Primary Human Trophoblasts Research Articles

Glucocorticoid regulation of System A amino acid transport in primary human trophoblast cells

Glucocorticoid regulation of System A amino acid transport in primary human trophoblast cells

Human trophoblast-derived hydrogen sulfide stimulates placental artery endothelial cell angiogenesis.

Endogenous hydrogen sulfide (H2S), mainly synthesized by cystathionine β-synthase (CBS) and cystathionine γ-lyase (CTH), has been implicated in regulating placental angiogenesis; however, the underlying mechanisms are unknown. This study was to test a hypothesis that trophoblasts synthesize H2S to promote placental angiogenesis. Human choriocarcinoma-derived BeWo cells expressed both CBS and CTH proteins, while the first trimester villous trophoblast-originated HTR-8/SVneo cells expressed CTH protein only. The H2S producing ability of BeWo cells was significantly inhibited by either inhibitors of CBS (carboxymethyl hydroxylamine hemihydrochloride, CHH) or CTH (β-cyano-L-alanine, BCA) and that in HTR-8/SVneo cells was inhibited by CHH only. H2S donors stimulated cell proliferation, migration, and tube formation in ovine placental artery endothelial cells (oFPAECs) as effectively as vascular endothelial growth factor. Co-culture with BeWo and HTR-8/SVneo cells stimulated oFPAEC migration, which was inhibited by CHH or BCA in BeWo but CHH only in HTR-8/SVneo cells. Primary human villous trophoblasts (HVT) were more potent than trophoblast cell lines in stimulating oFPAEC migration that was inhibited by CHH and CHH/BCA combination in accordance with its H2S synthesizing activity linked to CBS and CTH expression patterns. H2S donors activated endothelial nitric oxide synthase (NOS3), v-AKT murine thymoma viral oncogene homolog 1 (AKT1), and extracellular signal-activated kinase 1/2 (mitogen-activated protein kinase 3/1, MAPK3/1) in oFPAECs. H2S donor-induced NOS3 activation was blocked by AKT1 but not MAPK3/1 inhibition. In keeping with our previous studies showing a crucial role of AKT1, MAPK3/1, and NOS3/NO in placental angiogenesis, these data show that trophoblast-derived endogenous H2S stimulates placental angiogenesis, involving activation of AKT1, NOS3/NO, and MAPK3/1.

Invasive trophoblast promote stromal fibroblast decidualization via Profilin 1 and ALOX5

During the establishment of pregnancy, extravillous trophoblast (EVT) must invade into the uterine decidua to facilitate decidual artery remodelling to create the placental blood supply. The local decidual environment is thought to regulate trophoblast invasion, however these interactions are poorly defined in humans. Recent evidence in women suggests impaired decidualization is associated with miscarriage and preeclampsia. Primary human endometrial stromal cells (HESC) and first trimester extravillous trophoblast (EVTs) were used to assess the effect of EVT-secreted factors on HESC decidualization, adhesion, proliferation and migration. We determined the role of profilin (PFN)1, an EVT-secreted factor, on HESC function and identified a downstream target of PFN1. EVT-secreted factors induced HESC decidualization and enhanced decidualized HESC adhesion, proliferation and migration. Recombinant PFN1 enhanced methoxyprogesterone acetate-induced HESC decidualization and proliferation. PFN1 down-regulated the expression of lipoxygenase arachidonate 5-lipoxygenase (ALOX5) in HESC and THP-1 macrophages. ALOX5 localised to decidual cells and CD68+macrophages in 1st trimester decidua. This study demonstrated that EVT secretions, including PFN1, enhanced HESC decidualization and motility. This study has identified a new pathway that facilitates appropriate decidualization during the establishment of pregnancy.

