Caged Dexamethasone/Quercetin Nanoparticles, Formed of the Morphogenetic Active Inorganic Polyphosphate, are Strong Inducers of MUC5AC.

Inorganic polyphosphate (polyP) is a widely distributed polymer found from bacteria to animals, including marine species. This polymer exhibits morphogenetic as well as antiviral activity and releases metabolic energy after enzymatic hydrolysis also in human cells. In the pathogenesis of the coronavirus disease 2019 (COVID-19), the platelets are at the frontline of this syndrome. Platelets release a set of molecules, among them polyP. In addition, the production of airway mucus, the first line of body defense, is impaired in those patients. Therefore, in this study, amorphous nanoparticles of the magnesium salt of polyP (Mg-polyP-NP), matching the size of the coronavirus SARS-CoV-2, were prepared and loaded with the secondary plant metabolite quercetin or with dexamethasone to study their effects on the respiratory epithelium using human alveolar basal epithelial A549 cells as a model. The results revealed that both compounds embedded into the polyP nanoparticles significantly increased the steady-state-expression of the MUC5AC gene. This mucin species is the major mucus glycoprotein present in the secreted gel-forming mucus. The level of gene expression caused by quercetin or with dexamethasone, if caged into polyP NP, is significantly higher compared to the individual drugs alone. Both quercetin and dexamethasone did not impair the growth-supporting effect of polyP on A549 cells even at concentrations of quercetin which are cytotoxic for the cells. A possible mechanism of the effects of the two drugs together with polyP on mucin expression is proposed based on the scavenging of free oxygen species and the generation of ADP/ATP from the polyP, which is needed for the organization of the protective mucin-based mucus layer.

Effect of Korean Red Ginseng and Rg3 on Asian Sand Dust-Induced MUC5AC, MUC5B, and MUC8 Expression in Bronchial Epithelial Cells.

Korean Red ginseng (KRG), commonly used in traditional medicine, has anti-inflammatory, anti- oxidative, and anti-tumorigenic properties. Asian sand dust (ASD) is known to aggravate upper and lower airway inflammatory responses. BEAS-2B cells were exposed to ASD with or without KRG or ginsenoside Rg3. Mucin 5AC (MUC5AC), MUC5B, and MUC8 mRNA and protein expression levels were determined using quantitative RT-PCR and enzyme-linked immunosorbent assay. Nuclear factor kappa B (NF-κB), activator protein 1, and mitogen-activated protein kinase expression and activity were determined using western blot analysis. ASD induced MUC5AC, MUC5B, and MUC8 mRNA and protein expression in BEAS-2B cells, which was significantly inhibited by KRG and Rg3. Although ASD-induced mucin expression was associated with NF-κB and p38 mitogen-activated protein kinase (MAPK) activity, KRG and Rg3 significantly suppressed only ASD-induced NF-κB expression and activity. KRG and Rg3 inhibited ASD-induced mucin gene expression and protein production from bronchial epithelial cells. These results suggest that KRG and Rg3 have potential for treating mucus-producing airway inflammatory diseases.

Efficient RNP-directed Human Gene Targeting Reveals SPDEF Is Required for IL-13-induced Mucostasis.

Primary human bronchial epithelial cell (HBEC) cultures are a useful model for studies of lung health and major airway diseases. However, mechanistic studies have been limited by our ability to selectively disrupt specific genes in these cells. Here we optimize methods for gene targeting in HBECs by direct delivery of single guide RNA (sgRNA) and rCas9 (recombinant Cas9) complexes by electroporation, without a requirement for plasmids, viruses, or antibiotic selection. Variations in the method of delivery, sgRNA and rCas9 concentrations, and sgRNA sequences all had effects on targeting efficiency, allowing for predictable control of the extent of gene targeting and for near-complete disruption of gene expression. To demonstrate the value of this system, we targeted SPDEF, which encodes a transcription factor previously shown to be essential for the differentiation of MUC5AC-producing goblet cells in mouse models of asthma. Targeting SPDEF led to proportional decreases in MUC5AC expression in HBECs stimulated with IL-13, a central mediator of allergic asthma. Near-complete targeting of SPDEF abolished IL-13-induced MUC5AC expression and goblet cell differentiation. In addition, targeting of SPDEF prevented IL-13-induced impairment of mucociliary clearance, which is likely to be an important contributor to airway obstruction, morbidity, and mortality in asthma. We conclude that direct delivery of sgRNA and rCas9 complexes allows for predictable and efficient gene targeting and enables mechanistic studies of disease-relevant pathways in primary HBECs.

