Sex-specific phenotypical, functional and metabolic profiles of human term placenta macrophages.

BACKGROUND: Placental macrophages, Hofbauer cells (HBC) are the only fetal immune cell population within the stroma of healthy placenta along pregnancy. They are central players in maintaining immune tolerance during pregnancy. Immunometabolism emerged a few years ago as a new field that integrates cellular metabolism with immune responses, however, the immunometabolism of HBC has not been explored yet. Here we studied the sex-specific differences in the phenotypic, functional and immunometabolic profile of HBC. METHODS: HBC were isolated from human term placentas (N = 31, 16 from male and 15 female neonates). Ex vivo assays were carried out to assess active metabolic and endoplasmic reticulum stress pathways by flow cytometry, confocal microscopy, gene expression and in silico approaches. RESULTS: HBC from female placentas displayed a stronger M2 phenotype accompanied by high rates of efferocytosis majorly sustained on lipid metabolism. On the other hand, male HBC expressed a weaker M2 phenotype with higher glycolytic metabolism. LPS stimulation reinforced the glycolytic metabolism in male but not in female HBC. Physiological endoplasmic reticulum stress activates IRE-1 differently, since its pharmacological inhibition increased lipid mobilization, accumulation and efferocytosis only in female HBC. Moreover, differential sex-associated pathways accompanying the phenotypic and functional profiles of HBC appeared related to the placental villi environment. CONCLUSIONS: These results support sex-associated effects on the immunometabolism of the HBC and adds another layer of complexity to the intricate maternal-fetal immune interaction.

Placental macrophages are the only fetal immune cell population within the stroma of healthy placenta along pregnancy and play a central role in contributing to the correct functioning of the placenta for the development of the fetus. Alterations in their metabolism lead to failures in their functions which are associated to pregnancy complications. Although, sex-specific differences were found in placental adaptation to pregnancy complications and outcomes, but the metabolism associated to their functions of placenta macrophages and whether they are associated to the sex of the placenta have not been explored so far. Here we studied human term placenta macrophages with special focus on their metabolism associated with their functions. We found out that macrophages from female placenta got energy from fatty acids whereas male macrophages used glucose. Furthermore, when female macrophages were exposed to a bacterial component, their metabolism or cellular function did not change towards one associated with a classic profile, but male macrophages did. These results might contribute to gain more insight into the immune-placental interactions at term human pregnancy and provide new clues to begin personalizing the pregnancy according to the sex of the fetus in physiological or pregnancy complications of inflammatory nature.

Monocyte immunometabolic reprogramming in human pregnancy: contribution of trophoblast cells.

Immunometabolism research is uncovering the relationship between metabolic features and immune cell functions in physiological and pathological conditions. Normal pregnancy entails a fine immune and metabolic regulation of the maternal-fetal interaction to assist the energetic demands of the fetus with immune homeostasis maintenance. Here, we determined the immunometabolic status of monocytes of pregnant women compared with nonpregnant controls and its impact on monocyte anti-inflammatory functions such as efferocytosis. Monocytes from pregnant women (16-20 wk) and nonpregnant age-matched controls were studied. Single cell-based metabolic assays using freshly isolated monocytes from both groups were carried out in parallel with functional assays ex vivo to evaluate monocyte efferocytic capacity. On the other hand, various in vitro metabolic assays with human monocytes or monocyte-derived macrophages were designed to explore the effect of trophoblast cells in the profiles observed. We found that pregnancy alters monocyte metabolism and function. An increased glucose dependency and enhanced efferocytosis were detected in monocytes from pregnant women at resting states, compared with nonpregnant controls. Furthermore, monocytes display a reduced glycolytic response when stimulated with lipopolysaccharide (LPS). The metabolic profiling of monocytes at this stage of pregnancy was comparable with the immunometabolic phenotypes of human monocytes treated in vitro with human first trimester trophoblast cell conditioned media. These findings suggest that immunometabolic mechanisms are involved in the functional shaping of monocytes during pregnancy with a contribution of trophoblast cells. Results provide new clues for future hypotheses regarding pregnancies complicated by metabolic disorders.NEW & NOTEWORTHY Immunometabolism stands as a novel perspective to understand the complex regulation of the immune response and to provide small molecule-based therapies. By applying this approach to study monocytes during pregnancy, we found that these cells have a unique activation pattern. They rely more on glycolysis and show increased efferocytosis/IL-10 production, but they do not have the typical proinflammatory responses. We also present evidence that trophoblast cells can shape monocytes into this distinct immunometabolic profile.

