Airway Surface Liquid Research Articles

Synthesis and evaluation of tetrahydropyrazolopyridine inhibitors of anion exchange protein SLC26A4 (pendrin).

Pendrin is a transmembrane chloride/anion antiporter that is strongly upregulated in the airways in rhinoviral infection, asthma, cystic fibrosis and chronic rhinosinusitis. Based on its role in the regulation of airway surface liquid depth, pendrin inhibitors have potential indications for treatment of inflammatory airways diseases. Here, a completely regioselective route to tetrahydro-pyrazolopyridine pendrin inhibitors based on 1,3-diketone and substituted hydrazine condensation was been developed. Structure-activity relationships at the tetrahydropyridyl nitrogen were investigated using a focused library, establishing the privileged nature of N-phenyl ureas and improving inhibitor potency by greater than 2-fold.

Airway Surface Liquid Has Innate Antiviral Activity That Is Reduced in Cystic Fibrosis.

Although chronic bacterial infections and inflammation are associated with progressive lung disease in patients with cystic fibrosis (CF), much less is known regarding the contributions of respiratory viral infections to this process. Clinical studies suggest that antiviral host defenses may be compromised in individuals with CF, and CF airway epithelia exhibit impaired antiviral responses in vitro. Here, we used the CF pig model to test the hypothesis that the antiviral activity of respiratory secretions is reduced in CF. We developed an in vitro assay to measure the innate antiviral activity present in airway surface liquid (ASL) from CF and non-CF pigs. We found that tracheal and nasal ASL from newborn non-CF pigs exhibited dose-dependent inhibitory activity against several enveloped and encapsidated viruses, including Sendai virus, respiratory syncytial virus, influenza A, and adenovirus. Importantly, we found that the anti-Sendai virus activity of nasal ASL from newborn CF pigs was significantly diminished relative to non-CF littermate controls. This diminution of extracellular antiviral defenses appears to be driven, at least in part, by the differences in pH between CF and non-CF ASL. These data highlight the novel antiviral properties of native airway secretions and suggest the possibility that defects in extracellular antiviral defenses contribute to CF pathogenesis.

Regulation effects of naringin on diesel particulate matter-induced abnormal airway surface liquid secretion

BackgroundPM2.5 is closely related to the incidence and mortality of respiratory diseases. Diesel particulate matter (DPM) is the main component of particulate air pollution and an important source of PM2.5. Hypothesis/purposeThis study mainly explored the effect of DPM on airway surface liquid (ASL) secretion and the regulation of naringin in this process, to evaluate therapeutic potentials of naringin for the treatment of abnormal secretion of the respiratory tract caused by PM2.5. MethodsThe concentration of lysozyme was measured by Lysozyme Assay Kit. Total protein content was determined by the BCA Protein Assay Kit. The concentration of cAMP and MUC5AC, expressions of CFTR, AQP1, and AQP5 proteins were measured by ELISA. Expressions of CFTR, AQP1 and AQP5 mRNA were determined by qPCR. Amount of CFTR on the cell membrane was determined by immunofluorescence. ResultsThe in vitro and in vivo studies had indicated that DPM could inhibit ASL secretion and increased the viscosity of the liquid. Naringin had the functions to attenuate DPM-induced injury, reduce liquid viscosity by reducing MUC5AC and total protein secretion, increase DPM-induced CFTR, AQP1, and AQP5 mRNA and protein expression, positively regulate apical CFTR insertion and promote CFTR activation by increasing intracellular cAMP. ConclusionThese results demonstrated that naringin had regulating effects on the DPM-induced abnormal secretion of the respiratory tract.

Real-Time, Semi-Automated Fluorescent Measurement of the Airway Surface Liquid pH of Primary Human Airway Epithelial Cells.

