Mucin 5AC Production Research Articles

Surfactant protein D prevents mucin overproduction in airway goblet cells via SIRPα

Mucin overproduction is a common feature of chronic airway diseases such as asthma and chronic obstructive pulmonary disease (COPD), and exacerbates their underlying respiratory condition. Surfactant protein D (SP-D) protects against airway diseases through modulation of immune reactions, but whether it also exerts direct effects on airway epithelial cells has remained unclear. Therefore, we sought to investigate the inhibitory role of SP-D on mucin production in airway epithelial cells. We prepared air–liquid interface (ALI) cultures of human primary bronchial epithelial cells (HBECs), which recapitulated a well-differentiated human airway epithelium. Benzo(a)pyrene (BaP), a key toxicant in cigarette smoke, induced mucin 5AC (MUC5AC) production in ALI-cultured HBECs, airway secretory cell lines, and airway epithelia of mice. Then, the protective effects of SP-D against the BaP-induced mucin overproduction were examined. BaP increased MUC5AC production in ALI cultures of HBECs, and this effect was attenuated by SP-D. SP-D also suppressed the BaP-induced phosphorylation of extracellular signal-regulated kinase (ERK) and MUC5AC expression in NCI-H292 goblet-like cells, but not in NCI-H441 club-like cells. Signal regulatory protein α (SIRPα) was found to be expressed in HBECs and NCI-H292 cells but absent in NCI-H441 cells. In NCI-H292 cells, SP-D activated SH2 domain-containing tyrosine phosphatase-1 (SHP-1), downstream of SIRPα, and knockdown of SIRPα abolished the suppressive effects of SP-D on BaP-induced ERK phosphorylation and MUC5AC production. Consistent with these in vitro findings, intratracheal instillation of SP-D prevented the BaP-induced phosphorylation of ERK and Muc5ac expression in airway epithelial cells in a mouse model. SP-D acts directly on airway epithelial cells to inhibit mucin secretion through ligation of SIRPα and SHP-1-mediated dephosphorylation of ERK. Targeting of SIRPα is therefore a potential new therapeutic approach to suppression of mucin hypersecretion in chronic airway diseases such as COPD and asthma.

C-C Motif Chemokine Receptor 3-Mediated Extracellular Signal-Regulated Kinase 1/2 and p38 Mitogen-Activated Protein Kinase Signaling: Promising Targets for Human Airway Epithelial Mucin 5AC Induction by Eotaxin-2 and Eotaxin-3

Introduction: Eotaxin-2 and -3 of the C-C chemokine subfamily function as potent chemoattractant factors for eosinophil recruitment and various immune responses in allergic and inflammatory airway diseases. Mucin 5AC (MUC5AC), a major gel-forming secretory mucin, is overexpressed in airway inflammation. However, the association between mucin secretion and eotaxin-2/3 expression in the upper and lower airway epithelial cells has not been fully elucidated. Therefore, in this study, we investigated the effects of eotaxin-2/3 on MUC5AC expression and its potential signaling mediators. Methods: We analyzed the effects of eotaxin-2 and -3 on NCI-H292 human airway epithelial cells and primary human nasal epithelial cells (HNEpCs) via reverse transcription-polymerase chain reaction, enzyme-linked immunosorbent assay, and western blotting. Along with immunoblot analyses with specific inhibitors and small interfering RNA (siRNA), we explored the signaling pathway involved in MUC5AC expression following eotaxin-2/3 treatment. Results: In HCI-H292 cells, eotaxin-2/3 activated the mRNA expression and protein production of MUC5AC. A specific inhibitor of C-C motif chemokine receptor 3 (CCR3), SB328437, suppressed eotaxin-2/3-induced MUC5AC expression at both the mRNA and protein levels. Eotaxin-2/3 induced the phosphorylation of extracellular signal-regulated kinase (ERK)-1/2 and p38, whereas pretreatment with a CCR3 inhibitor significantly attenuated this effect. Induction of MUC5AC expression with eotaxin-2/3 was decreased by U0126 and SB203580, specific inhibitors of ERK1/2 and p38 mitogen-activated protein kinase (MAPK), respectively. In addition, cell transfection with ERK1/2 and p38 siRNAs inhibited eotaxin-2/3-induced MUC5AC expression. Moreover, specific inhibitors (SB328437, U0126, and SB203580) attenuated eotaxin-2/3-induced MUC5AC expression in HNEpCs. Conclusion: Our results imply that CCR3-mediated ERK1/2 and p38 MAPK are involved in the signal transduction of eotaxin-2/3-induced MUC5AC overexpression.

