Simon Y. Graeber

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.

Comparison of microbiomes from different niches of upper and lower airways in children and adolescents with cystic fibrosis.

Changes in the airway microbiome may be important in the pathophysiology of chronic lung disease in patients with cystic fibrosis. However, little is known about the microbiome in early cystic fibrosis lung disease and the relationship between the microbiomes from different niches in the upper and lower airways. Therefore, in this cross-sectional study, we examined the relationship between the microbiome in the upper (nose and throat) and lower (sputum) airways from children with cystic fibrosis using next generation sequencing. Our results demonstrate a significant difference in both α and β-diversity between the nose and the two other sampling sites. The nasal microbiome was characterized by a polymicrobial community while the throat and sputum communities were less diverse and dominated by a few operational taxonomic units. Moreover, sputum and throat microbiomes were closely related especially in patients with clinically stable lung disease. There was a high inter-individual variability in sputum samples primarily due to a decrease in evenness linked to increased abundance of potential respiratory pathogens such as Pseudomonas aeruginosa. Patients with chronic Pseudomonas aeruginosa infection exhibited a less diverse sputum microbiome. A high concordance was found between pediatric and adult sputum microbiomes except that Burkholderia was only observed in the adult cohort. These results indicate that an adult-like lower airways microbiome is established early in life and that throat swabs may be a good surrogate in clinically stable children with cystic fibrosis without chronic Pseudomonas aeruginosa infection in whom sputum sampling is often not feasible.

Comparison of Lung Clearance Index and Magnetic Resonance Imaging for Assessment of Lung Disease in Children with Cystic Fibrosis

Rationale: Early onset and progression of lung disease in children with cystic fibrosis (CF) indicates that sensitive noninvasive outcome measures are needed for diagnostic monitoring and early intervention clinical trials. The lung clearance index (LCI) and chest magnetic resonance imaging (MRI) were shown to detect early lung disease in CF; however, the relationship between the two measures remains unknown. Objectives: To correlate the LCI with abnormalities detected by MRI and compare the sensitivity of the two techniques to detect responses to therapy for pulmonary exacerbations in children with CF. Methods: LCI determined by age‐adapted multiple breath washout techniques and MRI studies were performed in 97 clinically stable children with CF across the pediatric age range (0.2‐21.1 yr). Furthermore, LCI (n = 26) or MRI (n = 10) were performed at the time of pulmonary exacerbation and after antibiotic therapy. MRI was evaluated using a dedicated morphofunctional score. Measurements and Main Results: The LCI correlated with the global MRI score as well as MRI‐defined airway wall abnormalities, mucus plugging, and abnormal lung perfusion in infants and toddlers (P < 0.05 to P < 0.001) and in older children (P < 0.001) with CF. LCI and MRI were sensitive to detect response to antibiotic therapy for pulmonary exacerbations. Conclusions: Our results indicate that LCI and MRI may be useful complementary tools for noninvasive monitoring and as quantitative endpoints in early intervention trials in children with CF. In this context, MRI enables detection of disease heterogeneity, including regional mucus plugging associated with abnormal lung perfusion in early CF lung disease. Clinical trial registered with www.clinicaltrials.gov (NCT 02270476).

Hypertonic Saline Is Effective in the Prevention and Treatment of Mucus Obstruction, but Not Airway Inflammation, in Mice with Chronic Obstructive Lung Disease

