Identification of pendrin as a common mediator for mucus production in bronchial asthma and chronic obstructive pulmonary disease

Excessive production of airway mucus is a cardinal feature of bronchial asthma and chronic obstructive pulmonary disease (COPD) and contributes to morbidity and mortality in these diseases. IL-13, a Th2-type cytokine, is a central mediator in the pathogenesis of bronchial asthma, including mucus overproduction. Using a genome-wide search for genes induced in airway epithelial cells in response to IL-13, we identified pendrin encoded by the SLC26A4 (PDS) gene as a molecule responsible for airway mucus production. In both asthma and COPD mouse models, pendrin was up-regulated at the apical side of airway epithelial cells in association with mucus overproduction. Pendrin induced expression of MUC5AC, a major product of mucus in asthma and COPD, in airway epithelial cells. Finally, the enforced expression of pendrin in airway epithelial cells in vivo, using a Sendai virus vector, rapidly induced mucus overproduction in the lumens of the lungs together with neutrophilic infiltration in mice. These findings collectively suggest that pendrin can induce mucus production in airway epithelial cells and may be a therapeutic target candidate for bronchial asthma and COPD.     

CFTR Delivery to 25% of Surface Epithelial Cells Restores Normal Rates of Mucus Transport to Human Cystic Fibrosis Airway Epithelium

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.     

CFTR Delivery to 25% of Surface Epithelial Cells Restores Normal Rates of Mucus Transport to Human Cystic Fibrosis Airway Epithelium

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.     

IL-4 receptor signaling in Clara cells is required for allergen-induced mucus production

Excessive mucus production is an important pathological feature of asthma. The Th2 cytokines IL-4 and IL-13 have both been implicated in allergen-induced mucus production, inflammation, and airway hyperreactivity. Both of these cytokines use receptors that contain the IL-4Ralpha subunit, and these receptors are expressed on many cell types in the lung. It has been difficult to determine whether allergen-induced mucus production is strictly dependent on direct effects of IL-4 and IL-13 on epithelial cells or whether other independent mechanisms exist. To address this question, we used a cell type-specific inducible gene-targeting strategy to selectively disrupt the IL-4Ralpha gene in Clara cells, an airway epithelial cell population that gives rise to mucus-producing goblet cells. Clara cell-specific IL-4Ralpha-deficient mice and control mice developed similar elevations in serum IgE levels, airway inflammatory cell numbers, Th2 cytokine production, and airway reactivity following OVA sensitization and challenge. However, compared with control mice, Clara cell-specific IL-4Ralpha-deficient mice were nearly completely protected from allergen-induced mucus production. Because only IL-13 and IL-4 are thought to signal via IL-4Ralpha, we conclude that direct effects of IL-4 and/or IL-13 on Clara cells are required for allergen-induced mucus production in the airway epithelium.     

IL13 activates autophagy to regulate secretion in airway epithelial cells

Cytokine modulation of autophagy is increasingly recognized in disease pathogenesis, and current concepts suggest that type 1 cytokines activate autophagy, whereas type 2 cytokines are inhibitory. However, this paradigm derives primarily from studies of immune cells and is poorly characterized in tissue cells, including sentinel epithelial cells that regulate the immune response. In particular, the type 2 cytokine IL13 (interleukin 13) drives the formation of airway goblet cells that secrete excess mucus as a characteristic feature of airway disease, but whether this process is influenced by autophagy was undefined. Here we use a mouse model of airway disease in which IL33 (interleukin 33) stimulation leads to IL13-dependent formation of airway goblet cells as tracked by levels of mucin MUC5AC (mucin 5AC, oligomeric mucus/gel forming), and we show that these cells manifest a block in mucus secretion in autophagy gene Atg16l1-deficient mice compared to wild-type control mice. Similarly, primary-culture human tracheal epithelial cells treated with IL13 to stimulate mucus formation also exhibit a block in MUC5AC secretion in cells depleted of autophagy gene ATG5 (autophagy-related 5) or ATG14 (autophagy-related 14) compared to nondepleted control cells. Our findings indicate that autophagy is essential for airway mucus secretion in a type 2, IL13-dependent immune disease process and thereby provide a novel therapeutic strategy for attenuating airway obstruction in hypersecretory inflammatory diseases such as asthma, chronic obstructive pulmonary disease, and cystic fibrosis lung disease. Taken together, these observations suggest that the regulation of autophagy by Th2 cytokines is cell-context dependent.     

