Airway Surface Liquid Research Articles

Nasal Mucociliary Clearance in Smokers: A Systematic Review.

Introduction Smoking is one of the most important causes of mortality and morbidity in the world, as it is related to the risk factor and etiology of respiratory-tract diseases. Long-term smoking causes both structural and functional damage in the respiratory airways, leading to changes in nasal mucociliary clearance (NMC). Objectives The aim of the present study was to look systematically into the current literature and carefully collect and analyze results to explore NMC in smokers. Data Synthesis Two independent reviewers conducted a literature search on some Electronic database: Pubmed, Medline, Ebsco, Springer Link, Science Direct, Scopus, and Proquest searching for articles fulfilling the inclusion and exclusion criteria. The lead author independently assessed the risk of bias of each of the included studies and discussed their assessments with the other two authors to achieve consensus. Of the 1,654 articles identified in the database search, 16 met the criteria for this review. Most of the articles (15 out of 16) showed the impairment of NMC in smokers. Conclusion The present systematic review suggests that there is an impairment of NMC in smokers. The impairment is not only observed in cigarette smoking, but also in passive smoking, bidi smoking, electronic smoking, and hookah smoking. The impairment of NMC in chronic exposure to smoking is caused by the ciliotoxic effect, hypersecretion and viscoelastic change of mucous, airway surface liquid depletion, increased oxidative stress, and deteriorations in the inflammatory and immune systems.

TMEM16A Potentiation: A Novel Therapeutic Approach for the Treatment of Cystic Fibrosis.

Rationale: Enhancing non–CFTR (cystic fibrosis transmembrane conductance regulator)-mediated anion secretion is an attractive therapeutic approach for the treatment of cystic fibrosis (CF) and other mucoobstructive diseases.Objectives: To determine the effects of TMEM16A potentiation on epithelial fluid secretion and mucociliary clearance.Methods: The effects of a novel low-molecular-weight TMEM16A potentiator (ETX001) were evaluated in human cell and animal models of airway epithelial function and mucus transport.Measurements and Main Results: Potentiating the activity of TMEM16A with ETX001 increased the Ca2+-activated Cl− channel activity and anion secretion in human bronchial epithelial (HBE) cells from patients with CF without impacting calcium signaling. ETX001 rapidly increased fluid secretion and airway surface liquid height in CF-HBE cells under both static conditions and conditions designed to mimic the shear stress associated with tidal breathing. In ovine models of mucus clearance (tracheal mucus velocity and mucociliary clearance), inhaled ETX001 was able to accelerate clearance both when CFTR function was reduced by administration of a pharmacological blocker and when CFTR was fully functional.Conclusions: Enhancing the activity of TMEM16A increases epithelial fluid secretion and enhances mucus clearance independent of CFTR function. TMEM16A potentiation is a novel approach for the treatment of patients with CF and non-CF mucoobstructive diseases.

Pharmacological Inhibition and Activation of the Ca2+ Activated Cl- Channel TMEM16A.

TMEM16A is a Ca2+ activated Cl− channel with important functions in airways, intestine, and other epithelial organs. Activation of TMEM16A is proposed as a therapy in cystic fibrosis (CF) to reinstall airway Cl− secretion and to enhance airway surface liquid (ASL). This CFTR-agnostic approach is thought to improve mucociliary clearance and lung function in CF. This could indeed improve ASL, however, mucus release and airway contraction may also be induced by activators of TMEM16A, particularly in inflamed airways of patients with asthma, COPD, or CF. Currently, both activators and inhibitors of TMEM16A are developed and examined in different types of tissues. Here we compare activation and inhibition of endogenous and overexpressed TMEM16A and analyze potential off-target effects. The three well-known blockers benzbromarone, niclosamide, and Ani9 inhibited both TMEM16A and ATP-induced Ca2+ increase by variable degrees, depending on the cell type. Niclosamide, while blocking Ca2+ activated TMEM16A, also induced a subtle but significant Ca2+ store release and inhibited store-operated Ca2+ influx. Niclosamide, benzbromarone and Ani9 also affected TMEM16F whole cell currents, indicating limited specificity for these inhibitors. The compounds Eact, cinnamaldehyde, and melittin, as well as the phosphatidylinositol diC8-PIP2 are the reported activators of TMEM16A. However, the compounds were unable to activate endogenous TMEM16A in HT29 colonic epithelial cells. In contrast, TMEM16A overexpressed in HEK293 cells was potently stimulated by these activators. We speculate that overexpressed TMEM16A might have a better accessibility to intracellular Ca2+, which causes spontaneous activity even at basal intracellular Ca2+ concentrations. Small molecules may therefore potentiate pre-stimulated TMEM16A currents, but may otherwise fail to activate silent endogenous TMEM16A.

