Airway Mucociliary Clearance Research Articles

Linking increased airway hydration, ciliary beating, and mucociliary clearance through ENaC inhibition.

Airway dehydration causes mucus stasis and bacterial overgrowth in cystic fibrosis and chronic bronchitis (CB). Rehydration by hypertonic saline is efficacious but suffers from a short duration of action. We tested whether epithelial sodium channel (ENaC) inhibition would rehydrate normal and dehydrated airways to increase mucociliary clearance (MCC) over a significant time frame. For this, we used a tool compound (Compound A), which displays nanomolar ENaC affinity and retention in the airway surface liquid (ASL). Using normal human bronchial epithelial cultures (HBECs) grown at an air-liquid interface, we evaluated in vitro potency and efficacy using short-circuit current (I(sc)) and ASL height measurements where it inhibited I(sc) and increased ASL height by ∼ 50% (0.052 μM at 6 h), respectively. The in vivo efficacy was investigated in a modified guinea pig tracheal potential difference model, where we observed an effective dose (ED50) of 5 μg/kg (i.t.), and by MCC measures in rats and sheep, where we demonstrated max clearance rates at 100 μg/kg (i.t.) and 75 μg/kg (i.t.), respectively. Acute cigarette smoke-induced ASL height depletion in HBECs was used to mimic the situation in patients with CB, and pretreatment prevented both cigarette smoke-induced ASL dehydration and lessened the decrease in ciliary beat frequency. Furthermore, when added after cigarette smoke exposure, Compound A increased the rate of ASL rehydration. In conclusion, Compound A demonstrated significant effects and a link between increased airway hydration, ciliary function, and MCC. These data support the hypothesis that ENaC inhibition may be efficacious in the restoration of mucus hydration and transport in patients with CB.

Inhibition of protein phosphatase 1 reverses alcohol induced ciliary dysfunction

Airway mucociliary clearance is a first-line defense of the lung against inhaled particles and debris. Among individuals with alcohol use disorders, there is an increase in lung diseases. We previously identified that prolonged alcohol exposure impairs mucociliary clearance, known as alcohol-induced ciliary dysfunction (AICD). Cilia-localized enzymes, known as the ciliary metabolon, are key to the pathogenesis of AICD. In AICD, cyclic nucleotide-dependent ciliary kinases, which modulate phosphorylation to regulate cilia beat, are desensitized. We hypothesized that alcohol activates cilia-associated protein phosphatase 1 (PP1) activity, driving phosphorylation changes of cilia motility regulatory proteins. To test this hypothesis we identified the effects of prolonged alcohol exposure on phosphatase activity, cilia beat, and kinase responsiveness and cilia-associated phosphorylation targets when stimulated by β-agonist or cAMP. Prolonged alcohol activated PP1 and blocked cAMP-dependent cilia beat and protein kinase A (PKA) responsiveness and phosphorylation of a 29-kDa substrate of PKA. Importantly, prolonged alcohol-induced phosphatase activation was inhibited by the PP1 specific inhibitor, inhibitor-2 (I-2), restoring cAMP-stimulated cilia beat and PKA responsiveness and phosphorylation of the 29-kDa substrate. The I-2 inhibitory effect persisted in tissue, cell, and isolated cilia-organelle models, highlighting the association of ciliary metabolon-localized enzymes to AICD. Prolonged alcohol exposure drives ciliary metabolon-localized PP1 activation. PP1 activation modifies phosphorylation of a 29-kDa protein related to PKA activity. These data reinforce our previous findings that alcohol is acting at the level of the ciliary metabolon to cause ciliary dysfunction and identifies PP1 as a therapeutic target to prevent or reverse AICD.

