RESUMEN
Selective repression of the antifibrotic gene CXCL10 contributes to tissue remodeling in idiopathic pulmonary fibrosis (IPF). We have previously reported that histone deacetylation and histone H3 lysine 9 (H3K9) methylation are involved in CXCL10 repression. In this study, we explored the role of H3K27 methylation and the interplay between the two histone lysine methyltransferases enhancer of zest homolog 2 (EZH2) and G9a in CXCL10 repression in IPF. By applying chromatin immunoprecipitation, Re-ChIP, and proximity ligation assays, we demonstrated that, like G9a-mediated H3K9 methylation, EZH2-mediated histone H3 lysine 27 trimethylation (H3K27me3) was significantly enriched at the CXCL10 promoter in fibroblasts from IPF lungs (F-IPF) compared with fibroblasts from nonfibrotic lungs, and we also found that EZH2 and G9a physically interacted with each other. EZH2 knockdown reduced not only EZH2 and H3K27me3 but also G9a and H3K9me3, and G9a knockdown reduced not only G9 and H3K9me3 but also EZH2 and H3K27me3. Depletion and inhibition of EZH2 and G9a also reversed histone deacetylation and restored CXCL10 expression in F-IPF. Furthermore, treatment of fibroblasts from nonfibrotic lungs with the profibrotic cytokine transforming growth factor-ß1 increased EZH2, G9a, H3K27me3, H3K9me3, and histone deacetylation at the CXCL10 promoter, similar to that observed in F-IPF, which was correlated with CXCL10 repression and was prevented by EZH2 and G9a knockdown. These findings suggest that a novel and functionally interdependent interplay between EZH2 and G9a regulates histone methylation-mediated epigenetic repression of the antifibrotic CXCL10 gene in IPF. This interdependent interplay may prove to be a target for epigenetic intervention to restore the expression of CXCL10 and other antifibrotic genes in IPF.
Asunto(s)
Quimiocina CXCL10/metabolismo , Proteína Potenciadora del Homólogo Zeste 2/metabolismo , Fibroblastos/enzimología , Antígenos de Histocompatibilidad/metabolismo , N-Metiltransferasa de Histona-Lisina/metabolismo , Fibrosis Pulmonar Idiopática/enzimología , Pulmón/enzimología , Estudios de Casos y Controles , Células Cultivadas , Quimiocina CXCL10/genética , Metilación de ADN , Regulación hacia Abajo , Proteína Potenciadora del Homólogo Zeste 2/genética , Represión Epigenética , Fibroblastos/efectos de los fármacos , Fibroblastos/patología , Antígenos de Histocompatibilidad/genética , N-Metiltransferasa de Histona-Lisina/genética , Humanos , Fibrosis Pulmonar Idiopática/genética , Fibrosis Pulmonar Idiopática/patología , Pulmón/efectos de los fármacos , Pulmón/patología , Regiones Promotoras Genéticas , Transducción de Señal , Factor de Crecimiento Transformador beta1/farmacologíaRESUMEN
BACKGROUND: There is excess cardiovascular mortality in patients with chronic obstructive pulmonary disease. Aortic stiffness, an independent predictor of cardiovascular risk, and systemic and airway inflammation are increased in patients with the disease. Statins modulate aortic stiffness and have anti-inflammatory properties. A proof-of-principle, double-blind, randomized trial determined if 6 weeks of simvastatin 20 mg once daily reduced aortic stiffness and systemic and airway inflammation in patients with chronic obstructive pulmonary disease. METHODS: Stable patients (n=70) were randomized to simvastatin (active) or placebo. Pre-treatment and post-treatment aortic stiffness, blood pressure, spirometry, and circulating and airway inflammatory mediators and lipids were measured. A predefined subgroup analysis was performed where baseline aortic pulse wave velocity (PWV) was >10 m/sec. RESULTS: Total cholesterol dropped in the active group. There was no significant change in aortic PWV between the active group and the placebo group (-0.7 m/sec, P=0.24). In those with aortic stiffness >10 m/sec (n=22), aortic PWV improved in the active group compared with the placebo group (-2.8 m/sec, P=0.03). Neither systemic nor airway inflammatory markers changed. CONCLUSION: There was a nonsignificant improvement in aortic PWV in those taking simvastatin 20 mg compared with placebo, but in those with higher baseline aortic stiffness (a higher risk group) a significant and clinically relevant reduction in PWV was shown.
