RESUMEN
RATIONALE: Accumulation of B cells and lymphoid follicles (LFs) has been described in chronic obstructive pulmonary disease (COPD) airways, but the functional status of lung B cells remains poorly known. OBJECTIVES: To characterize LFs for expression of IgA, the main mucosal antibody. METHODS: The presence of B cells and LFs, including intrafollicular IgA expression, were determined in the lung from patients with COPD (n = 37) versus control subjects (n = 34) by immunohistochemistry. We also evaluated follicular IgA responses in the lungs from mice infected with Pseudomonas aeruginosa (PAO1) (n = 10 per group) and in smoking mice. MEASUREMENTS AND MAIN RESULTS: Whereas in smokers B-cell numbers slightly increased, robust increases in B-cell and LF numbers (mainly in distal airways) were only observed in severe COPD. Most follicular B cells were IgM+ (70-80%), but IgA+ (and not IgG+) B-cell numbers were increased in LFs from severe COPD compared with control subjects (twofold, 44.7% vs. 25.2%), and this was significant in distal but not proximal airways. Follicular IgA response was also observed in PAO1-infected mouse lungs, but not after smoke exposure. Moreover, follicular IgA expression associated with expression of IL-21, which was very potent to activate immunoglobulin production in vitro. CONCLUSIONS: This study shows that IgA production occurs in peribronchiolar LFs from severe COPD, where IL-21-producing T cells are present, and presumably represents a feature of exacerbated mucosal adaptive immune responses against microbial and/or self-antigens.
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Inmunoglobulina A/metabolismo , Pulmón/inmunología , Enfermedad Pulmonar Obstructiva Crónica/inmunología , Estructuras Linfoides Terciarias/inmunología , Enfermedad Aguda , Animales , Linfocitos B/inmunología , Linfocitos B/metabolismo , Linfocitos B/patología , Estudios de Casos y Controles , Modelos Animales de Enfermedad , Ensayo de Inmunoadsorción Enzimática , Femenino , Técnica del Anticuerpo Fluorescente , Humanos , Interleucina-6/metabolismo , Interleucinas/metabolismo , Pulmón/metabolismo , Pulmón/patología , Masculino , Ratones Endogámicos C57BL , Persona de Mediana Edad , Enfermedad Pulmonar Obstructiva Crónica/metabolismo , Enfermedad Pulmonar Obstructiva Crónica/patología , Reacción en Cadena en Tiempo Real de la Polimerasa , Fumar/efectos adversos , Fumar/metabolismo , Fumar/patología , Estructuras Linfoides Terciarias/metabolismo , Estructuras Linfoides Terciarias/patologíaRESUMEN
RATIONALE: Asthma is associated with increased lung IgE production, but whether the secretory IgA system is affected in this disease remains unknown. OBJECTIVES: We explored mucosal IgA transport in human asthma and its potential regulation by T-helper cell type 2 inflammation. METHODS: Bronchial biopsies from asthma and control subjects were assayed for bronchial epithelial polymeric immunoglobulin receptor (pIgR) expression and correlated to T-helper cell type 2 biomarkers. Bronchial epithelium reconstituted in vitro from these subjects, on culture in air-liquid interface, was assayed for pIgR expression and regulation by IL-4/IL-13. MEASUREMENTS AND MAIN RESULTS: Downregulation of pIgR protein was observed in the bronchial epithelium from patients with asthma (P = 0.0002 vs. control subjects). This epithelial defect was not observed ex vivo in the cultured epithelium from patients with asthma. Exogenous IL-13 and IL-4 could inhibit pIgR expression and IgA transcytosis. Mechanistic experiments showed that autocrine transforming growth factor-ß mediates the IL-4/IL-13 effect on the pIgR, with a partial contribution of upregulated transforming growth factor-α/epidermal growth factor receptor. CONCLUSIONS: This study shows impaired bronchial epithelial pIgR expression in asthma, presumably affecting secretory IgA-mediated frontline defense as a result of type 2 immune activation of the transforming growth factor pathway.
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Asma/metabolismo , Asma/fisiopatología , Bronquios/metabolismo , Inmunoglobulina A Secretora/metabolismo , Inmunoglobulina A/metabolismo , Interleucina-4/metabolismo , Mucosa Respiratoria/metabolismo , Adulto , Anciano , Anciano de 80 o más Años , Femenino , Humanos , Masculino , Persona de Mediana EdadRESUMEN
Despite their relevance to mucosal defense, production of IgA and the function of lung B-cells remain unknown in chronic obstructive pulmonary disease (COPD). We assessed IgA synthesis in the lungs of COPD (n=28) and control (n=21) patients, and regulation of B-cells co-cultured with in vitro-reconstituted airway epithelium. In COPD lung tissue, synthesis of IgA1 was increased, which led to its accumulation in subepithelial areas. In vitro, the COPD bronchial epithelium imprinted normal human B-cells for increased production of IgA (mainly IgA1) and maturation into CD38(+) plasma cells. These effects were associated with upregulation of TACI (transmembrane activator and CAML interactor) and were observed under resting conditions, while being partly inhibited upon stimulation with cigarette smoke extract. Interleukin (IL)-6 and BAFF (B-cell activating factor)/APRIL (a proliferation-inducing ligand) were upregulated in the COPD epithelium and lung tissue, respectively; the IgA-promoting effect of the COPD bronchial epithelium was inhibited by targeting IL-6 and, to a lower extent, by blocking TACI. These data show that in COPD, the bronchial epithelium imprints B-cells with signals promoting maturation into IgA-producing plasma cells through the action of two epithelial/B-cell axes, namely the IL-6/IL-6 receptor and BAFF-APRIL/TACI pathways, while cigarette smoke partly counteracts this IgA-promoting effect.
