Chronic obstructive pulmonary disease (COPD) is the third leading cause of death in the US. The majority of COPD patients have symptoms of chronic bronchitis, which lacks specific therapies. A major impediment to therapeutic development has been the absence of animal models that recapitulate key clinical and pathologic features of human disease. Ferrets are well suited for the investigation of the significance of respiratory diseases, given prior data indicating similarities to human airway physiology and submucosal gland distribution. Here, we exposed ferrets to chronic cigarette smoke and found them to approximate complex clinical features of human COPD. Unlike mice, which develop solely emphysema, smoke-exposed ferrets exhibited markedly higher numbers of early-morning spontaneous coughs and sporadic infectious exacerbations as well as a higher level of airway obstruction accompanied by goblet cell metaplasia/hyperplasia and increased mucus expression in small airways, indicative of chronic bronchitis and bronchiolitis. Overall, we demonstrate the first COPD animal model exhibiting clinical and pathologic features of chronic bronchitis to our knowledge, providing a key advance that will greatly facilitate the preclinical development of novel treatments for this disease.

Transforming growth factor β (TGF-β), signaling induced by cigarette smoke (CS), plays an important role in the progression of airway diseases, like chronic bronchitis associated with chronic obstructive pulmonary disease (COPD), and in smokers. Chronic bronchitis is characterized by reduced mucociliary clearance (MCC). Cystic fibrosis transmembrane conductance regulator (CFTR) plays an important role in normal MCC. TGF-β and CS (via TGF-β) promote acquired CFTR dysfunction by suppressing CFTR biogenesis and function. Understanding the mechanism by which CS promotes CFTR dysfunction can identify therapeutic leads to reverse CFTR suppression and rescue MCC. TGF-β alters the microRNAome of primary human bronchial epithelium. TGF-β and CS upregulate miR-145-5p expression to suppress CFTR and the CFTR modifier, SLC26A9. miR-145-5p upregulation with a concomitant CFTR and SLC26A9 suppression was validated in CS-exposed mouse models. While miR-145-5p antagonism rescued the effects of TGF-β in bronchial epithelial cells following transfection, an aptamer to block TGF-β signaling rescues CS- and TGF-β-mediated suppression of CFTR biogenesis and function in the absence of any transfection reagent. These results demonstrate that miR-145-5p plays a significant role in acquired CFTR dysfunction by CS, and they validate a clinically feasible strategy for delivery by inhalation to locally modulate TGF-β signaling in the airway and rescue CFTR biogenesis and function.

BackgroundDysfunction in cystic fibrosis transmembrane conductance regulator (CFTR) can be elicited by cigarette smoke and is observed in patients with chronic bronchitis. We have previously demonstrated in human airway epithelial cell monolayers that roflumilast, a clinically approved phosphodiesterase 4 inhibitor that reduces the risk of exacerbations in chronic obstructive pulmonary disease patients with chronic bronchitis and a history of exacerbations, activates CFTR-dependent chloride secretion via a cAMP-mediated pathway, partially restores the detrimental effects of cigarette smoke on CFTR-mediated ion transport, and increases CFTR-dependent gastrointestinal fluid secretion in isolated murine intestine segments. Based on these findings, we hypothesized that roflumilast could improve CFTR-mediated chloride transport and induce secretory diarrhea in mice exhibiting cigarette smoke-induced CFTR dysfunction.MethodsA/J mice expressing wild type CFTR (+/+) were exposed to cigarette smoke or air with or without roflumilast and the effect of treatment on CFTR-dependent chloride transport was quantified using nasal potential difference (NPD) measurements in vivo and short-circuit current (Isc) analysis of trachea ex vivo. Stool specimen were collected and the wet/dry ratio measured to assess the effect of roflumilast on secretory diarrhea.ResultsAcute roflumilast treatment increased CFTR-dependent chloride transport in both smoke- and air-exposed mice (smoke, −2.0 ± 0.4 mV, 131.3 ± 29.3 μA/cm2, P < 0.01 and air, 3.9 ± 0.8 mV, 147.7 ± 38.0 μA/cm2, P < 0.01 vs. vehicle −0.3 ± 0.7 mV, 10.4 ± 7.0 μA/cm2). Oral administration of roflumilast over five weeks completely reversed the deleterious effects of cigarette smoke on CFTR function in smoke-exposed animals, in which CFTR-dependent chloride transport was 64% that of air controls (roflumilast, −15.22 ± 2.7 mV vs. air, −14.45 ± 1.4 mV, P < 0.05). Smoke exposure increased the wet/dry ratio of stool specimen to a level beyond which roflumilast had little additional effect.ConclusionsRoflumilast effectively rescues CFTR-mediated chloride transport in vivo, further implicating CFTR activation as a mechanism through which roflumilast benefits patients with bronchitis.

Rationale.The majority of chronic obstructive pulmonary disease (COPD) patients have chronic bronchitis, for which specific therapies are unavailable. Acquired cystic fibrosis transmembrane conductance regulator (CFTR) dysfunction is observed in chronic bronchitis, but has not been proven in a controlled animal model with airway disease. Furthermore, the potential of CFTR as a therapeutic target has not been tested in vivo, given limitations to rodent models of COPD. Ferrets exhibit cystic fibrosis-related lung pathology when CFTR is absent and COPD with bronchitis following cigarette smoke exposure.Objectives.To evaluate CFTR dysfunction induced by smoking and test its pharmacologic reversal by a novel CFTR potentiator, GLPG2196, in a ferret model of COPD with chronic bronchitis.Methods.Ferrets were exposed for six months to cigarette smoke to induce COPD and chronic bronchitis and then treated with eneral GLPG2196 once daily for one month. Electrophysiologic measurements of ion transport and CFTR function, assessment of mucociliary function by one-micron optical coherence tomography imaging and particle tracking microrhelogy, microcomputed tomography imaging, histopathological analysis, and quantification of CFTR protein and mRNA expression were used to evaluate mechanistic and pathophysiological changes.Measurements and Main Results.Following cigarette smoke exposure, ferrets exhibited CFTR dysfunction, increased mucus viscosity, delayed mucociliary clearance, airway wall thickening, and airway epithelial hypertrophy. In COPD ferrets, GLPG2196 treatment reversed CFTR dysfunction, increased mucus transport by decreasing mucus viscosity, and reduced brochial wall thickening and airway epithelial hypertrophy.Conclusions.The pharmacologic reversal of acquired CFTR dysfunction is beneficial against pathologic features of chronic bronchitis in a COPD ferret model.

Limited success achieved in translating basic science discoveries into clinical applications for chronic airway diseases is attributed to differences in respiratory anatomy and physiology, poor approximation of pathologic processes, and lack of correlative clinical endpoints between humans and laboratory animal models. Here, we discuss advantages of using ferrets (Mustela putorus furo) as a model for improved understanding of human airway physiology and demonstrate assays for quantifying airway epithelial ion transport in vivo and ex vivo, and establish air-liquid interface cultures of ferret airway epithelial cells as a complementary in vitro model for mechanistic studies. We present data here that establishes the feasibility of measuring these human disease endpoints in ferrets. Briefly, potential difference across the nasal and the lower airway epithelium in ferrets could be consistently assessed, were highly reproducible, and responsive to experimental interventions. Additionally, ferret airway epithelial cells were amenable to primary cell culture methods for in vitro experiments as was the use of ferret tracheal explants as an ex vivo system for assessing ion transport. The feasibility of conducting multiple assessments of disease outcomes supports the adoption of ferrets as a highly relevant model for research in obstructive airway diseases.