A combined ligand and target-based virtual screening strategy to repurpose drugs as putrescine uptake inhibitors with trypanocidal activity.

Chagas disease, also known as American trypanosomiasis, is a neglected tropical disease caused by the protozoa Trypanosoma cruzi, affecting nearly 7 million people only in the Americas. Polyamines are essential compounds for parasite growth, survival, and differentiation. However, because trypanosomatids are auxotrophic for polyamines, they must be obtained from the host by specific transporters. In this investigation, an ensemble of QSAR classifiers able to identify polyamine analogs with trypanocidal activity was developed. Then, a multi-template homology model of the dimeric polyamine transporter of T. cruzi, TcPAT12, was created with Rosetta, and then refined by enhanced sampling molecular dynamics simulations. Using representative snapshots extracted from the trajectory, a docking model able to discriminate between active and inactive compounds was developed and validated. Both models were applied in a parallel virtual screening campaign to repurpose known drugs as anti-trypanosomal compounds inhibiting polyamine transport in T. cruzi. Montelukast, Quinestrol, Danazol, and Dutasteride were selected for in vitro testing, and all of them inhibited putrescine uptake in biochemical assays, confirming the predictive ability of the computational models. Furthermore, all the confirmed hits proved to inhibit epimastigote proliferation, and Quinestrol and Danazol were able to inhibit, in the low micromolar range, the viability of trypomastigotes and the intracellular growth of amastigotes.

Broadening the spectrum of ivermectin: Its effect on Trypanosoma cruzi and related trypanosomatids.

Chagas disease is an endemic American parasitosis, caused by Trypanosoma cruzi. The current therapies, benznidazole (BZN) and nifurtimox (NFX), show limited efficacy and multiple side effects. Thus, there is a need to develop new trypanocidal strategies. Ivermectin (IVM) is a broad-spectrum antiparasitic drug with low human and veterinary toxicity with effects against T. brucei and Leishmania spp. Considering this and its relatively low cost, we evaluate IVM as a potential repurposed trypanocidal drug on T. cruzi and other trypanosomatids. We found that IVM affected, in a dose-dependent manner, the proliferation of T. cruzi epimastigotes as well as the amastigotes and trypomastigotes survival. The Selectivity Index for the amastigote stage with respect to Vero cells was 12. The IVM effect was also observed in Phytomonas jma 066 and Leishmania mexicana proliferation but not in Crithidia fasciculata. On the epimastigote stage, the IVM effect was trypanostatic at 50 µM but trypanocidal at 100 µM. The assays of the drug combinations of IVM with BNZ or NFX showed mainly additive effects among combinations. In silico studies showed that classical structures belonging to glutamate-gated Cl channels, the most common IVM target, are absent in kinetoplastids. However, we found in the studied trypanosomatid genomes one copy for putative IMPα and IMPß, potential targets for IVM. The putative IMPα genes (with 76% similarity) showed conserved Armadillo domains but lacked the canonical IMPß binding sequence. These results allowed us to propose a novel molecular target in T. cruzi and suggest IVM as a good candidate for drug repurposing in the Chagas disease context.

Cascade Ligand- and Structure-Based Virtual Screening to Identify New Trypanocidal Compounds Inhibiting Putrescine Uptake.

Chagas disease is a neglected tropical disease endemic to Latin America, though migratory movements have recently spread it to other regions. Here, we have applied a cascade virtual screening campaign combining ligand- and structure-based methods. In order to find novel inhibitors of putrescine uptake in Trypanosoma cruzi, an ensemble of linear ligand-based classifiers obtained by has been applied as initial screening filter, followed by docking into a homology model of the putrescine permease TcPAT12. 1,000 individual linear classifiers were inferred from a balanced dataset. Subsequently, different schemes were tested to combine the individual classifiers: MIN operator, average ranking, average score, average voting, with MIN operator leading to the best performance. The homology model was based on the arginine/agmatine antiporter (AdiC) from Escherichia coli as template. It showed 64% coverage of the entire query sequence and it was selected based on the normalized Discrete Optimized Protein Energy parameter and the GA341 score. The modeled structure had 96% in the allowed area of Ramachandran's plot, and none of the residues located in non-allowed regions were involved in the active site of the transporter. Positivity Predictive Value surfaces were applied to optimize the score thresholds to be used in the ligand-based virtual screening step: for that purpose Positivity Predictive Value was charted as a function of putative yields of active in the range 0.001-0.010 and the Se/Sp ratio. With a focus on drug repositioning opportunities, DrugBank and Sweetlead databases were subjected to screening. Among 8 hits, cinnarizine, a drug frequently prescribed for motion sickness and balance disorder, was tested against T. cruzi epimastigotes and amastigotes, confirming its trypanocidal effects and its inhibitory effects on putrescine uptake. Furthermore, clofazimine, an antibiotic with already proven trypanocidal effects, also displayed inhibitory effects on putrescine uptake. Two other hits, meclizine and butoconazole, also displayed trypanocidal effects (in the case of meclizine, against both epimastigotes and amastigotes), without inhibiting putrescine uptake.

