RESUMEN
17ß-Estradiol (E2) has been shown to regulate GM-CSF- or Flt3 ligand-driven dendritic cell (DC) development through estrogen receptor (ER) α signaling in myeloid progenitors. ERα regulates transcription of target genes through two distinct activation functions (AFs), AF-1 and AF-2, whose respective involvement varies in a cell type- or tissue-specific manner. In this study, we investigated the role of ERα AFs in the development and effector functions of inflammatory DCs, steady-state conventional DCs, and plasmacytoid DCs (pDC), using mouse lacking either AF-1 or AF-2. In agreement with previous works, we showed that E2 fostered the differentiation and effector functions of inflammatory DCs through ERα-dependent upregulation of IFN regulatory factor (IRF)-4 in GM-CSF-stimulated myeloid progenitors. Interestingly, whereas AF-1 was required for early IRF-4 upregulation in DC precursors, it was dispensable to enhance IRF-4 expression in differentiated DCs to a level compatible with the development of the more functional Ly6C(-) CD11b(+) DC subset. Presence of E2 had no effect on progenitors from either knock-in mice with 7-aa deletion in helix 12 of ERα, lacking AF-2, or ERα(-/-) mice. By contrast, in Flt3 ligand-driven DC differentiation, activation of AF-1 domain was required to promote the development of more functionally competent conventional DCs and pDCs. Moreover, lack of ERα AF-1 blunted the TLR7-mediated IFN-α response of female pDCs in vivo. Thus, our study demonstrates that ERα uses AF-1 differently in steady-state and inflammatory DC lineages to regulate their innate functions, suggesting that selective ER modulators could be used to target specific DC subsets.
Asunto(s)
Células Dendríticas/metabolismo , Estradiol/metabolismo , Receptor alfa de Estrógeno/metabolismo , Dominios y Motivos de Interacción de Proteínas , Animales , Antígenos CD11/metabolismo , Diferenciación Celular , Células Dendríticas/citología , Células Dendríticas/efectos de los fármacos , Receptor alfa de Estrógeno/química , Femenino , Factor Estimulante de Colonias de Granulocitos y Macrófagos/farmacología , Factores Reguladores del Interferón/genética , Factores Reguladores del Interferón/metabolismo , Masculino , Proteínas de la Membrana/farmacología , Ratones , Ratones Transgénicos , Fenotipo , Transducción de Señal , Receptores Toll-Like/metabolismoRESUMEN
Adaptor proteins positively or negatively regulate the T cell receptor for antigen (TCR) signaling cascade. We report that after TCR stimulation, the inhibitory adaptor downstream of kinase (Dok)-2 and its homologue Dok-1 are involved in a multimolecular complex including the lipid phosphatase Src homology 2 domain-containing inositol polyphosphate 5'-phosphatase (SHIP)-1 and Grb-2 which interacts with the membrane signaling scaffold linker for activation of T cells (LAT). Knockdown of LAT and SHIP-1 expression indicated that SHIP-1 favored recruitment of Dok-2 to LAT. Knockdown of Dok-2 and Dok-1 revealed their negative control on Akt and, unexpectedly, on Zap-70 activation. Our findings support the view that Dok-1 and -2 are critical elements of a LAT-dependent negative feedback loop that attenuates early TCR signal. Dok-1 and -2 may therefore exert a critical role in shaping the immune response and as gatekeepers for T cell tolerance.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas Adaptadoras Transductoras de Señales/fisiología , Proteína Adaptadora GRB2/fisiología , Activación de Linfocitos , Proteínas de la Membrana/metabolismo , Fosfoproteínas/fisiología , Monoéster Fosfórico Hidrolasas/fisiología , Complejo Receptor-CD3 del Antígeno de Linfocito T/fisiología , Transducción de Señal/inmunología , Proteínas Adaptadoras Transductoras de Señales/biosíntesis , Línea Celular Tumoral , Proteínas de Unión al ADN/fisiología , Regulación hacia Abajo/inmunología , Retroalimentación Fisiológica/inmunología , Humanos , Inositol Polifosfato 5-Fosfatasas , Células Jurkat , Ligandos , Proteínas de la Membrana/biosíntesis , Proteínas de la Membrana/fisiología , Fosfatidilinositol-3,4,5-Trifosfato 5-Fosfatasas , Fosforilación , Proteínas de Unión al ARN/fisiología , Complejo Receptor-CD3 del Antígeno de Linfocito T/metabolismo , Tirosina/metabolismoRESUMEN