Asunto(s)
Antiinflamatorios/uso terapéutico , Fármacos Cardiovasculares/uso terapéutico , Inhibidores de Hidroximetilglutaril-CoA Reductasas/uso terapéutico , Pulmón/efectos de los fármacos , Neumonía/tratamiento farmacológico , Enfermedad Pulmonar Obstructiva Crónica/tratamiento farmacológico , Simvastatina/uso terapéutico , Rigidez Vascular/efectos de los fármacos , Anciano , Anciano de 80 o más Años , Antiinflamatorios/efectos adversos , Biomarcadores/sangre , Fármacos Cardiovasculares/efectos adversos , Colesterol/sangre , Método Doble Ciego , Inglaterra , Femenino , Volumen Espiratorio Forzado , Humanos , Inhibidores de Hidroximetilglutaril-CoA Reductasas/efectos adversos , Mediadores de Inflamación/sangre , Pulmón/fisiopatología , Masculino , Persona de Mediana Edad , Neumonía/sangre , Neumonía/diagnóstico , Neumonía/fisiopatología , Enfermedad Pulmonar Obstructiva Crónica/sangre , Enfermedad Pulmonar Obstructiva Crónica/diagnóstico , Enfermedad Pulmonar Obstructiva Crónica/fisiopatología , Análisis de la Onda del Pulso , Simvastatina/efectos adversos , Espirometría , Factores de Tiempo , Resultado del Tratamiento , Capacidad VitalRESUMEN
RATIONALE: Nasopharyngeal carriage of Streptococcus pneumoniae is a prerequisite for invasive disease, but the majority of carriage episodes are asymptomatic and self-resolving. Interactions determining the development of carriage versus invasive disease are poorly understood but will influence the effectiveness of vaccines or therapeutics that disrupt nasal colonization. OBJECTIVES: We sought to elucidate immunological mechanisms underlying noninvasive pneumococcal nasopharyngeal carriage. METHODS: Pneumococcal interactions with human nasopharyngeal and bronchial fibroblasts and epithelial cells were investigated in vitro. A murine model of nasopharyngeal carriage and an experimental human pneumococcal challenge model were used to characterize immune responses in the airways during carriage. MEASUREMENTS AND MAIN RESULTS: We describe the previously unknown immunological basis of noninvasive carriage and highlight mechanisms whose perturbation may lead to invasive disease. We identify the induction of active transforming growth factor (TGF)-ß1 by S. pneumoniae in human host cells and highlight the key role for TGF-ß1 and T regulatory cells in the establishment and maintenance of nasopharyngeal carriage in mice and humans. We identify the ability of pneumococci to drive TGF-ß1 production from nasopharyngeal cells in vivo and show that an immune tolerance profile, characterized by elevated TGF-ß1 and high nasopharyngeal T regulatory cell numbers, is crucial for prolonged carriage of pneumococci. Blockade of TGF-ß1 signaling prevents prolonged carriage and leads to clearance of pneumococci from the nasopharynx. CONCLUSIONS: These data explain the mechanisms by which S. pneumoniae colonize the human nasopharynx without inducing damaging host inflammation and provide insight into the role of bacterial and host constituents that allow and maintain carriage.
