RESUMEN
An important feature of atopic asthma is the T cell-driven late phase reaction involving transient bronchoconstriction followed by development of airways hyperresponsiveness (AHR). Using a unique rat asthma model we recently showed that the onset and duration of the aeroallergen-induced airway mucosal T cell activation response in sensitized rats is determined by the kinetics of functional maturation of resident airway mucosal dendritic cells (AMDCs) mediated by cognate interactions with CD4+ T helper memory cells. The study below extends these investigations to chronic aeroallergen exposure. We demonstrate that prevention of ensuing cycles of T cell activation and resultant AHR during chronic exposure of sensitized rats to allergen aerosols is mediated by CD4+CD25+Foxp3+LAG3+ CTLA+CD45RC+ T cells which appear in the airway mucosa and regional lymph nodes within 24 h of initiation of exposure, and inhibit subsequent Th-mediated upregulation of AMDC functions. These cells exhibit potent regulatory T (T reg) cell activity in both in vivo and ex vivo assay systems. The maintenance of protective T reg activity is absolutely dependent on continuing allergen stimulation, as interruption of exposure leads to waning of T reg activity and reemergence of sensitivity to aeroallergen exposure manifesting as AMDC/T cell upregulation and resurgence of T helper 2 cytokine expression, airways eosinophilia, and AHR.
Asunto(s)
Asma/inmunología , Hiperreactividad Bronquial/inmunología , Hiperreactividad Bronquial/prevención & control , Activación de Linfocitos/inmunología , Mucosa Respiratoria/inmunología , Mucosa Respiratoria/patología , Linfocitos T Reguladores/inmunología , Animales , Asma/patología , Hiperreactividad Bronquial/patología , Células Cultivadas , Técnicas de Cocultivo , Modelos Animales de Enfermedad , Femenino , Masculino , RatasRESUMEN
As TNF is one of the earliest signals that can be detected in the leukocyte-derived inflammatory cascade which drives subsequent cytokine production, we are interested in determining whether TNF is one of the initiating factors controlling liver remodeling and regeneration following chronic liver damage. One of the early responses is the expression of lymphotoxin-ß by hepatic progenitor oval cells. The aim of this study was to determine whether hepatic expression of LT-ß was controlled by TNF and to understand the basis of this regulation. We previously showed that LT-ß expression is transcriptionally controlled via the TNF-induced, inflammatory NF-κB pathway in T lymphocytes. Here we show that TNF is able to upregulate LT-ß expression in hepatic cells at the transcriptional level by the binding of NF-κB p50/p65 heterodimers and Ets1 to their respective sites in the LT-ß promoter.
Asunto(s)
Hepatocitos/metabolismo , Linfotoxina beta/genética , FN-kappa B/metabolismo , Proteína Proto-Oncogénica c-ets-1/metabolismo , Factor de Necrosis Tumoral alfa/farmacología , Animales , Secuencia de Bases , Sitios de Unión , Proteína 1 de la Respuesta de Crecimiento Precoz/metabolismo , Células Hep G2 , Hepatocitos/efectos de los fármacos , Humanos , Linfotoxina beta/metabolismo , Ratones , Datos de Secuencia Molecular , Regiones Promotoras Genéticas/genética , Unión Proteica/efectos de los fármacos , Unión Proteica/genética , Multimerización de Proteína/efectos de los fármacos , Multimerización de Proteína/genética , Factor de Transcripción Sp1/metabolismo , Acetato de Tetradecanoilforbol/farmacología , Factor de Transcripción ReIA/metabolismo , Activación Transcripcional/efectos de los fármacos , Activación Transcripcional/genéticaRESUMEN
Severe asthma exacerbations in children requiring hospitalization are typically associated with viral infection and occur almost exclusively among atopics, but the significance of these comorbidities is unknown. We hypothesized that underlying interactions between immunoinflammatory pathways related to responses to aeroallergen and virus are involved, and that evidence of these interactions is detectable in circulating cells during exacerbations. To address this hypothesis we used a genomics-based approach involving profiling of PBMC subpopulations collected during exacerbation vs convalescence by microarray and flow cytometry. We demonstrate that circulating T cells manifest the postactivated "exhausted" phenotype during exacerbations, whereas monocyte/dendritic cell populations display up-regulated CCR2 expression accompanied by phenotypic changes that have strong potential for enhancing local inflammation after their recruitment to the atopic lung. Notably, up-regulation of FcepsilonR1, which is known to markedly amplify capacity for allergen uptake/presentation to Th2 effector cells via IgE-mediated allergen capture, and secondarily programming of IL-4/IL-13-dependent IL-13R(+) alternatively activated macrophages that have been demonstrated in experimental settings to be a potent source of autocrine IL-13 production. We additionally show that this disease-associated activation profile can be reproduced in vitro by cytokine exposure of atopic monocytes, and furthermore that IFN-alpha can exert both positive and negative roles in the process. Our findings suggest that respiratory viral infection in atopic children may initiate an atopy-dependent cascade that amplifies and sustains airway inflammation initiated by innate antiviral immunity via harnessing underlying atopy-associated mechanisms. These interactions may account for the unique susceptibility of atopics to severe viral-induced asthma exacerbations.
