RESUMEN
Biomedical research is multidisciplinary and often uses integrated approaches performing different experimental models with complementary functions. This approach is important to understand the pathogenetic mechanisms concerning the effects of environmental pollution on human health. The biological activity of the substances is investigated at least to three levels using molecular, cellular, and human tissue models. Each of these is able to give specific answers to experimental problems. A scientific approach, using biological methods (wet lab), cell cultures (cell lines or primary), isolated organs (three-dimensional cell cultures of primary epithelial cells), and animal organisms, including the human body, aimed to understand the effects of air pollution on the onset of diseases of the respiratory system. Biological methods are divided into three complementary models: in vitro, ex vivo, and in vivo. In vitro experiments do not require the use of whole organisms (in vivo study), while ex vivo experiments use isolated organs or parts of organs. The concept of complementarity and the informatic support are useful tools to organize, analyze, and interpret experimental data, with the aim of discussing scientific notions with objectivity and rationality in biology and medicine. In this scenario, the integrated and complementary use of different experimental models is important to obtain useful and global information that allows us to identify the effect of inhaled pollutants on the incidence of respiratory diseases in the exposed population. In this review, we focused our attention on the impact of air pollution in airway diseases with a rapid and descriptive analysis on the role of epithelium and on the experimental cell models useful to study the effect of toxicants on epithelial cells.
Asunto(s)
Contaminantes Atmosféricos , Contaminación del Aire , Contaminantes Atmosféricos/toxicidad , Contaminación del Aire/efectos adversos , Animales , Biomarcadores , Línea Celular , Células Epiteliales , Sistema RespiratorioRESUMEN
The role of PAR-1 expression and activation was described in epithelial cells from the central and distal airways of COPD patients using an ex vivo/in vitro model. PAR-1 immunoreactivity was studied in epithelial cells from surgical specimens of the central and distal airways of COPD patients and healthy control (HC). Furthermore, PAR-1 expression and activation were measured in both the human bronchial epithelial cell line (16HBE) and normal human bronchial epithelial cells (NHBEs) exposed to cigarette smoke extract (CSE) (10%) or thrombin. Finally, cell proliferation, apoptosis, and IL-8 release were detected in stimulated NHBEs. We identified higher levels of PAR-1 expression/activation in epithelial cells from the central airways of COPD patients than in HC. Active PAR-1 increased in epithelial cells from central and distal airways of COPD, with higher levels in COPD smokers (correlated with pack-years) than in COPD ex-smokers. 16HBE and NHBEs exposed to CSE or thrombin showed increased levels of active PAR-1 (localized in the cytoplasm) than baseline conditions, while NHBEs treated with thrombin or CSE showed increased levels of IL-8 proteins, with an additional effect when used in combination. Smoking habits generate the upregulation of PAR-1 expression/activation in airway epithelial cells, and promoting IL-8 release might affect the recruitment of infiltrating cells in the airways of COPD patients.
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Expresión Génica , Enfermedad Pulmonar Obstructiva Crónica/etiología , Enfermedad Pulmonar Obstructiva Crónica/metabolismo , Receptor PAR-1/genética , Receptor PAR-1/metabolismo , Mucosa Respiratoria/metabolismo , Apoptosis/genética , Biomarcadores , Estudios de Casos y Controles , Línea Celular , Proliferación Celular , Susceptibilidad a Enfermedades , Células Epiteliales/metabolismo , Técnica del Anticuerpo Fluorescente , Humanos , Interleucina-8/metabolismo , Enfermedad Pulmonar Obstructiva Crónica/patología , Mucosa Respiratoria/patología , Fumar/efectos adversosRESUMEN
IL-17A is overexpressed in the lung during acute neutrophilic inflammation. Acetylcholine (ACh) increases IL-8 and Muc5AC production in airway epithelial cells. We aimed to characterize the involvement of nonneuronal components of cholinergic system on IL-8 and Muc5AC production in bronchial epithelial cells stimulated with IL-17A. Bronchial epithelial cells were stimulated with recombinant human IL-17A (rhIL-17A) to evaluate the ChAT expression, the ACh binding and production, the IL-8 release, and the Muc5AC production. Furthermore, the effectiveness of PD098,059 (inhibitor of MAPKK activation), Bay11-7082 (inhibitor of IkBα phosphorylation), Hemicholinium-3 (HCh-3) (choline uptake blocker), and Tiotropium bromide (Spiriva®) (anticholinergic drug) was tested in our in vitro model. We showed that rhIL-17A increased the expression of ChAT, the levels of ACh binding and production, and the IL-8 and Muc5AC production in stimulated bronchial epithelial cells compared with untreated cells. The pretreatment of the cells with PD098,059 and Bay11-7082 decreased the ChAT expression and the ACh production/binding, while HCh-3 and Tiotropium decreased the IL-8 and Muc5AC synthesis in bronchial epithelial cells stimulated with rhIL-17A. IL-17A is involved in the IL-8 and Muc5AC production promoting, via NFκB and ERK1/2 pathway activation, the synthesis of ChAT, and the related activity of autocrine ACh in bronchial epithelial cells.
