in 3% H2O2 at room temperature for 15 min to remove endogenous peroxidase. Tissues sections were then blocked by incubating with 1% bovine serum albumin/phosphate buffered saline with Tween 20 (PBST) for 30 min, followed by incubation with mouse anti-human IL-24 (20 mg/L), IL-20R1 (20 mg/L), IL-20R2 (1∶50 dilution), and IL-22R1 (20 mg/L) monoclonal antibody or isotype control antibody overnight at 4°C. The next day, tissue sections were incubated with biotinylated secondary antibody, followed by addition of horseradish peroxidase-labeled avidin-biotin reagent and 3,3-diaminobenzidine (DAB) dye. Tissue sections were then rinsed with tap water, followed by hematoxylin counterstaining, dehydration, and xylene treatment. After that, tissue sections were sealed with neutral gum and observed under an inverted microscope (Olympus).
HTR-8/SVneo cells were seeded in 96-well plates with 2 × 104 cells per well, and then cultured for 24 h, followed by starvation treatment in DMEM-F12 supplemented with 1% FBS for 12 h. Then, recombinant human thymic stromal lymphopoietin (rhTSLP, 1 μg/L, 10 μg/L or 100 μg/L), or recombinant human CCL2 (rhCCL2, 1 μg/L, 10 μg/L or 100 μg/L was added and cells were cultured for another 72 h. Cells were then fixed in 4% paraformaldehyde at room temperature for 20 min and rinsed with 0.1% Triton. After that, blocking was performed by incubating with 150 mL LI-COR Odyssey Blocking Buffer at room temperature for 90 min, followed by incubation with mouse anti-human IL-24 (1∶50) monoclonal antibody, and rabbit anti-human GAPDH antibody (1∶80) overnight at 4°C. The next day, cells were rinsed with PBS, followed by incubation with corresponding IRDye ™ 700DX goat anti-mouse (red fluorescence) and IRDye ™ 800DX goat anti-rabbit (green fluorescence) fluorescence secondary antibody at room temperature in the dark for 30 min. After washing with PBST, Odyssey infrared fluorescence imaging system was used to scan and analyze results. Relative level of IL-24 = IL-24 absorbance value/corresponding GAPDH A value. Three replicate wells were set for each sample, and each experiment was repeated three times.
MTT cell viability assay
Human trophoblast cell line HTR-8/SVneo cells were made into a single-cell suspension, and seeded in 96-well plates (8 × 103/well) and cultured in DMEM containing 1% FBS for 12 h. Then, the vehicle (control group), different concentrations of rhIL-24 (1 μg/L, 10 μg/L, or 100 μg/L) or IL-24 neutralizing antibodies (aIL-24, 0.2 g/L, 1 g/L, or 5 g/L) and/or rhIL-24 (100 μg/L) was added into culture medium, and cells were cultured for another 48 h. Cell viability was subsequently analyzed by MTT colorimetric assay. The specific experimental procedure is as follows: after treatment, 20 μl of MTT (5 g/L) was added into each well and the incubation was continued at 37°C for 4 h. After that, culture was terminated and supernatant was carefully discarded. Then, 150 μl DMSO was added into each well, and the plates were shaken at room temperature for 10 min to fully dissolve the crystals. After that, absorbance value at 490 nm of each well was determined using a microplate reader. Trophoblast viability index = (A value of treatment group/A value of control group) × 100. Six replicate wells were set for each sample, and each experiment was repeated three times.
Experimental data were expressed as mean ± standard error mean (x¯ ± SEM) and analyzed by one-way ANOVA using SPSS 16.0 software system (SPSS Inc., Chicago, IL, USA). P < 0.05 was considered to be statistically significant.
| Results|| |
Expression of IL-24 and its receptors in villus tissue
The results of immunohistochemistry showed a strong positive expression of IL-24 and its receptors IL-20R2 and IL-22R1 in cytoplasm and membrane of villous trophoblast cells in normal early pregnancy, whereas there was a moderate positive expression of IL-20R1 [Figure 1]. Those data indicate that co-expression of IL-24 and its receptors in trophoblast may further regulate trophoblast biological behavior in an autocrine manner.