Serum and Glucocorticoid-Inducible Kinase1 Increases Plasma Membrane wt-CFTR in Human Airway Epithelial Cells by Inhibiting Its Endocytic Retrieval

BackgroundChloride (Cl) secretion by the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) located in the apical membrane of respiratory epithelial cells plays a critical role in maintenance of the airway surface liquid and mucociliary clearance of pathogens. Previously, we and others have shown that the serum and glucocorticoid-inducible kinase-1 (SGK1) increases wild type CFTR (wt-CFTR) mediated Cl transport in Xenopus oocytes by increasing the amount of wt-CFTR protein in the plasma membrane. However, the effect of SGK1 on the membrane abundance of wt-CFTR in airway epithelial cells has not been examined, and the mechanism whereby SGK1 increases membrane wt-CFTR has also not been examined. Thus, the goal of this study was to elucidate the mechanism whereby SGK1 regulates the membrane abundance of wt-CFTR in human airway epithelial cells.Methods and ResultsWe report that elevated levels of SGK1, induced by dexamethasone, increase plasma membrane abundance of wt-CFTR. Reduction of SGK1 expression by siRNA (siSGK1) and inhibition of SGK1 activity by the SGK inhibitor GSK 650394 abrogated the ability of dexamethasone to increase plasma membrane wt-CFTR. Overexpression of a constitutively active SGK1 (SGK1-S422D) increased plasma membrane abundance of wt-CFTR. To understand the mechanism whereby SGK1 increased plasma membrane wt-CFTR, we examined the effects of siSGK1 and SGK1-S442D on the endocytic retrieval of wt-CFTR. While siSGK1 increased wt-CFTR endocytosis, SGK1-S442D inhibited CFTR endocytosis. Neither siSGK1 nor SGK1-S442D altered the recycling of endocytosed wt-CFTR back to the plasma membrane. By contrast, SGK1 increased the endocytosis of the epidermal growth factor receptor (EGFR).ConclusionThis study demonstrates for the first time that SGK1 selectively increases wt-CFTR in the plasma membrane of human airway epithelia cells by inhibiting its endocytic retrieval from the membrane.

P239 A role for polycystins in airway mucociliary clearance?

Polycystins 1 and 2 are the gene products of the mutated genes PKD1 and PKD2 in autosomal dominant polycystic kidney disease (ADPKD). They are localised to primary cilia in the kidney and thought to detect fluid flow. Recently patients with ADPKD have been shown to have an increased incidence of radiological findings of bronchiectasis, an abnormal dilatation of the bronchioles involving impaired mucociliary clearance (Driscoll et al 2009). We hypothesise that proteins from the polycystin pathway are present in respiratory epithelium and that this pathway is defective in bronchiectasis. We demonstrate using immunoflourescent antibodies that the extracellular portion of polycystin 1 and the n terminal tail of polycystin 2 consistently localise to cilia of nasal epithelial cells from healthy individuals and that blocking polycystin 1 with antibodies can alter ciliary beat. Contrary to our hypothesis there were no differences in the distribution of these proteins in a group of patients with bronchiectasis (n = 13) In conclusion we have shown that polycystins 1 and 2 are present in the motile cilia of the airways and may be involved in ciliary beat frequency regulation. We did not find any evidence of disruption of the polycystin proteins in a small population of patients with bronchiectasis.

RGS21, a regulator of taste and mucociliary clearance?