Asunto(s)
Asma/inmunología , Asma/virología , Hipersensibilidad Inmediata/inmunología , Hipersensibilidad Inmediata/virología , Inmunidad Innata , Mediadores de Inflamación/metabolismo , Transducción de Señal/inmunología , Enfermedad Aguda , Infecciones por Adenovirus Humanos/inmunología , Infecciones por Adenovirus Humanos/metabolismo , Infecciones por Adenovirus Humanos/patología , Adolescente , Asma/patología , Niño , Preescolar , Femenino , Regulación Viral de la Expresión Génica/inmunología , Humanos , Hipersensibilidad Inmediata/patología , Inmunidad Innata/genética , Mediadores de Inflamación/fisiología , Gripe Humana/inmunología , Gripe Humana/metabolismo , Gripe Humana/patología , Masculino , Infecciones por Paramyxoviridae/inmunología , Infecciones por Paramyxoviridae/metabolismo , Infecciones por Paramyxoviridae/patología , Infecciones por Virus Sincitial Respiratorio/inmunología , Infecciones por Virus Sincitial Respiratorio/metabolismo , Infecciones por Virus Sincitial Respiratorio/patología , Transducción de Señal/genéticaRESUMEN
BACKGROUND: Dendritic cells (DCs) are important in allergic diseases such as asthma, although little is known regarding the mechanisms by which DCs induce T(H)2-polarized responses in atopic individuals. It has been suggested that intrinsic properties of allergens can directly stimulate T(H)2 polarizing functions of DCs, but little is known of the underlying mechanisms. OBJECTIVE: To identify novel genes expressed by house dust mite (HDM) allergen-exposed DCs. METHODS: We screened for allergen-induced gene expression by microarray, and validated differentially expressed genes at the mRNA and protein levels. RESULTS: Thrombomodulin (CD141, blood dendritic cell antigen 3) expression by microarray was higher on HDM-stimulated DCs from atopic (relative to nonatopic) individuals. These findings were confirmed at both the mRNA and protein levels in an independent group. Purified thrombomodulin(+) DCs induced a strongly T(H)2-polarized cytokine response by allergen-specific T cells compared with DCs lacking thrombomodulin. In vivo, thrombomodulin(+) circulating DCs were significantly more frequent in subjects with HDM allergy and asthma, compared with control subjects. Furthermore, thrombomodulin expression in blood leukocytes was higher in children with acute asthma than at convalescence 6 weeks later. CONCLUSION: Thrombomodulin expression on DCs may be involved in the pathogenesis of atopy and asthma.
