RESUMO
MicroRNAs that are overexpressed in cystic fibrosis (CF) bronchial epithelial cells (BEC) negatively regulate CFTR and nullify the beneficial effects of CFTR modulators. We hypothesized that it is possible to reverse microRNA-mediated inhibition of CFTR using CFTR-specific target site blockers (TSBs) and to develop a drug-device combination inhalation therapy for CF. Lead microRNA expression was quantified in a series of human CF and non-CF samples and in vitro models. A panel of CFTR 3' untranslated region (UTR)-specific locked nucleic acid antisense oligonucleotide TSBs was assessed for their ability to increase CFTR expression. Their effects on CFTR activity alone or in combination with CFTR modulators were measured in CF BEC models. TSB encapsulation in poly-lactic-co-glycolic acid (PLGA) nanoparticles was assessed as a proof of principle of delivery into CF BECs. TSBs targeting the CFTR 3' UTR 298-305:miR-145-5p or 166-173:miR-223-3p sites increased CFTR expression and anion channel activity and enhanced the effects of ivacaftor/lumacaftor or ivacaftor/tezacaftor in CF BECs. Biocompatible PLGA-TSB nanoparticles promoted CFTR expression in primary BECs and retained desirable biophysical characteristics following nebulization. Alone or in combination with CFTR modulators, aerosolized CFTR-targeting TSBs encapsulated in PLGA nanoparticles could represent a promising drug-device combination therapy for the treatment for CFTR dysfunction in the lung.
Assuntos
Brônquios/metabolismo , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Fibrose Cística/terapia , MicroRNAs/genética , Oligonucleotídeos/farmacologia , Adulto , Aminofenóis/farmacologia , Aminopiridinas/farmacologia , Benzodioxóis/farmacologia , Brônquios/citologia , Brônquios/efeitos dos fármacos , Células Cultivadas , Criança , Pré-Escolar , Fibrose Cística/genética , Fibrose Cística/metabolismo , Combinação de Medicamentos , Sinergismo Farmacológico , Células Epiteliais/citologia , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Feminino , Humanos , Indóis/farmacologia , Lactente , Masculino , Pessoa de Meia-Idade , Modelos Biológicos , Nanopartículas , Oligonucleotídeos/genética , Copolímero de Ácido Poliláctico e Ácido Poliglicólico/química , Quinolonas/farmacologiaRESUMO
Cystic fibrosis (CF) is an autosomal recessive genetic disorder arising from mutations to the cystic fibrosis transmembrane conductance regulator (CFTR) gene. Disruption to normal ion homeostasis in the airway results in impaired mucociliary clearance, leaving the lung more vulnerable to recurrent and chronic bacterial infections. The CF lung endures an excess of neutrophilic inflammation, and whilst neutrophil serine proteases are a crucial part of the innate host defence to infection, a surplus of neutrophil elastase (NE) is understood to create a net destructive effect. Alpha-1 antitrypsin (A1AT) is a key antiprotease in the control of NE protease activity but is ineffective in the CF lung due to the huge imbalance of NE levels. Therapeutic strategies to boost levels of protective antiproteases such as A1AT in the lung remain an attractive research strategy to limit the damage from excess protease activity. microRNAs are small non-coding RNA molecules that bind specific cognate sequences to inhibit expression of target mRNAs. The inhibition of miRNAs which target the SERPINA1 (A1AT-encoding gene) mRNA represents a novel therapeutic approach for CF inflammation. This could involve the delivery of antagomirs that bind and sequester the target miRNA, or target site blockers that bind miRNA recognition elements within the target mRNA to prevent miRNA interaction. Therefore, miRNA targeted therapies offer an alternative strategy to drive endogenous A1AT production and thus supplement the antiprotease shield of the CF lung.