BACKGROUND: The existence of a genetic basis for host responses to bacterial intramammary infections has been widely documented, but the underlying mechanisms and the genes are still largely unknown. Previously, two divergent lines of sheep selected for high/low milk somatic cell scores have been shown to be respectively susceptible and resistant to intramammary infections by Staphylococcus spp. Transcriptional profiling with an 15K ovine-specific microarray of the milk somatic cells of susceptible and resistant sheep infected successively by S. epidermidis and S. aureus was performed in order to enhance our understanding of the molecular and cellular events associated with mastitis resistance. RESULTS: The bacteriological titre was lower in the resistant than in the susceptible animals in the 48 hours following inoculation, although milk somatic cell concentration was similar. Gene expression was analysed in milk somatic cells, mainly represented by neutrophils, collected 12 hours post-challenge. A high number of differentially expressed genes between the two challenges indicated that more T cells are recruited upon inoculation by S. aureus than S. epidermidis. A total of 52 genes were significantly differentially expressed between the resistant and susceptible animals. Further Gene Ontology analysis indicated that differentially expressed genes were associated with immune and inflammatory responses, leukocyte adhesion, cell migration, and signal transduction. Close biological relationships could be established between most genes using gene network analysis. Furthermore, gene expression suggests that the cell turn-over, as a consequence of apoptosis/granulopoiesis, may be enhanced in the resistant line when compared to the susceptible line. CONCLUSIONS: Gene profiling in resistant and susceptible lines has provided good candidates for mapping the biological pathways and genes underlying genetically determined resistance and susceptibility towards Staphylococcus infections, and opens new fields for further investigation.
Asunto(s)
Perfilación de la Expresión Génica , Inmunidad Innata , Mastitis/veterinaria , Leche/citología , Enfermedades de las Ovejas/genética , Infecciones Estafilocócicas/veterinaria , Staphylococcus aureus , Staphylococcus epidermidis , Animales , Carga Bacteriana , Recuento de Células , Análisis por Conglomerados , Femenino , Redes Reguladoras de Genes , Leucocitos/patología , Mastitis/genética , Mastitis/inmunología , Mastitis/microbiología , Redes y Vías Metabólicas , Leche/inmunología , Leche/microbiología , Análisis de Secuencia por Matrices de Oligonucleótidos , Análisis de Componente Principal , Ovinos , Enfermedades de las Ovejas/inmunología , Enfermedades de las Ovejas/microbiología , Infecciones Estafilocócicas/genética , Infecciones Estafilocócicas/inmunologíaRESUMEN
BACKGROUND: Gene expression profiling studies of mastitis in ruminants have provided key but fragmented knowledge for the understanding of the disease. A systematic combination of different expression profiling studies via meta-analysis techniques has the potential to test the extensibility of conclusions based on single studies. Using the program Pointillist, we performed meta-analysis of transcription-profiling data from six independent studies of infections with mammary gland pathogens, including samples from cattle challenged in vivo with S. aureus, E. coli, and S. uberis, samples from goats challenged in vivo with S. aureus, as well as cattle macrophages and ovine dendritic cells infected in vitro with S. aureus. We combined different time points from those studies, testing different responses to mastitis infection: overall (common signature), early stage, late stage, and cattle-specific. RESULTS: Ingenuity Pathway Analysis of affected genes showed that the four meta-analysis combinations share biological functions and pathways (e.g. protein ubiquitination and polyamine regulation) which are intrinsic to the general disease response. In the overall response, pathways related to immune response and inflammation, as well as biological functions related to lipid metabolism were altered. This latter observation is consistent with the milk fat content depression commonly observed during mastitis infection. Complementarities between early and late stage responses were found, with a prominence of metabolic and stress signals in the early stage and of the immune response related to the lipid metabolism in the late stage; both mechanisms apparently modulated by few genes, including XBP1 and SREBF1.The cattle-specific response was characterized by alteration of the immune response and by modification of lipid metabolism. Comparison of E. coli and S. aureus infections in cattle in vivo revealed that affected genes showing opposite regulation had the same altered biological functions and provided evidence that E. coli caused a stronger host response. CONCLUSIONS: This meta-analysis approach reinforces previous findings but also reveals several novel themes, including the involvement of genes, biological functions, and pathways that were not identified in individual studies. As such, it provides an interesting proof of principle for future studies combining information from diverse heterogeneous sources.
