RESUMEN
Regulatory T (Treg) cells are essential for immune tolerance of embryo implantation and insufficient Treg cells are implicated in early pregnancy loss. An abortion-prone mouse model was utilized to evaluate the utility of IL-2 complexed with JES6-1 anti-IL-2 antibody (IL-2/JES6-1) to boost uterine Treg cells and improve reproductive success. IL-2/JES6-1 but not IL-2/IgG control administered in the peri-conception phase to CBA/J females mated with DBA/2 males elicited a >2-fold increase in the proportion of CD4+ T cells expressing FOXP3, and an increase in the ratio of FOXP3+ Treg cells to FOXP3- T conventional cells, in the uterus and its draining lymph nodes at embryo implantation that was sustained into mid-gestation. An attenuated phenotype was evident in both thymic-derived and peripheral Treg cells with elevated CTLA4, CD25, and FOXP3 indicating improved suppressive function, as well as increased proliferative marker Ki67. IL-2/JES6-1 treatment reduced fetal loss from 31% to 10%, but this was accompanied by a 6% reduction in late gestation fetal weight, despite comparable placental size and architecture. Similar effects of IL-2/JES6-1 on Treg cells and fetal growth were seen in CBA/J females with healthy pregnancies sired by BALB/c males. These findings show that expanding the uterine Treg cell pool through targeting IL-2 signaling is a strategy worthy of further investigation for mitigating immune-mediated fetal loss.
RESUMEN
Pregnancy depends on a state of maternal immune tolerance mediated by CD4+ regulatory T (Treg) cells. Uterine Treg cells release anti-inflammatory factors, inhibit effector immunity, and support adaptation of the uterine vasculature to facilitate placental development. Insufficient Treg cells or inadequate functional competence is implicated in infertility and recurrent miscarriage, as well as pregnancy complications preeclampsia, fetal growth restriction, and preterm birth, which stem from placental insufficiency. In this review we address an emerging area of interest in pregnancy immunology-the significance of metabolic status in regulating the Treg cell expansion required for maternal-fetal tolerance. We describe how hyperglycemia and insulin resistance affect T cell responses to suppress generation of Treg cells, summarize data that implicate a role for altered glucose metabolism in impaired maternal-fetal tolerance, and explore the prospect of targeting dysregulated metabolism to rebalance the adaptive immune response in women experiencing reproductive disorders.
Asunto(s)
Placenta , Nacimiento Prematuro , Femenino , Glucosa/metabolismo , Humanos , Tolerancia Inmunológica , Recién Nacido , Embarazo , Nacimiento Prematuro/metabolismo , Linfocitos T ReguladoresRESUMEN
Spontaneous preterm labor is frequently caused by an inflammatory response in the gestational tissues elicited by either infectious or sterile agents. In sterile preterm labor, the key regulators of inflammation are not identified, but platelet-activating factor (PAF) is implicated as a potential rate-limiting effector agent. Since Toll-like receptor (TLR)-4 can amplify PAF signaling, we evaluated whether TLR4 contributes to inflammation and fetal loss in a mouse model of PAF-induced sterile preterm labor, and whether a small-molecule TLR4 inhibitor, (+)-naltrexone, can mitigate adverse PAF-induced effects. The administration of carbamyl (c)-PAF caused preterm labor and fetal loss in wild-type mice but not in TLR4-deficient mice. Treatment with (+)-naltrexone prevented preterm delivery and alleviated fetal demise in utero elicited after cPAF administered by i.p. or intrauterine routes. Pups born after cPAF and (+)-naltrexone treatment exhibited comparable rates of postnatal survival and growth to carrier-treated controls. (+)-Naltrexone suppressed the cPAF-induced expression of inflammatory cytokine genes Il1b, Il6, and Il10 in the decidua; Il6, Il12b, and Il10 in the myometrium; and Il1b and Il6 in the placenta. These data demonstrate that the TLR4 antagonist (+)-naltrexone inhibits the inflammatory cascade induced by cPAF, preventing preterm birth and perinatal death. The inhibition of TLR4 signaling warrants further investigation as a candidate strategy for fetal protection and delay of preterm birth elicited by sterile stimuli.
