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RATIONALE: Aberrant expression of microRNAs (miRNAs) can have a detrimental role in disease pathogenesis. OBJECTIVES: To identify dysregulated miRNAs in lung tissue of patients with chronic obstructive pulmonary disease (COPD). METHODS: We performed miRNA and mRNA profiling using high throughput stem-loop reverse-transcriptase quantitative polymerase chain reaction and mRNA microarray, respectively, on lung tissue of 30 patients (screening cohort) encompassing 8 never-smokers, 10 smokers without airflow limitation, and 12 smokers with COPD. Differential expression of miRNA-218-5p (miR-218-5p) was validated by reverse-transcriptase quantitative polymerase chain reaction in an independent cohort of 71 patients, an in vivo murine model of COPD, and primary human bronchial epithelial cells. Localization of miR-218-5p was assessed by in situ hybridization. In vitro and in vivo perturbation of miR-218-5p combined with RNA sequencing and gene set enrichment analysis was used to elucidate its functional role in COPD pathogenesis. MEASUREMENTS AND MAIN RESULTS: Several miRNAs were differentially expressed among the different patient groups. Interestingly, miR-218-5p was significantly down-regulated in smokers without airflow limitation and in patients with COPD compared with never-smokers. Decreased pulmonary expression of miR-218-5p was validated in an independent validation cohort, in cigarette smoke-exposed mice, and in human bronchial epithelial cells. Importantly, expression of miR-218-5p strongly correlated with airway obstruction. Furthermore, cellular localization of miR-218-5p in human and murine lung revealed highest expression of miR-218-5p in the bronchial airway epithelium. Perturbation experiments with a miR-218-5p mimic or inhibitor demonstrated a protective role of miR-218-5p in cigarette smoke-induced inflammation and COPD. CONCLUSIONS: We highlight a role for miR-218-5p in the pathogenesis of COPD.
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MicroARNs/fisiología , Enfermedad Pulmonar Obstructiva Crónica/etiología , Adulto , Anciano , Animales , Bronquios/metabolismo , Estudios de Casos y Controles , Femenino , Perfilación de la Expresión Génica , Humanos , Pulmón/metabolismo , Masculino , Ratones , Persona de Mediana Edad , Análisis de Secuencia por Matrices de Oligonucleótidos , Enfermedad Pulmonar Obstructiva Crónica/genética , Enfermedad Pulmonar Obstructiva Crónica/metabolismo , Mucosa Respiratoria/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
BACKGROUND: Asthma is classified according to severity and inflammatory phenotype and is likely to be distinguished by specific microRNA (miRNA) expression profiles. OBJECTIVE: We sought to associate miRNA expression in sputum supernatants with the inflammatory cell profile and disease severity in asthmatic patients. METHODS: We investigated miRNA expression in sputum supernatants of 10 healthy subjects, 17 patients with mild-to-moderate asthma, and 9 patients with severe asthma using high-throughput, stem-loop, reverse transcriptase quantitative real-time PCR miRNA expression profiling (screening cohort, n = 36). Differentially expressed miRNAs were validated in an independent cohort (n = 60; 10 healthy subjects and 50 asthmatic patients). Cellular miRNA origin was examined by using in situ hybridization and reverse transcriptase quantitative real-time PCR. The functional role of miRNAs was assessed by using in silico analysis and in vitro transfecting miRNA mimics in human bronchial epithelial cells. RESULTS: In 2 independent cohorts expression of miR-629-3p, miR-223-3p, and miR-142-3p was significantly upregulated in sputum of patients with severe asthma compared with that in healthy control subjects and was highest in patients with neutrophilic asthma. Expression of the 3 miRNAs was associated with sputum neutrophilia, and miR-223-3p and miR-142-3p expression was associated also with airway obstruction (FEV1/forced vital capacity). Expression of miR-629-3p was localized in the bronchial epithelium, whereas miR-223-3p and miR-142-3p were expressed in neutrophils, monocytes, and macrophages. Transfecting human bronchial epithelial cells with miR-629-3p mimic induced epithelial IL-8 mRNA and protein expression. IL-1ß and IL-8 protein levels were significantly increased in sputum of patients with severe asthma and were positively associated with sputum neutrophilia. CONCLUSIONS: Expression of miR-223-3p, miR-142-3p, and miR-629-3p is increased in sputum of patients with severe asthma and is linked to neutrophilic airway inflammation, suggesting that these miRNAs contribute to this asthma inflammatory phenotype.
