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Rationale: Identification of the specific cell types expressing CFTR (cystic fibrosis [CF] transmembrane conductance regulator) is required for precision medicine therapies for CF. However, a full characterization of CFTR expression in normal human airway epithelia is missing. Objectives: To identify the cell types that contribute to CFTR expression and function within the proximal-distal axis of the normal human lung. Methods: Single-cell RNA (scRNA) sequencing (scRNA-seq) was performed on freshly isolated human large and small airway epithelial cells. scRNA in situ hybridization (ISH) and single-cell qRT-PCR were performed for validation. In vitro culture systems correlated CFTR function with cell types. Lentiviruses were used for cell type-specific transduction of wild-type CFTR in CF cells. Measurements and Main Results: scRNA-seq identified secretory cells as dominating CFTR expression in normal human large and, particularly, small airway superficial epithelia, followed by basal cells. Ionocytes expressed the highest CFTR levels but were rare, whereas the expression in ciliated cells was infrequent and low. scRNA ISH and single-cell qRT-PCR confirmed the scRNA-seq findings. CF lungs exhibited distributions of CFTR and ionocytes similar to those of normal control subjects. CFTR mediated Cl - secretion in cultures tracked secretory cell, but not ionocyte, densities. Furthermore, the nucleotide-purinergic regulatory system that controls CFTR-mediated hydration was associated with secretory cells and not with ionocytes. Lentiviral transduction of wild-type CFTR produced CFTR-mediated Cl - secretion in CF airway secretory cells but not in ciliated cells. Conclusions: Secretory cells dominate CFTR expression and function in human airway superficial epithelia. CFTR therapies may need to restore CFTR function to multiple cell types, with a focus on secretory cells.

Innate lymphoid and adaptive immune cells are known to regulate epithelial responses, including mucous cell metaplasia (MCM), but their roles in mucoinflammatory airway diseases, such as cystic fibrosis, remain unknown. Scnn1b transgenic ( Scnn1b -Tg + ) mice, which recapitulate cystic fibrosis-like mucoinflammatory airway disease, deficient in innate lymphoid ( Il2rg knockout mice [ Il2rg KO ]), adaptive immune ( Rag1 knockout mice [ Rag1 KO ]), or both systems ( Il2rg KO / Rag1 KO ), were employed to investigate their respective contributions in the pathogenesis of mucoinflammatory airway disease. As previously reported, immunocompetent Tg + juveniles exhibited spontaneous neonatal bacterial infections with robust mucoinflammatory features, including elevated expression of Th2 -associated markers accompanied by MCM, elevated MUC5B expression, and airway mucus obstruction. The bacterial burden was increased in Il2rg KO/ Tg + juveniles but returned to significantly lower levels in Il2rg KO / Rag1 KO /Tg + juveniles. Mechanistically, this improvement reflected reduced production of adaptive immunity-derived IL-10 and, in turn, increased activation of macrophages. Although all the mucoinflammatory features were comparable between the immunocompetent Tg + and Rag1 KO /Tg + juveniles, the Il2rg KO /Tg + and Il2rg KO / Rag1 KO /Tg + juveniles exhibited suppressed expression levels of Th2 markers, diminished MCM, suppressed MUC5B expression, and reduced mucus obstruction. Collectively, these data indicate that, in the context of airway mucus obstruction, the adaptive immune system suppresses antibacterial macrophage activation, whereas the innate lymphoid system contributes to MCM, mucin production, and mucus obstruction., (Copyright © 2020 by The American Association of Immunologists, Inc.)

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., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Airway mucus hyperproduction and fluid imbalance are important hallmarks of cystic fibrosis (CF), the most common life-shortening genetic disorder in Caucasians. Dysregulated expression and/or function of airway ion transporters, including cystic fibrosis transmembrane conductance regulator (CFTR) and epithelial sodium channel (ENaC), have been implicated as causes of CF-associated mucus hypersecretory phenotype. However, the contributory roles of other substances and transporters in the regulation of CF airway pathogenesis remain unelucidated. Here, we identified a novel connection between CFTR/ENaC expression and the intracellular Zn 2+ concentration in the regulation of MUC5AC, a major secreted mucin that is highly expressed in CF airway. CFTR-defective and ENaC-hyperactive airway epithelial cells specifically and highly expressed a unique, alternative splice isoform of the zinc importer ZIP2/SLC39A2 (ΔC-ZIP2), which lacks the C-terminal domain. Importantly, ΔC-ZIP2 levels correlated inversely with wild-type ZIP2 and intracellular Zn 2+ levels. Moreover, the splice switch to ΔC-ZIP2 as well as decreased expression of other ZIPs caused zinc deficiency, which is sufficient for induction of MUC5AC; while ΔC-ZIP2 expression per se induced ENaC expression and function. Thus, our findings demonstrate that the novel splicing switch contributes to CF lung pathology via the novel interplay of CFTR, ENaC, and ZIP2 transporters., (Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.)

