RESUMEN
Human rhinovirus (HRV) infections lead to exacerbations of lower airways disease in asthmatic patients but not in healthy individuals. However, underlying mechanisms remain to be completely elucidated. We hypothesized that the Th2-driven allergic environment enhances HRV-induced CC chemokine production, leading to asthma exacerbations. Ovalbumin (OVA)-sensitized and -challenged mice inoculated with HRV showed significant increases in the expression of lung CC chemokine ligand (CCL)-2/monocyte chemotactic protein (MCP)-1, CCL4/macrophage inflammatory protein (MIP)-1ß, CCL7/MCP-3, CCL19/MIP-3ß, and CCL20/MIP3α compared with mice treated with OVA alone. Inhibition of CCL2 with neutralizing antibody significantly attenuated HRV-induced airways inflammation and hyperresponsiveness in OVA-treated mice. Immunohistochemical stains showed colocalization of CCL2 with HRV in epithelial cells and CD68-positive macrophages, and flow cytometry showed increased CCL2(+), CD11b(+) cells in the lungs of OVA-treated, HRV-infected mice. Compared with lung macrophages from naïve mice, macrophages from OVA-exposed mice expressed significantly more CCL2 in response to HRV infection ex vivo. Pretreatment of mouse lung macrophages and BEAS-2B human bronchial epithelial cells with interleukin (IL)-4 and IL-13 increased HRV-induced CCL2 expression, and mouse lung macrophages from IL-4 receptor knockout mice showed reduced CCL2 expression in response to HRV, suggesting that exposure to these Th2 cytokines plays a role in the altered HRV response. Finally, bronchoalveolar macrophages from children with asthma elaborated more CCL2 upon ex vivo exposure to HRV than cells from nonasthmatic patients. We conclude that CCL2 production by epithelial cells and macrophages contributes to HRV-induced airway hyperresponsiveness and inflammation in a mouse model of allergic airways disease and may play a role in HRV-induced asthma exacerbations.
Asunto(s)
Quimiocina CCL2/inmunología , Células Epiteliales/inmunología , Hipersensibilidad/inmunología , Pulmón/inmunología , Macrófagos/inmunología , Rhinovirus/fisiología , Animales , Anticuerpos Neutralizantes/farmacología , Antígenos CD/genética , Antígenos CD/inmunología , Antígenos de Diferenciación Mielomonocítica/genética , Antígenos de Diferenciación Mielomonocítica/inmunología , Quimiocina CCL19/genética , Quimiocina CCL19/inmunología , Quimiocina CCL2/antagonistas & inhibidores , Quimiocina CCL2/genética , Quimiocina CCL20/genética , Quimiocina CCL20/inmunología , Quimiocina CCL4/genética , Quimiocina CCL4/inmunología , Quimiocina CCL7/genética , Quimiocina CCL7/inmunología , Niño , Modelos Animales de Enfermedad , Células Epiteliales/efectos de los fármacos , Células Epiteliales/patología , Expresión Génica , Humanos , Hipersensibilidad/patología , Hipersensibilidad/virología , Inflamación/inducido químicamente , Inflamación/inmunología , Inflamación/patología , Inflamación/virología , Interleucina-13/farmacología , Interleucina-4/farmacología , Pulmón/patología , Pulmón/virología , Macrófagos/efectos de los fármacos , Macrófagos/patología , Ratones , Ovalbúmina , Rhinovirus/patogenicidad , Células Th2/inmunologíaRESUMEN
We identified three consanguineous Austrian kindreds with 15 members affected by autosomal recessive childhood-onset severe retinal dystrophy, a genetically heterogeneous group of disorders characterized by degeneration of the photoreceptor cells. A whole-genome scan by microarray analysis of single-nucleotide polymorphisms (ref. 2) identified a founder haplotype and defined a critical interval of 1.53 cM on chromosome 14q23.3-q24.1 that contains the gene associated with this form of retinal dystrophy. RDH12 maps in this region and encodes a retinol dehydrogenase proposed to function in the visual cycle. A homozygous 677A-->G transition (resulting in Y226C) in RDH12 was present in all affected family members studied, as well as in two Austrian individuals with sporadic retinal dystrophy. We identified additional mutations in RDH12 in 3 of 89 non-Austrian individuals with retinal dystrophy: a 5-nucleotide deletion (806delCCCTG) and the transition 565C-->T (resulting in Q189X), each in the homozygous state, and 146C-->T (resulting in T49M) and 184C-->T (resulting in R62X) in compound heterozygosity. When expressed in COS-7 cells, Cys226 and Met49 variants had diminished and aberrant activity, respectively, in interconverting isomers of retinol and retinal. The severe visual impairment of individuals with mutations in RDH12 is in marked contrast to the mild visual deficiency in individuals with fundus albipunctatus caused by mutations in RDH5, encoding another retinal dehydrogenase. Our studies show that RDH12 is associated with retinal dystrophy and encodes an enzyme with a unique, nonredundant role in the photoreceptor cells.
