TRPV2 modulates mechanically Induced ATP Release from Human bronchial epithelial cells.

Repetitive bouts of coughing expose the large airways to significant cycles of shear stress. This leads to the release of alarmins and the tussive agent adenosine triphosphate (ATP) which may be modulated by the activity of ion channels present in the human airway. This study aimed to investigate the role of the transient receptor potential subfamily vanilloid member 2 (TRPV2) channel in mechanically induced ATP release from primary bronchial epithelial cells (PBECs).PBECs were obtained from individuals undergoing bronchoscopy. They were cultured in vitro and exposed to mechanical stress in the form of compressive and fluid shear stress (CFSS) or fluid shear stress (FSS) alone at various intensities. ATP release was measured using a luciferin-luciferase assay. Functional TRPV2 protein expression in human PBECs was investigated by confocal calcium imaging. The role of TRPV2 inhibition on FSS-induced ATP release was investigated using the TRPV2 inhibitor tranilast or siRNA knockdown of TRPV2. TRPV2 protein expression in human lung tissue was also determined by immunohistochemistry.ATP release was significantly increased in PBECs subjected to CFSS compared with control (unstimulated) PBECs (N = 3, ***P < 0.001). PBECs expressed functional TRPV2 channels. TRPV2 protein was also detected in fixed human lung tissue. ATP release from FFS stimulated PBECs was decreased by the TRPV2 inhibitor tranilast (N = 3, **P < 0.01) (vehicle: 159 ± 17.49 nM, tranilast: 25.08 ± 5.1 nM) or by TRPV2 siRNA knockdown (N = 3, *P < 0.05) (vehicle: 197 ± 24.52 nM, siRNA: 119 ± 26.85 nM).In conclusion, TRPV2 is expressed in the human airway and modulates ATP release from mechanically stimulated PBECs.

Aromatic pentaamide macrocycles bind anions with high affinity for transport across biomembranes.

The convergent positioning of functional groups in biomacromolecules leads to good binding, catalytic and transport capabilities. Synthetic frameworks capable of convergently locking functional groups with minimized conformational uncertainty-leading to similar properties-are highly desirable but rare. Here we report C5-symmetric aromatic pentaamide macrocycles synthesized in one pot from the corresponding monomers. Their crystal structures reveal a star-shaped, fully constrained backbone that causes ten alternating NH/CH hydrogen-bond donors and five large amide dipoles to orient towards the centre of the macrocycle. With a highly electropositive cavity in a high-energy unbound state, the macrocycles bind anions in a 1:1 stoichiometry in solution, with high affinity for halides and very high affinity for oxoanions. We demonstrate that such macrocycles are able to transport anions across lipid bilayers with a high chloride selectivity and restore the depleted airway surface liquid of cystic fibrosis airway cell cultures.

Chronic airway epithelial hypoxia exacerbates injury in muco-obstructive lung disease through mucus hyperconcentration.

Unlike solid organs, human airway epithelia derive their oxygen from inspired air rather than the vasculature. Many pulmonary diseases are associated with intraluminal airway obstruction caused by aspirated foreign bodies, virus infection, tumors, or mucus plugs intrinsic to airway disease, including cystic fibrosis (CF). Consistent with requirements for luminal O2, airway epithelia surrounding mucus plugs in chronic obstructive pulmonary disease (COPD) lungs are hypoxic. Despite these observations, the effects of chronic hypoxia (CH) on airway epithelial host defense functions relevant to pulmonary disease have not been investigated. Molecular characterization of resected human lungs from individuals with a spectrum of muco-obstructive lung diseases (MOLDs) or COVID-19 identified molecular features of chronic hypoxia, including increased EGLN3 expression, in epithelia lining mucus-obstructed airways. In vitro experiments using cultured chronically hypoxic airway epithelia revealed conversion to a glycolytic metabolic state with maintenance of cellular architecture. Chronically hypoxic airway epithelia unexpectedly exhibited increased MUC5B mucin production and increased transepithelial Na+ and fluid absorption mediated by HIF1α/HIF2α-dependent up-regulation of ß and γENaC (epithelial Na+ channel) subunit expression. The combination of increased Na+ absorption and MUC5B production generated hyperconcentrated mucus predicted to perpetuate obstruction. Single-cell and bulk RNA sequencing analyses of chronically hypoxic cultured airway epithelia revealed transcriptional changes involved in airway wall remodeling, destruction, and angiogenesis. These results were confirmed by RNA-in situ hybridization studies of lungs from individuals with MOLD. Our data suggest that chronic airway epithelial hypoxia may be central to the pathogenesis of persistent mucus accumulation in MOLDs and associated airway wall damage.

