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.

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.

VPAC1 and VPAC2 receptor deficiencies negatively influence pregnancy outcome through distinct and overlapping modulations of immune, trophoblast and vascular functions.

Pregnancy outcome relies on the maintenance of immune and metabolic homeostasis at the maternal fetal interface. Maternal and perinatal morbidity and mortality is associated with impaired placental development. Multiple regulatory effects of the endogenous-produced vasoactive intestinal peptide (VIP) on vascular, metabolic and immune functions at the maternal-fetal interface have been reported. Here we studied the involvement of the two primary high affinity receptors for VIP (VPAC1 and VPAC2) on maternal immune response, placental homeostasis and pregnancy outcome. Targeted disruption of each receptor gene led to altered placental structure, vascular and trophoblast functional markers and shaped the functional profiles of macrophages and neutrophils towards a proinflammatory state. Several changes in pregnant mice were receptor specific: ROS production elicited by VIP on neutrophils was selectively dependent on the presence of VPAC1 whereas apoptosis rate was associated with the VPAC2 deletion. In peritoneal macrophages from pregnant mice, levels of MHC-II, TLR2, and IL-10 were selectively altered in VPAC2 receptor-deficient mice, whereas IL-6 gene expression was reduced only in mice lacking VPAC1 receptors. Additionally, MMP9 mRNA in isolated TGCs was reduced in VPAC2 receptor deleted mice, while the percentage of IL-12 cells in post-phagocytosis macrophage cultures was selectively reduced in VPAC2 receptor deficient mice. The results indicate that manipulation of VPAC1 and VPAC2 receptor affects immune, vascular and metabolic environment at the maternal fetal interface. These mouse models offer new approaches to study pregnancy complications adding new perspectives to the development of VPAC receptor-selective drugs.

From decidualization to pregnancy progression: An overview of immune and metabolic effects of VIP.

BACKGROUND: A tight immune and metabolic regulation underlies the early maternal-placental interaction to assist the energetic dynamic demands of the fetus throughout pregnancy. The vasoactive intestinal peptide (VIP) holds biochemical, metabolic and immune properties consistent with a regulatory role during pregnancy. AIM: Here we overview critical aspects of embryo implantation and placental development with special focus on the immune and metabolic effects of VIP expressed by decidual and trophoblast cells. CONTENT: During decidualization, endometrial stromal cells undergo reticular stress and trigger unfolded protein response (UPR) that enable expansion of their endoplasmic reticulum and immunomodulatory factor synthesis. These processes appear differentially affected in recurrent abortion and in vitro fertilization failure suggesting their relevance in reproductive pathologies. Similarly, defective placentation associates with altered immune, vascular and trophoblast interaction resulting in complicated pregnancies that threaten maternal and neonatal health and underlie metabolic programming of adult life. We discuss the most recent research on decidual, trophoblast and immune cell interaction on the light of VIP regulation. Its role in decidualization and UPR associated with a sterile inflammatory response and angiogenesis is discussed. Evidence on VIP modulation of cytotrophoblast cell function, metabolism and immune profile is revised as well as the shaping of decidual leukocyte phenotype and function from decidualization to term. IMPLICATIONS: The broad spectrum of effects of VIP from implantation to term in normal and pathological conditions summarized here might contribute to the identification of novel biomarkers for diagnosis and pharmacological targeting.

SARS-CoV-2 infection negatively affects ovarian function in ART patients.

