RESUMO
Zika virus is a positive-sense single-stranded RNA virus, which can be transmitted across the placenta and has adverse effects on fetal development during pregnancy. The severity of these complications highlights the importance of prevention and treatment. However, no vaccines or drugs are currently available. In this study, we characterize the IFNß-mediated anti-viral response in trophoblast cells in order to identify critical components that are necessary for the successful control of viral replication and determine whether components of the IFN-induced response can be used as a replacement therapy for ZIKA virus infection during pregnancy. We identify and characterize interferon-stimulated gene 20 (ISG20) as playing a central role in controlling Zika virus infection in trophoblast cells and successfully establish a recombinant ISG20-Fc protein that effectively decreases viral titers in vitro and in vivo by maintaining its exonuclease activity and displaying potential immune modulatory functions. Recombinant ISG20-Fc has thus the potential to be further developed as an anti-viral treatment against ZIKA viral infection in high-risk populations, particularly in pregnant women.
Assuntos
Infecção por Zika virus , Zika virus , Antivirais/farmacologia , Exorribonucleases , Feminino , Humanos , Interferons , Placenta , Gravidez , Replicação Viral , Zika virus/genética , Infecção por Zika virus/tratamento farmacológicoRESUMO
Pregnant women have greater mortality and complications associated with viral infections compared with the general population, but the reason for the increased susceptibility is not well defined. Placenta type I IFN is an important immune modulator and protects the pregnancy. We hypothesized that loss of placental IFN affects the regulation of the maternal immune system, resulting in the differential response to infections observed in pregnancy. Pregnant mice lacking the IFN-α/ß receptor (IFNAR) became viremic and had higher mortality compared with nonpregnant animals. Notably, an embryo with functional IFN signaling alone was sufficient to rescue the pregnant IFNAR-/- dam from virus-associated demise. Placental IFN was also an important regulator of viral replication in placental tissue and significantly affected viral transmission to the fetus. These findings highlight the role of fetal/placental IFN in the modulation of viral infection in the mother and fetus.
Assuntos
Feto/imunologia , Infecções por Herpesviridae/imunologia , Interferon Tipo I/imunologia , Placenta/imunologia , Complicações Infecciosas na Gravidez/imunologia , Animais , Feminino , Genótipo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Gravidez , Rhadinovirus/imunologia , Infecções Tumorais por Vírus/imunologia , Carga ViralRESUMO
Chorioamnionitis, premature rupture of fetal membranes (FMs), and subsequent preterm birth are associated with local infection and inflammation, particularly IL-1ß production. Although bacterial infections are commonly identified, other microorganisms may play a role in the pathogenesis. Because viral pandemics, such as influenza, Ebola, and Zika, are becoming more common, and pregnant women are at increased risk for associated complications, this study evaluated the impact that viral infection had on human FM innate immune responses. This study shows that a herpes viral infection of FMs sensitizes the tissue to low levels of bacterial LPS, giving rise to an exaggerated IL-1ß response. Using an ex vivo human FM explant system and an in vivo mouse model of pregnancy, we report that the mechanism by which this aggravated inflammation arises is through the inhibition of the TAM receptor, MERTK, and activation of the inflammasome. The TAM receptor ligand, growth arrest specific 6, re-establishes the normal FM response to LPS by restoring and augmenting TAM receptor and ligand expression, as well as by preventing the exacerbated IL-1ß processing and secretion. These findings indicate a novel mechanism by which viruses alter normal FM immune responses to bacteria, potentially giving rise to adverse pregnancy outcomes.