Porphyromonas gingivalis outer membrane vesicles shape trophoblast cell metabolism impairing functions associated to adverse pregnancy outcome.

Periodontitis is proposed as a risk factor for preterm delivery, fetal growth restriction, and preeclampsia with severe consequences for maternal and neonatal health, but the biological mechanisms involved are elusive. Porphyromonas gingivalis gain access to the placental bed and impair trophoblast cell function, as assessed in murine and human pregnancy, suggesting a pathogenic role in adverse pregnancy and neonatal outcomes. P. gingivalis releases outer membrane vesicles (P. gingivalis OMV) during growth that spread to distant tissues and are internalized in host cells as described in metabolic, neurological, and vascular systemic diseases. Here we tested the hypothesis that P. gingivalis OMV internalized in trophoblast cells disrupt their metabolism leading to trophoblast and placenta dysfunction and adverse pregnancy outcomes. An in vitro design with human trophoblast cells incubated with P. gingivalis OMV was used together with ex vivo and in vivo approaches in pregnant mice treated with P. gingivalis OMV. P. gingivalis OMV modulated human trophoblast cell metabolism by reducing glycolytic pathways and decreasing total reactive oxygen species with sustained mitochondrial activity. Metabolic changes induced by P. gingivalis OMV did not compromise cell viability; instead, it turned trophoblast cells into a metabolic resting state where central functions such as migration and invasion were reduced. The effects of P. gingivalis OMV on human trophoblast cells were corroborated ex vivo in mouse whole placenta and in vivo in pregnant mice: P. gingivalis OMV reduced glycolytic pathways in the placenta and led to lower placental and fetal weight gain in vivo with reduced placental expression of the glucose transporter GLUT1. The present results point to OMV as a key component of P. gingivalis involved in adverse pregnancy outcomes, and even more, unveil a metabolic cue in the deleterious effect of P. gingivalis OMV on trophoblast cells and mouse pregnancy, providing new clues to understand pathogenic mechanisms in pregnancy complications and other systemic diseases.

Targeting first trimester trophoblast cell metabolism modulates its susceptibility to Zika virus infection.

In the last 15 years Zika virus (ZIKV) caused several outbreaks of increasing scale in Micronesia, South Pacific islands, and more recently in the Caribbean and South America. The severity of the clinical presentation in neonates from pregnant women infected with ZIKV during the last outbreak supports the relevance of unraveling the mechanism of infection and viral persistence in the placenta with local viral isolates. Here, we investigated the relevance of trophoblast metabolic rewiring for viral multiplication and the role of the vasoactive intestinal peptide (VIP) as an endogenous factor associated with placental restriction to ZIKV infection at early pregnancy. Our in vitro model demonstrated that ZIKV triggers metabolic rewiring in first trimester cytotrophoblast-derived cells by increasing glucose utilization as fuel to sustain its replication, decreasing long-chain polyunsaturated fatty acid uptake, and promoting lipid droplets accumulation to favor its multiplication. Of note, variations in nutrient availability modulated viral spread in trophoblast cultures. The presence of VIP during trophoblast infection impaired ZIKV infective particle production and viral replication, restoring cell migration and metabolism. Moreover, the blockade of endogenous VIP signaling increased viral particle production and the viral entry receptor AXL expression. These results highlight the potential role of VIP as an endogenous antiviral factor related to trophoblast cell permissiveness to ZIKV infection at early pregnancy.

Vasoactive intestinal peptide deficiency promotes ovarian dysfunction associated to a proinflammatory microenvironment reminiscent of premature aging.