Identification Of A Novel Histone Derived Antimicrobial Peptide In Airway Surface Liquid.

The airway surface liquid (ASL), a protective layer secreted by the airway epithelium, represents the first line of defence against inhaled infectious material. It contains a complex array of secreted proteins and peptides that aid the neutralisation and removal of inhaled microbes and toxicants. The ASL also contains many proteases that can cleave proteins to generate further bioactive peptides. We therefore hypothesised that the airway ASL sustained an extensive peptidome containing novel bioactive peptides. We examined the ASL peptide profile of normal human bronchial epithelial cells transformed with BMI‐1 (NHBE‐BMI1) and cell‐lines Calu3 (submucosal adenocarcinoma) and H441 (Clara cell like adenocarcinoma). ASL was acquired by washing the apical surface of epithelial monolayers grown at air‐liquid interface with PBS (100μl) at 0, 24 and 120 hours after exposure to hyperglycaemic (25mM glucose) or normoglycaemic (5mM glucose and 20mM mannitol) basolateral medium. The ASL peptides < 10kDa, were subsequently isolated and analysed using the Q Exactive™ HF‐X Hybrid Quadrupole‐Orbitrap™ Mass Spectrometer. The resulting spectra of native peptides and their subsequent fragments were analysed using the pNovo De novo sequencing tool. Our preliminary results identified 4765 unique peptides (NHBE‐BMI1: 646, Calu3: 3150, H441: 1197). Of these, 37 peptides were common to all samples regardless of glycaemic state. A number of histone derived peptides were identified in all samples and cell lines. These peptides had a proline‐alanine rich N‐terminal region and a cationic C‐terminal, similar to antimicrobial peptides from histone H1 found in Atlantic salmon skin mucous. Synthetic peptides were produced by SPOT synthesis and the antimicrobial activity tested against luminescent Pseudomonas aeruginosa over 18 hours at 37°C in (100mM Tris (pH 7) and 20mM glucose). One of the crude peptides elicited a dose dependent inhibition of bacterial luminescence at 4.63±1.34 μM (n=3), indicating no bacteria survived. Truncated variants of this peptide containing the alanine‐proline rich N‐terminal sequence or the cationic C‐terminal sequence were produced to determine if a specific region of the peptide was required for antimicrobial activity. The N and the C terminal peptides had their antimicrobial activity significantly decreased, only reducing bacterial luminescence by 28.06±2.27% and 35.28±9.45% respectively compared to the full‐length peptide; 99.99±0.001% (all normalised to untreated bacterial luminescence, n=4, p<0.0001, ±SD). This indicates that both regions are required for antimicrobial activity. An immunoblot of concentrated native ASL peptides and cell lysates showed the presence of known histone protein epitopes in our peptide of interest. Furthermore, we identified this histone‐derived peptide in sputum samples from both Cystic Fibrosis and normal healthy patients. Our preliminary data indicates that this novel histone derived peptide may serve an antimicrobial function in the ASL.Support or Funding InformationMedical Research Council London Intercollegiate Doctoral Training Programme