In recent years, the importance of mucosal surface pH in the airways has been highlighted by its ability to regulate airway surface liquid (ASL) hydration, mucus viscosity and activity of antimicrobial peptides, key parameters involved in innate defense of the lungs. This is of primary relevance in the field of chronic respiratory diseases such as cystic fibrosis (CF) where these parameters are dysregulated. While different groups have studied ASL pH both in vivo and in vitro, their methods report a relatively wide range of ASL pH values and even contradictory findings regarding any pH differences between non-CF and CF cells. Furthermore, their protocols do not always provide enough details in order to ensure reproducibility, most are low throughput and require expensive equipment or specialized knowledge to implement, making them difficult to establish in most labs. Here we describe a semi-automated fluorescent plate reader assay that enables the real-time measurement of ASL pH under thin film conditions that more closely resemble the in vivo situation. This technique allows for stable measurements for many hours from multiple airway cultures simultaneously and, importantly, dynamic changes in ASL pH in response to agonists and inhibitors can be monitored. To achieve this, the ASL of fully differentiated primary human airway epithelial cells (hAECs) are stained overnight with a pH-sensitive dye in order to allow for the reabsorption of the excess fluid to ensure thin film conditions. After fluorescence is monitored in the presence or absence of agonists, pH calibration is performed in situ to correct for volume and dye concentration. The method described provides the required controls to make stable and reproducible ASL pH measurements, which ultimately could be used as a drug discovery platform for personalized medicine, as well as adapted to other epithelial tissues and experimental conditions, such as inflammatory and/or host-pathogen models.

Cigarette and ENDS preparations differentially regulate ion channels and mucociliary clearance in primary normal human bronchial 3D cultures.

Cigarette smoking is known to disrupt the normal mucociliary function of the lungs, whereas the effect of electronic nicotine delivery systems (ENDS) is not completely understood. This study aimed to compare the effects of acute exposure of primary normal human bronchial epithelial (NHBE) 3D cultures at air-liquid interface to combustible cigarette and ENDS preparations on mucociliary function, including ion channel function, ciliary beat frequency (CBF), and airway surface liquid (ASL) height. Differentiated NHBE cultures were exposed to whole smoke-conditioned media (WS-CM) or total particulate matter (TPM) prepared from 3R4F reference cigarettes, whole aerosol-conditioned media (ACM) or e-TPM generated from a marketed ENDS product, or nicotine alone. We found that a dose of 7 μg/mL equi-nicotine units of cigarette TPM and WS-CM significantly decreased cystic fibrosis transmembrane conductance regulator (CFTR) and the epithelial sodium channel (ENaC) function, which regulates fluid homeostasis in the lung. Conversely, higher (56 µg/mL) equi-nicotine units of ENDS preparations or nicotine alone had no effect on CFTR and ENaC function. Despite a significant decrease in ion channel function, cigarette smoke preparations did not alter CBF and ASL. Similarly, ENDS preparations and nicotine alone had no effect on ASL and CBF. This study demonstrates that acute exposures of cigarette smoke preparations exert a notable inhibitory effect on CFTR and ENaC function compared with ENDS preparations. In summary, the functional assays described herein are potentially useful for tobacco product evaluations.

Modulation of glucose-related metabolic pathways controls glucose level in airway surface liquid and fight oxidative stress in cystic fibrosis cells.

Direct and indirect evidences show that elevated glucose concentrations in airway surface liquid (ASL) promote lung infection by pathogens, playing a role in the progression of the Cystic Fibrosis (CF) disease. The joint action of transporter/s for glucose and of the cellular enzymes is essential in order to try to lower ASL glucose level. Inside the cell, the glycolysis and the pentose phosphate pathway (PPP) compete for the utilization of glucose-6-phosphate (G6P), the product in which glucose, after entry within the cell and phosphorylation, is trapped. The study aims to clarify whether, modulating the activity of enzymatic proteins and/or the level of metabolites/cofactors, involved in intracellular glucose utilization, a lowering of the extracellular glucose level in CF occurs. Biochemical approaches have enabled us to understand i) how G6P is shunted between glycolysis and PPP and ii) that mitochondria, more than enzymes/cofactors participating to the two cell glucose utilization pathways, are protagonists of the scene in counteracting the high ASL glucose level as well as oxidative stress in CF.

