RESUMEN
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.
Asunto(s)
Trofoblastos , Infección por el Virus Zika , Virus Zika , Femenino , Humanos , Recién Nacido , Embarazo , Placenta/metabolismo , Primer Trimestre del Embarazo , Trofoblastos/metabolismo , Trofoblastos/virología , Replicación Viral , Células CultivadasRESUMEN
Normal placentation entails highly regulated interactions of maternal leukocytes with vascular and trophoblast cells to favor vascular transformation. Neutrophil activation and neutrophil extracellular trap (NET) formation associate with poor placentation and severe pregnancy complications. To deepen into the mechanisms of trophoblast-neutrophil interaction, we explored the effects of NETs on trophoblast cell function and, conversely, whether trophoblast cell-derived factors condition neutrophils to favor angiogenesis and anti-inflammatory signals required for fetal growth. NETs isolated from activated neutrophils hindered trophoblast cell migration. Trophoblast conditioned media prevented the effect as well as the vasoactive intestinal peptide (VIP) known to regulate trophoblast and neutrophil function. On the other hand, factors released by trophoblast cells and VIP shaped neutrophils to a proangiogenic profile with increased vascular endothelial growth factor synthesis and increased capacity to promote vascular transformation. Results presented here provide novel clues to reconstruct the interaction of trophoblast cells and neutrophils in vivo during placentation in humans.
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Autofagia/genética , Vasos Sanguíneos/crecimiento & desarrollo , Células Endoteliales/citología , Neovascularización Fisiológica/genética , Placentación/genética , Adulto , Vasos Sanguíneos/embriología , Movimiento Celular/genética , Implantación del Embrión/genética , Trampas Extracelulares/genética , Femenino , Humanos , Leucocitos/citología , Masculino , Neutrófilos/citología , Embarazo , Trofoblastos/citología , Péptido Intestinal Vasoactivo/farmacologíaRESUMEN
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.
Asunto(s)
Homeostasis/inmunología , Resultado del Embarazo , Trofoblastos/metabolismo , Péptido Intestinal Vasoactivo/metabolismo , Animales , Movimiento Celular , Femenino , Desarrollo Fetal , Masculino , Intercambio Materno-Fetal , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Placenta/metabolismo , Embarazo , Linfocitos T Reguladores/inmunología , Trofoblastos/citología , Péptido Intestinal Vasoactivo/genéticaRESUMEN
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.
Asunto(s)
Integrina alfaVbeta3/metabolismo , Trofoblastos/metabolismo , Apoptosis/efectos de los fármacos , Femenino , Humanos , Monocitos/metabolismo , Placenta/efectos de los fármacos , Placenta/metabolismo , Embarazo , Trofoblastos/efectos de los fármacos , Péptido Intestinal Vasoactivo/farmacologíaRESUMEN
PROBLEM: The decidualization process conditions monocytes to the immunosuppressive and tolerogenic dendritic cell (DC)-10 profile, a DC subset with high IL-10 production. Since the implantation process implies an embryo-endometrium-immune crosstalk, here we focused on the ability of embryonic soluble factors to modify decidual DC conditioning accordingly with its quality. METHOD OF STUDY: Human endometrial stromal cell line (HESC) decidualized with medroxyprogesterone and dibutyryl-cAMP (Dec) was stimulated with human embryo-conditioned media (ECM), classified as normal (ND) or impaired developed (ID) for 48 h (n = 18/group). Monocytes isolated from six healthy women were differentiated to DCs with rhGM-CSF+rhIL-4 in the presence/absence of conditioned media (CM) from decidualized cells stimulated with ECM or nontreated. RESULTS: We found that decidualized cells stimulated with ECM sustain a myeloid regulatory cell profile on monocyte-derived culture with increased frequency of CD1a-CD14+ and CD83+CD86low cells. ND-Dec sustained the higher expression of the DC-10 markers, HLA-G and IL-10 whereas ID-Dec diminished IL-10 production (ID-Dec: 135 ± 37.4 vs. Dec: 223.3 ± 49.9 pg/mL, p < 0.05). The treatment with ECM-Dec sustained a higher IL-10 production and prevented the increase of CD83/CD86 after LPS challenge regardless of embryo quality. Notably, TNF-α production increased in ID-Dec cultures (ID-Dec: 475.1 ± 134.7 vs. Dec: 347.5 ± 98 pg/mL, p < 0.05). CONCLUSIONS: Although remaining in a tolerogenic profile compatible with DC-10, DCs can differentially respond to decidual secreted factors based on embryo quality, changing their secretome. These results suggest that in the presence of arrested embryo, DCs could differentially shape the immunological microenvironment, contributing to arrested embryo clearance during the menstrual phase.