Asunto(s)
Antígenos Dermatofagoides/inmunología , Antígenos de Superficie/inmunología , Asma/inmunología , Células Dendríticas/inmunología , Células Dendríticas/patología , Células Th2/inmunología , Trombomodulina/inmunología , Adolescente , Adulto , Anciano , Antígenos Dermatofagoides/farmacología , Antígenos de Superficie/biosíntesis , Asma/metabolismo , Asma/patología , Niño , Preescolar , Convalecencia , Células Dendríticas/metabolismo , Femenino , Perfilación de la Expresión Génica/métodos , Regulación de la Expresión Génica/efectos de los fármacos , Regulación de la Expresión Génica/inmunología , Humanos , Masculino , Persona de Mediana Edad , Análisis de Secuencia por Matrices de Oligonucleótidos/métodos , Células Th2/patología , Trombomodulina/biosíntesisRESUMEN
Reporter assays are widely used in applications that require measurement of changes in gene expression over time (e.g. drug screening). With standard reporter vectors, the measurable effect of a treatment or compound (altered reporter activity) is substantially diluted and delayed, compared with its true effect (altered transcriptional activity). This problem is caused by the relatively long half-lives of both the reporter protein and its mRNA. As a result, the activities of compounds, ligands or treatments that have a relatively minor effect, or a substantial but transient effect, often remain undetected. To circumvent this problem, we introduced modular protein- and mRNA-destabilizing elements into a range of commonly used reporters. Our data show that both elements are required for maximal responses to both increases and decreases in transcriptional activity. The double-destabilized reporter vectors showed markedly improved performance in drug screening, kinetic assays and dose-response titrations.
Asunto(s)
Secuencias de Aminoácidos , Genes Reporteros , Secuencias Reguladoras de Ácido Ribonucleico , Transcripción Genética , Animales , Células CHO , Cricetinae , Cricetulus , Evaluación Preclínica de Medicamentos , Vectores Genéticos , Semivida , Células HeLa , Humanos , Proteínas/metabolismo , Estabilidad del ARN , ARN Mensajero/metabolismo , Proteínas Recombinantes de Fusión/análisis , Proteínas Recombinantes de Fusión/genética , Terminología como Asunto , TransfecciónRESUMEN
BACKGROUND: The timing of allergen sensitization is controversial, with conflicting evidence suggesting transplacental priming versus exclusively postnatal priming. Resolution of this question is important in relation to rational design of allergy prevention strategies, particularly the issue of allergen avoidance during pregnancy. OBJECTIVE: To elucidate the kinetics of sensitization in high-risk children during their first 2 years of life. METHODS: We prospectively studied house dust mite (HDM)-specific IgE and IgG(4) antibody production and associated T-cell immunity in a cohort of 200 high-risk infants. Parallel antibody studies tracked responses against a broader panel of inhalant and dietary allergens including peanut. RESULTS: HDM-induced T(H)2 responses in PBMC from 6 months onward, particularly IL-4 and IL-5, correlated increasingly strongly with sensitization outcomes at 2 years, and a contrasting negative relationship was observed with IFN-gamma response capacity. HDM-induced T-cell responses in cord blood, although common, were unrelated to subsequent sensitization. Transient HDM-IgE (and IgG(4)) production frequently peaked at 6 or 12 months before returning to baseline, which suggests the onset of protective tolerance. This finding contrasted with progressively increasing HDM-IgE titers in children sensitized by 2 years of age. Comparably contrasting patterns were observed in peanut-specific responses in sensitized versus nonsensitized children. CONCLUSION: Priming of T(H)2 responses associated with persistent HDM-IgE production occurs entirely postnatally, as HDM reactivity in cord blood seems nonspecific and is unrelated to subsequent development of allergen-specific T(H)2 memory or IgE. CLINICAL IMPLICATIONS: These findings question the scientific basis for existing recommendations for allergen avoidance by high-risk women during pregnancy.