ATP in airway surface liquid (ASL) controls mucociliary clearance functions via the activation of airway epithelial purinergic receptors. However, abnormally elevated ATP levels have been reported in inflamed airways, suggesting that excessive ATP in ASL contributes to airway inflammation. Despite these observations, little is known about the mechanisms of ATP accumulation in the ASL covering inflamed airways. In this study, links between cystic fibrosis (CF)-associated airway inflammation and airway epithelial ATP release were investigated. Primary human bronchial epithelial (HBE) cells isolated from CF lungs exhibited enhanced IL-8 secretion after 6 to 11 days, but not 28 to 35 days, in culture, compared with normal HBE cells. Hypotonic cell swelling-promoted ATP release was increased in 6- to 11-day-old CF HBE cells compared with non-CF HBE cells, but returned to normal values after 28 to 35 days in culture. The exposure of non-CF HBE cells to airway secretions isolated from CF lungs, namely, sterile supernatants of mucopurulent material (SMM), also caused enhanced IL-8 secretion and increased ATP release. The SMM-induced increase in ATP release was sensitive to Ca(2+) chelation and vesicle trafficking/exocytosis inhibitors, but not to pannexin inhibition. Transcript levels of the vesicular nucleotide transporter, but not pannexin 1, were up-regulated after SMM exposure. SMM-treated cultures displayed increased basal mucin secretion, but mucin secretion was not enhanced in response to hypotonic challenge after the exposure of cells to either vehicle or SMM. We propose that CF airway inflammation up-regulates the capacity of airway epithelia to release ATP via Ca(2+)-dependent vesicular mechanisms not associated with mucin granule secretion.

The combination of irradiated fibroblast feeder cells and Rho kinase inhibitor, Y-27632, conditionally induces an indefinite proliferative state in primary mammalian epithelial cells. These conditionally reprogrammed cells (CRCs) are karyotype-stable and nontumorigenic. Because self-renewal is a recognized property of stem cells, we investigated whether Y-27632 and feeder cells induced a stem-like phenotype. We found that CRCs share characteristics of adult stem cells and exhibit up-regulated expression of α6 and β1 integrins, ΔNp63α, CD44, and telomerase reverse transcriptase, as well as decreased Notch signaling and an increased level of nuclear β-catenin. The induction of CRCs is rapid (occurs within 2 d) and results from reprogramming of the entire cell population rather than the selection of a minor subpopulation. CRCs do not overexpress the transcription factor sets characteristic of embryonic or induced pluripotent stem cells (e.g., Sox2, Oct4, Nanog, or Klf4). The induction of CRCs is also reversible, and removal of Y-27632 and feeders allows the cells to differentiate normally. Thus, when CRCs from ectocervical epithelium or tracheal epithelium are placed in an air-liquid interface culture system, the cervical cells form a well differentiated stratified squamous epithelium, whereas the tracheal cells form a ciliated airway epithelium. We discuss the diagnostic and therapeutic opportunities afforded by a method that can generate adult stem-like cells in vitro without genetic manipulation.

Extracellular nucleotides are key components of the signaling network regulating airway clearance. They are released by the epithelium into the airway surface liquid (ASL) to stimulate cilia beating activity, mucus secretion and airway hydration. Understanding the factors affecting their availability for purinoceptor activation is an important step toward the development of new therapies for obstructive lung diseases. This chapter presents a mathematical model developed to gain predictive insights into the regulation of ASL nucleotide concentrations on human airway epithelia. The parameters were estimated from experimental data collected on polarized primary cultures of human nasal and bronchial epithelial cells. This model reproduces major experimental observations: (1) the independence of steady-state nucleotide concentrations on ASL height, (2) the impact of selective ectonucleotidase inhibitors on their steady-state ASL concentrations, (3) the changes in ASL composition caused by mechanical stress mimicking normal breathing, (4) and the differences in steady-state concentrations existing between nasal and bronchial epithelia. In addition, this model launched the study of nucleotide release into uncharted territories, which led to the discovery that airway epithelia release, not only ATP, but also ADP and AMP. This study shows that computational modeling, coupled to experimental validation, provides a powerful approach for the identification of key therapeutic targets for the improvement of airway clearance in obstructive respiratory diseases.

Extracellular ATP controls key aspects of lung function via activation of epithelial cell purinergic receptors, but how ATP is released from cells remains poorly understood. To identify mechanistic components upstream of ATP release, we examined the effect of selected G protein coupled-receptor activation on ATP release from lung epithelial cells. The protease-activated receptor (PAR) agonist thrombin elicited a rapid Ca(2+)-dependent release of ATP from A549 cells. In contrast, the P2Y(2) receptor agonist UTP caused negligible ATP release, despite promoting a robust Ca(2+) response. Agonist-elicited ATP release was associated with Rho activation and was reduced in cells transfected with dominant negative mutants of p115-Rho GEF or RhoA, and by inhibitors of Rho kinase (ROCK). However, RhoA activation alone did not promote ATP release if temporally separated from Ca(2+) mobilization. PAR3 was the only PAR subtype detected in A549 cells by reverse transcription-PCR. Transfection of cells with human PAR3 cDNA increased thrombin-promoted ATP release, inositol phosphate formation, and RhoA activation. Conversely, small interference RNA against PAR3 diminished thrombin-evoked responses. Thrombin-elicited ATP release was accompanied by an enhanced cellular uptake of propidium iodide in a Ca(2+)- and ROCK-dependent manner and was inhibited by connexin/pannexin hemichannel blockers. Our data suggest that thrombin promotes ATP release from A549 cells via Rho- and Ca(2+)-dependent activation of connexin/pannexin hemichannels. The relevance of these findings is highlighted by the observation that exposure of primary cultures of well differentiated human bronchial epithelial cells to thrombin resulted in robust ATP release, which was inhibited by ROCK inhibitors and by connexin/pannexin hemichannel blockers.