Asunto(s)
Acetilcolina/metabolismo , Células Epiteliales/efectos de los fármacos , Células Epiteliales/metabolismo , Interleucina-17/farmacología , Interleucina-8/metabolismo , Mucina 5AC/metabolismo , FN-kappa B/metabolismo , Comunicación Autocrina/efectos de los fármacos , Bronquios/citología , Línea Celular , Citometría de Flujo , Humanos , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Cigarette smoke is a risk factor for Chronic Obstructive Pulmonary Disease (COPD). Th-17 cytokines are involved in the pathogenesis of COPD. We aimed to evaluate the role of cigarette smoke on the expression of IL-17A, IL-17F and IL-17R in airways of COPD patients. Epithelial and subepithelial immunoreactivity for IL-17A, IL-17F and IL-17R was assessed in surgical specimens from COPD patients (n=15) and from healthy subjects (HC) (n=10) by immunohistochemistry. In vitro, human epithelial cell line 16HBE and A549 as well as PBMC from normal donors were stimulated with cigarette smoke extract (CSE) (0%, 2.5%, 5%, 10%) to evaluate the IL-17A, IL-17F and IL-17R expression by flow cytometry. Furthermore, rhIL-17A and CSE stimulation was evaluated on proliferation and apoptosis in 16HBE and in A549. In central and distal airways immunoreactivity for IL-17A, IL-17F and IL-17R significantly increased in the epithelium and IL-17A in the subepithelium from COPD than in HC. In distal airway, immunoreactivity for IL-17F increased in the subepithelium of COPD than in HC. IL-17A immunoreactivity positively correlate with IL-17R and total pack years in the epithelium from central and distal airways of COPD patients. In vitro, CSE stimulation significantly increased IL-17F and IL-17R in 16HBE (2.5%) and A549 (5%) while IL-17A and IL-17F in PBMC (10%). IL-17A and CSE stimulation, rather than CSE or rhIL-17A alone, significantly increased proliferation in 16HBE and apoptosis in A549. Cigarette smoke increases Th17 immunity in lung tissue of COPD patients, promoting the mechanism of proliferation and apoptosis in airway epithelial cells.
Asunto(s)
Interleucina-17/metabolismo , Pulmón/metabolismo , Nicotiana , Receptores de Interleucina-17/metabolismo , Humo , Anciano , Femenino , Humanos , Técnicas In Vitro , Masculino , Persona de Mediana Edad , Enfermedad Pulmonar Obstructiva Crónica/metabolismoRESUMEN
Airway epithelium alterations, including squamous cell metaplasia, characterize smokers with and without chronic obstructive pulmonary disease (COPD). The p21 regulates cell apoptosis and differentiation and its role in COPD is largely unknown. Molecules regulating apoptosis (cytoplasmic p21, caspase-3), cell cycle (nuclear p21), proliferation (Ki67/PCNA), and metaplasia (survivin) in central airways from smokers (S), smokers-COPD (s-COPD) and non-smokers (Controls) were studied. The role of cigarette smoke extracts (CSE) in p21, survivin, apoptosis (caspase-3 and annexin-V binding) and proliferation was assessed in a bronchial epithelial cell line (16HBE). Immunohistochemistry, image analysis in surgical samples and flow-cytometry and carboxyfluorescein succinimidyl ester proliferative assay in 16HBE with/without CSE were applied. Cytoplasmic and nuclear p21, survivin, and Ki67 expression significantly increased in large airway epithelium in S and in s-COPD in comparison to Controls. Caspase-3 was similar in all the studied groups. p21 correlated with epithelial metaplasia, PCNA, and Ki67 expression. CSE increased cytoplasmic p21 and survivin expression but not apoptosis and inhibited the cell proliferation in 16HBE. In large airway epithelium of smokers with and without COPD, the cytoplasmic p21 inhibits cell apoptosis, promotes cell proliferation and correlates with squamous cell metaplasia thus representing a potential pre-oncogenic hallmark.
Asunto(s)
Bronquios/fisiopatología , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/fisiología , Enfermedad Pulmonar Obstructiva Crónica/fisiopatología , Fumar/fisiopatología , Anciano , Bronquios/enzimología , Bronquios/metabolismo , Estudios de Casos y Controles , Caspasa 3/metabolismo , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/metabolismo , Epitelio/fisiopatología , Femenino , Humanos , Antígeno Ki-67/metabolismo , Masculino , Persona de Mediana Edad , Antígeno Nuclear de Célula en Proliferación/metabolismo , Enfermedad Pulmonar Obstructiva Crónica/enzimología , Enfermedad Pulmonar Obstructiva Crónica/metabolismoRESUMEN
The induction of nitric oxide synthase (iNOS) expression via the signal transducer and activator of transcription 1 (STAT-1) is involved in the mechanism of oxidative/nitrosative stress. We investigated whether acetylcholine (ACh) generates oxidative/nitrosative stress in bronchial epithelial cells during airway inflammation of COPD and evaluated the effects of Tiotropium, a once-daily antimuscarinic drug, and Olodaterol, a long-acting ß2-agonist on these mechanisms. Human bronchial epithelial cells (16-HBE) were stimulated (4h, 37°C) with induced sputum supernatants (ISSs) from healthy controls (HC) (n=10), healthy smokers (HS) (n=10) or COPD patients (n=10), as well as with ACh (from 1µM to 100µM). The activation of STAT-1 pathway (STAT-1Ser727 and STAT-1Tyr701) and iNOS was evaluated in the cell lysates by Western blot analysis as well as nitrotyrosine levels by ELISA, while reactive oxygen species (ROS) were evaluated by flow cytometry. Finally, the effect of Tiotropium (Spiriva®) (100nM), alone or in combination with Olodaterol (1nM), was tested in this model. ISSs from COPD patients significantly increased the phosphorylation of STAT-1Ser727 and STAT-1Tyr701, iNOS and ROS/Nitrotyrosine when compared with ISSs from HC or HS subjects in 16-HBE cells. Furthermore, synthetic ACh increased all these parameters in stimulated 16HBE when compared with untreated cells. Tiotropium and Olodaterol reduced the oxidative/nitrosative stress generated by ACh and ISSs. We concluded that ACh mediated the oxidative/nitrosative stress involving the STAT-1 pathway activation in human bronchial epithelial cells during COPD. ß2-Long acting and antimuscarinic drugs, normally used in the treatment of COPD as bronchodilator, might be able to control these cellular events.