Airway surface liquid acidification initiates host defense abnormalities in Cystic Fibrosis

Cystic fibrosis (CF) is caused by defective Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) protein. Morbidity is mainly due to early airway infection. We hypothesized that S. aureus clearance during the first hours of infection was impaired in CF human Airway Surface Liquid (ASL) because of a lowered pH. The ASL pH of human bronchial epithelial cell lines and primary respiratory cells from healthy controls (WT) and patients with CF was measured with a pH microelectrode. The antimicrobial capacity of airway cells was studied after S. aureus apical infection by counting surviving bacteria. ASL was significantly more acidic in CF than in WT respiratory cells. This was consistent with a defect in bicarbonate secretion involving CFTR and SLC26A4 (pendrin) and a persistent proton secretion by ATP12A. ASL demonstrated a defect in S. aureus clearance which was improved by pH normalization. Pendrin inhibition in WT airways recapitulated the CF airway defect and increased S. aureus proliferation. ATP12A inhibition by ouabain decreased bacterial proliferation. Antimicrobial peptides LL-37 and hBD1 demonstrated a pH-dependent activity. Normalizing ASL pH might improve innate airway defense in newborns with CF during onset of S. aureus infection. Pendrin activation and ATP12A inhibition could represent novel therapeutic strategies to normalize pH in CF airways.

Multiprobe Nuclear Imaging of the Cystic Fibrosis Lung as a Biomarker of Therapeutic Effect.

Background: Nuclear imaging biomarkers illustrate unique aspects of lung physiology and are useful for assessing therapeutic effects in cystic fibrosis (CF) lung disease. We have developed a multiprobe method to simultaneously measure mucociliary clearance (MCC) and paracellular absorption (ABS). MCC is a direct measure of mucus clearance. ABS has been related to airway surface liquid (ASL) absorption through previous in vitro studies. Methods: We describe baseline factors affecting MCC and ABS using data from a retrospective baseline group (n = 22) and the response of the measures to inhaled 7% hypertonic saline (HS) and dry powder mannitol using data from a prospective response group (n = 7). A retrospective healthy control group (n = 15) is also described. The baseline and control groups performed single measurements of MCC/ABS. The response group performed baseline measurements of MCC/ABS and measurements after each intervention. Results: ABS was correlated (Spearman's ρ = 0.51, p = 0.06) to sweat chloride, a systemic measure of cystic fibrosis transmembrane conductance regulator (CFTR) function, whereas MCC was not. Baseline MCC was depressed after Pseudomonas aeruginosa infection as we have previously described. MCC provided a more sensitive indication of therapeutic effect and indicated improved clearance with mannitol compared with HS. Conclusion: MCC provides a useful and well-established means of testing therapies directed at improving mucus clearance in the lung. ABS may provide a means of detecting local changes in ASL absorption and CFTR function in the lung. Both are useful tools for studying the key aspects of CF lung pathophysiology (ASL hyperabsorption and MCC depression) that link the basic genetic defects of CF to disease manifestations in the lung.

Proteolytic Cleavage by House Dust Mite Impairs SPLUNC1's Ability to Modulate TH2 Inflammation in the Airway

Lung epithelial cells are often on of the first lines of defense against inhaled allergens. They also work in concert with other airway cell types to orchestrate innate immune responses, keeping inflammation under control. In allergic asthma, allergens (e.g., HDM) disrupts the normal barrier function of epithelia, as well as its interaction with other airway cells via various mechanisms. Proteolytic enzyme activity of allergen components is thought to be one of top contributors of allergic inflammation in the lung. A large body of evidence supports that the biologic activity of HDM proteases augment allergic activity by 1) directly promoting IgE synthesis; 2) damaging the lung epithelium, increasing bronchial permeability; 3) degrading epithelial tight junctions and increasing accessibility to dendritic antigen presenting cells that lie beneath the epithelium; and 4) causing the release of pro‐inflammatory cytokines from epithelial cells, mast cells and basophils.Short palate, lung and nasal epithelial clone 1 (SPLUNC1) is a 256‐amino acid, secretive protein in polarized epithelia and is present at high levels in normal airway surface liquid (ASL). SPLUNC1 is a multi‐functional protein, which regulates ion channel activity, has anti‐microbial activity and affects surface tension. We observed that 1) SPLUNC1 is reduced in asthmatic patients' sputum as well as HDM challenged human bronchial epithelial cells (HBECs). 2)SPLUNC1 was degraded by HDM in both time‐ and dose‐ dependent manners. 3) SPLUNC1 knockout (KO) mice showed more severe inflammatory phenotypes in their airways when challenged with HDM, compared to their wildtype (WT) littermate controls.Overall, we conclude that SPLUNC1 is an epithelial‐derived factor that plays a major role in dampening TH2 inflammation in airways, which is deranged in asthma patients. SPLUNC1 deficiency is partially due to its proteolytical cleavage by HDM. Identifying strategies to increase SPLUNC1 resistance to HDM proteolytic activities or SPLUNC1 in ASL may lead to novel therapies to treat allergic asthma.Support or Funding InformationAmerican Asthma Foundation NCCU‐DUKE CTSIThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Distinct ion transport in the marsh rice rat (Oryzomys palustris) airway

