Susanne C. Schubert

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.

Airway Surface Liquid Volume Regulation Determines Different Airway Phenotypes in Liddle Compared with βENaC-overexpressing Mice

Studies in cystic fibrosis patients and mice overexpressing the epithelial Na+ channel β-subunit (βENaC-Tg) suggest that raised airway Na+ transport and airway surface liquid (ASL) depletion are central to the pathogenesis of cystic fibrosis lung disease. However, patients or mice with Liddle gain-of-function βENaC mutations exhibit hypertension but no lung disease. To investigate this apparent paradox, we compared the airway phenotype (nasal versus tracheal) of Liddle with CFTR-null, βENaC-Tg, and double mutant mice. In mouse nasal epithelium, the region that functionally mimics human airways, high levels of CFTR expression inhibited Liddle epithelial Nat channel (ENaC) hyperfunction. Conversely, in mouse trachea, low levels of CFTR failed to suppress Liddle ENaC hyperfunction. Indeed, Na+ transport measured in Ussing chambers (“flooded” conditions) was raised in both Liddle and βENaC-Tg mice. Because enhanced Na+ transport did not correlate with lung disease in these mutant mice, measurements in tracheal cultures under physiologic “thin film” conditions and in vivo were performed. Regulation of ASL volume and ENaC-mediated Na+ absorption were intact in Liddle but defective in βENaC-Tg mice. We conclude that the capacity to regulate Na+ transport and ASL volume, not absolute Na+ transport rates in Ussing chambers, is the key physiologic function protecting airways from dehydration-induced lung disease.

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.

Hypoxic Epithelial Necrosis Triggers Neutrophilic Inflammation via IL-1 Receptor Signaling in Cystic Fibrosis Lung Disease

RATIONALE In many organs, hypoxic cell death triggers sterile neutrophilic inflammation via IL-1R signaling. Although hypoxia is common in airways from patients with cystic fibrosis (CF), its role in neutrophilic inflammation remains unknown. We recently demonstrated that hypoxic epithelial necrosis caused by airway mucus obstruction precedes neutrophilic inflammation in Scnn1b-transgenic (Scnn1b-Tg) mice with CF-like lung disease. OBJECTIVES To determine the role of epithelial necrosis and IL-1R signaling in the development of neutrophilic airway inflammation, mucus obstruction, and structural lung damage in CF lung disease. METHODS We used genetic deletion and pharmacologic inhibition of IL-1R in Scnn1b-Tg mice and determined effects on airway epithelial necrosis; levels of IL-1α, keratinocyte chemoattractant, and neutrophils in bronchoalveolar lavage; and mortality, mucus obstruction, and structural lung damage. Furthermore, we analyzed lung tissues from 21 patients with CF and chronic obstructive pulmonary disease and 19 control subjects for the presence of epithelial necrosis. MEASUREMENTS AND MAIN RESULTS Lack of IL-1R had no effect on epithelial necrosis and elevated IL-1α, but abrogated airway neutrophilia and reduced mortality, mucus obstruction, and emphysema in Scnn1b-Tg mice. Treatment of adult Scnn1b-Tg mice with the IL-1R antagonist anakinra had protective effects on neutrophilic inflammation and emphysema. Numbers of necrotic airway epithelial cells were elevated and correlated with mucus obstruction in patients with CF and chronic obstructive pulmonary disease. CONCLUSIONS Our results support an important role of hypoxic epithelial necrosis in the pathogenesis of neutrophilic inflammation independent of bacterial infection and suggest IL-1R as a novel target for antiinflammatory therapy in CF and potentially other mucoobstructive airway diseases.

Bronchoalveolar sublineage specification of pluripotent stem cells: effect of dexamethasone plus cAMP-elevating agents and keratinocyte growth factor.