Asunto(s)
Acetilcolina/farmacología , Bronquios/efectos de los fármacos , Células Epiteliales/efectos de los fármacos , Estrés Oxidativo/efectos de los fármacos , Enfermedad Pulmonar Obstructiva Crónica/patología , Factor de Transcripción STAT1/metabolismo , Western Blotting , Bronquios/citología , Bronquios/metabolismo , Células Cultivadas , Agonistas Colinérgicos/farmacología , Células Epiteliales/citología , Células Epiteliales/metabolismo , Femenino , Humanos , Masculino , Persona de Mediana Edad , Óxido Nítrico Sintasa de Tipo II/metabolismo , Enfermedad Pulmonar Obstructiva Crónica/tratamiento farmacológico , Enfermedad Pulmonar Obstructiva Crónica/metabolismo , ARN Interferente Pequeño/genética , Especies Reactivas de Oxígeno/metabolismo , Factor de Transcripción STAT1/antagonistas & inhibidores , Factor de Transcripción STAT1/genética , Tirosina/análogos & derivados , Tirosina/metabolismoRESUMEN
IL-17A drives inflammation and oxidative stress, affecting the progression of chronic lung diseases (asthma, chronic obstructive pulmonary disease (COPD), lung cancer, and cystic fibrosis). Oleuropein (OLP) is a polyphenolic compound present in olive oil and widely included in the Mediterranean diet. It exerts antioxidant and anti-inflammatory activities, oxidative stress resistance, and anticarcinogenic effects with a conceivable positive impact on human health. We hypothesized that OLP positively affects the mechanisms of oxidative stress, apoptosis, DNA damage, cell viability during proliferation, and cell growth in alveolar epithelial cells and tested its effect in a human alveolar epithelial cell line (A549) in the presence of IL-17A. Our results show that OLP decreases the levels of oxidative stress (Reactive Oxygen Species, Mitochondrial membrane potential) and DNA damage (H2AX phosphorylation-ser139, Olive Tail Moment data) and increases cell apoptosis in A549 cells exposed to IL-17A. Furthermore, OLP decreases the number of viable cells during proliferation, the migratory potential (Scratch test), and the single cell capacity to grow within colonies as a cancer phenotype in A549 cells exposed to IL-17A. In conclusion, we suggest that OLP might be useful to protect lung epithelial cells from oxidative stress, DNA damage, cell growth, and cell apoptosis. This effect might be exerted in lung diseases by the downregulation of IL-17A activities. Our results suggest a positive effect of the components of olive oil on human lung health.
Asunto(s)
Apoptosis , Proliferación Celular , Daño del ADN , Interleucina-17 , Glucósidos Iridoides , Iridoides , Estrés Oxidativo , Humanos , Estrés Oxidativo/efectos de los fármacos , Interleucina-17/metabolismo , Glucósidos Iridoides/farmacología , Proliferación Celular/efectos de los fármacos , Células A549 , Daño del ADN/efectos de los fármacos , Apoptosis/efectos de los fármacos , Iridoides/farmacología , Especies Reactivas de Oxígeno/metabolismo , Antioxidantes/farmacología , Supervivencia Celular/efectos de los fármacos , Pulmón/efectos de los fármacos , Pulmón/metabolismo , Células Epiteliales/efectos de los fármacos , Células Epiteliales/metabolismo , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Aceite de Oliva/farmacología , Células Epiteliales Alveolares/efectos de los fármacos , Células Epiteliales Alveolares/metabolismoRESUMEN
We quantified TGF-ß1 and acetylcholine (ACh) concentrations in induced sputum supernatants (ISSs) from 18 healthy controls (HC), 22 healthy smokers (HS) and 21 COPDs. ISSs from HC, HS and COPD as well as rhTGF-ß1 were also tested in neutrophil adhesion and in mAChR2, mAChR3 and ChAT expression experiments in human bronchial epithelial cells (16-HBE). Finally, we evaluated the effects of Olodaterol (a novel inhaled ß(2)-adrenoceptor agonist) and Tiotropium Spiriva®, alone or in combination, on neutrophil adhesion and mAChRs and ChAT expression in stimulated 16-HBE. The results showed that 1) TGF-ß1 and ACh concentrations are increased in ISSs from COPD in comparison to HC and HS, and TGF-ß1 in HS is higher than in HC; 2) ISSs from COPD and HS caused increased neutrophil adhesion to 16-HBE when compared to ISSs from HC. The effect of ISSs from COPD was significantly reduced by TGF-ß1 depletion or by the pretreatment with Olodaterol or Tiotropium alone or in combination, while the effect of ISSs from HS was significantly reduced by the pretreatment with Olodaterol alone; 3) mAChR2, mAChR3 and ChAT expression was increased in 16-HBE stimulated with ISSs from COPD and TGF-ß1 depletion significantly reduced this effect on mAChR3 and ChAT expression; 4) rhTGF-ß1 increased mAChR2, mAChR3 and ChAT expression in 16-HBE; 5) Olodaterol did not affect the expression of mAChRs and ChAT in 16-HBE. Our findings support the use of ß2 long-acting and anticholinergic drugs to control the bronchoconstriction and TGF-ß1-mediated neutrophilic inflammation in COPD.