Mucus-targeting therapies of defective mucus clearance for cystic fibrosis: A short review.

In the lungs, defective CFTR associated with cystic fibrosis (CF) represents the nidus for abnormal mucus clearance in the airways and consequently a progressive lung disease. Defective CFTR-mediated Cl- secretion results in altered mucus properties, including concentration, viscoelasticity, and the ratio of the two mucins, MUC5B and MUC5AC. In the past decades, therapies targeting the CF mucus defect, directly or indirectly, have been developed; nevertheless, better treatments to prevent the disease progression are still needed. This review summarizes the existing knowledge on the defective mucus in CF disease and highlights it as a barrier to the development of future inhaled genetic therapies. The use of new mucus-targeting treatments is also discussed, focusing on their potential role to halt the progress of CF lung disease.

Mucus concentration-dependent biophysical abnormalities unify submucosal gland and superficial airway dysfunction in cystic fibrosis.

Cystic fibrosis (CF) is characterized by abnormal transepithelial ion transport. However, a description of CF lung disease pathophysiology unifying superficial epithelial and submucosal gland (SMG) dysfunctions has remained elusive. We hypothesized that biophysical abnormalities associated with CF mucus hyperconcentration provide a unifying mechanism. Studies of the anion secretion-inhibited pig airway model of CF revealed elevated SMG mucus concentrations, osmotic pressures, and SMG mucus accumulation. Human airway studies revealed hyperconcentrated CF SMG mucus with raised osmotic pressures and cohesive forces predicted to limit SMG mucus secretion/release. Using proline-rich protein 4 (PRR4) as a biomarker of SMG secretion, CF sputum proteomics analyses revealed markedly lower PRR4 levels compared to healthy and bronchiectasis controls, consistent with a failure of CF SMGs to secrete mucus onto airway surfaces. Raised mucus osmotic/cohesive forces, reflecting mucus hyperconcentration, provide a unifying mechanism that describes disease-initiating mucus accumulation on airway surfaces and in SMGs of the CF lung.

Physiology and pathophysiology of human airway mucus.

The mucus clearance system is the dominant mechanical host defense system of the human lung. Mucus is cleared from the lung by cilia and airflow, including both two-phase gas-liquid pumping and cough-dependent mechanisms, and mucus transport rates are heavily dependent on mucus concentration. Importantly, mucus transport rates are accurately predicted by the gel-on-brush model of the mucociliary apparatus from the relative osmotic moduli of the mucus and periciliary-glycocalyceal (PCL-G) layers. The fluid available to hydrate mucus is generated by transepithelial fluid transport. Feedback interactions between mucus concentrations and cilia beating, via purinergic signaling, coordinate Na+ absorptive vs Cl- secretory rates to maintain mucus hydration in health. In disease, mucus becomes hyperconcentrated (dehydrated). Multiple mechanisms derange the ion transport pathways that normally hydrate mucus in muco-obstructive lung diseases, e.g., cystic fibrosis (CF), chronic obstructive pulmonary disease (COPD), non-CF bronchiectasis (NCFB), and primary ciliary dyskinesia (PCD). A key step in muco-obstructive disease pathogenesis is the osmotic compression of the mucus layer onto the airway surface with the formation of adherent mucus plaques and plugs, particularly in distal airways. Mucus plaques create locally hypoxic conditions and produce airflow obstruction, inflammation, infection, and, ultimately, airway wall damage. Therapies to clear adherent mucus with hydrating and mucolytic agents are rational, and strategies to develop these agents are reviewed.

Airway Epithelial Nucleotide Release Contributes to Mucociliary Clearance.