Several organs, such as the heart, breasts, intestine, testes, and ovaries, have been reported to be target tissues of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. To date, no studies have demonstrated SARS-CoV-2 infection in the female reproductive system. In the present study, we investigated the effects of SARS-CoV-2 infection on ovarian function by comparing follicular fluid (FF) from control and recovered coronavirus disease 2019 (COVID-19) patients and by evaluating the influence of these FF on human endothelial and non-luteinized granulosa cell cultures. Our results showed that most FFs (91.3%) from screened post COVID-19 patients were positive for IgG antibodies against SARS-CoV-2. Additionally, patients with higher levels of IgG against SARS-CoV-2 had lower numbers of retrieved oocytes. While VEGF and IL-1ß were significantly lower in post COVID-19 FF, IL-10 did not differ from that in control FF. Moreover, in COV434 cells stimulated with FF from post COVID-19 patients, steroidogenic acute regulatory protein (StAR), estrogen-receptor ß (Erß), and vascular endothelial growth factor (VEGF) expression were significantly decreased, whereas estrogen-receptor α (ERα) and 3ß-hydroxysteroid dehydrogenase (3ß-HSD) did not change. In endothelial cells stimulated with post COVID-19 FF, we observed a decrease in cell migration without changes in protein expression of certain angiogenic factors. Both cell types showed a significantly higher γH2AX expression when exposed to post COVID-19 FF. In conclusion, our results describe for the first time that the SARS-CoV-2 infection adversely affects the follicular microenvironment, thus dysregulating ovarian function.

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.

Immunoregulation of the decidualization program: focus on the endoplasmic reticulum stress.

Decidualization denotes the reprogramming of endometrial stromal cells that includes the secretion of different mediators like cytokines, chemokines, and the selective recruitment of immune cells. This physiological process involves changes in the secretome of the endometrial stromal cells leading to the production of immunomodulatory factors. The increased amount of protein secretion is associated with a physiological endoplasmic reticulum (ER) stress and the resulting unfolded protein response (UPR), allowing the expansion of ER and the machinery to assist the protein folding. Notably, the signaling pathways involved in the ER stress and the UPR are interconnected with the onset of a sterile inflammatory response, as well as with angiogenesis. Both of these processes have a key role in decidualization and placentation, therefore, alterations in them could lead to pregnancy complications. In this review, we will discuss how the induction of ER stress and the UPR processes that accompanies the decidualization are associated with embryo implantation and whether they might condition pregnancy outcome. The ER stress activates/triggers sensing proteins which, among others, induces kinase/RNAse-TXNIP expression, activating the NLRP3 inflammasome. This multiprotein system allows caspase-1 activation, which catalyzes the cleavage of the inactive IL-1ß proform toward the mature secretory form, with pro-implantatory effects. However, the sterile inflammatory response should be later controlled in favor of a tolerogenic microenvironment to sustain pregnancy. In accordance, alterations of the ER stress and UPR processes can be reflected in recurrent implantation failures (RIF), recurrent pregnancy loss (RPL), or complications associated with deficient placentation, such as preeclampsia (PE).

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.

Vasoactive intestinal peptide shapes first-trimester placenta trophoblast, vascular, and immune cell cooperation.

BACKGROUND AND PURPOSE: Extravillous trophoblast (EVT) cells are responsible for decidual stromal invasion, vascular transformation, and the recruitment and functional modulation of maternal leukocytes in the first-trimester pregnant uterus. An early disruption of EVT function leads to placental insufficiency underlying pregnancy complications such as preeclampsia and fetal growth restriction. Vasoactive intestinal peptide (VIP) is a vasodilating and immune modulatory factor synthesized by trophoblast cells. However, its role in first-trimester placenta has not been explored. Here, we tested the hypothesis that VIP is involved in first-trimester EVT outgrowth, spiral artery remodelling, balancing angiogenesis, and maintenance of immune homeostasis. EXPERIMENTAL APPROACH: First-trimester placental tissue (five to nine weeks of gestation) was collected, and was used for EVT outgrowth experiments, immunofluorescence, isolation of decidual natural killer (dNK) cells and decidual macrophages (dMA), and functional assays. Peripheral blood monocytes were differentiated with GM-CSF and used for angiogenesis assays. KEY RESULTS: In decidua basalis, VIP+ EVT were observed sprouting from cell columns and lining spiral arterioles. EVT migrating from placental explants were also VIP+. VIP increased EVT outgrowth and IL-10 release, whereas it decreased pro-inflammatory cytokine production in EVT, dNK cells, and dMA. VIP disrupted endothelial cell networks, both directly and indirectly via an effect on macrophages. CONCLUSION AND IMPLICATIONS: The results suggest that VIP assists the progress of EVT invasion and vessel remodelling in first-trimester placental bed in an immunologically "silent" milieu. The effects of VIP in the present ex vivo human placental model endorse its potential as a therapeutic candidate for deep placentation disorders.