Role of Impaired CFTR Function in Pathogenesis of Pneumonia Among Alcohol Users

INTRODUCTIONImpairment of mucociliary clearance (MCC) increases the risk of respiratory infections among excessive alcohol users. The mechanisms underlying this impact of alcohol on MCC are not currently understood. Our previous in vitro studies indicated that alcohol decreases ion transport via CFTR channels, the defective protein that causes cystic fibrosis. Here, we have attempted to validate these findings in vivo and test the hypothesis that alcohol‐induced CFTR dysfunction reduces airway surface hydration and mucus transport thereby impairing airway defense against inhaled bacterial pathogens, in particular Klebsiella pneumoniae, to which alcohol users are disproportionately susceptible. Since CFTR dysfunction correlates with reduced PKA‐dependent CFTR phosphorylation and channel gating due to increased cAMP degradation by phosphodiesterase‐4B (PDE4B) enzymes, we also tested the effects of PDE4 inhibitor, roflumilast, on CFTR function and bacterial clearance in alcohol‐fed rats.METHODSAge and gender‐matched rats (CFTR+/+) were pair‐fed with Lieber‐DeCarli alcohol diet or isocaloric control diet for 4 weeks and with K. pneumoniae (KP, ATCC 43816). Subsets of rats were randomly treated with vehicle/roflumilast (5mg/kg/day) by oral gavage starting 7 days before the end of the alcohol diet. CFTR activity was measured with nasal potential difference (NPD) and MCC was assayed with radiographic clearance of Tc99. Airway surface liquid (ASL), periciliary layer (PCL), and ciliary beat frequency (CBF) of excised trachea were quantified with micro‐optical coherence tomography (μOCT).RESULTSAlcohol administration reduced CFTR ion transport by NPD. Clearance of Tc99 label in 30 min indicated delayed MCC in alcohol‐treated rats. Trachea excised from alcohol‐treated rats exhibited significantly decreased PCL depth, but no meaningful differences in ASL or CBF. Roflumilast treatment significantly increased CFTR activity in both control and alcohol‐administered rats. Increased CFTR activation by roflumilast resulted in augmented PCL depth, reduced mucus viscosity and significantly accelerated MCT rate. Compared to controls, histopathological examination of lung sections from alcohol‐treated animals exhibited more severe evidence of multifocal pneumonia with edema, fibrin deposition and numerous bacteria in alveolar spaces. Ongoing studies are determining the impact of roflumilast treatment on bacterial clearance and severity of pneumonia by K. pneumoniae.CONCLUSIONSAlcohol‐induced defects in CFTR ion transport resulted in reduced airway surface hydration and delayed mucus clearance in rat airways that was associated with diminished ability to clear bacterial pathogens. Acquired (non‐genetic) CFTR dysfunction may be a suitable pharmacologic target to improve mucus clearance impaired by chronic alcohol use. Administration of roflumilast may boost lung host defense against bacterial pathogens and improve pneumonia outcomes in individuals affected by excessive alcohol use.Support or Funding InformationNIH 1R01AA027528

Effect of continuous positive airway pressure treatment on permeability, inflammation and mucus production of human epithelial cells.

Continuous positive airway pressure (CPAP) therapy is the gold standard treatment for obstructive sleep apnoea, which affects millions of people worldwide. However, this therapy normally results in symptoms such as dryness, sneezing, rhinorrhoea, post-nasal drip, nasal congestion and epistaxis in the upper airways.Using bronchial epithelial (Calu-3) and nasal epithelial (RPMI 2650) cells in an in vitro respiratory model, this study, for the first time, investigates the effect of CPAP positive pressure on the human respiratory epithelial mechanisms that regulate upper airways lubrication characteristics. To understand how the epithelium and mucus are affected by this therapy, several parameters were determined before and after positive pressure application.This work demonstrates that the positive pressure not only compresses the cells, but also reduces their permeability and mucus secretion rate, thus drying the airway surface liquid layer and altering the mucus/water ratio. It is also observed that the respiratory epithelia is equally inflamed without or with external humidification during CPAP application.These findings clearly identify the causes of the side-effects reported by patients under CPAP therapy.

Effect of hyperglycaemia on the growth of lung pathogens in Artificial Sputum Media

Elevated glucose concentration in the airway surface liquid due to high blood glucose (hyperglycaemia) has been associated with the increased risk of developing pulmonary infection. It has recently been shown that lungs are colonisedby resident bacteria and are not truly sterile. Several chronic lung conditions, such as cystic fibrosis (CF) and chronic obstructive pulmonary disease (COPD), have been shown to induce changes in this bacterial population.Here we sought to develop an in vitro model using artificial sputum media (ASM) to investigate the effect of hyperglycaemia on Staphylococcus aureus (SA) and Pseudomonas aeruginosa (PA), two of the most common pathogens in CF patients.SA (ATCC29213) and PA (H174) were grown in the artificial sputum media (ASM) at varying glucose concentrations. Approximately 3×105 bacteria were added to 15 mL ASM and grown at 37°C with constant shaking. Two‐way ANOVA with Geisser‐Greenhouse correction and Pearsons correlation coefficients were used for statistical analysis.ASM supplemented with 4 mM glucose had a significant effect on the growth rate of both S. aureus and P. aeruginosa when compared with ASM with no glucose (p = 0.0028, n=5 for SA and p < 0.0001, n=5 for PA). Compared with growth in Mueller Hinton broth, only P. aeruginosa exhibited increased growth rate in the presence of 4 mM glucose (p = 0.0082, n = 3). Growth of both, S. aureus and P. aeruginosa, was correlated with the depletion of glucose in the media (p = 0.0002, n=5 for PA and 0.0038, n=5 for SA). The production of lactate by S. aureus was not related to the depletion of glucose as concentrations for both reached zero at the sixth hour of growth.As ASM mimics the nutrients available in the lungs, it facilitates the study of lung pathogens in the presence of glucose. The established in vitro assay in this study can be further utilised to examine the competition and/or co‐existence between multiple pathogens that are known to reside within the lungs.Support or Funding InformationResearch funded by MRC‐DTP