Complex immune regulation during pregnancy is required to ensure a successful pregnancy outcome. Vasoactive intestinal peptide (VIP) has local immunoregulatory effects on the ovary, uterus and maternal-fetal interface that favor a tolerogenic maternal microenvironment. Since the VIP Knockout (KO) mice are subfertile, we investigated the mechanisms underlying the effects of VIP deficiency on ovarian physiology and immune homeostasis. Therefore, we studied VIP KO, deficient (HT) and wild type (WT) female mice in estrus at 3 or 8 months of age. Young KO mice showed abnormal cycle timing and regularity associated with dysfunctional ovaries. Ovaries presented higher number of atretic follicles and reduced number of corpora lutea leading to a lower ovulation rates. Part of the VIP KO mice (25 %) failed to ovulate or ovulated oocytes incompetent to be fertilized (50 %). In particular, ovaries of young KO mice exhibited features of premature aging accompanied by a pro-inflammatory milieu with increased levels of IL-1ß. A unique macrophage subpopulation identified as "foamy macrophages" was found. On the other hand, aged VIP KO females did not gain body weight probably due to the sustained production of E2. Finally, the adoptive transfer of FOXP3+ cells to infertile VIP KO females resulted in their selective recruitment to the ovary. It increased FOXP3/RORγt and TGFß/IL-6 ratio improving ovarian microenvironment and pregnancy rate. The present results suggest that VIP contributes to ovarian homeostatic mechanisms required for a successful pregnancy.

Gingival crevicular fluid from pregnant women impairs trophoblast cell function and trophoblast-neutrophil interaction.

PROBLEM: A strong association between periodontitis and higher susceptibility to pregnancy complications like preeclampsia has been reported although the mechanisms remain elusive. Trophoblast cells modulate the recruitment and functional shaping of maternal leukocytes at early stages to sustain an antiinflammatory microenvironment and fetal growth. Neutrophil activation with reactive oxygen species (ROS) release is associated with preeclampsia. Our aim was to study the effect of the gingival crevicular fluid (GCF) from pregnant women on trophoblast cell function and trophoblast-neutrophil interaction. METHOD OF STUDY: Pregnant women at 16-20 weeks of gestation (n = 27) and non-pregnant women (n = 8) as the control group were studied for gingivoperiodontal clinical score evaluation and GCF collection. Total bacteria and common periodontal pathogens were analyzed in GCF samples. The effect of each GCF sample was tested on first trimester trophoblast-derived cells to assess cell migration, cytokine expression and glucose uptake. Also, the effect of GCF on human peripheral neutrophil chemoattraction by trophoblast cells and ROS formation was assessed. RESULTS: Gingival crevicular fluid from pregnant women reduced trophoblast cell migration, increased proinflammatory marker expression and glucose uptake. A significant correlation between gingivoperiodontal score and trophoblast dysfunction was observed. Upon conditioning of trophoblast cells with GCF, only the GCF from pregnant women stimulated neutrophil chemoattraction. Similarly, GCF from pregnant but not from non-pregnant controls stimulated ROS formation in neutrophils. CONCLUSIONS: Gingival crevicular fluid from pregnant women is deleterious for first trimester trophoblast cell function. These effects could lead to placental homeostasis disruption underlying a pathogenic mechanism of pregnancy complications associated to periodontal disease.

Growth impairment, increased placental glucose uptake and altered transplacental transport in VIP deficient pregnancies: Maternal vs. placental contributions.

