Bidirectional regulation between 1st trimester HTR8/SVneo trophoblast cells and in vitro differentiated Th17/Treg cells suggest a fetal-maternal regulatory loop in human pregnancy.
ABSTRACT: PROBLEM:During normal pregnancy, delicate crosstalk is established between fetus-derived trophoblasts and maternal immune cells to ensure maternal-fetal tolerance and successful placentation. Dysfunction in these interactions has been highly linked to certain pregnancy complications. METHOD OF STUDY:Naïve CD4+ T cells were cultivated with or without 1st trimester derived trophoblast cell line HTR8/SVneo cells in the absence or presence of T helper 17 (Th17) or regulatory (Treg)cell-inducing differentiation conditions. After 5 days of co-culture, HTR8/SVneo cells and CD4+ T cells were harvested and analyzed using flow cytometry. RESULTS:CD4+ T cells exposed to HTR8/SVneo cells showed enhanced induction of CD4+ Foxp3+ Treg cells with strong expression of TGF-β1 and inhibitory molecules (cytotoxic T lymphocyte-associated protein-4 [CTLA-4], T-cell immunoglobulin mucin-3 [Tim-3], and programmed cell death-1 [PD-1]). Though not effecting Th17 differentiation, exposure to HTR8/SVneo cells promoted increased expression of proliferative and apoptotic markers on Th17 cells. Co-culture with Th0 cells, or differentiated Th17 or Treg cells, down-regulated Caspase-3 and MMP-9 (but not MMP-2) expression in HTR8/SVneo cells, while promoting Ki67 expression. CONCLUSIONS:HTR8/SVneo cells regulated maternal CD4+ T-cell differentiation, resulting in the expansion of immunosuppressive Treg cells, while CD4+ T cells might promote the growth, and control the invasiveness of HTR8/SVneo cells. Thus, a bidirectional regulatory loop might exist between trophoblasts and maternal immune cell subsets, thereby promoting harmonious maternal-fetal crosstalk.
Project description:Maternal immune adaptation is required for a successful pregnancy to avoid rejection of the fetal-placental unit. Dendritic cells within the decidual microenvironment lock in a tolerogenic profile. However, how these tolerogenic DCs are induced and the underlying mechanisms are largely unknown. In this study, we show that human extravillous trophoblasts redirect the monocyte-to-DC transition and induce regulatory dendritic cells. DCs differentiated from blood monocytes in the presence of human extravillous trophoblast cell line HTR-8/SVneo displayed a DC-SIGN(+)CD14(+)CD1a(-) phenotype, similar with decidual DCs. HTR8-conditioned DCs were unable to develop a fully mature phenotype in response to LPS, and altered the cytokine secretory profile significantly. Functionally, conditioned DCs poorly induced the proliferation and activation of allogeneic T cells, whereas promoted CD4(+)CD25(+)Foxp3(+) Treg cells generation. Furthermore, the supernatant from DC and HTR-8/SVneo coculture system contained significant high amount of M-CSF and MCP-1. Using neutralizing antibodies, we discussed the role of M-CSF and MCP-1 during monocyte-to-DCs differentiation mediated by extravillous trophoblasts. Our data indicate that human extravillous trophoblasts play an important role in modulating the monocyte-to-DC differentiation through M-CSF and MCP-1, which facilitate the establishment of a tolerogenic microenvironment at the maternal-fetal interface.