Ablation of IL-33 Suppresses Th2 Responses but Is Accompanied by Sustained Mucus Obstruction in the Scnn1b Transgenic Mouse Model.

Cystic fibrosis is characterized by dehydration of the airway surface liquid layer with persistent mucus obstruction. Th2 immune responses are often manifested as increased mucous cell density (mucous cell metaplasia) associated with mucus obstruction. IL-33 is a known inducer of Th2 immune responses, but its roles in mucus obstruction and related phenotypes in a cystic fibrosis-like lung disease model (i.e., Scnn1b-Tg-positive [Tg+]) mouse, remain unclear. Accordingly, IL-33 knockout (IL-33KO) Tg+ mice were examined and compared with IL-33 heterozygous (IL-33HET) Tg+ mice. As compared with IL-33HET/Tg+ mice, IL-33KO/Tg+ mice had complete absence of bronchoalveolar lavage fluid eosinophilia, accompanied with significant reduction in bronchoalveolar lavage fluid concentration of IL-5, a cytokine associated with eosinophil differentiation and recruitment, and IL-4, a major Th2 cytokine. As compared with IL-33HET/Tg+ mice, IL-33KO/Tg+ mice had significantly reduced levels of Th2-associated gene signatures (Slc26a4, Clca1, Retnla, and Chi3l4), along with complete loss of intracellular mucopolysaccharide staining in the airway epithelium. As compared with IL-33HET/Tg+ mice, although the IL-33KO/Tg+ mice had significantly reduced levels of MUC5AC protein expression, they showed no reduction in the degree of mucus obstruction, MUC5B protein expression, bacterial burden, and neonatal mortality. Interestingly, the histological features, including subepithelial airway inflammation and alveolar space enlargement, were somewhat exaggerated in IL-33KO/Tg+ mice compared with IL-33HET/Tg+ mice. Taken together, our data indicate that although IL-33 modulates Th2 inflammatory responses and MUC5AC protein production, mucus obstruction is not dependent on IL-33.

Airway submucosal glands from cystic fibrosis swine suffer from abnormal ion transport across the serous acini, collecting duct, and ciliated duct.

The human airway is protected by an efficient innate defense mechanism that requires healthy secretion of airway surface liquid (ASL) to clear pathogens from the lungs. Most of the ASL in the upper airway is secreted by submucosal glands. In cystic fibrosis (CF), the function of airway submucosal glands is abnormal, and these abnormalities are attributed to anomalies in ion transport across the epithelia lining the different sections of the glands that function coordinately to produce the ASL. However, the ion transport properties of most of the anatomical regions of the gland have never been measured, and there is controversy regarding which segments express CFTR. This makes it difficult to determine the glandular abnormalities that may contribute to CF lung disease. Using a noninvasive, extracellular self-referencing ion-selective electrode technique, we characterized ion transport properties in all four segments of submucosal glands from wild-type and CFTR-/- swine. In wild-type airways, the serous acini, mucus tubules, and collecting ducts secrete Cl- and Na+ into the lumen in response to carbachol and forskolin stimulation. The ciliated duct also transports Cl- and Na+ but in the opposite direction, i.e., reabsorption from the ASL, which may contribute to lowering Na+ and Cl- activities in the secreted fluid. In CFTR-/- airways, the serous acini, collecting ducts, and ciliated ducts fail to transport ions after forskolin stimulation, resulting in the production of smaller volumes of ASL with normal Cl-, Na+, and K+ concentration.

Small Molecule Anion Carriers Correct Abnormal Airway Surface Liquid Properties in Cystic Fibrosis Airway Epithelia.