Glucose uptake by the placenta and its transfer to the fetus is a finely regulated process required for placental and fetal development. Deficient placentation is associated with pregnancy complications such as fetal growth restriction (FGR). The vasoactive intestinal peptide (VIP) has embryotrophic effects in mice and regulates human cytotrophoblast metabolism and function. Here we compared glucose uptake and transplacental transport in vivo by VIP-deficient placentas from normal or VIP-deficient maternal background. The role of endogenous VIP in placental glucose and amino acid uptake was also investigated. Wild type C57BL/6 (WT) or VIP+/- (VIP HT) females were mated with WT, VIP knock-out (VIP KO) or VIP HT males. Glucose uptake and transplacental transport were evaluated by the injection of the fluorescent d-glucose analogue 2-NBDG in pregnant mice at gestational day (gd) 17.5. Glucose and amino acid uptake in vitro by placental explants were measured with 2-NBDG or 14C-MeAIB respectively. In normal VIP maternal background, fetal weight was reduced in association with placental VIP deficiency, whereas placental weight was unaltered. Paradoxically, VIP+/- placentas presented higher glucose uptake and higher gene expression of GLUT1 and mTOR than VIP+/+ placentas. However, in a maternal VIP-deficient environment placental uptake and transplacental transport of glucose increased while fetal weights were unaffected, regardless of feto-placental genotype. Results point to VIP-deficient pregnancy in a normal background as a suitable FGR model with increased placental glucose uptake and transplacental transport. The apparently compensatory actions are unable to sustain normal fetal growth and could result in complications later in life.

Decidual factors and vasoactive intestinal peptide guide monocytes to higher migration, efferocytosis and wound healing in term human pregnancy.

AIM: To explore the functional profile of circulating monocytes and decidual macrophages at term human pregnancy and their contribution to tissue repair upon stimulation ex vivo with decidual factors and the vasoactive intestinal peptide (VIP). METHODS: Peripheral blood monocytes were isolated from pregnant and non-pregnant volunteers and tested in vitro with decidual explants from term placenta and VIP. The effect of VIP on decidual explants and the effect of its conditioned media on monocytes or decidual macrophages isolated by magnetic beads was carried out by RT-qPCR and ELISA for cytokines expression and release. Migration assays were performed in transwell systems. Efferocytosis was assessed in monocytes or decidual macrophages with CFSE-labelled autologous apoptotic neutrophils and quantified by flow cytometry. Monocyte and decidual macrophages wound healing capacity was evaluated using human endometrial stromal cell monolayers. Immunohistochemistry was performed in serial tissue sections of different placentas. RESULTS: VIP is expressed in the villi as well as in trophoblast giant cells distributed within the decidua of term placenta. VIP induced the expression of antiinflmammatory markers and monocyte chemoattractant CCL2 and CCL3 in decidual tissues. Monocytes presented higher migration towards decidual explants than CD4 and CD8 cells. VIP-conditioned monocytes displayed an enhanced efferocytosis and wound healing capacity comparable to that of decidual macrophages. Moreover limited efferocytosis of pregnant women monocytes was restored by VIP-induced decidual factors. CONCLUSION: Results show the conditioning of monocytes by decidual factors and VIP to sustain processes required for tissue repair and homeostasis maintenance in term placenta.

Zika virus infection of first trimester trophoblast cells affects cell migration, metabolism and immune homeostasis control.

Zika virus (ZIKV) re-emerged after circulating almost undetected for many years and the last spread in 2015 was the major outbreak reported. ZIKV infection was associated with congenital fetal growth anomalies such as microcephaly, brain calcifications, and low birth weight related to fetal growth restriction. In this study, we investigated the effect of ZIKV infection on first trimester trophoblast cell function and metabolism. We also studied the interaction of trophoblast cells with decidual immune populations. Results presented here demonstrate that ZIKV infection triggered a strong antiviral response in first trimester cytotrophoblast-derived cells, impaired cell migration, increased glucose uptake and GLUT3 expression, and reduced brain derived neurotrophic factor (BDNF) expression. ZIKV infection also conditioned trophoblast cells to favor a tolerogenic response since an increased recruitment of CD14+ monocytes bearing an anti-inflammatory profile, increased CD4+ T cells and NK CD56Dim and NK CD56Bright populations and an increment in the population CD4+ FOXP3+ IL-10+ cells was observed. Interestingly, when ZIKV infection of trophoblast cells occurred in the presence of the vasoactive intestinal peptide (VIP) there was lower detection of viral RNA and reduced toll-like receptor-3 and viperin messenger RNA expression, along with reduced CD56Dim cells trafficking to trophoblast conditioned media. The effects of ZIKV infection on trophoblast cell function and immune-trophoblast interaction shown here could contribute to defective placentation and ZIKV persistence at the fetal-maternal interface. The inhibitory effect of VIP on ZIKV infection of trophoblast cells highlights its potential as a candidate molecule to interfere ZIKV infection during early pregnancy.