Asunto(s)
Inmunoglobulina A/metabolismo , Interleucina-6/metabolismo , Enfermedad Pulmonar Obstructiva Crónica/inmunología , Enfermedad Pulmonar Obstructiva Crónica/patología , Proteína Activadora Transmembrana y Interactiva del CAML/metabolismo , Biomarcadores , Estudios de Casos y Controles , Células Cultivadas , Ensayo de Inmunoadsorción Enzimática , Células Epiteliales/inmunología , Células Epiteliales/metabolismo , Femenino , Citometría de Flujo , Humanos , Inmunohistoquímica , Masculino , Reacción en Cadena de la Polimerasa/métodos , ARN/metabolismo , Mucosa Respiratoria/citología , Mucosa Respiratoria/metabolismo , Sensibilidad y Especificidad , Transducción de Señal , Estadísticas no ParamétricasRESUMEN
In chronic obstructive pulmonary disease (COPD), epithelial changes and subepithelial fibrosis are salient features in conducting airways. Epithelial-to-mesenchymal transition (EMT) has been recently suggested in COPD, but the mechanisms and relationship to peribronchial fibrosis remain unclear. We hypothesised that de-differentiation of the COPD respiratory epithelium through EMT could participate in airway fibrosis and thereby, in airway obstruction. Surgical lung tissue and primary broncho-epithelial cultures (in air-liquid interface (ALI)) from 104 patients were assessed for EMT markers. Cell cultures were also assayed for mesenchymal features and for the role of transforming growth factor (TGF)-ß1. The bronchial epithelium from COPD patients showed increased vimentin and decreased ZO-1 and E-cadherin expression. Increased vimentin expression correlated with basement membrane thickening and airflow limitation. ALI broncho-epithelial cells from COPD patients also displayed EMT phenotype in up to 2 weeks of culture, were more spindle shaped and released more fibronectin. Targeting TGF-ß1 during ALI differentiation prevented vimentin induction and fibronectin release. In COPD, the airway epithelium displays features of de-differentiation towards mesenchymal cells, which correlate with peribronchial fibrosis and airflow limitation, and which are partly due to a TGF-ß1-driven epithelial reprogramming.
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Desdiferenciación Celular , Transición Epitelial-Mesenquimal , Regulación de la Expresión Génica , Enfermedad Pulmonar Obstructiva Crónica/fisiopatología , Anciano , Obstrucción de las Vías Aéreas , Antígenos CD , Bronquios/citología , Cadherinas/metabolismo , Células Epiteliales/citología , Femenino , Fibronectinas/metabolismo , Fibrosis/patología , Fibrosis/fisiopatología , Humanos , Técnicas In Vitro , Pulmón/patología , Masculino , Persona de Mediana Edad , Fenotipo , Mucosa Respiratoria/metabolismo , Factor de Crecimiento Transformador beta1/metabolismo , Vimentina/metabolismo , Proteína de la Zonula Occludens-1/metabolismoRESUMEN
We showed that IgA induces IL-10 in monocytes and dendritic cells. Because reciprocal inhibition exists between IL-10 and IL-12, we explored whether IgA could regulate this other immunoregulatory cytokine. In human monocytes and monocyte-derived dendritic cells preincubated with IFN-γ before stimulation by LPS, suppression of p40 and IL-12p70 production was observed upon IgA treatment during IFN-γ priming. Washout experiments and inhibition of IFN-γ-induced CXCL10 (IP-10) and FcγRI (CD64) indicated that inhibition by IgA occurred at both the LPS and IFN-γ levels. Inhibition was not affected by blockade of IL-10 or MAPK but involved FcαRI/CD89-mediated suppression of STAT1 phosphorylation. These data indicate that FcαRI ligation on human monocytes and dendritic cells inhibits IL-12 expression and type 1 activation by interfering with STAT1 activation.