The regulation of autophagy differentially affects Trypanosoma cruzi metacyclogenesis.

Autophagy is a cellular process required for the removal of aged organelles and cytosolic components through lysosomal degradation. All types of eukaryotic cells from yeasts to mammalian cells have the machinery to activate autophagy as a result of many physiological and pathological situations. The most frequent stimulus of autophagy is starvation and the result, in this case, is the fast generation of utilizable food (e.g. amino acids and basic nutrients) to maintain the vital biological processes. In some organisms, starvation also triggers other associated processes such as differentiation. The protozoan parasite Trypanosoma cruzi undergoes a series of differentiation processes throughout its complex life cycle. Although not all autophagic genes have been identified in the T. cruzi genome, previous works have demonstrated the presence of essential autophagic-related proteins. Under starvation conditions, TcAtg8, which is the parasite homolog of Atg8/LC3 in other organisms, is located in autophagosome-like vesicles. In this work, we have characterized the autophagic pathway during T. cruzi differentiation from the epimastigote to metacyclic trypomastigote form, a process called metacyclogenesis. We demonstrated that autophagy is stimulated during metacyclogenesis and that the induction of autophagy promotes this process. Moreover, with exception of bafilomycin, other classical autophagy modulators have similar effects on T. cruzi autophagy. We also showed that spermidine and related polyamines can positively regulate parasite autophagy and differentiation. We concluded that both polyamine metabolism and autophagy are key processes during T. cruzi metacyclogenesis that could be exploited as drug targets to avoid the parasite cycle progression.

Novel cruzipain inhibitors for the chemotherapy of chronic Chagas disease.

Despite current efforts worldwide to develop new medications against Chagas disease, only two drugs are available, nifurtimox and benznidazole. Both drugs require prolonged treatment and have multiple side effects and limited efficacy on adult patients chronically infected with Trypanosoma cruzi. Recently, computer-guided drug repositioning led to the discovery of the trypanocidal effects of clofazimine and benidipine. These compounds showed inhibitory effects on cruzipain, the major cysteine protease of T. cruzi, of different parasite stages and in a murine model of acute Chagas disease. The aim of this work was to determine the efficacy of these novel cruzipain inhibitors when administered in a murine model of chronic Chagas disease. Benidipine and clofazimine were able to reduce the parasite burden in cardiac and skeletal muscles of chronically infected mice compared with untreated mice as well as diminish the inflammatory process in these tissues. Further studies should be performed to study the synergism with benznidazole and nifurtimox in view of combined therapies.

Trypanosoma cruzi Polyamine Transporter: Its Role on Parasite Growth and Survival Under Stress Conditions.

Trypanosoma cruzi is the etiological agent of Chagas disease, a major health problem in Latin America. Polyamines are polycationic compounds that play a critical role as regulators of cell growth and differentiation. In contrast with other protozoa, T. cruzi is auxotrophic for polyamines because of its inability to synthesize putrescine due to the lack of both, arginine and ornithine decarboxylase; therefore, the intracellular availability of polyamines depends exclusively on transport processes. In this work, the polyamine transporter TcPAT12 was overexpressed in T. cruzi epimastigotes demonstrating that growth rates at different concentrations of polyamines strongly depend on the regulation of the polyamine transport. In addition, parasites overexpressing TcPAT12 showed a highly increased resistance to hydrogen peroxide and the trypanocidal drugs nifurtimox and benznidazole, which act by oxidative stress and interfering the synthesis of polyamine derivatives, respectively. Finally, the presence of putative polyamine transporters was analyzed in T. cruzi, Trypanosoma brucei, and Leishmania major genomes identifying 3-6 genes in these trypanosomatids.

Discovery of novel polyamine analogs with anti-protozoal activity by computer guided drug repositioning.

