RESUMEN
Our knowledge regarding immune-protective and immunopathogenic events in severe acute respiratory syndrome coronavirus (SARS-CoV) infection is limited, and little is known about the dynamics of the immune response at the primary site of disease. Here, an African green monkey (AGM) model was used to elucidate immune mechanisms that facilitate viral clearance but may also contribute to persistent lung inflammation following SARS-CoV infection. During primary infection, SARS-CoV replicated in the AGM lung for up to 10 days. Interestingly, lung inflammation was more prevalent following viral clearance, as leukocyte numbers peaked at 14 days postinfection (dpi) and remained elevated at 28 dpi compared to those of mock-infected controls. Lung macrophages but not dendritic cells were rapidly activated, and both cell types had high activation marker expression at late infection time points. Lung proinflammatory cytokines were induced at 1 to 14 dpi, but most returned to baseline by 28 dpi except interleukin 12 (IL-12) and gamma interferon. In SARS-CoV homologous rechallenge studies, 11 of the 12 animals were free of replicating virus at day 5 after rechallenge. However, incidence and severity of lung inflammation was not reduced despite the limited viral replication upon rechallenge. Evaluating the role of antibodies in immune protection or potentiation revealed a progressive increase in anti-SARS-CoV antibodies in lung and serum that did not correlate temporally or spatially with enhanced viral replication. This study represents one of the first comprehensive analyses of lung immunity, including changes in leukocyte populations, lung-specific cytokines, and antibody responses following SARS-CoV rechallenge in AGMs.
Asunto(s)
Coronavirus/inmunología , Neumonía/inmunología , Neumonía/virología , Síndrome Respiratorio Agudo Grave/inmunología , Síndrome Respiratorio Agudo Grave/virología , Replicación Viral , Animales , Anticuerpos Antivirales/inmunología , Línea Celular , Chlorocebus aethiops , Citocinas/metabolismo , Células Dendríticas/inmunología , Humanos , Inmunoglobulina A/inmunología , Inmunoglobulina G/inmunología , Pulmón/inmunología , Pulmón/patología , Pulmón/virología , Activación de Macrófagos/inmunología , Macrófagos/inmunología , Glicoproteínas de Membrana/inmunología , Monocitos/inmunología , Neumonía/patología , Síndrome Respiratorio Agudo Grave/metabolismo , Glicoproteína de la Espiga del Coronavirus , Linfocitos T/inmunología , Proteínas del Envoltorio Viral/inmunologíaRESUMEN
Macrophages are the primary lung phagocyte and are instrumental in maintenance of a sterile, noninflamed microenvironment. IFNs are produced in response to bacterial and viral infection, and activate the macrophage to efficiently counteract and remove pathogenic invaders. Respiratory syncytial virus (RSV) inhibits IFN-mediated signaling mechanisms in epithelial cells; however, the effects on IFN signaling in the macrophage are currently unknown. We investigated the effect of RSV infection on IFN-mediated signaling in macrophages. RSV infection inhibited IFN-beta- and IFN-gamma-activated transcriptional mechanisms in primary alveolar macrophages and macrophage cell lines, including the transactivation of important Nod-like receptor family genes, Nod1 and class II transactivator. RSV inhibited IFN-beta- and IFN-gamma-mediated transcriptional activation by two distinct mechanisms. RSV impaired IFN-beta-mediated signal transducer and activator of transcription (STAT)-1 phosphorylation through a mechanism that involves inhibition of tyrosine kinase 2 phosphorylation. In contrast, RSV-impaired transcriptional activation after IFN-gamma stimulation resulted from a reduction in the nuclear STAT1 interaction with the transcriptional coactivator, CBP, and was correlated with increased phosphorylation of STAT1beta, a dominant-negative STAT1 splice variant, in response to IFN-gamma. In support of this concept, overexpression of STAT1beta was sufficient to repress the IFN-gamma-mediated expression of class II transactivator. These results demonstrate that RSV inhibits IFN-mediated transcriptional activation in macrophages, and suggests that paramyxoviruses modulate an important regulatory mechanism that is critical in linking innate and adaptive immune mechanisms after infection.