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Antígenos CD/genética , Células Dendríticas/efectos de los fármacos , Interferón gamma/farmacología , Interleucina-12/antagonistas & inhibidores , Monocitos/efectos de los fármacos , Receptores Fc/genética , Antígenos CD/inmunología , Células Cultivadas , Quimiocina CXCL10/antagonistas & inhibidores , Quimiocina CXCL10/genética , Quimiocina CXCL10/inmunología , Células Dendríticas/citología , Células Dendríticas/inmunología , Citometría de Flujo , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Proteínas I-kappa B/genética , Proteínas I-kappa B/inmunología , Inmunoglobulina A/inmunología , Inmunoglobulina A/farmacología , Interferón gamma/inmunología , Interleucina-10/antagonistas & inhibidores , Interleucina-10/genética , Interleucina-10/inmunología , Interleucina-12/biosíntesis , Interleucina-12/genética , Interleucina-12/inmunología , Lipopolisacáridos/farmacología , Monocitos/citología , Monocitos/inmunología , Inhibidor NF-kappaB alfa , Fosforilación/efectos de los fármacos , Receptores Fc/inmunología , Receptores de IgG/genética , Receptores de IgG/inmunología , Factor de Transcripción STAT1/genética , Factor de Transcripción STAT1/inmunología , Transducción de Señal/efectos de los fármacosRESUMEN
RATIONALE: The generation of protective secretory IgA relies on the epithelial polymeric immunoglobulin receptor (pIgR). pIgR expression is reduced in chronic obstructive pulmonary disease (COPD), but correlation to disease severity and underlying mechanisms remains unknown. OBJECTIVES: To address the hypothesis that pIgR down-regulation in COPD concerns severe disease in relation to aberrant programming of the bronchial epithelium. METHODS: Surgical lung tissue and primary bronchial epithelium (cultured in air-liquid interface, ALI) obtained from a large series of patients (n = 116) were studied for pIgR expression and regulation. MEASUREMENTS AND MAIN RESULTS: pIgR immunostaining in the bronchial epithelium is decreased in severe COPD. In contrast, pIgR transcription was up-regulated in smokers with or without COPD. In ALI (vs. submerged) cultures, pIgR expression was strongly induced, whereas pIgR expression and IgA-transcytosis capacity were decreased in cultures from subjects with severe COPD as compared with control subjects. In addition, COPD cultures released more transforming growth factor-ß1 (TGF-ß1), reflecting increased epithelial TGF-ß1 immunostaining in COPD lung tissue. Finally, besides inducing epithelial dedifferentiation, exogenous TGF-ß1 dose-dependently inhibited pIgR production, whereas pIgR increased on blockade of TGF-ß1 activity during ALI differentiation. CONCLUSIONS: pIgR down-regulation in COPD correlates with disease severity, and the bronchial epithelium reconstituted in vitro from these patients retains its aberrant imprinting for pIgR expression. This study also links pIgR down-regulation to TGF-ß-driven reprogramming of the bronchial epithelium, which results in impaired lung IgA immunity in patients with COPD.
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Bronquios/metabolismo , Regulación hacia Abajo , Enfermedad Pulmonar Obstructiva Crónica/metabolismo , Receptores de Inmunoglobulina Polimérica/metabolismo , Mucosa Respiratoria/metabolismo , Factor de Crecimiento Transformador beta1/metabolismo , Anciano , Biomarcadores/metabolismo , Estudios de Casos y Controles , Femenino , Humanos , Inmunohistoquímica , Masculino , Persona de Mediana Edad , Estudios Prospectivos , Reacción en Cadena en Tiempo Real de la Polimerasa , Índice de Severidad de la Enfermedad , Fumar/metabolismo , Técnicas de Cultivo de Tejidos , Regulación hacia ArribaRESUMEN
Chronic obstructive pulmonary disease (COPD), a devastating and irreversible lung disease, causes structural and functional defects in the bronchial epithelium, the (ir)reversibility of which remains unexplored in vitro. This study aimed to investigate the persistence of COPD-related epithelial defects in long-term airway epithelial cultures derived from non-smokers, smokers, and COPD patients. Barrier function, polarity, cell commitment, epithelial-to-mesenchymal transition, and inflammation were evaluated and compared with native epithelium characteristics. The role of inflammation was explored using cytokines. We show that barrier dysfunction, compromised polarity, and lineage abnormalities observed in smokers and COPD persisted for up to 10 wk. Goblet cell hyperplasia was associated with recent cigarette smoke exposure. Conversely, increased IL-8/CXCL-8 release and abnormal epithelial-to-mesenchymal transition diminished over time. These ex vivo observations matched surgical samples' abnormalities. Cytokine treatment induced COPD-like changes in control cultures and reactivated epithelial-to-mesenchymal transition in COPD cells. In conclusion, these findings suggest that the airway epithelium of smokers and COPD patients retains a multidimensional memory of its original state and previous cigarette smoke-induced injuries, maintaining these abnormalities for extended periods.