Asunto(s)
Naltrexona/farmacología , Antagonistas de Narcóticos/farmacología , Trabajo de Parto Prematuro/metabolismo , Factor de Activación Plaquetaria/metabolismo , Receptor Toll-Like 4/metabolismo , Animales , Femenino , Ratones , Ratones Endogámicos BALB C , EmbarazoRESUMEN
BACKGROUND: The Oncotype DX 21-gene Recurrence Score is predictive of adjuvant chemotherapy benefit for women with early-stage, estrogen receptor (ER)-positive, HER2-negative breast cancer. In premenopausal women, fluctuations in estrogen and progesterone during the menstrual cycle impact gene expression in hormone-responsive cancers. However, the extent to which menstrual cycling affects the Oncotype DX 21-gene signature remains unclear. Here, we investigate the impact of ovarian cycle stage on the 21-gene signature using a naturally cycling mouse model of breast cancer. METHODS: ER-positive mammary tumours were dissected from naturally cycling Mmtv-Pymt mice at either the estrus or diestrus phase of the ovarian cycle. The Oncotype DX 21-gene signature was assessed through quantitative real time-PCR, and a 21-gene experimental recurrence score analogous to the Oncotype DX Recurrence Score was calculated. RESULTS: Tumours collected at diestrus exhibited significant differences in expression of 6 Oncotype DX signature genes (Ki67, Ccnb1, Esr1, Erbb2, Grb7, Bag1; p ≤ 0.05) and a significant increase in 21-gene recurrence score (21.8 ± 2.4; mean ± SEM) compared to tumours dissected at estrus (15.5 ± 1.9; p = 0.03). Clustering analysis revealed a subgroup of tumours collected at diestrus characterised by increased expression of proliferation- (p < 0.001) and invasion-group (p = 0.01) genes, and increased 21-gene recurrence score (p = 0.01). No correlation between ER, PR, HER2, and KI67 protein abundance measured by Western blot and abundance of mRNA for the corresponding gene was observed, suggesting that gene expression is more susceptible to hormone-induced fluctuation compared to protein expression. CONCLUSIONS: Ovarian cycle stage at the time of tissue collection critically affects the 21-gene signature in Mmtv-Pymt murine mammary tumours. Further studies are required to determine whether Oncotype DX Recurrence Scores in women are similarly affected by menstrual cycle stage.
Asunto(s)
Perfilación de la Expresión Génica/métodos , Genómica/métodos , Ciclo Menstrual/genética , Animales , Femenino , Neoplasias Mamarias Animales , Ratones , Ratones Transgénicos , Recurrencia Local de NeoplasiaRESUMEN
Regulatory T cells (Tregs) are essential for maternal tolerance in allogeneic pregnancy. In preeclampsia, Tregs are fewer and display aberrant phenotypes, particularly in the thymic Treg (tTreg) compartment, potentially because of insufficient priming to male partner alloantigens before conception. To investigate how tTregs as well as peripheral Tregs (pTregs) respond to male partner seminal fluid, Foxp3+CD4+ Tregs were examined in the uterus and uterus-draining lymph nodes in virgin estrus mice and 3.5 d postcoitum. Mating elicited 5-fold increases in uterine Tregs accompanied by extensive Treg proliferation in the uterus-draining lymph nodes, comprising 70% neuropilin 1+ tTregs and 30% neuropilin 1- pTregs. Proliferation marker Ki67 and suppressive competence markers Foxp3 and CTLA4 were induced after mating in both subsets, and Ki67, CTLA4, CD25, and GITR were higher in tTregs than in pTregs. Analysis by t-stochastic neighbor embedding confirmed phenotypically distinct tTreg and pTreg clusters, with the proportion of tTregs but not pTregs among CD4+ T cells expanding in response to seminal fluid. Bisulphite sequencing revealed increased demethylation of the Treg-specific demethylation region in the Foxp3 locus in tTregs but not pTregs after mating. These data show that tTregs and pTregs with distinct phenotypes both respond to seminal fluid priming, but the Foxp3 epigenetic signature is uniquely increased in tTregs. We conclude that reproductive tract tTregs as well as pTregs are sensitive to local regulation by seminal fluid, providing a candidate mechanism warranting evaluation for the potential to influence preeclampsia susceptibility in women.
Asunto(s)
Factores de Transcripción Forkhead/metabolismo , Semen/inmunología , Conducta Sexual Animal , Linfocitos T Reguladores/inmunología , Útero/inmunología , Animales , Antígeno CTLA-4/metabolismo , Proliferación Celular/fisiología , Epigénesis Genética , Femenino , Factores de Transcripción Forkhead/genética , Proteína Relacionada con TNFR Inducida por Glucocorticoide/metabolismo , Subunidad alfa del Receptor de Interleucina-2/metabolismo , Ganglios Linfáticos/citología , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Neuropilina-1/metabolismo , Preeclampsia/inmunología , Preeclampsia/patología , Embarazo , Timo/citología , Útero/citologíaRESUMEN
Corticosteroids have been utilised in the assisted reproduction setting with the expectation of suppressing aberrant immune activation and improving fertility in women. However, the effects of corticosteroids on fertility, and on pregnancy and offspring outcomes, are unclear. In this study, mice were administered prednisolone (1 mg/kg) or PBS daily in the pre-implantation phase, and effects on the adaptive immune response, the implantation rate, fetal development and postnatal outcomes were investigated. Prednisolone disrupted the expected expansion of CD4+ T cells in early pregnancy, inhibiting generation of both regulatory T cells (Treg cells) and effector T cells and suppressing IFNG required for T cell functional competence. Prednisolone caused an 8-20% increase in the embryo implantation rate and increased the number of viable pups per litter. In late gestation, fetal and placental weights were reduced in a litter size-dependent manner, and the canonical inverse relationship between litter size and fetal weight was lost. The duration of pregnancy was extended by ~ 0.5 day and birth weight was reduced by ~ 5% after prednisolone treatment. Viability of prednisolone-exposed offspring was comparable to controls, but body weight was altered in adulthood, particularly in male offspring. Thus, while prednisolone given in the pre-implantation phase in mice increases maternal receptivity to implantation and resource investment in fetal growth, there is a trade-off in long-term consequences for fetal development, birth weight and offspring health. These effects are associated with, and likely caused by, prednisolone suppression of the adaptive immune response at the outset of pregnancy.