Effect of MMP/ADAM inhibitors on goblet cell hyperplasia in cultured human bronchial epithelial cells

While epidermal growth factor receptor (EGFR) plays a pivotal role in the repair process of epithelial cells, it is also involved in the overproduction of mucus and goblet cell hyperplasia (GCH), which occurs in chronic airway diseases such as asthma. Among the EGFR ligands, transforming growth factor (TGF)-alpha is thought to be the most important in the synthesis of mucus. Pro-TGF-alpha is cleaved to give an active form by members of the matrix metalloproteinases (MMP)/a disintegrin and metalloproteinases (ADAM) family. Thus MMP/ADAM inhibitors might prevent GCH by inhibiting transactivation of EGFR. Upon stimulation of differentiating normal human bronchial epithelial (NHBE) cells by IL-13, GCH was induced. The mucin genes MUC5AC, MUC5B, and MUC2 were upregulated whereas the expression of ciliated cell markers was greatly repressed. GM6001, a broad-spectrum inhibitor for MMP/ADAM, inhibited IL-13-induced mucin gene expression and mucus production as measured by periodic acid-Schiff staining. This was accompanied by an inhibition of TGF-alpha release. These results suggest that MMP/ADAMs play a pivotal role in the development of GCH in lung epithelial cells.     

Effect of MMP/ADAM Inhibitors on Goblet Cell Hyperplasia in Cultured Human Bronchial Epithelial Cells

While epidermal growth factor receptor (EGFR) plays a pivotal role in the repair process of epithelial cells, it is also involved in the overproduction of mucus and goblet cell hyperplasia (GCH), which occurs in chronic airway diseases such as asthma. Among the EGFR ligands, transforming growth factor (TGF)-α is thought to be the most important in the synthesis of mucus. Pro-TGF-α is cleaved to give an active form by members of the matrix metalloproteinases (MMP)/a disintegrin and metalloproteinases (ADAM) family. Thus MMP/ADAM inhibitors might prevent GCH by inhibiting transactivation of EGFR. Upon stimulation of differentiating normal human bronchial epithelial (NHBE) cells by IL-13, GCH was induced. The mucin genes MUC5AC, MUC5B, and MUC2 were upregulated whereas the expression of ciliated cell markers was greatly repressed. GM6001, a broad-spectrum inhibitor for MMP/ADAM, inhibited IL-13-induced mucin gene expression and mucus production as measured by periodic acid-Schiff staining. This was accompanied by an inhibition of TGF-α release. These results suggest that MMP/ADAMs play a pivotal role in the development of GCH in lung epithelial cells.     

Decreased colonic mucus in rats with loperamide-induced constipation

Constipation is a risk factor of colorectal cancer. Mucin is a major component of lumenal mucus, which protects the colorectal mucosa against mechanical and chemical damage. The aim of this study was to evaluate mucus production and to quantitate lumen mucus in a rat model of spastic constipation. We induced constipation with loperamide (1.5 mg/kg), and histochemically evaluated mucus production and the thickness of the mucus layer at the fecal surface. We quantitated the mucus attached to the mucosal surface using colonic perfusion with N-acetylcysteine. While more feces remained in the colon, there was less fecal excretion and lower fecal water content in loperamide-administered rats than in control rats. Crypt epithelial cells contained less mucus in constipated rats than in control rats. The mucus layer at the fecal surface was thinner and less mucus was recovered from the mucosal surface in constipated rats than in control rats. Mucus production of crypt epithelial cells and mucus at the fecal and mucosal surface were reduced by loperamide-induced constipation.     

miRNA-34b/c regulates mucus secretion in RSV-infected airway epithelial cells by targeting FGFR1