Assuntos
Fibrose Cística/genética , MicroRNAs/genética , alfa 1-Antitripsina/genética , Antagomirs/farmacologia , Antagomirs/uso terapêutico , Fibrose Cística/metabolismo , Fibrose Cística/terapia , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Humanos , Elastase de Leucócito/metabolismo , MicroRNAs/antagonistas & inibidores , Terapia de Alvo Molecular , Regulação para Cima , alfa 1-Antitripsina/metabolismoRESUMO
Here we present miR-CATCHv2.0, an implemented experimental method that allows the identification of the microRNA species directly bound to an RNA of interest. After cross-linking of microRNA::RNA::Ago2 complexes using formaldehyde, the RNA is fragmented using sonication and then subjected to affinity purification using two sets of biotinylated tiling probes (ODD and EVEN). Finally, enriched microRNA species are retrieved by means of small RNA sequencing coupled with an ad hoc analytical workflow. In BRAFV600E mutant A375 melanoma cells, miR-CATCHv2.0 allowed us to identify 20 microRNAs that target X1, the most abundant isoform of BRAF mRNA. These microRNAs fall into different functional classes, according to the effect that they exert (decrease/increase in BRAFV600E mRNA and protein levels) and to the mechanism they use to achieve it (destabilization/stabilization of X1 mRNA or decrease/increase in its translation). microRNA-induced variations in BRAFV600E protein levels are most of the times coupled to consistent variations in pMEK levels, in melanoma cell proliferation in vitro and in sensitivity to the BRAF inhibitor vemurafenib in a xenograft model in zebrafish. However, microRNAs exist that uncouple the degree of activation of the ERK pathway from the levels of BRAFV600E protein. Our study proposes miR-CATCHv2.0 as an effective tool for the identification of direct microRNA-target interactions and, by using such a tool, unveils the complexity of the post-transcriptional regulation to which BRAFV600E and the ERK pathway are subjected in melanoma cells.
Assuntos
MicroRNAs/metabolismo , Proteínas Proto-Oncogênicas B-raf/genética , Software , Humanos , MicroRNAs/genética , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Estabilidade de RNA/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reprodutibilidade dos TestesRESUMO
Mutations in the Cystic Fibrosis Transmembrane Conductance Regulator (
Assuntos
Brônquios/metabolismo , Regulador de Condutância Transmembrana em Fibrose Cística/genética , MicroRNAs/metabolismo , Estabilidade de RNA , Mucosa Respiratória/metabolismo , Fator de Crescimento Transformador beta/farmacologia , Brônquios/citologia , Células Cultivadas , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Inativação Gênica , Humanos , MicroRNAs/genética , Mucosa Respiratória/efeitos dos fármacosRESUMO
Non-coding RNAs (ncRNAs) are an abundant class of RNAs that include small ncRNAs, long non-coding RNAs (lncRNA) and pseudogenes. The human ncRNA atlas includes thousands of these specialised RNA molecules that are further subcategorised based on their size or function. Two of the more well-known and widely studied ncRNA species are microRNAs (miRNAs) and lncRNAs. These are regulatory RNAs and their altered expression has been implicated in the pathogenesis of a variety of human diseases. Failure to express a functional cystic fibrosis (CF) transmembrane receptor (CFTR) chloride ion channel in epithelial cells underpins CF. Secondary to the CFTR defect, it is known that other pathways can be altered and these may contribute to the pathophysiology of CF lung disease in particular. For example, quantitative alterations in expression of some ncRNAs are associated with CF. In recent years, there has been a series of published studies exploring ncRNA expression and function in CF. The majority have focussed principally on miRNAs, with just a handful of reports to date on lncRNAs. The present study reviews what is currently known about ncRNA expression and function in CF, and discusses the possibility of applying this knowledge to the clinical management of CF in the near future.
Assuntos
Fibrose Cística/genética , RNA não Traduzido/fisiologia , Animais , Fibrose Cística/metabolismo , Fibrose Cística/fisiopatologia , Fibrose Cística/terapia , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Humanos , Imunidade Inata/genética , Inflamação/genética , MicroRNAs/fisiologia , Pseudogenes , Resposta a Proteínas não DobradasRESUMO
Cystic fibrosis is a genetic disease caused by mutations in the CFTR gene, whereas chronic obstructive pulmonary disease (COPD) is mainly caused by environmental factors (mostly cigarette smoking) on a genetically susceptible background. Although the etiology and pathogenesis of these diseases are different, both are associated with progressive airflow obstruction, airway neutrophilic inflammation, and recurrent exacerbations, suggesting common mechanisms. The airway epithelium plays a crucial role in maintaining normal airway functions. Major molecular and morphologic changes occur in the airway epithelium in both CF and COPD, and growing evidence suggests that airway epithelial dysfunction is involved in disease initiation and progression in both diseases. Structural and functional abnormalities in both airway and alveolar epithelium have a relevant impact on alteration of host defences, immune/inflammatory response, and the repair process leading to progressive lung damage and impaired lung function. In this review, we address the evidence for a critical role of dysfunctional airway epithelial cells in chronic airway inflammation and remodelling in CF and COPD, highlighting the common mechanisms involved in the epithelial dysfunction as well as the similarities and differences of the two diseases.
