Preeclampsia (PE) is a life-threatening pregnancy complication which is related to aggradation of risk regarding fetal and maternal morbidity and mortality. Dysregulation of systemic inflammatory response and dysfunction of trophoblast cells have been proposed to be involved in the development and progression of PE. Some studies have demonstrated that interleukin-33 (IL-33) is an immunomodulatory cytokine that is associated with the immune regulation of tumor cells. However, little is known whether IL-33 and its receptor ST2/IL-1 R4 could regulate trophoblast cells, which are associated with the pathogenesis of PE. In this study, our target is to explore the impact of IL-33 on trophoblast cells and elucidate its underlying pathophysiological mechanisms. Placental tissues from the severe PE group (
Preeclampsia (PE) is a pregnancy-specific disorder which is characterized by high blood pressure, proteinuria, and multiorgan functional disturbances [
Interleukin-33 (IL-33) is one of the members of the IL-1 cytokine family which is structurally presented in endothelial cells and epithelial cells of human tissues [
Several studies have shown that IL-33 is associated with tumorigenesis, metastasis, and proliferation of tumor cells [
Based on these researches demonstrating the role of IL-33 on tumor cells, as well as the similarity between tumor cells and trophoblast cells, we hypothesise that IL-33/ST2/IL-1 R4 will be associated with the function of trophoblast cells which may explain an association between IL-33/ST2/IL-1 R4 and PE.
The goal of our research is to explore whether the level of IL-33 was associated with the capability (proliferation, immigration, and invasion) of trophoblast cells. Our study has demonstrated the existence of the relationship between IL-33 and trophoblast cells. This further proved that the decreased IL-33 production was associated with the reduced functional capability of trophoblast cells. As a result, compromised placentation has been linked to the etiology of PE.
Pregnant women with severe PE (
Tissue samples were collected in the operation room and transported with ice to our laboratory immediately. After being washed with ice-cold phosphate-buffered saline (PBS) three times, the samples were snap frozen and stored at −80°C for Western blotting, or fixed before being embedded for immunohistochemical analysis. For the villus explant cultures, after being collected in aseptic conditions, samples were washed three times with sterile PBS, cut into 1–5 mm3 sections, and cultured immediately.
The following reagents were used in this study: mouse monoclonal antibody for IL-33 (ALX-804-840-C100, Enzo Life Sciences Inc., Farmingdale, New York, USA), rabbit polyclonal antibody for IL-33 (12372-1-AP, Proteintech Group Inc., Chicago, USA), goat polyclonal antibody for ST2/IL-1 R4 (AF523, R&D Systems, Minneapolis, USA), rabbit polyclonal antibody for ST2/IL-1 R4 (11920-1-AP, Proteintech Group, Chicago, USA), rabbit polyclonal antibody for TIMP1 (WL00869, Wanleibio, China), rabbit polyclonal antibody for TIMP2 (WL01209, Wanleibio, China),
Immunohistochemistry (IHC) was performed as follows [
The level of IL-33 expression in HTR8/SVneo cells (blank group, IL-33 shNC group, or shIL-33 group) and the expressive level of ST2/IL-1 R4 in villous explants treated with SNP were determined by immunofluorescence staining. Cells on the slides were first fixed for 30 min with paraformaldehyde at room temperature, and 5
The 48-well culture boards were coated with 200
Some villi were separated into small pieces (4–5 mm), cultured in DMEM/F12 (Gibco) containing 10% heat-treated fetal bovine serum (900-108, Gemini Bio-Products, California, USA), 1% penicillin-streptomycin (C0222, Beyotime Biotechnology, Shanghai, China), and 0.25
The human transformed primary extravillous trophoblast cell line, HTR8/SVneo, was kindly provided by Dr. C. H. Graham (Queen’s University, Kingston, ON, Canada). HTR8/SVneo cells were incubated in RPMI1640 (Gibco, Life Technologies Corporation, USA) containing 10% fetal bovine serum (Gemini Bio-Products, California, USA). Cells were transfected with shRNA and were separated into two groups: negative control group (shNC) and scrambled shRNA group (shIL-33).
Total RNA was extracted from pretreated cells and explanted by TRIpure reagents (RP1202, BioTeke, Beijing, China), and the concentration of RNA was quantified using ultraviolet spectroscopy (NanoDrop 2000, Thermo, USA). A PrimeScript RT reagent kit with a gDNA Eraser (RR047A, TaKaRa, Japan) was used for reverse transcription.
The knockdown cell invasion was evaluated using a matrigel invasion chamber. Firstly, the cell inserts (Costar, Cambridge, MA, USA) were coated with 100
Cell Counting Kit-8 (CCK-8, Bimake, USA) was used to evaluate cell proliferation. 8 × 103 HTR8/SVneo cells (blank group, IL-33 shNC group, or shIL-33 group) in 100
1 × 105 of HTR8/SVneo cells (normal group, IL-33 shNC group, or scrambled shRNA group) were seeded into six-well plates. When the cell density reached 70%–80%, 1 ml pipette tips were used to scratch across the cell monolayer, and the cells were gently washed with warm PBS (this point is defined as 0 hr). The plates were then cultured for further 24 hr. The migration was photographed at 0 hr and 24 hr. The area of the wound was analyzed using Image J software.