Cystic fibrosis (CF) is a genetic disease characterized by the lack of cystic fibrosis transmembrane conductance regulator (CFTR) protein expressed in epithelial cells. The resulting defective chloride and bicarbonate secretion and imbalance of the transepithelial homeostasis lead to abnormal airway surface liquid (ASL) composition and properties. The reduced ASL volume impairs ciliary beating with the consequent accumulation of sticky mucus. This situation prevents the normal mucociliary clearance, favouring the survival and proliferation of bacteria and contributing to the genesis of CF lung disease. Here, we have explored the potential of small molecules capable of facilitating the transmembrane transport of chloride and bicarbonate in order to replace the defective transport activity elicited by CFTR in CF airway epithelia. Primary human bronchial epithelial cells obtained from CF and non-CF patients were differentiated into a mucociliated epithelia in order to assess the effects of our compounds on some key properties of ASL. The treatment of these functional models with non-toxic doses of the synthetic anionophores improved the periciliary fluid composition, reducing the fluid re-absorption, correcting the ASL pH and reducing the viscosity of the mucus, thus representing promising drug candidates for CF therapy.

Losartan Rescues Inflammation-related Mucociliary Dysfunction in Relevant Models of Cystic Fibrosis.

Rationale: Despite therapeutic progress in treating cystic fibrosis (CF) airway disease, airway inflammation with associated mucociliary dysfunction remains largely unaddressed. Inflammation reduces the activity of apically expressed large-conductance Ca2+-activated and voltage-dependent K+ (BK) channels, critical for mucociliary function in the absence of CFTR (CF transmembrane conductance regulator).Objectives: To test losartan as an antiinflammatory therapy in CF using CF human bronchial epithelial cells and an ovine model of CF-like airway disease.Methods: Losartan's antiinflammatory effectiveness to rescue BK activity and thus mucociliary function was tested in vitro using primary, fully redifferentiated human airway epithelial cells homozygous for F508del and in vivo using a previously validated, now expanded pharmacologic sheep model of CF-like, inflammation-associated mucociliary dysfunction.Measurements and Main Results: Nasal scrapings from patients with CF showed that neutrophilic inflammation correlated with reduced expression of LRRC26 (leucine rich repeat containing 26), the γ subunit mandatory for BK function in the airways. TGF-β1 (transforming growth factor β1), downstream of neutrophil elastase, decreased mucociliary parameters in vitro. These were rescued by losartan at concentrations achieved by nebulization in the airway and oral application in the bloodstream: BK dysfunction recovered acutely and over time (the latter via an increase in LRRC26 expression), ciliary beat frequency and airway surface liquid volume improved, and mucus hyperconcentration and cellular inflammation decreased. These effects did not depend on angiotensin receptor blockade. Expanding on a validated and published nongenetic, CF-like sheep model, ewes inhaled CFTRinh172 and neutrophil elastase for 3 days, which resulted in prolonged tracheal mucus velocity reduction, mucus hyperconcentration, and increased TGF-β1. Nebulized losartan rescued both mucus transport and mucus hyperconcentration and reduced TGF-β1.Conclusions: Losartan effectively reversed CF- and inflammation-associated mucociliary dysfunction, independent of its angiotensin receptor blockade.

The Effects of the Anti-aging Protein Klotho on Mucociliary Clearance.

α-klotho (KL) is an anti-aging protein and has been shown to exert anti-inflammatory and anti-oxidative effects in the lung and pulmonary diseases such as chronic obstructive pulmonary disease (COPD) and cystic fibrosis. The current study investigated the direct effect of KL on the bronchial epithelium in regards to mucociliary clearance parameters. Primary human bronchial and murine tracheal epithelial cells, cultured, and differentiated at the air liquid interface (ALI), were treated with recombinant KL or infected with a lentiviral vector expressing KL. Airway surface liquid (ASL) volume, airway ion channel activities, and expression levels were analyzed. These experiments were paired with ex vivo analyses of mucociliary clearance in murine tracheas from klotho deficient mice and their wild type littermates. Our results showed that klotho deficiency led to impaired mucociliary clearance with a reduction in ASL volume in vitro and ex vivo. Overexpression or exogenous KL increased ASL volume, which was paralleled by increased activation of the large-conductance, Ca2+-activated, voltage-dependent potassium channel (BK) without effect on the cystic fibrosis transmembrane conductance regulator (CFTR). Furthermore, KL overexpression downregulated IL-8 levels and attenuated TGF-β-mediated downregulation of LRRC26, the γ subunit of BK, necessary for its function in non-excitable cells. In summary, we show that KL regulates mucociliary function by increasing ASL volume in the airways possibly due to underlying BK activation. The KL mediated BK channel activation may be a potentially important target to design therapeutic strategies in inflammatory airway diseases when ASL volume is decreased.