The effect of Notch signal pathway on PM2.5-induced Muc5ac in Beas-2B cells.

BACKGROUND: Atmospheric pollutants could induced over-expression of Muc5ac, which is a major pathological feature in acute exacerbation of Chronic Obstructive Pulmonary Disease (COPD) and fatal asthma. Notch signaling pathway could promote mucus cell proliferation and mucus secretion. However, the effects of Notch signaling pathway on the airway mucus secretion induced by PM2.5 remain unknown. In this study, we investigated the role of the Notch signaling pathway on Muc5ac by atmospheric PM2.5 in Beas-2B cell. METHODS: The mRNA and protein levels of the Notch1-4, downstream target gene Hes1 and Muc5ac in the Notch signaling pathway were detected by qPCR and western after Beas-2B cells were exposed to PM2.5 of different concentrations for 12h, 24h, and 48h. RESULTS: The longer the exposure time and the higher the concentration of PM2.5, the lower the survival rate of Beas-2B cells. The expressions of Hes1 and Muc5ac in mRNA and protein were significantly increased after PM2.5 exposure. Correlation analysis indicated that there was a positive correlation between the expression of Muc5ac and Hes1 in mRNA and protein. CONCLUSION: Atmospheric PM2.5 can induce the express of Muc5ac, the Notch signaling pathway may be involved in the regulation of Muc5ac by Hes1.

Bronchial Epithelial Calcium Metabolism Impairment in Smokers and Chronic Obstructive Pulmonary Disease. Decreased ORAI3 Signaling.

The airway epithelium represents a fragile environmental interface potentially disturbed by cigarette smoke (CS), the major risk factor for developing chronic obstructive pulmonary disease (COPD). CS leads to bronchial epithelial damage on ciliated, goblet, and club cells, which could involve calcium (Ca2+) signaling. Ca2+ is a key messenger involved in virtually all fundamental physiological functions, including mucus and cytokine secretion, cilia beating, and epithelial repair. In this study, we analyzed Ca2+ signaling in air-liquid interface-reconstituted bronchial epithelium from control subjects and smokers (with and without COPD). We further aimed to determine how smoking impaired Ca2+ signaling. First, we showed that the endoplasmic reticulum (ER) depletion of Ca2+ stores was decreased in patients with COPD and that the Ca2+ influx was decreased in epithelial cells from smokers (regardless of COPD status). In addition, acute CS exposure led to a decrease in ER Ca2+ release, significant in smoker subjects, and to a decrease in Ca2+ influx only in control subjects. Furthermore, the differential expression of 55 genes involved in Ca2+ signaling highlighted that only ORAI3 expression was significantly altered in smokers (regardless of COPD status). Finally, we incubated epithelial cells with an ORAI antagonist (GSK-7975A). GSK-7975A altered Ca2+ influx and ciliary beating, but not mucus and cytokine secretion or epithelial repair, in control subjects. Our data suggest that Ca2+ signaling is impaired in smoker epithelia (regardless of COPD status) and involves ORAI3. Moreover, ORAI3 is additionally involved in ciliary beating.

Obtusifolin isolated from the seeds of Cassia obtusifolia regulates the gene expression and production of MUC5AC mucin in airway epithelial cells via affecting NF-κB pathway.