Assuntos
Fibrose Cística/imunologia , Fibrose Cística/patologia , Doença Pulmonar Obstrutiva Crônica/imunologia , Doença Pulmonar Obstrutiva Crônica/patologia , Animais , Células Epiteliais/imunologia , Células Epiteliais/patologia , Humanos , Inflamação/imunologia , Inflamação/patologiaRESUMO
AIM: From birth to old age, males generally have poorer disease outcomes compared to females. Preterm infants display a marked gender disparity in disease outcomes, and the underlying mechanisms are not well delineated. Our aim was to review the literature on clinical outcomes between preterm infants of different genders and discuss the potential mechanisms underlying the differences observed. METHODS: A literature review was undertaken for experimental and clinical research related to gender differences in preterm outcomes. RESULTS: Preterm male infants appear to have consistently worse outcomes compared to females, and the aetiology of these differences, while mostly undetermined, is likely multifactorial. CONCLUSION: The male disadvantage in preterm outcomes is likely multifactorial with hormonal, genetic and immunological differences likely playing key roles. Gender is an important variable in preterm outcome and should be considered when designing clinical and experimental research.
RESUMO
BACKGROUND: Male neonates display poorer disease prognosis and outcomes compared with females. Immune genes which exhibit higher expression in umbilical cord blood (UCB) of females may contribute to the female immune advantage during infection and inflammation. The aim of this study was to quantify expression of Toll-like receptor (TLR) 4 signaling genes encoded on the X-chromosome in UCB from term female vs. male neonates. METHODS: UCB samples were collected from term neonates (n = 26) born by elective Caesarean section and whole blood was collected from adults (n = 20). Leukocyte RNA was isolated and used in quantitative PCR reactions for IκB kinase γ (IKKγ), Bruton's tyrosine kinase (BTK), and IL-1 receptor associated kinase (IRAK)1. IRAK1 protein was analyzed by Western blot and confocal microscopy. RESULTS: In neonates there was no significant difference in the relative expression of IKKγ or BTK mRNA between genders. IRAK1 gene and protein expression was significantly higher in female vs. male UCB, with increased cytosolic IRAK1 expression also evident in female UCB mononuclear cells. Adults had higher expression of all three genes compared with neonates. CONCLUSION: Increased expression of IRAK1 could be responsible, in part, for sex-specific responses to infection and subsequent immune advantage in female neonates.
Assuntos
Cromossomos Humanos X , Quinases Associadas a Receptores de Interleucina-1/genética , Transdução de Sinais/genética , Receptor 4 Toll-Like/genética , Adulto , Tirosina Quinase da Agamaglobulinemia , Fatores Etários , Feminino , Idade Gestacional , Humanos , Quinase I-kappa B/sangue , Quinase I-kappa B/genética , Quinases Associadas a Receptores de Interleucina-1/sangue , Leucócitos/metabolismo , Masculino , Proteínas Tirosina Quinases/sangue , Proteínas Tirosina Quinases/genética , Proteínas Ribossômicas/sangue , Proteínas Ribossômicas/genética , Fatores Sexuais , Nascimento a Termo , Receptor 4 Toll-Like/sangue , Adulto JovemRESUMO
BACKGROUND: Colonization by Aspergillus fumigatus in patients with cystic fibrosis (CF) can cause A fumigatus sensitization and/or allergic bronchopulmonary aspergillosis (ABPA), which affects pulmonary function and clinical outcomes. Recent studies show that specific allergens upregulate the surface-expressed basophil marker CD203c in sensitized subjects, a response that can be readily measured by using flow cytometry. OBJECTIVE: We sought to identify A fumigatus sensitization in patients with CF by using the basophil activation test (BAT). METHODS: Patients with CF attending Beaumont Hospital were screened for study inclusion. BAT was used to identify A fumigatus sensitization. Serologic (total and A fumigatus-specific IgE), pulmonary function, and body mass index measurements were performed. RESULTS: The BAT discriminates A fumigatus-sensitized from nonsensitized patients with CF. Persistent isolation of A fumigatus in sputum is a significant risk factor for A fumigatus sensitization. Levels of the A fumigatus-stimulated basophil activation marker CD203c inversely correlated with pulmonary function and body mass index in A fumigatus-sensitized but not nonsensitized patients with CF. Total and A fumigatus-specific IgE, but not IgG, levels are increased in A fumigatus-sensitized patients with CF and ABPA when compared with those in A fumigatus-sensitized and nonsensitized patients with CF without ABPA. Itraconazole treatment did not affect A fumigatus sensitization. CONCLUSION: Combining the BAT with routine serologic testing allows classification of patients with CF into 3 groups: nonsensitized, A fumigatus-sensitized, and ABPA. Accurate and prompt identification of A fumigatus-associated clinical status might allow early and targeted therapeutic intervention, potentially improving clinical outcomes.