Assuntos
Membranas Extraembrionárias/imunologia , Gammaherpesvirinae/imunologia , Infecções por Herpesviridae/imunologia , Herpesvirus Humano 2/imunologia , Inflamassomos/metabolismo , Nascimento Prematuro/imunologia , Proteínas Proto-Oncogênicas/metabolismo , Receptores Proteína Tirosina Quinases/metabolismo , Animais , Células Cultivadas , Corioamnionite , Feminino , Infecções por Herpesviridae/complicações , Humanos , Imunização , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Interleucina-1beta/metabolismo , Lipopolissacarídeos/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Gravidez , Nascimento Prematuro/etiologia , Proteínas Proto-Oncogênicas/genética , Receptores Proteína Tirosina Quinases/genética , c-Mer Tirosina QuinaseRESUMO
Preterm birth is the major cause of neonatal mortality and morbidity, and bacterial infections that ascend from the lower female reproductive tract are the most common route of uterine infection leading to preterm birth. The uterus and growing fetus are protected from ascending infection by the cervix, which controls and limits microbial access by the production of mucus, cytokines, and antimicrobial peptides. If this barrier is compromised, bacteria may enter the uterine cavity, leading to preterm birth. Using a mouse model, we demonstrate, to our knowledge for the first time, that viral infection of the cervix during pregnancy reduces the capacity of the female reproductive tract to prevent bacterial infection of the uterus. This is due to differences in susceptibility of the cervix to infection by virus during pregnancy and the associated changes in TLR and antimicrobial peptide expression and function. We suggest that preterm labor is a polymicrobial disease, which requires a multifactorial approach for its prevention and treatment.
Assuntos
Infecções Bacterianas/imunologia , Colo do Útero/imunologia , Infecções por Herpesviridae/imunologia , Doenças do Colo do Útero/virologia , Doenças Uterinas/imunologia , Animais , Infecções Bacterianas/microbiologia , Células Cultivadas , Colo do Útero/microbiologia , Colo do Útero/virologia , Infecções por Escherichia coli/imunologia , Infecções por Escherichia coli/microbiologia , Feminino , Hormônios Esteroides Gonadais/fisiologia , Infecções por Herpesviridae/virologia , Integrinas/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Gravidez , Nascimento Prematuro/etiologia , Receptores Toll-Like/metabolismo , Infecções por Ureaplasma/imunologia , Infecções por Ureaplasma/microbiologia , Doenças do Colo do Útero/imunologia , Doenças Uterinas/microbiologia , Doenças Uterinas/virologiaRESUMO
Pandemics pose a more significant threat to pregnant women than to the nonpregnant population and may have a detrimental effect on the well being of the fetus. We have developed an animal model to evaluate the consequences of a viral infection characterized by lack of fetal transmission. The experiments described in this work show that viral infection of the placenta can elicit a fetal inflammatory response that, in turn, can cause organ damage and potentially downstream developmental deficiencies. Furthermore, we demonstrate that viral infection of the placenta may sensitize the pregnant mother to bacterial products and promote preterm labor. It is critical to take into consideration the fact that during pregnancy it is not only the maternal immune system responding, but also the fetal/placental unit. Our results further support the immunological role of the placenta and the fetus affecting the global response of the mother to microbial infections. This is relevant for making decisions associated with treatment and prevention during pandemics.
Assuntos
Inflamação/imunologia , Trabalho de Parto Prematuro/imunologia , Placenta/imunologia , Rhadinovirus/imunologia , Animais , Infecções Bacterianas/complicações , Infecções Bacterianas/imunologia , Infecções Bacterianas/microbiologia , Linhagem Celular , Células Cultivadas , Citocinas/metabolismo , Feminino , Doenças Fetais/imunologia , Doenças Fetais/virologia , Feto/imunologia , Feto/virologia , Interações Hospedeiro-Patógeno/imunologia , Humanos , Imuno-Histoquímica , Inflamação/etiologia , Troca Materno-Fetal/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Células NIH 3T3 , Trabalho de Parto Prematuro/etiologia , Placenta/virologia , Doenças Placentárias/imunologia , Doenças Placentárias/virologia , Gravidez , Complicações Infecciosas na Gravidez/imunologia , Complicações Infecciosas na Gravidez/microbiologia , Complicações Infecciosas na Gravidez/virologia , Rhadinovirus/fisiologia , Receptor 3 Toll-Like/genética , Receptor 3 Toll-Like/imunologia , Receptor 3 Toll-Like/metabolismo , Viroses/complicações , Viroses/imunologia , Viroses/virologiaRESUMO
PROBLEM: It is unknown whether maternal cytokine production differs between twin and singleton gestations in the implantation phase. A difference in maternal serum cytokine concentrations in twins would imply a dose-response to the invading embryos, as opposed to a general immune reaction. METHOD OF STUDY: A prospective longitudinal cohort of women aged 18-45 at an academic fertility center undergoing in vitro fertilization and embryo transfer (IVF-ET) underwent routine collection of serial serum samples starting 9 days after ET and then approximately every 48 hours thereafter. Cryopreserved aliquots of these samples were assayed for interleukin-10 (IL-10), tumor necrosis factor-alpha (TNF-α), and C-X-C motif chemokine ligand 10 (CXCL10) using the SimplePlex immunoassay platform. Pregnancies were followed until delivery. Serial measures of serum concentrations of IL-10, CXCL10, and TNF-α in singleton or di-di twin pregnancies from 9 to 15 days after IVF-ET were compared. RESULTS: Maternal serum levels of CXCL10 are significantly lower in women with di-di twin pregnancies in early implantation compared to those with singleton gestation (day 9-11, P = .02). Serum levels of TNF-α and IL-10 were comparable at all studied time points (P > .05). CONCLUSION: Maternal serum levels of CXCL10 are significantly lower in the earliest implantation phase in di-di twins compared to singleton conceptions. Given the known anti-angiogenic role of CXCL10, we hypothesize that lower CXCL10 levels in twin implantations allow an environment that is conducive for the greater vascularization required for the establishment of dual placentation in di-di twins.