We investigated whether obtusin, obtusifolin, and cassiaside isolated from the seeds of Cassia obtusifolia inhibit the gene expression and production of airway mucin 5AC (MUC5AC). Confluent NCI-H292 cells were pretreated with obtusin, obtusifolin, or cassiaside for 30 min and then stimulated with epidermal growth factor (EGF), phorbol 12-myristate 13-acetate (PMA), or tumor necrosis factor-α (TNF-α) for 24 hr. The MUC5AC mucin gene expression was measured by reverse transcription-polymerase chain reaction. Production of MUC5AC mucin protein was measured by enzyme-linked immunosorbent assay. To elucidate the action mechanism of obtusifolin, effect of obtusifolin on PMA-induced nuclear factor kappa B (NF-κB) signaling pathway was investigated by western blot analysis. Obtusin, obtusifolin, or cassiaside inhibited the expression of MUC5AC mucin gene and the production of MUC5AC mucin protein, induced by EGF, PMA, or TNF-α. Obtusifolin inhibited PMA-induced activation (phosphorylation) of inhibitory kappa B kinase, and thus phosphorylation and degradation of inhibitory kappa B alpha. Obtusifolin inhibited PMA-induced nuclear translocation of NF-κB p65. These results suggest that obtusifolin can regulate the production and gene expression of mucin by acting on airway epithelial cells through regulation of NF-κB signaling pathway.

Activating Transcription Factor 3 Is Essential for Cigarette Smoke-Induced Mucin Expression via Interaction with Activator Protein-1.

Mucus hypersecretion is an important pathologic feature of chronic obstructive pulmonary disease. Activating transcription factor 3 (ATF3) is an adaptive-response gene that participates in various cellular processes. However, little is known about its role in cigarette smoke (CS)-induced mucus hyperproduction. This study aimed to investigate the role and molecular mechanisms of ATF3 in CS-induced Mucin 5AC (MUC5AC) expression. ATF3 was elevated in lung tissues of mice exposed to CS for 12 weeks. Treatment with CS extract significantly induced ATF3 expression and MUC5AC production in human bronchial epithelial cells, NCI-H292, and mouse tracheal epithelial cells. Interference of ATF3 significantly attenuated CS-induced MUC5AC expression in NCI-H292 and human bronchial epithelial cells. Mouse tracheal epithelial cells isolated from Atf3-/- mice also exhibited less MUC5AC production in response to CS extract treatment. In vivo, the Atf3-/- mice also displayed a significantly reduced mucus production relative to wild-type controls in response to chronic CS exposure. Furthermore, a chromatin immunoprecipitation assay revealed increased ATF3 binding to the MUC5AC promoter after CS treatment, and this transcriptional binding was significantly inhibited by knockdown of JUN, a subunit of activator protein-1. These results demonstrate that ATF3 may be involved in activator protein-1 signaling and transcriptional promotion of CS-induced MUC5AC expression in airway epithelial cells.

The dopamine D1 receptor is expressed and induces CREB phosphorylation and MUC5AC expression in human airway epithelium.

Background: Dopamine receptors comprise two subgroups, Gs protein-coupled "D1-like" receptors (D1, D5) and Gicoupled "D2-like" receptors (D2, D3, D4). In airways, both dopamine D1 and D2 receptors are expressed on airway smooth muscle and regulate airway smooth muscle force. However, functional expression of the dopamine D1 receptor has never been identified on airway epithelium. Activation of Gs-coupled receptors stimulate adenylyl cyclase leading to cyclic AMP (cAMP) production, which is known to induce mucus overproduction through the cAMP response element binding protein (CREB) in airway epithelial cells. We questioned whether the dopamine D1 receptor is expressed on airway epithelium, and whether it promotes CREB phosphorylation and MUC5AC expression. Methods: We evaluated the protein expression of the dopamine D1 receptor on native human airway epithelium and three sources of cultured human airway epithelial cells including primary cultured airway epithelial cells, the bronchial epithelial cell line (16HBE14o-), and the pulmonary mucoepidermoid carcinoma cell line (NCI-H292) using immunohistochemistry and immunoblotting. To characterize the stimulation of cAMP through the dopamine D1 receptor, 16HBE14o- cells and NCI-H292 cells were treated with dopamine or the dopamine D1 receptor agonists (SKF38393 or A68930) before cAMP measurements. The phosphorylation of CREB by A68930 in both 16HBE14o- and NCI-H292 cells was measured by immunoblot. The effect of dopamine or A68930 on the expression of MUC5AC mRNA and protein in NCI-H292 cells was evaluated by real-time PCR and immunofluorescence staining, respectively. Results: The dopamine D1 receptor protein was detected in native human airway epithelium and three sources of cultured human airway epithelial cells. Dopamine or the dopamine D1-like receptor agonists stimulated cAMP production in 16HBE14o- cells and NCI-H292 cells, which was reversed by the selective dopamine D1-like receptor antagonists (SCH23390 or SCH39166). A68930 significantly increased phosphorylation of CREB in both 16HBE14o- and NCI-H292 cells, which was attenuated by the inhibitors of PKA (H89) and MEK (U0126). Expression of MUC5AC mRNA and protein were also increased by either dopamine or A68930 in NCI-H292 cells. Conclusions: These results suggest that the activation of the dopamine D1 receptor on human airway epithelium could induce mucus overproduction, which could worsen airway obstructive symptoms.