Asunto(s)
Desarrollo Fetal/efectos de los fármacos , Placentación/efectos de los fármacos , Prednisolona/farmacología , Efectos Tardíos de la Exposición Prenatal , Linfocitos T/efectos de los fármacos , Animales , Diferenciación Celular/efectos de los fármacos , Femenino , Feto/efectos de los fármacos , Feto/inmunología , Edad Gestacional , Linfopoyesis/efectos de los fármacos , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Endogámicos CBA , Embarazo , Efectos Tardíos de la Exposición Prenatal/inducido químicamente , Efectos Tardíos de la Exposición Prenatal/inmunología , Efectos Tardíos de la Exposición Prenatal/fisiopatología , Linfocitos T/fisiologíaRESUMEN
Central to pregnancy success is a state of T cell tolerance to paternal antigens, which is initiated at conception. The role and regulation of specific phenotypes of CD8+ T cells in mediating pregnancy tolerance is not clear. This study aimed to investigate the impact on pregnancy outcome of altering the cytokine environment during maternal CD8+ T cell priming in early pregnancy. Transgenic Act-mOVA male mice were mated to C57BL/6 (B6) females to generate fetuses expressing ovalbumin (OVA) as a model paternal antigen. OVA-reactive CD8+ OT-I T cells were activated in vitro with OVA in the presence of either transforming growth factor-ß1 (TGFB1) plus interleukin-10 (IL10), or IL2, to mimic normal or dysregulated uterine conditions, respectively, and transferred into pregnant mice on gestational day 3.5. OT-I T cells activated with TGFB1 and IL10, like naive OT-I T cells, did not alter embryo implantation or fetal viability. In contrast, OT-I T cells activated with IL2 caused extensive fetal loss manifesting in mid-gestation. IL2-activated OT-I T cells expressed less FOXP3 and higher interferon-γ (IFNG) than cells activated with TGFB1 and IL10. Fetal loss did not occur in females mated with B6 males, demonstrating the antigen specificity of fetal loss, and was not abrogated by maternal genetic C1q deficiency indicating a mechanism independent of antibody-mediated cytotoxicity. These data indicate that alternative phenotypes generated in maternal CD8+ T cells at the time of priming with paternal antigens can impact pregnancy outcome, such that inappropriate activation of CD8+ T cells before implantation is capable of causing antigen-specific fetal loss later in pregnancy.
Asunto(s)
Aborto Espontáneo/inmunología , Linfocitos T CD8-positivos/inmunología , Implantación del Embrión/inmunología , Animales , Células Cultivadas , Complemento C1q/genética , Femenino , Tolerancia Inmunológica , Interleucina-2/inmunología , Activación de Linfocitos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Ovalbúmina/genética , Fenotipo , Embarazo , Especificidad del Receptor de Antígeno de Linfocitos TRESUMEN
There is ongoing interest in immune-suppressant corticosteroid drugs such as prednisolone to treat infertility in women with repeated IVF failure and recurrent miscarriage. The rationale draws on the pervasive but flawed view that immune activation is inconsistent with normal pregnancy. This ignores clear evidence that controlled inflammation and activation of the immune response is essential for embryo implantation. Generally, the immune response actively promotes reproductive success - by facilitating endometrial receptivity and tolerance of the foreign embryo, and promoting vascular adaptation to support placental morphogenesis. The peri-conception immune response also establishes developmental trajectories that can impact on fetal growth and gestational age at birth. Here, we describe immune changes accompanying conception that could be impeded by inappropriate corticosteroid administration. While women with specific clinical conditions may benefit from the anti-inflammatory and immune-deviating actions of prednisolone and related drugs, it is incorrect to assume a 'one-size-fits-all' approach. Better diagnostics and more preclinical studies are essential to define patient groups, build evidence for efficacy and fine-tune treatments so as not to inhibit essential actions of immune cells. We argue that unless overt immune pathology is evident, utilization of corticosteroids is not warranted and may be harmful. In most women, perturbing immune adaptation at implantation is expected to adversely influence placental development and impair immune-mediated quality control mechanisms, potentially elevating risk of altered fetal growth and developmental programming, congenital anomalies and preterm birth.
