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BACKGROUND: The primary underlying defect in cystic fibrosis (CF) is disrupted ion transport in epithelia throughout the body. It is unclear if symptoms such as airway hyperreactivity (AHR) and increased airway smooth muscle (ASM) volume in people with CF are due to inherent abnormalities in smooth muscle or are secondary to epithelial dysfunction. Transforming Growth Factor beta 1 (TGFß) is an established genetic modifier of CF lung disease and a known driver of abnormal ASM function. Prior studies have demonstrated that CF mice develop greater AHR, goblet cell hyperplasia, and ASM hypertrophy after pulmonary TGFß exposure. However, the mechanism driving these abnormalities in CF lung disease, specifically the contribution of CFTR loss in ASM, was unknown. METHODS: In this study, mice with smooth muscle-specific loss of CFTR function (Cftrfl/fl; SM-Cre mice) were exposed to pulmonary TGFß. The impact on lung pathology and physiology was investigated through examination of lung mechanics, Western blot analysis, and pulmonary histology. RESULTS: Cftrfl/fl; SM-Cre mice treated with TGFß demonstrated greater methacholine-induced AHR than control mice. However, Cftrfl/fl; SM-Cre mice did not develop increased inflammation, ASM area, or goblet cell hyperplasia relative to controls following TGFß exposure. CONCLUSIONS: These results demonstrate a direct smooth muscle contribution to CF airway obstruction mediated by TGFß. Dysfunction in non-epithelial tissues should be considered in the development of CF therapeutics, including potential genetic therapies.
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Asma , Fibrose Cística , Animais , Camundongos , Fibrose Cística/metabolismo , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Hiperplasia/metabolismo , Hiperplasia/patologia , Músculo Liso/metabolismo , Fator de Crescimento Transformador beta/metabolismoRESUMO
Neutrophil extracellular traps (NETs), a key component of early defense against microbial infection, are also associated with tissue injury. NET composition has been reported to vary with some disease states, but the composition and variability of NETs across many healthy subjects provide a critical comparison that has not been well investigated. We evaluated NETs from twelve healthy subjects of varying ages isolated from multiple blood draws over a three-and-one-half-year period to delineate the variability in extracellular DNA, protein, enzymatic activities, and susceptibility to protease inhibitors. We calculated correlations for NET constituents and loss of human bronchial epithelial barrier integrity, measured by transepithelial electrical resistance, after NET exposure. We found that although there was some variability within the same subject over time, the mean NET total DNA, dsDNA, protein, LDH, neutrophil elastase (NE), and proteinase 3 (PR3) in isolated NETs were consistent across subjects. NET serine protease activity varied considerably within the same donor from day to day. The mean NET cathepsin G and MPO were significantly different across donors. IL-8 > IL-1RA > G-CSF were the most abundant cytokines in NETs. There was no significant difference in the mean concentration or variability of IL-8, IL-1RA, G-CSF, IL-1α, IL-1ß, or TNF-α in different subjects' NETs. NET DNA concentration was correlated with increased NET neutrophil elastase activity and higher NET IL-1RA concentrations. The mean reduction in protease activity by protease inhibitors was significantly different across donors. NET DNA concentration correlated best with reductions in the barrier integrity of human bronchial epithelia. Defining NET concentration by DNA content correlates with other NET components and reductions in NET-driven epithelial barrier dysfunction, suggesting DNA is a reasonable surrogate measurement for these complex structures in healthy subjects.
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Armadilhas Extracelulares , Humanos , Voluntários Saudáveis , Proteína Antagonista do Receptor de Interleucina 1 , Interleucina-8 , Elastase de Leucócito , Fator Estimulador de Colônias de Granulócitos , DNA , Inibidores de ProteasesRESUMO
Rationale: In cystic fibrosis the major cause of morbidity and mortality is lung disease characterized by inflammation and infection. The influence of sphingolipid metabolism is poorly understood with a lack of studies using human airway model systems.Objectives: To investigate sphingolipid metabolism in cystic fibrosis and the effects of treatment with recombinant human acid ceramidase on inflammation and infection.Methods: Sphingolipids were measured using mass spectrometry in fully differentiated cultures of primary human airway epithelial cells and cocultures with Pseudomonas aeruginosa. In situ activity assays, Western blotting, and quantitative PCR were used to investigate function and expression of ceramidase and sphingomyelinase. Effects of treatment with recombinant human acid ceramidase on sphingolipid profile and inflammatory mediator production were assessed in cell cultures and murine models.Measurements and Main Results: Ceramide is increased in cystic fibrosis airway epithelium owing to differential function of enzymes regulating sphingolipid metabolism. Sphingosine, a metabolite of ceramide with antimicrobial properties, is not upregulated in response to P. aeruginosa by cystic fibrosis airway epithelia. Tumor necrosis factor receptor 1 is increased in cystic fibrosis epithelia and activates NF-κB signaling, generating inflammation. Treatment with recombinant human acid ceramidase, to decrease ceramide, reduced both inflammatory mediator production and susceptibility to infection.Conclusions: Sphingolipid metabolism is altered in airway epithelial cells cultured from people with cystic fibrosis. Treatment with recombinant acid ceramidase ameliorates the two pivotal features of cystic fibrosis lung disease, inflammation and infection, and thus represents a therapeutic approach worthy of further exploration.