Indoor Particulate Matter From Smoker Homes Induces Bacterial Growth, Biofilm Formation, and Impairs Airway Antimicrobial Activity. A Pilot Study.

Background: Particulate matter (PM) air pollution causes deleterious health effects; however, less is known about health effects of indoor air particulate matter (IAP).Objective: To understand whether IAP influences distinct mechanisms in the development of respiratory tract infections, including bacterial growth, biofilm formation, and innate immunity. Additionally, we tested whether IAP from Iowa houses of subjects with and without recent respiratory exacerbations recapitulated the National Institute of Standards and Technology (NIST) IAP findings.Methods: To test the effect of NIST and Iowa IAP on bacterial growth and biofilm formation, we assessed Staphylococcus aureus growth and Pseudomonas aeruginosa biofilm formation with and without the presence of IAP. To assess the effect of IAP on innate immunity, we exposed primary human airway surface liquid (ASL) to NIST, and Iowa IAP. Lastly, we tested whether specific metals may be responsible for effects on airway innate immunity.Results: NIST and Iowa IAP significantly enhanced bacterial growth and biofilm formation. NIST IAP (whole particle and the soluble portion) impaired ASL antimicrobial activity. IAP from one Iowa home significantly impaired ASL antimicrobial activity (p < 0.05), and five other homes demonstrated a trend (p ≤ 0.18) of impaired ASL antimicrobial activity. IAP from homes of subjects with a recent history of respiratory exacerbation tended (p = 0.09) to impair ASL antimicrobial activity more than IAP from homes of those without a history respiratory exacerbation. Aluminum and Magnesium impaired ASL antimicrobial activity, while copper was bactericidal. Combining metals varied their effect on ASL antimicrobial activity.Conclusions: NIST IAP and Iowa IAP enhanced bacterial growth and biofilm formation. ASL antimicrobial activity was impaired by NIST IAP, and Iowa house IAP from subjects with recent respiratory exacerbation tended to impair ASL antimicrobial activity. Individual metals may explain impaired ASL antimicrobial activity; however, antimicrobial activity in the presence of multiple metals warrants further study.

High-mobility group box-1 increases epithelial sodium channel activity and inflammation via the receptor for advanced glycation end products.

Cystic fibrosis (CF) lung disease persists and remains life-limiting for many patients. Elevated high-mobility group box-1 protein (HMGB-1) levels and epithelial sodium channel hyperactivity (ENaC) are hallmark features of the CF lung. The objective of this study was to better understand the pathogenic role of HMGB-1 signaling and ENaC in CF airway cells. We hypothesize that HMGB-1 links airway inflammation [via signaling to the receptor for advanced glycation end products (RAGE)] and airway surface liquid dehydration (via upregulation of ENaC) in the CF lung. We calculated equivalent short-current (Isc) and single-channel ENaC open probability (Po) in normal and CF human small airway epithelial cells (SAEC) in the presence and absence of human HMGB-1 peptide (0.5 μg/mL). In normal SAECs, HMGB-1 increased amiloride-sensitive Isc and elevated ENaC Po from 0.15 ± 0.03 to 0.28 ± 0.04 (P < 0.01). In CF SAECs, ENaC Po increased from 0.45 ± 0.06 to 0.73 ± 0.04 (P < 0.01). Pretreatment with 1 μM FPS-ZM1 (a RAGE inhibitor) attenuated all HMGB-1 effects on ENaC current in normal and CF SAECs. Confocal analysis of SAECs indicates that nuclear size and HMBG-1 localization can be impacted by ENaC dysfunction. Masson's trichrome labeling of mouse lung showed that intraperitoneally injected HMGB-1 significantly increased pulmonary fibrosis. Bronchoalveolar lavage fluid from HMGB-1-treated mice showed significant increases in IL-1β, IL-10, IL-6, IL-27, IL-17A, IFN-β, and granulocyte-macrophage colony-stimulating factor compared with vehicle-injected mice (P < 0.05). These studies put forth a new model in which HMGB-1 signaling to RAGE plays an important role in perpetuating ENaC dysfunction and inflammation in the CF lung.