Down-regulation of PDK4 is Critical for the Switch of Carbohydrate Catabolism during Syncytialization of Human Placental Trophoblasts

Pyruvate dehydrogenase kinase (PDK) is known as a gatekeeper directing the carbon flux into glycolysis via inhibition of the pyruvate dehydrogenase complex. During syncytialization of placental trophoblasts, both ATP production and oxygen consumption are increased to meet enhanced energetic demands by syntiotrophoblasts. We hypothesized that down-regulation of PDK expression may play a central role in the switch from glycolysis to oxidative phosphorylation (OXPHOS) during syncytialization. By using primary human trophoblasts, we demonstrated that PDK4 was the dominating PDK isoform in human cytotrophoblasts, and its abundance was substantially decreased upon syncytialization, which was accompanied by decreases in lactate production and increases in ATP production. Knock-down of PDK4 expression reduced lactate production and increased ATP production, while over-expression of PDK4 increased lactate production and decreased ATP production, indicating that down-regulation of PDK4 is key to the shift from glycolysis to OXPHOS during syncytialization. Moreover, human chorionic gonadotropin (hCG)/cAMP/PKA pathway was demonstrated to be involved in the down-regulation of PDK4 expression upon syncytialization. Taken together, our findings disclosed that down-regulation of PDK4 is critical for the metabolic shift from glycolysis to OXPHOS during syncytialization, which may be a prerequisite for the proper implementation of syncytiotrophoblast functions.

Human placenta expresses both peripheral and neuronal isoform of tryptophan hydroxylase

The role of placental serotonin has been an active topic of research notably because of its crucial role in brain development. However, which cell types synthesize serotonin in human placenta remains unknown. Moreover, it is not known if the two tryptophan hydroxylase isoforms (TPH1 and TPH2), the rate-limiting enzymes in serotonin biosynthesis, are expressed in placenta. Human placentas were obtained in first trimester or at term, and trophoblast cells were isolated and purified using a magnetic cell sorter and placed in primary culture. The tissue sublocalization of each TPH was determined by immunohistochemistry. TPH expression in primary villous trophoblasts was determined by PCR and immunoblotting, and serotonin secretion by LC-MS/MS. Villous cytotrophoblasts, syncytiotrophoblast, fetal capillaries, extravillous cytotrophoblasts, and decidual cells co-expressed TPH1 and TPH2. Moreover, mRNA and protein levels of both TPHs were detected in human primary trophoblast as well as in mouse placental tissues. Finally, human trophoblast cells were shown to produce serotonin de novo. This study demonstrates that both TPH1 and TPH2 are expressed in human and mouse placenta throughout pregnancy and helps to better understand the placental serotonin system, which is crucial for healthy pregnancy and fetal development. It is therefore important to further understand regulation of the placental serotonin system and how its disruption during pregnancy may impact the developing fetus and subsequent child programming.

TGF-β1 Inhibits Human Trophoblast Cell Invasion by Upregulating Connective Tissue Growth Factor Expression.

Appropriate trophoblast invasion into the maternal endometrium is essential for successful human implantation and placentation. Connective tissue growth factor (CTGF), also known as CCN2, is a matricellular protein that is expressed in the placenta. Interestingly, the CTGF expression levels in the placenta and serum from patients with severe preeclampsia or fetal growth restriction are higher than those from healthy controls. However, to date, the role of CTGF in the regulation of trophoblast cell invasion remains unclear. Transforming growth factor-β1 (TGF-β1) is a potent stimulator of CTGF expression and has been shown to inhibit trophoblast cell invasiveness. However, whether CTGF mediates TGF-β1-inhibited human trophoblast cell invasion is unknown. In the present study, we show that treatment with TGF-β1 upregulates CTGF expression in a human trophoblast cell line, HTR-8/SVneo, and in primary human trophoblast cells. Our results also demonstrate that the SMAD2/3 signaling pathways are required for TGF-β1-induced upregulation of CTGF. Importantly, CTGF knockdown attenuates TGF-β1-inhibited cell invasion. Furthermore, cell invasiveness is decreased by treatment with recombinant CTGF. These results provide evidence that CTGF mediates TGF-β1-inhibited human trophoblast cell invasion.