Asunto(s)
Portador Sano/inmunología , Infecciones Neumocócicas/inmunología , Streptococcus pneumoniae/inmunología , Linfocitos T Reguladores/inmunología , Factor de Crecimiento Transformador beta1/inmunología , Animales , Biomarcadores/sangre , Portador Sano/microbiología , Portador Sano/prevención & control , Humanos , Técnicas In Vitro , Ratones , Nasofaringe/inmunología , Infecciones Neumocócicas/microbiología , Infecciones Neumocócicas/prevención & control , Factores de TiempoRESUMEN
Selective silencing of the cyclooxygenase-2 (COX-2) gene with the loss of the antifibrotic mediator prostaglandin E2 contributes to the fibrotic process in idiopathic pulmonary fibrosis (IPF). This study explored the role of G9a- and enhancer of zeste homolog 2 (EZH2)-mediated methylation of histone H3 lysine 9 (H3K9me3) and histone H3 lysine 27 (H3K27me3) in COX-2 silencing in IPF. Chromatin immunoprecipitation (ChIP) and re-ChIP assays demonstrated marked increases in H3K9me3, H3K27me3, and DNA methylation, together with their respective modifying enzymes G9a, EZH2, and DNA methyltransferases (Dnmts) and respective binding proteins heterochromatin protein 1 (HP1), polycomb protein complex 1 (PRC1) and methyl CpG binding protein 2 (MeCP2), at the COX-2 promoter in lung fibroblasts from patients with IPF (F-IPFs) compared with fibroblasts from nonfibrotic lungs. HP1, EZH2, and MeCP2 in turn were associated with additional repressive chromatin modifiers in F-IPFs. G9a and EZH2 inhibitors and small interfering RNAs and the Dnmt1 inhibitor markedly reduced H3K9me3 (49-79%), H3K27me3 (44-81%), and DNA methylation (61-97%) at the COX-2 promoter. These reductions were correlated with increased histone H3 and H4 acetylation, resulting in COX-2 mRNA and protein reexpression in F-IPFs. Our results support a central role for G9a- and EZH2-mediated histone hypermethylation and a model of bidirectional, mutually reinforcing, and interdependent crosstalk between histone hypermethylation and DNA methylation in COX-2 epigenetic silencing in IPF.-Coward, W. R., Feghali-Bostwick, C. A., Jenkins, G., Knox, A. J., Pang, L. A central role for G9a and EZH2 in the epigenetic silencing of cyclooxygenase-2 in idiopathic pulmonary fibrosis.
Asunto(s)
Ciclooxigenasa 2/genética , Epigénesis Genética/genética , Silenciador del Gen/fisiología , Antígenos de Histocompatibilidad/metabolismo , N-Metiltransferasa de Histona-Lisina/metabolismo , Fibrosis Pulmonar Idiopática/genética , Fibrosis Pulmonar Idiopática/metabolismo , Complejo Represivo Polycomb 2/metabolismo , Acetilación , Adulto , Anciano , Células Cultivadas , Inmunoprecipitación de Cromatina/métodos , Ciclooxigenasa 2/metabolismo , Metilación de ADN/genética , Proteína Potenciadora del Homólogo Zeste 2 , Fibroblastos/metabolismo , Antígenos de Histocompatibilidad/genética , N-Metiltransferasa de Histona-Lisina/genética , Histonas/genética , Histonas/metabolismo , Humanos , Masculino , Persona de Mediana Edad , Complejo Represivo Polycomb 2/genética , Regiones Promotoras Genéticas/genética , ARN Mensajero/genética , Adulto JovenRESUMEN
The polysaccharide capsule and pneumolysin toxin are major virulence factors of the human bacterial pathogen Streptococcus pneumoniae. Colonization of the nasopharynx is asymptomatic but invasion of the lungs can result in invasive pneumonia. Here we show that the capsule suppresses the release of the pro-inflammatory cytokines CXCL8 (IL-8) and IL-6 from the human pharyngeal epithelial cell line Detroit 562. Release of both cytokines was much less from human bronchial epithelial cells (iHBEC) but levels were also affected by capsule. Pneumolysin stimulates CXCL8 release from both cell lines. Suppression of CXCL8 homologue (CXCL2/MIP-2) release by the capsule was also observed in vivo during intranasal colonization of mice but was only discernable in the absence of pneumolysin. When pneumococci were administered intranasally to mice in a model of long term, stable nasopharyngeal carriage, encapsulated S. pneumoniae remained in the nasopharynx whereas the nonencapsulated pneumococci disseminated into the lungs. Pneumococcal capsule plays a role not only in protection from phagocytosis but also in modulation of the pro-inflammatory immune response in the respiratory tract.