Asunto(s)
Oxidorreductasas de Alcohol/genética , Células Fotorreceptoras/enzimología , Degeneración Retiniana/genética , Adulto , Preescolar , Cromosomas Humanos Par 14 , Femenino , Efecto Fundador , Genes Recesivos , Humanos , Masculino , Mutación , Linaje , Polimorfismo de Nucleótido SimpleRESUMEN
Human rhinovirus is responsible for the majority of virus-induced asthma exacerbations. To determine the immunologic mechanisms underlying rhinovirus (RV)-induced asthma exacerbations, we combined mouse models of allergic airways disease and human rhinovirus infection. We inoculated OVA-sensitized and challenged BALB/c mice with rhinovirus serotype 1B, a minor group strain capable of infecting mouse cells. Compared with sham-infected, OVA-treated mice, virus-infected mice showed increased lung infiltration with neutrophils, eosinophils and macrophages, airway cholinergic hyperresponsiveness, and increased lung expression of cytokines including eotaxin-1/CCL11, IL-4, IL-13, and IFN-gamma. Administration of anti-eotaxin-1 attenuated rhinovirus-induced airway eosinophilia and responsiveness. Immunohistochemical analysis showed eotaxin-1 in the lung macrophages of virus-infected, OVA-treated mice, and confocal fluorescence microscopy revealed colocalization of rhinovirus, eotaxin-1, and IL-4 in CD68-positive cells. RV inoculation of lung macrophages from OVA-treated, but not PBS-treated, mice induced expression of eotaxin-1, IL-4, and IL-13 ex vivo. Macrophages from OVA-treated mice showed increased expression of arginase-1, Ym-1, Mgl-2, and IL-10, indicating a shift in macrophage activation status. Depletion of macrophages from OVA-sensitized and -challenged mice reduced eosinophilic inflammation and airways responsiveness following RV infection. We conclude that augmented airway eosinophilic inflammation and hyperresponsiveness in RV-infected mice with allergic airways disease is directed in part by eotaxin-1. Airway macrophages from mice with allergic airways disease demonstrate a change in activation state characterized in part by altered eotaxin and IL-4 production in response to RV infection. These data provide a new paradigm to explain RV-induced asthma exacerbations.
Asunto(s)
Quimiocina CCL11/inmunología , Macrófagos Alveolares/inmunología , Ovalbúmina/inmunología , Infecciones por Picornaviridae/inmunología , Animales , Antígenos CD/inmunología , Antígenos CD/metabolismo , Antígenos de Diferenciación Mielomonocítica/inmunología , Antígenos de Diferenciación Mielomonocítica/metabolismo , Hiperreactividad Bronquial/inmunología , Hiperreactividad Bronquial/metabolismo , Quimiocina CCL11/genética , Quimiocina CCL11/metabolismo , Citocinas/genética , Citocinas/inmunología , Citocinas/metabolismo , Eosinófilos/inmunología , Eosinófilos/metabolismo , Eosinófilos/patología , Células HeLa , Humanos , Inmunohistoquímica , Inflamación/inmunología , Inflamación/metabolismo , Mediadores de Inflamación/inmunología , Mediadores de Inflamación/metabolismo , Pulmón/inmunología , Pulmón/metabolismo , Pulmón/patología , Macrófagos Alveolares/metabolismo , Ratones , Ratones Endogámicos BALB C , Microscopía Confocal , Infecciones por Picornaviridae/virología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Rhinovirus/inmunologíaRESUMEN