Asunto(s)
Aborto Habitual/inmunología , Endometrio/inmunología , Inmunosupresores/uso terapéutico , Prednisolona/uso terapéutico , Técnicas Reproductivas Asistidas , Implantación del Embrión/inmunología , Femenino , Humanos , EmbarazoRESUMEN
Sphingosine 1-phosphate (S1P) is a bioactive lipid that can function both extracellularly and intracellularly to mediate a variety of cellular processes. Using lipid affinity matrices and a radiolabeled lipid binding assay, we reveal that S1P directly interacts with the transcription factor peroxisome proliferator-activated receptor (PPAR)γ. Herein, we show that S1P treatment of human endothelial cells (ECs) activated a luciferase-tagged PPARγ-specific gene reporter by â¼12-fold, independent of the S1P receptors. More specifically, in silico docking, gene reporter, and binding assays revealed that His323 of the PPARγ ligand binding domain is important for binding to S1P. PPARγ functions when associated with coregulatory proteins, and herein we identify that peroxisome proliferator-activated receptor-γ coactivator 1 (PGC1)ß binds to PPARγ in ECs and their progenitors (nonadherent endothelial forming cells) and that the formation of this PPARγ:PGC1ß complex is increased in response to S1P. ECs treated with S1P selectively regulated known PPARγ target genes with PGC1ß and plasminogen-activated inhibitor-1 being increased, no change to adipocyte fatty acid binding protein 2 and suppression of CD36. S1P-induced in vitro tube formation was significantly attenuated in the presence of the PPARγ antagonist GW9662, and in vivo application of GW9662 also reduced vascular development in Matrigel plugs. Interestingly, activation of PPARγ by the synthetic ligand troglitazone also reduced tube formation in vitro and in vivo. To support this, Sphk1(-/-)Sphk2(+/-) mice, with low circulating S1P levels, demonstrated a similar reduction in vascular development. Taken together, our data reveal that the transcription factor, PPARγ, is a bona fide intracellular target for S1P and thus suggest that the S1P:PPARγ:PGC1ß complex may be a useful target to manipulate neovascularization.
Asunto(s)
Células Endoteliales de la Vena Umbilical Humana/metabolismo , Lisofosfolípidos/metabolismo , Neovascularización Fisiológica/fisiología , PPAR gamma/metabolismo , Receptores de Lisoesfingolípidos/metabolismo , Esfingosina/análogos & derivados , Animales , Antígenos CD36/genética , Antígenos CD36/metabolismo , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Células HEK293 , Células Endoteliales de la Vena Umbilical Humana/citología , Humanos , Lisofosfolípidos/genética , Ratones , Ratones Noqueados , PPAR gamma/genética , Coactivador 1-alfa del Receptor Activado por Proliferadores de Peroxisomas gamma , Fosfotransferasas (Aceptor de Grupo Alcohol)/genética , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Inhibidor 1 de Activador Plasminogénico/genética , Inhibidor 1 de Activador Plasminogénico/metabolismo , Proteínas de Unión al ARN , Receptores de Lisoesfingolípidos/genética , Serpina E2/genética , Serpina E2/metabolismo , Esfingosina/genética , Esfingosina/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Células U937RESUMEN
Macrophages are abundant in the uterine stroma and are intimately juxtaposed with other cell lineages comprising the uterine epithelial and stromal compartments. We postulated that macrophages may participate in mediating or amplifying the effects of ovarian steroid hormones to facilitate the uterine remodeling that is a characteristic feature of every estrus cycle and is essential for pregnancy. Using the Cd11b-Dtr transgenic mouse model with an ovariectomy and hormone replacement strategy, we depleted macrophages to determine their role in hormone-driven proliferation of uterine epithelial and stromal cells and uterine vascular development. Following diphtheria toxin (DT) administration, approximately 85% of EMR1-positive (EMR1âº) macrophages, as well as 70% of CD11C⺠dendritic cells, were depleted from Cd11b-Dtr mice. There was no change in bromodeoxyuridine incorporation into epithelial cells induced to proliferate by administration of 17beta-estradiol (E2) to ovariectomized mice or into stromal cells induced to proliferate in response to E2 and progesterone (P4), and the resulting sizes and structures of the luminal epithelial and stromal cell compartments were not altered compared with those of leukocyte replete controls. Depletion of CD11B⺠myeloid cells failed to alter the density or pattern of distribution of uterine blood vessels, as identified by staining PECAM1-positive endothelial cells in the uterine stroma of E2- or E2 combined with P4 (E2P4)-treated ovariectomized mice. These experiments support the interpretation that macrophages are dispensable to regulation of proliferative events induced by steroid hormones in the cycling and early pregnant mouse uterus to establish the epithelial, stromal, and vascular architecture which is critical for normal reproductive competence.