Assuntos
Citocinas/metabolismo , Gravidez de Gêmeos/imunologia , Gravidez/imunologia , Adolescente , Adulto , Quimiocina CXCL10/genética , Quimiocina CXCL10/metabolismo , Estudos de Coortes , Citocinas/genética , Implantação Tardia do Embrião/imunologia , Feminino , Fertilização in vitro , Humanos , Masculino , Resultado da Gravidez , Estudos Prospectivos , Transcriptoma , Gêmeos , Adulto JovemRESUMO
Successful implantation requires the coordinated migration and invasion of trophoblast cells from out of the blastocyst and into the endometrium. This process relies on signals produced by cells in the maternal endometrium. However, the relative contribution of stroma cells remains unclear. The study of human implantation has major technical limitations, therefore the need of in vitro models to elucidate the molecular mechanisms. Using a recently described 3D in vitro models we evaluated the interaction between trophoblasts and human endometrial stroma cells (hESC), we assessed the process of trophoblast migration and invasion in the presence of stroma derived factors. We demonstrate that hESC promotes trophoblast invasion through the generation of an inflammatory environment modulated by TNF-α. We also show the role of stromal derived IL-17 as a promoter of trophoblast migration through the induction of essential genes that confer invasive capacity to cells of the trophectoderm. In conclusion, we describe the characterization of a cellular inflammatory network that may be important for blastocyst implantation. Our findings provide a new insight into the complexity of the implantation process and reveal the importance of inflammation for embryo implantation.
Assuntos
Movimento Celular , Implantação do Embrião , Endométrio/efeitos dos fármacos , Interleucina-17/metabolismo , Comunicação Parácrina/efeitos dos fármacos , Células Estromais/efeitos dos fármacos , Trofoblastos/metabolismo , Fator de Necrose Tumoral alfa/farmacologia , Adesão Celular , Diferenciação Celular , Linhagem Celular , Endométrio/imunologia , Endométrio/metabolismo , Feminino , Humanos , Interleucina-17/genética , Receptores Tipo I de Fatores de Necrose Tumoral/agonistas , Receptores Tipo I de Fatores de Necrose Tumoral/genética , Receptores Tipo I de Fatores de Necrose Tumoral/metabolismo , Via Secretória , Transdução de Sinais , Células Estromais/imunologia , Células Estromais/metabolismo , Trofoblastos/imunologiaRESUMO
Breast cancer recurrence after an initial favorable response to treatment is a major concern for patients who receive hormonal therapies. Additional therapies are necessary to extend the time of response, and ideally, these therapies should exhibit minimal toxicity. Our study described herein focuses on a non-toxic pro-apoptotic agent, TMS (2,4,3',5'-tetramethoxystilbene), which belongs to the Resveratrol family of stilbenes. Prior study demonstrated that TMS was more effective than Resveratrol for inducing apoptosis. Additionally, TMS was effective for invoking death of relapsing breast cancer cells. As TMS was effective for reducing tumor burden, we sought to determine the mechanism by which it achieved its effects. Microarray analysis demonstrated that TMS treatment increased tubulin genes as well as stress response and pro-apoptotic genes. Fractionation studies uncovered that TMS treatment causes cleavage of Bax from the p21 form to a truncated p18 form which is associated with the induction of potent apoptosis. Co-localization analysis of immunofluorescent studies showed that Bax moved from the cytosol to the mitochondria. In addition, the pro-apoptotic proteins Noxa and Bim (EL, L, and S) were increased upon TMS treatment. Cell lines reduced for Bax, Bim, and Noxa are compromised for TMS-mediated cell death. Electron microscopy revealed evidence of nuclear condensation, formation of apoptotic bodies and DAPI staining showed evidence of DNA fragmentation. TMS treatment was able to induce both caspase-independent and caspase-dependent death via the intrinsic death pathway.