Asunto(s)
Decidua , Células Dendríticas , Implantación del Embrión , Tolerancia Inmunológica , Humanos , Femenino , Células Dendríticas/inmunología , Células Dendríticas/metabolismo , Células Dendríticas/efectos de los fármacos , Implantación del Embrión/inmunología , Decidua/inmunología , Decidua/citología , Diferenciación Celular , Medios de Cultivo Condicionados , Interleucina-10/metabolismo , Adulto , Células del Estroma/inmunología , Células del Estroma/metabolismo , Células Cultivadas , Embrión de Mamíferos , Endometrio/inmunología , Endometrio/citología , Línea Celular , Monocitos/inmunología , EmbarazoRESUMEN
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.
Asunto(s)
Envejecimiento Prematuro , Péptido Intestinal Vasoactivo , Embarazo , Femenino , Ratones , Animales , Ratones Noqueados , Resultado del Embarazo , Factores de Transcripción ForkheadRESUMEN
Neutrophils release web like-structures known as neutrophil extracellular traps (NETs) that ensnare and kill microorganisms. These networks are constituted of a DNA scaffold with associated antimicrobial proteins, which are released to the extracellular space as an effective mechanism to fight against invading microorganisms. In parallel with this beneficial role to avoid microbial dissemination and wall off infections, accumulating evidence supports that under certain circumstances, NETs can exert deleterious effects in inflammatory, autoimmune, and thrombotic pathologies. Research on NET properties and their role in pathophysiological processes is a rapidly evolving and expanding field. Here, we describe a combination of methods to achieve a successful in vitro NET visualization, semiquantification, and isolation.
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Separación Celular/métodos , ADN/análisis , Trampas Extracelulares/metabolismo , Procesamiento de Imagen Asistido por Computador/métodos , Microscopía Fluorescente/métodos , Elastasa Pancreática/análisis , Peroxidasa/metabolismo , Humanos , Técnicas In VitroRESUMEN
The growth factor erythropoietin (Epo) has shown neuronal protective action in addition to its well known proerythroid activity. Furthermore, Epo has dealt with cellular inflammation by inhibiting the expression of several proinflammatory cytokines, such as IL-1 and TNF-alpha. The action of TNF can have both apoptotic and antiapoptotic consequences due to altered balance between different cell signalling pathways. This work has focused on the apoptotic effects of this cytokine and the potential protective action of Epo. The model we used was neuroblastoma SH-SY5Y cells cultured in the presence of 25 ng/ml TNF-alpha or pretreated with 25 U/ml Epo for 12 h before the addition of TNF-alpha. Apoptosis was evaluated by differential cell count after Hoechst staining, analysis of DNA ladder pattern, and measurement of caspase activity. Despite its ability to induce NF-kappaB nuclear translocation, TNF-alpha induced cell death, which was found to be associated to upregulation of TNF Receptor 1 expression. On the other hand, cells activated by Epo became resistant to cell death. Prevention of death receptor upregulation and caspase activation may explain this antiapoptotic effect of Epo, which may be also favoured by the induction of a higher expression of protective factors, such as Bcl-2 and NF-kappaB, through mechanisms involving Jak/STAT and PI3K signalling pathways.
Asunto(s)
Apoptosis , Eritropoyetina/farmacología , Factor de Necrosis Tumoral alfa/farmacología , Línea Celular Tumoral , Citoplasma/metabolismo , Humanos , FN-kappa B/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Receptores Tipo I de Factores de Necrosis Tumoral/metabolismo , Proteínas Recombinantes , Factor de Transcripción STAT5/metabolismo , Transducción de Señal/fisiología , Regulación hacia ArribaRESUMEN
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.
