Julia Duerr

Preventive but Not Late Amiloride Therapy Reduces Morbidity and Mortality of Lung Disease in βENaC-overexpressing Mice

RATIONALE Increased airway Na(+) absorption mediated by epithelial Na(+) channels (ENaC) is a characteristic abnormality in the pathogenesis of cystic fibrosis (CF) lung disease. However, inhalation therapy with the ENaC blocker amiloride did not have therapeutic benefits in patients with CF with established lung disease. OBJECTIVES We hypothesized that preventive inhibition of increased Na(+) absorption in a structurally normal lung may be required for effective therapy of CF lung disease in vivo, and that therapeutic effects of late amiloride intervention may be impeded by the chronic disease process. METHODS To test this hypothesis in vivo, we used the betaENaC-overexpression mouse as a model of CF lung disease and determined therapeutic effects of preventive versus late amiloride therapy on survival, airway mucus plugging, chronic bronchitis, and airway remodeling. MEASUREMENTS AND MAIN RESULTS We show that early intervention, i.e., from the first day of life, with the intranasal administration of amiloride significantly reduced pulmonary mortality, airway mucus obstruction, epithelial necrosis, goblet cell metaplasia, and airway inflammation in betaENaC-overexpressing mice. In contrast, consistent with previous human trials in patients with CF, amiloride administration did not have benefits if treatment was started after the development of CF-like lung disease in betaENaC-overexpressing mice. CONCLUSIONS We conclude that preventive inhibition of increased airway Na(+) absorption provides an effective therapy for CF-like lung disease in vivo. These results suggest that amiloride therapy may be an effective preventive therapy for patients with CF if initiated early in life before the onset of lung disease.

The ENaC-overexpressing mouse as a model of cystic fibrosis lung disease

Chronic lung disease remains the major cause of morbidity and mortality of cystic fibrosis (CF) patients. Cftr mutant mice developed severe intestinal obstruction, but did not exhibit the characteristic CF ion transport defects (i.e. deficient cAMP-dependent Cl(-) secretion and increased Na(+) absorption) in the lower airways, and failed to develop CF-like lung disease. These observations led to the generation of transgenic mice with airway-specific overexpression of the epithelial Na(+) channel (ENaC) as an alternative approach to mimic CF ion transport pathophysiology in the lung. Studies of the phenotype of βENaC-transgenic mice demonstrated that increased airway Na(+) absorption causes airway surface liquid (ASL) depletion, reduced mucus transport and a spontaneous CF-like lung disease with airway mucus obstruction and chronic airway inflammation. Here, we summarize approaches that can be applied for studies of the complex in vivo pathogenesis and preclinical evaluation of novel therapeutic strategies in this model of CF lung disease.

Lack of Neutrophil Elastase Reduces Inflammation, Mucus Hypersecretion, and Emphysema, but Not Mucus Obstruction, in Mice with Cystic Fibrosis–like Lung Disease

RATIONALE Recent evidence from clinical studies suggests that neutrophil elastase (NE) released in neutrophilic airway inflammation is a key risk factor for the onset and progression of lung disease in young children with cystic fibrosis (CF). However, the role of NE in the complex in vivo pathogenesis of CF lung disease remains poorly understood. OBJECTIVES To elucidate the role of NE in the development of key features of CF lung disease including airway inflammation, mucus hypersecretion, goblet cell metaplasia, bacterial infection, and structural lung damage in vivo. METHODS We used the Scnn1b-Tg mouse as a model of CF lung disease and determined effects of genetic deletion of NE (NE(-/-)) on the pulmonary phenotype. Furthermore, we used novel Foerster resonance energy transfer (FRET)-based NE reporter assays to assess NE activity in bronchoalveolar lavage from Scnn1b-Tg mice and sputum from patients with CF. MEASUREMENTS AND MAIN RESULTS Lack of NE significantly reduced airway neutrophilia, elevated mucin expression, goblet cell metaplasia, and distal airspace enlargement, but had no effect on airway mucus plugging, bacterial infection, or pulmonary mortality in Scnn1b-Tg mice. By using FRET reporters, we show that NE activity was elevated on the surface of airway neutrophils from Scnn1b-Tg mice and patients with CF. CONCLUSIONS Our results suggest that NE plays an important role in the in vivo pathogenesis and may serve as a therapeutic target for inflammation, mucus hypersecretion, and structural lung damage and indicate that additional rehydration strategies may be required for effective treatment of airway mucus obstruction in CF.

Allergic airway inflammation induces a pro-secretory epithelial ion transport phenotype in mice.