Assuntos
Aspergilose/etiologia , Aspergilose/metabolismo , Aspergillus/imunologia , Basófilos/metabolismo , Fibrose Cística/complicações , Diester Fosfórico Hidrolases/metabolismo , Pirofosfatases/metabolismo , Especificidade de Anticorpos/imunologia , Antifúngicos/farmacologia , Antifúngicos/uso terapêutico , Antígenos de Fungos/imunologia , Antígenos de Superfície/metabolismo , Aspergilose/diagnóstico , Aspergilose/tratamento farmacológico , Basófilos/imunologia , Biomarcadores , Índice de Massa Corporal , Fibrose Cística/fisiopatologia , Feminino , Volume Expiratório Forçado , Humanos , Imunoglobulina E/sangue , Imunoglobulina E/imunologia , Imunoglobulina G/sangue , Imunoglobulina G/imunologia , Imunofenotipagem , Contagem de Leucócitos , Masculino , Estudos Prospectivos , Escarro/imunologia , Escarro/microbiologiaRESUMO
Increasing levels of estrogen and progesterone are suggested to play a role in the gender switch in asthma prevalence during puberty. We investigated whether the process of sexual maturation in mice affects the development of lung inflammation in adulthood and the contributing roles of estrogen and progesterone during this process. By inducing ovalbumin-induced lung inflammation in sexually mature and immature (ovariectomized before sexual maturation) adult mice, we showed that sexually immature adult mice developed more eosinophilic lung inflammation. This protective effect of "puberty" appears to be dependent on estrogen, as estrogen supplementation at the time of ovariectomy protected against development of lung inflammation in adulthood whereas progesterone supplementation did not. Investigating the underlying mechanism of estrogen-mediated protection, we found that estrogen-treated mice had higher expression of the anti-inflammatory mediator secretory leukoprotease inhibitor (SLPI) and lower expression of the proasthmatic cytokine IL-33 in parenchymal lung tissue and that their expressions colocalized with type II alveolar epithelial cells (AECII). Treating AECII directly with SLPI significantly inhibited IL-33 production upon stimulation with ATP. Our data suggest that estrogen during puberty has a protective effect on asthma development, which is accompanied by induction of anti-inflammatory SLPI production and inhibition of proinflammatory IL-33 production by AECII.
Assuntos
Estrogênios/metabolismo , Pneumonia/metabolismo , Maturidade Sexual/fisiologia , Animais , Asma/metabolismo , Citocinas/imunologia , Citocinas/metabolismo , Modelos Animais de Doenças , Feminino , Camundongos Endogâmicos BALB C , Inibidor Secretado de Peptidases Leucocitárias/biossínteseRESUMO
Monocytes are critical to the pathogenesis of inflammatory bowel disease (IBD) as they infiltrate the mucosa and release cytokines that drive the inflammatory response. Ursodeoxycholic acid (UDCA), a naturally occurring bile acid with anti-inflammatory actions, has been proposed as a potential new therapy for IBD. However, its effects on monocyte function are not yet known. Primary monocytes from healthy volunteers or cultured U937 monocytes were treated with either the proinflammatory cytokine, TNFα (5 ng/ml) or the bacterial endotoxin, lipopolysaccharide (LPS; 1 µg/ml) for 24 h, in the absence or presence of UDCA (25-100 µM). IL-8 release into the supernatant was measured by ELISA. mRNA levels were quantified by qPCR and changes in cell signaling proteins were determined by Western blotting. Toxicity was assessed by measuring lactate dehydrogenase (LDH) release. UDCA treatment significantly attenuated TNFα-, but not LPS-driven, release of IL-8 from both primary and cultured monocytes. UDCA inhibition of TNFα-driven responses was associated with reduced IL-8 mRNA expression. Both TNFα and LPS stimulated NFκB activation in monocytes, while IL-8 release in response to both cytokines was attenuated by an NFκB inhibitor, BMS-345541. Interestingly, UDCA inhibited TNFα-, but not LPS-stimulated, NFκB activation. Finally, TNFα, but not LPS, induced phosphorylation of TNF receptor associated factor (TRAF2), while UDCA cotreatment attenuated this response. We conclude that UDCA specifically inhibits TNFα-induced IL-8 release from monocytes by inhibiting TRAF2 activation. Since such actions would serve to dampen mucosal immune responses in vivo, our data support the therapeutic potential of UDCA for IBD.