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Asma/genética , MicroARNs/metabolismo , Esputo/metabolismo , Adulto , Anciano , Asma/inmunología , Asma/metabolismo , Asma/fisiopatología , Bronquios/citología , Citocinas/metabolismo , Células Epiteliales/metabolismo , Femenino , Volumen Espiratorio Forzado , Humanos , Masculino , Persona de Mediana Edad , Neutrófilos/inmunología , FenotipoRESUMEN
The lung is an attractive target organ for inhalation of RNA therapeutics, such as small interfering RNA (siRNA). However, clinical translation of siRNA drugs for application in the lung is hampered by many extra- and intracellular barriers. We previously developed hybrid nanoparticles consisting of an siRNA-loaded nanosized hydrogel (nanogel) core coated with Curosurf®, a clinically used pulmonary surfactant. The surfactant shell was shown to markedly improve particle stability and promote intracellular siRNA delivery, both in vitro and in vivo. However, the full potential of siRNA nanocarriers is typically not reached as they are rapidly trafficked towards lysosomes for degradation and only a fraction of the internalized siRNA cargo is able to escape into the cytosol. We recently reported on the repurposing of widely applied cationic amphiphilic drugs (CADs) as siRNA delivery enhancers. Due to their physicochemical properties, CADs passively accumulate in the (endo)lysosomal compartment causing a transient permeabilization of the lysosomal membrane, which facilitates cytosolic drug delivery. In this work, we assessed a selection of cationic amphiphilic ß2-agonists (i.e., salbutamol, formoterol, salmeterol and indacaterol) for their ability to enhance siRNA delivery in a lung epithelial and macrophage cell line. These drugs are widely used in the clinic for their bronchodilating effect in obstructive lung disease. As opposed to the least hydrophobic drugs salbutamol and formoterol, the more hydrophobic long-acting ß2-agonist (LABA) salmeterol promoted siRNA delivery in both cell types for both uncoated and surfactant-coated nanogels, whereas indacaterol showed this effect solely in lung epithelial cells. Our results demonstrate the potential of both salmeterol and indacaterol to be repurposed as adjuvants for nanocarrier-mediated siRNA delivery to the lung, which could provide opportunities for drug combination therapy.
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Indanos , Polietilenglicoles , Polietileneimina , Surfactantes Pulmonares , Quinolonas , Surfactantes Pulmonares/química , Nanogeles , ARN Interferente Pequeño , Terapia Respiratoria , Xinafoato de Salmeterol , Albuterol , Fumarato de Formoterol , Adyuvantes Farmacéuticos , Administración por Inhalación , Adyuvantes Inmunológicos , TensoactivosRESUMEN
Lunsekimig is a novel, bispecific NANOBODY® molecule that inhibits both thymic stromal lymphopoietin (TSLP) and interleukin (IL)-13, two key mediators of asthma pathophysiology. In this first-in-human study, we evaluated the safety, tolerability, pharmacokinetics (PK), pharmacodynamics (PD), and immunogenicity of lunsekimig in healthy adult participants. Participants received single ascending doses (SAD) of lunsekimig (10-400 mg intravenous [IV] or 400 mg subcutaneous [SC]) (SAD part) or multiple ascending doses (MAD part) of lunsekimig (100 or 200 mg, every 2 weeks [Q2W] for three SC doses), or placebo. Overall, 48 participants were randomized 3:1 in the SAD part and 4:1 in the MAD part for lunsekimig or placebo. The primary endpoint was safety and tolerability. The secondary endpoints included PK, antidrug antibodies (ADAs) and total target measurement. Lunsekimig was well tolerated and common treatment-emergent adverse events were COVID-19, nasopharyngitis, injection site reactions, and headache. Lunsekimig showed dose-proportional increases in exposure and linear elimination. Mean t1/2z of lunsekimig was around 10 days across all IV and SC doses of the SAD and MAD parts of the study. Increases in the serum concentration of total TSLP and IL-13 for lunsekimig versus placebo indicated target engagement. ADA of low titers were detected in four (11.1%) participants who received lunsekimig in the SAD, and seven (43.8%) in the MAD. In conclusion, lunsekimig was well tolerated in healthy participants with a linear PK profile up to single 400 mg IV and SC dose and multiple doses of 100 and 200 mg SC Q2W, with low immunogenicity.