Chagas disease is a parasitic infection caused by the protozoa Trypanosoma cruzi that affects about 6 million people in Latin America. Despite its sanitary importance, there are currently only two drugs available for treatment: benznidazole and nifurtimox, both exhibiting serious adverse effects and limited efficacy in the chronic stage of the disease. Polyamines are ubiquitous to all living organisms where they participate in multiple basic functions such as biosynthesis of nucleic acids and proteins, proliferation and cell differentiation. T. cruzi is auxotroph for polyamines, which are taken up from the extracellular medium by efficient transporters and, to a large extent, incorporated into trypanothione (bis-glutathionylspermidine), the major redox cosubstrate of trypanosomatids. From a 268-compound database containing polyamine analogs with and without inhibitory effect on T. cruzi we have inferred classificatory models that were later applied in a virtual screening campaign to identify anti-trypanosomal compounds among drugs already used for other therapeutic indications (i.e. computer-guided drug repositioning) compiled in the DrugBank and Sweetlead databases. Five of the candidates identified with this strategy were evaluated in cellular models from different pathogenic trypanosomatids (T. cruzi wt, T. cruzi PAT12, T. brucei and Leishmania infantum), and in vitro models of aminoacid/polyamine transport assays and trypanothione synthetase inhibition assay. Triclabendazole, sertaconazole and paroxetine displayed inhibitory effects on the proliferation of T. cruzi (epimastigotes) and the uptake of putrescine by the parasite. They also interfered with the uptake of others aminoacids and the proliferation of infective T. brucei and L. infantum (promastigotes). Trypanothione synthetase was ruled out as molecular target for the anti-parasitic activity of these compounds.

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.

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.

High-throughput drug repositioning for the discovery of new treatments for Chagas disease.

Despite affecting around 8 million people worldwide and representing an economic burden above $7 billion/ year, currently approved medications to treat Chagas disease are still limited to two drugs, nifurtimox and benznidazole, which were developed more than 40 years ago and present important efficacy and safety limitations. Drug repositioning (i.e. finding second or further therapeutic indications for known drugs) has raised considerable interest within the international drug development community. There are many explanations to the current interest on drug repositioning including the possibility to partially circumvent clinical trials and the consequent saving in time and resources. It has been suggested as a particular attractive approach for the development of novel therapeutics for neglected diseases, which are usually driven by public or non-profit organizations. Here we review current computer-guided approaches to drug repositioning and reports on drug repositioning stories oriented to Chagas disease, with a focus on computer-guided drug repositioning campaigns.

Tissue transglutaminase on trophoblast cells as a possible target of autoantibodies contributing to pregnancy complications in celiac patients.

PROBLEM: Women with celiac disease (CD) are often affected by atypical presentations of the disease associated with reproductive disorders as a main extra-digestive complaint. Here, we analyzed if autoantibodies against tissue transglutaminase (tTG) in sera from CD patients with reproductive disorders could display direct effects through their interaction with tTG expressed on trophoblast cells and phagocytes inducing tissue damage and interfering in the clearance of trophoblast apoptotic bodies. METHOD OF STUDY: Sera from CD women with reproductive disorders were obtained, and their ability to induce apoptosis of Swan-71 (cytotrophoblast cell line) and to modulate the wound-healing and phagocytes process was tested. RESULTS: Swan-71 cells expressed tTG and CD sera displayed a significant decrease in trophoblast cell migration and a delay in injury healing on trophoblast cells, compared with those observed with control sera. Moreover, CD sera significantly reduced trophoblast cell proliferation and increased apoptosis levels in comparison with those observed in the control sera. Finally, autoantibodies against tTG interfere in the clearance of trophoblast apoptotic bodies through a mechanism involving MFG-E8 (milk fat globulin-EGF factor 8)-tTG binding. CONCLUSION: The anti-tTG antibodies might contribute to trophoblast damage and disrupt the phagocytosis process of apoptotic bodies that could promote a pro-inflammatory microenvironment.

Contribution of vasoactive intestinal peptide to immune homeostasis in trophoblast-maternal leukocyte interaction under LPS stimulation.