Mechanistic Target of Rapamycin Is a Novel Molecular Mechanism Linking Folate Availability and Cell Function

Folate deficiency has been linked to a wide range of disorders, including cancer, neural tube defects, and fetal growth restriction. Folate regulates cellular function mediated by its involvement in the synthesis of nucleotides, which are needed for DNA synthesis, and its function as a methyl donor, which is critical for DNA methylation. Here we review current data showing that folate sensing by mechanistic target of rapamycin (mTOR) constitutes a novel and distinct pathway by which folate modulates cell functions such as nutrient transport, protein synthesis, and mitochondrial respiration. The mTOR signaling pathway responds to growth factors and changes in nutrient availability to control cell growth, proliferation, and metabolism. mTOR exists in 2 complexes, mTOR complex (mTORC) 1 and mTORC2, which have distinct upstream regulators and downstream targets. Folate deficiency in pregnant mice caused a marked inhibition of mTORC1 and mTORC2 signaling in multiple maternal and fetal tissues, downregulation of placental amino acid transporters, and fetal growth restriction. In addition, folate deficiency in primary human trophoblast (PHT) cells resulted in inhibition of mTORC1 and mTORC2 signaling and decreased the activity of key amino acid transporters. Folate sensing by mTOR in PHT cells is independent of the accumulation of homocysteine and requires the proton-coupled folate transporter (PCFT; solute carrier 46A1). Furthermore, mTORC1 and mTORC2 regulate trophoblast folate uptake by modulating the cell surface expression of folate receptor α and the reduced folate carrier. These findings, which provide a novel link between folate availability and cell function, growth, and proliferation, may have broad biological significance given the critical role of folate in normal cell function and the multiple diseases that have been associated with decreased or excessive folate availability. Low maternal folate concentrations are linked to restricted fetal growth, and we propose that the underlying mechanisms involve trophoblast mTOR folate sensing resulting in inhibition of mTORC1 and mTORC2 and downregulation of placental amino acid transporters.

Multiple drug transporters mediate the placental transport of sulpiride

Sulpiride is a typical antipsychotic drug for the treatment of schizophrenia, depression and other psychological disorders. It has been proven that a small amount of sulpiride could cross the human placentausing anex vivoplacental perfusion model. However, the placental transfer mechanism has not been elucidated. Considering the structure of sulpiride, we speculated that the transporters expressed in placenta might be involved in sulpiride uptake across the blood-placenta barrier. The aim of our study was to determine which transporters contributed to the placental transfer of sulpiride. Our results revealed that sulpiride was a substrate of human organic cation transporter (hOCT) 3, human multidrug resistance protein (hMDR) 1 and human breast cancer resistance protein (hBCRP) using transfected cells expressing respective transporters. In addition, the accumulation of sulpiride in BeWo cells (a human choriocarcinoma cell line) was obviously affected by inhibitors of carnitine/organic cation transporter (OCTN) 2, MDR1 and BCRP. The accumulation of sulpiride in primary human trophoblast cells was obviously affected by inhibitors of OCT3, OCTN1 and OCTN2. The above results indicate that hOCTN1 and hOCTN2 likely contribute to the sulpiride uptake from maternal circulation to trophoblast cells, while hMDR1 and hBCRP mediate the efflux from trophoblast cells to maternal circulation, and hOCT3 probably is involved in the bidirectional transport of sulpiride between the placenta and fetal blood.