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Citocinas , Voluntarios Sanos , Interleucina-13 , Anticuerpos de Dominio Único , Linfopoyetina del Estroma Tímico , Humanos , Adulto , Masculino , Femenino , Interleucina-13/antagonistas & inhibidores , Interleucina-13/inmunología , Anticuerpos de Dominio Único/administración & dosificación , Anticuerpos de Dominio Único/inmunología , Anticuerpos de Dominio Único/efectos adversos , Persona de Mediana Edad , Citocinas/inmunología , Citocinas/sangre , Adulto Joven , Inyecciones Subcutáneas , Método Doble Ciego , Relación Dosis-Respuesta a Droga , Esquema de MedicaciónRESUMEN
Chronic obstructive pulmonary disease (COPD) is the third leading cause of morbidity and death worldwide. Inhalation of cigarette smoke (CS) is the major cause in developed countries. Current therapies have limited efficacy in controlling disease or halting its progression. Aberrant expression of microRNAs (miRNAs) is associated with lung disease, including COPD. We performed miRNA microarray analyses of the lungs of mice with CS-induced experimental COPD. miR-21 was the second highest up-regulated miRNA, particularly in airway epithelium and lung macrophages. Its expression in human lung tissue correlated with reduced lung function in COPD. Prophylactic and therapeutic treatment with a specific miR-21 inhibitor (Ant-21) inhibited CS-induced lung miR-21 expression in mice; suppressed airway macrophages, neutrophils, and lymphocytes; and improved lung function, as evidenced by decreased lung hysteresis, transpulmonary resistance, and tissue damping in mouse models of COPD. In silico analyses identified a potential miR-21/special AT-rich sequencebinding protein 1 (SATB1)/S100 calcium binding protein A9 (S100A9)/nuclear factor κB (NF-κB) axis, which was further investigated. CS exposure reduced lung SATB1 in a mouse model of COPD, whereas Ant-21 treatment restored SATB1 and reduced S100A9 expression and NF-κB activity. The beneficial effects of Ant-21 in mice were reversed by treatment with SATB1-targeting small interfering RNA. We have identified a pathogenic role for a miR-21/SATB1/S100A9/NF-κB axis in COPD and defined miR-21 as a therapeutic target for this disease.
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Calgranulina B , Proteínas de Unión a la Región de Fijación a la Matriz , MicroARNs , Enfermedad Pulmonar Obstructiva Crónica , Animales , Calgranulina B/genética , Calgranulina B/metabolismo , Pulmón/patología , Proteínas de Unión a la Región de Fijación a la Matriz/genética , Proteínas de Unión a la Región de Fijación a la Matriz/metabolismo , Ratones , MicroARNs/genética , MicroARNs/metabolismo , FN-kappa B/metabolismo , Enfermedad Pulmonar Obstructiva Crónica/genética , Enfermedad Pulmonar Obstructiva Crónica/metabolismoRESUMEN
Chronic obstructive pulmonary disease (COPD) is characterized by a progressive airflow limitation and is associated with a chronic inflammatory response in both airways and lungs. microRNAs (miRNAs) are often highly conserved between species and have an intricate role within homeostatic conditions and immune responses. Also, miRNAs are dysregulated in smoking-associated diseases. We investigated the miRNA profile of 523 miRNAs by stem-loop RT-qPCR in lung tissue and cell-free bronchoalveolar lavage (BAL) supernatant of mice exposed to air or cigarette smoke (CS) for 4 or 24 weeks. After 24 weeks of CS exposure, 31 miRNAs were differentially expressed in lung tissue and 78 in BAL supernatant. Next, we correlated the miRNA profiling data to inflammation in BAL and lung, obtained by flow cytometry or ELISA. In addition, we surveyed for overlap with newly assessed miRNA profiles in bronchial biopsies and with previously assessed miRNA profiles in lung tissue and induced sputum supernatant of smokers with COPD. Several miRNAs showed concordant differential expression between both species including miR-31*, miR-155, miR-218 and let-7c. Thus, investigating miRNA profiling data in different compartments and both species provided accumulating insights in miRNAs that may be relevant in CS-induced inflammation and the pathogenesis of COPD.