BACKGROUND/AIMS: The maternal-fetal interface is a unique immunological site that generates an adequate microenvironment during pregnancy, recognizing and eliminating infections and tolerating the trophoblast/placenta unit. For that purpose, trophoblast cells display several tolerogenic mechanisms to allow fetal survival, such as production of the neuropeptide vasoactive intestinal peptide (VIP). Here we investigated the contribution of VIP to maintain homeostasis at the maternal-placental interface under lipopolysaccharide (LPS) stimulation. METHODS: We performed cocultures between trophoblast cells (Swan-71 cell line) and maternal leukocytes obtained from fertile women as an in vitro model of maternal-placental interaction, and we focused on the effects of LPS on the modulation of VIP and their receptors (VPAC1 and VPAC2). RESULTS: VIP could prevent the upregulation of IL-6, MCP-1, and nitrite production and maintain the production of IL-10 and TGF-ß under LPS (10 µg/ml) stimulation after 48 h of coculture. To gain deeper insight into the mechanisms of how VIP could contribute to a tolerogenic microenvironment even in the presence of LPS, we investigated VIP production by maternal leukocytes and observed a significant increase in the frequency of CD4+VIP+ cells after interaction with Swan-71 cells in the presence of LPS. LPS increased VIP and inducible receptor VPAC2 expression directly on trophoblast cells in a dose- and time-dependent manner. CONCLUSIONS: The present results suggest that VIP might act as an additional homeostatic mechanism during early stages at the maternal-placental interface to control exacerbated inflammatory responses such as the ones observed in intrauterine infections.

Galectin-1 confers immune privilege to human trophoblast: implications in recurrent fetal loss.

Mechanisms accounting for the protection of the fetal semi-allograft from maternal immune cells remain incompletely understood. In previous studies, we showed that galectin-1 (Gal1), an immunoregulatory glycan-binding protein, hierarchically triggers a cascade of tolerogenic events at the mouse fetomaternal interface. Here, we show that Gal1 confers immune privilege to human trophoblast cells through the modulation of a number of regulatory mechanisms. Gal1 was mainly expressed in invasive extravillous trophoblast cells of human first trimester and term placenta in direct contact with maternal tissue. Expression of Gal1 by the human trophoblast cell line JEG-3 was primarily controlled by progesterone and pro-inflammatory cytokines and impaired T-cell responses by limiting T cell viability, suppressing the secretion of Th1-type cytokines and favoring the expansion of CD4(+)CD25(+)FoxP3(+) regulatory T (T(reg)) cells. Targeted inhibition of Gal1 expression through antibody (Ab)-mediated blockade, addition of the specific disaccharide lactose or retroviral-mediated siRNA strategies prevented these immunoregulatory effects. Consistent with a homeostatic role of endogenous Gal1, patients with recurrent pregnancy loss showed considerably lower levels of circulating Gal1 and had higher frequency of anti-Gal1 auto-Abs in their sera compared with fertile women. Thus, endogenous Gal1 confers immune privilege to human trophoblast cells by triggering a broad tolerogenic program with potential implications in threatened pregnancies.

Trophoblast cells induce a tolerogenic profile in dendritic cells.

BACKGROUND: Dendritic cells (DCs), which are biased toward a tolerogenic profile, play a pivotal role in tissue-remodeling processes and angiogenesis at the maternal-fetal interface. Here, we analyzed the effect of trophoblast cells on the functional profile of DCs to gain insight on the tolerogenic mechanisms underlying the human placental-maternal dialog at early stages of gestation. METHODS: DCs were differentiated from peripheral blood monocytes obtained from fertile women (n = 21), in the presence of interleukin (IL)-4 and granulocyte-macrophage colony-stimulating factor during 5 days in culture. Then, DCs were cultured with trophoblast cells (Swan-71 cell line obtained from normal cytotrophoblast, at 7 weeks) for 24 h and for an additional 24 h in the absence or presence of lipopolysaccharide (LPS) from Escherichia coli. DCs were recovered and used for flow cytometry, enzyme-linked immunosorbent assay, RT-PCR and suppression and migration assays. RESULTS: Trophoblast cells significantly prevented the increase in CD83 expression induced by LPS without affecting the expression of CD86, CD40 and human leukocyte antigen-DR (P < 0.05). Trophoblast cells significantly decreased the production of IL-12p70 and tumor necrosis factor-α, while it increased the production of IL-10 (P < 0.05). No changes were observed in the production of IL-6 and monocyte chemotactic protein-1. The culture of DCs with trophoblast cells, also suppressed the stimulation of the allogeneic response triggered by LPS (P < 0.05). Conditioned DCs were able to increase the frequency of CD4 + CD25 + Foxp3 cells and this effect was accompanied by an increase in indoleamine 2, 3-dioxygenase expression in DCs (P < 0.05). CONCLUSIONS: The interaction of DCs with trophoblast cells promotes the differentiation of DCs into cells with a predominantly tolerogenic profile that could contribute to a tolerogenic microenvironment at the maternal-fetal interface.