Mucociliary clearance (MCC) is a dominant component of pulmonary host defense. In health, the periciliary layer (PCL) is optimally hydrated, thus acting as an efficient lubricant layer over which the mucus layer moves by ciliary force. Airway surface dehydration and production of hyperconcentrated mucus is a common feature of chronic obstructive lung diseases such as cystic fibrosis (CF) and chronic bronchitis (CB). Mucus hydration is driven by electrolyte transport activities, which in turn are regulated by airway epithelial purinergic receptors. The activity of these receptors is controlled by the extracellular concentrations of ATP and its metabolite adenosine. Vesicular and conducted pathways contribute to ATP release from airway epithelial cells. In this study, we review the evidence leading to the identification of major components of these pathways: (a) the vesicular nucleotide transporter VNUT (the product of the SLC17A9 gene), the ATP transporter mediating ATP storage in (and release from) mucin granules and secretory vesicles; and (b) the ATP conduit pannexin 1 expressed in non-mucous airway epithelial cells. We further illustrate that ablation of pannexin 1 reduces, at least in part, airway surface liquid (ASL) volume production, ciliary beating, and MCC rates.

Effect of hypertonic saline on mucociliary clearance and clinical outcomes in chronic bronchitis.

BACKGROUND: Mucus dehydration and impaired mucus clearance are common features of cystic fibrosis (CF) and chronic obstructive pulmonary disease (COPD). In CF, inhaled hypertonic saline (HS) improves lung function and produces sustained increases in mucociliary clearance (MCC). We hypothesised that administration of HS (7% NaCl) twice daily for 2 weeks would improve clinical outcomes and produce sustained increases in MCC in COPD subjects with a chronic bronchitis (CB) phenotype. METHODS: Twenty-two CB subjects completed a double-blinded, crossover study comparing inhaled HS to a hypotonic control solution (0.12% saline) administered via nebuliser twice daily for 2 weeks. Treatment order was randomised. During each treatment period, symptoms and spirometry were measured. MCC was measured at baseline, shortly after initial study agent administration, and approximately 12 h after the final dose. RESULTS: HS was safe and well tolerated but overall produced no significant improvements in spirometry or patient-reported outcomes. CB subjects had slower baseline MCC than healthy subjects. The MCC rates over 60 min (Ave60Clr) in CB subjects following 2 weeks of HS were not different from 0.12% saline but were slower than baseline (Ave60Clr was 9.1±6.3% at baseline versus 5.3±6.9% after HS; p<0.05). Subgroup analyses determined that subjects with residual baseline central lung clearance (14 subjects) had improved spirometry and symptoms following treatment with HS, but not 0.12% saline, treatment. CONCLUSIONS: Inhaled HS appeared to be safe in a general CB population. A specific phenotypic subgroup may benefit from HS but requires additional study.

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.

Airway Mucus Hyperconcentration in Non-Cystic Fibrosis Bronchiectasis.

Rationale: Non-cystic fibrosis bronchiectasis is characterized by airway mucus accumulation and sputum production, but the role of mucus concentration in the pathogenesis of these abnormalities has not been characterized.Objectives: This study was designed to: 1) measure mucus concentration and biophysical properties of bronchiectasis mucus; 2) identify the secreted mucins contained in bronchiectasis mucus; 3) relate mucus properties to airway epithelial mucin RNA/protein expression; and 4) explore relationships between mucus hyperconcentration and disease severity.Methods: Sputum samples were collected from subjects with bronchiectasis, with and without chronic erythromycin administration, and healthy control subjects. Sputum percent solid concentrations, total and individual mucin concentrations, osmotic pressures, rheological properties, and inflammatory mediators were measured. Intracellular mucins were measured in endobronchial biopsies by immunohistochemistry and gene expression. MUC5B (mucin 5B) polymorphisms were identified by quantitative PCR. In a replication bronchiectasis cohort, spontaneously expectorated and hypertonic saline-induced sputa were collected, and mucus/mucin concentrations were measured.Measurements and Main Results: Bronchiectasis sputum exhibited increased percent solids, total and individual (MUC5B and MUC5AC) mucin concentrations, osmotic pressure, and elastic and viscous moduli compared with healthy sputum. Within subjects with bronchiectasis, sputum percent solids correlated inversely with FEV1 and positively with bronchiectasis extent, as measured by high-resolution computed tomography, and inflammatory mediators. No difference was detected in MUC5B rs35705950 SNP allele frequency between bronchiectasis and healthy individuals. Hypertonic saline inhalation acutely reduced non-cystic fibrosis bronchiectasis mucus concentration by 5%.Conclusions: Hyperconcentrated airway mucus is characteristic of subjects with bronchiectasis, likely contributes to disease pathophysiology, and may be a target for pharmacotherapy.