Asunto(s)
Acetilcolina/metabolismo , Agonistas de Receptores Adrenérgicos beta 2/uso terapéutico , Antagonistas Colinérgicos/uso terapéutico , Neutrófilos/efectos de los fármacos , Enfermedad Pulmonar Obstructiva Crónica/tratamiento farmacológico , Factor de Crecimiento Transformador beta1/metabolismo , Acetilcolina/análisis , Agonistas de Receptores Adrenérgicos beta 2/farmacología , Anciano , Análisis de Varianza , Benzoxazinas/farmacología , Benzoxazinas/uso terapéutico , Broncoconstricción/efectos de los fármacos , Broncodilatadores/farmacología , Broncodilatadores/uso terapéutico , Estudios de Casos y Controles , Adhesión Celular/efectos de los fármacos , Línea Celular Transformada , Colina O-Acetiltransferasa/metabolismo , Antagonistas Colinérgicos/farmacología , Quimioterapia Combinada , Células Epiteliales/efectos de los fármacos , Femenino , Citometría de Flujo , Humanos , Masculino , Persona de Mediana Edad , Enfermedad Pulmonar Obstructiva Crónica/metabolismo , Receptor Muscarínico M2/metabolismo , Receptor Muscarínico M3/metabolismo , Derivados de Escopolamina/farmacología , Derivados de Escopolamina/uso terapéutico , Fumar/efectos adversos , Esputo/química , Bromuro de Tiotropio , Factor de Crecimiento Transformador beta1/análisisRESUMEN
We aimed to evaluate the therapeutic effect of nebulized beclomethasone dipropionate (nBDP) on both allergic asthma and rhinitis. In a randomized, double-blind, placebo-controlled study, 40 children (mean age 10.7 ± 2.1 years) with allergic asthma and rhinitis received either nBDP (daily dose of 800 µg, administered twice daily) or placebo for 4 weeks (with a face mask), after a 2-week run-in period of clinical assessment. Nasal and oral fractional exhaled nitric oxide (FeNO) measurements together with pulmonary function tests, nasal and oral exhaled breath condensate (EBC) collection for pH and interleukin-5 (IL-5) measurements as well as nasal and bronchial symptom scores were obtained at baseline and after 4-week treatment. A significant improvement in oral FeNO, oral and nasal EBC IL-5 and nasal EBC pH was observed in the nBDP group when comparing the values with baseline, together with an improvement in symptom score of the visual analogue scale, nasal obstruction, sneezing, rhinorrhea, breathing difficulty, cough, wheezing and sleep disturbance (nBDP end treatment vs. baseline, Wilcoxon signed-rank test). nBDP was more effective than placebo (ANCOVA test) in improving [difference Δ = response after treatment at the last visit (active or placebo) - value at baseline] nasal pH, oral IL-5, oral FeNO, forced expiratory volume in 1 s, forced expiratory volume in 1 s/forced vital capacity, peek expiratory flow, visual analogue scale, breathing difficulty, cough, wheezing and sleep disturbance scores. No differences were observed between the nBDP and the placebo group for symptom score of rhinitis. nBDP is a useful treatment for airway inflammation and clinical status in children with concomitant allergic asthma and rhinitis.
Asunto(s)
Antiasmáticos/administración & dosificación , Asma/tratamiento farmacológico , Beclometasona/administración & dosificación , Rinitis Alérgica Perenne/tratamiento farmacológico , Administración por Inhalación , Adolescente , Agonistas de Receptores Adrenérgicos beta 2/administración & dosificación , Asma/metabolismo , Asma/fisiopatología , Pruebas Respiratorias , Niño , Método Doble Ciego , Femenino , Humanos , Inmunoglobulina E/sangre , Interleucina-5/metabolismo , Masculino , Nebulizadores y Vaporizadores , Óxido Nítrico/metabolismo , Proyectos Piloto , Pruebas de Función Respiratoria , Rinitis Alérgica Perenne/metabolismo , Rinitis Alérgica Perenne/fisiopatologíaRESUMEN
Autophagy is the key process by which the cell degrades parts of itself within the lysosomes. It maintains cell survival and homeostasis by removing molecules (particularly proteins), subcellular organelles, damaged cytoplasmic macromolecules, and by recycling the degradation products. The selective removal or degradation of mitochondria is a particular type of autophagy called mitophagy. Various forms of cellular stress (oxidative stress (OS), hypoxia, pathogen infections) affect autophagy by inducing free radicals and reactive oxygen species (ROS) formation to promote the antioxidant response. Dysfunctional mechanisms of autophagy have been found in different respiratory diseases such as chronic obstructive lung disease (COPD) and asthma, involving epithelial cells. Several existing clinically approved drugs may modulate autophagy to varying extents. However, these drugs are nonspecific and not currently utilized to manipulate autophagy in airway diseases. In this review, we provide an overview of different autophagic pathways with particular attention on the dysfunctional mechanisms of autophagy in the epithelial cells during asthma and COPD. Our aim is to further deepen and disclose the research in this direction to stimulate the develop of new and selective drugs to regulate autophagy for asthma and COPD treatment.
