Role of the SLC26A9 chloride channel as disease modifier and potential therapeutic target in cystic fibrosis

The solute carrier family 26, member 9 (SLC26A9) is an epithelial chloride channel that is expressed in several organs affected in patients with cystic fibrosis (CF) including the lungs, the pancreas, and the intestine. Emerging evidence suggests SLC26A9 as a modulator of wild-type and mutant CFTR function, and as a potential alternative target to circumvent the basic ion transport defect caused by deficient CFTR-mediated chloride transport in CF. In this review, we summarize in vitro studies that revealed multifaceted molecular and functional interactions between SLC26A9 and CFTR that may be implicated in normal transepithelial chloride secretion in health, as well as impaired chloride/fluid transport in CF. Further, we focus on recent genetic association studies and investigations utilizing genetically modified mouse models that identified SLC26A9 as a disease modifier and supported an important role of this alternative chloride channel in the pathophysiology of several organ manifestations in CF, as well as other chronic lung diseases such as asthma and non-CF bronchiectasis. Collectively, these findings and the overlapping endogenous expression with CFTR suggest SLC26A9 an attractive novel therapeutic target that may be exploited to restore epithelial chloride secretion in patients with CF irrespective of their CFTR genotype. In addition, pharmacological activation of SLC26A9 may help to augment the effect of CFTR modulator therapies in patients with CF carrying responsive mutations such as the most common disease-causing mutation F508del-CFTR. However, future research and development including the identification of compounds that activate SLC26A9-mediated chloride transport are needed to explore this alternative chloride channel as a therapeutic target in CF and potentially other muco-obstructive lung diseases

Role of the SLC26A9 Chloride Channel as Disease Modifier and Potential Therapeutic Target in Cystic Fibrosis

The solute carrier family 26, member 9 (SLC26A9) is an epithelial chloride channel that is expressed in several organs affected in patients with cystic fibrosis (CF) including the lungs, the pancreas, and the intestine. Emerging evidence suggests SLC26A9 as a modulator of wild-type and mutant CFTR function, and as a potential alternative target to circumvent the basic ion transport defect caused by deficient CFTR-mediated chloride transport in CF. In this review, we summarize in vitro studies that revealed multifaceted molecular and functional interactions between SLC26A9 and CFTR that may be implicated in normal transepithelial chloride secretion in health, as well as impaired chloride/fluid transport in CF. Further, we focus on recent genetic association studies and investigations utilizing genetically modified mouse models that identified SLC26A9 as a disease modifier and supported an important role of this alternative chloride channel in the pathophysiology of several organ manifestations in CF, as well as other chronic lung diseases such as asthma and non-CF bronchiectasis. Collectively, these findings and the overlapping endogenous expression with CFTR suggest SLC26A9 an attractive novel therapeutic target that may be exploited to restore epithelial chloride secretion in patients with CF irrespective of their CFTR genotype. In addition, pharmacological activation of SLC26A9 may help to augment the effect of CFTR modulator therapies in patients with CF carrying responsive mutations such as the most common disease-causing mutation F508del-CFTR. However, future research and development including the identification of compounds that activate SLC26A9-mediated chloride transport are needed to explore this alternative chloride channel as a therapeutic target in CF and potentially other muco-obstructive lung diseases

miRNA-221 is elevated in cystic fibrosis airway epithelial cells and regulates expression of ATF6.

Background MicroRNA (miRNA) and messenger RNA (mRNA) expression differs in cystic fibrosis (CF) versus non-CF bronchial epithelium. Here, the role of miRNA in basal regulation of the transcription factor ATF6 was investigated in bronchial epithelial cells in vitro and in vivo. Methods Using in silico analysis, miRNAs predicted to target the 3′untranslated region (3′UTR) of the human ATF6 mRNA were identified. Results Three of these miRNAs, miR-145, miR-221 and miR-494, were upregulated in F508del-CFTR homozygous CFBE41o- versus non-CF 16HBE14o- bronchial epithelial cells and also in F508del-CFTR homozygous or heterozygous CF (n = 8) versus non-CF (n = 9) bronchial brushings. ATF6 was experimentally validated as a molecular target of these miRNAs through the use of a luciferase reporter vector containing the full-length 3′UTR of ATF6. Expression of ATF6 was observed to be decreased in CF both in vivo and in vitro. miR-221 was also predicted to regulate murine ATF6, and its expression was significantly increased in native airway tissues of 6-week-old βENaC-overexpressing transgenic mice with CF-like lung disease versus wild-type littermates. Conclusions These results implicate miR-145, miR-221 and miR-494 in the regulation of ATF6 in CF bronchial epithelium, with miR-221 demonstrating structural and functional conservation between humans and mice. The altered miRNA expression evident in CF bronchial epithelial cells can affect expression of transcriptional regulators such as ATF6