Asunto(s)
Células Epiteliales/metabolismo , Interleucina-6/metabolismo , Interleucina-8/metabolismo , Faringe/citología , Polisacáridos Bacterianos/metabolismo , Streptococcus pneumoniae/metabolismo , Estreptolisinas/metabolismo , Animales , Proteínas Bacterianas/metabolismo , Bronquios/citología , Cápsulas , Línea Celular , Células Epiteliales/microbiología , Femenino , Humanos , Pulmón/citología , Ratones , Streptococcus pneumoniae/fisiologíaRESUMEN
The 10% of patients with the most severe asthma are responsible for a large part of healthcare expenditure and morbidity. Understanding the processes involved is key if new therapeutic approaches are to be developed. Evidence is accumulating that chronic diseases such as asthma are associated with temporal and spatial alterations in the pattern of inflammatory gene expression within the airways. Expression of these genes can be regulated by transcriptional, posttranscriptional, translational and epigenetic mechanisms. It is well established that binding of activated transcription factors to specific inducible gene promoter sites is tightly controlled by chromatin state as a result of histone modifications, particularly the balance between histone acetylation and deacetylation [1]. The interaction between transcription factors and the promoter is key to the diversification of gene expression in a time dependent manner leading to altered gene expression profiles. Alterations of the accessibility of transcription factors to the DNA can have residing effects upon gene transcription. This review will focus on the regulation of several groups of key genes which are involved in chronic airway inflammation and remodelling in asthma drawing mainly from our experience of studying these processes in airway smooth muscle cells. An overview is shown in figure 1.
Asunto(s)
Asma/genética , Regulación de la Expresión Génica , Mediadores de Inflamación/fisiología , Elementos Reguladores de la Transcripción/fisiología , Índice de Severidad de la Enfermedad , Animales , Asma/inmunología , Humanos , Inflamación/genética , Inflamación/inmunología , Transducción de Señal/fisiologíaRESUMEN
Idiopathic pulmonary fibrosis (IPF) is a progressive fibrotic lung disease with an appalling prognosis. The failure of anti-inflammatory therapies coupled with the observation that deranged epithelium overlies proliferative myofibroblasts to form the fibroblastic focus has lead to the emerging concept that IPF is a disease of deregulated epithelial-mesenchymal crosstalk. IPF is triggered by an as yet unidentified alveolar injury that leads to activation of transforming growth factor-ß (TGF-ß) and alveolar basement membrane disruption. In the presence of persisting injurious pathways, or disrupted repair pathways, activated TGF-ß can lead to enhanced epithelial apoptosis and epithelial-to-mesenchymal transition (EMT) as well as fibroblast, and fibrocyte, transformation into myofibroblasts which are resistant to apoptosis. The resulting deposition of excess disrupted matrix by these myofibroblasts leads to the development of IPF.
Asunto(s)
Antiinflamatorios/farmacología , Fibrosis Pulmonar Idiopática/fisiopatología , Miofibroblastos/patología , Factor de Crecimiento Transformador beta/metabolismo , Animales , Apoptosis , Membrana Basal/patología , Proliferación Celular , Humanos , Pronóstico , Alveolos Pulmonares/patologíaRESUMEN
Targeted repression of a subset of key genes involved in tissue remodeling is a cardinal feature of idiopathic pulmonary fibrosis (IPF). The mechanism is unclear but is potentially important in disease pathogenesis and therapeutic targeting. We have previously reported that defective histone acetylation is responsible for the repression of the antifibrotic cyclooxygenase-2 gene. Here we extended our study to the repression of another antifibrotic gene, the potent angiostatic chemokine gamma interferon (IFN-gamma)-inducible protein of 10 kDa (IP-10), in lung fibroblasts from patients with IPF. We revealed that this involved not only histone deacetylation, as with cyclooxygenase-2 repression, but also histone H3 hypermethylation, as a result of decreased recruitment of histone acetyltransferases and increased presence of histone deacetylase (HDAC)-containing repressor complexes, histone methyltransferases G9a and SUV39H1, and heterochromatin protein 1 at the IP-10 promoter, leading to reduced transcription factor binding. More importantly, treatment of diseased cells with HDAC or G9a inhibitors similarly reversed the repressive histone deacetylation and hypermethylation and restored IP-10 expression. These findings strongly suggest that epigenetic dysregulation involving interactions between histone deacetylation and hypermethylation is responsible for targeted repression of IP-10 and potentially other antifibrotic genes in fibrotic lung disease and that this is amenable to therapeutic targeting.