Asunto(s)
Asma , Enfermedad Pulmonar Obstructiva Crónica , Trastornos Respiratorios , Humanos , Mitofagia , Autofagia , Estrés Oxidativo , Células Epiteliales , LisosomasRESUMEN
AIMS: The lung epithelial cells form a physical barrier to the external environment acting as the first line of defence against potentially harmful environmental stimuli. These cells interact with several other cellular components, of which macrophages are some of the most relevant. We analysed the effects of the PBDE-47 on the microRNA cargo of THP-1 macrophage like derived small Extracellular Vesicles (sEVs) and the effects on A549 lung epithelial cells. MAIN METHODS: sEVs from M(LPS) THP-1 macrophage-like cells after PBDE-47 treatment (sEVsPBDE+LPS) were characterized by nanoparticle tracking analysis and their microRNA cargo studied by qPCR. Confocal microscopy was applied to study sEVs cellular uptake by A549 cells. The expression of tight junctions (TJs), adhesion molecules, inflammation markers and mucus production in A549 cultured in air liquid interface (ALI) conditions were studied by Real Time PCR and confocal microscopy. KEY FINDINGS: sEVsPBDE+LPS microRNA cargo analysis showed that the PBDE-47 modulated the expression of the miR-15a-5p, miR29a-3p, miR-143-3p and miR-122-5p. Furthermore, ALI cultured A549 cells incubated with sEVsPBDE+LPS showed that zonula occludens-1 (p ≤ 0.04), claudin (p ≤ 0.02), E-cadherin (p ≤ 0.006) and Vimentin (p ≤ 0.0008) mRNAs were increased in A549 cells after sEVsPBDE+LPS treatment. Indeed, Interleukin (IL)-8 (p ≤ 0.008) and mucin (MUC5AC and MUC5B) (p ≤ 0.03 and p ≤ 0.0001) mRNA expression were up- and down-regulated, respectively. SIGNIFICANCE: PBDE-47 treated macrophages secrete sEVs with altered microRNA cargo that affect the mRNA expression of TJs, adhesion molecules, cytokines and EMT markers damaging the normal function of the lung epithelium, potentially contributing to the development of lung diseases.
Asunto(s)
Vesículas Extracelulares , MicroARNs , Humanos , Lipopolisacáridos/farmacología , MicroARNs/metabolismo , Macrófagos/metabolismo , Epitelio/metabolismo , Vesículas Extracelulares/metabolismo , Pulmón/metabolismo , Inflamación/metabolismoRESUMEN
BACKGROUND: Nuclear factor-κB (NF-κB) is a transcriptional factor of different inflammatory patterns involved in asthma and chronic obstructive pulmonary disease (COPD) that is tightly controlled by IκB kinase (IKK) complex. OBJECTIVE: We investigated the dysregulation of IKK-driven NF-κB activation in patients with asthma and COPD. METHODS: We assessed IKKα and IKKß expression and activation, their regulation by glucocorticosteroids, and their involvement in IL-8 synthesis in PBMCs isolated from asthmatic patients, healthy smokers (HSs), patients with COPD, and control subjects. PBMCs from control subjects were stimulated with TNF-α and cigarette smoke extract in the presence or absence of fluticasone propionate (FP), L-glutathione reduced, or both, and IKK activation and IL-8 release were evaluated. RESULTS: IKKα activity was higher in patients with COPD and HSs than in asthmatic patients and control subjects. IKKß activity was higher in asthmatic patients, HSs, and patients with COPD than in control subjects. In vitro FP treatment induced inhibition of both IKKα and IKKß activity in PBMCs from asthmatic patients, patients with COPD, and HSs, although IKKß activity was more sensitive to FP than that of IKKα. FP reduced the IL-8 released from PBMCs of asthmatic patients, patients with COPD, and HSs, although IL-8 inhibition was higher in asthmatic patients than in patients with COPD and HSs. FP reduced IKKα and IKKß activities in TNF-α and cigarette smoke extract-treated PBMCs, with higher levels of inhibition for IKKß than IKKα activity. L-glutathione reduced improved the downregulatory effects of FP on IKKα and IL-8 levels. CONCLUSION: Based on differential activation of IKKα and IKKß, our findings suggest a different profile in the upstream regulation of the IKK-driven NF-κB system in asthmatic patients and patients with COPD. These differences in the regulation of the inflammatory process may explain, at least in part, the different pharmacologic responses in these patients.