Inhibition of TRPA1, Endoplasmic Reticulum Stress, Human Airway Epithelial Cell Damage, and Ectopic MUC5AC Expression by Vasaka (Adhatoda vasica; Malabar Nut) Tea.

This study tested whether a medicinal plant, Vasaka, typically consumed as a tea to treat respiratory malaise, could protect airway epithelial cells (AECs) from wood smoke particle-induced damage and prevent pathological mucus production. Wood/biomass smoke is a pneumotoxic air pollutant. Mucus normally protects the airways, but excessive production can obstruct airflow and cause respiratory distress. Vasaka tea pre- and co-treatment dose-dependently inhibited mucin 5AC (MUC5AC) mRNA induction by AECs treated with wood smoke particles. This correlated with transient receptor potential ankyrin-1 (TRPA1) inhibition, an attenuation of endoplasmic reticulum (ER) stress, and AEC damage/death. Induction of mRNA for anterior gradient 2, an ER chaperone/disulfide isomerase required for MUC5AC production, and TRP vanilloid-3, a gene that suppresses ER stress and wood smoke particle-induced cell death, was also attenuated. Variable inhibition of TRPA1, ER stress, and MUC5AC mRNA induction was observed using selected chemicals identified in Vasaka tea including vasicine, vasicinone, apigenin, vitexin, isovitexin, isoorientin, 9-oxoODE, and 9,10-EpOME. Apigenin and 9,10-EpOME were the most cytoprotective and mucosuppressive. Cytochrome P450 1A1 (CYP1A1) mRNA was also induced by Vasaka tea and wood smoke particles. Inhibition of CYP1A1 enhanced ER stress and MUC5AC mRNA expression, suggesting a possible role in producing protective oxylipins in stressed cells. The results provide mechanistic insights and support for the purported benefits of Vasaka tea in treating lung inflammatory conditions, raising the possibility of further development as a preventative and/or restorative therapy.

Gleditsia sinensis Lam. aqueous extract attenuates nasal inflammation in allergic rhinitis by inhibiting MUC5AC production through suppression of the STAT3/STAT6 pathway

Allergic rhinitis (AR), a chronic respiratory inflammatory disease, is among the most common chronic diseases reported worldwide. Mucus hypersecretion is a critical feature of AR pathogenesis. Although the Gleditsia sinensis extract has several beneficial effects on human health, its effects on allergic inflammation have not yet been investigated. In this study, we examined the effects of G. sinensis aqueous extract (GSAE) on nasal inflammation in an ovalbumin (OVA)-induced AR mouse model. GSAE was administered orally for 1 week and then the clinical nasal symptoms were evaluated. The levels of histamine, OVA-specific immunoglobulin (Ig) E, and interleukin (IL)-13 were measured in the serum using an enzyme-linked immunosorbent assay (ELISA). Inflammatory cells were then counted in the nasal lavage fluid (NALF) and histopathology in the nasal epithelium was evaluated. STAT3/STAT6 phosphorylation was examined in primary human nasal epithelial cells (HNEpCs) using western blot analysis. Oral administration of GSAE to OVA-induced AR mice alleviated nasal clinical symptoms and reduced OVA-specific immunoglobulin E, interleukin (IL)-13, and histamine levels. The accumulation of eosinophils in nasal lavage fluid, nasal mucosa, mast cells, goblet cells, and mucin 5AC (MUC5AC) in the nasal epithelium was also inhibited by GSAE. Treatment with GSAE inhibited the production of MUC5AC in IL-4/IL-13-stimulated primary human nasal epithelial cells through the signal transducer and activator of transcription (STAT)3/STAT6 signaling pathway. These results indicated that GSAE reduces nasal inflammation suggesting that it is a potential treatment option for AR.

MiR-214 Expression Is Elevated in Chronic Rhinosinusitis Mucosa and Regulates Lipopolysaccharide-Mediated Responses in Undifferentiated Human Nasal Epithelial Cell Culture.