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.

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.

VIP Promotes Recruitment of Tregs to the Uterine-Placental Interface During the Peri-Implantation Period to Sustain a Tolerogenic Microenvironment.

Uterine receptivity and embryo implantation are two main processes that need a finely regulated balance between pro-inflammatory and tolerogenic mediators to allow a successful pregnancy. The neuroimmune peptide vasoactive intestinal peptide (VIP) is a key regulator, and it is involved in the induction of regulatory T cells (Tregs), which are crucial in both processes. Here, we analyzed the ability of endogenous and exogenous VIP to sustain a tolerogenic microenvironment during the peri-implantation period, particularly focusing on Treg recruitment. Wild-type (WT) and VIP-deficient mice [heterozygous (HT, +/-), knockout (KO, -/-)], and FOXP3-knock-in-GFP mice either pregnant or in estrus were used. During the day of estrus, we found significant histological differences between the uterus of WT mice vs. VIP-deficient mice, with the latter exhibiting undetectable levels of FOXP3 expression, decreased expression of interleukin (IL)-10, and vascular endothelial growth factor (VEGF)c, and increased gene expression of the Th17 proinflammatory transcription factor RORγt. To study the implantation window, we mated WT and VIP (+/-) females with WT males and observed altered FOXP3, VEGFc, IL-10, and transforming growth factor (TGF)ß gene expression at the implantation sites at day 5.5 (d5.5), demonstrating a more inflammatory environment in VIP (+/-) vs. VIP (+/+) females. A similar molecular profile was observed at implantation sites of WT × WT mice treated with VIP antagonist at d3.5. We then examined the ability GFP-sorted CD4+ cells from FOXP3-GFP females to migrate toward conditioned media (CM) obtained from d5.5 implantation sites cultured in the absence/presence of VIP or VIP antagonist. VIP treatment increased CD4+FOXP3+ and decreased CD4+ total cell migration towards implantation sites, and VIP antagonist prevented these effects. Finally, we performed adoptive cell transfer of Tregs (sorted from FOXP3-GFP females) in VIP-deficient-mice, and we observed that FOXP3-GFP cells were mainly recruited into the uterus/implantation sites compared to all other tested tissues. In addition, after Treg transfer, we found an increase in IL-10 expression and VEGFc in HT females and allowed embryo implantation in KO females. In conclusion, VIP contributes to a local tolerogenic response necessary for successful pregnancy, preventing the development of a hostile uterine microenvironment for implantation by the selective recruitment of Tregs during the peri-implantation period.

Progesterone and VIP cross-talk enhances phagocytosis and anti-inflammatory profile in trophoblast-derived cells.

Trophoblast cells produce several inmmuneregulators like the Vasoactive Intestinal Peptide (VIP) and P4 targeting multiple circuits, and also display an intese phagocytic ability allowing embryo implantation in a tolerogenic context. Here, we explored whether P4 and VIP- crosstalk modulates trophoblast cell function, focus on the phagocytic ability and the immune homeostasis maintenance. P4 enhanced the phagocytosis in trophoblast-derived cells quantified by the engulfment of latex-beads or eryptotic erythrocytes. P4 and VIP modulated the balance of anti/pro-inflammatory mediators, increasing TGF-ß expression, with no changes in IL-1, IL-6, or nitrites production. This modulation was accompained by transcription factor expression changes that could turn on tolerogenic programs represented by increased PPAR-γ and decreased IRF-5 expression. Finally, P4 stimulated VPAC2 expression in trophoblast cells and VPAC2 over-expression enhanced phagocytosis mimicking P4-effect. Therefore, P4 and VIP network enhances the phagocytic ability of trophoblast-derived cells, through a mechanism involving VPAC2 accompained with an anti-inflammatory context.