Hypertonic solutions increase airway surface liquid (ASL) through cholinergically‐mediated epithelial cell ion transport and osmosis. Thus, mammals inhabiting salt marshes might have acquired adaptations, including greater dependence upon cholinergic mechanisms, for ASL regulation. To test this hypothesis, we investigated the airways of the rice rat, a medium‐sized rodent that inhabits salt marshes. We discovered unique ion transport properties, including minimal amiloride‐sensitive Na+ absorption and limited Cl− secretion through the cystic fibrosis transmembrane conductance regulator. In contrast, the cholinergic agonist carbachol induced a large Cl− secretory current that was blocked by the calcium‐activated chloride channel inhibitor CaCCinh‐A01. Rice rat airway cultures also secreted fluid in response to carbachol, and when challenged with 3.5% NaCl apically to mimic exposure to sea water aerosols, displayed a prolonged expansion of the ASL volume. These data suggest that the rice rat airway might possess unique ion transport mechanisms to facilitate survival in the harsh salt marsh environment.Support or Funding InformationHL119560, 0T2TR001983, DE0237863This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Furin Inhibition as a Mechanism to Reduce Aberrant ENaC‐Mediated Sodium Transport and Rehydrate the Airways in Cystic Fibrosis Lung Disease

The epithelial sodium channel (ENaC) is found in a number of tissues including lung epithelia and is activated through proteolytic cleavage of its α and γ subunits. Dysregulation of ENaC, secondary to the loss of functional CFTR in cystic fibrosis (CF), leads to increased Na+ absorption which contributes to airways dehydration, thickened mucus and impaired mucociliary clearance (MCC). Pro‐protein convertases such as furin are implicated in the activation of a distinct pool of ENaC as it passes through the biosynthetic pathway (1). Furin cleaves α‐ENaC at two sites releasing a small inhibitory peptide from the extracellular loop, partially activating the channel, and subsequently cleaves γ‐ENaC. These actions ‘prime’ the channel for cleavage at a secondary site on the γ subunit by cell‐surface channel activating proteases such as prostasin, trypsin and neutrophil elastase (2–4). Furin, therefore plays a key role in the proteolytic regulation of ENaC‐mediated sodium absorption and presents a promising therapeutic target for CF lung disease.The aim of this first‐in‐kind study was to determine the effect of two chemically different, highly selective furin inhibitors (BOS‐981 and BOS‐318) on ENaC signaling and airway surface liquid (ASL) height, using primary human CF bronchial epithelial cells (HBEs).Fully differentiated HBEs homozygous for F508‐del, grown at air‐liquid interface, were used to determine ENaC activity via equivalent current (Ieq) readings obtained using a 24‐well Transepithelial Current Clamp (TECC‐24) system. ASL height was measured using confocal microscopy.We report BOS‐981 and BOS‐318 to be potent inhibitors of furin. Both compounds, applied basolaterally, significantly reduced ENaC‐mediated Na+ absorption as measured by an acute reduction in amiloride‐sensitive Ieq after immediate treatment, which was sustained after longer (48 hr) exposure. The compounds also protected ENaC against cell surface activation by neutrophil elastase and CF sputum supernatant. Furthermore, the reduction in ENaC activity after a 48 hr treatment correlated with an increase in ASL height.These ground‐breaking results demonstrate that highly selective inhibition of furin by the novel inhibitors BOS‐981 and BOS‐318 presents a promising mechanism for the reduction of aberrant ENaC‐mediated Na+ absorption. This strategy has the potential to rehydrate the airways, restore effective MCC and reduce the devastating pulmonary decline observed in CF.Support or Funding InformationThis work was supported by funding from the MRC Proximity to Discovery: Industry Engagement FundThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Inhibition of ENaC Activity by Novel Peptide Trypsin‐Like Inhibitors Derived from Amphibian Skin Secretions