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Imunossupressores/farmacologia , Interleucina-8/metabolismo , Monócitos/efeitos dos fármacos , Fator de Necrose Tumoral alfa/farmacologia , Ácido Ursodesoxicólico/farmacologia , Regulação da Expressão Gênica , Humanos , Imunidade nas Mucosas/efeitos dos fármacos , Interleucina-8/genética , Interleucina-8/imunologia , Lipopolissacarídeos/farmacologia , Monócitos/imunologia , Monócitos/metabolismo , NF-kappa B/metabolismo , Fosforilação , Transdução de Sinais/efeitos dos fármacos , Fator 2 Associado a Receptor de TNF/metabolismo , Células U937RESUMO
RATIONALE: Anaerobic bacteria are present in large numbers in the airways of people with cystic fibrosis (PWCF). In the gut, anaerobes produce short-chain fatty acids (SCFAs) that modulate immune and inflammatory processes. OBJECTIVES: To investigate the capacity of anaerobes to contribute to cystic fibrosis (CF) airway pathogenesis via SCFAs. METHODS: Samples of 109 PWCF were processed using anaerobic microbiological culture with bacteria present identified by 16S RNA sequencing. SCFA levels in anaerobic supernatants and bronchoalveolar lavage (BAL) were determined by gas chromatography. The mRNA and/or protein expression of two SCFA receptors, GPR41 and GPR43, in CF and non-CF bronchial brushings and 16HBE14o(-) and CFBE41o(-) cells were evaluated using reverse transcription polymerase chain reaction, Western blot analysis, laser scanning cytometry, and confocal microscopy. SCFA-induced IL-8 secretion was monitored by ELISA. MEASUREMENTS AND MAIN RESULTS: Fifty-seven (52.3%) of 109 PWCF were anaerobe positive. Prevalence increased with age, from 33.3% to 57.7% in PWCF younger (n = 24) and older (n = 85) than 6 years of age. All evaluated anaerobes produced millimolar concentrations of SCFAs, including acetic, propionic, and butyric acids. SCFA levels were higher in BAL samples of adults than in those of children. GPR41 levels were elevated in CFBE41o(-) versus 16HBE14o(-) cells; CF versus non-CF bronchial brushings; and 16HBE14o(-) cells after treatment with cystic fibrosis transmembrane conductance regulator inhibitor CFTR(inh)-172, CF BAL, or inducers of endoplasmic reticulum stress. SCFAs induced a dose-dependent and pertussis toxin-sensitive IL-8 response in bronchial epithelial cells, with a higher production of IL-8 in CFBE41o(-) than in 16HBE14o(-) cells. CONCLUSIONS: This study illustrates that SCFAs contribute to excessive production of IL-8 in CF airways colonized with anaerobes via up-regulated GPR41.
Assuntos
Bactérias Anaeróbias , Fibrose Cística/microbiologia , Ácidos Graxos/biossíntese , Adolescente , Adulto , Fatores Etários , Western Blotting , Líquido da Lavagem Broncoalveolar/microbiologia , Criança , Pré-Escolar , Cromatografia Gasosa , Regulador de Condutância Transmembrana em Fibrose Cística/análise , Ensaio de Imunoadsorção Enzimática , Células Epiteliais/microbiologia , Feminino , Humanos , Lactente , Masculino , Pessoa de Meia-Idade , Reação em Cadeia da Polimerase , Mucosa Respiratória/microbiologia , Regulação para Cima , Adulto JovemRESUMO
BACKGROUND: Women with cystic fibrosis are at increased risk for mucoid conversion of Pseudomonas aeruginosa, which contributes to a sexual dichotomy in disease severity. METHODS: We evaluated the effects of estradiol and its metabolite estriol on P. aeruginosa in vitro and in vivo and determined the effect of estradiol on disease exacerbations in women with cystic fibrosis. RESULTS: Estradiol and estriol induced alginate production in P. aeruginosa strain 01 and in clinical isolates obtained from patients with and those without cystic fibrosis. After prolonged exposure to estradiol, P. aeruginosa adopted early mucoid morphology, whereas short-term exposure inhibited bacterial catalase activity and increased levels of hydrogen peroxide, which is potentially damaging to DNA. Consequently, a frameshift mutation was identified in mucA, a key regulator of alginate biosynthesis in P. aeruginosa. In vivo levels of estradiol correlated with infective exacerbations in women with cystic fibrosis, with the majority occurring during the follicular phase (P<0.05). A review of the Cystic Fibrosis Registry of Ireland revealed that the use of oral contraceptives was associated with a decreased need for antibiotics. Predominantly nonmucoid P. aeruginosa was isolated from sputum during exacerbations in the luteal phase (low estradiol). Increased proportions of mucoid bacteria were isolated during exacerbations occurring in the follicular phase (high estradiol), with a variable P. aeruginosa phenotype evident in vivo during the course of the menstrual cycle corresponding to fluctuating estradiol levels. CONCLUSIONS: Estradiol and estriol induced mucoid conversion of P. aeruginosa in women with cystic fibrosis through a mutation of mucA in vitro and were associated with selectivity for mucoid isolation, increased exacerbations, and mucoid conversion in vivo. (Funded by the Molecular Medicine Ireland Clinician-Scientist Fellowship Programme.).