Asunto(s)
Interferón-alfa/inmunología , Interferón beta/inmunología , Interferón gamma/inmunología , Macrófagos Alveolares/inmunología , Infecciones por Virus Sincitial Respiratorio/inmunología , Virus Sincitiales Respiratorios/inmunología , Transcripción Genética/inmunología , Inmunidad Adaptativa , Animales , Proteína de Unión a CREB/inmunología , Proteína de Unión a CREB/metabolismo , Línea Celular , Femenino , Inmunidad Innata , Interferón-alfa/biosíntesis , Interferón beta/biosíntesis , Interferón gamma/biosíntesis , Macrófagos Alveolares/metabolismo , Macrófagos Alveolares/virología , Ratones , Ratones Endogámicos BALB C , Proteína Adaptadora de Señalización NOD1/inmunología , Proteína Adaptadora de Señalización NOD1/metabolismo , Fosforilación/inmunología , Infecciones por Virus Sincitial Respiratorio/metabolismo , Infecciones por Virus Sincitial Respiratorio/virología , Virus Sincitiales Respiratorios/metabolismo , Factor de Transcripción STAT1/inmunología , Factor de Transcripción STAT1/metabolismo , Transducción de Señal/inmunología , TYK2 Quinasa/inmunología , TYK2 Quinasa/metabolismoRESUMEN
Epithelial-specific Ets (ESE) transcription factors, consisting of ESE-1, ESE-2, and ESE-3, are constitutively expressed in distinct epithelia of mucosal tissues, including the lung. Each ESE member exhibits alternative splicing and yields at least two isoforms (a and b) with transcriptional targets largely unidentified. The studies described herein define a novel role for ESE transcription factors in transactivation of the human lysozyme gene (LYZ), an essential component of innate defense in lung epithelia. Of the six ESE isoforms, ESE-1a and ESE-1b transactivated LYZ promoter in reporter gene assays, whereas only ESE-1b dramatically upregulated transcription of endogenous LYZ in both nonpulmonary and pulmonary epithelial cells. Importantly, ESE-1a and ESE-1b could transactivate the LYZ promoter in cultured primary airway epithelial cells. ESE-2 and ESE-3 isoforms were unable to substantially transactivate the lysozyme promoter or upregulate transcription of endogenous LYZ. Two functional consensus Ets sites located in the proximal 130-bp LYZ promoter were responsive to ESE-1b as identified by site-directed mutagenesis and DNA binding assays. Short hairpin RNA attenuation of endogenous ESE-1b mRNA levels in lung epithelia resulted in decreased LYZ transcription. Furthermore, ESE-1 antibody specifically enriched the 130-bp proximal LYZ promoter in chromatin immunoprecipitation analyses. These findings define a novel role for ESE transcription factors in regulating lung innate defense and suggest distinct regulatory functions for ESE family members.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Epitelio/metabolismo , Pulmón/metabolismo , Muramidasa/genética , Proteínas Proto-Oncogénicas/metabolismo , Factores de Transcripción/metabolismo , Transcripción Genética , Activación Transcripcional , Células Cultivadas , Inmunoprecipitación de Cromatina , Proteínas de Unión al ADN/antagonistas & inhibidores , Proteínas de Unión al ADN/genética , Ensayo de Cambio de Movilidad Electroforética , Humanos , Pulmón/citología , Muramidasa/metabolismo , Regiones Promotoras Genéticas/genética , Proteínas Proto-Oncogénicas/antagonistas & inhibidores , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas c-ets , Interferencia de ARN , ARN Mensajero , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factores de Transcripción/antagonistas & inhibidores , Factores de Transcripción/genéticaRESUMEN
The c-Myb transcription factor controls differentiation and proliferation in hematopoietic and other cell types and has latent transforming activity, but little is known about its regulation during the cell cycle. Here, c-Myb was identified as part of a protein complex from human T cells containing the cyclin-dependent kinase (CDK) CDK6. Assays using model reporter constructs as well as endogenous target genes showed that the activity of c-Myb was inhibited by cyclin D1 plus CDK4 or CDK6 but stimulated by expression of the CDK inhibitors p16 Ink4a, p21 Cip1, or p27 Kip1. Mapping experiments identified a highly conserved region in c-Myb which, when transferred to the related A-Myb transcription factor, also rendered it responsive to CDKs and p27. The results suggest that c-Myb activity is directly regulated by cyclin D1 and CDKs and imply that c-Myb activity is regulated during the cell cycle in hematopoietic cells.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Ciclina D1/metabolismo , Quinasas Ciclina-Dependientes/metabolismo , Proteínas Proto-Oncogénicas c-myb/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Secuencia de Aminoácidos , Animales , Ciclo Celular , Proteínas de Ciclo Celular/genética , Línea Celular , Pollos , Ciclina D1/genética , Quinasa 4 Dependiente de la Ciclina , Quinasa 6 Dependiente de la Ciclina , Inhibidor p27 de las Quinasas Dependientes de la Ciclina , Quinasas Ciclina-Dependientes/antagonistas & inhibidores , Quinasas Ciclina-Dependientes/genética , Humanos , Ratones , Datos de Secuencia Molecular , Unión Proteica , Inhibidores de Proteínas Quinasas/farmacología , Estructura Terciaria de Proteína , Proteínas Proto-Oncogénicas/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-myb/química , Proteínas Proto-Oncogénicas c-myb/genética , Codorniz , Alineación de Secuencia , Activación Transcripcional , Proteínas Supresoras de Tumor/genéticaRESUMEN