Dysfunction of CFTR in cystic fibrosis (CF) airway epithelium perturbs the normal regulation of ion transport, leading to a reduced volume of airway surface liquid (ASL), mucus dehydration, decreased mucus transport, and mucus plugging of the airways. CFTR is normally expressed in ciliated epithelial cells of the surface and submucosal gland ductal epithelium and submucosal gland acinar cells. Critical questions for the development of gene transfer strategies for CF airway disease are what airway regions require CFTR function and how many epithelial cells require CFTR expression to restore normal ASL volume regulation and mucus transport to CF airway epithelium? An in vitro model of human CF ciliated surface airway epithelium (CF HAE) was used to test whether a human parainfluenza virus (PIV) vector engineered to express CFTR (PIVCFTR) could deliver sufficient CFTR to CF HAE to restore mucus transport, thus correcting the CF phenotype. PIVCFTR delivered CFTR to >60% of airway surface epithelial cells and expressed CFTR protein in CF HAE approximately 100-fold over endogenous levels in non-CF HAE. This efficiency of CFTR delivery fully corrected the basic bioelectric defects of Cl(-) and Na(+) epithelial ion transport and restored ASL volume regulation and mucus transport to levels approaching those of non-CF HAE. To determine the numbers of CF HAE surface epithelial cells required to express CFTR for restoration of mucus transport to normal levels, different amounts of PIVCFTR were used to express CFTR in 3%-65% of the surface epithelial cells of CF HAE and correlated to increasing ASL volumes and mucus transport rates. These data demonstrate for the first time, to our knowledge, that restoration of normal mucus transport rates in CF HAE was achieved after CFTR delivery to 25% of surface epithelial cells. In vivo experimentation in appropriate models will be required to determine what level of mucus transport will afford clinical benefit to CF patients, but we predict that a future goal for corrective gene transfer to the CF human airways in vivo would attempt to target at least 25% of surface epithelial cells to achieve mucus transport rates comparable to those in non-CF airways., Competing Interests: The authors have declared that no competing interests exist.

In the airways, adenine nucleotides support a complex signaling network mediating host defenses. Released by the epithelium into the airway surface liquid (ASL) layer, they regulate mucus clearance through P2 (ATP) receptors, and following surface metabolism through P1 (adenosine; Ado) receptors. The complexity of ASL nucleotide regulation provides an ideal subject for biochemical network modeling. A mathematical model was developed to integrate nucleotide release, the ectoenzymes supporting the dephosphorylation of ATP into Ado, Ado deamination into inosine (Ino), and nucleoside uptake. The model also includes ecto-adenylate kinase activity and feed-forward inhibition of Ado production by ATP and ADP. The parameters were optimized by fitting the model to experimental data for the steady-state and transient concentration profiles generated by adding ATP to polarized primary cultures of human bronchial epithelial (HBE) cells. The model captures major aspects of ATP and Ado regulation, including their >4-fold increase in concentration induced by mechanical stress mimicking normal breathing. The model also confirmed the independence of steady-state nucleotide concentrations on the ASL volume, an important regulator of airway clearance. An interactive approach between simulations and assays revealed that feed-forward inhibition is mediated by selective inhibition of ecto-5'-nucleotidase. Importantly, the model identifies ecto-adenylate kinase as a key regulator of ASL ATP and proposes novel strategies for the treatment of airway diseases characterized by impaired nucleotide-mediated clearance. These new insights into the biochemical processes supporting ASL nucleotide regulation illustrate the potential of this mathematical model for fundamental and clinical research.

In cystic fibrosis (CF), the absence of functional CFTR leads to dysregulated Na(+) absorption across airway epithelia. We established an in vitro model of dysregulated Na(+) absorption by treating polarized normal human bronchial epithelial cells (HBEs) with nystatin (Nys), a polyene antibiotic that enables monovalent cations to permeate biological membranes. Acute mucosal Nys produced a rapid increase in short circuit current (I(sc)) that reflected increased transepithelial Na(+) absorption and required Na(+)/K(+)ATPase activity. The acute increase in I(sc) was associated with increased mucosal liquid absorption. Prolonged mucosal Nys treatment resulted in sustained Na(+) hyperabsorption, associated with increased mucosal liquid absorption in comparison with naïve (nontreated, kept under air-liquid interface conditions) or vehicle-treated cultures. Nys treatment was not toxic. Increased lactate accumulation in Nys-treated culture media suggested a higher metabolic rate associated with the higher energy demand for Na(+) transport. After chronic Nys treatment, the increased I(sc) was rapidly lost when the cultures were mounted in Ussing chambers, indicating that Nys could be rapidly removed from the apical membrane. Importantly, chronic Nys treatment promoted sustained mucosal liquid depletion and caused mucus dehydration, compaction, and adhesion to the apical surface of Nys-treated cultures. We conclude that mucosal Nys treatment of HBEs provides a simple in vitro model to recapitulate the Na(+) and volume hyperabsorptive features of CF airway epithelia.