Preferential Generation of 15-HETE-PE Induced by IL-13 Regulates Goblet Cell Differentiation in Human Airway Epithelial Cells.

Type 2-associated goblet cell hyperplasia and mucus hypersecretion are well known features of asthma. 15-Lipoxygenase-1 (15LO1) is induced by the type 2 cytokine IL-13 in human airway epithelial cells (HAECs) in vitro and is increased in fresh asthmatic HAECs ex vivo. 15LO1 generates a variety of products, including 15-hydroxyeicosatetraenoic acid (15-HETE), 15-HETE-phosphatidylethanolamine (15-HETE-PE), and 13-hydroxyoctadecadienoic acid (13-HODE). In this study, we investigated the 15LO1 metabolite profile at baseline and after IL-13 treatment, as well as its influence on goblet cell differentiation in HAECs. Primary HAECs obtained from bronchial brushings of asthmatic and healthy subjects were cultured under air-liquid interface culture supplemented with arachidonic acid and linoleic acid (10 µM each) and exposed to IL-13 for 7 days. Short interfering RNA transfection and 15LO1 inhibition were applied to suppress 15LO1 expression and activity. IL-13 stimulation induced expression of 15LO1 and preferentially generated 15-HETE-PE in vitro, both of which persisted after removal of IL-13. 15LO1 inhibition (by short interfering RNA and chemical inhibitor) decreased IL-13-induced forkhead box protein A3 (FOXA3) expression and enhanced FOXA2 expression. These changes were associated with reductions in both mucin 5AC and periostin. Exogenous 15-HETE-PE stimulation (alone) recapitulated IL-13-induced FOXA3, mucin 5AC, and periostin expression. The results of this study confirm the central importance of 15LO1 and its primary product, 15-HETE-PE, for epithelial cell remodeling in HAECs.

IL-4-induced caveolin-1-containing lipid rafts aggregation contributes to MUC5AC synthesis in bronchial epithelial cells.

BACKGROUND: Mucus overproduction is an important feature of asthma. Interleukin (IL)-4 is required for allergen-induced airway inflammation and mucus production. MUC5AC gene expression is regulated by transcript factors NF-κB. The intracellular Ca2+ ([Ca2+]i) signal is required for activation of NF-κB. The transient receptor potential canonical 1 (TRPC1) channel has been shown to contribute for agonist-stimulated Ca2+ influx in some types of cells. However, the relationships among IL-4, TRPC1 and mucus overproduction in bronchial epithelial cells (BECs) in asthma are poorly understood. METHODS: BECs were isolated from large bronchial airway of rats and used as cell model. To present changes of lipid raft, caveolin-1 and TRPC1, immunofluorescence staining and sucrose gradient centrifugation were performed. [Ca2+]i was measured after loading with Fura-2. NF-κB activities were measured by an ELISA-based assay. MUC5AC mRNA and protein levels were detected by real-time quantitative RT-PCR, ELISA analysis and immunofluorescence staining respectively. RESULTS: IL-4 induced Ca2+ influx in BECs, and this was blocked by a Ca2+ influx inhibitor (2-APB). 2-APB also prevented MUC5AC protein synthesis induced by IL-4. Depletion of extracellular Ca2+ resulted in partial decrease in expression of MUC5AC in IL-4 treated cells. NF-κB rather than STAT6 activation mediated IL-4-induced MUC5AC protein synthesis. Then the mechanism of Ca2+ influx was investigated. Immunofluorescence staining and sucrose gradient centrifugation revealed that caveolin-1-containing lipid rafts aggregation was involved in TRPC1 activation and Ca2+ influx in BECs. Lastly, the data revealed that blocking lipid rafts aggregation exactly prevented Ca2+ influx, NF-κB activation and MUC5AC synthesis induced by IL-4. CONCLUSIONS: Our results indicate that IL-4-induced caveolin-1-containing lipid rafts aggregation at least partly contributes to MUC5AC synthesis in BECs.