Transport and fate of inhaled particles after deposition onto the airway surface liquid: A 3D numerical study

In this paper, a numerical investigation is carried out to provide insights into the fate of inhaled aerosols after their deposition on the lung lining fluid in both healthy and diseased states. Pulmonary drug delivery is a well-known non-invasive route of administration compared to intravenous delivery. Aerosol particles are formulated and used as drug carriers, which are then sent to the airways using aerosol drug delivery devices. This approach is useful for site-specific treatment of lung diseases, treatment of central nervous system (CNS) disorders and a variety of other diseases. Bioavailability of the inhaled therapeutic particles after landing on the airway lining fluid can be significantly altered by the lung muco-ciliary clearance, a process through which hairlike structures known as cilia beat in a harmonised manner and induce the mucus in the proximal direction, leading to an effective clearance of the foreign inhaled particles entrapped by this sticky layer from the airways. Here, we set up a 3D computational model of ciliary arrays interacting with periciliary liquid film (i.e. confined between the epithelium and mucus layer) and a detailed analysis is conducted to better understand the fate of drug nanoparticles that are able to penetrate the mucus. Consistent with clinical findings, we find that the actions of cilia result in a low rate of drug retention and absorption by the pulmonary tissues in healthy lungs. However, under conditions associated with abnormal ciliary beats, the retention time of particles is notably increased at the site of release. Nonetheless, the results associated with some of the ciliary impairments reveal that deposition of drug aerosols on the ciliated cells may still be a significant challenge. These findings have potentially important implications on the modification of therapeutic drug particles to achieve a higher absorption rate.

Inhibition of ATP hydrolysis restores airway surface liquid production in cystic fibrosis airway epithelia

Inhibition of ATP hydrolysis restores airway surface liquid production in cystic fibrosis airway epithelia

Protease Inhibitors: Candidate Drugs to Inhibit Severe Acute Respiratory Syndrome Coronavirus 2 Replication.

The number of patients infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has rapidly increased, although the WHO declared a pandemic. However, drugs that function against SARS-CoV-2 have not been established. SARS-CoV-2 has been suggested to bind angiotensin-converting enzyme 2, the receptor of the SARS coronavirus. SARS coronavirus and coronavirus 229E, the cause of the common cold, replicate through cell-surface and endosomal pathways using a protease, the type II transmembrane protease. To examine the effects of protease inhibitors on the replication of coronavirus 229E, we pretreated primary cultures of human nasal epithelial (HNE) cells with camostat or nafamostat, each of which has been used for the treatment of pancreatitis and/or disseminated intravascular coagulation. HNE cells were then infected with coronavirus 229E, and viral titers in the airway surface liquid of the cells were examined. Pretreatment with camostat (0.1-10 μg/mL) or nafamostat (0.01-1 μg/mL) reduced the titers of coronavirus 229E. Furthermore, a significant amount of type II transmembrane protease protein was detected in the airway surface liquid of HNE cells. Additionally, interferons have been reported to have antiviral effects against SARS coronavirus. The additive effects of interferons on the inhibitory effects of other candidate drugs to treat SARS-CoV-2 infection, such as lopinavir, ritonavir and favipiravir, have also been studied. These findings suggest that protease inhibitors of this type may inhibit coronavirus 229E replication in human airway epithelial cells at clinical concentrations. Protease inhibitors, interferons or the combination of these drugs may become candidate drugs to inhibit the replication of SARS-CoV-2.

Inhibition of ATP hydrolysis restores airway surface liquid production in cystic fibrosis airway epithelia.

Airway surface dehydration is a pathological feature of cystic fibrosis (CF) lung disease. CF is caused by mutations in the CF transmembrane conductance regulator (CFTR), a cyclic AMP-regulated Cl- channel controlled in part by the adenosine A2B receptor. An alternative CFTR-independent mechanism of fluid secretion is regulated by ATP via the P2Y2 receptor (P2Y2R) that activates Ca2+-regulated Cl- channels (CaCC/TMEM16) and inhibits Na+ absorption. However, due to rapid ATP hydrolysis, steady-state ATP levels in CF airway surface liquid (ASL) are inadequate to maintain P2Y2R-mediated fluid secretion. Therefore, inhibiting airway epithelial ecto-ATPases to increase ASL ATP levels constitutes a strategy to restore airway surface hydration in CF. Using [γ32P]ATP as radiotracer, we assessed the effect of a series of ATPase inhibitory compounds on the stability of physiologically occurring ATP concentrations. We identified the polyoxometalate [Co4(H2O)2(PW9O34)2]10- (POM-5) as the most potent and effective ecto-ATPase inhibitor in CF airway epithelial cells. POM-5 caused long-lasting inhibition of ATP hydrolysis in airway epithelia, which was reversible upon removal of the inhibitor. Importantly, POM-5 markedly enhanced steady-state levels of released ATP, promoting increased ASL volume in CF cell surfaces. These results provide proof of concept for ecto-ATPase inhibitors as therapeutic agents to restore hydration of CF airway surfaces. As a test of this notion, cell-free sputum supernatants from CF subjects were studied and found to have abnormally elevated ATPase activity, which was markedly inhibited by POM-5.