Rhinovirus (RV), a ssRNA virus of the picornavirus family, is a major cause of the common cold as well as asthma and chronic obstructive pulmonary disease exacerbations. Viral dsRNA produced during replication may be recognized by the host pattern recognition receptors TLR-3, retinoic acid-inducible gene (RIG)-I, and melanoma differentiation-associated gene (MDA)-5. No study has yet identified the receptor required for sensing RV dsRNA. To examine this, BEAS-2B human bronchial epithelial cells were infected with intact RV-1B or replication-deficient UV-irradiated virus, and IFN and IFN-stimulated gene expression was determined by quantitative PCR. The separate requirements of RIG-I, MDA5, and IFN response factor (IRF)-3 were determined using their respective small interfering RNAs (siRNA). The requirement of TLR3 was determined using siRNA against the TLR3 adaptor molecule Toll/IL-1R homologous region-domain-containing adapter-inducing IFN-beta (TRIF). Intact RV-1B, but not UV-irradiated RV, induced IRF3 phosphorylation and dimerization, as well as mRNA expression of IFN-beta, IFN-lambda1, IFN-lambda2/3, IRF7, RIG-I, MDA5, 10-kDa IFN-gamma-inducible protein/CXCL10, IL-8/CXCL8, and GM-CSF. siRNA against IRF3, MDA5, and TRIF, but not RIG-I, decreased RV-1B-induced expression of IFN-beta, IFN-lambda1, IFN-lambda2/3, IRF7, RIG-I, MDA5, and inflammatory protein-10/CXCL10 but had no effect on IL-8/CXCL8 and GM-CSF. siRNAs against MDA5 and TRIF also reduced IRF3 dimerization. Finally, in primary cells, transfection with MDA5 siRNA significantly reduced IFN expression, as it did in BEAS-2B cells. These results suggest that TLR3 and MDA5, but not RIG-I, are required for maximal sensing of RV dsRNA and that TLR3 and MDA5 signal through a common downstream signaling intermediate, IRF3.
Asunto(s)
ARN Helicasas DEAD-box/inmunología , Células Epiteliales/inmunología , Infecciones por Picornaviridae/inmunología , Mucosa Respiratoria/inmunología , Receptor Toll-Like 3/inmunología , Western Blotting , Línea Celular , Proteína 58 DEAD Box , ARN Helicasas DEAD-box/biosíntesis , ARN Helicasas DEAD-box/genética , Células Epiteliales/virología , Expresión Génica , Humanos , Helicasa Inducida por Interferón IFIH1 , ARN Bicatenario/inmunología , ARN Interferente Pequeño , Receptores Inmunológicos , Receptores de Reconocimiento de Patrones/biosíntesis , Receptores de Reconocimiento de Patrones/genética , Receptores de Reconocimiento de Patrones/inmunología , Mucosa Respiratoria/virología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Rhinovirus/inmunología , Transducción de Señal/inmunología , Receptor Toll-Like 3/biosíntesis , Receptor Toll-Like 3/genéticaRESUMEN
RDH12 codes for a member of the family of short-chain alcohol dehydrogenases/reductases proposed to function in the visual cycle that supplies the chromophore 11-cis retinal to photoreceptor cells. Mutations in RDH12 cause severe and progressive childhood onset autosomal-recessive retinal dystrophy, including Leber congenital amaurosis. We generated Rdh12 knockout mice, which exhibited grossly normal retinal histology at 10 months of age. Levels of all-trans and 11-cis retinoids in dark- and light-adapted animals and scotopic and photopic electroretinogram (ERG) responses were similar to those for the wild type, as was recovery of the ERG response following bleaching, for animals matched for an Rpe65 polymorphism (p.L450M). Lipid peroxidation products and other measures of oxidative stress did not appear to be elevated in Rdh12(-/-) animals. RDH12 was localized to photoreceptor inner segments and the outer nuclear layer in both mouse and human retinas by immunohistochemistry. The present findings, together with those of earlier studies showing only minor functional deficits in mice deficient for Rdh5, Rdh8, or Rdh11, suggest that the activity of any one isoform is not rate limiting in the visual response.