Short Chain Fatty Acid Acetate Protects against Ethanol-Induced Acute Gastric Mucosal Lesion in Mice.

Short chain fatty acids acetate and propionate have been demonstrated protective function in the intestinal mucosa. However, their impact on gastric mucosa has not yet been elucidated. The current study aimed to investigate the potential protective effects of acetate and propionate against ethanol-induced gastric mucosal lesion and the underlying mechanism in mice. ICR mice were orally treated with acetate and propionate, respectively, 30 min prior to the establishment of gastric mucosal injury model by challenge with absolute ethanol. The gastric samples were collected for the detection of oxidative, inflammatory and apoptotic related parameters. Acetate, but not propionate, attenuated the severity of gastric mucosal damage as evidenced by the gross changes of gastric mucosa, pathological aberrations. Acetate alleviated oxidative stress as shown by the increase in glutathione (GSH) content and superoxide dismutase (SOD) activities, and the decrease of malondialdehyde (MDA) level. The elevated concentrations of interleukin (IL)-1ß, tumor necrosis factor (TNF)-α and IL-6, and the activation of nuclear factor-kappaB (NF-κB) p65 by ethanol stimulation was also reduced by acetate. Moreover, the anti-inflammatory factors, IL-4, LXA4 and IL-10, were up-regulated in acetate treated group. With respect to gastric mucosal apoptosis, acetate suppressed caspase-3 activity and BAX expression in favor of cell survival. These favorable actions were maybe associated with up-regulation of the gastric MUC5AC, the key defense factor of gastric mucosal system. These findings accentuate the gastroprotective actions of acetate in ethanol-induced gastric injury which were mediated via concerted multi-prolonged actions, including suppression of gastric oxidation, inflammation and apoptosis and promotion of MUC5AC expression.

Differences between gastric signet-ring cell carcinoma and poorly differentiated adenocarcinoma: A comparison of histopathologic features determined by mucin core protein and trefoil factor family peptide immunohistochemistry.

We investigated differences between the pathological features of gastric signet-ring cell carcinoma (sig) and poorly differentiated adenocarcinoma (por) by examining the expressions of the trefoil factor family peptides (TFFs) and mucin core proteins (MUCs). Ninety-seven tissues of 97 gastric cancer patients were selected for this study. After gastrectomy, the major histopathologic types were determined to be sig, solid-type poorly differentiated adenocarcinoma (por1), non-solid type poorly differentiated adenocarcinoma (por2), and well-differentiated tubular adenocarcinoma (tub1). We evaluated the prevalence of positive staining for MUCs (MUC5AC and MUC2) and TFFs (TFF1 and TFF3) and assessed the correlation between MUCs and TFFs in each histopathological type. The rate of MUC2 expression significantly differed between sig and por2 (50.0% vs 11.7%, P = 0.011). TFF3 expression in sig significantly differed from TFF3 expression in both por2 (100% vs 17.6%, P < 0.0001) and por1 (100% vs 33.3%, P = 0.0004). MUC5AC and TFF1 expressions were significantly correlated in por1 (r = 0.705, P = 0.002), por2 (r = 0.535, P = 0.0009), and tub1 (r = 0.470, P = 0.0034), while MUC2 and TFF3 expressions were significantly correlated only in sig (r = 0.593, P = 0.040). The expression and correlation patterns of the TFFs and MUCs suggest that the histopathologic features of gastric sig differ from those of por.

Tedizolid inhibits MUC5AC production induced by methicillin-resistant Staphylococcus aureus in human airway epithelial cells.