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Enfermedad Pulmonar Obstructiva Crónica , Fumadores , Humanos , Células Epiteliales , Células Cultivadas , Epitelio , Citocinas , InflamaciónRESUMEN
RATIONALE: Intrinsic asthma was described by Rackemann as asthma without allergy. Local IgE production has been documented in intrinsic asthma, but antigen specificity of this response remains elusive. OBJECTIVES: We investigated (1) the presence of dust mite-specific IgE in sputum of patients with intrinsic asthma, (2) their clinical/immunological relevance, and (3) their functionality. METHODS: Specific IgE to Dermatophagoides pteronyssinus (Der p) and to recombinant major allergens (rDer p1 and rDer p2) were assayed by ELISA in sputum samples from patients with intrinsic versus atopic asthma and control subjects. Whole-lung challenge was performed with Der p for clinical and inflammatory readouts. Functionality of local IgE to trigger effector cells was assessed using basophil activation test (surface expression of CD203c). MEASUREMENTS AND MAIN RESULTS: Both total IgE and Der p-specific IgE levels are increased in patients with intrinsic asthma compared with healthy nonatopic patients. However, no immediate asthmatic responses were observed in patients with intrinsic asthma after Der p exposure. These sputum Der p-specific IgE do, however, recognize major allergens Der p1 and Der p2 and are able to trigger activation of blood basophils from atopic donors. CONCLUSIONS: We confirm that IgE production occurs in intrinsic asthma and show that part of this IgE recognizes Der p antigens. However, this IgE reactivity does not translate into clinical responses to Der p exposure, despite specificity to major allergens and functionality to activate effector cells in vitro. We postulate that a second signal that promotes IgE-mediated asthmatic responses through FcεRI is lacking in intrinsic asthma.
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Anticuerpos Antiidiotipos/inmunología , Asma/inmunología , Dermatophagoides pteronyssinus/inmunología , Inmunoglobulina E/inmunología , Esputo/inmunología , Adulto , Anciano , Animales , Especificidad de Anticuerpos , Ensayo de Inmunoadsorción Enzimática , Femenino , Volumen Espiratorio Forzado/inmunología , Humanos , Inmunoensayo , Masculino , Persona de Mediana Edad , Estadísticas no Paramétricas , Adulto JovenAsunto(s)
Células Dendríticas/inmunología , Inmunoglobulina A/inmunología , Péptidos y Proteínas de Señalización Intercelular/inmunología , Receptores Notch/inmunología , Transducción de Señal/inmunología , Proteínas Adaptadoras Transductoras de Señales , Adulto , Anciano , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/metabolismo , Proteínas de Unión al Calcio , Técnicas de Cocultivo , Citocinas/inmunología , Citocinas/metabolismo , Células Dendríticas/metabolismo , Ensayo de Inmunoadsorción Enzimática , Femenino , Citometría de Flujo , Expresión Génica/inmunología , Humanos , Inmunoglobulina A/metabolismo , Inmunoglobulina G/inmunología , Péptidos y Proteínas de Señalización Intercelular/genética , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Masculino , Persona de Mediana Edad , Modelos Inmunológicos , Receptores Notch/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Rinitis Alérgica/genética , Rinitis Alérgica/inmunología , Rinitis Alérgica/metabolismo , Células TH1/inmunología , Células TH1/metabolismo , Células Th2/inmunología , Células Th2/metabolismoAsunto(s)
Alérgenos/inmunología , Antígenos Dermatofagoides/inmunología , Asma/inmunología , Moléculas de Adhesión Celular/inmunología , Inmunoglobulina E/inmunología , Adulto , Anciano , Animales , Asma/sangre , Asma/fisiopatología , Bronquios/inmunología , Eosinófilos/citología , Eosinófilos/inmunología , Células Epiteliales/inmunología , Femenino , Volumen Espiratorio Forzado , Humanos , Inmunoglobulina E/sangre , Recuento de Leucocitos , Masculino , Persona de Mediana Edad , Mucosa Respiratoria/inmunología , Pruebas Cutáneas , Esputo/inmunologíaRESUMEN
BACKGROUND: In cystic fibrosis, the respiratory epithelium is the target tissue of both the genetic abnormality of the disease and of external aggressions, notably by pathogens (Pseudomonas aeruginosa). A detailed characterisation of the cystic fibrosis bronchial epithelium is however lacking, as most previous studies focused on the nasal epithelium or on cell lines. This study aimed to characterise the abnormal phenotype and epithelial-to-mesenchymal transition in cystic fibrosis bronchial epithelium and to evaluate in cell cultures whether abnormalities persist ex vivo. METHODS: Explant lung tissues (n = 44) were assessed for bronchial epithelial cell phenotyping by immunostaining. Human bronchial epithelial cells were derived from basal cells isolated from cystic fibrosis patients or control donors and cultured in air-liquid interface for 2, 4 or 6 weeks. RESULTS: Enhanced mucin 5AC and decreased ß-tubulin expression were observed in cystic fibrosis airways reflecting a decreased ciliated/goblet cell ratio, associated with increased number of vimentin-positive cells, indicating epithelial-to-mesenchymal transition process. These features were recapitulated in vitro, in cystic fibrosis-derived reconstituted epithelium. However, they were not induced by CFTR inhibition or Pseudomonas infection, and most abnormalities tended to disappear in long-term culture (6 weeks) except for increased fibronectin release, an epithelial-to-mesenchymal transition marker. CONCLUSIONS: This study provides new insights into airway epithelial changes in cystic fibrosis, which are imprinted through an acquired mechanism that we could not relate to CFTR function.