Recent evidence suggests that inadequate hydration of airway surfaces is a common mechanism in the pathogenesis of airway mucus obstruction. Inhaled hypertonic saline (HS) induces osmotic water flux, improving hydration of airway surfaces. However, trials in patients with obstructive lung diseases are limited. The aim of this study was to investigate effects of HS on mucus obstruction and airway inflammation in the prevention and treatment of obstructive lung disease in vivo. We, therefore, used the β-epithelial Na(+) channel (βENaC)-overexpressing mouse as a model of chronic obstructive lung disease and determined effects of preventive and late therapy with 3% HS and 7% HS on pulmonary mortality, airway mucus obstruction, and inflammation. We found that preventive treatment with 3% HS and 7% HS improved growth, reduced mortality, and reduced mucus obstruction in neonatal βENaC-overexpressing mice. In adult βENaC-overexpressing mice with chronic lung disease, mucus obstruction was significantly reduced by 7% HS, but not by 3% HS. Treatment with HS triggered airway inflammation with elevated keratinocyte chemoattractant levels and neutrophils in airways from wild-type mice, but reduced keratinocyte chemoattractant in chronic neutrophilic inflammation in adult βENaC-overexpressing mice. Our data demonstrate that airway surface rehydration with HS provides an effective preventive and late therapy of mucus obstruction with no consistent effects on inflammation in chronic lung disease. These results suggest that, through mucokinetic effects, HS may be beneficial for patients with a spectrum of obstructive lung diseases, and that additional strategies are required for effective treatment of associated airway inflammation.

Early cystic fibrosis lung disease: Role of airway surface dehydration and lessons from preventive rehydration therapies in mice.

Cystic fibrosis (CF) lung disease starts in the first months of life and remains one of the most common fatal hereditary diseases. Early therapeutic interventions may provide an opportunity to prevent irreversible lung damage and improve outcome. Airway surface dehydration is a key disease mechanism in CF, however, its role in the in vivo pathogenesis and as therapeutic target in early lung disease remains poorly understood. Mice with airway-specific overexpression of the epithelial Na(+) channel (βENaC-Tg) recapitulate airway surface dehydration and phenocopy CF lung disease. Recent studies in neonatal βENaC-Tg mice demonstrated that airway surface dehydration produces early mucus plugging in the absence of mucus hypersecretion, which triggers airway inflammation, promotes bacterial infection and causes early mortality. Preventive rehydration therapy with hypertonic saline or amiloride effectively reduced mucus plugging and mortality in neonatal βENaC-Tg mice. These results support clinical testing of preventive/early rehydration strategies in infants and young children with CF.

Early detection and sensitive monitoring of CF lung disease: Prospects of improved and safer imaging: Early Detection and Sensitive Monitoring of CF

Recent imaging studies using chest computed tomography (CT) in presymptomatic infants and young children with cystic fibrosis (CF) diagnosed by newborn screening presented compelling evidence of early onset and progression of structural lung damage in CF. These data argue persuasively that non‐invasive outcome measures for early detection and sensitive monitoring of lung disease applicable in the clinical setting will be instrumental for further improvement of clinical care and the development of early intervention therapies that have the potential to prevent irreversible lung damage. In this context, the use of CT imaging for early detection and long‐term monitoring has the disadvantage of the risk to induce malignancies due to cumulating ionizing radiation exposure. More recently, magnetic resonance imaging (MRI) has emerged as an alternative radiation‐free imaging technique for quantitative assessment of CF lung disease. In addition to structural lung damage, chest MRI enables non‐invasive assessment of abnormalities in lung perfusion and ventilation characteristically associated with mucus plugging in CF lung disease. Here, we review recent developments and the prospects of MRI for improved and safer imaging with a focus on recent studies that support its utility as a sensitive non‐invasive outcome measure of early lung disease in young children with CF. Pediatr Pulmonol. 2016;51:S49–S60.

Effects of Lumacaftor–Ivacaftor Therapy on Cystic Fibrosis Transmembrane Conductance Regulator Function in Phe508del Homozygous Patients with Cystic Fibrosis