Asunto(s)
Apoptosis/inmunología , Quimiocina CCL5/fisiología , Leucocitos Mononucleares/inmunología , Linfocitos T/inmunología , Trofoblastos/inmunología , Aborto Habitual/inmunología , Western Blotting , Línea Celular , Proliferación Celular , Quimiocina CCL5/metabolismo , Medios de Cultivo Condicionados , Implantación del Embrión/inmunología , Femenino , Citometría de Flujo , Factores de Transcripción Forkhead/metabolismo , Humanos , Leucocitos Mononucleares/fisiología , Intercambio Materno-Fetal/inmunología , Embarazo , Receptores CCR1/metabolismo , Receptores CCR5/metabolismo , Linfocitos T/citología , Linfocitos T/fisiología , Trofoblastos/metabolismo , Trofoblastos/fisiologíaRESUMEN
Among several factors known to modulate embryo implantation and survival, uterine quiescence and neovascularization, maternal immunotolerance through the Th1/Th2 cytokine balance towards a Th2 profile, local regulatory T-cell (Treg) activation, and high levels of progesterone were assigned a prominent role. Vasoactive intestinal peptide (VIP) is a neuroimmunopeptide that has anti-inflammatory effects, promotes Th2 cytokines and CD4(+)CD25(+)FOXP3(+) Treg activation, and stimulates exocrine secretion, smooth muscle relaxation, and vasodilatation favoring uterus quiescence. The goal of the present work was to explore the participation of VIP in the implantation sites of normal and pregnant prediabetic nonobese diabetic (NOD) females, a mouse strain that spontaneously develops an autoimmune exocrinopathy similar to Sjögren's syndrome. Our results indicate a reduction in litter size from the third parturition onwards in the NOD female lifespan with increased resorption rates. Progesterone systemic levels were significantly decreased in pregnant NOD mice compared with BALB/c mice, although the allogeneic response to progesterone by spleen cells was not impaired. VIP receptors, Vipr1 and Vipr2 (Vpac1 and Vpac2), were expressed at the implantation sites and VIP induced leukemia inhibitory factor (LIF) and Treg marker expression in both strains; however, a reduced Vip expression was found in NOD implantation sites. We conclude that the reduced birth rate at 16-week-old NOD mice with a Th1 systemic cytokine profile involves resorption processes with a lower expression of VIP at the sites of implantation, which acts as a local inducer of pro-implantatory LIF and Treg activation.
Asunto(s)
Implantación del Embrión/inmunología , Factores Inmunológicos/fisiología , Estado Prediabético , Péptido Intestinal Vasoactivo/fisiología , Animales , Diabetes Gestacional/genética , Diabetes Gestacional/inmunología , Diabetes Gestacional/metabolismo , Diabetes Gestacional/patología , Implantación del Embrión/efectos de los fármacos , Implantación del Embrión/genética , Pérdida del Embrión/genética , Pérdida del Embrión/inmunología , Pérdida del Embrión/metabolismo , Pérdida del Embrión/patología , Femenino , Factores Inmunológicos/farmacología , Tamaño de la Camada , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Endogámicos NOD , Estado Prediabético/genética , Estado Prediabético/metabolismo , Estado Prediabético/patología , Embarazo , Receptores de Tipo II del Péptido Intestinal Vasoactivo/genética , Receptores de Tipo II del Péptido Intestinal Vasoactivo/metabolismo , Receptores de Tipo II del Péptido Intestinal Vasoactivo/fisiología , Receptores de Tipo I del Polipéptido Intestinal Vasoactivo/genética , Receptores de Tipo I del Polipéptido Intestinal Vasoactivo/metabolismo , Linfocitos T Reguladores/inmunología , Linfocitos T Reguladores/metabolismo , Linfocitos T Reguladores/patología , Péptido Intestinal Vasoactivo/genética , Péptido Intestinal Vasoactivo/metabolismo , Péptido Intestinal Vasoactivo/farmacologíaRESUMEN
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.
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Comunicación Celular/fisiología , Homeostasis/inmunología , Linfocitos T Reguladores/inmunología , Trofoblastos/metabolismo , Péptido Intestinal Vasoactivo/metabolismo , Apoptosis/inmunología , Trampas Extracelulares/inmunología , Femenino , Humanos , Inflamación/inmunología , Macrófagos/inmunología , Monocitos/inmunología , Neutrófilos/inmunología , EmbarazoRESUMEN
Since apoptosis appeared to be related to neurodegenerative processes, neuroprotection has been involved in investigation of therapeutic approaches focused upon pharmacological agents to prevent neuronal programmed cell death. In this regard, erythropoietin (Epo) seems to play a critical role. The present work was focused on the study of the Epo protective effect upon human neuroblastoma SH-SY5Y cells subjected to differentiation by staurosporine. Under this condition, profuse neurite outgrowth was accompanied by programmed cell death (35% of apoptotic cells by Hoechst assay, showing characteristic DNA ladder pattern). A previous treatment with recombinant human Epo (rHuEpo) increased the expression of the specific receptor for Epo while prevented apoptosis. Simultaneously, morphological changes in neurite elongation and interconnection induced by staurosporine were blocked by Epo. These Epo effects proved to be associated to the induction of Bcl-xL at the mRNA and protein levels (RT-PCR and Western blot after immunoprecipitation) and were mediated by activation of pathways inhibited by wortmannin. In conclusion, the fact that both events induced by staurosporine, cell apoptosis and differentiation, were prevented in SH-SY5Y cells previously exposed to rHuEpo suggests interrelated signaling pathways triggered by the Epo/EpoR interaction.