Adenosine is a multifaceted signaling molecule mediating key aspects of innate and immune lung defenses. However, abnormally high airway adenosine levels exacerbate inflammatory lung diseases. This study identifies the mechanisms regulating adenosine elimination from the apical surface of human airway epithelia. Experiments conducted on polarized primary cultures of nasal and bronchial epithelial cells showed that extracellular adenosine is eliminated by surface metabolism and cellular uptake. The conversion of adenosine to inosine was completely inhibited by the adenosine deaminase 1 (ADA1) inhibitor erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA). The reaction exhibited Km and Vmax values of 24 microM and 0.14 nmol x min(-1) x cm(-2). ADA1 (not ADA2) mRNA was detected in human airway epithelia. The adenosine/mannitol permeability coefficient ratio (18/1) indicated a minor contribution of paracellular absorption. Adenosine uptake was Na+-dependent and was inhibited by the concentrative nucleoside transporter (CNT) blocker phloridzin but not by the equilibrative nucleoside transporter (ENT) blocker dipyridamole. Apparent Km and Vmax values were 17 microM and 7.2 nmol x min(-1) x cm(-2), and transport selectivity was adenosine = inosine = uridine > guanosine = cytidine > thymidine. CNT3 mRNA was detected throughout the airways, while CNT2 was restricted to nasal epithelia. Inhibition of adenosine elimination by EHNA or phloridzin raised apical adenosine levels by >3-fold and stimulated IL-13 and MCP-1 secretion by 6-fold. These responses were reproduced by the adenosine receptor agonist 5'-(N-ethylcarboxamido)adenosine (NECA) and blocked by the adenosine receptor antagonist, 8-(p-sulfophenyl) theophylline (8-SPT). This study shows that adenosine elimination on human airway epithelia is mediated by ADA1, CNT2, and CNT3, which constitute important regulators of adenosine-mediated inflammation.

Extracellular ATP and its metabolite adenosine regulate mucociliary clearance in airway epithelia. Little has been known, however, regarding the actual ATP and adenosine concentrations in the thin ( approximately 7 microm) liquid layer lining native airway surfaces and the link between ATP release/metabolism and autocrine/paracrine regulation of epithelial function. In this study, chimeric Staphylococcus aureus protein A-luciferase (SPA-luc) was bound to endogenous antigens on primary human bronchial epithelial (HBE) cell surface and ATP concentrations assessed in real-time in the thin airway surface liquid (ASL). ATP concentrations on resting cells were 1-10 nm. Inhibition of ecto-nucleotidases resulted in ATP accumulation at a rate of approximately 250 fmol/min/cm2, reflecting the basal ATP release rate. Following hypotonic challenge to promote cell swelling, cell-surface ATP concentration measured by SPA-luc transiently reached approximately 1 microm independent of ASL volume, reflecting a transient 3-log increase in ATP release rates. In contrast, peak ATP concentrations measured in bulk ASL by soluble luciferase inversely correlated with volume. ATP release rates were intracellular calcium-independent, suggesting that non-exocytotic ATP release from ciliated cells, which dominate our cultures, mediated hypotonicity-induced nucleotide release. However, the cystic fibrosis transmembrane conductance regulator (CFTR) did not participate in this function. Following the acute swelling phase, HBE cells exhibited regulatory volume decrease which was impaired by apyrase and facilitated by ATP or UTP. Our data provide the first evidence that ATP concentrations at the airway epithelial surface reach the range for P2Y2 receptor activation by physiological stimuli and identify a role for mucosal ATP release in airway epithelial cell volume regulation.

Inefficient adenoviral vector (AdV)-mediated gene transfer to the ciliated respiratory epithelium has hindered gene transfer strategies for the treatment of cystic fibrosis lung disease. In part, the inefficiency is due to an absence of the coxsackie B and adenovirus type 2 and 5 receptor (CAR) from the apical membranes of polarized epithelia. In this study, using an in vitro model of human ciliated airway epithelium, we show that providing a glycosylphosphatidylinositol (GPI)-linked AdV receptor (GPI-CAR) at the apical surface did not significantly improve AdV gene transfer efficiency because the lumenal surface glycocalyx limited the access of AdV to apical GPI-CAR. The highly glycosylated tethered mucins were considered to be significant glycocalyx components that restricted AdV access because proteolytic digestion and inhibitors of O-linked glycosylation enhanced AdV gene transfer. To determine whether these in vitro observations are relevant to the in vivo situation, we generated transgenic mice expressing GPI-CAR at the surface of the airway epithelium, crossbred these mice with mice that were genetically devoid of tethered mucin type 1 (Muc1), and tested the efficiency of gene transfer to murine airways expressing apical GPI-human CAR (GPI-hCAR) in the presence and absence of Muc1. We determined that AdV gene transfer to the murine airway epithelium was inefficient even in GPI-hCAR transgenic mice but that the gene transfer efficiency improved in the absence of Muc1. However, the inability to achieve a high gene transfer efficiency, even in mice with a deletion of Muc1, suggested that other glycocalyx components, possibly other tethered mucin types, also provide a significant barrier to AdV interacting with the airway lumenal surface.