MTOR folate sensing links folate availability to trophoblast cell function

Folate deficiency during pregnancy is associated with restricted fetal growth, although the underlying mechanisms are poorly understood. Here we show that mechanistic target of rapamycin (mTOR) functions as a folate sensor in primary human trophoblast (PHT) cells. Folate sensing by mTOR in PHT cells involves both mTOR Complex 1 and 2 and requires the proton-coupled folate transporter. We report a previously unknown molecular mechanism by which folate regulates trophoblast cell function. Because mTOR is a positive regulator of placental amino acid transport and mitochondrial function, placental mTOR folate sensing may constitute the mechanistic link between maternal folate status and fetal growth. These findings provide new insight into how folate influences human cell physiology and may have implications for our understanding of how altered folate availability causes diseases such as fetal growth restriction, fetal malformations and cancer. Folate is a water-soluble B vitamin that is essential for cellular methylation reactions and DNA synthesis and repair. Low maternal folate levels in pregnancy are associated with fetal growth restriction, but the underlying mechanisms are poorly understood. Mechanistic target of rapamycin (mTOR) links nutrient availability to cell growth and function by regulating gene expression and protein translation. Here we show that mTOR functions as a folate sensor in primary human trophoblast (PHT) cells. Folate deficiency in PHT cells caused inhibition of mTOR signalling and decreased the activity of key amino acid transporters. Folate sensing by mTOR in PHT cells involves both mTOR Complex 1 and 2 and requires the proton-coupled folate transporter (PCFT, SLC46A1). The involvement of PCFT in mTOR folate sensing is not dependent on its function as a plasma membrane folate transporter. Increasing levels of homocysteine had no effect on PHT mTOR signalling, suggesting that mTOR senses low folate rather than high homocysteine. In addition, we demonstrate that maternal serum folate is positively correlated to placental mTORC1 and mTORC2 signalling activity in human pregnancy. We have identified a previously unknown molecular link between folate availability and cell function involving PCFT and mTOR signalling. We propose that mTOR folate sensing in trophoblast cells matches placental nutrient transport, and therefore fetal growth, to folate availability. These findings may have implications for our understanding of how altered folate availability causes human diseases such as fetal growth restriction, fetal malformations and cancer.

Fatty acid and lipid profiles in primary human trophoblast over 90 h in culture

Little is known about the mechanisms underlying the preferential transport of long chain polyunsaturated fatty acids (LCPUFA) to the fetus by the syncytiotrophoblast and the role of cytotrophoblasts in placental lipid metabolism and transport. We studied primary human trophoblast (PHT) cells cultured for 90h to determine the fatty acid and lipid composition of cytotrophoblast (18h culture) and syncytiotrophoblast (90h culture) cells. In cultured PHT total lipid fatty acids were significantly (P < 0.05) reduced at 90h compared to 18h in culture including lower levels of palmitic acid (PA, 16:0, −37%), palmitoleic acid (POA, 16:1n-7, −30%), oleic acid (OA, 18:1n-9, −31%), LCPUFA arachidonic acid (AA, 20:4n-6, −28%) and α-linolenic acid (ALA, 18:3n-3, −55%). In major lipid classes, OA and most of the n-3 and n-6 LCPUFA were markedly lower at 90h in TG (−57 to −76%; p < 0.05). In the cellular NEFA, n-6 LCPUFA, dihomo-γ-linolenic acid (DGLA, 20:3n-6) and AA were both reduced by −51% and DHA was −55% lower (p < 0.05) at 90h. In contrast, phospholipid FA content did not change between cytotrophoblasts and syncytiotrophoblast except for OA, which decreased by −62% (p < 0.05). Decreasing PHT TG and NEFA lipid content at 90h in culture is likely due to processes related to differentiation such as alterations in lipase activity that occur as cytotrophoblast cells differentiate. We speculate that syncytiotrophoblast prioritizes PL containing AA and DHA for transfer to the fetus by mobilizing FA from storage lipids.

Glyburide treatment in gestational diabetes is associated with increased placental glucose transporter 1 expression and higher birth weight