Chronic rhinosinusitis (CRS) is an inflammatory disorder of the upper airways. MicroRNAs (miRs) are reported to regulate several diverse physiological and pathological processes. This study aimed to evaluate the impact of miR-214 on lipopolysaccharide (LPS)-mediated inflammation, and mucin 5AC (MUC5AC) expression in human nasal epithelial cells. The expression of miR-214 was detected in CRS with polyps (CRSwNP) and CRS without polyps (CRSsNP) tissues. Cells were treated with LPS and a miR-214 inhibitor. The level of miR-214 was detected by quantitative real-time reverse transcriptase-PCR (qRT-PCR). The inflammatory cytokines (IL-6, IL-8, TNF, and IL-1β) and MUC5AC production were determined by qRT-PCR and ELISA. MUC5AC protein level was detected using western blot. Similarly, we determined the relationship between miR-214 and Sirtuin 1 (SIRT1) using the Dual luciferase activity assay. miR-214 was increased in CRSwNP and CRSsNP tissues. LPS triggered the expression of miR-214, while miR-214 inhibition diminished the level of miR-214. MiR-214 inhibition prevented LPS-mediated the production of inflammatory cytokines. LPS treatment augmented MUC5AC mRNA, protein levels, and secretion, whereas miR-214 loss inhibited MUC5AC production in the presence of LPS. SIRT1 is a direct target of miR-214. Impairing SIRT1 by siRNA (siSIRT1) or EX527 (a selective SIRT1 inhibitor) reversed the effects of miR-214 inhibitor on inflammation and MUC5AC expression. Furthermore, miR-214 depression inhibited the STAT3/GDF15 pathway via targeting SIRT1. Upregulation of STAT3 or GDF15 partly abolished the anti-inflammatory roles of miR-214 inhibitor. Taken together, miR-214 regulates LPS-mediated inflammation and MUC5AC expression via targeting SIRT1, and STAT3/GDF15 may involve in the regulation of miR-214 inhibitor on inflammation and MUC5AC expression.

Inhibition of microRNA-328 Increases Ocular Mucin Expression and Conjunctival Goblet Cells.

We previously reported anti-miR-328 therapy for dry eye disease (DED). Since decreased mucin secretion is a risk factor for DED, we aimed to explore whether anti-miR-328 affects mucin expression and goblet cells. MiR-328 was increased in goblet cells when they were under desiccating stress or treated with benzalkonium chloride (BAC), both of which are risk factors for DED. Based on bioinformatics tool results, miR-328 was predicted to directly target the transcription factor CREB1 that has been known to promote the expression of mucin5AC. The inhibitory effect of miR-328 on CREB1 was confirmed by the transfection assay. A miR-328 binding site on the CREB1 gene was confirmed by the luciferase assay. Furthermore, anti-miR-328 increased CREB1 and mucin5AC in cultured goblet cells according to qPCR, Western blot, and IF staining experiments. Anti-miR-328 increased mucin5AC secretion from the cultured goblet cells based on an ELISA assay for the cultured medium. Finally, impression cytology data revealed anti-miR-328 increased conjunctival goblet cells in the DED rabbits induced by BAC. In conclusion, anti-miR-328 increases CREB1 expression leading to an increase in mucin5AC production and secretion. Furthermore, anti-miR-328 also increases conjunctival goblet cells. These results warrant the further development of anti-miR-328 therapy for DED.

Home Dust Mites Promote MUC5AC Hyper-Expression by Modulating the sNASP/TRAF6 Axis in the Airway Epithelium.

House dust mites (HDMs) are a common source of respiratory allergens responsible for allergic asthma and innate immune responses in human diseases. Since HDMs are critical factors in the triggering of allergen-induced airway mucosa from allergic asthma, we aimed to investigate the mechanisms of Toll-like receptors (TLR) in the signaling of the HDM extract that is involved in mucus hypersecretion and airway inflammation through the engagement of innate immunity. Previously, we reported that the somatic nuclear autoantigenic sperm protein (sNASP)/tumor necrosis factor receptor-associated factor 6 (TRAF6) axis controls the initiation of TLRs to maintain the homeostasis of the innate immune response. The present study showed that the HDM extract stimulated the biogenesis of Mucin 5AC (MUC5AC) in bronchial epithelial cells via the TLR2/4 signaling pathway involving MyD88 and TRAF6. Specifically, sNASP binds to TRAF6 in unstimulated bronchial epithelial cells to prevent the activation of TRAF6-depenedent kinases. Upon on HDMs’ stimulation, sNASP is phosphorylated, leading to the activation of TRAF6 downstream of the p38 MAPK and NF-κB signaling pathways. Further, NASP-knockdown enhanced TRAF6 signaling and MUC5AC biogenesis. In the HDM-induced mouse asthma model, we found that the HDM extract promoted airway hyperresponsiveness (AHR), MUC5AC, and allergen-specific IgE production as well as IL-5 and IL-13 for recruiting inflammatory cells. Treatment with the PEP-NASP peptide, a selective TRAF6-blocking peptide, ameliorated HDM-induced asthma in mice. In conclusion, this study indicated that the sNASP/TRAF6 axis plays a regulatory role in asthma by modulating mucus overproduction, and the PEP-NASP peptide might be a potential target for asthma treatment.