Asunto(s)
Asma/patología , Regulación de la Expresión Génica , Quinasa I-kappa B/metabolismo , Leucocitos Mononucleares/inmunología , FN-kappa B/metabolismo , Enfermedad Pulmonar Obstructiva Crónica/patología , Adulto , Asma/tratamiento farmacológico , Asma/metabolismo , Broncodilatadores/uso terapéutico , Activación Enzimática , Femenino , Glucocorticoides/uso terapéutico , Humanos , Interleucina-8/metabolismo , Masculino , Persona de Mediana Edad , Enfermedad Pulmonar Obstructiva Crónica/tratamiento farmacológico , Enfermedad Pulmonar Obstructiva Crónica/metabolismo , FumarRESUMEN
Inflammation of the human lung is mediated in response to different stimuli (e.g., physical, radioactive, infective, pro-allergenic, or toxic) such as cigarette smoke and environmental pollutants. These stimuli often promote an increase in different inflammatory activities in the airways, manifesting themselves as chronic diseases (e.g., allergic airway diseases, asthma chronic bronchitis/chronic obstructive pulmonary disease, or even lung cancer). Non-coding RNA (ncRNAs) are single-stranded RNA molecules of few nucleotides that regulate the gene expression involved in many cellular processes. ncRNA are molecules typically involved in the reduction of translation and stability of the genes of mRNAs s. They regulate many biological aspects such as cellular growth, proliferation, differentiation, regulation of cell cycle, aging, apoptosis, metabolism, and neuronal patterning, and influence a wide range of biologic processes essential for the maintenance of cellular homeostasis. The relevance of ncRNAs in the pathogenetic mechanisms of respiratory diseases has been widely established and in the last decade many papers were published. However, once their importance is established in pathogenetic mechanisms, it becomes important to further deepen the research in this direction. In this review we describe several of most recent knowledge concerning ncRNA (overall miRNAs) expression and activities in the lung.
RESUMEN
Inflammation of the human lung is mediated in response to different stimuli (e.g., physical, radioactive, infective, pro-allergenic or toxic) such as cigarette smoke and environmental pollutants. They often promote an increase in inflammatory activities in the airways that manifest themselves as chronic diseases (e.g., allergic airway diseases, asthma, chronic bronchitis/chronic obstructive pulmonary disease (COPD) or even lung cancer). Increased levels of oxidative stress (OS) reduce the antioxidant defenses, affect the autophagy/mitophagy processes, and the regulatory mechanisms of cell survival, promoting inflammation in the lung. In fact, OS potentiate the inflammatory activities in the lung, favoring the progression of chronic airway diseases. OS increases the production of reactive oxygen species (ROS), including superoxide anions (O2-), hydroxyl radicals (OH) and hydrogen peroxide (H2O2), by the transformation of oxygen through enzymatic and non-enzymatic reactions. In this manner, OS reduces endogenous antioxidant defenses in both nucleated and non-nucleated cells. The production of ROS in the lung can derive from both exogenous insults (cigarette smoke or environmental pollution) and endogenous sources such as cell injury and/or activated inflammatory and structural cells. In this review, we describe the most relevant knowledge concerning the functional interrelation between the mechanisms of OS and inflammation in airway diseases.
RESUMEN
The airway epithelium is a dynamic tissue that undergoes slow but constant renewal. Dysregulation of airway epithelial function related to cigarette smoke exposure plays an important role in the pathophysiology of COPD. Oct4 is a transcription factor responsible for maintaining cellular self-renewal and regeneration, and CD146 and CD105/Endoglin are adhesion molecules involved in cell proliferation, differentiation, epithelial-mesenchymal-transition and tissue remodeling. Bronchial biopsy specimens (BBs) were obtained from 7 healthy controls (HC) and 10 COPD and subjected to paraffin embedding; BBs from HC were also used for epithelial cell expansion and pHBEC/ALI (air-liquid interface) culture. pHBEC/ALI were exposed to cigarette smoke extract (CSE) for 7, 14 and 21 days. In BBs, Oct4, CD146 and CD105 were evaluated by immunohistochemistry. In pHBEC/ALI, the expression of Oct4, CD146, CD105 and acetyl-αtubulin was evaluated by Western Blot, MUC5AC and IL-8 measurements by ELISA. The Oct4 epithelial immunoreactivity was lower in COPD than in HC, whilst CD146 and CD105 expression was higher in COPD than in HC. In pHBEC/ALI, Transepithelial Electrical Resistance values, measured over 7 to 21 days of differentiation, decreased by 18% (2.5% CSE) and 29% (5% CSE) compared to untreated samples. Oct4 and acetyl-αtubulin were induced after one-week differentiation and downregulated by CSE in reconstituted epithelium; CD146, CD105, MUC5AC and IL-8 were increased by CSE. Oct4 de-regulation and CD146 and CD105 overexpression, induced by cigarette smoke exposure, might play a role in airway epithelial dysfunction by causing changes in self-renewal and mesenchymal transition mechanisms, leading to alteration of epithelium homeostasis and abnormal tissue remodeling involved in progression of COPD.
