Marcus A. Mall

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.

Antimicrobial Peptides (AMPs) represent one of the most durable and effective defence of multicellular organisms against bacterial infections. These cationic and amphipathic peptides represent promising leads for the development of antibiotics combatting the resistance of bacteria against antibiotics. However, their clinical applications have often been limited by an inadequate margin of safety [1]. A prodrug approach can overcome a toxicity barrier in drug delivery. Prodrugs of AMPs can be generated by transiently reducing or annulling their net positive charges by attaching a negative promoiety through a linker which can be degraded by an enzyme (bacterial or human) confined to sites of infection. For example, neutrophil elastase (NE), a human protease involved in chronic airway inflammation and infections associated with cystic fibrosis (CF), can restore the cationic property of AMPs modified with oligo-glutamate promoieties. Consequently, their bactericidal activities against the CF pathogen P. aeruginosa are restored by NE in CF bronchoalveolar lavage fluids. The potential of this prodrug approach in reducing the safety barrier in the clinical use of AMPs has also been demonstrated in vivo, in a murine model of lung delivery [2]. In parallel, a novel class of peptidomimetics with antimicrobial activities similar to AMPs, against Gram-positive bacteria, has been developed. Their spectrum of activity is currently extended to Gram-negative organisms. [1] Zasloff M. Antimicrobial peptides of multicellular organisms. Nature. 2002, 415(6870):389-395. [2] Forde É, Schütte A, Reeves E, Greene C, Humphreys H, Mall M, Fitzgerald-Hughes D, Devocelle M. Differential In Vitro and In Vivo Toxicities of Antimicrobial Peptide Prodrugs for Potential Use in Cystic Fibrosis. Antimicrob Agents Chemother. 2016, 60(5):2813-2821.

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.

Chronic lung disease remains the major cause of morbidity and mortality in patients with cystic fibrosis (CF). Recent studies in young children with CF diagnosed by newborn screening identified neutrophil elastase (NE), a major product released from neutrophils in inflamed airways, as a key risk factor for the onset and early progression of CF lung disease. However, the understanding of how NE and potentially other proteases contribute to the complex in vivo pathogenesis of CF lung disease remains limited. In this review, we summarize recent progress in this area based on studies in βENaC-overexpressing (βENaC-Tg) mice featuring CF-like lung disease and novel protease-specific Förster resonance energy transfer (FRET) sensors for localization and quantification of protease activity in the lung. These studies demonstrated that NE is implicated in several key features of CF lung disease such as neutrophilic airway inflammation, mucus hypersecretion, and structural lung damage in vivo. Furthermore, these studies identified macrophage elastase (matrix metalloproteinase 12 (MMP12)) as an additional protease contributing to early lung damage in βENaC-Tg mice. Collectively, these results suggest that NE and MMP12 released from activated neutrophils and macrophages in mucus-obstructed airways play important pathogenetic roles and may serve as potential therapeutic targets to prevent and/or delay irreversible structural lung damage in patients with CF.

Airway mucus obstruction is a key feature of cystic fibrosis (CF) and chronic obstructive pulmonary disease (COPD). The thin layer of mucus that covers healthy airway surfaces has important protective functions in lung defense. However, excess mucus produces airflow obstruction and provides a nidus for bacterial infection and inflammation. Despite its importance in pathogenesis, understanding of the mechanisms underlying airway mucus obstruction, as well as therapeutic options, remain limited. Studies in the rare genetic disease CF identified airway surface dehydration due to cystic fibrosis transmembrane conductance regulator (CFTR) gene dysfunction as an important disease mechanism that may explain mucus stasis and plugging in a spectrum of muco-obstructive lung diseases, including COPD. This concept is supported by the phenotype of the beta-epithelial Na+ channel-transgenic mouse that exhibits airway surface dehydration and develops a spontaneous lung disease that shares key features with CF and COPD, such as airway mucus plugging, chronic neutrophilic inflammation, and structural lung damage. Furthermore, preclinical testing demonstrated that hydration strategies, including osmotically active hypertonic saline and preventive inhibition of the amiloride-sensitive epithelial Na+ channel are effective in unplugging airways in this mouse model of chronic obstructive lung disease. On the other hand, genetic deletion of neutrophil elastase, a potent stimulus for mucus hypersecretion, reduced goblet cell metaplasia and mucin expression but had no effect on mucus obstruction in vivo. Collectively, these studies demonstrate that airway surface dehydration is sufficient to produce mucus obstruction even in the absence of mucus hypersecretion and support further clinical testing of hydrating agents as a promising therapeutic strategy to unplug mucus in CF and COPD.

Recent studies identified the SLC26A9 Cl⁻ channel as a modifier and potential therapeutic target in cystic fibrosis (CF). However, understanding of the regulation of SLC26A9 in epithelia remains limited and cellular models with stable expression for biochemical and functional studies are missing. We, therefore, generated Fisher rat thyroid (FRT) epithelial cells with stable expression of HA-tagged SLC26A9 via retroviral transfection and characterized SLC26A9 expression and function using Western blotting, immunolocalization, whole cell patch-clamp, and transepithelial bioelectric studies in Ussing chambers. We demonstrate stable expression of SLC26A9 in transfected FRT (SLC26A9-FRT) cells on the mRNA and protein level. Immunolocalization and Western blotting detected SLC26A9 in different intracellular compartments and to a lesser extent at the cell surface. Whole cell patch-clamp recordings demonstrated significantly increased constitutive Cl⁻ currents in SLC26A9-FRT compared with control-transduced FRT (Control-FRT) cells ( P < 0.01). Similar, transepithelial measurements showed that the basal short circuit current was significantly increased in SLC26A9-FRT vs. Control-FRT cell monolayers ( P < 0.01). SLC26A9-mediated Cl⁻ currents were increased by cAMP-dependent stimulation (IBMX and forskolin) and inhibited by GlyH-101, niflumic acid, DIDS, and 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB), as well as RNAi knockdown of WNK1 implicated in epithelial osmoregulation. Our results support that these novel epithelial cells with stable expression of SLC26A9 will be a useful model for studies of pharmacological regulation including the identification of activators of SLC26A9 Cl⁻ channels that may compensate deficient cystic fibrosis transmembrane regulator (CFTR)-mediated Cl⁻ secretion and serve as an alternative therapeutic target in patients with CF and potentially other muco-obstructive lung diseases.