Asunto(s)
Quimiocina CXCL10/metabolismo , Histonas/metabolismo , Fibrosis Pulmonar Idiopática/metabolismo , Proteínas Represoras/metabolismo , Acetilación/efectos de los fármacos , Línea Celular , Quimiocina CXCL10/genética , Homólogo de la Proteína Chromobox 5 , Proteínas Cromosómicas no Histona/metabolismo , Citocinas/farmacología , Histona Acetiltransferasas/metabolismo , Inhibidores de Histona Desacetilasas/farmacología , Histona Desacetilasas/metabolismo , Histona Metiltransferasas , N-Metiltransferasa de Histona-Lisina/antagonistas & inhibidores , N-Metiltransferasa de Histona-Lisina/metabolismo , Humanos , Fibrosis Pulmonar Idiopática/genética , Metilación/efectos de los fármacos , Regiones Promotoras Genéticas/genética , Unión Proteica/efectos de los fármacos , ARN Mensajero/genética , ARN Mensajero/metabolismo , Factores de Transcripción/metabolismoRESUMEN
Diminished cyclooxygenase 2 (COX-2) expression in fibroblasts, with a resultant defect in the production of the antifibrotic mediator prostaglandin E(2), plays a key role in the pathogenesis of idiopathic pulmonary fibrosis (IPF). Here, we have characterized the molecular mechanism. We found that COX-2 mRNA levels in fibroblasts from patients with IPF (F-IPF) were significantly lower than those in fibroblasts from nonfibrotic lungs (F-NL) after transforming growth factor beta1 and interleukin-1beta treatment but that COX-2 mRNA degradation rates were similar, suggesting defective transcription. A reporter gene assay showed that there were no clear differences between F-IPF and F-NL in transcription factor involvement and activation in COX-2 gene transcription. However, a chromatin immunoprecipitation assay revealed that transcription factor binding to the COX-2 promoter in F-IPF was reduced compared to that in F-NL, an effect that was dynamically linked to reduced histone H3 and H4 acetylation due to decreased recruitment of histone acetyltransferases (HATs) and increased recruitment of transcriptional corepressor complexes to the COX-2 promoter. The treatment of F-IPF with histone deacetylase (HDAC) inhibitors together with cytokines increased histone H3 and H4 acetylation. Both HDAC inhibitors and the overexpression of HATs restored cytokine-induced COX-2 mRNA and protein expression in F-IPF. The results demonstrate that epigenetic abnormality in the form of histone hypoacetylation is responsible for diminished COX-2 expression in IPF.
Asunto(s)
Ciclooxigenasa 2/genética , Regulación Enzimológica de la Expresión Génica , Histonas/metabolismo , Fibrosis Pulmonar Idiopática/genética , Acetilación/efectos de los fármacos , Western Blotting , Células Cultivadas , Ciclooxigenasa 2/metabolismo , Dinoprostona/metabolismo , Fibroblastos/citología , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Histona Acetiltransferasas/genética , Histona Acetiltransferasas/metabolismo , Histona Desacetilasas/genética , Histona Desacetilasas/metabolismo , Humanos , Fibrosis Pulmonar Idiopática/enzimología , Fibrosis Pulmonar Idiopática/patología , Interleucina-1beta/farmacología , Luciferasas/genética , Luciferasas/metabolismo , Regiones Promotoras Genéticas/genética , Unión Proteica/efectos de los fármacos , Estabilidad del ARN/efectos de los fármacos , ARN Mensajero/genética , ARN Mensajero/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factores de Transcripción/metabolismo , Transfección , Factor de Crecimiento Transformador beta1/farmacologíaRESUMEN
The cathepsin B inhibitor, benzyloxycarbonyl-phenyl-alanyl-fluoromethylketone (z-FA-FMK) at nontoxic doses was found to be immunosuppressive and repressed human T cell proliferation induced by mitogens and IL-2 in vitro. We showed that z-FA-FMK suppresses the secretion of IL-2 and IFN-gamma as well as the expression of IL-2R alpha-chain (CD25) in activated T cells, whereas the expression of the early activated T cell marker, CD69, was unaffected. Furthermore, z-FA-FMK blocks NF-kappaB activation, inhibits T cell blast formation, and prevents cells from entering and leaving the cell cycle. z-FA-FMK inhibits the processing of caspase-8 and caspase-3 to their respective subunits in resting T cells stimulated through the Ag receptor, but has no effect on the activation of these caspases during Fas-induced apoptosis in proliferating T cells. When administered in vivo, z-FA-FMK significantly increased pneumococcal growth in both lungs and blood, compared with controls, in a mouse model of intranasal pneumococcal infection. Because host response to bronchopneumonia in mice is T cell dependent, our collective results demonstrated that z-FA-FMK is immunosuppressive in vitro and in vivo.