|Figure 1: IL-24 and its receptors expression in villi from women with normal pregnancy by immunohistochemistry. IL-24: Interleukin-24.|
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rhCCL2 inhibits the expression of IL-24 in HTR-8/SVneo cells
In-cell Western assay showed that rhTSLP treatment did not affect IL-24 expression in HTR-8/SVneo cells (P > 0.05) [Figure 2]a. Compared with control group, rhCCL2 treatment significantly down-regulated IL-24 expression in HTR-8/SVneo cells (P < 0.001) [Figure 2]b. These results suggest that CCL2 inhibits IL-24 expression in trophoblasts.
|Figure 2: Expression of IL-24 in HTR 8/SVneo cells by in cell western assay. (a) Expression of IL-24 in HTR 8/SVneo cells after stimulation with rhTSLP; (b) Expression of IL-24 in HTR 8/SVneo cells after stimulation with rhCCL2. Red fluorescence: IL-24; Green fluorescence: GAPDH; *P<0.001 compared with the control group. IL-24: Interleukin 24; rhTSLP: recombinant human thymic stromal lymphopoietin; rhCCL2: recombinant human CCL2.|
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IL-24 inhibits HTR-8/SVneo cell viability in vitro
Treatment with rhIL-24 (1 ng/mL, 10 ng/mL, or 100 ng/mL) significantly inhibited cell viability (P < 0.001) of HTR-8/SVneo cells in a dose-dependent manner (P < 0.001) [Figure 3]a. In contrast, aIL-24 (1 mg/mL or 5 mg/mL) increased cell viability of HTR-8/SVneo cells [Figure 3]b. These results suggest that both exogenous and endogenous IL-24 can inhibit HTR-8/SVneo cell viability in vitro.
|Figure 3: The viability of HTR 8/SCneo cells by MTT assay. (a) The viability of HTR 8/SCneo cells after stimulation with rhIL-24; (b) The viability of HTR 8/SCneo cells after stimulation with aIL-24. *P<0.05, †P<0.01, ‡P<0.001, compared with the control group. rhIL-24: recombinant human Interleukin 24; aIL-24: IL-24 neutralizing antibodies.|
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CCL2 enhances the viability of HTR-8/SVneo cells by downregulating IL-24
Treatment with rhCCL2 significantly enhanced the viability of HTR-8/SVneo cells (P < 0.01). On the contrary, rhIL-24 significantly inhibited the viability of HTR-8/SVneo cells (P < 0.001). When treatment with rhCCL2 plus rhIL-24, the viability of HTR-8/SVneo cells was not significantly different from that of control group (P > 0.05), and showed significant differences compared with the application of rhCCL2 or rhIL-24 alone (P < 0.001) [Figure 4]. Those data suggest that CCL2 can enhance the viability of human trophoblast cell line HTR-8 by inhibiting the secretion of IL-24.
|Figure 4: The viability of HTR 8/SCneo cells after stimulation with rhCCL2 and rhIL-24 by MTT assay. *P<0.01, †P<0.001, compared with the control group, ‡P<0.001, compared with the rhCCL2 group, §P<0.001, compared with the rhIL-24 group. rhCCL2: recombinant human CCL2; rhIL-24: recombinant human IL-24.|
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| Discussion|| |
Members of the IL-10 cytokine family include IL-10, IL-19, IL-20, IL-22, IL-26, and IL-24, of which human MDA-7/IL-24 gene is located on chromosome 1q32-33 and encodes a secretory protein composed of 206 amino acid residues. IL-24 has two pairs of heterodimeric receptors IL-20R1/IL-20R2 and IL-22R1/IL-20R2. IL-24 exerts a series of anti-tumor effects through membrane receptor-mediated or nonreceptor-mediated apoptotic pathways, such as inducing apoptosis in various tumor cells and stimulatory the sensitivity of tumor cells to radiation therapy without affecting normal cells. In addition, IL-24 has the ability to inhibit tumor growth and neovascularization as well as modulate immune response.,,,
Our previous study have showed that human decidual stromal cells in pregnancy co-express IL-24 and receptors IL-20R1, IL-20R2, and IL-22R1. IL-24 inhibits decidual stromal cell proliferation and promotes apoptosis by binding to IL-20R2/IL-22R1 but not IL-20R1/IL-20R2. In this study, we detected that trophoblasts in villus tissue also co-express IL-24 and its receptor in normal early pregnancy stage. Treatment with rhIL-24 significantly inhibited HTR-8/SVneo cell viability in vitro, whereas aIL-24 significantly enhanced HTR-8/SVneo cell viability, suggesting that both endogenous and exogenous IL-24 can inhibit trophoblast viability. Given the relatively low level of IL-20R1 expression in trophoblasts, we hypothesized that the inhibitory effect of IL-24 on trophoblast viability may also be achieved through receptors IL-20R2/IL-22R1 signaling, but the possible mechanism still need to be further studied. In addition, considering that IL-24 can promote the apoptosis of tumor cells and decidual stromal cells; therefore, IL-24 may also have the same function in regulating trophoblast apoptosis. However, more studies are needed to confirm this hypothesis.