Use of glyburide in gestational diabetes (GDM) has raised concerns about fetal and neonatal side effects, including increased birth weight. Placental nutrient transport is a key determinant of fetal growth, however the effect of glyburide on placental nutrient transporters is largely unknown. We hypothesized that glyburide treatment in GDM pregnancies is associated with increased expression of nutrient transporters in the syncytiotrophoblast plasma membranes.We collected placentas from GDM pregnancies who delivered at term and were treated with either diet modification (n = 15) or glyburide (n = 8). Syncytiotrophoblast microvillous (MVM) and basal (BM) plasma membranes were isolated and expression of glucose (glucose transporter 1; GLUT1), amino acid (sodium-coupled neutral amino acid transporter 2; SNAT2 and L-type amino acid transporter 1; LAT1) and fatty acid (fatty acid translocase; FAT/CD36, fatty acid transporter 2 and 4; FATP2, FATP4) transporters was determined by Western blot. Additionally, we determined GLUT1 expression by confocal microscopy in cultured primary human trophoblasts (PHT) after exposure to glyburide.Birth weight was higher in the glyburide-treated group as compared to diet-treated GDM women (3764 ± 126 g vs. 3386 ± 75 g; p < 0.05). GLUT1 expression was increased in both MVM (+50%; p < 0.01) and BM (+75%; p < 0.01). In contrast, MVM FAT/CD36 (−65%; p = 0.01) and FATP2 (−65%; p = 0.02) protein expression was reduced in mothers treated with glyburide. Glyburide increased membrane expression of GLUT1 in a dose-dependent manner in cultured PHT.This data is the first to show that glyburide increases GLUT1 expression in syncytiotrophoblast MVM and BM in GDM pregnancies, and may promote transplacental glucose delivery contributing to fetal overgrowth.

A microfluidics assay to study invasion of human placental trophoblast cells.

Pre-eclampsia, fetal growth restriction and stillbirth are major pregnancy disorders throughout the world. The underlying pathogenesis of these diseases is defective placentation characterized by inadequate invasion of extravillous placental trophoblast cells into the uterine arteries. How trophoblast invasion is controlled remains an unanswered question but is influenced by maternal uterine immune cells called decidual natural killer cells. Here, we describe an in vitro microfluidic invasion assay to study the migration of primary human trophoblast cells. Each experiment can be performed with a small number of cells making it possible to conduct research on human samples despite the challenges of isolating primary trophoblast cells. Cells are exposed to a chemical gradient and tracked in a three-dimensional microenvironment using real-time high-resolution imaging, so that dynamic readouts on cell migration such as directionality, motility and velocity are obtained. The microfluidic system was validated using isolated trophoblast and a gradient of granulocyte-macrophage colony-stimulating factor, a cytokine produced by activated decidual natural killer cells. This microfluidic model provides detailed analysis of the dynamics of trophoblast migration compared to previous assays and can be modified in future to study in vitro how human trophoblast behaves during placentation.

The expression level of C19MC miRNAs in early pregnancy and in response to viral infection

IntroductionWe have previously shown that miRNAs produced from the Chromosome 19 MiRNA Cluster (C19MC), which are expressed almost exclusively in primate trophoblasts and are released into the maternal circulation, reduce viral replication in non-placental cells and can modulate migratory behavior of extravillous trophoblast. We sought to define the expression pattern of C19MC miRNA in early pregnancy and in response to viral infection in vitro and in vivo. MethodsWe prospectively followed women undergoing in vitro fertilization (IVF) and determined their blood level of C19MC miRNA using RT-qPCR. To examine the effect of viral exposure on C19MC miRNAs expression, we used three systems: (1) a transgenic mouse overexpressing the C19MC cluster and exposed to Togaviridae during pregnancy, (2) cultured primary human trophoblasts exposed to Vesicular Stomatitis Virus in vitro, and (3) amniotic fluid from women exposed to cytomegalovirus during pregnancy. ResultsIn 27 IVF pregnancies, C19MC miRNAs were detected as early as 2 weeks after implantation, and their levels increased thereafter. There was no change in C19MC miRNA expression levels in the mouse placenta in response to viral exposure. Similarly, Vesicular Stomatitis Virus infection of primary human trophoblast did not selectively increase C19MC miRNA expression. C19MC miRNA expression in the amniotic fluid was not affected by vertical transmission of cytomegalovirus. DiscussionThe expression of C19MC miRNAs in maternal circulation very early in pregnancy suggests a role in the establishment of the maternal-fetal interface. The levels of C19MC miRNA are not influenced by diverse types of viral infection.