Asunto(s)
Endometrio/inmunología , Estradiol/metabolismo , Ciclo Estral , Macrófagos/inmunología , Progesterona/metabolismo , Útero/inmunología , Remodelación Vascular , Animales , Biomarcadores/metabolismo , Proliferación Celular , Toxina Diftérica , Endometrio/irrigación sanguínea , Endometrio/citología , Endometrio/metabolismo , Endotelio Vascular/citología , Endotelio Vascular/inmunología , Endotelio Vascular/metabolismo , Femenino , Terapia de Reemplazo de Hormonas , Inmunohistoquímica , Terapia de Inmunosupresión , Macrófagos/citología , Macrófagos/metabolismo , Ratones Transgénicos , Ovariectomía , Organismos Libres de Patógenos Específicos , Útero/irrigación sanguínea , Útero/citología , Útero/metabolismoRESUMEN
Regulatory T (Treg) cell defects are implicated in disorders of embryo implantation and placental development, but the origins of Treg cell dysfunction are unknown. Here, we comprehensively analyzed the phenotypes and transcriptional profile of peripheral blood Treg cells in individuals with early pregnancy failure (EPF). Compared to fertile subjects, EPF subjects had 32% fewer total Treg cells and 54% fewer CD45RA+CCR7+ naive Treg cells among CD4+ T cells, an altered Treg cell phenotype with reduced transcription factor FOXP3 and suppressive marker CTLA4 expression, and lower Treg:Th1 and Treg:Th17 ratios. RNA sequencing demonstrated an aberrant gene expression profile, with upregulation of pro-inflammatory genes including CSF2, IL4, IL17A, IL21, and IFNG in EPF Treg cells. In silico analysis revealed 25% of the Treg cell dysregulated genes are targets of FOXP3. We conclude that EPF is associated with systemic Treg cell defects arising due to disrupted FOXP3 transcriptional control and loss of lineage fidelity.
RESUMEN
Each ovarian cycle, the mammary gland epithelium rotates through a sequence of hormonally regulated cell proliferation, differentiation and apoptosis. These studies investigate the role of macrophages in this cellular turnover. Macrophage populations and their spatial distribution were found to fluctuate across the cycle. The number of macrophages was highest at diestrus, and the greatest number of macrophages in direct contact with epithelial cells occurred at proestrus. The physiological necessity of macrophages in mammary gland morphogenesis during the estrous cycle was demonstrated in Cd11b-Dtr transgenic mice. Ovariectomised mice were treated with estradiol and progesterone to stimulate alveolar development, and with the progesterone receptor antagonist mifepristone to induce regression of the newly formed alveolar buds. Macrophage depletion during alveolar development resulted in a reduction in both ductal epithelial cell proliferation and the number of alveolar buds. Macrophage depletion during alveolar regression resulted in an increased number of branch points and an accumulation of TUNEL-positive cells. These studies show that macrophages have two roles in the cellular turnover of epithelial cells in the cycling mammary gland; following ovulation, they promote the development of alveolar buds in preparation for possible pregnancy, and they remodel the tissue back to its basic architecture in preparation for a new estrous cycle.
Asunto(s)
Epitelio/metabolismo , Ciclo Estral/fisiología , Macrófagos/metabolismo , Glándulas Mamarias Animales/citología , Glándulas Mamarias Animales/metabolismo , Animales , Epitelio/efectos de los fármacos , Estradiol/sangre , Estradiol/farmacología , Estrógenos/farmacología , Ciclo Estral/efectos de los fármacos , Femenino , Citometría de Flujo , Antagonistas de Hormonas/farmacología , Inmunohistoquímica , Etiquetado Corte-Fin in Situ , Glándulas Mamarias Animales/efectos de los fármacos , Ratones , Ratones Endogámicos C57BL , Mifepristona/farmacología , Progesterona/sangre , Progesterona/farmacología , Progestinas/farmacología , Receptores de Progesterona/antagonistas & inhibidoresRESUMEN
Progesterone (P4) is essential for embryo implantation, but the extent to which the pro-gestational effects of P4 depend on the maternal immune compartment is unknown. Here, we investigate whether regulatory T cells (Treg cells) act to mediate luteal phase P4 effects on uterine receptivity in mice. P4 antagonist RU486 administered to mice on days 0.5 and 2.5 postcoitum to model luteal phase P4 deficiency caused fewer CD4+Foxp3+ Treg cells and impaired Treg functional competence, along with dysfunctional uterine vascular remodeling and perturbed placental development in midgestation. These effects were linked with fetal loss and fetal growth restriction, accompanied by a Th1/CD8-skewed T cell profile. Adoptive transfer at implantation of Treg cells - but not conventional T cells - alleviated fetal loss and fetal growth restriction by mitigating adverse effects of reduced P4 signaling on uterine blood vessel remodeling and placental structure and by restoring maternal T cell imbalance. These findings demonstrate an essential role for Treg cells in mediating P4 effects at implantation and indicate that Treg cells are a sensitive and critical effector mechanism through which P4 drives uterine receptivity to support robust placental development and fetal growth.