Maternal-derived cells (decidual stromal cells and immune cells), fetal-derived cells (extravillous trophoblast cells that invade the decidua), and various cytokines, growth factors and hormones produced by these cells together constitute the maternal-fetal interface special immune microenvironment.,,, This unique immune microenvironment is bound to maintain a successful pregnancy by participating in the regulation of trophoblast biological behavior and coordinating the interaction of various functional cells at the maternal-fetal interface. Many cytokines are known to be involved in the regulation of trophoblast biological functions, such as viability, proliferation, and invasion.,, Our previous studies have confirmed that trophoblast cells can express CCL2/CCR2 and TSLP/TSLPR, and both CCL2 and TSLP can promote the proliferation and invasion of trophoblast cells through autocrine., In this study, we further observed that in vitro rhTSLP stimulation did not affect IL-24 expression in HTR-8/SVneo cells, whereas CCL2 significantly down-regulated IL-24 expression in HTR-8/SVneo cells. Our previous studies showed that unlike progestin and human chorionic gonadotropin, estrogen inhibited the secretion of IL-24 by decidual stromal cells through ERβ, thereby promoting the growth of decidual stromal cells. Therefore, estrogen may be involved in the regulation of IL-24 expression in trophoblast, but further studies are needed to confirm it.
In this study, we also observed that the enhancing effect of CCL2 on the viability of HTR-8/SVneo cells in vitro was significantly reversed by IL-24, suggesting that CCL2 secreted by trophoblasts may further down-regulate IL-24 expression and further enhance cell viability. Our previous study showed that decidual stromal cells secreted high levels of CCL2. High levels of CCL2, on one hand, promote proliferation of decidual stromal cells; on the other hand, CCL2 promote the formation and maintenance of maternal-fetal immune tolerance by inducing Th2 type immunity at the maternal-fetal interface. Based on our findings and previous studies, we speculate that CCL2 secreted by decidual stromal cells may inhibit the expression of IL-24 in trophoblasts through a paracrine manner and further participate in the regulation of trophoblast viability, i.e., CCL2/IL-24 was used as a media between decidual stromal cells and trophoblast cells to achieve an interactive dialogue and the regulation of trophoblast viability. However, this hypothesis remains to be further studied. In addition, it has been shown that IL-24 can stimulate human leukocytes to secrete cytokines such as IL-6, IFN-γ, and tumor necrosis factor and other cytokines, suggesting that CCL2 can induce and maintain Th2-type immunity at the maternal-fetal interface possibly by downregulating IL-24 expression.
In summary, CCL2 may enhance the cell viability of trophoblasts in vitro by down-regulating the expression of IL-24, and may further participate in blastocyst implantation and placental development. This mechanism of action of CCL2 will help us to understand the regulation of blastocyst implantation and trophoblast invasion. Subsequent studies should focus on the comparison of expression levels of IL-24 and its receptor in patients with normal pregnancy and abnormal trophoblastic diseases, so to further assess the potential value of IL-24 in the diagnosis and treatment of pregnancy-related diseases and trophoblastic diseases.
Financial support and sponsorship
This study was supported by the National Natural Science Foundation of China (NSFC) (81571509, 31671200) and the Program for Zhuoxue of Fudan University.
Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2], [Figure 3], [Figure 4]