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Immunometabolism is an emerging growing field that focuses on the role of cellular metabolism in the regulation of immune cell function and fate. Thus, proliferation, differentiation, activation, and function of immune cell populations are modulated by reprogramming their fueling and metabolic pathways. Pregnancy entails a fine immune and metabolic regulation of the maternal-fetal interaction to assist the energetic demands of the fetus where trophoblast cells have a central role. Maternal neutrophil functional shaping by trophoblast cells has been proposed though their metabolic conditioning during pregnancy has not been studied yet. Here, we explored the effects of trophoblast-derived factors on the metabolic rewiring of neutrophils from nonpregnant women and its impact on central functions like reactive oxygen species (ROS) production, neutrophil extracellular trap (NET) release, and migration. In parallel, the immunometabolic status and function of neutrophils isolated from pregnant women (16-20 weeks) was compared with nonpregnant age-matched control samples. Trophoblast-derived factors induced glucose uptake and lipid droplet accumulation without activating ROS production or NET release. Conditioned media from trophoblast cells also inhibited PMA-induced NETosis partly by impairing glucose uptake in neutrophils. In turn, neutrophils from pregnant women had increased basal ROS production, lipid accumulation, and glucose uptake compared to neutrophils from nonpregnant women, accompanied by a higher release of PMA-induced NETs. Interestingly, PMA-induced NETs was blocked by a fatty acid oxidation inhibitor in neutrophils from pregnant women indicating the contribution of fatty acid metabolism to neutrophil activity during pregnancy. Results are consistent with immunometabolic mechanisms underlying the functional shaping of neutrophils during pregnancy and point out the contribution of trophoblast-derived factors to their metabolic profiling. These findings provide novel immunometabolic clues to understand immune homeostasis maintenance during pregnancy and raise the clinical potential of monitoring neutrophil metabolism during normal and complicated pregnancies., (© 2024 Wiley Periodicals LLC.)

Decidualization denotes the process of inflammatory reprogramming of endometrial stromal cells (EnSC) into specialized decidual cells (DC). During this process, EnSC are subjected to endoplasmic reticulum (ER) stress as well as acute cellular senescence. Both processes contribute to the proinflammatory mid-luteal implantation window and their dysregulation has been implicated in reproductive failure. Here, we evaluated the link between ER stress, decidual differentiation and senescence. In-silico analysis identified HSPA5 gene, codifying the ER chaperone BiP, as a potentially critical regulator of cell fate divergence of decidualizing EnSC into anti-inflammatory DC and pro-inflammatory senescent decidual cells (snDC). Knockdown of HSPA5 in primary EnSC resulted both in decreased expression of DC marker genes and attenuated induction of senescence associated β-galactosidase activity, a marker of snDC. Stalling of the decidual reaction upon HSPA5 knockdown was apparent at 8 days of differentiation and was preceded by the upregulation of ER stress associated proteins IRE1α and PERK. Further, HSPA5 knockdown impaired colony-forming unit activity of primary EnSC, indicative of loss of cellular plasticity. Together, our results point to a key role for HSPA5/BiP in decidual transformation of EnSCs and highlight the importance of constraining ER stress levels during this process., (© 2024. The Author(s).)

Background: Placental macrophages, Hofbauer cells (HBC) are the only fetal immune cell population within the stroma of healthy placenta along pregnancy. They are central players in maintaining immune tolerance during pregnancy. Immunometabolism emerged a few years ago as a new field that integrates cellular metabolism with immune responses, however, the immunometabolism of HBC has not been explored yet. Here we studied the sex-specific differences in the phenotypic, functional and immunometabolic profile of HBC., Methods: HBC were isolated from human term placentas (N = 31, 16 from male and 15 female neonates). Ex vivo assays were carried out to assess active metabolic and endoplasmic reticulum stress pathways by flow cytometry, confocal microscopy, gene expression and in silico approaches., Results: HBC from female placentas displayed a stronger M2 phenotype accompanied by high rates of efferocytosis majorly sustained on lipid metabolism. On the other hand, male HBC expressed a weaker M2 phenotype with higher glycolytic metabolism. LPS stimulation reinforced the glycolytic metabolism in male but not in female HBC. Physiological endoplasmic reticulum stress activates IRE-1 differently, since its pharmacological inhibition increased lipid mobilization, accumulation and efferocytosis only in female HBC. Moreover, differential sex-associated pathways accompanying the phenotypic and functional profiles of HBC appeared related to the placental villi environment., Conclusions: These results support sex-associated effects on the immunometabolism of the HBC and adds another layer of complexity to the intricate maternal-fetal immune interaction., (© 2024. The Author(s).)