Project description:Preeclampsia is a pregnancy-related disease with increasing maternal and perinatal morbidity and mortality worldwide. Defective trophoblast invasion is considered to be a major factor in the pathophysiological mechanism of preeclampsia. Heparanase, the only endo-?-glucuronidase in mammalian cells, has been shown to be abnormally expressed in the placenta of preeclampsia patients in our previous study. The biological role and potential mechanism of heparanase in trophoblasts remain unclear. In the present study, stably transfected HTR8/SVneo cell lines with heparanase overexpression or knockdown were constructed. The effect of heparanase on cellular proliferation, apoptosis, invasion, tube formation, and potential pathways in trophoblasts was explored. Our results showed that overexpression of heparanase promoted proliferation and invasion. Knockdown of heparanase suppressed proliferation, invasion, and tube formation but induced apoptosis. These findings reveal that downregulation of heparanase may contribute to defective placentation and plays a crucial role in the pathogenesis of preeclampsia. Furthermore, increased activation of p38 MAPK in heparanase-knockdown HTR8/SVneo cell was shown by MAPK pathway phosphorylation array and Western blotting assay. After pretreatment with 3 specific p38 MAPK inhibitors (BMS582949, SB203580, or BIRB796), inadequate invasion in heparanase-knockdown HTR8/SVneo cell was rescued. That indicates that knockdown of heparanase decreases HTR8/SVneo cell invasion through excessive activation of the p38 MAPK signaling pathway. Our study suggests that heparanase can be a potential predictive biomarker for preeclampsia at an early stage of pregnancy and represents a promising therapeutic target for the treatment of preeclampsia.
Project description:Placental trophoblast invasion involves a cellular transition from epithelial to mesenchymal phenotype. Cytotrophoblasts undergo epithelial to mesenchymal transition (EMT) when differentiating into extravillous trophoblasts and gaining the capacity of invasion. In this research, we investigated the role of DNA methylation in trophoblasts during this EMT. First, using BeWo and HTR8/SVneo cell lines as models of cytotrophoblasts and extravillous trophoblasts, respectively, we analyzed the gene expression and DNA methylation status of the known epithelial marker genes, E-Cadherin and Cytokeratin7. We found that, in HTR8/SVneo cells, both genes were silenced and their promoters hypermethylated, as compared with the high-level gene expression and promoter hypomethylation observed in BeWo cells. This result suggests that dynamic DNA methylation of epithelial marker genes plays a critical role in the trophoblast EMT process. To verify these results, we treated HTR8/SVneo cells with 5-aza-dC, a known inhibitor of DNA methyltransferase, for three days. Five-Aza-dC treatment significantly increased the expression of epithelial marker genes and slightly decreased the expression of mesenchymal genes, as detected by qRT-PCR, immunocytochemistry and Western blot. Furthermore, 5-aza-dC treated HTR8/SVneo cells changed their morphology from mesenchymal into epithelial phenotype, indicating that 5-aza-dC induced mesenchymal to epithelial transition. Lastly, we examined the effect of 5-aza-dC on trophoblast migration and invasion capacity. We applied 5-aza-dC to HTR8/SVneo cells in trans-well cell migration and invasion assays and found that 5-aza-dC treatment decreased trophoblast migration and invasion capacity. In conclusion, DNA methylation of epithelial marker genes represents a molecular mechanism for the process of trophoblast EMT.
Project description:DICER is a key rate-limiting enzyme in the canonical miRNAs biogenesis pathway, and DICER and DICER-dependent miRNAs have been proved to play essential roles in many physiological and pathological processes. However, whether DICER is involved in placentation has not been studied. Successful spiral artery remodelling is one of the key milestones during placentation, which depends mostly on the invasion of trophoblasts and the crosstalk between trophoblasts and endothelial cells. In the present study, we show that DICER knockdown impairs the invasion ability of both primary extravillous trophoblasts (EVT) and HTR8/SVneo (HTR8) cell lines. The decreased invasion of HTR8 cells upon DICER knockdown (sh-Dicer) was partly due to the up-regulation of miR-16-2-3p, which led to a reduced expression level of the collagen type 1 alpha 2 chain (COL1A2) protein. Moreover, microvesicles (MVs) can be secreted by HTR8 cells and promote the tube formation ability of human umbilical cord vein endothelial cells (HUVECs). However, conditioned medium and MVs derived from sh-Dicer HTR8 cells have an anti-angiogenic effect, due to reduced angiogenic factors and increased anti-angiogenic miRNAs (including let-7d, miR-1-6-2 and miR-15b), respectively. In addition, reduced protein expression of DICER is found in PE placenta by immunoblotting and immunohistochemistry. In summary, our study uncovered a novel DICER-miR-16-2-COL1A2 mediated pathway involved in the invasion ability of EVT, and DICER-containing MVs mediate the pro-angiogenic effect of trophoblast-derived conditioned medium on angiogenesis, implying the involvement of DICER in the pathogenesis of PE.