Asunto(s)
Catepsina B/antagonistas & inhibidores , Proliferación Celular/efectos de los fármacos , Inhibidores de Cisteína Proteinasa/farmacología , Dipéptidos/farmacología , Inmunosupresores/farmacología , Cetonas/farmacología , Infecciones Neumocócicas/inmunología , Linfocitos T/efectos de los fármacos , Linfocitos T/enzimología , Animales , Apoptosis/efectos de los fármacos , Apoptosis/inmunología , Células Cultivadas , Dipéptidos/administración & dosificación , Femenino , Inhibidores de Crecimiento/farmacología , Humanos , Inmunosupresores/administración & dosificación , Cetonas/administración & dosificación , Ratones , Infecciones Neumocócicas/enzimología , Streptococcus pneumoniae/efectos de los fármacos , Streptococcus pneumoniae/crecimiento & desarrollo , Linfocitos T/inmunologíaRESUMEN
Statins, which inhibit 3-hydroxy-3-methylglutaryl CoA reductase, have been shown recently to promote proinflammatory responses. We show in this study that both atorvastatin and simvastatin induced proinflammatory responses in mitogen-activated PBMCs by increasing the number of T cells secreting IFN-gamma. This is abolished by the presence of mevalonate, suggesting that statins act specifically by blocking the mevalonate pathway for cholesterol synthesis to promote the proinflammatory response. Both statins at low concentrations induced a dose-dependent increase in the number of IFN-gamma-secreting T cells in mitogen-activated PBMCs, whereas at higher concentrations the effect was abolished. The proinflammatory effect of statins was not seen in purified T cells per se activated with mitogen. However, conditioned medium derived from statin-treated PBMCs enhanced the number of IFN-gamma-secreting cells in activated purified T cells. This effect was not blocked by mevalonate, but was abolished by neutralizing Abs to IL-18 and IL-12. Similarly, the up-regulation of IFN-gamma-secreting T cells in PBMCs costimulated with statins and mitogens was blocked by the neutralizing anti-IL-18 and anti-IL-12. We showed that simvastatin stimulates the secretion of IL-18 and IL-1beta in monocytes. Active caspase-1, which is required for the processing and secretion of IL-18 and IL-1beta, was activated in simvastatin-treated monocytes. This was blocked by mevalonate and the caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp (OMe) fluoromethylketone. Taken together, the proinflammatory response mediated by statins in activated PBMCs is mediated mainly via the activation of caspase-1 and IL-18 secretion in the monocytes and to a lesser extent by IL-12.
Asunto(s)
Caspasa 1/metabolismo , Ácidos Heptanoicos/farmacología , Interleucina-18/metabolismo , Monocitos/efectos de los fármacos , Monocitos/metabolismo , Pirroles/farmacología , Simvastatina/farmacología , Linfocitos T/efectos de los fármacos , Clorometilcetonas de Aminoácidos/farmacología , Anticuerpos/inmunología , Atorvastatina , Antígenos CD28/inmunología , Complejo CD3/inmunología , Células Cultivadas , Activación Enzimática/efectos de los fármacos , Humanos , Inflamación/inducido químicamente , Inflamación/inmunología , Inflamación/metabolismo , Interferón gamma/metabolismo , Interleucina-1/metabolismo , Interleucina-12/metabolismo , Ácido Mevalónico/farmacología , Mitosis/efectos de los fármacos , Monocitos/citología , Solubilidad , Linfocitos T/citología , Linfocitos T/metabolismoRESUMEN
The airway epithelium is the first cellular component of the lung to be encountered by the particles and pathogens present in inhaled air. In addition to its role as a physical barrier, the immunological activity of the airway epithelium is an essential part of the pulmonary immune system. This means that the symptoms of lung diseases that involve immunological mechanisms are frequently exacerbated by infection of the airway epithelium with respiratory viruses. The virus-induced enhancement of immunological activity in infected epithelial cells is well characterized. However, the effects that contaminants of inhaled air have upon the infectivity and replication of respiratory viruses and the inflammation they cause, are comparatively unknown. In this study, we have shown that pre-exposure of airway epithelial cells to bacterial lipopolysaccharides or a proteolytically active house dust mite allergen, is able to, respectively, inhibit or enhance the level of cellular infection with respiratory syncytial virus and similarly alter virus-induced expression of the inflammatory chemokine interleukin-8. These results suggest that respiratory syncytial virus infection and the inflammation caused by respiratory syncytial virus may be modified by the biologically active contaminants of indoor air.