Extracellular nucleotides are among the most potent mediators of mucociliary clearance (MCC) in human lungs. However, clinical trials revealed that aerosolized nucleotides provide only a transient improvement of MCC to patients diagnosed with cystic fibrosis (CF). In this study, we identified the mechanism that eliminates extracellular nucleotides from human airways. Polarized primary cultures of human bronchial epithelial cells were impermeable to extracellular nucleotides but rapidly dephosphorylated ATP into ADP, AMP, and adenosine. The half-life of a therapeutic ATP concentration (0.1 mm) was approximately 20 s within the periciliary liquid layer. The mucosal epithelial surface eliminated P2 receptor agonists (ATP = UTP > ADP > UDP) at 3-fold higher rates than the serosal surface. We also showed that mucosal (not serosal) ectoATPase activity increases toward areas most susceptible to airway obstruction (nose < bronchi << bronchioles). Bronchial cultures from patients with CF, primary ciliary dyskinesia, or alpha1-antitrypsin deficiency exhibited 3-fold higher mucosal (not serosal) ectoATPase activity than normal cultures. Time course experiments indicated that CF enhances ATP elimination and adenosine accumulation on the mucosal surface. Furthermore, nonspecific alkaline phosphatase was identified as the major regulator of airway nucleotide concentrations in CF, primary ciliary dyskinesia, and alpha1-antitrypsin deficiency. The ectoAT-Pase activity and mRNA expression of mucosally restricted nonspecific alkaline phosphatase were 3-fold higher on bronchial cultures from these patients than from healthy subjects. This study demonstrates that the duration of nucleotide-mediated MCC is limited by epithelial ectonucleotidases throughout human airways, with the efficiency of this mechanism enhanced in chronic inflammatory lung diseases, including CF.

Epithelial tight junctions (TJs) provide an important route for passive electrolyte transport across airway epithelium and provide a barrier to the migration of toxic materials from the lumen to the interstitium. The possibility that TJ function may be perturbed by airway inflammation originated from studies reporting (1) increased levels of the proinflammatory cytokines interleukin-8 (IL-8), tumor necrosis factor alpha (TNF-alpha), interferon gamma (IFN-gamma), and IL-1beta in airway epithelia and secretions from cystic fibrosis (CF) patients and (2) abnormal TJ strands of CF airways as revealed by freeze-fracture electron microscopy. We measured the effects of cytokine exposure of CF and non-CF well-differentiated primary human airway epithelial cells on TJ properties, including transepithelial resistance, paracellular permeability to hydrophilic solutes, and the TJ proteins occludin, claudin-1, claudin-4, junctional adhesion molecule, and ZO-1. We found that whereas IL-1beta treatment led to alterations in TJ ion selectivity, combined treatment of TNF-alpha and IFN-gamma induced profound effects on TJ barrier function, which could be blocked by inhibitors of protein kinase C. CF bronchi in vivo exhibited the same pattern of expression of TJ-associated proteins as cultures exposed in vitro to prolonged exposure to TNF-alpha and IFN-gamma. These data indicate that the TJ of airway epithelia exposed to chronic inflammation may exhibit parallel changes in the barrier function to both solutes and ions.

5'-Nucleoside triphosphates (NTP) are present in the liquid covering airway surfaces and mediate important physiologic events through their interaction with P2-nucleotide receptors. Activation of airway P2Y(2) receptors, for example, stimulates ciliary beat frequency, chloride/liquid secretion, and goblet cell degranulation. We, therefore, have studied the metabolic pathways that regulate the concentration of nucleotides on airway surfaces. Stimulation of submucosal gland secretion in the nose was previously found to decrease the concentration of 5'-adenosine triphosphate (ATP) in nasal lavage samples due to the presence of a secreted 5'-nucleoside triphosphatase (NTPase). In this study, gland secretions were further studied and found to also contain adenylate kinase (AK) and nucleoside diphosphokinase (NDPK) activities. Ecto-AK and ecto-NDPK activities were also detected in well-differentiated cultures of superficial nasal epithelia, which reflected a combination of cell-associated and released (into culture media) AK and NDPK activities. This study demonstrates that "ecto-kinases" on airway surfaces (1) emanate from different enzyme families, including both AK and NDPK; (2) are expressed at superficial epithelial surfaces and in submucosal glands; and (3) may be important regulators of nucleotide concentrations on airway surfaces.