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Líquido del Lavado Bronquioalveolar , Perfilación de la Expresión Génica , Pulmón/patología , MicroARNs/genética , Enfermedad Pulmonar Obstructiva Crónica/genética , Fumar/genética , Investigación Biomédica Traslacional , Aire , Animales , Regulación hacia Abajo/genética , Inflamación/genética , Inflamación/patología , Masculino , Ratones Endogámicos C57BL , MicroARNs/metabolismo , Estadísticas no Paramétricas , Regulación hacia Arriba/genéticaRESUMEN
INTRODUCTION: Airway surface dehydration, caused by an imbalance between secretion and absorption of ions and fluid across the epithelium and/or increased epithelial mucin secretion, impairs mucociliary clearance. Recent evidence suggests that this mechanism may be implicated in chronic obstructive pulmonary disease (COPD). However, the role of airway surface dehydration in the pathogenesis of cigarette smoke (CS)-induced COPD remains unknown. OBJECTIVE: We aimed to investigate in vivo the effect of airway surface dehydration on several CS-induced hallmarks of COPD in mice with airway-specific overexpression of the ß-subunit of the epithelial Na⺠channel (ßENaC). METHODS: ßENaC-Tg mice and wild-type (WT) littermates were exposed to air or CS for 4 or 8 weeks. Pathological hallmarks of COPD, including goblet cell metaplasia, mucin expression, pulmonary inflammation, lymphoid follicles, emphysema and airway wall remodelling were determined and lung function was measured. RESULTS: Airway surface dehydration in ßENaC-Tg mice aggravated CS-induced airway inflammation, mucin expression and destruction of alveolar walls and accelerated the formation of pulmonary lymphoid follicles. Moreover, lung function measurements demonstrated an increased compliance and total lung capacity and a lower resistance and hysteresis in ßENaC-Tg mice, compared to WT mice. CS exposure further altered lung function measurements. CONCLUSIONS: We conclude that airway surface dehydration is a risk factor that aggravates CS-induced hallmarks of COPD.
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Deshidratación/patología , Enfermedad Pulmonar Obstructiva Crónica/patología , Mucosa Respiratoria/metabolismo , Contaminación por Humo de Tabaco/efectos adversos , Animales , Células Cultivadas , Deshidratación/etiología , Deshidratación/metabolismo , Canales Epiteliales de Sodio/genética , Canales Epiteliales de Sodio/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Mucinas/metabolismo , Enfermedad Pulmonar Obstructiva Crónica/metabolismo , Mucosa Respiratoria/patología , Fumar/efectos adversosRESUMEN
The inflammatory cytokine TNF-α is a central mediator in many immune-mediated diseases, such as Crohn's disease (CD), spondyloarthritis (SpA) and chronic obstructive pulmonary disease (COPD). Epidemiologic studies have shown that cigarette smoking (CS) is a prominent common risk factor in these TNF-dependent diseases. We exposed TNFΔARE mice; in which a systemic TNF-α overexpression leads to the development of inflammation; to 2 or 4 weeks of air or CS. We investigated the effect of deregulated TNF expression on CS-induced pulmonary inflammation and the effect of CS exposure on the initiation and progression of gut and joint inflammation. Upon 2 weeks of CS exposure, inflammation in lungs of TNFΔARE mice was significantly aggravated. However, upon 4 weeks of CS-exposure, this aggravation was no longer observed. TNFΔARE mice have no increases in CD4+ and CD8+ T cells and a diminished neutrophil response in the lungs after 4 weeks of CS exposure. In the gut and joints of TNFΔARE mice, 2 or 4 weeks of CS exposure did not modulate the development of inflammation. In conclusion, CS exposure does not modulate gut and joint inflammation in TNFΔARE mice. The lung responses towards CS in TNFΔARE mice however depend on the duration of CS exposure.