Assuntos
Antineoplásicos Fitogênicos/farmacologia , Apoptose/efeitos dos fármacos , Neoplasias da Mama/metabolismo , Estilbenos/farmacologia , Proteína X Associada a bcl-2/metabolismo , Apoptose/genética , Proteínas Reguladoras de Apoptose/genética , Proteínas Reguladoras de Apoptose/metabolismo , Proteína 11 Semelhante a Bcl-2 , Neoplasias da Mama/genética , Neoplasias da Mama/ultraestrutura , Caspases/metabolismo , Linhagem Celular Tumoral , Fragmentação do DNA , Relação Dose-Resposta a Droga , Ativação Enzimática , Feminino , Perfilação da Expressão Gênica/métodos , Regulação Neoplásica da Expressão Gênica , Humanos , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Microscopia Eletrônica de Transmissão , Análise de Sequência com Séries de Oligonucleotídeos , Transporte Proteico , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Interferência de RNA , Fatores de Tempo , Tubulina (Proteína)/genética , Tubulina (Proteína)/metabolismo , Proteína X Associada a bcl-2/genéticaRESUMO
The maternal-fetal interface possesses innate immune strategies to protect against infections. We previously reported that prior viral infection of human fetal membranes (FMs) in vitro and mouse FMs in vivo sensitized the tissue to low dose bacterial LPS leading to augmented inflammation. The objective of this study was to examine FM production of type I interferons (IFNs) and IFN-stimulated genes (ISGs) in the context of this polymicrobial model. Human FM explants and pregnant C57BL/6 mice were treated with or without low dose LPS following exposure to media or the γ-herpes virus, MHV-68. FM RNA was analyzed by qRT-PCR for type I IFNs, ISGs, upstream signaling, and MHV-68 open reading frames (ORFs). Pre-exposure to MHV-68 followed by LPS treatment inhibited the ability of LPS to induce human FM type I IFNs (IFNA, IFNB); ISGs (OAS, MxA, APOBEC3G) and upstream signaling mediators (RIG-I, TBK-1). Signaling mediators IRF-3 and IRF-7 were also reduced. In mouse FMs, pre-exposure to MHV-68 followed by LPS treatment reduced the ability of LPS to upregulate Ifna, Ifnb, Mxa, Irf7, and also reduced Irf3. MHV-68 infection of FMs induced ORF45 which targets IRF-7, and this was further augmented in response to a combination of MHV-68 and LPS. Together, these findings indicate that a viral infection blunts FM type I IFN production and signaling in response to LPS leading to a suppressed ISG response. Our studies suggest that a viral infection inhibits this protective FM response by negatively regulating IRF-7 through ORF45, leaving the maternal-fetal interface vulnerable to further viral attack.