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Ceramidase Ácida/metabolismo , Ceramidase Ácida/farmacologia , Fibrose Cística/tratamento farmacológico , Pneumonia/diagnóstico , Infecções por Pseudomonas/diagnóstico , Esfingolipídeos/metabolismo , Adolescente , Células Epiteliais Alveolares/efeitos dos fármacos , Animais , Western Blotting/métodos , Células Cultivadas , Criança , Fibrose Cística/diagnóstico , Humanos , Inflamação/diagnóstico , Inflamação/tratamento farmacológico , Espectrometria de Massas/métodos , Camundongos , Pneumonia/tratamento farmacológico , Reação em Cadeia da Polimerase/métodos , Infecções por Pseudomonas/tratamento farmacológico , Sensibilidade e Especificidade , Índice de Gravidade de Doença , Adulto JovemRESUMO
Cystic fibrosis (CF) is a lethal genetic disease characterized by progressive lung damage and airway obstruction. The majority of patients demonstrate airway hyperresponsiveness (AHR), which is associated with more rapid lung function decline. Recent studies in the neonatal CF pig demonstrated airway smooth muscle (ASM) dysfunction. These findings, combined with observed CF transmembrane conductance regulator (CFTR) expression in ASM, suggest that a fundamental defect in ASM function contributes to lung function decline in CF. One established driver of AHR and ASM dysfunction is transforming growth factor (TGF) ß1, a genetic modifier of CF lung disease. Prior studies demonstrated that TGFß exposure in CF mice drives features of CF lung disease, including goblet cell hyperplasia and abnormal lung mechanics. CF mice displayed aberrant responses to pulmonary TGFß, with elevated PI3K signaling and greater increases in lung resistance compared with controls. Here, we show that TGFß drives abnormalities in CF ASM structure and function through PI3K signaling that is enhanced in CFTR-deficient lungs. CF and non-CF mice were exposed intratracheally to an adenoviral vector containing the TGFß1 cDNA, empty vector, or PBS only. We assessed methacholine-induced AHR, bronchodilator response, and ASM area in control and CF mice. Notably, CF mice demonstrated enhanced AHR and bronchodilator response with greater ASM area increases compared with non-CF mice. Furthermore, therapeutic inhibition of PI3K signaling mitigated the TGFß-induced AHR and goblet cell hyperplasia in CF mice. These results highlight a latent AHR phenotype in CFTR deficiency that is enhanced through TGFß-induced PI3K signaling.
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Fibrose Cística/enzimologia , Fibrose Cística/fisiopatologia , Fosfatidilinositol 3-Quinases/metabolismo , Hipersensibilidade Respiratória/enzimologia , Hipersensibilidade Respiratória/fisiopatologia , Transdução de Sinais , Fator de Crescimento Transformador beta/efeitos adversos , Agonistas Adrenérgicos beta/farmacologia , Albuterol/farmacologia , Animais , Broncoconstrição/efeitos dos fármacos , Células Caliciformes/patologia , Hiperplasia , Pulmão/fisiopatologia , Camundongos Endogâmicos C57BL , Músculo Liso/efeitos dos fármacos , Músculo Liso/fisiopatologia , Inibidores de Fosfoinositídeo-3 Quinase/farmacologia , Transdução de Sinais/efeitos dos fármacosRESUMO
Neutrophil extracellular traps (NETs) provide host defense but can contribute to the pathobiology of diverse human diseases. We sought to determine the extent and mechanism by which NETs contribute to human airway cell inflammation. Primary normal human bronchial epithelial cells (HBEs) grown at air-liquid interface and wild-type (wt)CFBE41o- cells (expressing wtCFTR) were exposed to cell-free NETs from unrelated healthy volunteers for 18 h in vitro. Cytokines were measured in the apical supernatant by Luminex, and the effect on the HBE transcriptome was assessed by RNA sequencing. NETs consistently stimulated IL-8, TNF-α, and IL-1α secretion by HBEs from multiple donors, with variable effects on other cytokines (IL-6, G-CSF, and GM-CSF). Expression of HBE RNAs encoding IL-1 family cytokines, particularly IL-36 subfamily members, was increased in response to NETs. NET exposure in the presence of anakinra [recombinant human IL-1 receptor antagonist (rhIL-1RA)] dampened NET-induced changes in IL-8 and TNF-α proteins as well as IL-36α RNA. rhIL-36RA limited the increase in expression of proinflammatory cytokine RNAs in HBEs exposed to NETs. NETs selectively upregulate an IL-1 family cytokine response in HBEs, which enhances IL-8 production and is limited by rhIL-1RA. The present findings describe a unique mechanism by which NETs may contribute to inflammation in human lung disease in vivo. NET-driven IL-1 signaling may represent a novel target for modulating inflammation in diseases characterized by a substantial NET burden.