Reineckia carnea Alleviates the Production of Inflammatory Cytokines and MUC5AC in Rats with Chronic Obstructive Pulmonary Disease.

Background Reineckia carnea (RC), a perennial evergreen herb which belongs to Reineckia Kunth (Liliaceae), can be used for clearing the lungs and relieving cough, reducing phlegm and anti-inflammatory effects. Moreover, chronic obstructive pulmonary disease (COPD) is characterized by airway and lung inflammation and increased secretion of airway mucus. Therefore, RC has the potential to treat COPD. Methods NR8383 cells were cultured and treated with various concentrations of RC (100 mg/mL, 10 mg/mL, 1 mg/mL, 100 μg/mL, 10 μg/mL, 1 μg/mL, 100 ng/mL, and 10 ng/mL). Cell viability and levels of interleukin (IL)-1β, cyclooxygenase-2 (COX-2), and prostaglandin E2 (PGE2) in the cell culture supernatant or rat serum were analyzed using CCK-8 and enzyme-linked immunosorbent assay (ELISA), respectively. Sprague Dawley rats were assigned to mock, COPD model, RC (0.67 g/kg, 1.35 g/kg, and 2.7 g/kg), and ambroxol (5.4 mg/kg) groups. Western blot and quantitative polymerase chain reaction (qPCR) analyses were used to evaluate the protein and mRNA expression levels of mucin 5AC (MUC5AC) and Toll-like receptor 4 (TLR4). Results The results showed that Reineckia carnea (RC) extract (RCE) inhibited the proliferation of NR8383 cells and suppressed the production of IL-1β, PGE2, and COX-2 in NR8383 cells. Moreover, RCE decreased the levels of IL-1β, PGE2, and COX-2 in the serum of rats with COPD and alleviated the expression of TLR4 and MUC5AC induced by COPD in rat lung tissue. Conclusion RCE alleviated COPD by inhibiting the expression of COPD-induced inflammatory cytokines and MUC5AC in rats.

Effect of Hyperoside on the Gene Expression and Production of Mucin and EGFR-p44/42-Sp1signaling Pathway in Airway Epithelial Cells

Hyperoside is an anti-inflammatory natural product derived from a medicinal plant Houttuynia cordata, empirically used as remedies for controlling the pulmonary inflammatory diseases in folk medicine. In this study, hyperoside was investigated for its potential effect on the gene expression and production of airway mucin 5AC (MUC5AC). Human airway epithelial NCI-H292 cells were pretreated with hyperoside for 30 min and stimulated with epidermal growth factor (EGF) for the following 24 h. For the elucidation of the action mechanism of hyperoside, EGFinduced epidermal growth factor receptor (EGFR) signaling pathway was investigated. Hyperoside inhibited EGF-induced gene expression and production of MUC5AC mucin through regulating the phosphorylation of EGFR, phosphorylation of extracellular signal-regulated kinase (ERK) 1/2 (p44/42), and the nuclear expression of specificity protein-1 (Sp1). These results suggest hyperoside could regulate the gene expression and production of MUC5AC by modulating the EGFR signaling pathway in human airway epithelial cells.

Bee Venom Prevents Mucin 5AC Production through Inhibition of AKT and SPDEF Activation in Airway Epithelia Cells.