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.

Decoding the chemokine network that links leukocytes with decidual cells and the trophoblast during early implantation.

Chemokine network is central to the innate and adaptive immunity and entails a variety of proteins and membrane receptors that control physiological processes such as wound healing, angiogenesis, embryo growth and development. During early pregnancy, the chemokine network coordinates not only the recruitment of different leukocyte populations to generate the maternal-placental interface, but also constitutes an additional checkpoint for tissue homeostasis maintenance. The normal switch from a pro-inflammatory to an anti-inflammatory predominant microenvironment characteristic of the post-implantation stage requires redundant immune tolerance circuits triggered by key master regulators. In this review we will focus on the recruitment and conditioning of maternal immune cells to the uterus at the early implantation period with special interest on high plasticity macrophages and dendritic cells and their ability to induce regulatory T cells. We will also point to putative immunomodulatory polypeptides involved in immune homeostasis maintenance at the maternal-placental interface.

VIP treatment prevents embryo resorption by modulating efferocytosis and activation profile of maternal macrophages in the CBAxDBA resorption prone model.

Successful embryo implantation occurs followed by a local pro-inflammatory response subsequently shifted toward a tolerogenic one. VIP (vasoactive intestinal peptide) has embryotrofic, anti-inflammatory and tolerogenic effects. In this sense, we investigated whether the in vivo treatment with VIP contributes to an immunosuppressant local microenvironment associated with an improved pregnancy outcome in the CBA/J × DBA/2 resorption prone model. Pregnancy induced the expression of VIP, VPAC1 and VPAC2 in the uterus from CBA/J × DBA/2 mating females on day 8.5 of gestation compared with non-pregnant mice. VIP treatment (2 nmol/mouse i.p.) on day 6.5 significantly increased the number of viable implantation sites and improved the asymmetric distribution of implanted embryos. This effect was accompanied by a decrease in RORγt and an increase in TGF-ß and PPARγ expression at the implantation sites. Moreover, VIP modulated the maternal peritoneal macrophages efferocytosis ability, tested using latex beads-FITC or apoptotic thymocytes, displaying an increased frequency of IL-10-producer F4/80 cells while did not modulate TNF-α and IL-12 secretion. The present data suggest that VIP treatment increases the number of viable embryos associated with an increase in the efferocytic ability of maternal macrophages which is related to an immunosuppressant microenvironment.

Trophoblast cells primed with vasoactive intestinal peptide enhance monocyte migration and apoptotic cell clearance through αvß3 integrin portal formation in a model of maternal-placental interaction.