Primary Human Placental Trophoblasts are Permissive for Zika Virus (ZIKV) Replication

Zika virus (ZIKV) is an emerging mosquito-borne (Aedes genus) arbovirus of the Flaviviridae family. Although ZIKV has been predominately associated with a mild or asymptomatic dengue-like disease, its appearance in the Americas has been accompanied by a multi-fold increase in reported incidence of fetal microcephaly and brain malformations. The source and mode of vertical transmission from mother to fetus is presumptively transplacental, although a causal link explaining the interval delay between maternal symptoms and observed fetal malformations following infection has been missing. In this study, we show that primary human placental trophoblasts from non-exposed donors (n = 20) can be infected by primary passage ZIKV-FLR isolate, and uniquely allowed for ZIKV viral RNA replication when compared to dengue virus (DENV). Consistent with their being permissive for ZIKV infection, primary trophoblasts expressed multiple putative ZIKV cell entry receptors, and cellular function and differentiation were preserved. These findings suggest that ZIKV-FLR strain can replicate in human placental trophoblasts without host cell destruction, thereby serving as a likely permissive reservoir and portal of fetal transmission with risk of latent microcephaly and malformations.

Lipopolysaccharide and double stranded viral RNA mediate insulin resistance and increase system a amino acid transport in human trophoblast cells in vitro

IntroductionInflammation and underlying low-grade maternal infection can impair insulin signalling and upregulate nutrient transport in the placenta which contribute to fetal overgrowth associated with GDM and/or obese pregnancies. There are, however, no studies on the role of infection on placental nutrient transport in pregnancies complicated by GDM and/or obesity. Thus, the aims of this study were to determine the effect of the bacterial product lipopolysaccharide (LPS) or the viral dsRNA analogue polyinosinic:polycytidylic acid (poly(I:C)) on the insulin signalling pathway and amino acid transport in primary human trophoblast cells. MethodsHuman primary villous trophoblast cells were treated with LPS or poly(I:C). Protein expression of insulin signalling pathway proteins, insulin receptor (IR)-β, insulin receptor substrate (IRS)-1 and protein kinase B (also known as Akt), and phosphatidylinositol-4,5-bisphosphate 3-kinase p85α subunit (PI3K-p85α) protein were assessed by Western blotting. Glucose and amino acid uptake were assessed by radiolabelled assay. Western blotting and qRT-PCR were used to determine amino acid transporter protein and mRNA expression, respectively. ResultsLPS and poly(I:C) significantly decreased phosphorylation of IR-β, IRS-1, Akt, total PI3K-p85α protein expression and glucose uptake. LPS and poly(I:C) also significantly increased expression of System A amino acid transporters SNAT1 and SNAT2, and System A-mediated uptake of amino acids. DiscussionLPS and poly(I:C) induces insulin resistance and increases amino acid uptake in human primary trophoblast cells. This suggests that the presence of low-grade maternal infection can contribute to excess placental nutrient availability and promote fetal overgrowth in pregnancies complicated by GDM and/or obesity.