Assuntos
Estradiol/farmacologia , Estriol/farmacologia , Polissacarídeos Bacterianos/biossíntese , Infecções por Pseudomonas/tratamento farmacológico , Pseudomonas aeruginosa/efeitos dos fármacos , Alginatos , Fibrose Cística/microbiologia , Estradiol/uso terapêutico , Feminino , Regulação Bacteriana da Expressão Gênica , Ácido Glucurônico/biossíntese , Ácido Glucurônico/genética , Ácidos Hexurônicos , Humanos , Irlanda , Fenótipo , Polissacarídeos Bacterianos/genética , Gravidez , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/metabolismo , Sistema de RegistrosRESUMO
Biomarkers are quantifiable indicators of disease. These surrogates should be specific, sensitive, predictive, robust and easily accessible. A major class of RNA described as non-coding RNA fulfils many of these criteria, and recent studies have demonstrated that the two major subclasses of non-coding RNA, long non-coding RNA and, in particular, microRNA are promising potential biomarkers. The ability to detect non-coding RNAs in biofluids has highlighted their usefulness as non-invasive markers of lung disease. Because expression of specific non-coding RNAs is altered in many lung diseases and their levels in the circulation often reflect the changes in expression of their lung-specific counterparts, exploiting these biomolecules as diagnostic tools seems an obvious goal. New technology is driving developments in this area and there has been significant recent progress with respect to lung cancer diagnostics. The non-coding RNA biomarker field represents a clear example of modern-day bench-to-bedside research.
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Biomarcadores Tumorais/metabolismo , Pneumopatias/diagnóstico , Pneumopatias/metabolismo , MicroRNAs/metabolismo , RNA Longo não Codificante/metabolismo , Humanos , Pneumopatias/genéticaRESUMO
Interleukin (IL)-8 levels are higher than normal in cystic fibrosis (CF) airways, causing neutrophil infiltration and non-resolving inflammation. Overexpression of microRNAs that target IL-8 expression in airway epithelial cells may represent a therapeutic strategy for cystic fibrosis. IL-8 protein and mRNA were measured in cystic fibrosis and non-cystic fibrosis bronchoalveolar lavage fluid and bronchial brushings (n=20 per group). miRNAs decreased in the cystic fibrosis lung and predicted to target IL-8 mRNA were quantified in ßENaC-transgenic, cystic fibrosis transmembrane conductance regulator (Cftr)-/- and wild-type mice, primary cystic fibrosis and non-cystic fibrosis bronchial epithelial cells and a range of cystic fibrosis versus non-cystic fibrosis airway epithelial cell lines or cells stimulated with lipopolysaccharide, Pseudomonas-conditioned medium or cystic fibrosis bronchoalveolar lavage fluid. The effect of miRNA overexpression on IL-8 protein production was measured. miR-17 regulates IL-8 and its expression was decreased in adult cystic fibrosis bronchial brushings, ßENaC-transgenic mice and bronchial epithelial cells chronically stimulated with Pseudomonas-conditioned medium. Overexpression of miR-17 inhibited basal and agonist-induced IL-8 protein production in F508del-CFTR homozygous CFTE29o(-) tracheal, CFBE41o(-) and/or IB3 bronchial epithelial cells. These results implicate defective CFTR, inflammation, neutrophilia and mucus overproduction in regulation of miR-17. Modulating miR-17 expression in cystic fibrosis bronchial epithelial cells may be a novel anti-inflammatory strategy for cystic fibrosis and other chronic inflammatory airway diseases.
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Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Fibrose Cística/imunologia , Células Epiteliais/metabolismo , Interleucina-8/metabolismo , MicroRNAs/metabolismo , Infiltração de Neutrófilos , Adulto , Animais , Brônquios/citologia , Líquido da Lavagem Broncoalveolar , Contagem de Células , Linhagem Celular , Fibrose Cística/genética , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Feminino , Humanos , Interleucina-8/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , MicroRNAs/genética , Pessoa de Meia-Idade , Adulto JovemRESUMO
Expression of the cystic fibrosis transmembrane conductance regulator (CFTR) is altered in individuals with the ΔF508 CFTR mutation. We previously reported differential expression of microRNA (miRNA) in CF airway epithelium; however, the role of miRNA in regulation of CFTR expression here remains unexplored. In this study, we investigated the role of upregulated miRNAs in CFTR regulation in vivo in bronchial brushings from individuals homozygous or heterozygous for ΔF508 CFTR, validated our observations in vitro, and assessed the impact of defective chloride ion conductance, genotype, and colonization status on miRNA expression. miRNA target prediction was performed in silico, and expression of miRNA and target genes were measured by quantitative real-time PCR and/or Western blotting. Overexpression and inhibition studies were performed with pre-miRs or antimiRs, respectively, and a luciferase reporter gene was used to elucidate direct miRNA-target interactions. miR-145, miR-223, and miR-494 were upregulated in CF versus non-CF bronchial brushings and cell lines; in ΔF508 CFTR homozygotes versus heterozygotes; in subjects positive for P. aeruginosa; and in cells treated with a CFTR inhibitor or IL-1ß. Reciprocal downregulation or upregulation of CFTR gene and/or protein expression was observed after miRNA manipulation and direct miRNA-target relationships demonstrated via a reporter system containing a wild type or mutated full-length CFTR 3' untranslated region. Increased expression of miR-145, miR-223, and miR-494 in vivo in bronchial epithelium of individuals carrying the ΔF508 CFTR mutation correlates with decreased CFTR expression. Defective CFTR function, Pseudomonas colonization, and inflammation may affect miRNA expression and contribute to the regulation of ΔF508 CFTR.