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Brônquios/citologia , Células Epiteliais/metabolismo , Armadilhas Extracelulares/metabolismo , Interleucina-1/metabolismo , Interleucina-8/metabolismo , Adulto , Linhagem Celular , Células Epiteliais/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Mediadores da Inflamação/metabolismo , Proteína Antagonista do Receptor de Interleucina 1/farmacologia , Elastase de Leucócito/metabolismo , Peroxidase/metabolismo , Proteínas Recombinantes/farmacologia , Transdução de Sinais/efeitos dos fármacos , Transcrição Gênica/efeitos dos fármacosRESUMO
Cystic fibrosis (CF) produces variable lung disease phenotypes that are, in part, independent of the CF transmembrane conductance regulator ( CFTR) genotype. Transforming growth factor-ß (TGFß) is the best described genetic modifier of the CF phenotype, but its mechanism of action is unknown. We hypothesized that TGFß is sufficient to drive pathognomonic features of CF lung disease in vivo and that CFTR deficiency enhances susceptibility to pathological TGFß effects. A CF mouse model and littermate controls were exposed intratracheally to an adenoviral vector containing the TGFß1 cDNA (Ad-TGFß), empty vector, or PBS only. Studies were performed 1 wk after treatment, including lung mechanics, collection of bronchoalveolar lavage fluid, and analysis of lung histology, RNA, and protein. CF and non-CF mice showed similar weight loss, inflammation, goblet cell hyperplasia, and Smad pathway activation after Ad-TGFß treatment. Ad-TGFß produced greater abnormalities in lung mechanics in CF versus control mice, which was uniquely associated with induction of phosphoinositide 3-kinase and mitogen-activated protein kinase signaling. CFTR transcripts were reduced, and epithelial sodium channel transcripts were increased in CF and non-CF mice, whereas the goblet cell transcription factors, forkhead ortholog A3 and SAM-pointed domain-containing ETS-like factor, were increased in non-CF but not CF mice following Ad-TGFß treatment. Pulmonary TGFß1 expression was sufficient to produce pulmonary remodeling and abnormalities in lung mechanics that were associated with both shared and unique cell signaling pathway activation in CF and non-CF mice. These results highlight the multifunctional impact of TGFß on pulmonary pathology in vivo and identify cellular-response differences that may impact CF lung pathology.
Assuntos
Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Fibrose Cística/metabolismo , Regulação da Expressão Gênica , Células Caliciformes/metabolismo , Pulmão/metabolismo , Fator de Crescimento Transformador beta1/biossíntese , Adenoviridae , Animais , Fibrose Cística/genética , Fibrose Cística/patologia , Fibrose Cística/fisiopatologia , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Células Caliciformes/patologia , Hiperplasia , Inflamação/genética , Inflamação/metabolismo , Inflamação/patologia , Pulmão/patologia , Pulmão/fisiopatologia , Camundongos , Camundongos Transgênicos , Transdução Genética , Fator de Crescimento Transformador beta1/genéticaRESUMO
RATIONALE: Cystic Fibrosis (CF) progresses through recurrent infection and inflammation, causing permanent lung function loss and airway remodeling. CT scans reveal abnormally low-density lung parenchyma in CF, but its microstructural nature remains insufficiently explored due to clinical CT limitations. To this end, diffusion-weighted 129Xe MRI is a non-invasive and validated measure of lung microstructure. In this work, we investigate microstructural changes in people with CF (pwCF) relative to age-matched, healthy subjects using comprehensive imaging and analysis involving pulmonary-function tests (PFTs), and 129Xe MRI. METHODS: 38 healthy subjects (age 6-40; 17.2 ± 9.5 years) and 39 pwCF (age 6-40; 15.6 ± 8.0 years) underwent 129Xe-diffusion MRI and PFTs. The distribution of diffusion measurements (i.e., apparent diffusion coefficients (ADC) and morphometric parameters) was assessed via linear binning (LB). The resulting volume percentages of bins were compared between controls and pwCF. Mean ADC and morphometric parameters were also correlated with PFTs. RESULTS: Mean whole-lung ADC correlated significantly with age (P < 0.001) for both controls and CF, and with PFTs (P < 0.05) specifically for pwCF. Although there was no significant difference in mean ADC between controls and pwCF (P = 0.334), age-adjusted LB indicated significant voxel-level diffusion (i.e., ADC and morphometric parameters) differences in pwCF compared to controls (P < 0.05). CONCLUSIONS: 129Xe diffusion MRI revealed microstructural abnormalities in CF lung disease. Smaller microstructural size may reflect compression from overall higher lung density due to interstitial inflammation, fibrosis, or other pathological changes. While elevated microstructural size may indicate emphysema-like remodeling due to chronic inflammation and infection.