Respiratory syncytial virus (RSV) infection in airway epithelial cells is the main cause of bronchiolitis in children. Excessive mucus secretion is one of the primary symbols in RSV related lower respiratory tract infections (RSV-related LRTI). However, the pathological processes of mucus hypersecretion in RSV-infected airway epithelial cells remains unclear. The current study explores the involvement of miR-34b/miR-34c in mucus hypersecretion in RSV-infected airway epithelial cells by targeting FGFR1. First, miR-34b/miR-34c and FGFR1 mRNA were quantified by qPCR in throat swab samples and cell lines, respectively. Then, the luciferase reporters’ assay was designed to verify the direct binding between FGFR1 and miR-34b/miR-34c. Finally, the involvement of AP-1 signalling was assessed by western blot. This study identified that miR-34b/miR-34c was involved in c-Jun-regulated MUC5AC production by targeting FGFR1 in RSV-infected airway epithelial cells. These results provide some useful insights into the molecular mechanisms of mucus hypersecretion which may also bring new potential strategies to improve mucus hypersecretion in RSV disease.     

Otitis Media in Sperm-Associated Antigen 6 (Spag6)-Deficient Mice

Mammalian SPAG6 protein is localized to the axoneme central apparatus, and it is required for normal flagella and cilia motility. Recent studies demonstrated that the protein also regulates ciliogenesis and cilia polarity in the epithelial cells of brain ventricles and trachea. Motile cilia are also present in the epithelial cells of the middle ear and Eustachian tubes, where the ciliary system participates in the movement of serous fluid and mucus in the middle ear. Cilia defects are associated with otitis media (OM), presumably due to an inability to efficiently transport fluid, mucus and particles including microorganisms. We investigated the potential role of SPAG6 in the middle ear and Eustachian tubes by studying mice with a targeted mutation in the Spag6 gene. SPAG6 is expressed in the ciliated cells of middle ear epithelial cells. The orientation of the ciliary basal feet was random in the middle ear epithelial cells of Spag6-deficient mice, and there was an associated disrupted localization of the planar cell polarity (PCP) protein, FZD6. These features are associated with disordered cilia orientation, confirmed by scanning electron microscopy, which leads to uncoordinated cilia beating. The Spag6 mutant mice were also prone to develop OM. However, there were no significant differences in bacterial populations, epithelial goblet cell density, mucin expression and Eustachian tube angle between the mutant and wild-type mice, suggesting that OM was due to accumulation of fluid and mucus secondary to the ciliary dysfunction. Our studies demonstrate a role for Spag6 in the pathogenesis of OM in mice, possibly through its role in the regulation of cilia/basal body polarity through the PCP-dependent mechanisms in the middle ear and Eustachian tubes.     

Selective loss of sialic acid from rat small intestinal epithelial cells during postnatal development: demonstration with lectin-gold techniques

Post-embedding lectin-gold cytochemistry was employed to investigate the distribution of sialic acid and fucose residues in rat small intestinal epithelial cells during postnatal development. During the suckling phase (postnatal day 1) the apical and basolateral plasma membranes of epithelial cells, as well as the goblet cell mucus was intensely stained with the sialic acid-specific Sambucus nigra L. lectin I-gold complex (SNL I-g). By the weaning period (postnatal day 23), the entire villus contained both SNL I-g-positive and negative cells. In adult small intestine, the plasma membranes of all epithelial cells were unreactive with SNL I-g; however, abundant staining was detectable in goblet cell mucus, cells of the lamina propria, and smooth muscle cells. The distribution of fucose residues as detected with a Ulex europaeus lectin I-gold complex (UEL I-g) was virtually opposite that of sialic acid. At postnatal day 1, staining was restricted to goblet cell mucus, whereas by postnatal day 23, a portion of epithelial cells displayed UEL I-g binding sites along the apical and basolateral plasma membranes. In the adult, the apical and basolateral plasma membranes of all epithelial cells, as well as goblet cell mucus were stained with UEL I-g. These results support biochemical data demonstrating a shift from sialylation to fucosylation of intestinal microvillar glycoconjugates during the weaning phase of postnatal development. Moreover, the results indicate that rather than a general decrease in cellular sialylation, specific individual cells at all positions along the crypt-to-villus axis become devoid of sialic acid.     