Patient-derived tissue samples, pretreated cells, and villi were lysed by RIPA buffer (Beyotime Biotechnology, China). Protein concentrations were determined by BCA Protein Assay Kit (Beyotime Biotechnology, China). Protein extracted from above samples (20
A gelatin zymography assay was used to determine the gelatinolytic activity. After the gel was prepared, 3
Data is recorded as mean ± standard deviation (SD). The statistical difference was assessed by
The expression of IL-33 was remarkably lower in women with PE compared with controls who had a normal pregnancy, while no remarkable distinction was observed in the expression of ST2/IL-1 R4 (Figure
Expression and distribution of IL-33 and ST2/IL-1 R4 in placental tissues. (a) IL-33 was localized in syncytiotrophoblast (STB) cells, cytotrophoblast (CTB) cells, trophoblast columns (TC) of placental villi (A–C), and some extravillous trophoblasts (EVT) in maternal decidual cells (E–G). D, H, K, and N were the negative controls (NEG). CK7 is a marker for STB and TC. HLA-G is a marker for EVT. The IL-33 in women with normal (I, J) or preeclamptic pregnancies (L, M) was localized in trophoblast cells (200x; scale bar, 200
Sodium nitroprusside (SNP), which produces reactive oxygen species, was used to stimulate villous explants to simulate the pathophysiological status of PE [
Western blot demonstrating the level of IL-33 in villous explants treated with SNP at different concentrations and immunofluorescence images demonstrating the expression of ST2/IL-1 R4 in these groups. (a) The level of IL-33 was high in low concentrations of SNP and then decreased continuously with increasing concentrations of SNP. (b, c) Fluorescence specific to ST2/IL-1 R4 is red, and the nuclei were stained by DAPI (blue) (200x; scale bar, 200
The expression of IL-33 was observed in HTR8/SVneo which suggested the role of IL-33 in the regulation of cell behavior (Figure
Immunofluorescence images, Western blot, and qRT-PCR were used to evaluate the transfection efficiency of shRNA targeting IL-33 in HTR8/SVneo cells. (a) Fluorescence specific to IL-33 is red, and the nuclei were stained by DAPI (blue) (400x; scale bar, 100
Treatment with IL-33 shRNA weakened the proliferation, invasion, and migration of HTR8/SVneo cells. (a, b) IL-33 knockdown significantly decreased the area of the wound in the wound healing assay (40x; scale bar, 50
ShRNA targeting IL-33 significantly decreased IL-33 expression (Figure
Knockdown of IL-33 decreased the outgrowth of the human villous explants. (a, b) Western blot and qRT-PCR were used to evaluate the transfection efficiency of shRNA targeting IL-33 in villous explants. (c, d) The outgrowth of villous explants treated with shRNA was photographed at 24 hr (Aa, Ee), 48 hr (Bb, Ff), 72 hr (Cc, Gg), and 96 hr (Dd, Hh) by a light microscope and fluorescence microscope (40x; scale bar, 50
The result of the gelatin zymography demonstrated that the activity of MMP2/9 was decreased in the knockdown group when compared with the scrambled shRNA group (Figure
IL-33 knockdown decreased the activity of MMP2/9 and increased the utterance of TIMP1/2. (a) Image of gelatin zymography assay (GZ) using the medium of HTR8/SVneo cells disturbed by shRNA. Western blot (WB) was used to test the utterance of TIMP1/2 in proteins from these HTR/SVneo cells. (b) Image of the gelatin zymography assay using the medium of villous explants disturbed by shRNA. Western blot was used to test the
Previous research findings have revealed that IL-33 dysfunction is associated with pregnancy complications, such as PE [
A former study that explored the expression of IL-33/ST2 in pregnancies affected by PE found that there was no difference in circulating IL-33 or the placental levels of IL-33 between healthy and PE participants [
Several studies have shown that the onset of PE is due to the impaired behavior of placental trophoblast cells in early pregnancy. The proliferation and invasion of trophoblast cells have been demonstrated previously to be enhanced by exogenous IL-33 [
Previous research has determined that the expression of MMPs is the key modulator in retrogradation of the extracellular matrix (ECM) to promote the invasion and migration of trophoblast cells [
Circulating ST2 has been found to be significantly increased in PE, while ST2L, the transmembrane form of ST2, was increased but without statistical significance [
In a word, our research proved that IL-33 was expressed in trophoblast cells and its expression was impaired in PE. Additionally, IL-33 could be related to trophoblast cell proliferation, migration, and invasion and might be a regulatory factor in the pathogenesis of PE.
The authors declare that they have no conflicts of interest.
The authors are grateful to all the pregnant women who participated in their study. The authors also thank the support of the Canada-China-New Zealand Joint Laboratory of Maternal and Fetal Medicine and the Laboratory of Lipid & Glucose Metabolism, The First Affiliated Hospital of Chongqing Medical University, Chongqing, China. This research was assisted by the 111 Project (Yuwaizhuan (2016) 32) and National Natural Science Foundation of China (nos. 81370732, 81571453, and 81650110522).
Supplemental Figure 1: immunofluorescence images demonstrated the expression of ST2/IL-1 R4 in the shNC group and the shIL-33 group. (A-B) Fluorescence specific to ST2/IL-1 R4 is red, and the nuclei was stained by DAPI (blue) (200x. Scale bar, 200