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Bronquios/citología , Fibrosis Quística/metabolismo , Mucosa Respiratoria/citología , Adulto , Biomarcadores/metabolismo , Diferenciación Celular , Femenino , Humanos , Masculino , Persona de Mediana Edad , Mucina 5AC/metabolismo , Tubulina (Proteína)/metabolismoRESUMEN
As previously reported by others for immunoglobulin (Ig)G, we observed that IgA can induce interleukin (IL)-10 expression in human monocytes. In this study, we explored the molecular mechanisms of IL-10 induction by IgA in monocytes and monocyte-derived dendritic cells (MD-DCs). Monomeric IgA induced IL-10 production in monocytes and this production was further increased upon IgA cross-linking. Similar IL-10 responses were observed in monocytes and autologous MD-DCs, and were inhibited (by approximately 77%) by preincubation with a blocking mAb to FcalphaRI. IL-10 induction by IgA correlated with activation of MAPKinases ERK1/2, p38 and JNK, whereas only p38-inhibitor SB-203580 inhibited IL-10 induction. Upon IgA stimulation, AP-1, NFkappaB and Sp1 transcription factors were activated and inhibitors of NFkappaB and of Sp1 suppressed IgA-driven transcriptional activation of IL-10. In addition, p38 MAPK activation appeared that it was required to control nuclear translocation of NFkappaB and Sp1 upon IgA stimulation. Therefore, in human monocytes and MD-DCs the mechanisms of IL-10 induction by IgA involve p38 MAPK-dependent recruitment of both NFkappaB and Sp1.
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Células Dendríticas/inmunología , Interleucina-10/metabolismo , Monocitos/inmunología , Receptores Fc/inmunología , Proteínas Quinasas p38 Activadas por Mitógenos/inmunología , Western Blotting , Células Dendríticas/efectos de los fármacos , Células Dendríticas/enzimología , Relación Dosis-Respuesta Inmunológica , Inhibidores Enzimáticos/farmacología , Ensayo de Inmunoadsorción Enzimática , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Inmunoglobulina A/inmunología , Inmunoglobulina A/farmacología , Monocitos/efectos de los fármacos , Monocitos/enzimología , Receptores Fc/efectos de los fármacos , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal , Factores de TiempoRESUMEN
Neutrophils have a dual affect on epithelial pIgR/SC, the critical receptor for transcellular routing of mucosal IgA, but mechanisms of pIgR/SC upregulation remain elusive. Requirements of cytokine, redox, and signalling pathways for pIgR/SC production were assessed in human bronchial epithelial (Calu-3) cells cocultured with increasing numbers of blood neutrophils. Increased SC production was observed after incubation for 48 hrs with intermediate neutrophil numbers (1.25 to 2.5 x 10(6)), was favoured by the elastase inhibitor SLPI, and correlated with increased TGF-beta production. Exogenous TGF-beta stimulated SC production with a maximal effect at 48 hrs and both TGF-beta- and neutrophil-driven SC upregulation were dependent on redox balance and p38 MAP-kinase activation. This paper shows that activated neutrophils could upregulate epithelial pIgR/SC production through TGF-beta-mediated activation of a redox-sensitive and p38 MAPK-dependent pathway. An imbalance between the two neutrophil-driven opposite mechanisms (SC upregulation and SC degradation) could lead to downregulation of pIgR/SC, as observed in severe COPD.
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Bronquios/inmunología , Neutrófilos/inmunología , Receptores de Inmunoglobulina Polimérica/inmunología , Mucosa Respiratoria/inmunología , Componente Secretorio/inmunología , Factor de Crecimiento Transformador beta/inmunología , Bronquios/citología , Línea Celular , Células Epiteliales/inmunología , Humanos , Oxidación-Reducción , Mucosa Respiratoria/citología , Mucosa Respiratoria/metabolismo , Inhibidor Secretorio de Peptidasas Leucocitarias/farmacología , Estadísticas no Paramétricas , Factor de Crecimiento Transformador beta/biosíntesis , Factor de Crecimiento Transformador beta/farmacología , Regulación hacia Arriba/efectos de los fármacosRESUMEN
BACKGROUND: Chronic obstructive pulmonary disease (COPD) is a devastating lung disease, mainly due to cigarette smoking, which represents the third cause of mortality worldwide. The mechanisms driving its epithelial salient features remain largely elusive. We aimed to evaluate the activation and the role of the canonical, ß-catenin-dependant WNT pathway in the airway epithelium from COPD patients. METHODS: The WNT/ß-catenin pathway was first assessed by WNT-targeted RNA sequencing of the air/liquid interface-reconstituted bronchial epithelium from COPD and control patients. Airway expression of total and active ß-catenin was assessed in lung sections, as well as WNT components in laser-microdissected airway epithelium. Finally, we evaluated the role of WNT at the bronchial epithelial level by modulating the pathway in the reconstituted COPD epithelium. FINDINGS: We show that the WNT/ß-catenin pathway is upregulated in the COPD airway epithelium as compared with that of non-smokers and control smokers, in targeted RNA-sequencing of in vitro reconstituted airway epithelium, and in situ in lung tissue and laser-microdissected epithelium. Extrinsic activation of this pathway in COPD-derived airway epithelium inhibited epithelial differentiation, polarity and barrier function, and induced TGF-ß-related epithelial-to-mesenchymal transition (EMT). Conversely, canonical WNT inhibition increased ciliated cell numbers, epithelial polarity and barrier function, whilst inhibiting EMT, thus reversing COPD features. INTERPRETATION: In conclusion, the aberrant reactivation of the canonical WNT pathway in the adult airway epithelium recapitulates the diseased phenotype observed in COPD patients, suggesting that this pathway or its downstream effectors could represent a future therapeutic target. FUNDING: This study was supported by the Fondation Mont-Godinne, the FNRS and the WELBIO.