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.

Adhesive and Cohesive Peel Force Measurement of Human Airway Mucus.

In health, the high-speed airflow associated with cough represents a vital backup mechanism for clearing accumulated mucus from our airways. However, alterations in the mucus layer in cystic fibrosis (CF) and chronic obstructive pulmonary disease (COPD) leads to the mucus layer adhered to the airway surfaces, representing the nidus of chronic lung infection. To understand what is different about diseased mucus and why cough clearance is defective, there is a need for techniques to quantify the strength of the interactions limiting the ability of airflow to strip mucus from the airway surface (i.e., adhesive strength) or tear mucus apart (i.e., cohesive strength). To overcome the issues with measuring these properties in a soft (i.e., low elastic modulus) mucus layer, we present here novel peel-testing technologies capable of quantifying the mucus adhesive strength on cultured airway cells and cohesive strength of excised mucus samples. While this protocol focuses on measurements of airway mucus, this approach can easily be adapted to measuring adhesive/cohesive properties of other soft biological materials.

IL-1ß dominates the promucin secretory cytokine profile in cystic fibrosis.

Cystic fibrosis (CF) lung disease is characterized by early and persistent mucus accumulation and neutrophilic inflammation in the distal airways. Identification of the factors in CF mucopurulent secretions that perpetuate CF mucoinflammation may provide strategies for novel CF pharmacotherapies. We show that IL-1ß, with IL-1α, dominated the mucin prosecretory activities of supernatants of airway mucopurulent secretions (SAMS). Like SAMS, IL-1ß alone induced MUC5B and MUC5AC protein secretion and mucus hyperconcentration in CF human bronchial epithelial (HBE) cells. Mechanistically, IL-1ß induced the sterile α motif-pointed domain containing ETS transcription factor (SPDEF) and downstream endoplasmic reticulum to nucleus signaling 2 (ERN2) to upregulate mucin gene expression. Increased mRNA levels of IL1B, SPDEF, and ERN2 were associated with increased MUC5B and MUC5AC expression in the distal airways of excised CF lungs. Administration of an IL-1 receptor antagonist (IL-1Ra) blocked SAMS-induced expression of mucins and proinflammatory mediators in CF HBE cells. In conclusion, IL-1α and IL-1ß are upstream components of a signaling pathway, including IL-1R1 and downstream SPDEF and ERN2, that generate a positive feedback cycle capable of producing persistent mucus hyperconcentration and IL-1α and/or IL-1ß-mediated neutrophilic inflammation in the absence of infection in CF airways. Targeting this pathway therapeutically may ameliorate mucus obstruction and inflammation-induced structural damage in young CF children.

The Cystic Fibrosis-Like Airway Surface Layer Is not a Significant Barrier for Delivery of Eluforsen to Airway Epithelial Cells.

Background: Eluforsen (previously known as QR-010) is a 33-mer antisense oligonucleotide under development for oral inhalation in cystic fibrosis (CF) patients with the delta F508 mutation. Previous work has shown that eluforsen restores CF transmembrane conductance regulator (CFTR) function in vitro and in vivo. To be effective, eluforsen has first to reach its primary target, the lung epithelial cells. Therefore, it has to diffuse through the CF airway surface layer (ASL), which in CF is characterized by the presence of thick and viscous mucus, impaired mucociliary clearance, and persistent infections. The goal of this study was to assess delivery of eluforsen through CF-like ASL. Methods and Results: First, air-liquid interface studies with cultured primary airway epithelial cells revealed that eluforsen rapidly diffuses through CF-like mucus at clinically relevant doses when nebulized once or repeatedly, over a range of testing doses. Furthermore, eluforsen concentrations remained stable in CF patient sputum for at least 48 hours, and eluforsen remained intact in the presence of various inhaled CF medications for at least 24 hours. When testing biodistribution of eluforsen after orotracheal administration in vivo, no differences in lung, liver, trachea, and kidney eluforsen concentration were observed between mice with a CF-like lung phenotype (ENaC-overexpressing mice) and control wild-type (WT) littermates. Also, eluforsen was visualized in the airway epithelial cell layer of CF-like muco-obstructed mice and WT littermates. Finally, studies of eluforsen uptake and binding to bacteria prevalent in CF lungs, and diffusion through bacterial biofilms showed that eluforsen was stable and not absorbed by, or bound to bacteria. In addition, eluforsen was found to be able to penetrate Pseudomonas aeruginosa biofilms. Conclusions: The thickened and concentrated CF ASL does not constitute a significant barrier for delivery of eluforsen, and feasibility of oral inhalation of eluforsen is supported by these data.