Asunto(s)
Progesterona , Linfocitos T Reguladores , Humanos , Embarazo , Femenino , Animales , Ratones , Progesterona/farmacología , Placenta , Retardo del Crecimiento Fetal , Implantación del Embrión/fisiología , Desarrollo FetalRESUMEN
Uterine dendritic cells (DCs) are critical for activating the T cell response mediating maternal immune tolerance of the semiallogeneic fetus. GM-CSF (CSF2), a known regulator of DCs, is synthesized by uterine epithelial cells during induction of tolerance in early pregnancy. To investigate the role of GM-CSF in regulating uterine DCs and macrophages, Csf2-null mutant and wild-type mice were evaluated at estrus, and in the periconceptual and peri-implantation periods. Immunohistochemistry showed no effect of GM-CSF deficiency on numbers of uterine CD11c(+) cells and F4/80(+) macrophages at estrus or on days 0.5 and 3.5 postcoitum, but MHC class II(+) and class A scavenger receptor(+) cells were fewer. Flow cytometry revealed reduced CD80 and CD86 expression by uterine CD11c(+) cells and reduced MHC class II in both CD11c(+) and F4/80(+) cells from GM-CSF-deficient mice. CD80 and CD86 were induced in Csf2(-/-) uterine CD11c(+) cells by culture with GM-CSF. Substantially reduced ability to activate both CD4(+) and CD8(+) T cells in vivo was evident after delivery of OVA Ag by mating with Act-mOVA males or transcervical administration of OVA peptides. This study shows that GM-CSF regulates the efficiency with which uterine DCs and macrophages activate T cells, and it is essential for optimal MHC class II- and class I-mediated indirect presentation of reproductive Ags. Insufficient GM-CSF may impair generation of T cell-mediated immune tolerance at the outset of pregnancy and may contribute to the altered DC profile and dysregulated T cell tolerance evident in infertility, miscarriage, and preeclampsia.
Asunto(s)
Células Dendríticas/inmunología , Células Dendríticas/metabolismo , Factor Estimulante de Colonias de Granulocitos y Macrófagos/fisiología , Activación de Linfocitos/inmunología , Proteínas Gestacionales/fisiología , Subgrupos de Linfocitos T/inmunología , Útero/inmunología , Útero/metabolismo , Secuencia de Aminoácidos , Animales , Células Presentadoras de Antígenos/inmunología , Células Presentadoras de Antígenos/metabolismo , Endometrio/embriología , Endometrio/inmunología , Endometrio/metabolismo , Epítopos de Linfocito T/inmunología , Femenino , Factor Estimulante de Colonias de Granulocitos y Macrófagos/deficiencia , Factor Estimulante de Colonias de Granulocitos y Macrófagos/genética , Antígenos de Histocompatibilidad Clase I/metabolismo , Antígenos de Histocompatibilidad Clase II/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Datos de Secuencia Molecular , Especificidad de Órganos/inmunología , Embarazo , Proteínas Gestacionales/deficiencia , Proteínas Gestacionales/genética , Subgrupos de Linfocitos T/metabolismo , Útero/embriologíaRESUMEN
Immune cells are essential for endometrial receptivity to embryo implantation and early placental development. They exert tissue-remodeling and immune regulatory roles-acting to promote epithelial attachment competence, regulate the differentiation of decidual cells, remodel the uterine vasculature, control and resolve inflammatory activation, and suppress destructive immunity to paternally inherited alloantigens. From a biological perspective, the endometrial immune response exerts a form of "quality control"-it promotes implantation success when conditions are favorable but constrains receptivity when physiological circumstances are not ideal. Women with recurrent implantation failure and recurrent miscarriage may exhibit altered numbers or disturbed function of certain uterine immune cell populations-most notably uterine natural killer cells and regulatory T cells. Preclinical and animal studies indicate that deficiencies or aberrant activation states in these cells can be causal in the pathophysiological mechanisms of infertility. Immune cells are, therefore, targets for diagnostic evaluation and therapeutic intervention. However, current diagnostic tests are overly simplistic and have limited clinical utility. To be more informative, they need to account for the full complexity and reflect the range of perturbations that can occur in uterine immune cell phenotypes and networks. Moreover, safe and effective interventions to modulate these cells are in their infancy, and personalized approaches matched to specific diagnostic criteria will be needed. Here we summarize current biological understanding and identify knowledge gaps to be resolved before the promise of therapies to target the uterine immune response can be fully realized.