Assuntos
Membranas Extraembrionárias/metabolismo , Gammaherpesvirinae/fisiologia , Infecções por Herpesviridae/imunologia , Interferon Tipo I/metabolismo , Animais , Células Cultivadas , Feminino , Humanos , Proteínas Imediatamente Precoces/genética , Tolerância Imunológica , Inflamação , Fator Regulador 3 de Interferon/genética , Fator Regulador 7 de Interferon/genética , Interferon Tipo I/genética , Lipopolissacarídeos/imunologia , Camundongos , Camundongos Endogâmicos C57BL , GravidezRESUMO
INTRODUCTION: The limited cell number of primary trophoblasts and contamination of trophoblast cell lines promote us to develop a novel stable trophoblast cell line. METHOD OF STUDY: Primary trophoblast cells were isolated from first-trimester placenta and telomerase-induced immortalization was used to immortalize these cells. Subsets of cells were then evaluated by flow cytometry using CK7, HLA-G, CD45 and CD14, specific markers for trophoblast cells, extra-villous trophoblast, pan leucocyte and monocyte/macrophage, respectively. Immunofluorescence staining and immunocytochemistry were used to detect CK7 expression in trophoblast cells. The level of secreted human Chorionic Gonadotropin (hCG) was measured by electrochemiluminescence (ECL). The Bio-Plex MAGPIX System was used to analyze the cytokines and chemokines produced by AL07 cell line. RESULTS: We were able to isolate primary trophoblast cells from several first-trimester placentas. One clone, AL07 trophoblast cells, isolated from a week 7 placenta, was morphologically stable and positive for the expression of CK7 by immunofluorescence and immunocytochemistry staining. Characterization of AL07 cells reveled that they are CD45 or CD14 negative and had constitutive secretion of hCG and low HLA-G expression. Furthermore, clone AL07 secret high levels of several cytokines and chemokines, including IL-6, IL-8 and VEGF, and moderately secreted MCP-1 IP-10 and RANTES. DISCUSSION: We report the successful isolation, immortalization and characterization of AL07 cells, a novel cell clone isolated from first trimester human placenta. The clone is free of contamination of immune cells, and exhibits similar cytokine profile as other trophoblast cell lines. This new cytotrophoblast-like AL07 cell, can be a valuable tool for in-vitro trophoblast studies in the future.
Assuntos
Linhagem Celular , Primeiro Trimestre da Gravidez , Trofoblastos , Gonadotropina Coriônica/metabolismo , Citocinas/metabolismo , Feminino , Humanos , GravidezRESUMO
PROBLEM: Embryo implantation and placentation require a careful immunological balance. Cytokines such as IL-10 and TNFα have been implicated as markers of dysregulation, but have only been studied at a single time point or after a pregnancy loss. Our objective was to determine normative patterns of serum levels of IL-10 and TNFα and their ratio throughout the first trimester in healthy pregnancies and to determine if this pattern differs from pregnancy loss. METHOD OF STUDY: Two prospective longitudinal cohorts of gravidae including in vitro fertilization (IVF) and naturally conceived pregnancies with serial blood draws. Cytokines were assayed using Simple Plex. In the IVF cohort, we monitored from the implantation day up to 6 weeks of gestation; whereas in the naturally conceived cohort, sample collection began at 4 weeks and throughout the whole first trimester. RESULTS: IL-10 concentrations in normal pregnancies were significantly higher than in pregnancies ending in a loss starting at 6-8 weeks of gestation, while TNFα concentrations were significantly lower in normal than in pregnancies ending in a loss starting at 3-5 of gestation weeks. The IL-10 to TNFα ratio in normal pregnancies was significantly higher from 4 to 9 weeks compared to pregnancies that were lost (t test, P < .05). Changes were observed before any symptoms of miscarriage were present. CONCLUSION: We provide evidences of differences in early immunomodulation in healthy pregnancies vs those destined to end in first-trimester loss. The ratio of IL-10 to TNFα rises significantly higher in viable pregnancies as early as 4.5 weeks compared to pregnancies loss.
Assuntos
Aborto Espontâneo/imunologia , Interleucina-10/sangue , Primeiro Trimestre da Gravidez/imunologia , Fator de Necrose Tumoral alfa/sangue , Aborto Espontâneo/sangue , Adulto , Estudos de Coortes , Feminino , Fertilização in vitro , Humanos , Gravidez , Primeiro Trimestre da Gravidez/sangueRESUMO
The process of implantation, trophoblast invasion and placentation demand continuous adaptation and modifications between the trophoblast (embryonic) and the decidua (maternal). Within the decidua, the maternal immune system undergoes continued changes, as the pregnancy progress, in terms of the cell population, phenotype and production of immune factors, cytokines and chemokines. Human chorionic gonadotropin (hCG) is one of the earliest hormones produced by the blastocyst and has potent immune modulatory effects, especially in relation to T cells. We hypothesized that trophoblast-derived hCG modulates the immune population present at the maternal fetal interface by modifying the cytokine profile produced by the stromal/decidual cells. Using in vitro models from decidual samples we demonstrate that hCG inhibits CXCL10 expression by inducing H3K27me3 histone methylation, which binds to Region 4 of the CXCL10 promoter, thereby suppressing its expression. hCG-induced histone methylation is mediated through EZH2, a functional member of the PRC2 complex. Regulation of CXCL10 expression has a major impact on the capacity of endometrial stromal cells to recruit CD8 cells. We demonstrate the existence of a cross talk between the placenta (hCG) and the decidua (CXCL10) in the control of immune cell recruitment. Alterations in this immune regulatory function, such as during infection, will have detrimental effects on the success of the pregnancy.