Asunto(s)
Infecciones por Escherichia coli/veterinaria , Perfilación de la Expresión Génica , Enfermedades de las Cabras/genética , Mastitis Bovina/genética , Mastitis/veterinaria , Enfermedades de las Ovejas/genética , Infecciones Estafilocócicas/veterinaria , Infecciones Estreptocócicas/veterinaria , Animales , Bovinos , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Infecciones por Escherichia coli/genética , Femenino , Enfermedades de las Cabras/microbiología , Cabras , Mastitis/genética , Mastitis/microbiología , Mastitis Bovina/microbiología , Redes y Vías Metabólicas , Análisis de Secuencia por Matrices de Oligonucleótidos , Factores de Transcripción del Factor Regulador X , Ovinos , Enfermedades de las Ovejas/microbiología , Infecciones Estafilocócicas/genética , Proteína 1 de Unión a los Elementos Reguladores de Esteroles/genética , Proteína 1 de Unión a los Elementos Reguladores de Esteroles/metabolismo , Infecciones Estreptocócicas/genética , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
Sex hormones influence immune responses and the development of autoimmune diseases including MS and its animal model, EAE. Although it has been previously reported that ovariectomy could worsen EAE, the mechanisms implicated in the protective action of endogenous ovarian hormones have not been addressed. In this report, we now show that endogenous estrogens limit EAE development and CNS inflammation in adult female mice through estrogen receptor α expression in the host non-hematopoietic tissues. We provide evidence that the enhancing effect of gonadectomy on EAE development was due to quantitative rather than qualitative changes in effector Th1 or Th17 cell recruitment into the CNS. Consistent with this observation, adoptive transfer of myelin oligodendrocyte glycoprotein-specific encephalitogenic CD4(+) T lymphocytes induced more severe EAE in ovariectomized mice as compared to normal female mice. Finally, we show that gonadectomy accelerated the early recruitment of inflammatory cells into the CNS upon adoptive transfer of encephalitogenic CD4(+) T cells. Altogether, these data show that endogenous estrogens, through estrogen receptor α, exert a protective effect on EAE by limiting the recruitment of blood-derived inflammatory cells into the CNS.
Asunto(s)
Encéfalo/patología , Linfocitos T CD4-Positivos/metabolismo , Encefalomielitis Autoinmune Experimental/inmunología , Receptor alfa de Estrógeno/metabolismo , Estrógenos/inmunología , Traslado Adoptivo , Animales , Encéfalo/inmunología , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/patología , Linfocitos T CD4-Positivos/trasplante , Movimiento Celular/genética , Células Cultivadas , Citoprotección/genética , Citoprotección/inmunología , Modelos Animales de Enfermedad , Encefalomielitis Autoinmune Experimental/genética , Encefalomielitis Autoinmune Experimental/cirugía , Receptor alfa de Estrógeno/genética , Receptor alfa de Estrógeno/inmunología , Femenino , Humanos , Mediadores de Inflamación/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Esclerosis Múltiple , Proteínas de la Mielina , Glicoproteína Asociada a Mielina/inmunología , Glicoproteína Mielina-Oligodendrócito , OvariectomíaRESUMEN
Whereas studies on dendritic cells in rodents rely largely on bone marrow-derived dendritic cells (BM-DCs), no data are available about BM-DCs in sheep, a species that is largely used for immunology and transplantation studies. We have developed a culture protocol to produce ovine BM-DCs, using 6x(His)-tagged recombinant GM-CSF which was purified from baculovirus-infected insect cells. When ovine bone marrow progenitors were cultured in the presence of recombinant GM-CSF, large numbers of CD11c-positive cells were generated after 6-7 days. The phenotypic appearance of BM-DCs was assessed by flow cytometry and electron microscopy. Two DC subsets were identified that expressed different levels of MHC class II molecules, differed in receptor-mediated endocytosis, and could be separated on CD11b expression. When separated cells were incubated with microbial products, they react differently to those that are considered the TLR2 and TLR4 agonists in other species. Indeed, although CD11b(int/hi) cells were partially resistant to maturation induced by lipoteichoic acid or lipopolysaccharide, MHC class II upregulation was observed on CD11b(dull) cells. Moreover, these cells had strong stimulatory capacity for CD4 T cells when assayed in allogeneic reactions. This protocol will help analyzing ovine DC interactions with pathogens, and enables future studies on the development of vaccines.