Asunto(s)
Apoptosis/efectos de los fármacos , Diferenciación Celular/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Eritropoyetina/farmacología , Neuroblastoma/patología , Estaurosporina/farmacología , Western Blotting , Relación Dosis-Respuesta a Droga , Electroforesis , Eritropoyetina/genética , Humanos , Cinética , Microscopía Fluorescente , Neuroblastoma/metabolismo , Pruebas de Precipitina , ARN Mensajero/análisis , Receptores de Eritropoyetina/metabolismo , Proteínas Recombinantes , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Proteína bcl-X/metabolismoRESUMEN
The spontaneous non obese diabetic (NOD) mouse model of Sjögren's syndrome provides a valuable tool to study the onset and progression of both autoimmune response and secretory dysfunction. Vasoactive intestinal peptide (VIP) is a neuro and immunopeptide with prosecretory effect in salivary glands and anti-inflammatory actions in various models of autoimmune disease. Our purpose was to analyze the response of peritoneal macrophages to an inflammatory stimulus during the decline of salivary secretion in NOD mice and the potential anti-inflammatory effect of VIP. We present evidence of an increased nitric oxide production by peritoneal macrophages of NOD mice in basal and lipopolysaccharide (LPS)+IFN-gamma-stimulated conditions and a lower IL-10 response to LPS compared with normal BALB/c mice. VIP inhibited LPS-induced TNF-alpha, IL-12 and nitrites accumulation in NOD macrophages while it increased IL-10 production. VIP effect was prevented by an anti-IL-10 monoclonal antibody and it showed an additive effect on exogenously added IL-10 only in NOD mice. The inhibitory effect of VIP-induced IL-10 on nitrites was mediated by COX metabolites mostly in NOD cells as indomethacine inhibited both the increase in IL-10 and the reduction of nitrites exerted by VIP. We conclude that both PGE2 and VIP inhibit nitric oxide production and increase IL-10 induced by LPS in NOD macrophages and VIP effect is mediated through an increase of COX metabolites and IL-10.
Asunto(s)
Antiinflamatorios/farmacología , Macrófagos Peritoneales/efectos de los fármacos , Péptido Intestinal Vasoactivo/farmacología , Animales , Antiinflamatorios/inmunología , Dinoprostona/inmunología , Dinoprostona/farmacología , Femenino , Interferón gamma/inmunología , Interleucina-10/inmunología , Interleucina-12/inmunología , Lipopolisacáridos/inmunología , Macrófagos Peritoneales/inmunología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos NOD , Óxido Nítrico/inmunología , Nitritos/inmunología , Péptido Intestinal Vasoactivo/inmunologíaRESUMEN
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.
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Macrófagos/efectos de los fármacos , Macrófagos/fisiología , Fagocitosis/efectos de los fármacos , Péptido Intestinal Vasoactivo/farmacología , Animales , Microambiente Celular/efectos de los fármacos , Citocinas/biosíntesis , Implantación del Embrión/efectos de los fármacos , Implantación del Embrión/fisiología , Femenino , Masculino , Ratones , Ratones Endogámicos CBA , Ratones Endogámicos DBA , Miembro 3 del Grupo F de la Subfamilia 1 de Receptores Nucleares/metabolismo , Receptores de Péptido Intestinal Vasoactivo/genética , Receptores de Péptido Intestinal Vasoactivo/metabolismo , Péptido Intestinal Vasoactivo/administración & dosificaciónRESUMEN
Nitric oxide is an intracellular and diffusible messenger of neurotransmitters involved in salivary secretion, as well as an inflammatory mediator in salivary gland diseases. It is synthesized by three different isoforms of nitric oxide synthase (NOS), each subject to a fine transcriptional, post-transcriptional and/or post-translational regulation. Our purpose was to study the possible mechanisms leading to NOS downregulation in submandibular glands of normal mice and in the nonobese diabetic (NOD) mouse model of salivary dysfunction with lower NOS activity. NOS activity and cGMP accumulation were determined by radioassays in submandibular glands of both mice in the presence of the protein kinase inhibitors KN-93 and bisindolylmaleimide. NOS I mRNA and protein expression and localization were assessed by RT-PCR, Western blot and immunohistochemistry. A downregulatory effect of calcium-calmodulin kinase II (CaMK II) on NOS activity in submandibular glands of both NOD and BALB/c mice was observed. Our results are consistent with a physiological regulation of NOS activity by this kinase but not by PKC in normal BALB/c mice. They are also supportive of a role for CaMK II in the lack of detectable NOS activity in submandibular glands of NOD mice. KN-93 also restored cGMP accumulation in NOD submandibular glands. The downregulation of NOS in NOD mice seems to be mainly mediated by this kinase rather than the result of a lower expression or different cellular localization of the enzyme. It was not related to different substrate or cofactors availability either.