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The mammalian target of rapamycin (mTOR) signaling pathway integrates environmental cues, promotes cell growth/differentiation, and regulates immune responses. Although inhibition of mTOR with rapamycin has potent immunosuppressive activity, mixed effects have been reported in OVA-induced models of allergic asthma. We investigated the impact of two rapamycin treatment protocols on the major characteristics of allergic asthma induced by the clinically relevant allergen, house dust mite (HDM). In protocol 1, BALB/c mice were exposed to 10 intranasal HDM doses over a period of 24 d and treated with rapamycin simultaneously during the sensitization/exposure period. In protocol 2, rapamycin was administered after the mice had been sensitized to HDM (i.p. injection) and prior to initiation of two intranasal HDM challenges over 4 d. Airway hyperreactivity (AHR), IgE, inflammatory cells, cytokines, leukotrienes, goblet cells, and activated T cells were assessed. In protocol 1, rapamycin blocked HDM-induced increases in AHR, inflammatory cell counts, and IgE, as well as attenuated goblet cell metaplasia. In protocol 2, rapamycin blocked increases in AHR, IgE, and T cell activation and reduced goblet cell metaplasia, but it had no effect on inflammatory cell counts. Increases in IL-13 and leukotrienes were also blocked by rapamycin, although increases in IL-4 were unaffected. These data demonstrated that rapamycin can inhibit cardinal features of allergic asthma, including increases in AHR, IgE, and goblet cells, most likely as a result of its ability to reduce the production of two key mediators of asthma: IL-13 and leukotrienes. These findings highlight the importance of the mTOR pathway in allergic airway disease.
Assuntos
Asma/tratamento farmacológico , Hiper-Reatividade Brônquica/tratamento farmacológico , Células Caliciformes/efeitos dos fármacos , Imunoglobulina E/biossíntese , Imunossupressores/farmacologia , Sirolimo/farmacologia , Animais , Asma/imunologia , Western Blotting , Hiper-Reatividade Brônquica/imunologia , Separação Celular , Citocinas/biossíntese , Modelos Animais de Doenças , Feminino , Citometria de Fluxo , Células Caliciformes/imunologia , Hipersensibilidade/tratamento farmacológico , Hipersensibilidade/imunologia , Imuno-Histoquímica , Camundongos , Camundongos Endogâmicos BALB C , Pyroglyphidae/imunologia , Reação em Cadeia da Polimerase em Tempo Real , Transdução de Sinais/imunologia , Serina-Treonina Quinases TOR/antagonistas & inibidoresRESUMO
Neutrophil Extracellular Traps (NETs), a key component of early defense against microbial infection, are also associated with tissue injury. NET composition has been reported to vary with some disease states, but the composition and variability of NETs across many healthy subjects provides a critical comparison that has not been well investigated. We evaluated NETs from twelve healthy subjects of varying ages isolated from multiple blood draws over a three and one half-year period to delineate the variability in extracellular DNA, protein, enzymatic activities, and susceptibility to protease inhibitors. We calculated correlations for NET constituents and loss of human bronchial epithelial barrier integrity, measured by transepithelial electrical resistance, after NET exposure. We found that although there was some variability within the same subject over time, the mean numbers of neutrophils, protein, LDH, serine protease activities, and cytokines IL-8, IL-1RA, and G-CSF in isolated NETs were consistent across subjects. Total DNA and double stranded DNA content in NETs were different across donors. NETs had little or no TNFα, IL-17A, or GM-CSF. NET DNA concentration correlated with increased NET neutrophil elastase activity and higher NET IL-1RA concentrations. NET serine protease activity varied considerably within the same donor from day-to-day. Mean response to protease inhibitors was significantly different across donors. NET DNA concentration correlated best with reductions in barrier integrity of human bronchial epithelia. Defining NET concentration by DNA content correlates with other NET components and reductions in NET-driven epithelial barrier dysfunction, suggesting DNA is a reasonable surrogate measurement for these complex structures in healthy subjects.
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Increases in the epidermal growth factor receptor (EGFR) have been associated with the severity of airway thickening in chronic asthmatic subjects, and EGFR signaling is induced by asthma-related cytokines and inflammation. The goal of this study was to determine the role of EGFR signaling in a chronic allergic model of asthma and specifically in epithelial cells, which are increasingly recognized as playing an important role in asthma. EGFR activation was assessed in mice treated with intranasal house dust mite (HDM) for 3 wk. EGFR signaling was inhibited in mice treated with HDM for 6 wk, by using either the drug erlotinib or a genetic approach that utilizes transgenic mice expressing a mutant dominant negative epidermal growth factor receptor in the lung epithelium (EGFR-M mice). Airway hyperreactivity (AHR) was assessed by use of a flexiVent system after increasing doses of nebulized methacholine. Airway smooth muscle (ASM) thickening was measured by morphometric analysis. Sensitization to HDM (IgG and IgE), inflammatory cells, and goblet cell changes were also assessed. Increased EGFR activation was detected in HDM-treated mice, including in bronchiolar epithelial cells. In mice exposed to HDM for 6 wk, AHR and ASM thickening were reduced after erlotinib treatment and in EGFR-M mice. Sensitization to HDM and inflammatory cell counts were similar in all groups, except neutrophil counts, which were lower in the EGFR-M mice. Goblet cell metaplasia with HDM treatment was reduced by erlotinib, but not in EGFR-M transgenic mice. This study demonstrates that EGFR signaling, especially in the airway epithelium, plays an important role in mediating AHR and remodeling in a chronic allergic asthma model.