Asunto(s)
Marcación de Gen , Retinal-Deshidrogenasa/genética , Retinal-Deshidrogenasa/metabolismo , Visión Ocular/fisiología , Oxidorreductasas de Alcohol , Animales , Electrorretinografía , Regulación de la Expresión Génica , Humanos , Inmunohistoquímica , Ratones , Estrés Oxidativo , Células Fotorreceptoras de Vertebrados/citología , Células Fotorreceptoras de Vertebrados/enzimología , Células Fotorreceptoras de Vertebrados/ultraestructura , Transporte de Proteínas , ARN Mensajero/genética , ARN Mensajero/metabolismo , Retinal-Deshidrogenasa/deficiencia , Retinal-Deshidrogenasa/inmunología , Retinoides/análisis , Visión Ocular/genéticaRESUMEN
RATIONALE: Infection with rhinovirus (RV) triggers exacerbations of asthma and chronic obstructive lung disease. OBJECTIVES: We sought to develop a mouse model of RV employing RV1B, a minor group serotype that binds to the low-density lipoprotein receptor. METHODS: C57BL/6 mice were inoculated intranasally with RV1B, replication-deficient ultraviolet (UV)-irradiated RV1B, or RV39, a major group virus. MEASUREMENTS AND MAIN RESULTS: Viral RNA was present in the lungs of RV1B-treated mice, but not in those exposed to UV-irradiated RV1B or RV39. Lung homogenates of RV-treated mice contained infectious RV 4 days after inoculation. RV1B exposure induced neutrophilic and lymphocytic airway inflammation, as well as increased lung expression of KC, macrophage-inflammatory protein-2, and IFN-alpha and IFN-beta. RV1B-exposed mice showed airway hyperresponsiveness 1 and 4 days after inoculation. UV-irradiated RV1B induced modest neutrophilic airway inflammation and hyperresponsiveness 1 day after exposure. Both RV1B and UV-irradiated RV1B, but not RV39, increased lung phosphorylation of Akt. Confocal immunofluorescence showed colocalization of RV1B and phospho-Akt in the airway epithelium. Finally, pretreatment with the phosphatidylinositol 3-kinase inhibitor LY294002 attenuated chemokine production and neutrophil infiltration. CONCLUSIONS: We conclude that RV1B induces airway inflammation in vivo. Evidence is presented that viral replication occurs in vivo and is required for maximal responses. On the other hand, viral replication was not required for a subset of RV-induced responses, including neutrophilic inflammation, airway hyperresponsiveness, and Akt phosphorylation. Finally, phosphatidylinositol 3-kinase/Akt signaling is required for maximal RV1B-induced airway neutrophilic inflammation, likely via its essential role in virus internalization.
Asunto(s)
Asma , Fosfatidilinositol 3-Quinasas/metabolismo , Infecciones por Picornaviridae/inmunología , Neumonía/virología , Enfermedad Pulmonar Obstructiva Crónica , Rhinovirus/patogenicidad , Animales , Asma/complicaciones , Asma/virología , Hiperreactividad Bronquial/inmunología , Hiperreactividad Bronquial/virología , Quimiocinas/metabolismo , Quimiotaxis , Modelos Animales de Enfermedad , Células Epiteliales/inmunología , Células Epiteliales/virología , Femenino , Humanos , Ratones , Neutrófilos/inmunología , Proteína Oncogénica v-akt/inmunología , Proteína Oncogénica v-akt/metabolismo , Infecciones por Picornaviridae/fisiopatología , Neumonía/inmunología , Enfermedad Pulmonar Obstructiva Crónica/complicaciones , Enfermedad Pulmonar Obstructiva Crónica/virología , Transducción de SeñalRESUMEN
PURPOSE: Mutations in the MERTK gene are responsible for retinal degeneration in the Royal College of Surgeons (RCS) rat and are a cause of human autosomal recessive retinitis pigmentosa (RP). This study reports the identification and functional analysis of novel MERTK mutations to provide information regarding whether they are causative of severe rod-cone degeneration in a young patient. METHODS: MERTK missense variants identified by single-strand conformational polymorphism (SSCP) and sequence analysis were introduced into expression constructs and used to transfect HEK293T cells. Recombinant protein expression was assayed with anti-MERTK and anti-phosphotyrosine antibodies. Protein turnover was assayed in pulse-chase studies of 35S-methionine incorporation. Transcript levels were determined by quantitative RT-PCR. RESULTS: Three MERTK sequence variants were identified in a patient with rod-cone dystrophy: R722X in exon 16 and R865W in exon 19 on the paternal allele and R844C in exon 19 on the maternal allele. The R844C sequence change affects an evolutionarily conserved amino acid residue and was not detected in unaffected individuals. In transfected HEK293Tcells, wild-type (wt) and W865 MERTK were expressed at equivalent levels and present in the plasma membrane, stimulated tyrosine phosphorylation, and induced significant rounding of the cell bodies. In contrast, C844 MERTK was expressed at low levels and did not stimulate tyrosine phosphorylation. In addition, the relative stability of C844 MERTK was significantly less than wt in assays of protein turnover. At age 13, the patient had 20/60 and 20/200 acuities, tunnel vision of 5 degrees centrally, and a far temporal peripheral crescent bilaterally, and ERGs were nondetectable. The fundi showed bull's-eye macular atrophy and widespread RPE thinning. CONCLUSIONS: The present study reports the identification of R844C, the first putative pathogenic MERTK missense mutation that results in severe retinal degeneration with childhood onset when in compound heterozygous form with a R722X allele. The loss of function of C844 MERTK is probably due to decreased protein stability.