Asunto(s)
Células de la Médula Ósea/citología , Antígeno CD11b/biosíntesis , Células Dendríticas/citología , Factor Estimulante de Colonias de Granulocitos y Macrófagos/farmacología , Células Madre/citología , Animales , Células de la Médula Ósea/inmunología , Células de la Médula Ósea/metabolismo , Antígeno CD11b/inmunología , Células Cultivadas , Células Dendríticas/inmunología , Células Dendríticas/metabolismo , Endocitosis/efectos de los fármacos , Endocitosis/inmunología , Citometría de Flujo/métodos , Antígenos de Histocompatibilidad Clase II/biosíntesis , Antígenos de Histocompatibilidad Clase II/inmunología , Lipopolisacáridos/farmacología , Proteínas Recombinantes , Roedores , Ovinos , Células Madre/inmunología , Células Madre/metabolismo , Ácidos Teicoicos/farmacología , Factores de Tiempo , Receptor Toll-Like 2/agonistas , Receptor Toll-Like 2/biosíntesis , Receptor Toll-Like 2/inmunología , Receptor Toll-Like 4/agonistas , Receptor Toll-Like 4/biosíntesis , Receptor Toll-Like 4/inmunología , Inmunología del Trasplante/efectos de los fármacos , Vacunas/inmunologíaRESUMEN
BACKGROUND: Staphylococcus aureus is a major pathogen of humans and animals and emerging antibiotic-resistant strains have further increased the concern of this health issue. Host genetics influence susceptibility to S. aureus infections, and the genes determining the outcome of infections should be identified to find alternative therapies to treatment with antibiotics. Here, we used outbred animals from a divergent selection based on susceptibility towards Staphylococcus infection to explore host immunogenetics. METHODOLOGY/PRINCIPAL FINDINGS: We investigated how dendritic cells respond to heat-inactivated S. aureus and whether dendritic cells from animals showing different degrees of susceptibility had distinct gene expression profiles. We measured gene expression levels of in vitro S. aureus-stimulated bone marrow-derived dendritic cells at three different time points (0, 3 and 8 hrs) by using 15 k ovine Agilent microarrays. Furthermore, differential expression of a selected number of genes was confirmed by RT-qPCR. Gene signatures of stimulated DCs were obtained and showed that genes involved in the inflammatory process and T helper cell polarization were highly up-regulated upon stimulation. Moreover, a set of 204 genes were statistically differentially expressed between susceptible and resistant animals, and grouped them according to their predisposition to staphylococcal infection. Interestingly, over-expression of the C1q and Ido1 genes was observed in the resistant line and suggested a role of classical pathway of complement and early regulation of inflammation pathways, respectively. On the contrary, over expression of genes involved in the IL1R pathway was observed in susceptible animals. Furthermore, the leucocyte extravasation pathway was also found to be dominant in the susceptible line. CONCLUSION/SIGNIFICANCE: We successfully obtained Staphylococcus aureus associated gene expression of ovine BM-DC in an 8-hour kinetics experiment. The distinct transcriptional profiles of dendritic cells obtained from resistant and susceptible animals may explain susceptibility towards S. aureus infections in a broader context.