Butyrate enemas upregulate Muc genes expression but decrease adherent mucus thickness in mice colon

Colonic mucosal protection is provided by the mucus gel, mainly composed of mucins. Several factors can modulate the formation and the secretion of mucins, and among them butyrate, an end-product of carbohydrate fermentation. However, the specific effect of butyrate on the various colonic mucins, and the consequences in terms of the mucus layer thickness are not known. Our aim was to determine whether butyrate modulates colonic MUC genes expression in vivo and whether this results in changes in mucus synthesis and mucus layer thickness. Mice received daily for 7 days rectal enemas of butyrate (100 mM) versus saline. We demonstrated that butyrate stimulated the gene expression of both secreted (Muc2) and membrane-linked (Muc1, Muc3, Muc4) mucins. Butyrate especially induced a 6-fold increase in Muc2 gene expression in proximal colon. However, butyrate enemas did not modify the number of epithelial cells containing the protein Muc2, and caused a 2-fold decrease in the thickness of adherent mucus layer. Further studies should help understanding whether this last phenomenon, i.e. the decrease in adherent mucus gel thickness, results in a diminished protective function or not.     

IL-4 induces mucin gene expression and goblet cell metaplasia in vitro and in vivo

Goblet cell metaplasia and mucus hypersecretion are important features in the pathogenesis of asthma. The cytokine IL-4 has been shown to play a role in animal models of asthma, where it induces Th2 lymphocyte differentiation and B lymphocyte IgE class switch. IL-4 has also been implicated in the differentiation of goblet cells via effects on lymphocytes and eosinophils. In this study we hypothesized that IL-4 induces airway epithelial cell mucin gene expression and mucous glycoconjugate production by direct action on these cells. In vitro, cultured airway epithelial cells (NCI-H292) expressed IL-4R constitutively, and IL-4 (10 ng/ml) induced MUC2 gene expression and mucous glycoconjugate production. In vivo, mouse airway epithelial cells expressed IL-4R constitutively, and IL-4 (250 ng) increased MUC5 gene expression and Alcian blue/periodic acid-Schiff-positive staining at 24 h; IL-4 did not increase inflammatory cell numbers in airway tissue or in bronchoalveolar lavage. TNF-alpha and IL-1beta levels in bronchoalveolar lavage were not increased in response to IL-4 instillation. These results indicate that airway epithelial cells express IL-4R constitutively and that IL-4 directly induces the differentiation of epithelium into mucous glycoconjugate-containing goblet cells.     

Efficient gene transfer to airway epithelium using recombinant Sendai virus

Clinical studies of gene therapy for cystic fibrosis (CF) suggest that the key problem is the efficiency of gene transfer to the airway epithelium. The availability of relevant vector receptors, the transient contact time between vector and epithelium, and the barrier function of airway mucus contribute significantly to this problem. We have recently developed recombinant Sendai virus (SeV) as a new gene transfer agent. Here we show that SeV produces efficient transfection throughout the respiratory tract of both mice and ferrets in vivo, as well as in freshly obtained human nasal epithelial cells in vitro. Gene transfer efficiency was several log orders greater than with cationic liposomes or adenovirus. Even very brief contact time was sufficient to produce this effect, and levels of expression were not significantly reduced by airway mucus. Our investigations suggest that SeV may provide a useful new vector for airway gene transfer.     

Autophagy is essential for ultrafine particle-induced inflammation and mucus hyperproduction in airway epithelium

Environmental ultrafine particulate matter (PM) is capable of inducing airway injury, while the detailed molecular mechanisms remain largely unclear. Here, we demonstrate pivotal roles of autophagy in regulation of inflammation and mucus hyperproduction induced by PM containing environmentally persistent free radicals in human bronchial epithelial (HBE) cells and in mouse airways. PM was endocytosed by HBE cells and simultaneously triggered autophagosomes, which then engulfed the invading particles to form amphisomes and subsequent autolysosomes. Genetic blockage of autophagy markedly reduced PM-induced expression of inflammatory cytokines, e.g. IL8 and IL6, and MUC5AC in HBE cells. Mice with impaired autophagy due to knockdown of autophagy-related gene Becn1 or Lc3b displayed significantly reduced airway inflammation and mucus hyperproduction in response to PM exposure in vivo. Interference of the autophagic flux by lysosomal inhibition resulted in accumulated autophagosomes/amphisomes, and intriguingly, this process significantly aggravated the IL8 production through NFKB1, and markedly attenuated MUC5AC expression via activator protein 1. These data indicate that autophagy is required for PM-induced airway epithelial injury, and that inhibition of autophagy exerts therapeutic benefits for PM-induced airway inflammation and mucus hyperproduction, although they are differentially orchestrated by the autophagic flux.     