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Enfermedad Pulmonar Obstructiva Crónica/metabolismo , Mucosa Respiratoria/metabolismo , Proteínas Wnt/metabolismo , Vía de Señalización Wnt , Adulto , Anciano , Biomarcadores , Estudios de Casos y Controles , Diferenciación Celular , Células Cultivadas , Susceptibilidad a Enfermedades , Células Epiteliales/metabolismo , Transición Epitelial-Mesenquimal/genética , Femenino , Expresión Génica , Regulación de la Expresión Génica , Humanos , Masculino , Persona de Mediana Edad , Modelos Biológicos , Enfermedad Pulmonar Obstructiva Crónica/etiología , Enfermedad Pulmonar Obstructiva Crónica/patología , Pruebas de Función Respiratoria , Mucosa Respiratoria/patología , Fumar/efectos adversos , Proteínas Wnt/genética , beta Catenina/metabolismoRESUMEN
BACKGROUND: In cystic fibrosis (CF), recurrent infections suggest impaired mucosal immunity but whether production of secretory immunoglobulin A (S-IgA) is impaired remains elusive. S-IgA is generated following polymeric immunoglobulin receptor (pIgR)-mediated transepithelial transport of dimeric (d-)IgA and represents a major defence through neutralisation of inhaled pathogens like Pseudomonas aeruginosa (Pa). METHODS: Human lung tissue (n = 74), human sputum (n = 118), primary human bronchial epithelial cells (HBEC) (cultured in air-liquid interface) (n = 19) and mouse lung tissue and bronchoalveolar lavage were studied for pIgR expression, IgA secretion and regulation. FINDINGS: Increased epithelial pIgR immunostaining was observed in CF lung explants, associated with more IgA-producing plasma cells, sputum and serum IgA, especially Pa-specific IgA. In contrast, pIgR and IgA transport were downregulated in F508del mice, CFTR-inhibited HBEC, and CF HBEC. Moreover, the unfolded protein response (UPR) due to F508del mutation, inhibited IgA transport in Calu-3 cells. Conversely, pIgR expression and IgA secretion were strongly upregulated following Pa lung infection in control and F508del mice, through an inflammatory host response involving interleukin-17. INTERPRETATION: A complex regulation of IgA secretion occurs in the CF lung, UPR induced by CFTR mutation/dysfunction inhibiting d-IgA transcytosis, and Pa infection unexpectedly unleashing this secretory defence mechanism. FUNDING: This work was supported by the Forton's grant of the King Baudouin's Foundation, Belgium, the Fondazione Ricerca Fibrosi Cistica, Italy, and the Fonds National de la Recherche Scientifique, Belgium.