Maintenance of Elective Patient Care at Berlin University Children's Hospital During the COVID-19 Pandemic

Background: In Germany, so far the COVID-19 pandemic evolved in two distinct waves, the first beginning in February and the second in July, 2020. The Berlin University Children's Hospital at Charité (BCH) had to ensure treatment for children not infected and infected with SARS-CoV-2. Prevention of nosocomial SARS-CoV-2 infection of patients and staff was a paramount goal. Pediatric hospitals worldwide discontinued elective treatments and established a centralized admission process. Methods: The response of BCH to the pandemic adapted to emerging evidence. This resulted in centralized admission via one ward exclusively dedicated to children with unclear SARS-CoV-2 status and discontinuation of elective treatment during the first wave, but maintenance of elective care and decentralized admissions during the second wave. We report numbers of patients treated and of nosocomial SARS-CoV-2 infections during the two waves of the pandemic. Results: During the first wave, weekly numbers of inpatient and outpatient cases declined by 37% (p < 0.001) and 29% (p = 0.003), respectively. During the second wave, however, inpatient case numbers were 7% higher (p = 0.06) and outpatient case numbers only 6% lower (p = 0.25), compared to the previous year. Only a minority of inpatients were tested positive for SARS-CoV-2 by RT-PCR (0.47% during the first, 0.63% during the second wave). No nosocomial infection of pediatric patients by SARS-CoV-2 occurred. Conclusion: In contrast to centralized admission via a ward exclusively dedicated to children with unclear SARS-CoV-2 status and discontinuation of elective treatments, maintenance of elective care and decentralized admission allowed the almost normal use of hospital resources, yet without increased risk of nosocomial infections with SARS-CoV-2. By this approach unwanted sequelae of withheld specialized pediatric non-emergency treatment to child and adolescent health may be avoided

DMBT1 is upregulated in cystic fibrosis, affects ciliary motility, and is reduced by acetylcysteine

Background Cystic fibrosis (CF) is the most common genetic disorder in the Caucasian population. Despite remarkable improvements in morbidity and mortality during the last decades, the disease still limits survival and reduces quality of life of affected patients. Moreover, CF still represents substantial economic burden for healthcare systems. Inflammation and infection already start in early life and play important roles in pulmonary impairment. The aim of this study is to analyze the potential role of DMBT1, a protein with functions in inflammation, angiogenesis, and epithelial differentiation, in CF. Results Immunohistochemically DMBT1 protein expression was upregulated in lung tissues of CF patients compared to healthy controls. Additionally, pulmonary expression of Dmbt1 was approximately 6-fold increased in an established transgenic mouse model of CF-like lung disease (ENaC tg) compared to wild-type mice as detected by qRT-PCR. Since acetylcysteine (ACC) has been shown to reduce inflammatory markers in the airways, its potential influence on DMBT1 expression was analyzed. A549 cells stably transfected with an expression plasmid encoding the largest (8kb) DMBT1 variant (DMBT1+ cells) or an empty vector control (DMBT1- cells) and incubated with ACC both showed significantly reduced DMBT1 concentrations in the culture medium (p = 0.0001). To further elucidate the function of DMBT1 in pulmonary airways, respiratory epithelial cells were examined by phase contrast microscopy. Addition of human recombinant DMBT1 resulted in altered cilia motility and irregular beat waves (p < 0.0001) suggesting a potential effect of DMBT1 on airway clearance. Conclusions DMBT1 is part of inflammatory processes in CF and may be used as a potential biomarker for CF lung disease and a potential tool to monitor CF progression. Furthermore, DMBT1 has a negative effect on ciliary motility thereby possibly compromising airway clearance. Application of ACC, leading to reduced DMBT1 concentrations, could be a potential therapeutic option for CF patients

Elastase activity on sputum neutrophils correlates with severity of lung disease in cystic fibrosis