The airway epithelium is a central effector tissue in allergic inflammation and T-helper cell (Th) type 2-driven epithelial responses, such as mucus hypersecretion contribute to airflow obstruction in allergic airway disease. Previous in vitro studies demonstrated that Th2 cytokines also act as potent modulators of epithelial ion transport and fluid secretion, but the in vivo effect of allergic inflammation on airway ion transport remains unknown. We, therefore, induced allergic inflammation by intratracheal instillation of Aspergillus fumigatus extract or interleukin-13 in mice and determined effects on ion transport in native tracheal and bronchial tissues. We demonstrate that allergic inflammation enhanced basal Cl- secretion in both airway regions and inhibited epithelial Na+ channel (ENaC)-mediated Na+ absorption and increased Ca2+-dependent Cl- secretion in bronchi. Allergen-induced alterations in bronchial ion transport were associated with reduced transcript levels of &agr;-, &bgr;- and &ggr;ENaC, and were largely abrogated in signal transducer and activator of transcription (Stat)6-/- mice. Our studies demonstrate that Th2-dependent airway inflammation produced a pro-secretory ion transport phenotype in vivo, which was largely Stat6-dependent. These results suggest that Th2-mediated fluid secretion may improve airway surface hydration and clearance of mucus that is hypersecreted in allergic airway diseases such as asthma, and identify epithelial Stat6 signalling as a potential therapeutic target to promote mucus hydration and airway clearance.

The K+ Channel Opener 1-EBIO Potentiates Residual Function of Mutant CFTR in Rectal Biopsies from Cystic Fibrosis Patients

Background The identification of strategies to improve mutant CFTR function remains a key priority in the development of new treatments for cystic fibrosis (CF). Previous studies demonstrated that the K+ channel opener 1-ethyl-2-benzimidazolone (1-EBIO) potentiates CFTR-mediated Cl− secretion in cultured cells and mouse colon. However, the effects of 1-EBIO on wild-type and mutant CFTR function in native human colonic tissues remain unknown. Methods We studied the effects of 1-EBIO on CFTR-mediated Cl− secretion in rectal biopsies from 47 CF patients carrying a wide spectrum of CFTR mutations and 57 age-matched controls. Rectal tissues were mounted in perfused micro-Ussing chambers and the effects of 1-EBIO were compared in control tissues, CF tissues expressing residual CFTR function and CF tissues with no detectable Cl− secretion. Results Studies in control tissues demonstrate that 1-EBIO activated CFTR-mediated Cl− secretion in the absence of cAMP-mediated stimulation and potentiated cAMP-induced Cl− secretion by 39.2±6.7% (P<0.001) via activation of basolateral Ca2+-activated and clotrimazole-sensitive KCNN4 K+ channels. In CF specimens, 1-EBIO potentiated cAMP-induced Cl− secretion in tissues with residual CFTR function by 44.4±11.5% (P<0.001), but had no effect on tissues lacking CFTR-mediated Cl−conductance. Conclusions We conclude that 1-EBIO potentiates Cl−secretion in native CF tissues expressing CFTR mutants with residual Cl− channel function by activation of basolateral KCNN4 K+ channels that increase the driving force for luminal Cl− exit. This mechanism may augment effects of CFTR correctors and potentiators that increase the number and/or activity of mutant CFTR channels at the cell surface and suggests KCNN4 as a therapeutic target for CF.

Airway Mucus Obstruction Triggers Macrophage Activation and Matrix Metalloproteinase 12–Dependent Emphysema

Whereas cigarette smoking remains the main risk factor for emphysema, recent studies in β-epithelial Na(+) channel-transgenic (βENaC-Tg) mice demonstrated that airway surface dehydration, a key pathophysiological mechanism in cystic fibrosis (CF), caused emphysema in the absence of cigarette smoke exposure. However, the underlying mechanisms remain unknown. The aim of this study was to elucidate mechanisms of emphysema formation triggered by airway surface dehydration. We therefore used expression profiling, genetic and pharmacological inhibition, Foerster resonance energy transfer (FRET)-based activity assays, and genetic association studies to identify and validate emphysema candidate genes in βENaC-Tg mice and patients with CF. We identified matrix metalloproteinase 12 (Mmp12) as a highly up-regulated gene in lungs from βENaC-Tg mice, and demonstrate that elevated Mmp12 expression was associated with progressive emphysema formation, which was reduced by genetic deletion and pharmacological inhibition of MMP12 in vivo. By using FRET reporters, we show that MMP12 activity was elevated on the surface of airway macrophages in bronchoalveolar lavage from βENaC-Tg mice and patients with CF. Furthermore, we demonstrate that a functional polymorphism in MMP12 (rs2276109) was associated with severity of lung disease in CF. Our results suggest that MMP12 released by macrophages activated on dehydrated airway surfaces may play an important role in emphysema formation in the absence of cigarette smoke exposure, and may serve as a therapeutic target in CF and potentially other chronic lung diseases associated with airway mucus dehydration and obstruction.