STUDY HYPOTHESIS: Is apoptotic cell phagocytosis by monocytes modulated by pathways elicited by vasoactive intestinal peptide (VIP) action on trophoblast? STUDY FINDING: Targeting trophoblast cells with VIP induces monocyte migration, polarization to anti-inflammatory phenotypes and apoptotic trophoblast cell clearance which involves increased αvß3 integrin expression on phagocytic cells and binding to thrombospondin 1. WHAT IS KNOWN ALREADY: Monocytes recruited to the maternal-placental interface interact with trophoblast cells and differentiate to alternatively activated macrophages involved in the silent clearance of apoptotic cells. Vasoactive intestinal peptide (VIP) is an immunomodulatory polypeptide synthesized at the human placenta that can target both trophoblast cells and monocytes/macrophages. Integrin αvß3 and thrombospondin 1 are involved in the formation of a phagocytic portal for the immunosuppressant clearance of apoptotic cells. STUDY DESIGN, SAMPLES/MATERIALS, METHODS: This is a laboratory-based study studying monocytes isolated from peripheral blood of healthy women (n = 33) and their interaction in vitro with first trimester trophoblast cell lines. Peripheral blood monocytes were isolated from healthy volunteers by Percoll gradient and tested in co-culture settings with first trimester trophoblast cell lines (Swan 71 and HTR8) or with trophoblast cell conditioned media obtained in the presence or absence of 10 or 100 nM VIP. The effect of VIP-conditioned media on monocyte migration was assessed through transwell systems and monocyte/macrophage phenotype was determined by flow cytometry. Phagocytosis of apoptotic cells and the mechanisms involved in phagocytic portal formation were assessed by flow cytometry, confocal microscopy, immunological blockade and RT-PCR. MAIN RESULTS AND THE ROLE OF CHANCE: Exposing cells to 100 nM VIP increased the migration of monocytes toward trophoblast cell conditioned media (VIP conditioned medium) (P < 0.05 versus conditioned media from cells not exposed to VIP) and contributed to the monocytes acquiring an anti-inflammatory profile with increased CD39 and IL-10 expression (P < 0.05). Phagocytosis of apoptotic trophoblast cells by monocytes and monocyte-differentiated macrophages was increased by VIP conditioned medium (P < 0.05 versus media conditioned in the absence of VIP or direct addition of 100 nM VIP). The boosting effect of VIP conditioned medium on phagocytosis involved increased expression and re-localization of αvß3 integrin on phagocytic cells along with enhanced expression of thrombospondin 1 on trophoblast cells. LIMITATIONS, REASONS FOR CAUTION: The conclusions are based on in vitro experiments with monocytes drawn from peripheral blood of healthy individuals and trophoblast cell lines and we were unable to ascertain that these mechanisms operate similarly in vivo. We cannot rule out a differential behavior of either trophoblast cells targeted in vivo with VIP, or primary cultures of first trimester trophoblast cells assayed in vitro. WIDER IMPLICATIONS OF THE FINDINGS: The results presented provide new clues for immune and trophoblast cell pharmacological targeting in pregnancy complications of immunopathologic nature. STUDY FUNDING/COMPETING INTERESTS: This work was funded by the National Agency of Sciences and Technology ANPCyT (PICT 2011-0144), National Research Council CONICET (PIP 602/2012) and University of Buenos Aires (UBACyT 20020130100040BA) to C.P.L. The authors have no conflicts of interest to disclose.

VIP boosts regulatory T cell induction by trophoblast cells in an in vitro model of trophoblast-maternal leukocyte interaction.

Inducible regulatory T cells (Tregs) exert a timely and efficient immunosuppressive action at the critical peri-implantation stage essential for maternal tolerance to the conceptus. Vasoactive intestinal peptide (VIP) promotes anti-inflammatory and tolerogenic profiles through binding to VIP receptors on immune cells. We evaluated whether VIP produced by trophoblast cells induces Tregs during the early interaction of maternal leukocytes with trophoblast cells, thus contributing to maternal tolerance. We used an in vitro model of maternal leukocyte-trophoblast cell interaction represented by cocultures of fertile women's PBMCs with a human trophoblast cell line (Swan-71) and evaluated the effect of VIP added exogenously and of the endogenous polypeptide. VIP increased the frequency of CD4(+)CD25(+)FoxP3(+) cells after coculture, and these cells were able to suppress the maternal alloresponse. VIP also increased the frequency of CD4(+)IL10(+) and CD4(+)TGFß(+) cells, but it did not modulate IFN-γ or IL-17 production. Swan-71 secreted VIP, and their coculture with maternal PBMCs significantly increased the frequency of Tregs. This effect was even more pronounced if the trophoblast cells had been pretreated with VIP. In both situations, the VIP antagonist prevented the increase in the frequency of CD4(+)Foxp3(+) cells, reflecting a specific effect of the polypeptide after the interaction with Swan-71 cells. Finally, the increase in CD4(+)CD25(+)FoxP3(+) frequency was prevented by an anti-TGF-ß Ab and a VIP antagonist. These results suggest that VIP could have an active role in the immunoregulatory processes operating in the maternal-placental interface by contributing to the induction of Tregs through a mechanism involving TGF-ß1.