Augmentation of Cystic Fibrosis Transmembrane Conductance Regulator Function in Human Bronchial Epithelial Cells via SLC6A14-Dependent Amino Acid Uptake. Implications for Treatment of Cystic Fibrosis.

SLC6A14-mediated l-arginine transport has been shown to augment the residual anion channel activity of the major mutant, F508del-CFTR, in the murine gastrointestinal tract. It is not yet known if this transporter augments residual and pharmacological corrected F508del-CFTR in primary airway epithelia. We sought to determine the role of l-arginine uptake via SLC6A14 in modifying F508del-CFTR channel activity in airway cells from patients with cystic fibrosis (CF). Human bronchial epithelial (HBE) cells from lung explants of patients without CF (HBE) and those with CF (CF-HBE) were used for H3-flux, airway surface liquid, and Ussing chamber studies. We used α-methyltryptophan as a specific inhibitor for SLC6A14. CFBE41o-, a commonly used CF airway cell line, was employed for studying the mechanism of the functional interaction between SLC6A14 and F508del-CFTR. SLC6A14 is functionally expressed in CF-HBE cells. l-arginine uptake via SLC6A14 augmented F508del-CFTR function at baseline and after treatment with lumacaftor. SLC6A14-mediated l-arginine uptake also increased the airway surface liquid in CF-HBE cells. Using CFBE41o cells, we showed that the positive SLC6A14 effect was mainly dependent on the nitric oxide (NO) synthase activity, nitrogen oxides, including NO, and phosphorylation by protein kinase G. These finding were confirmed in CF-HBE, as inducible NO synthase inhibition abrogated the functional interaction between SLC6A14 and pharmacological corrected F508del-CFTR. In summary, SLC6A14-mediated l-arginine transport augments residual F508del-CFTR channel function via a noncanonical, NO pathway. This effect is enhanced with increasing pharmacological rescue of F508del-CFTR to the membrane. The current study demonstrates how endogenous pathways can be used for the development of companion therapy in CF.

A Novel AAV-mediated Gene Delivery System Corrects CFTR Function in Pigs.

Cystic fibrosis is an autosomal-recessive disease that is caused by a mutant CFTR (cystic fibrosis transmembrane conductance regulator) gene and is characterized by chronic bacterial lung infections and inflammation. Complementation with functional CFTR normalizes anion transport across the airway surface. Adeno-associated virus (AAV) is a useful vector for gene therapy because of its low immunogenicity and ability to persist for months to years. However, because its episomal expression may decrease after cell division, readministration of the AAV vector may be required. To overcome this, we designed an integrating AAV-based CFTR-expressing vector, termed piggyBac (PB)/AAV, carrying CFTR flanked by the terminal repeats of the piggyBac transposon. With codelivery of the piggyBac transposase, PB/AAV can integrate into the host genome. Because of the packaging constraints of AAV, careful consideration was required to ensure that the vector would package and express its CFTR cDNA cargo. In this short-term study, PB/AAV-CFTR was aerosolized to the airways of CF pigs in the absence of the transposase. Two weeks later, transepithelial Cl- current was restored in freshly excised tracheal and bronchial tissue. Additionally, we observed an increase in tracheal airway surface liquid pH and bacterial killing in comparison with untreated CF pigs. Airway surface liquid from primary airway cells cultured from treated CF pigs exhibited increased pH correlating with decreased viscosity. Together, these results show that complementing CFTR in CF pigs with PB/AAV rescues the anion transport defect in a large-animal CF model. Delivery of this integrating viral vector system to airway progenitor cells could lead to persistent, life-long expression in vivo.