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 + CD86 low 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., (© 2024 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

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., (© 2023 Wiley Periodicals LLC.)

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-1β. 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 TGFβ/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., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022. Published by Elsevier B.V.)

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.

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., (© 2019 Wiley Periodicals, Inc.)

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.

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., (© 2018 New York Academy of Sciences.)

During decidualization, endometrial stromal cells undergo reticular stress (RS) and unfolded protein response (UPR), allowing the endoplasmic reticulum-expansion and immunomodulators production. Physiological RS generates the activation of sensing proteins, inflammasome activation and mature-IL-1β secretion, associated with pro-implantatory effects. We focus on the impact of RS and UPR on decidualized cells and whether they induce a physiological sterile inflammatory response through IL-1β production. Human endometrial stromal cell line (HESC) after decidualization treatment with MPA + dibutyryl-cAMP (Dec) increased the expression of RS-sensors (ATF6, PERK and IRE1α) and UPR markers (sXBP1 and CHOP) in comparison with Non-dec cells. Then we found increased NLRP3 expression in Dec cells compared with Non-dec cells. In fact STF-083010 (an IRE1α inhibitor) prevented this increase. Downstream, increased levels of active caspase-1 on Dec cells were detected by FAM-Flica Caspase-1 associated with an increase in IL-1β production. Moreover, the treatment with STF-083010 decreased the invasion index observed in Dec cells, evaluated by an in vitro model of implantation. In endometrial biopsies from recurrent spontaneous abortion patients an increased expression of IRE1α was found in comparison with fertile women; while recurrent implantation failure samples showed a lower expression of sXBP1, TXNIP and NLRP3 than fertile women, suggesting that RS/UPR tenors might condition endometrial receptivity.

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-β 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.

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 αvβ3 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 αvβ3 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 αvβ3 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., (© The Author 2015. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

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., (Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.)

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 TGFβ 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 TGFβ 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., (© 2011 John Wiley & Sons A/S.)

Sjögren's syndrome (SS) is a chronic autoimmune disease characterized by a progressive oral and ocular dryness that correlates poorly with the autoimmune damage of the glands. It has been proposed that a loss of homeostatic equilibrium in the glands is partly responsible for salivary dysfunction with acinar cells involved actively in the pathogenesis of SS. The non-obese diabetic (NOD) mouse model of Sjögren's syndrome develops secretory dysfunction and early loss of glandular homeostatic mechanisms, with mild infiltration of the glands. Based on the vasodilator, prosecretory and trophic effects of the vasoactive intestinal peptide (VIP) on acini as well as its anti-inflammatory properties we hypothesized that the local expression of VIP/vasoactive intestinal peptide receptor (VPAC) system in salivary glands could have a role in acinar cell apoptosis and macrophage function thus influencing gland homeostasis. Here we show a progressive decline of VIP expression in submandibular glands of NOD mice with no changes in VPAC receptor expression compared with normal mice. The deep loss of endogenous VIP was associated with a loss of acinar cells through apoptotic mechanisms that could be induced further by tumour necrosis factor (TNF)-α and reversed by VIP through a cyclic adenosine-5'-monophosphate (cAMP)/protein kinase A (PKA)-mediated pathway. The clearance of apoptotic acinar cells by macrophages was impaired for NOD macrophages but a shift from inflammatory to regulatory phenotype was induced in macrophages during phagocytosis of apoptotic acinar cells. These results support that the decline in endogenous VIP/VPAC local levels might influence the survival/apoptosis intracellular set point in NOD acinar cells and their clearance, thus contributing to gland homeostasis loss., (© 2011 The Authors. Clinical and Experimental Immunology © 2011 British Society for Immunology.)

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., (Copyright © 2010 Elsevier Ireland Ltd. All rights reserved.)

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.

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.

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.

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.

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.