Maternal Type 1 diabetes activates stress response in early placenta

IntroductionHuman pregnancy and in particular the first trimester, is a period highly susceptible towards adverse insults such as oxidative stress, which may lead to inadequate embryonic and feto-placental development. Diabetes mellitus is associated with increased oxidative stress caused by hyperglycemia, reactive oxygen species (ROS) production and inflammatory signals. In pregnancy, diabetes elevates the risk for early pregnancy loss, preeclampsia and fetal growth restriction, pathologies that origin from early placental maldevelopment. We hypothesized that maternal Type 1 diabetes mellitus (T1DM) induces oxidative stress in the first trimester human placenta. MethodsWe quantified stress induced, cytoprotective proteins, i.e. heat shock protein (HSP)70 and heme oxygenase (HO)-1 and determined protein modifications as markers for oxidation and glycation, i.e. levels of 4-hydroxynonenal (HNE) or Nε-(carboxymethyl)lysine (CML) modified proteins. Moreover, we measured expression levels of enzymes involved in antioxidant defense in the first trimester (week 7–9) placenta of normal and T1DM women by immunoblot and real-time qPCR. Primary human trophoblasts were isolated from first trimester placenta and the effects of oxygen, hyperglycemia and the pro-inflammatory cytokine tumor necrosis factor (TNF)-α on levels of HSP70 and HO-1 were analyzed. ResultsHSP70 (+19.9± 10.1%) and HO-1 (+63.5± 14.5%) were elevated (p < 0.05) in first trimester placenta of T1DM women when compared to normal women. However, levels of HNE or CML modified proteins were unchanged. Also, expression of most antioxidant enzymes was unchanged, with only superoxide dismutase 3 (SOD3) being upregulated by 3.0-fold (p < 0.05). In isolated primary trophoblasts, HSP70 and HO-1 were upregulated by increasing oxygen tension, but not by hyperglycemia or TNF-α. ConclusionAlthough protein oxidation and glycation was not elevated, we infer that T1DM increases placental cellular stress in the first trimester. Elevated stress in early placenta of T1DM women may contribute to disturbances in placental development.

Correction: Hepatitis C Virus Sensing by Human Trophoblasts Induces Innate Immune Responses and Recruitment of Maternal NK Cells: Potential Implications for Limiting Vertical Transmission.

Hepatitis C virus (HCV) is the world's most common blood-borne viral infection for which there is no vaccine. The rates of vertical transmission range between 3 and 6% with odds 90% higher in the presence of HIV coinfection. Prevention of vertical transmission is not possible because of lack of an approved therapy for use in pregnancy or an effective vaccine. Recently, HCV has been identified as an independent risk factor for preterm delivery, perinatal mortality, and other complications. In this study, we characterized the immune responses that contribute to the control of viral infection at the maternal-fetal interface (MFI) in the early gestational stages. In this study, we show that primary human trophoblast cells and an extravillous trophoblast cell line (HTR8), from first and second trimester of pregnancy, express receptors relevant for HCV binding/entry and are permissive for HCV uptake. We found that HCV-RNA sensing by human trophoblast cells induces robust upregulation of type I/III IFNs and secretion of multiple chemokines that elicit recruitment and activation of decidual NK cells. Furthermore, we observed that HCV-RNA transfection induces a proapoptotic response within HTR8 that could affect the morphology of the placenta. To our knowledge, for the first time, we demonstrate that HCV-RNA sensing by human trophoblast cells elicits a strong antiviral response that alters the recruitment and activation of innate immune cells at the MFI. This work provides a paradigm shift in our understanding of HCV-specific immunity at the MFI as well as novel insights into mechanisms that limit vertical transmission but may paradoxically lead to virus-related pregnancy complications.

Multiple SLC and ABC Transporters Contribute to the Placental Transfer of Entecavir.

Entecavir (ETV), a nucleoside analog with high efficacy against hepatitis B virus, is recommended as a first-line antiviral drug for the treatment of chronic hepatitis B. However, scant information is available on the use of ETV in pregnancy. To better understand the safety of ETV in pregnant women, we aimed to demonstrate whether ETV could permeate placental barrier and the underlying mechanism. Our study showed that small amount of ETV could permeate across placenta in mice. ETV accumulation in activated or nonactivated BeWo cells (treated with or without forskolin) was sharply reduced in the presence of 100 µM of adenosine, cytidine, and in Na+ free medium, indicating that nucleoside transporters possibly mediate the uptake of ETV. Furthermore, ETV was proved to be a substrate of concentrative nucleoside transporter (CNT) 2 and CNT3, of organic cation transporter (OCT) 3, and of breast cancer resistance protein (BCRP) using transfected cells expressing respective transporters. The inhibition of ETV uptake in primary human trophoblast cells further confirmed that equilibrative nucleoside transporter (ENT) 1/2, CNT2/3, OCT3, and organic cation/carnitine transporter (OCTN) 2 might be involved in ETV transfer in human placenta. Therefore, ETV uptake from maternal circulation to trophoblast cells was possibly transported by CNT2/3, ENT1/2, and OCTN2, whereas ETV efflux from trophoblast cells to fetal circulation was mediated by OCT3, and efflux from trophoblast cells to maternal circulation might be mediated by BCRP, multidrug resistance-associated protein 2, and P-glycoprotein. The information obtained in the present study may provide a basis for the use of ETV in pregnancy.