Respiratory progenitors can be efficiently generated from pluripotent stem cells (PSCs). However, further targeted differentiation into bronchoalveolar sublineages is still in its infancy, and distinct specifying effects of key differentiation factors are not well explored. Focusing on airway epithelial Clara cell generation, we analyzed the effect of the glucocorticoid dexamethasone plus cAMP-elevating agents (DCI) on the differentiation of murine embryonic and induced pluripotent stem cells (iPSCs) into bronchoalveolar epithelial lineages, and whether keratinocyte growth factor (KGF) might further influence lineage decisions. We demonstrate that DCI strongly induce expression of the Clara cell marker Clara cell secretory protein (CCSP). While KGF synergistically supports the inducing effect of DCI on alveolar markers with increased expression of surfactant protein (SP)-C and SP-B, an inhibitory effect on CCSP expression was shown. In contrast, neither KGF nor DCI seem to have an inducing effect on ciliated cell markers. Furthermore, the use of iPSCs from transgenic mice with CCSP promoter-dependent lacZ expression or a knockin of a YFP reporter cassette in the CCSP locus enabled detection of derivatives with Clara cell typical features. Collectively, DCI was shown to support bronchoalveolar specification of mouse PSCs, in particular Clara-like cells, and KGF to inhibit bronchial epithelial differentiation. The targeted in vitro generation of Clara cells with their important function in airway protection and regeneration will enable the evaluation of innovative cellular therapies in animal models of lung diseases.

18F-FDG PET in a 10-year-old female patient with subacute sclerosing panencephalitis

1 Medical PET Group-Biological Imaging (E0601), Clinical Cooperation Unit Nuclear Medicine, German Cancer Research Center, Im Neuenheimer Feld 280, 69120 Heidelberg, Germany 2 Pediatric Clinics, Universitätsklinikum Mannheim, University of Heidelberg, Mannheim, Germany 3 Department of Clinical Radiology, Universitätsklinikum Mannheim, University of Heidelberg, Mannheim, Germany 4 Department of Nuclear Medicine, University of Heidelberg, Heidelberg, Germany

WS10.2 Hypoxic epithelial necrosis triggers neutrophilic inflammation via IL-1 receptor signaling in cystic fibrosis-like lung disease

In many organs, hypoxic cell death triggers sterile neutrophilic inflammation via Interleukin-1 receptor (IL-1R) signaling. Although hypoxia is common in airways from patients with cystic fibrosis (CF), its role in neutrophilic inflammation remains unknown. We recently demonstrated that hypoxic epithelial necrosis due to airway mucus obstruction precedes neutrophilic inflammation in Scnn1b -transgenic ( Scnn1b -Tg) mice with CF-like lung disease. To determine the role of hypoxic epithelial necrosis and IL-1R signaling in the development of neutrophilic inflammation, mucus obstruction and structural lung damage in CF lung disease, we used genetic deletion and pharmacological inhibition of IL-1R in Scnn1b -Tg mice and studied effects on airway epithelial necrosis, levels of IL-1α, keratinocyte chemoattractant (KC), neutrophils in bronchoalveolar lavage and mortality, mucus obstruction and emphysema. We evaluated lung tissues from patients with CF and COPD and controls for airway epithelial necrosis and mucus obstruction. Lack of IL-1R abrogated airway neutrophilia and reduced mortality, mucus obstruction and emphysema in Scnn1b -Tg mice. Treatment with IL-1R antagonist anakinra in adult Scnn1b -Tg mice showed similar protective effects. Numbers of necrotic airway epithelial cells were elevated and correlated with mucus obstruction in patients with CF and COPD. Our results support an important role of hypoxic epithelial necrosis in the pathogenesis of neutrophilic airway inflammation independent of bacterial infection and suggest IL-1R as a novel target for antiinflammatory therapy in CF and potentially other muco-obstructive airway diseases. Supported by DFG (DFG MA 2081/3–2).

57* Generation of new lung-specific tet-dependent activator mice for tight and quantitative control of conditional gene expression in the murine lung

57* Generation of new lung-specific tet-dependent activator mice for tight and quantitative control of conditional gene expression in the murine lung J. Duerr1, M. Gruner1, S.C. Schubert1, U. Haberkorn2, H. Bujard3, M. Mall1. 1University of Heidelberg, Department of Pediatrics III, Division of Pediatric Pulmonology and Cystic Fibrosis Center, Heidelberg, Germany; 2University of Heidelberg, Department of Nuclear Medicine, Heidelberg, Germany; 3University of Heidelberg, Zentrum fur Molekulare Biologie, Heidelberg, Germany