The innate immune system plays an important role in early immunity against respiratory tract infection. Although airway epithelial cells produce mucus to eliminate pathogens and irritants, hypersecretion of mucus is harmful for the host as it may cause airway obstruction and inhibit influx of antimicrobial agents. It has been reported that several antimicrobial agents have an immunomodulatory effect in vitro and in vivo, but little is known about whether tedizolid, a novel oxazolidinone, can modulate immune responses. In this study, we evaluated whether tedizolid can suppress MUC5AC production in human airway epithelial cells stimulated by methicillin-resistant Staphylococcus aureus (MRSA). Compared with the control, tedizolid significantly inhibited MUC5AC protein production and mRNA overexpression at concentrations of both 2 and 10 µg/mL (representative of trough and peak concentrations in human epithelial lining fluid). Among the mitogen-activated protein kinase inhibitors tested, only extracellular signal-regulated protein kinase 1/2 (ERK1/2) phosphorylation was inhibited by tedizolid as indicated by western blot analysis. These results indicate that tedizolid inhibits the overproduction of MUC5AC protein by inhibiting phosphorylation of ERK1/2. This study revealed that tedizolid suppresses excessive mucin production in human airway epithelial cells. The immunomodulatory effect of tedizolid may improve outcomes in patients with severe respiratory infectious diseases caused by MRSA.

Suppression of MUC5AC expression in human bronchial epithelial cells by interferon-γ.

BACKGROUND: Excessive mucin secretion in the airway is an important feature of airway inflammatory diseases. MUC5AC expression is regulated by a variety of stimuli such as cytokines. Little is known about the role of interferon (IFN)-γ in MUC5AC expression in human bronchial epithelial cells. METHODS: Human pulmonary mucoepidermoid carcinoma cell line (NCI-H292) and normal human bronchial epithelial (NHBE) cells were used to assess the effects of IFN-γ on MUC5AC transcription. RESULTS: Transforming growth factor (TGF)-α and double-stranded RNA (polyI:C)-induced MUC5AC mRNA and protein expression was repressed by IFN-γ in a concentration-dependent manner. IFN-γ showed limited effects on TGF-α and polyI:C-induced activation of epidermal growth factor receptor (EGFR) and extracellular signal-regulated kinase (ERK). A chromatin immunoprecipitation assay indicated that Sp1 bound to its cognate sequence located on the MUC5AC promoter. The Sp1 inhibitor mithramycin A inhibited MUC5AC mRNA expression, implying a critical role for Sp1 in MUC5AC induction. Importantly, IFN-γ impeded Sp1 binding to the MUC5AC promoter. CONCLUSIONS: These results suggest that IFN-γ represses MUC5AC expression, disturbing binding of Sp1 to its target sequences.

Early-Life Intranasal Colonization with Nontypeable Haemophilus influenzae Exacerbates Juvenile Airway Disease in Mice.

Accumulating evidence suggests a connection between asthma development and colonization with nontypeable Haemophilus influenzae (NTHi). Specifically, nasopharyngeal colonization of human infants with NTHi within 4 weeks of birth is associated with an increased risk of asthma development later in childhood. Monocytes derived from these infants have aberrant inflammatory responses to common upper respiratory bacterial antigens compared to those of cells derived from infants who were not colonized and do not go on to develop asthma symptoms in childhood. In this study, we hypothesized that early-life colonization with NTHi promotes immune system reprogramming and the development of atypical inflammatory responses. To address this hypothesis in a highly controlled model, we tested whether colonization of mice with NTHi on day of life 3 induced or exacerbated juvenile airway disease using an ovalbumin (OVA) allergy model of asthma. We found that animals that were colonized on day of life 3 and subjected to induction of allergy had exacerbated airway disease as juveniles, in which exacerbated airway disease was defined as increased cellular infiltration into the lung, increased amounts of inflammatory cytokines interleukin-5 (IL-5) and IL-13 in lung lavage fluid, decreased regulatory T cell-associated FOXP3 gene expression, and increased mucus production. We also found that colonization with NTHi amplified airway resistance in response to increasing doses of a bronchoconstrictor following OVA immunization and challenge. Together, the murine model provides evidence for early-life immune programming that precedes the development of juvenile airway disease and corroborates observations that have been made in human children.