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Remodelação das Vias Aéreas/fisiologia , Asma/fisiopatologia , Hiper-Reatividade Brônquica/complicações , Células Epiteliais/enzimologia , Receptores ErbB/metabolismo , Transdução de Sinais , Animais , Asma/complicações , Asma/parasitologia , Asma/patologia , Hiper-Reatividade Brônquica/parasitologia , Hiper-Reatividade Brônquica/patologia , Hiper-Reatividade Brônquica/fisiopatologia , Doença Crônica , Modelos Animais de Doenças , Ativação Enzimática , Células Epiteliais/patologia , Receptores ErbB/antagonistas & inibidores , Células Caliciformes/patologia , Inflamação/complicações , Inflamação/patologia , Pulmão/parasitologia , Pulmão/patologia , Pulmão/fisiopatologia , Metaplasia , Camundongos , Músculo Liso/patologia , Pyroglyphidae/fisiologiaRESUMO
BACKGROUND: Two functional measurements (multiple breath washout [MBW] and hyperpolarized 129Xe ventilation magnetic resonance imaging [129Xe MRI]) have been shown to be more sensitive to cystic fibrosis (CF) lung obstruction than traditional spirometry. However, functional techniques may be sensitive to different underlying structural abnormalities. The purpose of this study was to determine relationships between these functional markers, their pathophysiology, and 1-year clinical outcomes. METHODS: Spirometry, MBW, 129Xe MRI, and ultrashort echo-time (UTE) MRI were obtained in a same-day assessment of 27 pediatric CF patients (ages 11.5±5.0) who had not begun CFTR modulator therapies. UTE MRI was scored for structural abnormalities and functional metrics obtained via spirometry, MBW and 129Xe MRI. 1-year outcomes (ΔFEV1 and pulmonary exacerbations), during which ≈50% initiated modulator therapy, were obtained from the electronic medical record. RESULTS: MBW, 129Xe MRI, and UTE MRI detected clinically significant disease in more subjects (>78%) compared to spirometry (<30%). UTE MRI suggests increased odds of bronchial changes when mucus plugging is present in the same lobe. MBW and 129Xe MRI correlated best with mucus plugging, while spirometry correlated best with consolidations. Bronchial abnormalities were associated with future pulmonary exacerbations. CONCLUSIONS: MBW, 129Xe MRI, and UTE MRI are more sensitive for detection of pediatric CF lung disease when compared to spirometry. MBW and 129Xe MRI correlated with structural abnormalities which occur in early CF disease, suggesting MBW and 129Xe MRI are valuable tools in mild CF lung disease that can guide clinical decision making.
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Fibrose Cística/diagnóstico por imagem , Fibrose Cística/fisiopatologia , Imageamento por Ressonância Magnética/métodos , Espirometria , Criança , Feminino , Humanos , Masculino , Estudos Prospectivos , Sensibilidade e Especificidade , Isótopos de XenônioRESUMO
BACKGROUND: Cystic fibrosis (CF) patients develop severe lung disease including chronic airway infections, neutrophilic inflammation, and progressive fibrotic remodeling in airways. However, cellular and molecular processes that regulate excessive collagen deposition in airways in these patients remain unclear. Fibrocytes are bone marrow (BM)-derived mesenchymal cells that express the hematopoietic cell marker CD45, and mesenchymal cell markers and implicated in collagen deposition in several fibrotic diseases. It is unknown whether fibrocytes accumulate in the lungs of CF patients, so the current study evaluates the presence of fibrocytes in the fibrotic lesions of airways in explanted CF lungs compared to non-CF unused donor lungs (control). METHODS: We used immunofluorescence staining to determine if fibrocytes accumulate in explanted CF lungs compared to healthy donor lungs. Simultaneously, we evaluated cells collected by bronchoalveolar lavage (BAL) in CF patients using multi-color flow cytometry. Finally, we analyzed transcripts differentially expressed in fibrocytes isolated from the explanted CF lungs compared to control to assess fibrocyte-specific pro-fibrotic gene networks. RESULTS: Our findings demonstrate fibrocyte accumulation in CF lungs compared to non-CF lungs. Additionally, fibrocytes were detected in the BAL of all CF children. Transcriptomic analysis of fibrocytes identified dysregulated genes associated with fibrotic remodeling in CF lungs. CONCLUSIONS: With significantly increased fibrocytes that show increased expression of pro-fibrotic gene transcripts compared to control, our findings suggest an intervention for fibrotic remodeling as a potential therapeutic target in CF.