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Proteínas Quinasas Dependientes de Calcio-Calmodulina/antagonistas & inhibidores , Óxido Nítrico/biosíntesis , Inhibidores de Proteínas Quinasas/farmacología , Enfermedades de las Glándulas Salivales/enzimología , Glándula Submandibular/metabolismo , Animales , Proteínas Quinasas Dependientes de Calcio-Calmodulina/fisiología , Modelos Animales de Enfermedad , Femenino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos NOD , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiología , Glándula Submandibular/efectos de los fármacosRESUMEN
The non-obese diabetic (NOD) mouse is chosen among other experimental models to study autoimmune sialadenitis resembling Sjögren's syndrome (SS), because of its unique characteristic of developing salivary dysfunction. Based on the deep loss of nitric oxide synthase (NOS) activity in parotid glands of NOD mice observed from early stages of disease and the inhibitory effect of nitric oxide (NO) donors on amylase secretion in normal salivary glands, our goal was to investigate whether parotid glands from NOD mice lacking NOS activity presented this regulatory mechanism of amylase secretion. We found that parotid glands from NOD mice lack nitric oxide-mediated regulation of amylase secretion in response to VIP stimulation. The lack of regulation might be assigned to the loss of NOS activity as derived from the results with NOS inhibitors and increasing concentrations of VIP. These functional differences observed in NOD vs. BALB/c parotid glands occur in the absence of immune infiltrates in exocrine tissue, and it is not related to cAMP accumulation. NO-mediated regulation of amylase secretion was not observed in BALB/c submandibular glands to the same extent as described in parotid glands and was absent in submandibular glands of NOD mice.
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Amilasas/metabolismo , Fármacos Gastrointestinales/farmacología , Óxido Nítrico/fisiología , Glándula Parótida/metabolismo , Péptido Intestinal Vasoactivo/farmacología , Animales , AMP Cíclico/metabolismo , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos NOD , Óxido Nítrico Sintasa/metabolismo , Glándula Parótida/enzimologíaRESUMEN
Autoimmunity is a feature of celiac disease (CD) with tissue transglutaminase (tTG) as a major autoantigen. A correlation between gynecological-obstetric disorders in CD patients and the presence of circulating antibodies anti-tTG that inhibited tTG activity was reported. Serum anti-tTG antibodies were detected in a non-obese diabetic (NOD) mouse model of type I insulin-dependent diabetes mellitus and Sjögren's syndrome, two comorbid states with CD. Since pregnancy complications have been described in NOD mice, we evaluated the ability of anti-tTG antibodies to affect the functions of tTG relevant to the normal course of an early pregnancy like extracellular matrix assembling and apoptotic cell phagocytosis by macrophages. Circulating IgG antibodies against tTG were detected in NOD mice with titers that decreased at early pregnancy; interestingly, the in vitro transamidating activity of tTG was reduced by NOD serum samples. Particularly, anti-tTG antibody inhibited apoptotic cell phagocytosis by peritoneal macrophages from pregnant NOD mice that express the enzyme on surface. Evidence provided support for a role for anti-tTG antibodies through reduced transamidating activity and reduced apoptotic cell clearance by the macrophages of pregnant NOD mice.