Asunto(s)
Contaminantes Atmosféricos/farmacología , Contaminación del Aire Interior , Neumonía Viral/inmunología , Infecciones por Virus Sincitial Respiratorio/inmunología , Virus Sincitiales Respiratorios/patogenicidad , Antígenos Dermatofagoides/farmacología , Proteínas de Artrópodos , Técnicas de Cultivo de Célula , Línea Celular , Cisteína Endopeptidasas , Susceptibilidad a Enfermedades , Células Epiteliales/inmunología , Células Epiteliales/virología , Humanos , Interleucina-8/metabolismo , Lipopolisacáridos/farmacología , Neumonía Viral/patología , Infecciones por Virus Sincitial Respiratorio/patología , Virus Sincitiales Respiratorios/fisiología , Replicación Viral/efectos de los fármacosRESUMEN
The proteolytic activities frequently associated with sources of allergens and parasite secretions have been suggested as important immunomodulators. We have investigated whether the protease activity of the house dust mite allergen Der p1 and the secreted proteases of the hookworm Necator americanus are able to directly induce type 2 cytokine production by basophils. Der p1 and the secretions of N. americanus induced interleukin (IL)-4, IL-5, and IL-13 but not interferon-gamma mRNA in KU812 basophils. Enzyme-linked immunosorbent assay confirmed that IL-4 and IL-13 were secreted. A nonproteolytic antigen failed to induce cytokine expression, and preincubation of Der p1 or N. americanus secretions with protease inhibitors inhibited cytokine expression. Data were confirmed using basophils purified from human peripheral blood. We speculate that this innate mechanism may contribute to the development of a cytokine milieu that could promote immunoglobulin E synthesis, eosinophil recruitment, and the development of type 2 T cells.
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Basófilos/inmunología , Citocinas/biosíntesis , Endopeptidasas/inmunología , Helmintos/enzimología , Pyroglyphidae/enzimología , Animales , Antígenos Dermatofagoides/inmunología , Antígenos Dermatofagoides/farmacología , Proteínas de Artrópodos , Basófilos/metabolismo , Cisteína Endopeptidasas , Citocinas/efectos de los fármacos , Endopeptidasas/farmacología , Regulación de la Expresión Génica/efectos de los fármacos , Regulación de la Expresión Génica/inmunología , Helmintos/inmunología , Humanos , Interferón gamma , Interleucina-13/biosíntesis , Interleucina-13/metabolismo , Interleucina-4/biosíntesis , Interleucina-4/metabolismo , Interleucina-5/biosíntesis , Necator americanus/enzimología , Necator americanus/inmunología , Pyroglyphidae/inmunología , Células Th2/inmunologíaRESUMEN
The generation of cytokines, particularly TNF-alpha, by mast cells is crucial for the initiation of the allergic response. A key transcription factor involved in the synthesis of TNF-alpha is NF-kappaB. Using a mAb specific for the activated form of NF-kappaB, immunocytochemistry, confocal microscopy, and gel shift assays have been used in conjunction to localize this transcription factor to human lung mast cells and to study its activation. Activation of mast cells with stem cell factor (10 ng/ml) and anti-IgE (1 micro g/ml) induced maximal activation of NF-kappaB at 4 and 2 h, respectively. In contrast, with TNF-alpha (5 ng/ml) maximal activation occurred within 15 min. Parallel falls in IkappaB were demonstrated. Confocal microscopy demonstrated the localization of the activated form of NF-kappaB to the nuclei of activated mast cells. NF-kappaB activation was verified using a gel shift assay. A supershift assay showed mast cell NF-kappaB to be composed primarily of p50 with smaller amounts of p65. No interaction with Abs for Rel-A, c-Rel, Rel-B, and p52 was seen. Immunocytochemistry and ELISAs showed TNF-alpha to be stored within mast cells and released into the extracellular environment following activation. The possible participation of TNF-alpha generated by mast cells in NF-kappaB activation by anti-IgE was investigated using a blocking Ab for TNF-alpha. The blocking Ab reduced NF-kappaB activation by anti-IgE by >50%, suggesting that the release of preformed mast cell-associated TNF-alpha acts as a positive autocrine feedback signal to augment NF-kappaB activation and production of further cytokine, including GM-CSF and IL-8.