Decidualization Process Induces Maternal Monocytes to Tolerogenic IL-10-Producing Dendritic Cells (DC-10).

Decidualization is a process that involves phenotypic and functional changes of endometrial stromal cells to sustain endometrial receptivity and the participation of immunoregulatory factors to maintain immune homeostasis. In this context, tolerogenic dendritic cells (DCs) can induce regulatory T cells, which are essential to manage the pro- to anti-inflammatory transition during embryo implantation. Recently, Myeloid Regulatory Cells (MRCs) were proposed as immunosuppressants and tolerance-inducer cells, including the DC-10 subset. This novel and distinctive subset has the ability to produce IL-10 and to induce type 1 regulatory T cells (Tr1) through an HLA-G pathway. Here we focus on the impact of the decidualization process in conditioning peripheral monocytes to MRCs and the DC-10 subset, and their ability to induce regulatory T cells. An in vitro model of decidualization with the human endometrial stromal cell line (HESC), decidualized by medroxyprogesterone and dibutyryl-cAMP was used. Monocytes isolated from peripheral blood mononuclear cells from healthy women were cultured with rhGM-CSF + rhIL-4 and then, the effect of conditioned media from decidualized (Dec-CM) and non-decidualized cells (Non-dec-CM) was tested on monocyte cultures. We found that Dec-CM inhibited the differentiation to the CD1a+CD14- immature DC profile in a concentration-dependent manner. Dec-CM also significantly increased the frequency of CD83+CD86low and HLA-DR+ cells in the monocyte-derived culture. These markers, associated with the increased production of IL-10, are consistent with a MRCs tolerogenic profile. Interestingly, Dec-CM treatment displayed a higher expression of the characteristic markers of the tolerogenic DC-10 subset, HLA-G and ILT2/CD85j; while this modulation was not observed in cultures treated with Non-dec-CM. Moreover, when monocyte cultures with Dec-CM were challenged with LPS, they sustained a higher IL-10 production and prevented the increase of CD83, CD86, IL-12p70, and TNF-α expression. Finally, the DC-10 subset was able to induce a CD4+HLA-G+ regulatory T cells subset. These results suggest that the decidualization process might induce different subsets of MRCs, like DC-10, able to induce regulatory T cells as a novel CD4+HLA-G+ subset which might play an immunoregulatory role in embryo implantation.

Vasoactive Intestinal Peptide induces glucose and neutral amino acid uptake through mTOR signalling in human cytotrophoblast cells.

The transport of nutrients across the placenta involves trophoblast cell specific transporters modulated through the mammalian target of rapamycin (mTOR). The vasoactive intestinal peptide (VIP) has embryotrophic effects in mice and regulates human cytotrophoblast cell migration and invasion. Here we explored the effect of VIP on glucose and System A amino acid uptake by human trophoblast-derived cells (Swan 71 and BeWo cell lines). VIP activated D-glucose specific uptake in single cytotrophoblast cells in a concentration-dependent manner through PKA, MAPK, PI3K and mTOR signalling pathways. Glucose uptake was reduced in VIP-knocked down cytotrophoblast cells. Also, VIP stimulated System A amino acid uptake and the expression of GLUT1 glucose transporter and SNAT1 neutral amino acid transporter. VIP increased mTOR expression and mTOR/S6 phosphorylation whereas VIP silencing reduced mTOR mRNA and protein expression. Inhibition of mTOR signalling with rapamycin reduced the expression of endogenous VIP and of VIP-induced S6 phosphorylation. Our findings support a role of VIP in the transport of glucose and neutral amino acids in cytotrophoblast cells through mTOR-regulated pathways and they are instrumental for understanding the physiological regulation of nutrient sensing by endogenous VIP at the maternal-foetal interface.

Control of the inflammatory response during pregnancy: potential role of VIP as a regulatory peptide.