Asunto(s)
Alérgenos/inmunología , Exposición a Riesgos Ambientales/efectos adversos , Hipersensibilidad/inmunología , Sistema Inmunológico/enzimología , Efectos Tardíos de la Exposición Prenatal/inmunología , Animales , Preescolar , Femenino , Sangre Fetal/citología , Sangre Fetal/inmunología , Humanos , Inmunoglobulina E/sangre , Inmunoglobulina G/sangre , Lactante , Recién Nacido , Interferón gamma/inmunología , Interleucina-4/inmunología , Interleucina-5/inmunología , Leucocitos Mononucleares/inmunología , Embarazo , Pyroglyphidae/inmunología , Factores de Riesgo , Células Th2/inmunologíaRESUMEN
BACKGROUND: Lymphotoxin-beta (LT-beta) plays an important role in inflammation and its promoter contains a functional nuclear factor-kappaB (NF-kappaB) element, rendering it a likely target of pro-inflammatory cytokines. Inflammatory cytokines play a central role in liver regeneration resulting from acute or chronic liver injury, with interleukin (IL)-6 signaling essential for liver regeneration induced by partial hepatectomy. In hepatic oval cells observed following chronic liver injury, LT-beta levels are upregulated, suggesting a link between LT-beta and liver regeneration. RESULTS: The expression of LT-beta in hepatic oval cell and hepatocellular carcinoma cell lines was further investigated, along with its responsiveness to IL-6 and IL-1beta. Key regulatory cis-acting elements of the LT-beta promoter that mediate IL-6 responsiveness (Sp/BKLF, Ets, NF-kappaB and Egr-1/Sp1) and IL-1beta responsiveness (NF-kappaB and Ets) of hepatic LT-beta expression were identified. The novel binding of basic Kruppel-like factor (BKLF) proteins to an apparent composite Sp/BKLF site of the LT-beta promoter was shown to mediate IL-6 responsiveness. Binding of NF-kappaB p65/p50 heterodimers and Ets-related transcription factors to their respective sites mediates responsiveness to IL-1beta. CONCLUSION: The identification of IL-6 and IL-1beta as activators of LT-beta supports their involvement in LT-beta signaling in liver regeneration associated with chronic liver damage.
Asunto(s)
Regulación de la Expresión Génica/efectos de los fármacos , Hepatocitos/fisiología , Interleucina-1/farmacología , Interleucina-6/farmacología , Linfotoxina-alfa/genética , Proteínas de la Membrana/genética , Animales , Secuencia de Bases , Carcinoma Hepatocelular , Línea Celular Transformada , Línea Celular Tumoral , Proteínas de Unión al ADN/metabolismo , Proteína 1 de la Respuesta de Crecimiento Precoz , Regulación de la Expresión Génica/inmunología , Hepatocitos/citología , Humanos , Proteínas Inmediatas-Precoces/metabolismo , Factores de Transcripción de Tipo Kruppel , Neoplasias Hepáticas , Linfotoxina-alfa/metabolismo , Linfotoxina beta , Proteínas de la Membrana/metabolismo , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , FN-kappa B/metabolismo , Subunidad p50 de NF-kappa B , Regiones Promotoras Genéticas/genética , Precursores de Proteínas/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas c-ets , Transducción de Señal/inmunología , Factor de Transcripción Sp1/metabolismo , Factor de Transcripción ReIA , Factores de Transcripción/metabolismo , Transcripción Genética/efectos de los fármacos , Transcripción Genética/inmunologíaRESUMEN
Lymphotoxin-beta (LT-beta) is a transmembrane protein expressed mainly on cells of the lymphoid lineage. It associates with LT-alpha on the cell surface to form the heterotrimeric LTalpha1,beta2 complex, which binds the LT-beta receptor. Membrane lymphotoxin is a crucial signal for the appropriate development of lymph nodes and Peyer's patches, and in the formation of B and T cell compartments in the spleen. In this study we report the characterization of mechanisms governing both basal as well as PMA- and TNF-inducible regulation of the human LT-beta promoter. Using a Jurkat T cell line, induction with either PMA or TNF resulted in an increase in mRNA levels compared with uninduced values. This induction corresponded to an increase in transcriptional activity of the human LT-beta promoter. Mutational and deletion analysis demonstrated the importance of Ets and NF-kappaB motifs in the regulation of basal transcription. Furthermore, the ability of PMA to induce activity was lost in the Ets mutant constructs. Interestingly, the same mutation had little effect on the ability of TNF to induce transcription of the LT-beta promoter. TNF inducibility was localized to the NF-kappaB site positioned at -83 of the promoter sequence. Thus, it appears that the Ets site, although playing a major role in PMA induction, did not mediate TNF inducibility. Therefore, our study suggests that alternative signaling pathways may be present to induce the expression of LT-beta in response to different immunological or inflammatory stimuli.