IL-13 induces mucus metaplasia, which causes airway obstruction in asthma. Bee venom (BV) and its components have shown anti-inflammatory effects in allergic diseases such as atopic dermatitis and asthma. In this study, we investigated the effect of BV on IL-13-induced mucus metaplasia through activation of the signal transducer and activator of transcription (STAT6), and regulation of SAM-pointed domain containing Ets-like factor (SPDEF) and forkhead box A2 (FOXA2) in the airway epithelia cell line A549. In A549 cells, BV (1.0 µg/mL) inhibited IL-13 (10 ng/mL)-induced AKT phosphorylation, increase in SPDEF protein expression, and decrease in FOXA2 protein expression—but not STAT6 phosphorylation. BV also prevented the IL-13-induced increase in mucin 5AC (MUC5AC) mRNA and protein expression. Moreover, we observed that inhibition of phosphoinositide 3 kinase (PI3K)/AKT using LY294002 (50 µM) could reverse the alterations in FOXA2 and MUC5AC expression -by IL-13 and BV. However, LY294002 did not affect IL-13- and BV-induced changes in SPDEF expression. These findings indicate that BV inhibits MUC5AC production through the regulation of SPDEF and FOXA2. The inhibition of MUC5AC production through FOXA2 is mediated via the suppression of PI3K/AKT activation by BV. BV may be helpful in the prevention of mucus metaplasia in asthma.

Integrin β1 subunit regulates cellular and secreted MUC5AC and MUC5B production in NCI–H292 human lung epithelial cells

The surface of the human respiratory tract is covered with a mucus layer containing mucin 5AC (MUC5AC) and mucin 5B (MUC5B) as the main components. This layer contributes to biological defense by eliminating irritants, but excessive MUC5AC secretion by the airway epithelial cells exacerbates asthma. Therefore, regulating mucin production is important for asthma treatment. In this study, the effects of integrin β1 subunit on MUC5AC and MUC5B production were examined in NCI–H292 human lung cancer epithelial cells. When integrin β1 was overexpressed, cellular and secreted MUC5AC levels were decreased, whereas cellular MUC5B production was increased. Conversely, integrin β1 depletion using siRNA increased cellular and secreted MUC5AC production, but decreased cellular MUC5B production. Further, the activity of extracellular signal-regulated kinase (ERK), which promotes MUC5AC production, was decreased by integrin β1 overexpression and increased by its depletion. These results suggest that integrin β1 suppresses MUC5AC production and promotes MUC5B production by downregulating ERK.

Amygdalin Attenuates Airway Epithelium Apoptosis, Inflammation, and Epithelial-Mesenchymal Transition through Restraining the TLR4/NF-κB Signaling Pathway on LPS-Treated BEAS-2B Bronchial Epithelial Cells

Background: Cough-variant asthma (CVA) is a special type of asthma, solely manifesting with coughing. Studies suggest that airway inflammation is associated with CVA pathogenesis. Amygdalin is found to have an anti-inflammatory potential, while how it affects CVA remains unexplored. Methods: Cytotoxicity delivered by various concentrations of LPS and amygdalin on BEAS-2B cells was determined by Cell Counting Kit-8 assay. CVA in vitro models were established via LPS exposure on BEAS-2B cells which underwent amygdalin pretreatment. Cell apoptosis was determined by flow cytometry. Production of tumor necrosis factor (TNF)-α, interleukin (IL)-6, IL-8, and mucin 5AC (MUC5AC) in BEAS-2B cells was measured by ELISA and qRT-PCR. Expressions of TLR4, E-cadherin, N-cadherin, α-smooth muscle actin (SMA), vimentin, phosphorylated-p65 (p-p65), p65, phosphorylated-IκBα (p-IκBα), and IκBα in BEAS-2B cells were measured by qRT-PCR or Western blot. Results: LPS and high concentrations of amygdalin (over 600 μg/mL) decreased BEAS-2B cell toxicity. Exposure to LPS inhibited toxicity, enhanced apoptosis; and promoted production of TNF-α, IL-6, IL-8, and MUC5AC, increased the levels of N-Cadherin, α-SMA, vimentin, p-p65, and p-IκBα, and decreased the levels of E-cadherin and IκBα in BEAS-2B cells. Amygdalin pretreatment counteracted the effects of LPS on BEAS-2B cells. Overexpressing TLR4 reversed amygdalin-exerted effects in LPS-exposed BEAS-2B cells. Conclusion: Amygdalin attenuated airway epithelium apoptosis, inflammation and epithelial-mesenchymal transition through restraining the TLR4/NF-κB signaling pathway in CVA.