A network of cell-cell communications through contact and soluble factors supports the maternal-placental interaction and provides a suitable environment for fetal growth. Trophoblast cells take center stage at these loops: they interact with maternal leukocytes to sustain the varying demands of gestation, and they synthesize hormones, cytokines among other factors that contribute to the maintenance of immune homeostasis. Here, we discuss vasoactive intestinal peptide (VIP) and its potential as a regulatory neuropeptide in pregnancy. VIP is synthesized by trophoblast cells; it regulates trophoblast cell function and interaction with the major immune cell populations present in the pregnant uterus. VIP activity produces an anti-inflammatory microenvironment by modulating the functional profile of monocytes, macrophages, and regulatory T cells. Trophoblast VIP inhibits neutrophil extracellular trap formation and accelerates neutrophil apoptosis, enabling their silent clearance by phagocytic cells. The effects of VIP on the trophoblast-immune interaction are consistent with its regulatory role throughout pregnancy for immune homeostasis maintenance. These observations may provide new clues for pharmacological targeting of pregnancy complications associated with exacerbated inflammation.

Trophoblast VIP deficiency entails immune homeostasis loss and adverse pregnancy outcome in mice.

Immune homeostasis maintenance throughout pregnancy is critical for normal fetal development. Trophoblast cells differentiate into an invasive phenotype and contribute to the transformation of maternal arteries and the functional shaping of decidual leukocyte populations. Insufficient trophoblast invasion, inadequate vascular remodeling, and a loss of immunologic homeostasis are associated with pregnancy complications, such as preeclampsia and intrauterine growth restriction. Vasoactive intestinal peptide (VIP) is a pleiotropic neuropeptide synthetized in trophoblasts at the maternal-placental interface. It regulates the function of trophoblast cells and their interaction with decidual leukocytes. By means of a murine model of pregnancy in normal maternal background with VIP-deficient trophoblast cells, here we demonstrate that trophoblast VIP is critical for trophoblast function: VIP gene haploinsufficiency results in lower matrix metalloproteinase 9 expression, and reduced migration and invasion capacities. A reduced number of regulatory T cells at the implantation sites along with a lower expression of proangiogenic and antiinflammatory markers were also observed. Findings detected in the implantation sites at early stages were followed by an abnormal placental structure and lower fetal weight. This effect was overcome by VIP treatment of the early pregnant mice. Our results support the relevance of trophoblast-synthesized VIP as a critical factor in vivo for trophoblast-cell function and immune homeostasis maintenance in mouse pregnancy.-Hauk, V., Vota, D., Gallino, L., Calo, G., Paparini, D., Merech, F., Ochoa, F., Zotta, E., Ramhorst, R., Waschek, J., Leirós, C. P. Trophoblast VIP deficiency entails immune homeostasis loss and adverse pregnancy outcome in mice.

Vasoactive Intestinal Peptide modulates trophoblast-derived cell line function and interaction with phagocytic cells through autocrine pathways.

Trophoblast cells migrate and invade the decidual stroma in a tightly regulated process to maintain immune homeostasis at the maternal-placental interface during the first weeks of pregnancy. Locally synthesized factors modulate trophoblast cell function and their interaction with maternal leukocytes to promote the silent clearance of apoptotic cells. The vasoactive intestinal peptide (VIP) is a pleiotropic polypeptide with trophic and anti-inflammatory effects in murine pregnancy models. We explored the effect of VIP on two human first trimester trophoblast cell lines, particularly on their migration, invasiveness and interaction with phagocytic cells, and the signalling and regulatory pathways involved. We found that VIP enhanced trophoblast cell migration and invasion through the activation of high affinity VPAC receptors and PKA-CRE signalling pathways. VIP knocked-down trophoblast cells showed reduced migration in basal and leukemic inhibitor factor (LIF)-elicited conditions. In parallel, VIP-silenced trophoblast cells failed to induce the phagocytosis of apoptotic bodies and the expression of immunosuppressant markers by human monocytes. Our results suggest that VIP-mediated autocrine pathways regulate trophoblast cell function and contribute to immune homeostasis maintenance at placentation and may provide new clues for therapeutic intervention in pregnancies complicated by defective deep placentation.