Modulation and recruitment of inducible regulatory T cells by first trimester trophoblast cells.

PROBLEM The specialized regulatory T-cells (Treg) population, essential for maternal tolerance of the fetus, performs its suppressive actions in the critical peri-implantation phase of pregnancy. In the present work, we investigated whether trophoblast cells are able to induce Treg recruitment, differentiation, and whether these mechanisms are modified by a bacterial or viral infection. METHOD OF STUDY Human T-regulatory cells were differentiated from naïve CD45RA(+) CCR7(+) cells obtained from peripheral blood mononuclear cells cultured with IL-2 and TGFß over 5 days. Induction of iTregs (CD4(+) Foxp3(+) cells) was evaluated using low serum conditioned media (LSCM), obtained from two first trimester trophoblast cell lines, Swan-71 and HTR8. Coculture experiments were carried out using transwell assays where trophoblast cells were in the absence or presence of PGN, LPS, or Poly [I:C]. Cytokine production was measured by multiplex analysis. RESULTS Trophoblast cells constitutively secrete high levels of TGFß and induced a significant increase of Foxp3 expression accompanied by a specific T-reg cytokine profile. Moreover, trophoblast cells were able to recruit iTregs in a specific manner. CONCLUSION We demonstrate that trophoblast cells have an active role on the recruitment and differentiation of iTregs, therefore, contributing to the process of immune regulation at the placental-maternal interface.

Modulation of maternal LIF producers T cells by trophoblast and paternal antigens.

PROBLEM: Fetal implantation enhances the production of essential growth factors such as LIF (leukaemia inhibitory factor), hence we investigated the contribution of maternal CD4 cells, activated by paternal or trophoblast antigens and its modulation by VIP (vasoactive intestinal peptide) and progesterone. METHOD OF STUDY: We performed co cultures of trophoblast cells (Swan-71 cell line) or paternal antigens and PBMCs from patients with recurrent spontaneous abortions (RSA) and fertile women. RESULT: Fertile-CD4(+) LIF(+) cells were increased by VIP and progesterone in response to paternal and trophoblast antigens. Also MMP-9 activity was decreased and pSTAT3/STAT3 ratio was increased. RSA patients have decreased levels of LIF expression which could not be modulated by VIP and progesterone and displayed a reduced number of endometrial infiltrated CD4(+) LIF(+) cells compared with fertile women. CONCLUSION: The decrease of CD4(+) LIF(+) cells in RSA patients could be related with the exacerbated inflammatory response observed in the maternal-fetal dialogue model.

Modulation of macrophage inflammatory profile in pregnant nonobese diabetic (NOD) mice.

During normal early pregnancy circulating monocytes are recruited to the maternal-placental interface where they differentiate to macrophages expressing different functional phenotypes for the maintenance of tissue homeostasis. Pregnancy in the nonobese diabetic (NOD) mouse model presents some pathological features in the pre-diabetic stage. The aim of this work was to analyze the functional profile of peritoneal macrophages faced with inflammatory and phagocytic stimuli in early pregnant pre-diabetic NOD mice and their modulation by vasoactive intestinal peptide (VIP). Pregnant NOD mouse macrophages showed no basal NFκB activation, lower IL-12 and nitrites production compared with the macrophages from non-pregnant NOD mice. Their pro-inflammatory aberrant response to LPS and apoptotic cell challenge was reduced and VIP inhibited macrophage residual deleterious responses to apoptotic cells. A functional phenotype switch in macrophages during pregnancy in NOD mice and a promoting effect of VIP towards this regulatory phenotype would be in line with the central role of macrophages in the maternal-placental dialogue.

Immunomodulatory effects of chemokines during the early implantation window.

Successful implantation requires a functionally normal embryo at the blastocyst stage and a receptive endometrium as well as adequate communication between them throughout the implantation process. This cross-talk is highly regulated by a number of different kinds of molecules. Particularly, chemokines, small polypeptides that attract specific leukocyte subsets by binding to cell-surface receptors, are also required to maintain immune-privileged sites as the feto-maternal interface. Chemokines expression involves an interdependent network with the absence of a single chemokine affecting the expression of multiple other chemokines, we have chosen to focus on just two representative examples: RANTES (regulated on normal T cell expressed and secreted) and MCP-1 (Monocyte chemo-attractant protein). Here, we present updated information on their expression levels and regulation on three different levels: 1) systemic effects on maternal allogeneic response; 2) local effects on endometrial cells; and 3) during an early stage of the feto-maternal dialogue. For each of the three levels, we analyzed data from both fertile women and patients having experienced recurrent spontaneous abortions as representative of physiological and pathological situations respectively.