Asunto(s)
Células de la Médula Ósea/metabolismo , Células Dendríticas/metabolismo , Perfilación de la Expresión Génica/métodos , Transcriptoma , Animales , Células de la Médula Ósea/inmunología , Células de la Médula Ósea/microbiología , Células Cultivadas , Células Dendríticas/inmunología , Células Dendríticas/microbiología , Resistencia a la Enfermedad/genética , Femenino , Predisposición Genética a la Enfermedad/genética , Interacciones Huésped-Patógeno , Calor , Análisis de Secuencia por Matrices de Oligonucleótidos , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Ovinos , Infecciones Estafilocócicas/genética , Infecciones Estafilocócicas/inmunología , Infecciones Estafilocócicas/microbiología , Staphylococcus aureus/inmunología , Staphylococcus aureus/fisiología , Vacunas Atenuadas/inmunologíaRESUMEN
Myxoma virus (MYXV), a leporide-specific poxvirus, represents an attractive candidate for the generation of safe and non-replicative vaccine vectors for other species. With the aim of developing new recombinant vaccines for ruminants, we evaluated the safety and the immunogenicity of recombinant MYXV in sheep. In vitro studies indicated that ovine primary fibroblasts were not permissive for MYXV and that infection of ovine peripheral blood mononuclear cells occurred at a low rate. Although non-specific activation significantly improved the susceptibility of lymphocytes, MYXV infection remained abortive. Histological and immunohistochemical examination at the inoculation sites revealed the development of an inflammatory process and allowed the detection of sparse infected cells in the dermis. In addition, inoculated sheep developed an antibody response directed against MYXV and the product of the transgene. Overall, these results provide the first line of evidence on the potential of MYXV as a viral vector for ruminants.
Asunto(s)
Vectores Genéticos/fisiología , Myxoma virus/fisiología , Vacunación/métodos , Vacunas Virales , Animales , Anticuerpos Antivirales/sangre , Antígenos Virales/genética , Antígenos Virales/inmunología , Células Cultivadas , Fibroblastos/virología , Vectores Genéticos/administración & dosificación , Virus de la Enfermedad Hemorrágica del Conejo/inmunología , Inyecciones Intradérmicas , Leucocitos Mononucleares/virología , Myxoma virus/patogenicidad , Conejos , Virus Reordenados/fisiología , Ovinos , Piel/virología , Especificidad de la Especie , Vacunación/veterinaria , Vacunas Sintéticas/administración & dosificación , Vacunas Sintéticas/inmunología , Proteínas Estructurales Virales/genética , Proteínas Estructurales Virales/inmunología , Vacunas Virales/administración & dosificación , Virulencia , Replicación ViralRESUMEN
Conventional dendritic cells enter lymph nodes by migrating from peripheral tissues via the lymphatic route, whereas plasmacytoid dendritic cells (pDC), also called IFN-producing cells (IPC), are described to gain nodes from blood via the high endothelial venules. We demonstrate here that IPC/pDC migrate in the afferent lymph of two large mammals. In sheep, injection of type A CpG oligodinucleotide (ODN) induced lymph cells to produce type I IFN. Furthermore, low-density lymph cells collected at steady state produced type I IFN after stimulation with type A CpG ODN and enveloped viruses. Sheep lymph IPC were found within a minor B(neg)CD11c(neg) subset expressing CD45RB. They presented a plasmacytoid morphology, expressed high levels of TLR-7, TLR-9, and IFN regulatory factor 7 mRNA, induced IFN-gamma production in allogeneic CD4(pos) T cells, and differentiated into dendritic cell-like cells under viral stimulation, thus fulfilling criteria of bona fide pDC. In mini-pig, a CD4(pos)SIRP(pos) subset in afferent lymph cells, corresponding to pDC homologs, produced type I IFN after type A CpG-ODN triggering. Thus, pDC can link innate and acquired immunity by migrating from tissue to draining node via lymph, similarly to conventional dendritic cells.