Motile cilia of airway epithelial cells help to expel harmful inhaled material. Activation of bitterant-responsive G protein-coupled receptors (GPCRs) is believed to potentiate cilia beat frequency and mucociliary clearance. In this study, we investigated whether regulator of G protein signaling-21 (RGS21) has the potential to modulate signaling pathways connected to airway mucociliary clearance, given that RGS proteins modulate GPCR signaling by acting as GTPase-accelerating proteins (GAPs) for the Gα subunits of heterotrimeric G proteins. This is a pilot investigation to determine if RGS21, a potential tastant specific RGS gene, is expressed in sinonasal mucosa, and to determine its specific Gα substrate using in vitro biochemical assays with purified proteins. Rgs21 expression in sinonasal mucosa was determined using quantitative, real-time PCR and a transgenic mouse expressing RFP from the Rgs21 promoter. Rgs21 was cloned, over-expressed, and purified using multistep protein chromatography. Biochemical and biophysical assays were used to determine if RGS21 could bind and accelerate the hydrolysis of GTP on heterotrimeric Gα subunits. Rgs21 was expressed in sinonasal mucosa and lingual epithelium. Purified recombinant protein directly bound and accelerated GTP hydrolysis on Gα subunits. Rgs21 is expressed in sinonasal mucosa, is amenable to purification as a recombinant protein, and can bind to Gα(i/o/q) subunits. Furthermore, RGS21 can accelerate the hydrolysis rate of GTP on Gαi subunits. This provides evidence that RGS21 may be a negative regulator of bitterant responses. Future studies will be needed to determine the physiological role of this protein in mucociliary clearance.

Adverse Effects of Immunosuppressant Drugs upon Airway Epithelial Cell and Mucociliary Clearance: Implications for Lung Transplant Recipients

Optimal post-transplantation immunosuppression is critical to the survival of the graft and the patient after lung transplantation. Immunosuppressant agents target various aspects of the immune system to maximize graft tolerance while minimizing medication toxicities and side effects. The vast majority of patients receive maintenance immunosuppressive therapy consisting of a triple-drug regimen including a calcineurin inhibitor, a cell cycle inhibitor and a corticosteroid. Although these immunosuppressant drugs are frequently used after transplantation and to control inflammatory processes, limited data are available with regard to their effects on cells other than those from the immunological system. Notably, the airway epithelial cell is of interest because it may contribute to development of bronchiolitis obliterans through production of pro-inflammatory cytokines. This review focuses the current armamentarium of immunosuppressant drugs used after lung transplantation and their main side effects upon airway epithelial cells and mucociliary clearance.

Immunosuppression Effects on Airway Mucociliary Clearance: Comparison Between Two Triple Therapies

Tacrolimus and mycophenolate have now become the most widely used combination for maintenance immunosuppressive regimens after lung transplantation in comparison with cyclosporine and azathioprine. However, limited information is available with respect to their effects on cells, other than those from the immunologic compartment. We hypothesized that different triple therapies could have different effects on airway mucociliary clearance, playing an important role in respiratory infections observed after lung transplantation. Ninety rats were assigned to three groups (n = 30 each): control = vehicle, therapy 1 = tacrolimus + mycophenolate + prednisone, and therapy 2 = cyclosporine + azathioprine + prednisone. After 7, 15, or 30 days of treatment by gavage, the animals were killed and the following parameters were studied: mucus transportability, ciliary beating frequency, mucociliary transport velocity, and neutral and acid mucus production. There was a significant decrease in ciliary beating frequency, mucociliary transport velocity, and neutral mucus production in all immunosuppressed animals; indeed, both therapies, mainly therapy 1, caused an increase in acid mucus production for as long as 15 days of treatment. Both triple therapies impaired airway mucociliary clearance of rats, but therapy 1 had a more deleterious effect. These data suggest that these undesirable effects can contribute to the high incidence of respiratory infections observed in patients undergoing lung transplantation.

Short Palate, Lung, and Nasal Epithelial Clone–1 Is a Tightly Regulated Airway Sensor in Innate and Adaptive Immunity

Short palate, lung, and nasal epithelial clone-1 (SPLUNC1) is a protein abundantly expressed by the respiratory epithelium of the proximal lower respiratory tract, a site of great environmental exposure. Previous studies showed that SPLUNC1 exerts antimicrobial effects, regulates airway surface liquid and mucociliary clearance, and suppresses allergic airway inflammation. We studied SPLUNC1 to gain insights into its role in host defense. In the lower respiratory tract, concentrations of SPLUNC1 are high under basal conditions. In models of pneumonia caused by common respiratory pathogens, and in Th1-induced and Th2-induced airway inflammation, SPLUNC1 secretion is markedly reduced. Pathogen-associated molecular patterns and IFN-γ act directly on airway epithelial cells to inhibit SPLUNC1 mRNA expression. Thus, SPLUNC1 is quickly suppressed during infection, in response to an insult on the epithelial surface. These experiments highlight the finely tuned fluctuations of SPLUNC1 in response to exposures in the respiratory tract, and suggest that the loss of SPLUNC1 is a crucial feature of host defense across air-breathing animal species.