Project description:Extravillous trophoblasts (EVTs) characterize the invasion of the maternal decidua under low oxygen and poor nutrition at the early feto-maternal interface to establish a successful pregnancy. We previously reported that autophagy in EVTs was activated under 2% O2 in vitro, and autophagy activation was also observed in EVTs at the early feto-maternal interface in vivo. Here, we show that autophagy is an energy source for the invasion of EVTs. Cobalt chloride (CoCl2), which induces hypoxia inducible factor 1? (HIF1?) overexpression, activated autophagy in HTR8/SVneo cells, an EVT cell line. The number of invading HTR8-ATG4B(C74A) cells, an autophagy-deficient EVT cell line, was markedly reduced by 81 percent with the CoCl2 treatment through the suppression of MMP9 level, although CoCl2 did not affect the cellular invasion of HTR8-mStrawberry cells, a control cell line. HTR8-ATG4B(C74A) cells treated with CoCl2 showed a decrease in cellular adenosine triphosphate (ATP) levels and a compensatory increase in the expression of purinergic receptor P2X ligand-gated ion channel 7 (P2RX7), which is stimulated with ATP, whereas HTR8-mStrawberry cells maintained cellular ATP levels and did not affect P2RX7 expression. Furthermore, the decreased invasiveness of HTR8-ATG4B(C74A) cells treated with CoCl2 was neutralized by ATP supplementation to the level of HTR8-ATG4B(C74A) cells treated without CoCl2. These results suggest that autophagy plays a role in maintaining homeostasis by countervailing HIF1?-mediated cellular energy consumption in EVTs.
Project description:Preeclampsia, a relatively common pregnancy disorder, is a major contributor to maternal mortality and morbidity worldwide. An elevation in microRNA-210 (miR-210) expression in the placenta has been reported to be associated with preeclampsia. Our bioinformatic analysis showed that thrombospondin type I domain containing 7A (THSD7A) is a predicted target for miR-210. The aim of this study was to determine whether miR-210 is involved in preeclampsia through its targeting of THSD7A in human placental trophoblasts. In preeclamptic placental tissues, THSD7A levels were significantly downregulated, and were inversely correlated with the levels of miR-210. THSD7A was validated as a direct target of miR-210 using quantitative real time PCR (qRT-PCR), Western blotting, and dual luciferase assays in HTR8/SVneo cells. Transwell insert invasion assays showed that THSD7A mediated the invasion-inhibitory effect of miR-210 in HTR8/SVneo cells. Interestingly, hypoxia markedly increased miR-210 expression while suppressing THSD7A expression in a time-dependent manner in HTR8/SVneo cells. This study provides novel data on the function of THSD7A in human placental cells, and extends our knowledge of how miR-210 is involved in the development of the preeclampsia.
Project description:Communication between the maternal uterus and the embryo is vital for a successful pregnancy. Exosomes, subtypes of extracellular vesicles comprising many bioactive factors regulate the early stages of pregnancy, specifically during embryo implantation. Nevertheless, the mechanism by which exosomal microRNAs (miRNAs) derived from placental trophoblasts regulate embryo implantation remains elusive. Herer, we isolated and identified exosomes derived from placental trophoblasts cells (HTR8/SVneo). Subsequently, we evaluated the loading miRNA in exosomes by small RNA sequencing. This study provides novel insights into the mechanism of trophoblasts cells-derived exosomes during embryo implantation. Overall design: Three HTR8/Svneo cells and three HTR8/SVneo cells-derived exosomes samples were collected and sequenced using Illumina HiSeq. The miRNAs in cells and exosomes were mapped.