The A-Myb and c-Myb transcription factors share a highly conserved DNA-binding domain and activate the same promoters in reporter gene assays. However, the two proteins have distinct biological activities, and expressing them individually in human cells leads to the activation of distinct sets of endogenous genes, suggesting that each protein has a unique transcriptional specificity. Here, the structural and functional features of the Myb proteins were compared, using assays of endogenous gene expression to measure changes in specificity. When the Myb proteins were tested in different cell types, they activated unique and nearly nonoverlapping sets of genes in each cellular context. Deletion and domain swap experiments identified small, discreet positive and negative elements in A-Myb and c-Myb that were required for the regulation of specific genes, such as DHRS2, DSIPI, and mim-1. The results suggest that individual functional elements in the transcriptional activation domains are responsible for activating specific cellular genes in a context-specific manner. The results also have important implications for interpreting results from reporter gene assays, which fail to detect the differences in activity identified through endogenous gene assays, and fusion protein constructs that alter the transcriptional activation domains and the activities of the Myb proteins.
Asunto(s)
Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Proteínas Proto-Oncogénicas c-myb/metabolismo , Factores de Transcripción/metabolismo , Animales , Pollos , Genes Reporteros , Humanos , Mutación , Análisis de Secuencia por Matrices de Oligonucleótidos , Estructura Terciaria de Proteína , Proteínas Proto-Oncogénicas c-myb/genética , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Factores de Transcripción/genéticaRESUMEN
The c-Myb transcription factor regulates cellular differentiation and proliferation and is regulated by complex mechanisms that control its repressed oncogenic activity. The transcriptional activity of c-Myb is regulated by the serine/threonine protein kinase Pim-1. Here, we show that Pim-1 is able to interact with c-Myb and the closely related transcription factor A-Myb, via direct interactions with the highly conserved Myb DNA binding domain. Pim-1 associated with Myb both in cells and in vitro, and phosphorylated the Myb DNA binding domain, suggesting that it regulates Myb protein activity by direct phosphorylation.
Asunto(s)
Diferenciación Celular/genética , División Celular/genética , Células Eucariotas/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-myb/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Transcripción Genética/genética , Animales , Sitios de Unión/genética , Células Cultivadas , Embrión de Pollo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Células Eucariotas/citología , Fosforilación , Proteínas Serina-Treonina Quinasas/genética , Estructura Terciaria de Proteína/genética , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas c-myb/genética , Proteínas Proto-Oncogénicas c-pim-1 , Transducción de Señal/genéticaRESUMEN
The c-Myb, A-Myb and B-Myb transcription factors have nearly identical DNA-binding domains, activate the same reporter gene constructs in animal cells, but have different biological roles. The Myb proteins are often coexpressed in the same cells, raising questions about whether they activate similar or distinct gene expression profiles, and whether they cooperate or compete in regulating the same promoters. Here, recombinant adenoviruses were used to express each protein in human mammary cells, and then microarray assays were used to assess global changes in gene expression. Each Myb protein induced a unique and specific set of changes, displaying activities far more complex than revealed by standard reporter gene assays. These results have important implications for the roles of various Myb proteins in normal and transformed human cells, for regulatory pathways that might modify their activities and for the importance of acquired mutations that may qualitatively alter their functions in tumors.