Assuntos
Regulador de Condutância Transmembrana em Fibrose Cística/genética , Fibrose Cística/genética , Regulação da Expressão Gênica , MicroRNAs/genética , Mucosa Respiratória/metabolismo , Regiões 3' não Traduzidas , Adulto , Sequência de Bases , Linhagem Celular , Cloretos/metabolismo , Fibrose Cística/metabolismo , Fibrose Cística/microbiologia , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Feminino , Expressão Gênica , Perfilação da Expressão Gênica , Técnicas de Silenciamento de Genes , Genótipo , Humanos , Espaço Intracelular/metabolismo , Pulmão/metabolismo , Pulmão/microbiologia , Masculino , MicroRNAs/química , MicroRNAs/metabolismo , Pseudomonas aeruginosa/metabolismo , Mucosa Respiratória/microbiologia , Mucosa Respiratória/patologiaRESUMO
RATIONALE: Retention of abnormal α1-antitrypsin (AAT) activates the unfolded protein response in AAT-deficient monocytes. The regulatory role of microRNAs (miRNAs) in unfolded protein responses and chronic obstructive pulmonary disease pathogenesis has not been investigated. OBJECTIVES: To investigate miRNA expression and function in MM and ZZ monocytes and identify miRNA(s) regulating the unfolded protein response. METHODS: Peripheral blood monocytes were isolated from asymptomatic and symptomatic MM and ZZ individuals for miRNA expression profiling and pyrosequencing analysis. miRNA/gene and protein expression was measured with quantitative polymerase chain reaction and Western blotting. Overexpression and inhibition studies were performed with pre-miR or anti-miR, respectively. Luciferase reporter genes were used to elucidate direct miRNA-target interactions. Inflammatory cytokines were detected using the Meso Scale Discovery Plex assays. MEASUREMENTS AND MAIN RESULTS: Forty-three miRNAs were differentially expressed, with miR-199a-5p most highly up-regulated in asymptomatic ZZ versus MM monocytes. miR-199a-2 promoter hypermethylation inhibits miR-199a-5p expression and was increased in symptomatic MM and ZZ monocytes compared with asymptomatic counterparts. GRP78, activating transcription factor 6, p50, and p65 were increased in symptomatic versus asymptomatic ZZ monocytes. Reciprocal down- or up-regulation of these markers was observed after miRNA modulation. Direct miR-199a-5p targeting of activating transcription factor 6, p50, and p65 by miR-199a-5p was demonstrated using luciferase reporter systems. Overexpression of miR-199a-5p also decreased other arms of the UPR and expression of cytokines that are not putative targets. CONCLUSIONS: miR-199a-5p is a key regulator of the unfolded protein response in AAT-deficient monocytes, and epigenetic silencing of its expression regulates this process in chronic obstructive pulmonary disease.