Sperm-associated antigen-17 gene is essential for motile cilia function and neonatal survival.

Primary ciliary dyskinesia (PCD), resulting from defects in cilia assembly or motility, is caused by mutations in a number of genes encoding axonemal proteins. PCD phenotypes are variable, and include recurrent respiratory tract infections, bronchiectasis, hydrocephaly, situs inversus, and male infertility. We generated knockout mice for the sperm-associated antigen-17 (Spag17) gene, which encodes a central pair (CP) protein present in the axonemes of cells with "9 + 2" motile cilia or flagella. The targeting of Spag17 resulted in a severe phenotype characterized by immotile nasal and tracheal cilia, reduced clearance of nasal mucus, profound respiratory distress associated with lung fluid accumulation and disruption of the alveolar epithelium, cerebral ventricular expansion consistent with emerging hydrocephalus, failure to suckle, and neonatal demise within 12 hours of birth. Ultrastructural analysis revealed the loss of one CP microtubule in approximately one quarter of tracheal cilia axonemes, an absence of a C1 microtubule projection, and other less frequent CP structural abnormalities. SPAG6 and SPAG16 (CP proteins that interact with SPAG17) were increased in tracheal tissue from SPAG17-deficient mice. We conclude that Spag17 plays a critical role in the function and structure of motile cilia, and that neonatal lethality is likely explained by impaired airway mucociliary clearance.

SGK1 Increases Plasma Membrane CFTR in Human Airway Epithelial Cells by Inhibiting Its Endocytic Retrieval

Chloride secretion by CFTR located in the apical membrane of airway epithelial cells (AECs) plays a critical role in maintenance of the airway surface liquid and mucociliary clearance of respiratory pathogens and debris. Previously, others and we have shown that the serum and glucocorticoid‐inducible kinase1 (SGK1) increases the amount of wt‐CFTR protein in the plasma membrane of Xenopus oocytes by an unknown mechanism. The goal of this study was to elucidate the mechanism whereby SGK1 regulates the membrane abundance of CFTR in human AECs. We report that elevated levels of SGK1, induced by dexamethasone, increases plasma membrane abundance of CFTR. Reduction of SGK1 expression by siRNA (siSGK1) or inhibition of SGK1 activity by GSK‐650394 abrogated the ability of dexamethasone to increase plasma membrane CFTR. By contrast, overexpression of a constitutively active SGK1 (SGK1–422D) increased plasma membrane abundance of CFTR. To examine the mechanism whereby SGK1 increased plasma membrane CFTR the effects of siSGK1 and SGK1‐S442D on the endocytic retrieval of CFTR was examined. Whereas siSGK1 increased CFTR endocytosis, SGK1‐ S442D inhibited CFTR endocytosis. By contrast, neither siSGK1 nor SGK1‐S442D altered the recycling of endocytosed CFTR back to the plasma membrane. This work demonstrates that SGK1 increases CFTR in the plasma membrane of human airway epithelia by inhibiting its endocytic retrieval.