Roles of mucus adhesion and cohesion in cough clearance.

Clearance of intrapulmonary mucus by the high-velocity airflow generated by cough is the major rescue clearance mechanism in subjects with mucoobstructive diseases and failed cilial-dependent mucus clearance, e.g., subjects with cystic fibrosis (CF) or chronic obstructive pulmonary disease (COPD). Previous studies have investigated the mechanical forces generated at airway surfaces by cough but have not considered the effects of mucus biophysical properties on cough efficacy. Theoretically, mucus can be cleared by cough from the lung by an adhesive failure, i.e., breaking mucus-cell surface adhesive bonds and/or by cohesive failure, i.e., directly fracturing mucus. Utilizing peel-testing technologies, mucus-epithelial surface adhesive and mucus cohesive strengths were measured. Because both mucus concentration and pH have been reported to alter mucus biophysical properties in disease, the effects of mucus concentration and pH on adhesion and cohesion were compared. Both adhesive and cohesive strengths depended on mucus concentration, but neither on physiologically relevant changes in pH nor bicarbonate concentration. Mucus from bronchial epithelial cultures and patient sputum samples exhibited similar adhesive and cohesive properties. Notably, the magnitudes of both adhesive and cohesive strength exhibited similar velocity and concentration dependencies, suggesting that viscous dissipation of energy within mucus during cough determines the efficiency of cough clearance of diseased, hyperconcentrated, mucus. Calculations of airflow-induced shear forces on airway mucus related to mucus concentration predicted substantially reduced cough clearance in small versus large airways. Studies designed to improve cough clearance in subjects with mucoobstructive diseases identified reductions of mucus concentration and viscous dissipation as key therapeutic strategies.

Pathological mucus and impaired mucus clearance in cystic fibrosis patients result from increased concentration, not altered pH.

Cystic fibrosis (CF) is a recessive genetic disease that is characterised by airway mucus plugging and reduced mucus clearance. There are currently alternative hypotheses that attempt to describe the abnormally viscous and elastic mucus that is a hallmark of CF airways disease, including: 1) loss of CF transmembrane regulator (CFTR)-dependent airway surface volume (water) secretion, producing mucus hyperconcentration-dependent increased viscosity, and 2) impaired bicarbonate secretion by CFTR, producing acidification of airway surfaces and increased mucus viscosity.A series of experiments was conducted to determine the contributions of mucus concentration versus pH to the rheological properties of airway mucus across length scales from the nanoscopic to macroscopic.For length scales greater than the nanoscopic, i.e. those relevant to mucociliary clearance, the effect of mucus concentration dominated over the effect of airway acidification.Mucus hydration and chemical reduction of disulfide bonds that connect mucin monomers are more promising therapeutic approaches than alkalisation.

An integrated mathematical epithelial cell model for airway surface liquid regulation by mechanical forces.

A robust method based on reverse engineering was utilized to construct the ion-channel conductance functions for airway epithelial sodium channels (ENaC), the cystic fibrosis transmembrane conductance regulator (CFTR), and calcium-activated chloride channels (CaCC). The ion-channel conductance models for both normal (NL) and cystic fibrosis (CF) airway epithelia were developed and then coupled to an adenosine triphosphate (ATP) metabolism model and a fluid transport model (collectively called the integrated cell model) to investigate airway surface liquid (ASL) volume regulation and hence mucus concentration, by mechanical forces in NL and CF human airways. The epithelial cell models for NL and CF required differences in Cl- secretion (decreased in CF) and Na+ absorption (raised in CF) to reproduce behaviors similar to in vitro epithelial cells exposed to mechanical forces (cyclic shear stress, cyclic compressive pressure and cilial strain) and selected modulators of ion channels and ATP release. The epithelial cell models were then used to investigate the effects of mechanical forces and evaporative flux on ASL and mucus homeostasis in both NL and CF airway epithelia. Because of reduced CF ASL volumes, CF mucus concentrations increased and produced a greater dependence of ASL volume regulation on cilia-mucus-ATP release interactions in CF than NL epithelial nodules. Similarly, the CF model was less tolerant to evaporation induced ASL volume reduction at all ATP release rates than the NL model. Consequently, this reverse engineered model appears to provide a robust tool for investigating CF pathophysiology and novel therapies.