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Fibrosis Quística/inmunología , Inmunidad , Inmunoglobulina A/inmunología , Pulmón/inmunología , Adulto , Anciano , Animales , Biomarcadores , Línea Celular , Fibrosis Quística/genética , Fibrosis Quística/metabolismo , Fibrosis Quística/patología , Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Retículo Endoplásmico/metabolismo , Femenino , Expresión Génica , Humanos , Inmunoglobulina A/sangre , Inmunoglobulina A Secretora/inmunología , Inmunohistoquímica , Pulmón/metabolismo , Pulmón/patología , Pulmón/ultraestructura , Masculino , Ratones , Persona de Mediana Edad , Mutación , Receptores de Inmunoglobulina Polimérica/metabolismo , Mucosa Respiratoria/inmunología , Mucosa Respiratoria/metabolismo , Mucosa Respiratoria/patología , Esputo/inmunologíaRESUMEN
Small cell lung cancer (SCLC) has a poor prognosis. Focal adhesion kinase (FAK) is a non-receptor tyrosine kinase regulating cell proliferation, survival, migration, and invasion, which is overexpressed and/or activated in several cancers, including SCLC. We wanted to determine whether FAK contributes to SCLC aggressive behavior. We first evaluated the effect of FAK small-molecule inhibitor PF-573,228 in NCI-H82, NCI-H146, NCI-H196, and NCI-H446 SCLC cell lines. PF-573,228 (0.1-5 µmol/L) inhibited FAK activity by decreasing phospho-FAK (Tyr397), without modifying total FAK expression. PF-573,228 decreased proliferation, decreased DNA synthesis, induced cell-cycle arrest in G2-M phases, and increased apoptosis in all cell lines. PF-573,228 also decreased motility in adherent cell lines. To make sure that these effects were not off-target, we then used a genetic method to inhibit FAK in NCI-H82 and NCI-H446, namely stable transduction with FAK shRNA and/or FAK-related nonkinase (FRNK), a splice variant lacking the N-terminal and kinase domains. Although FAK shRNA transduction decreased total and phospho-FAK (Tyr397) expression, it did not affect proliferation, DNA synthesis, or progression through cell cycle. However, restoration of FAK-targeting (FAT) domain (attached to focal adhesion complex where it inhibits pro-proliferative proteins such as Rac-1) by FRNK transduction inhibited proliferation, DNA synthesis, and induced apoptosis. Moreover, although FAK shRNA transduction increased active Rac1 level, FRNK reexpression in cells previously transduced with FAK shRNA decreased it. Therefore, FAK appears important in SCLC biology and targeting its kinase domain may have a therapeutic potential, while targeting its FAT domain should be avoided to prevent Rac1-mediated protumoral activity.
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Quinasa 1 de Adhesión Focal/metabolismo , Neoplasias Pulmonares/enzimología , Inhibidores de Proteínas Quinasas/farmacología , Quinolonas/farmacología , Carcinoma Pulmonar de Células Pequeñas/enzimología , Sulfonas/farmacología , Adhesión Celular/efectos de los fármacos , Ciclo Celular/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Quinasa 1 de Adhesión Focal/antagonistas & inhibidores , Quinasa 1 de Adhesión Focal/genética , Humanos , Neoplasias Pulmonares/tratamiento farmacológico , Fosforilación/efectos de los fármacos , ARN Interferente Pequeño/farmacología , Carcinoma Pulmonar de Células Pequeñas/tratamiento farmacológicoRESUMEN
In COPD, epithelial changes are prominent features in the airways, such as goblet cell hyperplasia and squamous metaplasia. In contrast, it remains unclear whether ciliated cells are reduced and which pathways dysregulate epithelial differentiation. We hypothesized that bronchial epithelial cell lineage specification is dysregulated in COPD because of an aberrant reprogramming through transforming growth factor (TGF)-ß1. Surgical lung tissue from 81 COPD and 61 control (smokers and non-smokers) patients was assessed for bronchial epithelial cell phenotyping by immunohistochemistry, both in situ and in vitro in reconstituted air-liquid interface (ALI) cultures. The role of TGF-ß1 was studied in vitro. COPD epithelium in large airways, when compared to controls, showed decreased ß-tubulin IV + ciliated cells (4.4%, 2.5-8.8% versus 8.5%, 6.3-11.8% of surface staining, median and IQR, p = 0.0009) and increased MUC5AC + goblet cells (34.8%, 24.4-41.9% versus 10.3%, 5.1-17.6%, p < 0.0001). Both features were recapitulated in the ALI-cultured epithelium from COPD patients. Exogenous TGF-ß1 reduced mucociliary differentiation while neutralizing TGF-ß1 during ALI increased both specialized cell types. The COPD airway epithelium displays altered differentiation for ciliated cells, which recapitulates in vitro, at least in part through TGF-ß1.
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Bronquios/patología , Cilios/patología , Enfermedad Pulmonar Obstructiva Crónica/patología , Mucosa Respiratoria/patología , Anciano , Células Cultivadas , Femenino , Humanos , Masculino , Persona de Mediana Edad , Factor de Crecimiento Transformador beta1/análisisRESUMEN
A decrease in hemoglobin affinity for oxygen is considered an adaptive mechanism against tissue hypoxia. Obstructive sleep apnea-hypopnea syndrome (OSAHS) is characterized by recurrent episodes of apnea and hypopnea resulting in arterial oxygen desaturations during sleep. Maillard et al. (10) observed a right shift of the oxyhemoglobin dissociation curve (ODC) and an increase in 2,3-diphosphoglycerate (2,3-DPG) concentration ([2,3-DPG]) in 15 patients with severe OSAHS, but some had slight daytime arterial hypoxemia while breathing room air. The aim of our study was to measure the ODC and 2,3-DPG concentrations in a group of subjects normoxemic during daytime referred to our sleep laboratory for suspicion of snoring or OSAHS. The patients were recruited during a period of 6 mo. All arterial and venous blood samples were taken early in the morning within 1 h of awakening following a full-night polysomnography. ODC and 2,3-DPG were analyzed in 88 patients: 56 OSAHS (oxygen desaturation index: 27.5 +/- 24.5) and 32 non-OSAHS. We found a significant correlation between the P50 and 2,3-DPG levels in the 88 patients: r = 0.502, P < 0.001. We observed no difference between OSAHS and non-OSAHS for the P50 and for [2,3-DPG]. There was no correlation between the severity of OSAHS and either P50 or [2,3-DPG]. Finally, there was no change in these parameters measured at baseline, after 3 days and after 1 mo of treatment by nasal continuous positive airway pressure in 7 patients with OSAHS. We conclude that patients with OSAHS who are normoxemic during daytime have comparable oxyhemoglobin affinity than nonapneic subjects.