Asunto(s)
Aborto Habitual , Placenta , Aborto Habitual/diagnóstico , Animales , Implantación del Embrión/fisiología , Endometrio/fisiología , Femenino , Humanos , Embarazo , ÚteroRESUMEN
Regulatory T (Treg) cells are a specialized CD4+ T cell subpopulation that are essential for immune homeostasis, immune tolerance, and protection against autoimmunity. There is evidence that sex-steroid hormones estrogen and progesterone modulate Treg cell abundance and phenotype in women. Since natural oscillations in these hormones are modified by hormonal contraceptives, we examined whether oral contraception (OC) use impacts Treg cells and related T cell populations. T cells were analyzed by multiparameter flow cytometry in peripheral blood collected across the menstrual cycle from healthy women either using OC or without hormonal contraception and from age-matched men. Compared to naturally cycling women, women using OC had fewer Treg cells and an altered Treg cell phenotype. Notably, Treg cells exhibiting a strongly suppressive phenotype, defined by high FOXP3, CD25, Helios, HLADR, CTLA4, and Ki67, comprised a lower proportion of total Treg cells, particularly in the early- and mid-cycle phases. The changes were moderate compared to more substantial differences in Treg cells between women and men, wherein women had fewer Treg cells-especially of the effector memory Treg cell subset-associated with more T helper type 1 (Th1) cells and CD8+ T cells and lower Treg:Th1 cell and Treg:CD8+ T cell ratios than men. These findings imply that OC can modulate the number and phenotype of peripheral blood Treg cells and raise the possibility that Treg cells contribute to the physiological changes and altered disease susceptibility linked with OC use.
Asunto(s)
Factores de Transcripción Forkhead , Linfocitos T Reguladores , Anticoncepción , Femenino , Factores de Transcripción Forkhead/metabolismo , Hormonas/metabolismo , Humanos , Fenotipo , Linfocitos T Reguladores/metabolismoRESUMEN
Regulatory T (Treg) cells facilitate maternal immune tolerance of the semiallogeneic conceptus in early pregnancy, but the origin and regulation of these cells at embryo implantation is unclear. During the preimplantation period, factors in the seminal fluid delivered at coitus cause expansion of a CD4(+)CD25(+) putative Treg cell population in the para-aortic lymph nodes draining the uterus. Using flow cytometry, immunohistochemistry, and real-time quantitative PCR (qPCR) for the signature Treg cell transcription factor FOXP3, we confirmed the identity of the expanded lymph node population as FOXP3(+) Treg cells and showed that this is accompanied by a comparable increase in the uterus of FOXP3(+) Treg cells and expression of Foxp3 mRNA by Day 3.5 postcoitum. Seminal plasma was necessary for uterine Treg cell accumulation, as mating with seminal vesicle-deficient males failed to elicit an increase in uterine Treg cells. Furthermore seminal fluid induced expression of mRNA encoding the Treg chemokine CCL19 (MIP3beta), which acts through the CCR7 receptor to regulate Treg cell recruitment and retention in peripheral tissues. Glandular and luminal epithelial cells were identified as the major cellular origins of uterine CCL19, and exposure to both seminal plasma and sperm was required for maximum expression. Together, these results indicate that Treg cells accumulate in the uterus prior to embryo implantation and that seminal fluid is a key regulator of the uterine Treg cell population, operating by both increasing the pool of available Treg cells and promoting their CCL19-mediated recruitment from the circulation into the implantation site.
Asunto(s)
Quimiocina CCL19/metabolismo , Factores de Transcripción Forkhead/metabolismo , Regulación de la Expresión Génica/fisiología , Semen/metabolismo , Linfocitos T Reguladores/metabolismo , Útero/citología , Animales , Quimiocina CCL19/genética , Femenino , Factores de Transcripción Forkhead/genética , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Útero/fisiologíaRESUMEN
Alcohol-induced proinflammatory central immune signaling has been implicated in the chronic neurotoxic actions of alcohol, although little work has examined if these non-neuronal actions contribute to the acute behavioral responses elicited by alcohol administration. The present study examined if acute alcohol-induced sedation (loss of righting reflex, sleep time test) and motor impairment (rotarod test) were influenced by acute alcohol-induced microglial-dependent central immune signaling. Inhibition of acute alcohol-induced central immune signaling, through the reduction of proinflammatory microglial activation with minocycline, or by blocking interleukin-1 (IL-1) receptor signaling using IL-1 receptor antagonist (IL-1ra), reduced acute alcohol-induced sedation in mice. Mice treated with IL-1ra recovered faster from acute alcohol-induced motor impairment than control animals. However, minocycline led to greater motor impairment induced by alcohol, implicating different mechanisms in alcohol-induced sedation and motor impairment. At a cellular level, IκBα protein levels in mixed hippocampal cells responded rapidly to alcohol in a time-dependent manner, and both minocycline and IL-1ra attenuated the elevated levels of IκBα protein by alcohol. Collectively these data suggest that alcohol is capable of rapid modification of proinflammatory immune signaling in the brain and this contributes significantly to the pharmacology of alcohol.