Regulation of Viral Infection-induced Airway Remodeling Cytokine Production by the TLR3-EGFR Signaling Pathway in Human Bronchial Epithelial Cells.

Toll-like receptor 3 (TLR3) is involved in the virus-induced pulmonary inflammatory response, but its role in airway remodeling after viral infection is unclear. We explored the role of TLR3 in poly(I:C)-induced inflammatory cytokines and mucin 5AC (MUC5AC) production in human bronchial epithelial cells by Western blotting, RT-PCR and ELISA. The expression of TLR3, MUC5AC, Matrixmetalloproteinase (MMP9), Transforming growth factor (TGF-ß1) and Vascular endothelial growth factor (VEGF) in human bronchial epithelial cells increased in a dose-dependent manner after exposure to poly(I:C), and this effect was inhibited by treatment with TLR3 siRNA. The phosphorylation of epithelial growth factor receptor (EGFR)/ERK/P38 Mitogen-activated protein kinases (MAPK) proteins increased after poly(I:C) treatment, and inhibition of this signaling pathway decreased TLR3 expression and MUC5AC and TGF-ß1 production in human bronchial epithelial cells. The TLR3-EGFR signaling pathway is involved in the production of airway remodeling cytokines after virus infection. Inhibiting EGFR and its signaling pathway may be a therapeutic strategy for modifying airway remodeling.

Expression and characterization of a novel recombinant version of the secreted human mucin MUC5AC in airway cell lines.

Molecular manipulation and expression of mucins, large glycoproteins that provide the structural framework of mucus, are challenging due to mucins' size and numerous domains, including variable number tandem repeat (VNTRs) regions that are sites of O-glycosylation. Only individual human mucin domains have been expressed in mammalian cells. We produced recombinant versions of MUC5AC, a major secreted mucin in the respiratory tract, encoding the N-terminus, C-terminus, N- and C-termini together, and N- and C-termini interspersed with two native tandem repeat sequences (N+2TR+C) in both tracheal and bronchial cell lines. The latter protein contains all of the functional domains required for the biosynthesis and secretion of glycosylated mucin. The N-terminus protein was found in monomeric and higher molecular mass forms suggesting that secreted MUC5AC may form a branched netlike structure analogous to that described for MUC2. At the C-terminus, proteins underwent cleavage, polymerization, and glycosylation. Thus, they appear to undergo pivotal processing steps as predicted for native MUC5AC, which is analogous to that for other individual recombinant mucin domains. Secretion occurred when cells were grown on transwell filter inserts but not on plastic, indicating that the extracellular environment likely plays a role in mucin processing. The secreted N+2TR+C protein differed in molecular mass from the intracellular form, indicating that additional processing occurred. These recombinant proteins, expressed in different backgrounds, can potentially address the role of different mucin domains on MUC5AC processing and function as well as the role of MUC5AC in health and disease.

The levels of Brachyspira hyodysenteriae binding to porcine colonic mucins differ between individuals, and binding is increased to mucins from infected pigs with de novo MUC5AC synthesis.

Brachyspira hyodysenteriae colonizes the pig colon, resulting in mucohemorrhagic diarrhea and growth retardation. Fecal mucus is a characteristic feature of swine dysentery; therefore, we investigated how the mucin environment changes in the colon during infection with B. hyodysenteriae and how these changes affect this bacterium's interaction with mucins. We isolated and characterized mucins, the main component of mucus, from the colon of experimentally inoculated and control pigs and investigated B. hyodysenteriae binding to these mucins. Fluorescence microscopy revealed a massive mucus induction and disorganized mucus structure in the colon of pigs with swine dysentery. Quantitative PCR (qPCR) and antibody detection demonstrated that the mucus composition of pigs with swine dysentery was characterized by de novo expression of MUC5AC and increased expression of MUC2 in the colon. Mucins from the colon of inoculated and control pigs were isolated by two steps of isopycnic density gradient centrifugation. The mucin densities of control and inoculated pigs were similar, whereas the mucin quantity was 5-fold higher during infection. The level of B. hyodysenteriae binding to mucins differed between pigs, and there was increased binding to soluble mucins isolated from pigs with swine dysentery. The ability of B. hyodysenteriae to bind, measured in relation to the total mucin contents of mucus in sick versus healthy pigs, increased 7-fold during infection. Together, the results indicate that B. hyodysenteriae binds to carbohydrate structures on the mucins as these differ between individuals. Furthermore, B. hyodysenteriae infection induces changes to the mucus niche which substantially increase the amount of B. hyodysenteriae binding sites in the mucus.