Impaired mucociliary clearance (MCC) constitutes the main hallmark of the chronic bronchitis phenotype in COPD. Whilst aberrant processes involved in mucus secretion and ciliary function are important drivers of abnormal MCC, dysregulation of airway hydration appears to be the principal determinant affecting this disorder (1). The hydration status of the airways is largely governed by interplay between the epithelial sodium channel (ENaC) and the cystic fibrosis transmembrane conductance regulator (CFTR). ENaC is activated by trypsin‐like (TL), channel activating proteases (CAPs) and work carried out in cystic fibrosis has shown that inhibition of these proteases increases airways surface liquid (ASL) volume and normalises MCC (2).The aim of this study was to characterise a number of natural TL peptide inhibitors, derived from amphibian skin secretions (frog peptides), based on their ability to inhibit CAPs and regulate ENaC function.Initial screening established an inhibitory profile for each frog peptide against known recombinant CAPs using relevant fluorogenic substrate‐based activity assays. The peptides were then added to fully differentiated human primary bronchial epithelial cells (HBEs) grown at air‐liquid interface (ALI) and ENaC activity determined by obtaining equivalent short circuit current (Ieq) readings using the Transepithelial Current Clamp system (TECC‐24; EP Devices).When examined against recombinant proteases (rTrypsin, rHAT, rMatriptase, rProstasin, rFurin), the frog peptides showed different inhibitory profiles with no single molecule demonstrating overall superiority. Of these, QUB‐2136 rapidly inhibited amiloride‐sensitive Ieq when applied apically to HBEs, to a greater degree than the other compounds investigated. Interestingly, we note that for several of these frog peptides, including QUB‐2136, inactivation of ENaC was so acute that the compounds behave more like direct channel blockers rather than acting indirectly through inhibition of the CAPs.We report for the first time novel biologically active natural peptides, derived from amphibian skin secretions, to be potent inhibitors of CAPs and rapid inactivators of ENaC‐mediated Na+ transport in airway cells. These compounds represent a novel strategy towards ENaC inhibition which has potential to rehydrate the airways, restore effective MCC and reduce the devastating pulmonary decline observed in CF, irrespective of CFTR mutation. Importantly, this strategy could be effective for other chronic airways disease patients such as COPD and non‐CF bronchiectasis.Support or Funding InformationBREATH (Border and REgions Airways Training Hub) is funded by the EU's INTERREG Va programme managed by the Special EU Programmes Body (SEUPB).This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Can enhancing the expression and activity of the Cystic Fibrosis Transmembrane Regulator improve efficacy of gene therapy for Cystic Fibrosis?

Cystic Fibrosis, an autosomal recessive genetic disease, is the most common cause of respiratory disease in children of the Caucasian population. Due to the monogenetic nature of the disease and the accessibility of the airways to nebulizers, it is a desirable candidate for gene therapy/editing. It has been suggested that ~10% of endogenous cystic fibrosis transmembrane regulator (CFTR) expression within target cells in vivo is sufficient for prevention of lung disease. With gene editing technology gaining traction it is important for CF researchers to understand how much functional CFTR is required to prevent the development of lung disease and how to make this more achievable.To answer this question we used two models: cultures of mixed CF:non‐CF primary epithelial cells (a model for gene correction) and cultures of CF cells transduced with CFTR under expression of a high activity promoter (a model for gene therapy). Cultures were grown on semipermeable membranes and differentiated at airway liquid interface. Ussing chamber, Airway surface liquid height and immunohistochemistry were performed as functional readouts. It was observed that essential CFTR functions increased in a linear fashion in CF:WT primary epithelial mixes (CFTRinhIsc μA/cm2: R2=0.91, n=28) whilst only ~16% transduction of a CF culture with CFTR fully restored ion channel activity and airway surface liquid height (n=6).Our data shows that overexpression of CFTR is a more effective method for restoring function in a CF culture. Therefore, we have since developed an enhanced CFTR gene with ~7 fold greater processing in to protein and dramatically increased anion transport activity as shown by a YFP quenching assay (n=3). It is our hope that this enhanced gene could be used as a tool for a more effective gene therapy.Support or Funding InformationCystic Fibrosis Trust UKSt. George's University LondonUniversity of North Carolina, Chapel HillThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