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. [ABSTRACT FROM AUTHOR]

Purinergic regulation of airway innate defence activities is in part achieved by the release of nucleotides from epithelial cells. However, the mechanisms of airway epithelial nucleotide release are poorly understood. We have previously demonstrated that ATP is released from ionomycin-stimulated airway epithelial goblet cells coordinately with mucin exocytosis, suggesting that ATP is released as a co-cargo molecule from mucin-containing granules. We now demonstrate that protease-activated-receptor (PAR) agonists also stimulate the simultaneous release of mucins and ATP from airway epithelial cells. PAR-mediated mucin and ATP release were dependent on intracellular Ca2+ and actin cytoskeleton reorganization since BAPTA AM, cytochalasin D, and inhibitors of Rho and myosin light chain kinases blocked both responses. To test the hypothesis that ATP is co-released with mucin from mucin granules, we measured the nucleotide composition of isolated mucin granules purified based on their MUC5AC and VAMP-8 content by density gradients. Mucin granules contained ATP, but the levels of ADP and AMP within granules exceeded by nearly 10-fold that of ATP. Consistent with this finding, apical secretions from PAR-stimulated cells contained relatively high levels of ADP/AMP, which could not be accounted for solely based on ATP release and hydrolysis. Thus, mucin granules contribute to ATP release and also are a source of extracellular ADP and AMP. Direct release of ADP/AMP from mucin granules is likely to provide a major source of airway surface adenosine to signal in a paracrine faction ciliated cell A2b receptors to activate ion/water secretion and appropriately hydrate goblet cell-released mucins. [ABSTRACT FROM AUTHOR]

Dysfunction of CFTR in cystic fibrosis (CF) airway epithelium perturbs the normal regulation of ion transport, leading to a reduced volume of airway surface liquid (ASL), mucus dehydration, decreased mucus transport, and mucus plugging of the airways. CFTR is normally expressed in ciliated epithelial cells of the surface and submucosal gland ductal epithelium and submucosal gland acinar cells. Critical questions for the development of gene transfer strategies for CF airway disease are what airway regions require CFTR function and how many epithelial cells require CFTR expression to restore normal ASL volume regulation and mucus transport to CF airway epithelium? An in vitro model of human CF ciliated surface airway epithelium (CF HAE) was used to test whether a human parainfluenza virus (PIV) vector engineered to express CFTR (PIVCFTR) could deliver sufficient CFTR to CF HAE to restore mucus transport, thus correcting the CF phenotype. PIVCFTR delivered CFTR to >60% of airway surface epithelial cells and expressed CFTR protein in CF HAE approximately 100-fold over endogenous levels in non-CF HAE. This efficiency of CFTR delivery fully corrected the basic bioelectric defects of Cl- and Na+ epithelial ion transport and restored ASL volume regulation and mucus transport to levels approaching those of non-CF HAE. To determine the numbers of CF HAE surface epithelial cells required to express CFTR for restoration of mucus transport to normal levels, different amounts of PIVCFTR were used to express CFTR in 3%-65% of the surface epithelial cells of CF HAE and correlated to increasing ASL volumes and mucus transport rates. These data demonstrate for the first time, to our knowledge, that restoration of normal mucus transport rates in CF HAE was achieved after CFTR delivery to 25% of surface epithelial cells. In vivo experimentation in appropriate models will be required to determine what level of mucus transport will afford clinical benefit to CF patients, but we predict that a future goal for corrective gene transfer to the CF human airways in vivo would attempt to target at least 25% of surface epithelial cells to achieve mucus transport rates comparable to those in non-CF airways. [ABSTRACT FROM AUTHOR]

Prolonged macrolide antibiotic therapy at low doses improves clinical outcome in patients affected with diffuse panbronchiolitis and cystic fibrosis. Consensus is building that the therapeutic effects are due to anti-inflammatory, rather than anti-microbial activities, but the mode of action is likely complex. To gain insights into how the macrolide azithromycin (AZT) modulates inflammatory responses in airways, well-differentiated primary cultures of human airway epithelia were exposed to AZT alone, an inflammatory stimulus consisting of soluble factors from cystic fibrosis airways, or AZT followed by the inflammatory stimulus. RNA microarrays were conducted to identify global and specific gene expression changes. Analysis of gene expression changes revealed that the AZT treatment alone altered the gene profile of the cells, primarily by significantly increasing the expression of lipid/cholesterol genes and decreasing the expression of cell cycle/mitosis genes. The increase in cholesterol biosynthetic genes was confirmed by increased filipin staining, an index of free cholesterol, after AZT treatment. AZT also affected genes with inflammatory annotations, but the effect was variable (both up- and down-regulation) and gene specific. AZT pretreatment prevented the up-regulation of some genes, such as MUC5AC and MMP9, triggered by the inflammatory stimulus, but the up-regulation of other inflammatory genes, e.g., cytokines and chemokines, such as interleukin-8, was not affected. On the other hand, HLA genes were increased by AZT. Notably, secreted IL-8 protein levels did not reflect mRNA levels, and were, in fact, higher after AZT pretreatment in cultures exposed to the inflammatory stimulus, suggesting that AZT can affect inflammatory pathways other than by altering gene expression. These findings suggest that the specific effects of AZT on inflamed and non-inflamed airway epithelia are likely relevant to its clinical activity, and their apparent complexity may help explain the diverse immunomodulatory roles of macrolides. [ABSTRACT FROM AUTHOR]