Asunto(s)
Fumar Cigarrillos/efectos adversos , Endoglina/metabolismo , Transición Epitelial-Mesenquimal , Factor 3 de Transcripción de Unión a Octámeros/metabolismo , Enfermedad Pulmonar Obstructiva Crónica/patología , Sistema Respiratorio/patología , Adulto , Anciano , Antígeno CD146/genética , Antígeno CD146/metabolismo , Estudios de Casos y Controles , Diferenciación Celular , Endoglina/genética , Femenino , Humanos , Masculino , Persona de Mediana Edad , Factor 3 de Transcripción de Unión a Octámeros/genética , Enfermedad Pulmonar Obstructiva Crónica/inducido químicamente , Enfermedad Pulmonar Obstructiva Crónica/genética , Enfermedad Pulmonar Obstructiva Crónica/metabolismo , Sistema Respiratorio/efectos de los fármacos , Sistema Respiratorio/metabolismoRESUMEN
AIMS: We aimed to investigate the effect of PBDEs (47, 99, 209) on cellular events involved in epigenetic modification, inflammation, and epithelial mesenchymal transition (EMT). MATERIALS AND METHODS: We studied: 1) ERK1/2 phosphorylation; 2) Enhancer of Zester Homolog 2 (EZH2); 3) Histone H3 tri-methylated in lysine 27 (H3K27me3); 4) K-RAS; 5) silencing disabled homolog 2-interacting protein gene (DAB2IP), 6) let-7a; 7) Muc5AC/Muc5B, and 8) IL-8 in a 3D in vitro model of epithelium obtained with primary Normal Human Bronchial Epithelial cells (pNHBEs) or A549 cell line, chronically exposed to PBDEs (47, 99, 209). KEY FINDINGS: PBDEs (10 nM, 100 nM and 1 µM) increased ERK1/2 phosphorylation, and EZH2, H3K27me3, and K-RAS protein expression, while decreased DAB2IP and Let-7a transcripts in pNHBEs ALI culture. Furthermore PBDEs (47, 99) (100 nM) increased Muc5AC and Muc5B mRNA, and PBDE 47 (100 nM) IL-8 mRNA via EZH2 in pNHBEs. Finally, PBDEs (100 nM) affected EZH2, H3K27me3, K-RAS protein expression, and DAB2IP, Let-7a transcripts and cell invasion in A549 cells. Gsk343 (methyltransferase EZH2 inhibitor) (1 mM) and U0126 (inhibitor of MEK1/2) (10 µM) were used to show the specific effect of PBDEs. SIGNIFICANCE: PBDE inhalation might promote inflammation/cancer via EZH2 methyltransferase activity and H3K27me3, k-RAS and ERk1/2 involvement, generating adverse health outcomes of the human lung.
Asunto(s)
Proteína Potenciadora del Homólogo Zeste 2/metabolismo , Células Epiteliales , Retardadores de Llama/administración & dosificación , Éteres Difenilos Halogenados/efectos adversos , Neoplasias Pulmonares , Proteínas de Neoplasias/metabolismo , Mucosa Respiratoria , Células A549 , Anciano , Células Epiteliales/enzimología , Células Epiteliales/patología , Femenino , Retardadores de Llama/farmacología , Éteres Difenilos Halogenados/farmacología , Humanos , Inflamación/inducido químicamente , Inflamación/metabolismo , Inflamación/patología , Neoplasias Pulmonares/inducido químicamente , Neoplasias Pulmonares/enzimología , Neoplasias Pulmonares/patología , Masculino , Persona de Mediana Edad , Mucosa Respiratoria/enzimología , Mucosa Respiratoria/patologíaRESUMEN
Cigarette smoke is the main cause of chronic obstructive pulmonary disease (COPD), where it can contribute to the observed airway inflammation. PGE(2) is produced within human airways, and both pro- and anti-inflammatory activities have been reported. We quantitated PGE(2) concentrations in induced sputum supernatants from different groups of subjects and correlated the obtained values to neutrophil infiltration as well as to the expression of cyclooxygenase-2 (COX-2). Cigarette smoke extract (CSE) was used to evaluate the effect of smoking on COX-2 and PGE(2) receptor expression as well as on PGE(2) release in neutrophils and alveolar macrophages (AM) obtained from normal donors. The effects of PGE(2) and of PGE receptor agonists and antagonists were evaluated on the adhesion of neutrophil to a human bronchial epithelial cell line (16HBE). PGE(2) levels, COX-2 expression, and neutrophil infiltration were significantly higher in normal smokers and COPD smokers (P < 0.0001) compared with controls and COPD former smokers. Induced sputum supernatant caused neutrophil adhesion to 16HBE that was significantly reduced, in COPD smokers only, by PGE(2) immunoprecipitation. In vitro experiments confirmed that CSE increased PGE(2) release and COX-2 and PGE(2) receptor expression in neutrophils and AM; PGE(2) enhanced the adhesion of neutrophils to 16HBE, and a specific E-prostanoid 4 (EP(4)) receptor antagonist blunted its effect. These results suggest that CSE promote the induction of COX-2 and contributes to the proinflammatory effects of PGE(2) in the airways of COPD subjects.