Tacrolimus Causes Airway Mucociliary Clearance Impairment

Purpose According to ISHLT Registry, the vast majority of lung transplant patients receive a tacrolimus-based maintenance immunosuppressant therapy. However, its side effects on respiratory tract are not very well studied. We hypothesized that tacrolimus could impair airway mucociliary clearance. Methods and Materials Twenty Wistar rats were assigned into two groups: Control (n=10): saline solution; and TAC (n=10): tacrolimus (1 mg/kg/day). After 15 days of therapy by gavage, animals were killed and in situ mucociliary transport velocity (MCTV) and ciliary beating frequency (CBF) were measured by microscopic direct view of airway ciliated epithelium. Mucus production by goblet cells was quantified in periodic acid Schiff and alcian blue stained slides of the tracheobronchial tissue. This study was support by Sao Paulo Research Foundation - Fapesp. Results There was a significant decrease in MCTV (1.77±0.50 and 0.73±0.34 mm/min, Control and TAC, respectively, p Conclusions Our study showed that TAC plays an important role on the impairment of the mucociliary clearance either by decreasing CBF and MCTV or by increasing mucus production. This impairment can be related with the high level of respiratory infection observed during the first 30 days after lung transplantation and must receive good attention from clinicians.

Abstract 19282: NAT10 Mutaion Causes Ciliary Aplisia and Congenital Heart Disease Associated with Heterotaxy

Introduction: We recently showed patients with CHD associated with heterotaxy can exhibit respiratory ciliary dysfunction (CD) similar to that of patients with primary ciliary dyskinesia (PCD) - a ciliopathy with airway mucociliary clearance defects. Exome sequencing analysis revealed known PCD causing mutation and rare coding variants in known PCD genes were enriched in heterotaxy patients with CD, suggesting a common etiology for heterotaxy and motile cilia dysfunction. Further interrogation for novel variants in non-PCD genes have uncovered one patient with a homozygous mutation in the acetyltransferase NAT10 . In this study, we investigated the hypothesis that NAT10 may play an essential role in ciliogenesis and is required for left-right patterning. Methods and Results: Exome sequencing was carried out in heterotaxy patients with ciliary dysfunction. Coding variants were filtered against dbSNP and 1000 genome databases. Novel homozygous coding variant in NAT10 (M638V) was identified in one heterotaxy patient with ciliary aplasia. This mutation is in a highly conserved residue of the HAT domain and is predicted to be deleterious. As NAT10 is known to acetylate tubulin, a key structural component of cilia, this seems likely to be disease causing. Using human hertRPE cells, we showed shRNA knockdown of NAT10 resulted in the inhibition of primary cilia formation. We further did NAT10 shRNA knockdown in ciliated human airway epithelial cells and showed complete repression of ciliogenesis. In contrast, reciliating airway epithelial cells treated with control shRNA formed normal motile cilia. Further analysis of zebrafish embryos after Nat10 morpholino injections showed laterality defects consistent with heterotaxy, confirming a requirement for NAT10 in left-right patterning. Conclusion: We identified a novel homozygous NAT10 mutation in heterotaxy patient with ciliary aplasia. We showed NAT10 is required for both formation of nonmotile primary cilia and motile cilia in the airway. Studies in zebrafish embryos further suggest NAT10 is required for motile cilia function regulating left-right patterning. Together these findings support the conclusion that NAT10 mutation can cause heterotaxy and ciliary aplasia phenotypes.

Effects of second hand smoke on airway secretion and mucociliary clearance

The airway acts as the first defense against inhaled pathogens and particulate matter from the environment. One major way for the airway to clear inhaled foreign objects is through mucociliary clearance (MCC), an important component of the respiratory innate immune defense against lung disease. MCC is characterized by the upward movement of mucus by ciliary motion that requires a balance between the volume and composition of the mucus, adequate periciliary liquid (PCL) volume, and normal ciliary beat frequency (CBF). Airway surface fluid (ASL) is a thin layer liquid that consists of the highly viscous mucus upper “gel” layer, and the watery lubricating lower “sol” layer. Mucus production, secretion and clearance are considered to play a critical role in maintenance of airway health because it maintains hydration in the airway and traps particulates, bacteria, and viruses. Different types of epithelial cells, including secretory cells, and ciliated cells, contribute to the MCC function. Cigarette smoke (CS) contains chemicals and particulates that significantly affect airway secretion. Active and passive CS-induced chronic obstructive pulmonary disease (COPD) is frequently associated with hyperplasia of goblet cells and submucosal glands (SMGs), thus increasing the secretory capacity of the airways that impairs MCC.