Cystic fibrosis (CF) transmembrane conductance regulator (CFTR)dependent airway epithelial bicarbonate transport is hypothesized to participate in airway surface liquid pH regulation and contribute to lung defense. We measured pH and ionic composition in apical surface liquid (ASL) on polarized normal (NL) and CF primary bronchial epithelial cell cultures under basal conditions, after cAMP stimulation, and after challenge with luminal acid loads. Under basel conditions, CF epithelia acidified ASL more rapidly than NL epithelia. Two ASL pH regulatory paths that contributed to basal pH were identified in the apical membrane of airway epithelia, and their activities were measured. We detected a ouabain-sensitive (nongastric) H[SUP+],K[SUP+]-ATPase that acidified ASL, but its activity was not different in NL and CF cultures. We also detected the following evidence for a CFTR-dependent HCO[SUP-][SUB3] secretory pathway that was defective in CF: (1) ASL [HCO[SUP-][SUB3]] was higher in NL than CF ASL; (ii) activating CFTR with forskolin β-isobutyl-l-methylxanthine alkalinized NL ASL but acidified CF ASL; and (iii) NL airway epithelia more rapidly and effectively alkalinized ASL in response to a luminal acid challenge than CF epithelia. We conclude that cultured human CF bronchial epithelial pHAsL is abnormally regulated under basal conditions because of absent CFTRdependent HCO[SUP-][SUB3] secretion and that this defect can lead to an impaired capacity to respond to airway conditions associated with acidification of ASL. [ABSTRACT FROM AUTHOR]

Investigates the effect of purinergic receptor agonists on arachidonic acid release in (3H)arachidonic acid-prelabeled human airway epithelial cells. Uptake of (3H)arachidonic acid; Effect of adenosine triphosphate on the accumulation of (3H)arachidonic acid, (3H)diacylglycerol and (3H)phosphatidic acid; Specificity of nucleotides.

ATP released from airway epithelial cells promotes purinergic receptor-regulated mucociliary clearance activities necessary for innate lung defense. Cell swelling-induced membrane stretch/strain is a common stimulus that promotes airway epithelial ATP release, but the mechanisms transducing cell swelling into ATP release are incompletely understood. Using knockdown and knockout approaches, we tested the hypothesis that pannexin 1 mediates ATP release from hypotonically swollen airway epithelia and investigated mechanisms regulating this activity. Well differentiated primary cultures of human bronchial epithelial cells subjected to hypotonic challenge exhibited enhanced ATP release, which was paralleled by the uptake of the pannexin probe propidium iodide. Both responses were reduced by pannexin 1 inhibitors and by knocking down pannexin 1. Importantly, hypotonicity-evoked ATP release from freshly excised tracheas and dye uptake in primary tracheal epithelial cells were impaired in pannexin 1 knockout mice. Hypotonicity-promoted ATP release and dye uptake in primary well differentiated human bronchial epithelial cells was accompanied by RhoA activation and myosin light chain phosphorylation and was reduced by the RhoA dominant negative mutant RhoA(T19N) and Rho and myosin light chain kinase inhibitors. ATP release and Rho activation were reduced by highly selective inhibitors of transient receptor potential vanilloid 4 (TRPV4). Lastly, knocking down TRPV4 impaired hypotonicity-evoked airway epithelial ATP release. Our data suggest that TRPV4 and Rho transduce cell membrane stretch/strain into pannexin 1-mediated ATP release in airway epithelia. [ABSTRACT FROM AUTHOR]

A balance sheet describing the integrated homeostasis of secretion, absorption, and surface movement of liquids on pulmonary surfaces has remained elusive. It remains unclear whether the alveolus exhibits an intra-alveolar ion/liquid transport physiology or whether it secretes ions/liquid that may communicate with airway surfaces. Studies employing isolated human alveolar type II (AT2) cells were utilized to investigate this question. Human AT2 cells exhibited both epithelial Na+ channel-mediated Na+ absorption and cystic fibrosis transmembrane conductance regulator-mediated Cl- secretion, both significantly regulated by extracellular nucleotides. In addition, we observed in normal AT2 cells an absence of cystic fibrosis transmembrane conductance regulator regulation of epithelial Na+ channel activity and an absence of expression/activity of reported calcium-activated chloride channels (TMEM16A, Bestrophin-1, ClC2, and SLC26A9), both features strikingly different from normal airway epithelial cells. Measurements of alveolar surface liquid volume revealed that normal AT2 cells: 1) achieved an extracellular nucleotide concentration-dependent steady state alveolar surface liquid height of ~4 μm in vitro; 2) absorbed liquid when the lumen was flooded; and 3) secreted liquid when treated with UTP or forskolin or subjected to cyclic compressive stresses mimicking tidal breathing. Collectively, our studies suggest that human AT2 cells in vitro have the capacity to absorb or secrete liquid in response to local alveolar conditions. [ABSTRACT FROM AUTHOR]