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Fibrose Cística/patologia , Pulmão/patologia , Células-Tronco Mesenquimais/fisiologia , Adolescente , Estudos de Casos e Controles , Técnicas de Cultura de Células , Criança , Pré-Escolar , Fibrose Cística/metabolismo , Feminino , Humanos , Antígenos Comuns de Leucócito/metabolismo , Pulmão/metabolismo , MasculinoRESUMO
Transforming growth factor (TGF)-alpha and its receptor, the epidermal growth factor receptor, are induced after lung injury and are associated with remodeling in chronic pulmonary diseases, such as pulmonary fibrosis and asthma. Expression of TGF-alpha in the lungs of adult mice causes fibrosis, pleural thickening, and pulmonary hypertension, in addition to increased expression of a transcription factor, early growth response-1 (Egr-1). Egr-1 was increased in airway smooth muscle (ASM) and the vascular adventitia in the lungs of mice conditionally expressing TGF-alpha in airway epithelium (Clara cell secretory protein-rtTA(+/-)/[tetO](7)-TGF-alpha(+/-)). The goal of this study was to determine the role of Egr-1 in TGF-alpha-induced lung disease. To accomplish this, TGF-alpha-transgenic mice were crossed to Egr-1 knockout (Egr-1(ko/ko)) mice. The lack of Egr-1 markedly increased the severity of TGF-alpha-induced pulmonary disease, dramatically enhancing airway muscularization, increasing pulmonary fibrosis, and causing greater airway hyperresponsiveness to methacholine. Smooth muscle hyperplasia, not hypertrophy, caused the ASM thickening in the absence of Egr-1. No detectable increases in pulmonary inflammation were found. In addition to the airway remodeling disease, vascular remodeling and pulmonary hypertension were also more severe in Egr-1(ko/ko) mice. Thus, Egr-1 acts to suppress epidermal growth factor receptor-mediated airway and vascular muscularization, fibrosis, and airway hyperresponsiveness in the absence of inflammation. This provides a unique model to study the processes causing pulmonary fibrosis and ASM thickening without the complicating effects of inflammation.
Assuntos
Hiper-Reatividade Brônquica/fisiopatologia , Proteína 1 de Resposta de Crescimento Precoce/fisiologia , Pulmão/patologia , Fibrose Pulmonar/patologia , Fator de Crescimento Transformador alfa/fisiologia , Resistência das Vias Respiratórias , Albuterol/farmacologia , Animais , Hiper-Reatividade Brônquica/induzido quimicamente , Hiper-Reatividade Brônquica/genética , Células Cultivadas/efeitos dos fármacos , Células Cultivadas/patologia , Modelos Animais de Doenças , Proteína 1 de Resposta de Crescimento Precoce/biossíntese , Proteína 1 de Resposta de Crescimento Precoce/genética , Receptores ErbB/antagonistas & inibidores , Fibroblastos/citologia , Humanos , Hiperplasia , Complacência Pulmonar , Cloreto de Metacolina/toxicidade , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Músculo Liso/patologia , Músculo Liso Vascular/efeitos dos fármacos , Músculo Liso Vascular/patologia , Artéria Pulmonar/citologia , Fibrose Pulmonar/genética , Fibrose Pulmonar/fisiopatologia , Proteínas Recombinantes de Fusão/fisiologia , Fator de Crescimento Transformador alfa/efeitos adversos , Redução de PesoRESUMO
INTRODUCTION: Cystic fibrosis (CF) is a genetic disease characterized by progressive lung disease. Most CF therapies focus on treating secondary pulmonary complications rather than addressing the underlying processes inducing airway remodeling and ineffective response to infection. Transforming growth factor beta (TGFß) is a cytokine involved in fibrosis, inflammation, and injury response as well as a genetic modifier and biomarker of CF lung disease. Targeting the TGFß pathway has been pursued in other diseases, but the mechanism of TGFß effects in CF is less well understood. Areas covered: In this review, we discuss CF lung disease pathogenesis with a focus on potential links to TGFß. TGFß signaling in lung health and disease is reviewed. Recent studies investigating TGFß's impact in CF airway epithelial cells are highlighted. Finally, an overview of potential therapies to target TGFß signaling relevant to CF are addressed. Expert opinion: The broad impact of TGFß signaling on numerous cellular processes in homeostasis and disease is both a strength and a challenge to developing TGFß dependent therapeutics in CF. We discuss the challenges inherent in developing TGFß-targeted therapy, identifying appropriate patient populations, and questions regarding the timing of treatment. Future directions for research into TGFß focused therapeutics are discussed.