Assuntos
Quimiocina CXCL10/genética , Gonadotropina Coriônica/imunologia , Decídua/imunologia , Epigênese Genética , Histonas/imunologia , Adulto , Linfócitos T CD8-Positivos/citologia , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Linhagem Celular , Quimiocina CXCL10/imunologia , Decídua/citologia , Decídua/metabolismo , Feminino , Humanos , Imunomodulação , Metilação , Placentação , Gravidez , Adulto JovemRESUMO
Decidual macrophages are in close contact with trophoblast cells during placenta development, and an appropriate crosstalk between these cellular compartments is crucial for the establishment and maintenance of a healthy pregnancy. During different phases of gestation, macrophages undergo dynamic changes to adjust to the different stages of fetal development. Trophoblast-secreted factors are considered the main modulators responsible for macrophage differentiation and function. However, the phenotype of these macrophages induced by trophoblast-secreted factors and the factors responsible for their polarization has not been elucidated. In this study, we characterized the phenotype and function of human trophoblast-induced macrophages. Using in vitro models, we found that human trophoblast-educated macrophages were CD14+ CD206+ CD86- and presented an unusual transcriptional profile in response to TLR4/LPS activation characterized by the expression of type I IFN-ß expression. IFN-ß further enhances the constitutive production of soluble programmed cell death ligand 1 (PD-L1) from trophoblast cells. PD-1 blockage inhibited trophoblast-induced macrophage differentiation. Soluble PD-L1 (sPD-L1) was detected in the blood of pregnant women and increased throughout the gestation. Collectively, our data suggest the existence of a regulatory circuit at the maternal fetal interface wherein IFN-ß promotes sPD-L1 expression/secretion by trophoblast cells, which can then initiate a PD-L1/PD-1-mediated macrophage polarization toward an M2 phenotype, consequently decreasing inflammation. Macrophages then maintain the expression of sPD-L1 by the trophoblasts through IFN-ß production induced through TLR4 ligation.
Assuntos
Antígeno B7-H1/imunologia , Diferenciação Celular/imunologia , Ativação de Macrófagos , Macrófagos/imunologia , Gravidez/imunologia , Trofoblastos/imunologia , Antígenos CD/imunologia , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular , Feminino , Humanos , Interferon beta/imunologia , Lipopolissacarídeos/farmacologia , Macrófagos/citologia , Receptor 4 Toll-Like/imunologia , Trofoblastos/citologiaRESUMO
PROBLEM: Embryo implantation depends on the interactions between the developing embryo and the maternal endometrium. Signals originating from the decidua play a critical role in the process of implantation and trophoblast invasion; however, the molecular mechanisms mediating this interaction are poorly understood. The objective of this study was to develop in vitro models that would mimic the processes of attachment, migration, and early invasion of the trophoblast. METHODS OF STUDY: First trimester trophoblast cells (Sw.71 cells) were cultured in low attachment plates to form blastocyst-like spheroids (BLS). Epithelial-mesenchymal transition (EMT) characterization during BLS formation was determined by RT-PCR and Western Blot. The two 3D in vitro culture models consist of (a) trophoblast migration: BLS cultured in suspension (b) trophoblast invasion: human endometrium stromal cells (HESC) plated in the bottom of a 96-well plate, covered by Matrigel and BLS transferred on top. Matrigel was used to mimic the human endometrial extracellular matrix. RESULTS: Using 3D cell culture systems and real-time imaging, we are able to determine the impact of endometrial factors on trophoblast cell function. Endometrial stromal cells promote blastocyst-like spheroid migration of trophoblast cells and invasion of the extracellular matrix. CONCLUSION: We report the characterization of 3D in vitro models to evaluate the interaction between endometrial cells and trophoblast during the process of migration and invasion. The models are useful tools in order to further study the molecular mechanism of embryo-maternal uterine cells interactions.