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Etanol/farmacología , Interleucina-1/metabolismo , Microglía/efectos de los fármacos , Actividad Motora/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Análisis de Varianza , Animales , Conducta Animal/efectos de los fármacos , Conducta Animal/fisiología , Western Blotting , Células Cultivadas , Relación Dosis-Respuesta a Droga , Hipocampo/efectos de los fármacos , Hipocampo/metabolismo , Masculino , Ratones , Ratones Endogámicos BALB C , Microglía/metabolismo , Minociclina/farmacología , Actividad Motora/fisiología , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Fosforilación/efectos de los fármacos , Fosforilación/fisiología , Receptores Tipo I de Interleucina-1/antagonistas & inhibidores , Receptores Tipo I de Interleucina-1/metabolismo , Reflejo de Enderezamiento/efectos de los fármacos , Reflejo de Enderezamiento/fisiología , Prueba de Desempeño de Rotación con Aceleración Constante , Transducción de Señal/fisiología , Sueño/efectos de los fármacos , Sueño/fisiologíaRESUMEN
The events that generate T cell-mediated immune tolerance in early pregnancy are ill-defined. To investigate the significance of seminal fluid Ags in activating maternal T cells, and define the underlying Ag presentation pathways, OVA-specific T cells were adoptively transferred to female mice inseminated by males ubiquitously expressing membrane-bound OVA. OVA-reactive CD8(+) OT-I and CD4(+) OT-II T cells transferred to mated recipients expressed activation markers CD25 and CD69 and proliferated vigorously in the para-aortic lymph nodes, but not in distal lymph nodes or spleen, and OT-I T cells expressed IFN-gamma and IL-2. In contrast, OT-I T cells transferred later in pregnancy or up to 10 days postpartum expressed CD25 and CD69 and proliferated in all peripheral lymphoid tissues examined. OVA Ag was present predominantly in the plasma fraction of seminal fluid, and seminal plasma, but not sperm, was necessary for T cell proliferation. Female H-2K(b) bone marrow-derived cells expressing TAP were essential for OT-I T cell proliferation, but responses were not elicited by OVA Ag presented by paternal MHC in seminal fluid or associated with placental cells. This study shows that at conception, seminal fluid drives activation and expansion of paternal Ag-reactive CD4(+) and CD8(+) T cell populations, and female APCs have an essential role in cross-presenting Ag to CD8(+) T cells via a TAP-dependent pathway. Delivery of paternal Ags and immune-deviating cytokines by seminal fluid at conception may activate Ag-dependent CD4(+) and CD8(+) regulatory T cells mediating tolerance of pregnancy.
Asunto(s)
Antígenos/inmunología , Reactividad Cruzada/inmunología , Inmunidad Innata/inmunología , Activación de Linfocitos/inmunología , Embarazo/inmunología , Semen/inmunología , Linfocitos T/inmunología , Animales , Células Presentadoras de Antígenos/citología , Células Presentadoras de Antígenos/inmunología , Médula Ósea/inmunología , Diferenciación Celular/inmunología , Proliferación Celular , Células Cultivadas , Citocinas/inmunología , Femenino , Cinética , Masculino , Ratones , Ratones Noqueados , Conducta Sexual AnimalRESUMEN
Maternal immune adaptation to accommodate pregnancy depends on sufficient availability of regulatory T (Treg) cells to enable embryo implantation. Toll-like receptor 4 is implicated as a key upstream driver of a controlled inflammatory response, elicited by signals in male partner seminal fluid, to initiate expansion of the maternal Treg cell pool after mating. Here, we report that mice with null mutation in Tlr4 (Tlr4-/-) exhibit impaired reproductive outcomes after allogeneic mating, with reduced pregnancy rate, elevated mid-gestation fetal loss, and fetal growth restriction, compared to Tlr4+/+ wild-type controls. To investigate the effects of TLR4 deficiency on early events of maternal immune adaptation, TLR4-regulated cytokines and immune regulatory microRNAs were measured in the uterus at 8 h post-mating by qPCR, and Treg cells in uterus-draining lymph nodes were evaluated by flow cytometry on day 3.5 post-coitum. Ptgs2 encoding prostaglandin-endoperoxide synthase 2, cytokines Csf2, Il6, Lif, and Tnf, chemokines Ccl2, Cxcl1, Cxcl2, and Cxcl10, and microRNAs miR-155, miR-146a, and miR-223 were induced by mating in wild-type mice, but not, or to a lesser extent, in Tlr4-/- mice. CD4+ T cells were expanded after mating in Tlr4+/+ but not Tlr4-/- mice, with failure to expand peripheral CD25+FOXP3+ NRP1- or thymic CD25+FOXP3+ NRP1+ Treg cell populations, and fewer Treg cells expressed Ki67 proliferation marker and suppressive function marker CTLA4. We conclude that TLR4 is an essential mediator of the inflammation-like response in the pre-implantation uterus that induces generation of Treg cells to support robust pregnancy tolerance and ensure optimal fetal growth and survival.