Airway surface liquid pH is not acidic in children with cystic fibrosis.

Modulation of airway surface liquid (ASL) pH has been proposed as a therapy for cystic fibrosis (CF). However, evidence that ASL pH is reduced in CF is limited and conflicting. The technical challenges associated with measuring ASL pH in vivo have precluded accurate measurements in humans. In order to address this deficiency, ASL pH was measured in vivo in children using a novel luminescent technology integrated with fibre-optic probes. Here we show that ASL pH in children with CF is similar to that of children without CF. Findings were supported by highly controlled direct pH measurements in primary human airway epithelial cell culture models, which also suggest that the potential ASL pH gradient produced by defective apical ion transport is balanced out by paracellular shunting of acid/base. Thus, reduced baseline ASL pH is unlikely to be an important pathobiological factor in early CF lung disease.

Sialic acid-to-urea ratio as a measure of airway surface hydration.

Although airway mucus dehydration is key to pathophysiology of cystic fibrosis (CF) and other airways diseases, measuring mucus hydration is challenging. We explored a robust method to estimate mucus hydration using sialic acid as a marker for mucin content. Terminal sialic acid residues from mucins were cleaved by acid hydrolysis from airway samples, and concentrations of sialic acid, urea, and other biomarkers were analyzed by mass spectrometry. In mucins purified from human airway epithelial (HAE), sialic acid concentrations after acid hydrolysis correlated with mucin concentrations (r2 = 0.92). Sialic acid-to-urea ratios measured from filters applied to the apical surface of cultured HAE correlated to percent solids and were elevated in samples from CF HAEs relative to controls (2.2 ± 1.1 vs. 0.93 ± 1.8, P < 0.01). Sialic acid-to-urea ratios were elevated in bronchoalveolar lavage fluid (BALF) from ß-epithelial sodium channel (ENaC) transgenic mice, known to have reduced mucus hydration, and mice sensitized to house dust mite allergen. In a translational application, elevated sialic acid-to-urea ratios were measured in BALF from young children with CF who had airway infection relative to those who did not (5.5 ± 3.7 vs. 1.9 ± 1.4, P < 0.02) and could be assessed simultaneously with established biomarkers of inflammation. The sialic acid-to-urea ratio performed similarly to percent solids, the gold standard measure of mucus hydration. The method proved robust and has potential to serve as flexible techniques to assess mucin hydration, particularly in samples like BALF in which established methods such as percent solids cannot be utilized.

Mucus Hyperconcentration as a Unifying Aspect of the Chronic Bronchitic Phenotype.

Abnormalities in mucus production and qualitative properties such as mucus hydration are central to the pathophysiology of airway disease including cystic fibrosis, asthma, and chronic bronchitis. In vitro air-liquid interface epithelial cell cultures demonstrate direct relationships between mucociliary transport, periciliary liquid (PCL) height, and mucus concentration (expressed as percent solids or partial osmotic pressure). In health, the osmotic modulus/pressure of the PCL exceeds that of the mucus layer, resulting in efficient, low-friction movement of mucus. In disease, through multiple mechanisms, the osmotic pressure of the mucus begins to exceed basal PCL values, resulting in compression of the cilia and slowing of mucus transport. The in vivo data in both cystic fibrosis and chronic bronchitis parallel in vitro data demonstrating that when mucus osmotic pressure is increased, mucociliary clearance is decreased. In chronic bronchitis, there is a direct correlation between FEV1 and percent solids of mucus, demonstrating a strong relationship between disease progression and mucus abnormalities. Animal models, based mechanistically on raised sodium absorption (and therefore water absorption) from airway surfaces, mimic the pathophysiology of chronic obstructive pulmonary disease. Collectively, these data suggest the importance of mucus concentration in the pathogenesis of airway disease. It is important to understand the precise mechanisms that result in mucus hyperconcentration, for example, mucin overproduction versus abnormal regulation of ion/water transport, which may be unique to and characteristic of each disease phenotype. The measurement of mucus concentration may be a simple method to diagnose chronic bronchitis, monitor its progression, and serve as a biomarker for development of new therapies.