Abstract 16678: Multigenic Etiology of CHD With Heterotaxy Involving PCD and Cilia Genes

Introduction: We recently showed patients with CHD associated with heterotaxy can exhibit respiratory ciliary dysfunction (CD) similar to that of patients with primary ciliary dyskinesia (PCD) - a ciliopathy with airway mucociliary clearance defects. As both PCD and heterotaxy have a 1 in 10,000 incidence, the finding of CD in heterotaxy patients likely reflects the dual role of motile cilia in airway clearance and left-right patterning. Thus, we hypothesize heterotaxy may arise from mutations in PCD genes and other cilia related ciliome genes. Methods and Results: CHD patients with heterotaxy, 13 with CD and 13 without CD (no-CD), were sequence captured followed by SOLiD sequencing at 40X coverage to identify sequence variants in all 13 PCD genes and nearly 1000 other ciliome genes. Coding variants were filtered against dbSNP and the 1000 Genomes databases, and novel coding variants (NCV) were validated by Sanger sequencing. Analysis of the 13 PCD genes revealed 10 NCV in 7 CD patients, and 5 NCV in 3 no-CD patients. The 3 PCD patients had 5 NCV, with 2 siblings having homozygous CCDC39 mutation (1072delA) known to cause PCD. Interestingly, CD-heterotaxy patient 9002 was heterozygous for the most common pathogenic DNAI1 mutation, IVS1+2_3insT, a disease NCV not seen in the general population. As PCD is a recessive disorder, we evaluated NCVs found in DNAH6 , a motor dynein gene not yet demonstrated to cause PCD. DNAH6 NCVs were found in 9002 and one other CD and one PCD patient, but not in any no-CD patient. Dnah6 morpholino (MO) knockdown (KD) in zebrafish embryos showed laterality defects and phenotypes consistent with heterotaxy, suggesting a role in CD and heterotaxy. Conclusion: Our study showed heterotaxy patients with CD can have heterozygous NCVs in PCD genes, including known disease causing DNAI1 mutation. Functional analysis with zebrafish MO showed DNAH6 KD can cause heterotaxy. We propose DNAH6/DNAI1 double heterozygous mutation may contribute to CD and heterotaxy.

CFTR Modulation by the Tobacco Smoke Toxin Acrolein

Objective: Tobacco-smoke exposure inhibits transepithelial Cl-secretion—a major determinant of airway surface liquid hydration and mucociliary clearance (MCC). The objective of the current study was to evaluate the effects of acrolein exposure (a tobacco smoke toxin) on Cl-transport through the major apical anion channel CFTR in sinonasal epithelium. Method: Primary murine nasal septal (MNSE, wild type, and transgenic CFTR-/-) and human sinonasal epithelial (HSNE) cultures were exposed to acrolein in Ussing chambers, and effects on Cl-secretion were investigated using pharmacologic manipulation. Cellular cAMP signaling and cytotoxicity were also investigated. Results: Acrolein-stimulated Cl-secretion (ΔISC - change in short-circuit current in µA/cm2) at low concentrations (100 µM, 15.8 ± 2.2 vs 2.4 ± 0.8 (control); p-transport at 300 µM (13.3 ± 1.2 vs 19.9 ± 1.0; p-secretion was solely reliant upon the presence of CFTR (confirmed in transgenic CFTR -/- MNSE), but independent of cAMP signaling. Inhibition at higher concentrations was not secondary to cellular cytotoxicity, indicating direct effects on the CFTR. Conclusion: Decreased MCC is a major contributing feature to chronic rhinosinusitis. The present study demonstrated that acrolein has complex but direct interactions with CFTR. Robust inhibition of Cl-transport at higher concentrations indicates the potential contribution of this toxin to decreased MCC in individuals with chronic tobacco smoke exposure.