Assuntos
Blastocisto/citologia , Endométrio/fisiologia , Troca Materno-Fetal/fisiologia , Gravidez , Esferoides Celulares/fisiologia , Células Estromais/fisiologia , Trofoblastos/fisiologia , Comunicação Celular , Técnicas de Cultura de Células , Movimento Celular , Células Cultivadas , Implantação do Embrião , Desenvolvimento Embrionário , Feminino , HumanosRESUMO
PROBLEM: A successful outcome to pregnancy is critically dependent on the initiation of maternal immune tolerance before embryo implantation. Cells of embryonic origin that come in contact with the uterine microenvironment can exert influence over the phenotype and function of immune cells to facilitate robust implantation; however, what influence they may have on B cells remains unknown. In this study, we investigate the effect of human trophoblast cells on B-cell phenotype and the subsequent effect on peri-implantation events. METHOD OF STUDY: We cultured purified human B cells with the first-trimester human trophoblast cell line Swan 71 to investigate trophoblast-B-cell interactions and utilized trophoblast spheroids in an in vitro implantation model of migration and invasion. RESULTS: Trophoblast-educated B cells or TE-B cells were found to consist of B cells in committed lineages such as plasmablasts and memory B cells, as well as increased proportions in subsets of CD24hi CD27+ regulatory B cells and CD19+ IL-10+ B cells. Conditioned media from the TE-B cells showed reduced production of pro-inflammatory cytokines that influenced the T-cell proliferation and cytokine production. Using trophoblast spheroids, we assessed the role of TE-B cells in trophoblast invasion and migration. Our results demonstrate a protective effect of TE-B-conditioned media against deleterious inflammation as evidenced by survival of the trophoblast spheroid in the presence of an immune assault and promotion of a migratory phenotype. CONCLUSION: We posit that trophoblast-mediated education of B cells leads to their acquisition of properties capable of modulating inflammation in the uterine environment during the peri-implantation period.
Assuntos
Linfócitos B Reguladores/imunologia , Linfócitos T/imunologia , Trofoblastos/imunologia , Linfócitos B Reguladores/patologia , Linhagem Celular , Técnicas de Cocultura , Feminino , Humanos , Memória Imunológica , Inflamação/imunologia , Inflamação/patologia , Linfócitos T/patologia , Trofoblastos/patologiaRESUMO
Pregnancy is a unique immunologic and microbial condition that requires an adequate level of awareness to provide a fast and protective response against pathogens as well as to maintain a state of tolerance to paternal antigens. Dysregulation of inflammatory pathways in the placenta triggered by pathogens is one of the main factors responsible for pregnancy complications. Type I IFNs are key molecules modulating immune responses at the level of the placenta and are crucial for protection of the pregnancy via their antiviral and immune modulatory properties. In this study, we elucidate the mechanisms controlling the basal expression of IFNß and its negative feedback. Using in vitro and in vivo animal models, we found that TLR signaling maintains basal IFNß levels through the TLR4-MyD88-independent TBK/IRF3 signaling pathway. We describe the role of the TAM receptor Axl in the regulation of IFNß function in human and mouse trophoblast cells. The absence of TAM receptors in vivo is associated with fetal demise due to dysregulation of IFNß expression and its pro-apoptotic downstream effectors. Collectively, our data describe a feedback signaling pathway controlling the expression and function of IFNß in the trophoblast that is essential for an effective response during viral and microbial infections.
Assuntos
Interferon beta/metabolismo , Placenta/imunologia , Trofoblastos/imunologia , Proteínas Adaptadoras de Transporte Vesicular/genética , Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Animais , Células Cultivadas , Feminino , Regulação da Expressão Gênica , Humanos , Fator Regulador 3 de Interferon/genética , Fator Regulador 3 de Interferon/metabolismo , Interferon beta/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Gravidez , Primeiro Trimestre da Gravidez , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Receptores Proteína Tirosina Quinases/genética , Receptores Proteína Tirosina Quinases/metabolismo , Receptor 4 Toll-Like/genética , Receptor 4 Toll-Like/metabolismo , c-Mer Tirosina Quinase/genética , c-Mer Tirosina Quinase/metabolismo , Receptor Tirosina Quinase AxlRESUMO
The comparison of the immunological state of pregnancy to an immunosuppressed host-graft model continues to lead research and clinical practice to ill-defined approaches. This Review discusses recent evidence that supports the idea that immunological responses at the receptive maternal-fetal interface are not simply suppressed but are instead highly dynamic. We discuss the crucial role of trophoblast cells in shaping not only the way in which immune cells respond to the invading blastocyst but also how they collectively react to external stimuli. We also discuss the role of the microbiota in promoting a tolerogenic maternal immune system and highlight how subclinical viral infections can disrupt this status quo, leading to pregnancy complications.