Assuntos
Inativação Gênica , MicroRNAs/metabolismo , Doença Pulmonar Obstrutiva Crônica/genética , Resposta a Proteínas não Dobradas/genética , Deficiência de alfa 1-Antitripsina/genética , Adulto , Doenças Assintomáticas , Biomarcadores/metabolismo , Western Blotting , Citocinas/metabolismo , Chaperona BiP do Retículo Endoplasmático , Feminino , Perfilação da Expressão Gênica , Humanos , Masculino , Metilação , Pessoa de Meia-Idade , Monócitos/metabolismo , Fenótipo , Reação em Cadeia da Polimerase , Doença Pulmonar Obstrutiva Crônica/complicações , Doença Pulmonar Obstrutiva Crônica/metabolismo , Análise de Sequência de RNA , Estresse Fisiológico , Resposta a Proteínas não Dobradas/fisiologia , Regulação para Cima , Deficiência de alfa 1-Antitripsina/complicações , Deficiência de alfa 1-Antitripsina/metabolismoRESUMO
RATIONALE: Cathepsin S (CTSS) activity is increased in bronchoalveolar lavage (BAL) fluid from patients with cystic fibrosis (CF). This activity contributes to lung inflammation via degradation of antimicrobial proteins, such as lactoferrin and members of the ß-defensin family. OBJECTIVES: In this study, we investigated the hypothesis that airway epithelial cells are a source of CTSS, and mechanisms underlying CTSS expression in the CF lung. METHODS: Protease activity was determined using fluorogenic activity assays. Protein and mRNA expression were analyzed by ELISA, Western blotting, and reverse-transcriptase polymerase chain reaction. MEASUREMENTS AND MAIN RESULTS: In contrast to neutrophil elastase, CTSS activity was detectable in 100% of CF BAL fluid samples from patients without Pseudomonas aeruginosa infection. In this study, we identified epithelial cells as a source of pulmonary CTSS activity with the demonstration that CF airway epithelial cells express and secrete significantly more CTSS than non-CF control cells in the absence of proinflammatory stimulation. Furthermore, levels of the transcription factor IRF-1 correlated with increased levels of its target gene CTSS. We discovered that miR-31, which is decreased in the CF airways, regulates IRF-1 in CF epithelial cells. Treating CF bronchial epithelial cells with a miR-31 mimic decreased IRF-1 protein levels with concomitant knockdown of CTSS expression and secretion. CONCLUSIONS: The miR-31/IRF-1/CTSS pathway may play a functional role in the pathogenesis of CF lung disease and may open up new avenues for exploration in the search for an effective therapeutic target.
Assuntos
Líquido da Lavagem Broncoalveolar/química , Catepsinas/metabolismo , Fibrose Cística/genética , Fator Regulador 1 de Interferon/metabolismo , MicroRNAs/metabolismo , Mucosa Respiratória/metabolismo , Adolescente , Biomarcadores/metabolismo , Western Blotting , Líquido da Lavagem Broncoalveolar/microbiologia , Linhagem Celular , Criança , Pré-Escolar , Fibrose Cística/metabolismo , Fibrose Cística/microbiologia , Regulação para Baixo , Ensaio de Imunoadsorção Enzimática , Humanos , Lactente , Peptídeo Hidrolases/metabolismo , Infecções por Pseudomonas/complicações , Infecções por Pseudomonas/diagnóstico , Infecções por Pseudomonas/metabolismo , Pseudomonas aeruginosa/isolamento & purificação , Mucosa Respiratória/microbiologia , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
RATIONALE: Severe α1-antitrypsin deficiency (typically PiZZ homozygosity) is associated with a significantly increased risk of airflow obstruction and emphysema but the risk of chronic obstructive pulmonary disease (COPD) in PiMZ heterozygotes remains uncertain. OBJECTIVES: This was a family-based study to determine the risk of COPD in PiMZ individuals. METHODS: We compared 99 PiMM and 89 PiMZ nonindex subjects recruited from 51 index probands who were confirmed PiMZ heterozygotes and also had a diagnosis of COPD Global Initiative for Chronic Obstructive Lung Disease stage II-IV. The primary outcome measures of interest were quantitative variables of pre- and post-bronchodilator FEV1/FVC ratio, FEV1 (liters), FEV1 (% predicted), forced expiratory flow midexpiratory phase (FEF25-75; liters per second), FEF25-75 (% predicted), and a categorical outcome of COPD. MEASUREMENTS AND MAIN RESULTS: PiMZ heterozygotes compared with PiMM individuals had a reduced median (interquartile range) post-bronchodilator FEV1 (% predicted) (92.0 [75.6-105.4] vs. 98.6 [85.5-109.7]; P = 0.04), FEV1/FVC ratio (0.75 [0.66-0.79] vs. 0.78 [0.73-0.83]; P = 0.004), and FEF25-75 (% predicted) (63.84 [38.45-84.35] vs. 72.8 [55.5-97.7]; P = 0.0013) compared with PiMM individuals. This effect was abrogated in never-smoking and accentuated in ever-smoking PiMZ individuals. PiMZ heterozygosity was associated with an adjusted odds ratio for COPD of 5.18 (95% confidence interval, 1.27-21.15; P = 0.02) and this was higher (odds ratio, 10.65; 95% confidence interval, 2.17-52.29; P = 0.004) in ever-smoking individuals. CONCLUSIONS: These results indicate that PiMZ heterozygotes have significantly more airflow obstruction and COPD than PiMM individuals and cigarette smoke exposure exerts a significant modifier effect.