AP2α is essential for MUC8 gene expression in human airway epithelial cells

Mucins are high molecular weight proteins that make up the major components of mucus. Hypersecretion of mucus is a feature of several chronic inflammatory airway diseases. MUC8 is an important component of airway mucus, and its gene expression is upregulated in nasal polyp epithelium. Little is known about the molecular mechanisms of MUC8 gene expression. We first observed overexpression of activator protein‐2alpha (AP2α) in human nasal polyp epithelium. We hypothesized that AP2α overexpression in nasal polyp epithelium correlates closely with MUC8 gene expression. We demonstrated that phorbol 12‐myristate 13‐acetate (PMA) treatment of the airway epithelial cell line NCI‐H292 increases MUC8 gene and AP2α expression. In this study, we sought to determine which signal pathway is involved in PMA‐induced MUC8 gene expression. The results show that the protein kinase C and mitogen‐activating protein/ERK kinase (MAPK) pathways modulate MUC8 gene expression. PD98059 or ERK1/2 siRNA and RO‐31‐8220 or PKC siRNA significantly suppress AP2α as well as MUC8 gene expression in PMA‐treated cells. To verify the role of AP2α, we specifically knocked down AP2α expression with siRNA. A significant AP2α knock‐down inhibited PMA‐induced MUC8 gene expression. While dominant negative AP2α decreased PMA‐induced MUC8 gene expression, overexpressing wildtype AP2α increased MUC8 gene expression. Furthermore, using lentiviral vectors for RNA interference in human nasal polyp epithelial cells, we confirmed an essential role for AP2α in MUC8 gene expression. From these results, we concluded that PMA induces MUC8 gene expression through a mechanism involving PKC, ERK1/2, and AP2α activation in human airway epithelial cells. J. Cell. Biochem. 110: 1386–1398, 2010. © 2010 Wiley‐Liss, Inc.     

The role of Mycobacterium avium complex fibronectin attachment protein in adherence to the human respiratory mucosa

Mycobacterium avium complex (MAC) are opportunistic respiratory pathogens that infect non-immunocompromised patients with established lung disease, although they can also cause primary infections. The ability to bind fibronectin is conserved among many mycobacterial species. We have investigated the adherence of a sputum isolate of MAC to the mucosa of organ cultures constructed with human tissue and the contribution of M. avium fibronectin attachment protein (FAP) to the process. MAC adhered to fibrous, but not globular mucus, and to extracellular matrix (ECM) in areas of epithelial damage, but not to intact extruded cells and collagen fibres. Bacteria occasionally adhered to healthy unciliated epithelium and to cells that had degenerated exposing their contents, but never to ciliated cells. The results obtained with different respiratory tissues were similar. Two ATCC strains of MAC gave similar results. There was a significant reduction (P < 0.05) in the number of bacteria adhering to ECM after preincubation of bacteria with fibronectin and after preincubation of the tissue with M. avium FAP in a concentration-dependant manner. The number of bacteria adhering to fibrous mucus was unchanged. Immunogold labelling demonstrated fibronectin in ECM as well as in other areas of epithelial damage, but only ECM bound FAP. A Mycobacterium smegmatis strain had the same pattern of adherence to the mucosa as MAC. When the FAP gene was deleted, the strain demonstrated reduced adherence to ECM, and adherence was restored when the strain was transfected with an M. avium FAP expression construct. We conclude that MAC adheres to ECM in areas of epithelial damage via FAP and to mucus with a fibrous appearance via another adhesin. Epithelial damage exposing ECM and poor mucus clearance will predispose to MAC airway infection.