A key aspect of the lung's innate defense system is the ability of the superficial epithelium to regulate airway surface liquid (ASL) volume to maintain a 7-µm periciliary liquid layer (PCL), which is required for cilia to beat and produce mucus flow. The mechanisms whereby airway epithelia regulate ASL height to ≥ 7 µm are poorly understood. Using bumetanide as an inhibitor of Cl- secretion, and nystatin as an activator of Na+ absorption, we found that a coordinated "blending" of both Cl- secretion and Na+ absorption must occur to effect ASL volume homeostasis. We then investigated how ASL volume status is regulated by the underlying epithelia. Cilia were not critical to this process as (a) ASL volume was normal in cultures from patients with primary ciliary dyskinesia with immotile cilia, and (b) in normal cultures that had not yet undergone ciliogenesis. However, we found that maneuvers that mimic deposition of excess ASL onto the proximal airways, which occurs during mucociliary clearance and after glandular secretion, acutely stimulated Na+ absorption, suggesting that volume regulation was sensitive to changes in concentrations of soluble mediators in the ASL rather than alterations in ciliary beating. To investigate this hypothesis further, we added potential "soluble mediators" to the ASL. ASL volume regulation was sensitive to a channel-activating protein (CAP; trypsin) and a CAP inhibitor (aprotinin), which regulated Na+ absorption via changes in epithelial Na+ channel (ENaC) activity in both normal and cystic fibrosis cultures. ATP was also found to acutely regulate ASL volume by inducing secretion in normal and cystic fibrosis (CF) cultures, while its metabolite adenosine (ADO) evoked secretion in normal cultures but stimulated absorption in CF cultures. Interestingly, the amount of ASL/Cl- secretion elicited by ATP/ADO was influenced by the level of CAP-induced Na+ absorption, suggesting that there are important interactions between the soluble regulators which finely tune ASL volume. [ABSTRACT FROM AUTHOR]

Mucoid, mucA mutant Pseudomonas aeruginosa cause chronic lung infections in cystic fibrosis (CF) patients and are refractory to phagocytosis and antibiotics. Here we show that mucoid bacteria perish during anaerobic exposure to 15 mM nitrite (NO2) at pH 6.5, which mimics CF airway mucus. Killing required a pH lower than 7, implicating formation of nitrous acid (HNO2) and NO, that adds NO equivalents to cellular molecules. Eighty-seven percent of CF isolates possessed mucA mutations and were killed by HNO2 (3-log reduction in 4 days). Furthermore, antibiotic-resistant strains determined were also equally sensitive to HNO2. More importantly, HNO2 killed mucoid bacteria (a) in anaerobic biofilms; (b) in vitro in ultrasupernatants of airway secretions derived from explanted CF patient lungs; and (c) in mouse lungs in vivo in a pH-dependent fashion, with no organisms remaining after daily exposure to HNO2 for 16 days. HNO2 at these levels of acidity and NO2 also had no adverse effects on cultured human airway epithelia in vitro. In summary, selective killing by HNO2 may provide novel insights into the important clinical goal of eradicating mucoid P. aeruginosa from the CF airways. [ABSTRACT FROM AUTHOR]

Background: Cystic fibrosis is a monogenic disease that deranges multiple systems of ion transport in the airways, culminating in chronic infection and destruction of the lung. The introduction of a normal copy of the cystic fibrosis transmembrane conductance regulator (CFTR) gene into the airway epithelium through gene transfer is an attractive approach to correcting the underlying defects in patients with cystic fibrosis. We tested the feasibility of gene therapy using adenoviral vectors in the nasal epithelium of such patients. Methods: An adenoviral vector containing the normal CFTR complementary DNA in four logarithmically increasing doses (estimated multiplicity of infection, 1, 10, 100, and 1000), or vehicle alone, was administered in a randomized, blinded fashion to the nasal epithelium of 12 patients with cystic fibrosis. Gene transfer was quantitated by molecular techniques that detected the expression of CFTR messenger RNA and by functional measurements of transepithelial potential differences (PDs) to assess abnormalities of ion transport specific to cystic fibrosis. The safety of this treatment was monitored by nasal lavage and biopsy to assess inflammation and vector replication. Results: The adenoviral vector was detected in nasal-lavage fluid by culture, the polymerase chain reaction (PCR), or both in a dose-dependent fashion for up to eight days after vector administration. There was molecular evidence of gene transfer by reverse-transcriptase PCR assays or in situ hybridization in five of six patients treated at the two highest doses. However, the percentage of epithelial cells transfected by the vector was very low (<1 percent), and measurement of PD across the epithelium revealed no significant restoration of chloride transport or normalization of sodium transport. At the lower doses of vector, there were no toxic effects. However, at the highest dose there was mucosal inflammation in two of three patients. Conclusions: In patients with cystic fibrosis, adenoviral-vector–mediated transfer of the CFTR gene did not correct functional defects in nasal epithelium, and local inflammatory responses limited the dose of adenovirus that could be administered to overcome the inefficiency of gene transfer. (N Engl J Med 1995;333:823-31.) [ABSTRACT FROM AUTHOR]