Inherent Differences in Nasal and Tracheal Ciliary Function in Response to Pseudomonas aeruginosa Challenge

Sinonasal mucosal biofilms are recognized as contributors to the pathogenesis of chronic rhinosinusitis (CRS). Attachment of bacteria to the sinonasal surface is an initial step in biofilm formation. A critical defense against this occurrence is mucociliary clearance (MCC). To ascertain whether the ciliary component of MCC is uniform throughout the airway we compared ciliary beat frequency (CBF) in the murine nasal septum and trachea at baseline and after challenge with Pseudomonas aeruginosa, a common pathogen of CRS. Murine septal and tracheal air-liquid interface cultures were evaluated for basal and stimulated CBF after exposure to control or conditioned media from Pseudomonas. Additionally, the attachment of Pseudomonas to nasal and tracheal cultures was assessed after pretreatment with control or conditioned media. Basal CBF is significantly slower in primary nasal airway cultures compared with tracheal airway cultures. Tracheal airway cultures show resistance to Pseudomonas secreted ciliotoxins not evident in nasal septal cultures. Furthermore, after challenge with viable Pseudomonas, significantly more bacteria attach to the nasal cultures compared with the tracheal cultures. Using primary murine nasal and tracheal airway cultures we show inherent differences in cilia function and increased susceptibility of the upper airway to attachment by Pseudomonas. Understanding the differences between upper and subglottic airway mucociliary clearance should lead to novel approaches in the management of upper airway infection.

TRPV4-Mediated Calcium Influx into Human Bronchial Epithelia upon Exposure to Diesel Exhaust Particles

BackgroundHuman respiratory epithelia function in airway mucociliary clearance and barrier function and have recently been implicated in sensory functions.ObjectiveWe investigated a link between chronic obstructive pulmonary disease (COPD) pathogenesis and molecular mechanisms underlying Ca2+ influx into human airway epithelia elicited by diesel exhaust particles (DEP).Methods and ResultsUsing primary cultures of human respiratory epithelial (HRE) cells, we determined that these cells possess proteolytic signaling machinery, whereby proteinase-activated receptor-2 (PAR-2) activates Ca2+-permeable TRPV4, which leads to activation of human respiratory disease–enhancing matrix metalloproteinase-1 (MMP-1), a signaling cascade initiated by diesel exhaust particles (DEP), a globally relevant air pollutant. Moreover, we observed ciliary expression of PAR-2, TRPV4, and phospholipase-Cβ3 in human airway epithelia and their DEP-enhanced protein–protein complex formation. We also found that the chronic obstructive pulmonary disease (COPD)–predisposing TRPV4P19S variant enhances Ca2+ influx and MMP 1 activation, providing mechanistic linkage between man-made air pollution and human airway disease.ConclusionDEP evoked protracted Ca2+ influx via TRPV4, enhanced by the COPD-predisposing human genetic polymorphism TRPV4P19S. This mechanism reprograms maladaptive inflammatory and extracellular-matrix–remodeling responses in human airways. The novel concept of air pollution–responsive ciliary signal transduction from PAR-2 to TRPV4 in human respiratory epithelia will accelerate rationally targeted therapies, possibly via the inhalatory route.

Allosteric Inhibition of the Epithelial Na+ Channel through Peptide Binding at Peripheral Finger and Thumb Domains

The epithelial Na(+) channel (ENaC) mediates the rate-limiting step in transepithelial Na(+) transport in the distal segments of the nephron and in the lung. ENaC subunits are cleaved by proteases, resulting in channel activation due to the release of inhibitory tracts. Peptides derived from these tracts inhibit channel activity. The mechanism by which these intrinsic inhibitory tracts reduce channel activity is unknown, as are the sites where these tracts interact with other residues within the channel. We performed site-directed mutagenesis in large portions of the predicted periphery of the extracellular region of the α subunit and measured the effect of mutations on an 8-residue inhibitory tract-derived peptide. Our data show that the inhibitory peptide likely binds to specific residues within the finger and thumb domains of ENaC. Pairwise interactions between the peptide and the channel were identified by double mutant cycle experiments. Our data suggest that the inhibitory peptide has a specific peptide orientation within its binding site. Extended to the intrinsic inhibitory tract, our data suggest that proteases activate ENaC by removing residues that bind at the finger-thumb domain interface.