Inhibition of placental mTOR signaling provides a link between placental malaria and reduced birthweight

BackgroundPlacental Plasmodium falciparum malaria can trigger intervillositis, a local inflammatory response more strongly associated with low birthweight than placental malaria infection alone. Fetal growth (and therefore birthweight) is dependent on placental amino acid transport, which is impaired in placental malaria-associated intervillositis. Here, we tested the hypothesis that mechanistic target of rapamycin (mTOR) signaling, a pathway known to regulate amino acid transport, is inhibited in placental malaria-associated intervillositis, contributing to lower birthweight.MethodsWe determined the link between intervillositis, mTOR signaling activity, and amino acid uptake in tissue biopsies from both uninfected placentas and malaria-infected placentas with and without intervillositis, and in an in vitro model using primary human trophoblast (PHT) cells.ResultsWe demonstrated that (1) placental mTOR activity is lower in cases of placental malaria with intervillositis, (2) placental mTOR activity is negatively correlated with the degree of inflammation, and (3) inhibition of placental mTOR activity is associated with reduced placental amino acid uptake and lower birthweight. In PHT cells, we showed that (1) inhibition of mTOR signaling is a mechanistic link between placental malaria-associated intervillositis and decreased amino acid uptake and (2) constitutive mTOR activation partially restores amino acid uptake.ConclusionsOur data support the concept that inhibition of placental mTOR signaling constitutes a mechanistic link between placental malaria-associated intervillositis and decreased amino acid uptake, which may contribute to lower birthweight. Restoring placental mTOR signaling in placental malaria may increase birthweight and improve neonatal survival, representing a new potential therapeutic approach.

Buprenorphine, Norbuprenorphine, R-Methadone, and S-Methadone Upregulate BCRP/ABCG2 Expression by Activating Aryl Hydrocarbon Receptor in Human Placental Trophoblasts.

Opioid dependence during pregnancy is a rising concern. Maintaining addicted pregnant women on long-acting opioid receptor agonist is the most common strategy to manage drug abuse in pregnant women. Methadone (MET) and buprenorphine (BUP) are widely prescribed for opiate maintenance therapy. Norbuprenorphine (NBUP) is the primary active metabolite of BUP. These medications can cross the placenta to the fetus, leading to postpartum neonatal abstinence syndrome. Despite their use during pregnancy, little is known about the cellular changes in the placenta brought about by these drugs. In this study, we showed that BUP, NBUP, and MET at clinically relevant plasma concentrations significantly induced BCRP mRNA up to 10-fold in human model placental JEG3 and BeWo cells and in primary human villous trophoblasts, and this induction was abrogated by CH223191, an aryl hydrocarbon receptor (AhR)-specific antagonist. These drugs increased AhR recruitment onto the AhR-response elements and significantly induced breast cancer resistance protein (BCRP) gene transcription. AhR overexpression further increased BCRP mRNA and protein expression. Knockdown of AhR by shRNA decreased BCRP expression, and this decrease was reversed by rescuing AhR expression. Finally, induction of BCRP expression in JEG3 and BeWo cells was accompanied by an increase in its efflux activity. Collectively, we have demonstrated, for the first time, that BUP, NBUP, and MET are potent AhR agonists and can induce BCRP in human placental trophoblasts by activating AhR. Given the critical role of BCRP in limiting fetal exposure to drugs and xenobiotics, long-term use of these medications may affect fetal drug exposure by altering BCRP expression in human placenta.