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Oxihemoglobinas/metabolismo , Apnea Obstructiva del Sueño/metabolismo , 2,3-Difosfoglicerato/sangre , Adulto , Análisis de los Gases de la Sangre , Índice de Masa Corporal , Presión de las Vías Aéreas Positiva Contínua , Femenino , Humanos , Hipoxia/metabolismo , Masculino , Persona de Mediana Edad , Organofosfatos/sangre , Oxígeno/sangre , Polisomnografía , Apnea Obstructiva del Sueño/terapiaRESUMEN
Caprine herpesvirus 1 (CpHV-1) is responsible of systemic infection in neonatal kids as well as abortion and fertility disorders in adult goats. This virus is closely related to bovine herpesvirus 1 (BoHV-1) which causes infectious bovine rhinotracheitis. Glycoprotein D (gD) mediates important functions in alphaherpesviruses and is also a main immunogen. The sequence of CpHV-1 gD gene and the biochemical properties of its translation product were analyzed and compared to those of BoHV-1 and other alphaherpesviruses. A relatively high homology was found between CpHV-1 and BoHV-1 glycoproteins D amino acid sequences (similarity of 68.8%). Moreover, six cysteine residues are conserved by CpHV-1 gD and the other studied alphaherpesviruses. CpHV-1 gD has a molecular mass similar to BoHV-1 gD and contains complex N-linked oligosaccharides. In contrast to the BoHV-1 gD, CpHV-1 gD is expressed as a late protein. In spite of the observed differences which could influence its biological functions, CpHV-1 gD shares most characteristics with other alphaherpesviruses and especially BoHV-1.
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Glicoproteínas/genética , Varicellovirus/química , Varicellovirus/genética , Proteínas del Envoltorio Viral/química , Proteínas del Envoltorio Viral/genética , Alphaherpesvirinae/química , Alphaherpesvirinae/genética , Secuencia de Aminoácidos , Animales , Bovinos , Línea Celular , Secuencia Conservada , Cisteína/genética , ADN Viral/química , ADN Viral/genética , Regulación Viral de la Expresión Génica , Glicoproteínas/química , Datos de Secuencia Molecular , Peso Molecular , Oligosacáridos/análisis , Oligosacáridos/química , Análisis de Secuencia de ADN , Homología de Secuencia de AminoácidoRESUMEN
STUDY OBJECTIVES: To trace the entire oxyhemoglobin dissociation curve (ODC) in a cohort of cirrhotic patients in stable condition who were candidates for orthotopic liver transplantation (OLT). DESIGN: Prospective cohort study. SETTING: A large academic hospital. PATIENTS AND METHODS: We traced the entire ODC in whole blood in standard conditions (pH 7.4; PCO2, 40 mm Hg; temperature, 37 degrees C) for 50 cirrhotic candidates for OLT (27 men and 23 women) and 50 age- and height-matched healthy subjects (27 men and 23 women). All subjects were nonsmokers or ex-smokers for at least 5 years. We also measured 2,3 diphosphoglycerate (2,3 DPG) in RBCs, plasma ions, and arterial blood gases in all subjects according to standard methods. Mixed venous blood was also obtained from the 50 cirrhotic patients. RESULTS: Mean ODC was the same in the two groups. However, for the cirrhotic patients, the dispersion of the PO2 values of oxygen saturation percentage (SO2%) from 20 to 80% was significantly larger (p < 0.01 to p < 0.0001). In the cirrhotic patients, the mean PO2 for half-saturation of hemoglobin (P50) was 7.11 + 0.14 mEq/L chloride (p < 0.001) plus 0.36 mEq/L inorganic phosphate (p < 0.05) plus 0.25 micromol/gram of hemoglobin (gHb) 2,3 DPG (p < 0.00002) in absolute numerical values. Sodium, potassium, and calcium, three plasma ions disturbed in cirrhotic patients, did not contribute to determine the mean P50. DISCUSSION: In patients with cirrhosis, increased dispersion of PO2 values for a given level of SO2% may be related to four factors: (1) an observed alteration of the enzymes controlling the phosphoglycerate shunt; (2) hypothyroidism, which may affect 7 to 20% of patients with primary biliary cirrhosis; (3) the type of ongoing treatment, eg, diuretics and/or propranolol; and (4) plasma ion disturbances. CONCLUSIONS: We describe the ODC by three indexes: shape, position, and an index of dispersion of the PO2 values for a given level of SO2%. In addition, when the latter is increased, we suggest that other factors than pH, temperature, carbon dioxide, and inorganic phosphates are acting on the position of the ODC.