Project description:<h4>Problem</h4>Does aquaporin 3 (AQP3) affect the migration and invasion of human extravillous trophoblast (HTR8/Svneo) cells?<h4>Method of study</h4>A lentivirus infection system was used to construct stable cell lines with either AQP3 knockdown or overexpression. RT-PCR and western blotting were used to verify the efficiencies of AQP3 knockdown or overexpression in HTR8/Svneo cells at mRNA and protein levels, respectively. Cell Counting Kit-8 and flow cytometry assays were used to detect the influence of AQP3 knockdown or overexpression on proliferation and apoptosis of HTR8/Svneo cells. In addition, wound healing and Transwell invasion assays were used to detect the effects of AQP3 knockdown or overexpression on migration and invasion capabilities of HTR8/Svneo cells. An Agilent gene chip was used to screen for significant differentially expressed genes after AQP3 knockdown. Finally, mechanisms by which AQP3 influences the migration and invasion of HTR8/Svneo cells were explored using bioinformatic analysis.<h4>Results</h4>Compared with controls, migration and invasion capabilities of HTR8/Svneo cells were significantly reduced after AQP3 knockdown, and significantly increased after AQP3 overexpression. Subsequent bioinformatic analysis of gene chip expression profiles indicated downregulation of genes related to adhesion such as PDGF-B, as well as signaling pathways (such as PIK3/AKT, NF-κB, and TNF) after AQP3 knockdown.<h4>Conclusions</h4>AQP3 could significantly promote migration and invasion capabilities of human extravillous trophoblasts, it may mediate embryo invasion and adhesion to endometrium by regulating PDGF-B, PIK3/AKT signaling pathways, although this requires further verification.
Project description:Communication between the maternal uterus and the embryo is vital for a successful pregnancy. Exosomes, subtypes of extracellular vesicles comprising many bioactive factors, regulate the early stages of pregnancy, specifically during embryo implantation. Nevertheless, the mechanism by which exosomal microRNAs (miRNAs) derived from placental trophoblasts regulate embryo implantation remains elusive. We isolated and identified exosomes derived from placental trophoblast cells (HTR8/SVneo). Subsequently, we evaluated the loading miRNA in exosomes by small RNA sequencing. Consequently, we showed that trophoblast cell-derived exosomes could transfer to endometrial epithelial cells. Besides, these exosomes promoted the epithelial-mesenchymal transition (EMT) as well as migration of endometrial cells and were implicated in the regulation of inflammation. Further, the specific miRNAs were screened in exosomes, and as a result, miRNA (miR)-1290 was enriched specifically in exosomes. miR-1290 promoted the expression of inflammatory factors (interleukin [IL]-6 and IL-8) and migration of endometrial epithelial cells. In addition, exosomal miR-1290 promoted angiogenesis in vitro. More importantly, by targeting LHX6, trophoblast HTR8/SVneo cell-derived exosomal miR-1290 promoted the EMT process of endometrial epithelial cell HEC-1-A. Altogether, our findings provide novel insights into the mechanism of trophoblast cell-derived exosomes during embryo implantation. Graphical abstract Trophoblast cell-derived exosomal miR-1290 delivered to endometrial epithelial cells and target LHX6, thereby promoting the EMT process. This mechanism is important for successful embryo implantation.
Project description:Successful human pregnancy requires the maternal immune system to recognize and tolerate the semi-allogeneic fetus. Myeloid-derived suppressor cells (MDSCs), which are capable of inhibiting T-cell responses, are highly increased in the early stages of pregnancy. Although recent reports indicate a role for MDSCs in fetal-maternal tolerance, little is known about the expansion of MDSCs during pregnancy. In the present study, we demonstrated that the trophoblast cell line HTR8/SVneo could instruct peripheral CD14(+) myelomonocytic cells toward a novel subpopulation of MDSCs, denoted as CD14(+)HLA-DR(-/low) cells, with suppressive activity and increased expression of IDO1, ARG-1, and COX2. After interaction with HTR8/SVneo cells, CD14(+) myelomonocytic cells secrete high levels of CCL2, promoting the expression of signal transducer and activator of transcription 3. We utilized a neutralizing monoclonal antibody to reveal the prominent role of CCL2 in the induction of CD14(+)HLA-DR(-/low) MDSCs. In combination, the results of the present study support a novel role for the cross-talk between the trophoblast cell line HTR8/SVneo and maternal CD14(+) myelomonocytic cells in initiating MDSCs induction, prompting a tolerogenic immune response to ensure a successful pregnancy.