VIP modulates the pro-inflammatory maternal response, inducing tolerance to trophoblast cells.

BACKGROUND AND PURPOSE: Successful embryo implantation is followed by a local pro-inflammatory and Th1 response, subsequently controlled by a Th2 response. Vasoactive intestinal peptide (VIP) has anti-inflammatory effects and promotes tolerogenic/Th2 responses while favouring embryonic development. We investigated the potential regulatory role of VIP on human trophoblast cells, maternal pro-inflammatory responses and trophoblast-maternal leukocyte interactions. EXPERIMENTAL APPROACH: We tested VIP effects directly on a trophoblast cell line (Swan 71 cells) and after co-culture with maternal peripheral blood mononuclear cells (PBMCs) as models of the feto-maternal dialogue. We also co-cultured maternal and paternal PBMCs to test effects of endogenous VIP on maternal alloresponses. KEY RESULTS: Swan 71 cells express VPAC(1) receptors and VIP induced their proliferation and the expression of leukaemia inhibitor factor, a pro-implantatory marker. After interaction with trophoblast cells, VIP increased Foxp3, the proportion of CD4+CD25+Foxp3+ cells within maternal PBMCs and transforming growth factor beta expression. Also, during the trophoblast-maternal PBMCs interaction, VIP reduced pro-inflammatory mediators [interleukin (IL)-6, monocyte chemoattractant protein 1, nitric oxide], while increasing IL-10. Trophoblast cells produced VIP which dose-dependently suppressed allomaternal responses, accompanied by reduced expression of the T cell transcription factor, T-bet. CONCLUSIONS AND IMPLICATIONS: Vasoactive intestinal peptide induced pro-implantatory markers and trophoblast cell proliferation, while controlling the initial pro-inflammatory response, by increasing maternal regulatory T cells and anti-inflammatory cytokines. As an autocrine regulatory peptide VIP might contribute to fetal survival through two mechanisms; a direct trophic effect on trophoblast cells and an immunomodulatory effect that favours tolerance to fetal antigens.

A potential tolerogenic immune mechanism in a trophoblast cell line through the activation of chemokine-induced T cell death and regulatory T cell modulation.

BACKGROUND: Successful implantation is followed by a local pro-inflammatory and Th1 response, subsequently controlled by Th2. Regulated upon activation, normal T cell expressed and secreted (RANTES) promotes a Th1 response and is implicated as a physiologic tolerogenic factor; therefore, we studied its potential role in the trophoblast-maternal leukocyte dialog. METHODS: We performed co-cultures of immortalized trophoblast cell line (Swan 71) and peripheral blood mononuclear cells (PBMCs) from fertile women (n = 23) or with recurrent spontaneous abortions (n = 18, RSA). After 24 and 48 h, supernatant and cells were analyzed by enzyme-linked immunosorbent assay, fluorescence-activated cell sorting, Western blot and apoptosis assay. To investigate the physiological effects at peripheral level, we co-cultured maternal and paternal PBMCs with conditioned media from Swan cells and progesterone. RESULTS: Following interaction of maternal PBMCs and trophoblast cells, RANTES production increased (P < 0.05) and was accompanied by low levels of interferon gamma, interleukin-12 p70 and high levels of tumor necrosis factor-alpha, nitrites and leukemia-inhibitory factor. RANTES production resulted in elevated apoptosis of potentially deleterious maternal CD3+ lymphocytes, accompanied by a decrease in the proliferative maternal response. During fetal-maternal dialog, the anti-RANTES antibody significantly reduced the frequency of CD4+CD25+Foxp3+ cells (P < 0.05) and was associated with trophoblast cell survival. However, co-cultures of Swan cells and RSA-PBMCs displayed a differential RANTES kinetics, lower levels of regulatory T cells (Tregs) and CD3+annexin-V+cells, accompanied by higher levels of apoptotic trophoblast cells. CONCLUSIONS: RANTES promotes an adequate pro-implantatory microenvironment that influences trophoblast cell survival and modulates the balance of maternal Treg/T effector lymphocytes in favor of maternal tolerance.