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Fibrose Cística/tratamento farmacológico , Desenho de Fármacos , Fator de Crescimento Transformador beta/metabolismo , Remodelação das Vias Aéreas/fisiologia , Animais , Fibrose Cística/genética , Fibrose Cística/fisiopatologia , Humanos , Terapia de Alvo Molecular , Transdução de Sinais/fisiologiaRESUMO
Traditional pulmonary therapies for cystic fibrosis (CF) target the downstream effects of CF transmembrane conductance regulator (CFTR) dysfunction (the cause of CF). Use of one such therapy, ß-adrenergic bronchodilators (such as albuterol), is nearly universal for airway clearance. Conversely, novel modulator therapies restore function to select mutant CFTR proteins, offering a disease-modifying treatment. Recent trials of modulators targeting F508del-CFTR, the most common CFTR mutation, suggest that chronic ß-agonist use may undermine clinical modulator benefits. We therefore sought to understand the impact of chronic or excess ß-agonist exposure on CFTR activation in human airway epithelium. The present studies demonstrate a greater than 60% reduction in both wild-type and modulator-corrected F508del-CFTR activation following chronic exposure to short- and long-acting ß-agonists. This reduction was due to reduced cellular generation of cAMP downstream of the ß-2 adrenergic receptor-G protein complex. Our results point towards a posttranscriptional reduction in adenylyl cyclase function as the mechanism of impaired CFTR activation produced by prolonged ß-agonist exposure. ß-Agonist-induced CFTR dysfunction was sufficient to abrogate VX809/VX770 modulation of F508del-CFTR in vitro. Understanding the clinical relevance of our observations is critical for CF patients using these drugs, and for investigators to inform future CFTR modulator drug trials.
Assuntos
Agonistas de Receptores Adrenérgicos beta 2/farmacologia , Aminofenóis/farmacologia , Aminopiridinas/farmacologia , Benzodioxóis/farmacologia , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Fibrose Cística/tratamento farmacológico , Quinolonas/farmacologia , Mucosa Respiratória/efeitos dos fármacos , Agonistas de Receptores Adrenérgicos beta 2/uso terapêutico , Albuterol/farmacologia , Albuterol/uso terapêutico , Aminofenóis/uso terapêutico , Aminopiridinas/uso terapêutico , Benzodioxóis/uso terapêutico , Linhagem Celular , Cílios/efeitos dos fármacos , Cílios/patologia , AMP Cíclico/metabolismo , Fibrose Cística/genética , Fibrose Cística/patologia , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Interações Medicamentosas , Células Epiteliais/citologia , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/patologia , Humanos , Mutação , Quinolonas/uso terapêutico , Mucosa Respiratória/citologia , Mucosa Respiratória/patologia , Fatores de TempoRESUMO
INTRODUCTION: Mutations in the cystic fibrosis transmembrane conductance regulator protein (CFTR) cause cystic fibrosis (CF), a disease with life threatening pulmonary and gastrointestinal manifestations. Recent breakthrough therapies restore function to select disease-causing CFTR mutations. Ivacaftor is a small molecule that increases the open channel probability of certain CFTR mutations, producing clear evidence of bioactivity and efficacy in pediatric CF patients. CFTR modulators represent a significant advancement in CF treatment. Extending these therapies to young CF patients is proposed to have the greatest long term impact, potentially preventing later disease. AREAS COVERED: Here we summarize the research experience of CFTR modulators in pediatrics, focusing on ivacaftor and highlighting challenges in pediatric studies. As a result of these studies, ivacaftor has been approved in CF patients age 2 years and older who have one of ten CFTR mutations. EXPERT OPINION: Conducting studies in young CF patients presents unique challenges, including small numbers of patients and difficulty selecting sensitive biomarkers and meaningful outcome measures. Adverse events may be more pronounced in children and deserve special attention. Ongoing efforts must focus on expanding and validating new biomarkers, innovative study design, and thorough monitoring of adverse events in children treated with CFTR modulators.
RESUMO
Airway hyperreactivity (AHR) and remodeling are cardinal features of asthma and chronic obstructive pulmonary disease. New therapeutic targets are needed as some patients are refractory to current therapies and develop progressive airway remodeling and worsening AHR. The mammalian target of rapamycin (mTOR) is a key regulator of cellular proliferation and survival. Treatment with the mTOR inhibitor rapamycin inhibits inflammation and AHR in allergic asthma models, but it is unclear if rapamycin can directly inhibit airway remodeling and AHR, or whether its therapeutic effects are entirely mediated through immunosuppression. To address this question, we utilized transforming growth factor-α (TGF-α) transgenic mice null for the transcription factor early growth response-1 (Egr-1) (TGF-α Tg/Egr-1(ko/ko) mice). These mice develop airway smooth muscle thickening and AHR in the absence of altered lung inflammation, as previously reported. In this study, TGF-α Tg/Egr-1(ko/ko) mice lost body weight and developed severe AHR after 3 wk of lung-specific TGF-α induction. Rapamycin treatment prevented body weight loss, airway wall thickening, abnormal lung mechanics, and increases in airway resistance to methacholine after 3 wk of TGF-α induction. Increases in tissue damping and airway elastance were also attenuated in transgenic mice treated with rapamycin. TGF-α/Egr-1(ko/ko) mice on doxycycline for 8 wk developed severe airway remodeling. Immunostaining for α-smooth muscle actin and morphometric analysis showed that rapamycin treatment prevented airway smooth muscle thickening around small airways. Pentachrome staining, assessments of lung collagen and fibronectin mRNA levels, indicated that rapamycin also attenuated fibrotic pathways induced by TGF-α expression for 8 wk. Thus rapamycin reduced airway remodeling and AHR, demonstrating an important role for mTOR signaling in TGF-α-induced/EGF receptor-mediated reactive airway disease.