High mobility group box‐1 increases epithelial sodium channel activity and inflammation in small airway epithelial cells

In Cystic Fibrosis (CF) patients, inherited defects in anion transport leads to hyperactive epithelial Na+ channel (ENaC) activity. ENaC dysfunction in CF lung is known to cause airway surface liquid dehydration. Thick and dry mucus, in turn, leads to an inflammatory lung phenotype that often occurs without detection of infection in CF patients. Many investigative groups have reported that airway tissue and fluid from murine models exhibiting the CF lung phenotype (Scnn1b‐Tr mice), as well as CF patients, have increased levels of high mobility group box‐1 (HMGB‐1). HMGB‐1 is a cytokine mediator of inflammation and is a biomarker for severity of CF lung disorder. Herein, we hypothesize that HMGB‐1 plays an important role in the pathogenic regulation of ENaC, inflammation, and fibrosis in sterile airways. To begin testing this hypothesis, we measured transepithelial sodium current (Isc) and single channel ENaC open probability (Po) in human small airway epithelial cells (SAEC) in the presence or absence of 1 mg/mL human HMGB‐1 peptide. Electrophysiological measurements show that HMGB‐1 significantly increased amiloride‐sensitive ENaC Isc from −9.7±1.4 μA/cm2 to −18.4±2.9 μA/cm2 in confluent monolayers of SAEC (n=12; p=0.01) within 30 min. Subsequent amiloride treatment of the same SEAC monolayers decreased Isc 71%. Single channel measurements were conducted in primary SAEC obtained from 4 separate donor lungs in the cell‐attached configuration; HMGB‐1 increased ENaC Po from 0.18±0.02 to 0.35±0.04 (n=17; p<0.01) in single channel analysis. Mean absolute total cell counts were significantly higher in bronchoalveolar lavage fluid (BALF) from mice intraperitoneally (IP) injected with HMBG‐1 (10 mg in 1 mL saline/kg) vs vehicle injected mice. Total immune cell counts were 27.4×103± 8.8×103 (in HMGB‐1) vs. 5.2×103±1.3X103 (in vehicle) injected mice (n=8 counts from 2 independent studies; p<0.05). Masson's Trichrome labeling of IP injected mice show that HMGB‐1 significantly increases pulmonary fibrosis (see Fig 1). Flow cytometric analysis of HMGB‐1 and vehicle injected mice showed significant increase in IL‐1β, IL‐10, IL‐6, IL‐27, IL‐17A, IFN‐β, and GM‐CSF (n=3; p<0.05; see Fig 2). Together, our data suggests that HMGB‐1 plays an important role in the CF lung phenotype; additional studies are underway to strengthen these observations. Impact: Successful completion of our work can lead to novel therapies needed to counteract ENaC dysfunction and inflammation in the CF lung.Support or Funding InformationThis work was supported by NIH 1 R01 HL137033‐01A1 awarded to MH.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Concurrent absorption and secretion of airway surface liquids and bicarbonate secretion in human bronchioles.

Although small airways account for the largest fraction of the total conducting airway surfaces, the epithelial fluid and electrolyte transport in small, native airway epithelia has not been well characterized. Investigations have been limited, no doubt, by the complex tissue architecture as well as by its inaccessibility, small dimensions, and lack of applicable assays, especially in human tissues. To better understand how the critically thin layer of airway surface liquid (ASL) is maintained, we applied a "capillary"-Ussing chamber (area ≈1 mm2) to measure ion transport properties of bronchioles with diameters of ~2 mm isolated from resected specimens of excised human lungs. We found that the small human airway, constitutively and concurrently, secretes and absorbs fluid as observed in porcine small airways (50). We found that the human bronchiolar epithelium is also highly anion selective and constitutively secretes bicarbonate ( ), which can be enhanced pharmacologically by cAMP as well as Ca2+-mediated agonists. Concurrent secretion and absorption of surface liquid along with secretion help explain how the delicate volume of the fluid lining the human small airway is physiologically buffered and maintained in a steady state that avoids desiccating or flooding the small airway with ASL.