Assuntos
Gravidez/imunologia , Células Apresentadoras de Antígenos , Células Dendríticas/imunologia , Feminino , Feto/imunologia , Humanos , Troca Materno-FetalRESUMO
In the early 1950s, Medawar recognized for the first time the unique immunology of the maternal/ fetal interface and its potential relevance for transplantation. In his original work, he described the fetal allograft analogy whereby the fetus may be viewed as a semi-allogeneic conceptus that has evaded rejection by the maternal immune system. Although numerous hypotheses have been proposed to prove this observation, none have demonstrated that the maternal immune system is antagonist to the invading trophoblast. In the present article, we have reviewed recent studies demonstrating the expression and function of Toll-like receptors (TLRs) in trophoblast cells, and, based on these data, we propose an alternative view for maternal/fetal immune interactions.
Assuntos
Placenta/citologia , Placenta/imunologia , Proteínas da Gravidez/fisiologia , Receptores Toll-Like/fisiologia , Trofoblastos/imunologia , Animais , Feminino , Humanos , Placenta/metabolismo , Gravidez , Proteínas da Gravidez/biossíntese , Receptores Toll-Like/biossínteseRESUMO
PROBLEM: Quantitative measurement of proteins in bodily fluids or cellular preparations is critical for the evaluation of biomarkers or the study of complex cellular processes. While immunoassays are the most common quantitative approach used so far, they are not practical for the evaluation of multiple proteins. Microfluidic technology allows a fine spatial control in immobilizing proteins and biomolecules inside microchannels, eliminating cross-reactivity between competing analytes, and allowing rapid and sensitive detection of targeted antigens for multiple applications. We report the characterization and validation of the Simple Plex(™) platform for the detection and quantification of cytokines and chemokines from human and mouse samples. METHOD: Cytokine and chemokine expression levels were determined using Simple Plex cartridges from ProteinSimple. Serum samples were obtained from the Yale Biorepository. RESULTS: Our data demonstrate an excellent correlation between the results obtained with Simple Plex and conventional immunoassays such as ELISA and Luminex. CONCLUSION: We describe the characterization and validation of Simple Plex, a novel multi-analyte, automated microfluidic platform that allows the evaluation of cytokines and chemokines from human and mice biological samples. Simple Plex showed significant advantages over traditional approaches in terms of low sample volume requirements, sensitivity and dynamic range, coefficient of variation, and reproducibility.
Assuntos
Quimiocinas/sangue , Citocinas/sangue , Dispositivos Lab-On-A-Chip , Microfluídica/instrumentação , Animais , Automação Laboratorial , Ensaio de Imunoadsorção Enzimática , Humanos , Camundongos , Microfluídica/métodos , Miniaturização , Variações Dependentes do Observador , Padrões de Referência , Reprodutibilidade dos Testes , Sensibilidade e EspecificidadeRESUMO
PROBLEM: Preterm birth (PTB) affects approximately 12% of pregnancies and at least 50% of cases have no known risk factors. We hypothesize that subclinical viral infections of the placenta are a factor sensitizing women to intrauterine bacterial infection. Specifically, we propose that viral-induced placental IFN-ß inhibition results in a robust inflammatory response to low concentrations of bacteria. METHODS: Human trophoblast SW.71, C57BL/6, and interferon (IFN) receptor knockout animals were used to determine IFN function. Illumina and Bio-Rad microarrays identified pathways. RESULTS: Inhibiting the IFN-ß pathway resulted in a significant increase in inflammatory cytokines such as IL-1B in response to LPS. Twist was positively correlated with IFN-ß expression and STAT3 phosphorylation and overexpressing Twist reduced IL-1B. Treating IFNAR-/- mice with low-dose LPS at E15.5 caused preterm birth. CONCLUSION: IFN-ß was identified as a key modulator of placental inflammation and, importantly, is commonly affected by viruses. We propose dysregulation of IFN-ß is a major determinant for preterm birth associated with polymicrobial infection.