A functional interaction between progesterone, Th2 cytokines and a suitable balance between nitric oxide and prostaglandins in the uterus is considered to have a major role in the success of embryo implantation and pregnancy. Non-obese diabetic (NOD) mice offer a suitable model to study the modulatory role of Th1 cytokines on uterus signalling and function, since at the prediabetic stage they develop a spontaneous Th1 autoimmune response against exocrine glands similar to Sjögren's syndrome. Vasoactive intestinal peptide (VIP) is a vasoactive neuro- and immunopeptide that promotes Th2 profiles and contributes to the smooth muscle relaxation and vasodilation. The aim of the present study was to investigate the activities of nitric oxide synthase and cyclo-oxygenase and the effect of VIP in the uterus of NOD mice with an emerging Th1 cytokine response. We present evidence of a reduced basal and VIP-stimulated activity of both enzymes in the uterus of NOD mice compared with normal BALB/c mice in proestrus. An altered functional interaction between both enzymes is also present in NOD mice at the time when increased levels of serum interleukin (IL)-12 and tumour necrosis factor-alpha but not interferon (IFN)-gamma or IL-10 were detected. We conclude that signalling alterations in uteri of NOD mice are simultaneous to the onset of a systemic Th1 cytokine response.

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.

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.

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.

Muscarinic receptors play an important role in secretory and vasodilator responses in rat salivary glands. Nitric oxide synthase (NOS) appears to be one of the multiple effectors coupled to muscarinic receptors in both submandibular and sublingual glands although some differences have been found depending on the gland studied. First, submandibular glands had a lower basal activity of nitric oxide synthase than sublingual glands and the concentration-response curve for carbachol was bell-shaped in the former but not in sublingual glands. Second, cGMP levels displayed a similar profile to that observed for NOS activity in both glands. Third, protein kinase C also coupled to muscarinic receptor activation in the glands might have a regulatory effect on nitric oxide production since its activity was higher in basal conditions in submandibular than sublingual glands and it also increased in the presence of the agonist at a concentration that inhibited NOS activity in submandibular glands. The effects appear to be partly related to the expression of a minor population of M(1) receptors in submandibular glands absent in sublingual as determined in binding and signaling experiments with the muscarinic receptor antagonist pirenzepine.

Addition of recombinant rat interferon-gamma (IFN-gamma) to beating rat atria decreased the contractile strength in a dose-dependent manner. The effect was specific of IFN-gamma since it was abrogated by monoclonal anti-rat IFN-gamma. It required the activation of the cholinergic system of the heart as inhibition of both nicotinic (10(-7) M hexametonium) and muscarinic cholinoceptors (10(-7) M atropine) prevented the reaction. Hemicholinium (2 x 10(-5) M) and tetrodotoxin (5 x 10(-7) M) also reduced the response. Likewise, IFN-gamma potentiated the action of the muscarinic agonist carbachol. IFN-gamma simulated the biological effect of cholinergic agonists because: (a) it increased cGMP formation; (b) it decreased cAMP formation; and (c) it reduced heart contractility at doses that can be considered physiologic. IFN-gamma also modified the muscarinic receptor by interfering with the binding of the radiolabelled antagonist quinuclidinyl benzilate [( 3H]QNB). It is suggested that IFN-gamma binding to IFN-gamma receptors in the heart may lead to a cholinergic response by interaction of both receptor systems on the surface of atrial cells.

The presence of alpha-adrenergic receptors (absent in normal lymphocytes) has been demonstrated in transformed human lymphocytes of the Raji cell line. Binding properties of beta-adrenergic receptors were similar to those reported for normal lymphocytes. A single population of alpha 2-adrenergic receptors was characterized in intact Raji lymphoblasts by binding and saturation assays with the alpha 2-adrenergic antagonist yohimbine. Competition curves with [3H]yohimbine indicate the presence of typical alpha 2-adrenoceptors. Reaction of Raji with the alpha 2-adrenergic agonist clonidine (10(-6) M) stimulated their growth rate. In contrast, the alpha 1-adrenergic agonist methoxamine (10(-6) M) had no effect. Previous work indicates that Raji can actively produce thromboxanes (TX) and that these decreased atrium contractility. In agreement with these results and with the binding studies, it is now shown that clonidine stimulation enhanced the negative inotropic effects of Raji on isolated rat atria. This reaction was prevented by incubation of Raji with yohimbine (10(-6) M) but not with the alpha 1-adrenergic antagonist prazosin (10(-6) M) or the beta-adrenergic antagonist propranolol (10(-7) M). The biologic effect of Raji on rat atria was probably due to production of cyclooxygenase metabolites of arachidonic acid, because it was blocked by preincubation of the cells with the cyclooxygenase inhibitors indomethacin (10(-6) M) and aspirin (10(-4) M) or the thromboxane synthetase inhibitors nictindol (10(-5) M) and imidazole (10(-4) M).(ABSTRACT TRUNCATED AT 250 WORDS)