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Apoptosis/inmunología , Enfermedad Celíaca/inmunología , Proteínas de Unión al GTP/inmunología , Macrófagos/inmunología , Fagocitosis/inmunología , Transglutaminasas/inmunología , Animales , Anticuerpos/sangre , Anticuerpos/inmunología , Autoantígenos/inmunología , Diabetes Mellitus Tipo 1/sangre , Diabetes Mellitus Tipo 1/inmunología , Femenino , Inmunoglobulina G/sangre , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos NOD , Embarazo , Resultado del Embarazo , Proteína Glutamina Gamma Glutamiltransferasa 2 , Síndrome de Sjögren/sangre , Síndrome de Sjögren/inmunología , Útero/inmunologíaRESUMEN
Macrophages at the maternal-placental interface coordinate opposite demands under the control of trophoblast cells such as the response against pathogens on one hand, and apoptotic cell clearance and wound healing with the production of suppressor cytokines. Here, we investigated whether trophoblast cells induce maternal monocyte activation towards an alternative activated macrophage profile and whether bacterial or viral stimuli modulate their migratory properties. We used an in vitro model of the maternal-placental interface represented by co-cultures of CD14+ cells isolated from fertile women with first trimester trophoblast cell line (Swan-71 cells) in the presence or absence of pathogen associated molecular pattern (PAMP) stimuli lipopolysaccharide (LPS), peptidoglycan (PGN) or poly [I:C]). Maternal CD14+ cells showed increased CD16 and CD39 expression, both markers associated to an alternative activation profile, with no changes in CD80 expression after trophoblast cell interaction. These changes were accompanied by increased IL-10 and decreased IL-12 production by CD14+ cells. After stimulation with LPS, PGN or poly [I:C], monocytes co-cultured with trophoblast cells had lower production of TNF-α and IL-1ß compared with non co-cultured monocytes. Interestingly, monocyte migration towards trophoblast cells was prevented in the presence of LPS or PGN but not after 24h of stimulation with poly [I:C]. LPS or PGN also decreased CCR5, CXCL-8 and CCL5 expression. Finally, trophoblast cells co-cultured with monocytes in the presence of pathological stimuli failed to increase chemokine expression, indicating a bidirectional effect. In conclusion, trophoblast might 'instruct' maternal monocytes to express an alternative activation profile and restrain their early recruitment under pathological threats as one of the first strategies to avoid potential tissue damage at the maternal-placental interface.
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Lipopolisacáridos/farmacología , Monocitos/efectos de los fármacos , Peptidoglicano/farmacología , Poli I-C/farmacología , Trofoblastos/efectos de los fármacos , Antígenos CD/genética , Antígenos CD/metabolismo , Apirasa/genética , Apirasa/metabolismo , Comunicación Celular/efectos de los fármacos , Movimiento Celular/efectos de los fármacos , Quimiocina CCL5/genética , Quimiocina CCL5/metabolismo , Técnicas de Cocultivo , Femenino , Proteínas Ligadas a GPI/genética , Proteínas Ligadas a GPI/metabolismo , Expresión Génica/efectos de los fármacos , Humanos , Interleucina-1beta/genética , Interleucina-1beta/metabolismo , Interleucina-8/genética , Interleucina-8/metabolismo , Receptores de Lipopolisacáridos/genética , Receptores de Lipopolisacáridos/metabolismo , Monocitos/citología , Monocitos/metabolismo , Receptores CCR5/genética , Receptores CCR5/metabolismo , Receptores de IgG/genética , Receptores de IgG/metabolismo , Trofoblastos/citología , Trofoblastos/metabolismo , Factor de Necrosis Tumoral alfa/genética , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
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.
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Diferenciación Celular/inmunología , Movimiento Celular/inmunología , Tolerancia Inmunológica , Leucocitos Mononucleares/inmunología , Linfocitos T Reguladores/inmunología , Trofoblastos/inmunología , Antígenos CD4/genética , Antígenos CD4/inmunología , Diferenciación Celular/efectos de los fármacos , Línea Celular , Movimiento Celular/efectos de los fármacos , Técnicas de Cocultivo , Medios de Cultivo Condicionados/farmacología , Cámaras de Difusión de Cultivos , Femenino , Citometría de Flujo , Factores de Transcripción Forkhead/genética , Factores de Transcripción Forkhead/inmunología , Humanos , Interleucina-2/inmunología , Interleucina-2/farmacología , Leucocitos Mononucleares/citología , Leucocitos Mononucleares/efectos de los fármacos , Leucocitos Mononucleares/metabolismo , Embarazo , Primer Trimestre del Embarazo , Linfocitos T Reguladores/citología , Linfocitos T Reguladores/efectos de los fármacos , Linfocitos T Reguladores/metabolismo , Factor de Crecimiento Transformador beta/inmunología , Factor de Crecimiento Transformador beta/farmacología , Trofoblastos/citología , Trofoblastos/efectos de los fármacos , Trofoblastos/metabolismoRESUMEN
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.