Neutrophil elastase (NE) is a key risk factor for severity of cystic fibrosis (CF) lung disease. Recent studies identified increased NE activity on the surface of airway neutrophils from CF-like mice and patients with CF. However, the role of surface-bound NE in CF lung disease remains unknown. We determined the relationship between surface-bound NE activity and severity of lung disease in CF.Surface-bound NE activity was measured on sputum neutrophils from 35 CF patients and eight healthy controls using novel lipidated Förster resonance energy transfer reporters and correlated with free NE activity, neutrophil counts, interleukin-8, myeloperoxidase and antiproteases in sputum supernatant, and with lung function parameters.Surface-bound NE activity was increased in CF compared to healthy controls (p&lt;0.01) and correlated with free NE activity (p&lt;0.05) and other inflammation markers (p&lt;0.001). Surface-bound and free NE activity correlated with forced expiratory volume in 1 s % predicted (p&lt;0.01 and p&lt;0.05), but only surface-bound NE activity correlated with plethysmographic functional residual capacity % pred (p&lt;0.01) in patients with CF.We demonstrate that surface-bound NE activity on airway neutrophils correlates with severity of lung disease in patients with CF. Our results suggest that surface-bound NE activity may play an important role in the pathogenesis and serve as novel biomarker in CF lung disease.</jats:p

Contrast agent-free functional magnetic resonance imaging with matrix pencil decomposition to quantify abnormalities in lung perfusion and ventilation in patients with cystic fibrosis

BackgroundPrevious studies showed that contrast-enhanced (CE) morpho-functional magnetic resonance imaging (MRI) detects abnormalities in lung morphology and perfusion in patients with cystic fibrosis (CF). Novel matrix pencil decomposition MRI (MP-MRI) enables quantification of lung perfusion and ventilation without intravenous contrast agent administration.ObjectivesTo compare MP-MRI with established morpho-functional MRI and spirometry in patients with CF.MethodsThirty-nine clinically stable patients with CF (mean age 21.6 ± 10.7 years, range 8–45 years) prospectively underwent morpho-functional MRI including CE perfusion MRI, MP-MRI and spirometry. Two blinded chest radiologists assessed morpho-functional MRI and MP-MRI employing the validated chest MRI score. In addition, MP-MRI data were processed by automated software calculating perfusion defect percentage (QDP) and ventilation defect percentage (VDP).ResultsMP perfusion score and QDP correlated strongly with the CE perfusion score (both r = 0.81; p &lt; 0.01). MP ventilation score and VDP showed strong inverse correlations with percent predicted FEV1 (r = −0.75 and r = −0.83; p &lt; 0.01). The comparison of visual and automated parameters showed that both MP perfusion score and QDP, and MP ventilation score and VDP were strongly correlated (r = 0.74 and r = 0.78; both p &lt; 0.01). Further, the MP perfusion score and MP ventilation score, as well as QDP and VDP were strongly correlated (r = 0.88 and r = 0.86; both p &lt; 0.01).ConclusionMP-MRI detects abnormalities in lung perfusion and ventilation in patients with CF without intravenous or inhaled contrast agent application, and correlates strongly with the well-established CE perfusion MRI score and spirometry. Automated analysis of MP-MRI may serve as quantitative noninvasive outcome measure for diagnostic monitoring and clinical trials

An optimized protocol for assessment of sputum macrorheology in health and muco-obstructive lung disease

Background: Airway mucus provides important protective functions in health and abnormal viscoelasticity is a hallmark of muco-obstructive lung diseases such as cystic fibrosis (CF). However, previous studies of sputum macrorheology from healthy individuals and patients with CF using different experimental protocols yielded in part discrepant results and data on a systematic assessment across measurement settings and conditions remain limited. Objectives: The aim of this study was to develop an optimized and reliable protocol for standardized macrorheological measurements of airway mucus model systems and native human sputum from healthy individuals and patients with muco-obstructive lung disease. Methods: Oscillatory rheological shear measurements were performed using bovine submaxillary mucin (BSM) at different concentrations (2% and 10% solids) and sputum samples from healthy controls (n = 10) and patients with CF (n = 10). Viscoelastic properties were determined by amplitude and frequency sweeps at 25°C and 37°C with or without solvent trap using a cone-plate geometry. Results: Under saturated atmosphere, we did not observe any temperature-dependent differences in 2% and 10% BSM macrorheology, whereas in the absence of evaporation control 10% BSM demonstrated a significantly higher viscoelasticity at 37°C. Similarly, during the measurements without evaporation control at 37°C we observed a substantial increase in the storage modulus G′ and the loss modulus G″ of the highly viscoelastic CF sputum but not in the healthy sputum. Conclusion: Our data show systematically higher viscoelasticity of CF compared to healthy sputum at 25°C and 37°C. For measurements at the higher temperature using a solvent trap to prevent evaporation is essential for macrorheological analysis of mucus model systems and native human sputum. Another interesting finding is that the viscoelastic properties are not much sensitive to the applied experimental deformation and yield robust results despite their delicate consistency. The optimized protocol resulting from this work will facilitate standardized quantitative assessment of abnormalities in viscoelastic properties of airway mucus and response to muco-active therapies in patients with CF and other muco-obstructive lung diseases

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

AbstractChronic 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 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