Tiotropium attenuates IL-13-induced goblet cell metaplasia of human airway epithelial cells.

BACKGROUND: It has been shown that acetylcholine is both a neurotransmitter and acts as a local mediator, produced by airway cells including epithelial cells. In vivo studies have demonstrated an indirect role for acetylcholine in epithelial cell differentiation. Here, we aimed to investigate direct effects of endogenous non-neuronal acetylcholine on epithelial cell differentiation. METHODS: Human airway epithelial cells from healthy donors were cultured at an air-liquid interface (ALI). Cells were exposed to the muscarinic antagonist tiotropium (10 nM), interleukin (IL)-13 (1, 2 and 5 ng/mL), or a combination of IL-13 and tiotropium, during or after differentiation at the ALI. RESULTS: Human airway epithelial cells expressed all components of the non-neuronal cholinergic system, suggesting acetylcholine production. Tiotropium had no effects on epithelial cell differentiation after air exposure. Differentiation into goblet cells was barely induced after air exposure. Therefore, IL-13 (1 ng/mL) was used to induce goblet cell metaplasia. IL-13 induced MUC5AC-positive cells (5-fold) and goblet cells (14-fold), as assessed by histochemistry, and MUC5AC gene expression (105-fold). These effects were partly prevented by tiotropium (47-92%). Goblet cell metaplasia was induced by IL-13 in a dose-dependent manner, which was inhibited by tiotropium. In addition, tiotropium reversed goblet cell metaplasia induced by 2 weeks of IL-13 exposure. IL-13 decreased forkhead box protein A2 (FoxA2) expression (1.6-fold) and increased FoxA3 (3.6-fold) and SAM-pointed domain-containing ETS transcription factor (SPDEF) (5.2-fold) expression. Tiotropium prevented the effects on FoxA2 and FoxA3, but not on SPDEF. CONCLUSIONS: We demonstrate that tiotropium has no effects on epithelial cell differentiation after air exposure, but inhibits and reverses IL-13-induced goblet cell metaplasia, possibly via FoxA2 and FoxA3. This indicates that non-neuronal acetylcholine contributes to goblet cell differentiation by a direct effect on epithelial cells.

Omega-3 PUFA docosahexaenoic acid decreases LPS-stimulated MUC5AC production by altering EGFR-related signaling in NCI-H292 cells.

Chronic obstructive pulmonary disease (COPD) is an inflammatory process characterized by airway mucus hypersecretion. Lipopolysaccharides (LPS) are known to stimulate the production of mucin 5AC (MUC5AC) via epidermal growth factor receptor (EGFR) in human airway cells. Noteworthy, we have previously demonstrated that EGFR/Rac1/reactive oxygen species (ROS)/matrix metalloproteinase 9 (MMP-9) is a key signaling cascade regulating MUC5AC production in airway cells challenged with LPS. Various reports have shown an inverse association between the intake of polyunsaturated fatty acids (PUFA) of the n-3 (omega-3) family or fish consumption and COPD. In the present study, we investigated the influence of docosahexaenoic acid (DHA), one of the most important omega-3 PUFA contained in fish oil, on the production of MUC5AC in LPS-challenged human airway cells NCI--H292. Our results indicate that DHA is capable of counteracting MUC5AC overproduction in LPS-stimulated cells by abrogating both EGFR phosphorylation and its downstream signaling pathway. This signaling pathway not only includes Rac1, ROS and MMP-9, but also NF-κB, since we have found that ROS require NF-κB activity to induce MMP-9 secretion and activation.