Use of a new-generation reverse tetracycline transactivator system for quantitative control of conditional gene expression in the murine lung

Conditional regulation of gene expression by the combined use of a lung-specific promoter and the tetracycline-regulated system provides a powerful tool for studying gene function in lung biology and disease pathogenesis in a development-independent fashion. However, the original version of the reverse tetracycline-dependent transactivator (rtTA) exhibited limited doxycycline sensitivity and residual affinity to its promoter (P(tet)), producing leaky transgene expression in the absence of doxycycline. These limitations impeded the use of this system in studying gene dosage effects in pulmonary pathogenesis and repair mechanisms in the diseased lung. Therefore, we used a new-generation rtTA, rtTA2(s)-M2, with no basal activity and increased doxycycline sensitivity, and the rat Clara cell secretory protein (CCSP) promoter to target its expression to pulmonary epithelia in mice. Novel CCSP-rtTA2(s)-M2 founder lines were crossed, with bi-transgenic reporter mice expressing luciferase and Cre recombinase. Background activity, doxycycline sensitivity, tissue and cell-type specificity, inducibility, and reversibility of doxycycline-dependent gene expression were determined by luciferase activity, immunohistochemistry, morphometry, and bioluminescence measurements in neonatal and adult lungs. We generated two distinct novel CCSP-rtTA2(s)-M2 activator mouse lines that confer tight and doxycycline dose-dependent regulation of transgene expression, with high inducibility, complete reversibility, and no background activity, in airway and alveolar epithelia. We conclude that rtTA2(s)-M2 enables quantitative control of conditional gene expression in respiratory epithelia of the murine lung, and that the new CCSP-rtTA2(s)-M2 activator mouse lines will be useful in the further elucidation of the pathogenesis of complex lung diseases and in studies of lung repair.

Airway mucus, inflammation and remodeling: emerging links in the pathogenesis of chronic lung diseases

Airway mucus obstruction is a hallmark of many chronic lung diseases including rare genetic disorders such as cystic fibrosis (CF) and primary ciliary dyskinesia, as well as common lung diseases such as asthma and chronic obstructive pulmonary disease (COPD), which have emerged as a leading cause of morbidity and mortality worldwide. However, the role of excess airway mucus in the in vivo pathogenesis of these diseases remains poorly understood. The generation of mice with airway-specific overexpression of epithelial Na+ channels (ENaC), exhibiting airway surface dehydration (mucus hyperconcentration), impaired mucociliary clearance (MCC) and mucus plugging, led to a model of muco-obstructive lung disease that shares key features of CF and COPD. In this review, we summarize recent progress in the understanding of causes of impaired MCC and in vivo consequences of airway mucus obstruction that can be inferred from studies in βENaC-overexpressing mice. These studies confirm that mucus hyperconcentration on airway surfaces plays a critical role in the pathophysiology of impaired MCC, mucus adhesion and airway plugging that cause airflow obstruction and provide a nidus for bacterial infection. In addition, these studies support the emerging concept that excess airway mucus per se, probably via several mechanisms including hypoxic epithelial necrosis, retention of inhaled irritants or allergens, and potential immunomodulatory effects, is a potent trigger of chronic airway inflammation and associated lung damage, even in the absence of bacterial infection. Finally, these studies suggest that improvement of mucus clearance may be a promising therapeutic strategy for a spectrum of muco-obstructive lung diseases.

Airway Surface Dehydration Aggravates Cigarette Smoke-Induced Hallmarks of COPD in Mice.

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 β-subunit of the epithelial Na+ channel (βENaC). Methods βENaC-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 βENaC-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 βENaC-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.

Lung arginase expression and activity is increased in cystic fibrosis mouse models

The activity of arginase is increased in airway secretions of patients with cystic fibrosis (CF). Downstream products of arginase activity may contribute to CF lung disease. We hypothesized that pulmonary arginase expression and activity would be increased in mouse models of CF and disproportionally increased in CF mice with Pseudomonas aeruginosa pneumonia. Expression of arginase isoforms in lung tissue was quantified with reverse transcriptase-PCR in naive cystic fibrosis transmembrane conductance regulator (Cftr)-deficient mice and β-epithelial sodium channel-overexpressing [β-ENaC-transgenic (Tg)] mice. An isolated lung stable isotope perfusion model was used to measure arginase activity in Cftr-deficient mice before and after intratracheal instillation of Pseudomonas aeruginosa. The expression of arginase-2 in lung was increased in adult Cftr-deficient animals and in newborn β-ENaC-Tg. Arginase-1 lung expression was normal in Cftr-deficient and in newborn β-ENaC-Tg mice, but was increased in β-ENaC-Tg mice at age 1, 3, and 6 wk. Arginase activity was significantly higher in lung (5.0 ± 0.7 vs. 3.2 ± 0.3 nmol·(-1)·h(-1), P = 0.016) and airways (204.6 ± 49.8 vs. 79.3 ± 17.2 nmol·(-1)·h(-1), P = 0.045) of naive Cftr-deficient mice compared with sex-matched wild-type littermate controls. Infection with Pseudomonas aeruginosa resulted in a far greater increase in lung arginase activity in Cftr-deficient mice (10-fold) than in wild-type controls (6-fold) (P = 0.01). This is the first ex vivo characterization of arginase expression and activity in CF mouse lung and airways. Our data show that pulmonary arginase expression and activity is increased in CF mice, especially with Pseudomonas aeruginosa infections.