Construction of a Suite of Computable Biological Network Models Focused on Mucociliary Clearance in the Respiratory Tract.

Mucociliary clearance (MCC), considered as a collaboration of mucus secreted from goblet cells, the airway surface liquid layer, and the beating of cilia of ciliated cells, is the airways’ defense system against airborne contaminants. Because the process is well described at the molecular level, we gathered the available information into a suite of comprehensive causal biological network (CBN) models. The suite consists of three independent models that represent (1) cilium assembly, (2) ciliary beating, and (3) goblet cell hyperplasia/metaplasia and that were built in the Biological Expression Language, which is both human-readable and computable. The network analysis of highly connected nodes and pathways demonstrated that the relevant biology was captured in the MCC models. We also show the scoring of transcriptomic data onto these network models and demonstrate that the models capture the perturbation in each dataset accurately. This work is a continuation of our approach to use computational biological network models and mathematical algorithms that allow for the interpretation of high-throughput molecular datasets in the context of known biology. The MCC network model suite can be a valuable tool in personalized medicine to further understand heterogeneity and individual drug responses in complex respiratory diseases.

Inhaled medications in cystic fibrosis beyond antibiotics.

Structural lung disease begins very early in children with cystic fibrosis (CF), often in the first three months of life. Inhaled medications represent an attractive therapeutic approach in CF that are routinely used as early intervention strategies. Two aerosolized solutions, hypertonic saline and dornase alfa, have significant potential benefits by improving mucociliary clearance, with minimal associated side-effects. In particular, they favor rehydration of airway surface liquid and cleavage of extracellular DNA in the airways, respectively, consequently reducing rate of pulmonary disease exacerbations. Indirect anti-inflammatory effects have been documented for both drugs, addressing each of the three interrelated elements in the vicious cycle of lung disease in CF: airway obstruction, inflammation and infection. This short review aimed to summarize the main papers that support potential clinical impact of inhaled solutions on pulmonary disease in CF.

Airways Surface Liquid and Ion Transport - The Mechanism Maintained Patency

The airway epithelium is covered with a thin, liquid protective layer - Airways Surface Liquid (ASL) [1].

Interleukin-Mediated Pendrin Transcriptional Regulation in Airway and Esophageal Epithelia.

Pendrin (SLC26A4), a Cl−/anion exchanger, is expressed at high levels in kidney, thyroid, and inner ear epithelia, where it has an essential role in bicarbonate secretion/chloride reabsorption, iodide accumulation, and endolymph ion balance, respectively. Pendrin is expressed at lower levels in other tissues, such as airways and esophageal epithelia, where it is transcriptionally regulated by the inflammatory cytokines interleukin (IL)-4 and IL-13 through a signal transducer and activator of transcription 6 (STAT6)-mediated pathway. In the airway epithelium, increased pendrin expression during inflammatory diseases leads to imbalances in airway surface liquid thickness and mucin release, while, in the esophageal epithelium, dysregulated pendrin expression is supposed to impact the intracellular pH regulation system. In this review, we discuss some of the recent findings on interleukin-mediated transcriptional regulation of pendrin and how this dysregulation impacts airway and esophagus epithelial homeostasis during inflammatory diseases.

Lumacaftor-rescued F508del-CFTR has a modified bicarbonate permeability

Deletion of phenylalanine at position 508, F508del, the most frequent mutation among Cystic fibrosis (CF) patients, destabilizes the protein, thus causing both a folding and a trafficking defect, resulting in a dramatic reduction in expression of CFTR. In vitro treatment with lumacaftor produces an enhancement of anion transport in cells. We studied the permeability properties of the CFTR mutant F508del treated with the corrector lumacaftor, showing that the rescued protein has selectivity properties different than the wild type CFTR, showing an augmented bicarbonate permeability. This difference would indicate a diverse conformation of the rescued F508del-CFTR, that is plausibly reflected on an improper regulation of the airway surface liquid, lessening the efficacy of the corrector. Our findings rather support the idea that a combination of correctors would be required to address the CFTR-dependent bicarbonate permeability.