Asunto(s)
Dinoprostona/metabolismo , Infiltración Neutrófila/inmunología , Prostaglandina-Endoperóxido Sintasas/metabolismo , Enfermedad Pulmonar Obstructiva Crónica , Humo/efectos adversos , Fumar/efectos adversos , Anciano , Adhesión Celular/fisiología , Femenino , Humanos , Macrófagos/citología , Macrófagos/inmunología , Masculino , Persona de Mediana Edad , Neutrófilos/citología , Neutrófilos/inmunología , Isoformas de Proteínas/metabolismo , Enfermedad Pulmonar Obstructiva Crónica/enzimología , Enfermedad Pulmonar Obstructiva Crónica/etiología , Enfermedad Pulmonar Obstructiva Crónica/inmunología , Receptores de Prostaglandina E/metabolismo , Esputo/química , Esputo/citologíaRESUMEN
Environmental agents, including viral and bacterial infectious agents, are involved in the alteration of physicochemical and biological parameters in the nasal epithelium. Hyaluronan (HA) has an important role in the regulation of tissue healing properties. High molecular weight HA (HMW-HA) shows greater anti-inflammatory responses than medium molecular weight HA (MMW-HA) and low molecular weight HA (LMW-HA). We investigated the effect of HMW-HA, MMW-HA and LMW-HA on the regulation of physicochemical and biological parameters in an "in vitro" model that might mimic viral infections of the nasal epithelium. Human nasal epithelial cell line RPMI2650 was stimulated with double-stranded RNA (dsRNA) Poly(I:C) for 5 days in air-liquid-interface (ALI) culture (3D model of airway tissue). dsRNA Poly(I:C) treatment significantly decreased transepithelial electrical resistance (TEER) in the stratified nasal epithelium of RPMI2650 and increased pH values, rheological parameters (elastic G' and viscous G"), and Muc5AC and Muc5B production in the apical wash of ALI culture of RPMI2650 in comparison to untreated cells. RPMI2650 treated with dsRNA Poly(I:C) in the presence of HMW-HA showed lower pH values, Muc5AC and Muc5B production, and rheological parameters, as well as increased TEER values in ALI culture, compared to cells treated with Poly(I:C) alone or pretreated with LMW-HA and MMW-HA. Our 3D "in vitro" model of epithelium suggests that HMW-HA might be a coadjuvant in the pharmacological treatment of viral infections, allowing for the control of some physicochemical and biological properties affecting the epithelial barrier of the nose during infection.
RESUMEN
Polybrominated diphenyl ethers (PBDEs) are widespread as flame-retardants in different types of consumer products. PBDEs present in the air or dust and their inhalation can damage human health by influencing the respiratory system. We evaluated the effects of environment relevant concentrations (0.01-1 µM) of PBDE-47, PBDE-99 and PBDE-209 on the mechanism of oxidative stress, dysregulation of cell proliferation, apoptosis, and DNA damage and repair (in term of H2AX phosphorylation ser139) in an in-vitro/ex-vivo model of bronchial epithelial cells. PBDEs (-47, -99 and -209) at the environment relevant concentrations (0.01 and 1 µM) induce oxidative stress (in term of NOX-4 expression as well as ROS and JC-1 production), activate the mechanism of DNA-damage and repair affecting Olive Tail length (comet assay) production and H2AX phosphorylation (ser139) in normal human bronchial epithelial cells. Furthermore PBDEs, although do not affect cell viability, induce cell apoptosis and single cell capacity to grow into a colony (like a cancer phenotype) in bronchial epithelial cells. Finally, PBDE-47 had a greater effect than -99 and -209. PBDE-47, -99 and -209 congeners exert cytotoxic and genotoxic effects, and play a critical role in the dysregulation of oxidative stress, damaging DNA and the related gene expression in bronchial epithelial cells. Our findings might suggest that PBDEs inhalation might have adverse effect on human health regarding pulmonary diseases in the areas of environmental pollution.
Asunto(s)
Bronquios/patología , Células Epiteliales/efectos de los fármacos , Retardadores de Llama/toxicidad , Éteres Difenilos Halogenados/toxicidad , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Daño del ADN/efectos de los fármacos , Células Epiteliales/metabolismo , Retardadores de Llama/análisis , Éteres Difenilos Halogenados/análisis , Humanos , Estrés Oxidativo/efectos de los fármacosRESUMEN
Brominated flame-retardant (BFRs) exposure promotes multiple adverse health outcomes involved in oxidative stress, inï¬ammation, and tissues damage. We investigated BFR effects, known as polybrominated diphenyl ethers (PBDEs) (47, 99 and 209) in an air-liquid-interface (ALI) airway tissue derived from A549â¯cell line, and compared with ALI culture of primary human bronchial epithelial cells (pHBEC). The cells, exposed to PBDEs (47, 99 and 209) (0.01-1⯵M) for 24â¯h, were studied for IL-8, Muc5AC and Muc5B (mRNAs and proteins) production, as well as NOX-4 (mRNA) expression. Furthermore, we evaluated tight junction (TJ) integrity by Trans-Epithelial Electrical Resistance (TEER) measurements, and zonula occludens-1 (ZO-1) expression in the cells, and pH variations and rheological properties (elastic G', and viscous Gâ³, moduli) in apical washes of ALI cultures. N-acetylcysteine (NAC) (10â¯mM) effects were tested in our experimental model of A549â¯cells. PBDEs (47, 99 and 209) exposure decreased TEER, ZO-1 and pH values, and increased IL-8, Muc5AC, Muc5B (mRNAs and proteins), NOX-4 (mRNA), and rheological parameters (G', Gâ³) in ALI cultures of A549â¯cell line and pHBEC. NAC inhibited PBDE effects in A549â¯cells. PBDE inhalation might impairs human health of the lungs inducing oxidative stress, inflammatory response, loss of barrier integrity, unchecked mucus production, as well as altered physicochemical and biological properties of the fluids in airway epithelium. The treatment with anti-oxidants restored the negative effects of PBDEs in epithelial cells.