Asunto(s)
Regulación de la Expresión Génica/fisiología , Mutación Missense , Células Fotorreceptoras de Vertebrados/patología , Proteínas Proto-Oncogénicas , Proteínas Tirosina Quinasas Receptoras/genética , Degeneración Retiniana/genética , Adolescente , Arginina , Western Blotting , Cisteína , Femenino , Variación Genética , Humanos , Riñón/embriología , Riñón/metabolismo , Fosforilación , Polimorfismo Conformacional Retorcido-Simple , Proteínas Tirosina Quinasas Receptoras/metabolismo , Degeneración Retiniana/metabolismo , Degeneración Retiniana/patología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transfección , Tirosina/metabolismo , Tirosina Quinasa c-MerRESUMEN
Retinoid dehydrogenases/reductases catalyze key oxidation-reduction reactions in the visual cycle that converts vitamin A to 11-cis retinal, the chromophore of the rod and cone photoreceptors. It has recently been shown that mutations in RDH12, encoding a retinol dehydrogenase, result in severe and early-onset autosomal recessive retinal dystrophy (arRD). In a cohort of 1011 individuals diagnosed with arRD, we have now identified 20 different disease-associated RDH12 mutations, of which 16 are novel, in a total of 22 individuals (2.2%). Haplotype analysis suggested a founder mutation for each of the three common mutations: p.L99I, p.T155I and c.806_810delCCCTG. Patients typically presented with early disease that affected the function of both rods and cones and progressed to legal blindness in early adulthood. Eleven of the missense variants identified in our study exhibited profound loss of catalytic activity when expressed in transiently transfected COS-7 cells and assayed for ability to convert all-trans retinal to all-trans retinol. Loss-of-function appeared to result from decreased protein stability, as expression levels were significantly reduced. For the p.T49M variant, differing activity profiles were associated with each of the alleles of the common p.R161Q RDH12 polymorphism, suggesting that genetic background may act as a modifier of mutation effect. A locus (LCA3) for Leber congenital amaurosis, a severe, early-onset form of arRD, maps close to RDH12 on chromosome 14q24. Haplotype analysis in the family in which LCA3 was mapped excluded RDH12 as the LCA3 gene and thus suggests the presence of a novel arRD gene in this region.
Asunto(s)
Oxidorreductasas de Alcohol/genética , Oxidorreductasas de Alcohol/metabolismo , Mutación , Degeneración Retiniana/genética , Retinaldehído/biosíntesis , Oxidorreductasas de Alcohol/química , Animales , Ceguera/genética , Células COS , Chlorocebus aethiops , Femenino , Genes Recesivos , Haplotipos/genética , Humanos , Masculino , Datos de Secuencia Molecular , Mutación Missense , Atrofia Óptica Hereditaria de Leber/genética , Linaje , Polimorfismo Genético , Retinaldehído/metabolismoRESUMEN
Uniparental disomy (UPD) is a rare condition in which a diploid offspring carries a chromosomal pair from a single parent. We now report the first two cases of UPD resulting in retinal degeneration. We identified an apparently homozygous loss-of-function mutation of RPE65 (1p31) in one retinal dystrophy patient and an apparently homozygous loss-of-function mutation of MERTK (2q14.1) in a second retinal dystrophy patient. In both families, the gene defect was present in the patient's heterozygous father but not in the patient's mother. Analysis of haplotypes in each nuclear kindred, by use of DNA polymorphisms distributed along both chromosomal arms, indicated the absence of the maternal allele for all informative markers tested on chromosome 1 in the first patient and on chromosome 2 in the second patient. Our results suggest that retinal degeneration in these individuals is due to apparently complete paternal isodisomy involving reduction to homoallelism for RPE65 or MERTK loss-of-function alleles. Our findings provide evidence for the first time, in the case of chromosome 2, and confirm previous observations, in the case of chromosome 1, that there are no paternally imprinted genes on chromosomes 1 and 2 that have a major effect on phenotype.