Coupled Nucleotide and Mucin Hypersecretion from Goblet-Cell Metaplastic Human Airway Epithelium

Adenosine triphosphate (ATP) and its metabolite adenosine regulate airway mucociliary clearance via activation of purinoceptors. In this study, we investigated the contribution of goblet cells to airway epithelial ATP release. Primary human bronchial epithelial (HBE) cultures, typically dominated by ciliated cells, were induced to develop goblet cell metaplasia by infection with respiratory syncytial virus (RSV) or treatment with IL-13. Under resting conditions, goblet-cell metaplastic cultures displayed enhanced mucin secretion accompanied by increased rates of ATP release and mucosal surface adenosine accumulation as compared with nonmetaplastic control HBE cultures. Intracellular calcium chelation [1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetraacetoxymethyl ester] or disruption of the secretory pathways (nocodazole, brefeldin A, and N-ethylmaleimide) decreased mucin secretion and ATP release in goblet-cell metaplastic HBE cultures. Conversely, stimuli that triggered calcium-regulated mucin secretion (e.g., ionomycin or UTP) increased luminal ATP release and adenyl purine accumulation in control and goblet-cell metaplastic HBE cultures. Goblet cell-associated ATP release was not blocked by the connexin/pannexin hemichannel inhibitor carbenoxolone, suggesting direct nucleotide release from goblet cell vesicles rather than the hemichannel insertion. Collectively, our data demonstrate that nucleotide release is increased by goblet cell metaplasia, reflecting, at least in part, a mechanism tightly associated with goblet cell mucin secretion. Increased goblet cell nucleotide release and resultant adenosine accumulation provide compensatory mechanisms to hydrate mucins by paracrine stimulation of ciliated cell ion and water secretion and maintain mucociliary clearance, and to modulate inflammatory responses.

The effect and mechanism of action of carbocysteine on airway bacterial load in rats chronically exposed to cigarette smoke

Carbocysteine (S-carboxymethylcysteine) is a mucoactive drug with in vitro free radical scavenging and anti-inflammatory properties. Several clinical trials have indicated that carbocysteine reduces exacerbation rates in COPD. In the present study, the effect of carbocysteine on the airway load of Haemophilus influenzae was assessed in rats chronically exposed to cigarette smoke (CS). In addition, the effects of carbocysteine on airway mucus hypersecretion and mucociliary clearance (MCC) associated with the adherence and clearance of H. influenzae were investigated. Wistar rats were randomly divided into control, carbocysteine vehicle, CS exposure and carbocysteine treatment groups. After 12 weeks, rats were selected for quantitative inoculation of H. influenzae. BAL fluid and lungs were collected aseptically after 3 h for quantitative culture of H. influenzae. MCC was measured by quantifying the clearance of (99m)Tc-Sc. Goblet cell metaplasia and the presence of mucoid matter were evaluated by Alcian blue/periodic acid-Schiff staining. Mucin 5AC (Muc5AC) expression was detected by western blotting and real-time reverse transcription-PCR. Exposure to CS increased airway H. influenzae load, aggravated mucus hypersecretion and delayed MCC. Treatment with carbocysteine decreased airway H. influenzae load, and attenuated airway mucus hypersecretion, with improved MCC associated with adherence and clearance of H. influenzae. These results suggest that carbocysteine may be beneficial in patients with COPD by increasing the clearance of bacteria and decreasing bacterial load.