Assuntos
Remodelação das Vias Aéreas/efeitos dos fármacos , Hiper-Reatividade Brônquica/tratamento farmacológico , Pneumopatias/tratamento farmacológico , Pneumopatias/fisiopatologia , Sirolimo/farmacologia , Remodelação das Vias Aéreas/genética , Remodelação das Vias Aéreas/fisiologia , Animais , Hiper-Reatividade Brônquica/genética , Hiper-Reatividade Brônquica/fisiopatologia , Proteína 1 de Resposta de Crescimento Precoce/deficiência , Proteína 1 de Resposta de Crescimento Precoce/genética , Proteína 1 de Resposta de Crescimento Precoce/fisiologia , Receptores ErbB/fisiologia , Pneumopatias/genética , Pneumopatias/patologia , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Modelos Biológicos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Transdução de Sinais , Serina-Treonina Quinases TOR/fisiologia , Fator de Crescimento Transformador alfa/genética , Fator de Crescimento Transformador alfa/fisiologiaRESUMO
Transforming growth factor-alpha (TGF-alpha) and its receptor, the epithelial growth factor receptor (EGFR), have been associated with lung remodeling in premature infants with bronchopulmonary dysplasia (BPD). The goal of this study was to target TGF-alpha overexpression to the saccular phase of lung morphogenesis and determine early alterations in gene expression. Conditional lung-specific TGF-alpha bitransgenic mice and single-transgene control mice were generated. TGF-alpha overexpression was induced by doxycycline (Dox) treatment from embryonic day 16.5 (E16.5) to E18.5. After birth, all bitransgenic pups died by postnatal day 7 (P7). Lung histology at E18.5 and P1 showed abnormal lung morphogenesis in bitransgenic mice, characterized by mesenchymal thickening, vascular remodeling, and poor apposition of capillaries to distal air spaces. Surfactant levels (saturated phosphatidylcholine) were not reduced in bitransgenic mice. Microarray analysis was performed after 1 or 2 days of Dox treatment during the saccular (E17.5, E18.5) and alveolar phases (P4, P5) to identify genes induced by EGFR signaling that were shared or unique to each phase. We found 196 genes to be altered (>1.5-fold change; P < 0.01 for at least 2 time points), with only 32% similarly altered in both saccular and alveolar phases. Western blot analysis and immunostaining showed that five genes selected from the microarrays (egr-1, SP-B, SP-D, S100A4, and pleiotrophin) were also increased at the protein level. Pathological changes in TGF-alpha-overexpressing mice bore similarities to premature infants born in the saccular phase who develop BPD, including remodeling of the distal lung septae and arteries.
Assuntos
Displasia Broncopulmonar/patologia , Displasia Broncopulmonar/fisiopatologia , Alvéolos Pulmonares/crescimento & desenvolvimento , Fator de Crescimento Transformador alfa/genética , Fator de Crescimento Transformador alfa/metabolismo , Animais , Peso Corporal , Displasia Broncopulmonar/mortalidade , Diferenciação Celular , Receptores ErbB/metabolismo , Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Recém-Nascido , Camundongos , Camundongos Transgênicos , Microscopia Eletrônica de Transmissão , Miocárdio/patologia , Análise de Sequência com Séries de Oligonucleotídeos , Tamanho do Órgão , Alvéolos Pulmonares/patologia , Alvéolos Pulmonares/fisiologia , Ratos , Mucosa Respiratória/crescimento & desenvolvimento , Mucosa Respiratória/fisiologia , Mucosa Respiratória/ultraestrutura , Transcrição GênicaRESUMO
Lens regeneration in the adult newt is a classic example of replacing a lost organ by the process of transdifferentiation. After lens removal, the pigmented epithelial cells of the dorsal iris proliferate and dedifferentiate to form a lens vesicle, which subsequently differentiates to form a new lens. In searching for factors that control this remarkable process, we investigated the expression and role of hedgehog pathway members. These molecules are known to affect retina and pigment epithelium morphogenesis and have been recently shown to be involved in repair processes. Here we show that Shh, Ihh, ptc-1, and ptc-2 are expressed during lens regeneration. The expression of Shh and Ihh is quite unique since these genes have never been detected in lens. Interestingly, both Shh and Ihh are only expressed in the regenerating and developing lens, but not in the intact lens. Interfering with the hedgehog pathway results in considerable inhibition of the process of lens regeneration, including decreased cell proliferation as well as interference with lens fiber differentiation in the regenerating lens vesicle. Down-regulation of ptc-1 was also observed when inhibiting the pathway. These results provide the first evidence of a novel role for the hedgehog pathway in specific regulation of the regenerating lens.