Rationale: The combination of the CFTR (cystic fibrosis transmembrane conductance regulator) corrector lumacaftor with the potentiator ivacaftor has been approved for the treatment of patients with cystic fibrosis homozygous for the Phe508del CFTR mutation. The phase 3 trials examined clinical outcomes but did not evaluate CFTR function in patients. Objectives: To examine the effect of lumacaftor‐ivacaftor on biomarkers of CFTR function in Phe508del homozygous patients with cystic fibrosis aged 12 years and older. Methods: This prospective observational study assessed clinical outcomes including FEV1% predicted and body mass index, and CFTR biomarkers including sweat chloride concentration, nasal potential difference, and intestinal current measurement before and 8‐16 weeks after initiation of lumacaftor‐ivacaftor. Measurements and Main Results: A total of 53 patients were enrolled in the study, and 52 patients had baseline and follow‐up measurements. After initiation of lumacaftor‐ivacaftor sweat chloride concentrations were reduced by 17.8 mmol/L (interquartile range [IQR], −25.9 to −6.1; P < 0.001), nasal potential difference showed partial rescue of CFTR function in nasal epithelia to a level of 10.2% (IQR, 0.0‐26.1; P < 0.011), and intestinal current measurement showed functional improvement in rectal epithelia to a level of 17.7% of normal (IQR, 10.8‐29.0; P < 0.001). All patients improved in at least one CFTR biomarker, but no correlations were found between CFTR biomarker responses and clinical outcomes. Conclusions: Lumacaftor‐ivacaftor results in partial rescue of Phe508del CFTR function to levels comparable to the lower range of CFTR activity found in patients with residual function mutations. Functional improvement was detected even in the absence of short‐term improvement of FEV1% predicted and body mass index. Clinical trial registered with www.clinicaltrials.gov (NCT02807415).

One time quantitative PCR detection of Pseudomonas aeruginosa to discriminate intermittent from chronic infection in cystic fibrosis

BACKGROUND Chronic airway infection with Pseudomonas aeruginosa is a major risk factor of progression of lung disease in patients with cystic fibrosis (CF). Chronic P. aeruginosa infection evolves from intermittent infection that is amenable to antibiotic eradication, whereas chronically adapted P. aeruginosa becomes resistant to antibiotic therapy. Discrimination of intermittent versus chronic infection is therefore of high therapeutic relevance, yet the available diagnostic methods are only partly satisfactory. The aim of the present study was, therefore, to evaluate the usage of quantitative PCR (qPCR) to measure pathogen abundance and to discriminate between intermittent and chronic Pseudomonas infection in patients with CF. METHOD Using an established qPCR protocol, we analyzed the abundance of P. aeruginosa in 141 throats swabs and 238 sputa from CF patients with intermittent or chronic infection with P. aeruginosa, as determined by standard culture based diagnostics. RESULTS We observed a large increase of abundance of P. aeruginosa in throat swabs and sputum samples from patients with chronic compared to intermittent infections with P. aeruginosa. The data show that abundance of P. aeruginosa as measured by qPCR is a valuable tool to discriminate intermittent from chronic infection. Of note, P. aeruginosa burden seems more sensitive than mucoidity phenotype to discriminate chronic from intermittent strains. Furthermore we observed that molecular detection in throat swabs was linked to a viable culture in the sputum when sputum was available. This result is of special interest in young patients with cystic fibrosis that often cannot expectorate sputum. We also observed that qPCR in comparison to culture detected the infection earlier. CONCLUSION The results suggest that qPCR detection and quantification of P. aeruginosa is a precious tool to be added to the diagnostic toolbox in cystic fibrosis.

Three-center feasibility of lung clearance index in infants and preschool children with cystic fibrosis and other lung diseases

BACKGROUND Lung clearance index (LCI) detects early ventilation inhomogeneity and has been suggested as sensitive endpoint in multicenter intervention trials in infants and preschoolers with cystic fibrosis (CF). However, the feasibility of multicenter LCI in this age group has not been determined. We, therefore, investigated the feasibility of LCI in infants and preschoolers with and without CF in a three-center setting. METHODS Following central training, standardized SF6-MBW measurements were performed in 73 sedated children (10 controls, 49 with CF and 14 with other lung diseases), mean age 2.3±1.2years across three centers, and data were analyzed centrally. RESULTS Overall success rate of LCI measurements was 91.8% ranging from 78.9% to 100% across study sites. LCI was increased in patients with CF (P<0.05) and with other lung diseases (P<0.05) compared to controls. CONCLUSION Our results support feasibility of LCI as multicenter endpoint in clinical trials in infants and preschoolers with CF.