Telmisartan mitigates lipopolysaccharide (LPS)-induced production of mucin 5AC (MUC5AC) through increasing suppressor of cytokine signaling 1 (SOCS1)

ABSTRACT Acute lung injury (ALI) is a serious clinical pulmonary disease. The pathogenesis of ALI is related to the excessive release of inflammatory factors and upregulation of mucin 5AC (MUC5AC). Telmisartan is a novel antihypertension agent that exerts promising anti-inflammatory effects. The purpose of this study is to investigate whether Telmisartan has a protective role in lipopolysaccharide (LPS)-induced MUC5AC expression and to explore the underlying mechanism in human bronchial epithelial cells. Firstly, the decreased cell viability, elevated release of lactate dehydrogenase (LDH), and excessively released inflammatory factors tumor necrosis factor-α (TNF-α), interleukin- 6 (IL-6), and transforming growth factor-β (TGF)-β in bronchial BEAS-2B epithelial cells induced by stimulation with LPS were significantly reversed by the introduction of Telmisartan. Secondly, the upregulated MUC5AC and downregulated suppressor of cytokine signaling 1 (SOCS1) caused by stimulation with LPS were dramatically reversed by Telmisartan. Notably, treatment with Telmisartan attenuated LPS-induced activation of nuclear factor κ-B (NF-κB). Lastly, silencing of SOCS1 abolished the protective effects of Telmisartan against LPS-induced production of MUC5AC and the activation of NF-κB. Based on these findings, we conclude that Telmisartan displayed a protective effect against LPS by improving mitochondrial function, mitigating inflammatory response, and reducing the production of mucin 5AC by regulating the SOCS1/NF-κB axis in human bronchial epithelial cells.

Mechanism of azithromycin in airway diseases.

Azithromycin (AZM) has been used to treat chronic inflammatory airway diseases because it regulates cell–cell contact between airway epithelial cells. Airway mucus hypersecretion is an important component of chronic respiratory diseases. Mucin 5AC (MUC5AC) is the major mucin produced by airway epithelial cells, and hypersecretion of MUC5AC is a sign of various pulmonary inflammatory diseases. Recently, it was found that matrix metallopeptidase 9 is involved in mucus hypersecretion. Moreover, AZM can inhibit the ability of TNF-α-to induce interleukin (IL)-8 production. This review focuses on the effects on AZM that may be beneficial in inhibiting MUC5AC, matrix metalloprotease-9 and IL-8 production in airway epithelial cells. In addition, recent studies have begun to assess activation of mitogen-activated protein kinase (MAPK) signaling pathways in response to AZM. Understanding these new developments may be helpful for clinicians.

Dupilumab-Associated Mucin Deficiency (DAMD).

PurposeDupilumab, a monoclonal antibody directed against the interleukin-4 receptor subunit α (IL-4Rα) of IL-4 and IL-13, is increasingly being used to control atopic disease. Dupilumab use has been associated with a poorly understood conjunctivitis. In this study, we sought to investigate the hypothesis that dupilumab use and the associated IL-13 blockade causes a relative ocular mucin deficiency.MethodsTear levels of mucin 5ac (Muc5AC) and total tear protein levels were evaluated from 28 eyes of 14 patients. Bilateral tear samples were acquired from seven patients on dupilumab and seven patients with no exposure to dupilumab. Study subjects were age and gender matched. In addition to tear samples, photographic documentation of ocular surface findings and a questionnaire of ocular surface symptoms were obtained. Between-group mean differences were calculated.ResultsCompared with control, ocular Muc5AC levels normalized to total tear protein was statistically significantly lower. The average Muc5AC levels for persons on dupilumab was 1.54 ± 0.58 ng/mg and that of controls was 7.99 ± 1.16 ng/ mg. Persons on dupilumab reported a statistically increased occurrence of ocular fatigue/eye strain, uncomfortable sensation, pain, red eye, and itching.ConclusionsThis study demonstrates for the first time, a relative deficiency of Muc5AC in patients on dupilumab.Translational RelevanceThe results of this study support the previously reported role of IL-13 in increasing goblet cell density and associated Muc5AC production. Further efforts are underway to better understand the relative contribution of Muc5AC deficiency in the overall presentation of conjunctivitis associated with dupilumab use.

Esculetin inhibits histamine-induced expression of inflammatory cytokines and mucin in nasal epithelial cells.

Allergic rhinitis (AR) is a type of respiratory disease closely associated with chronic inflammation. Esculetin is a natural coumarin derivative and has been reported to possess anti-allergic and anti-inflammatory effects. However, the roles of esculetin in AR have not been studied. In this study, we aimed to examine the effect of esculetin on AR using an in vitro model. The human nasal epithelial cells (HNEpC) were stimulated by histamine for 24hours with or without the pretreatment of esculetin. The mRNA levels and production of inflammatory cytokines including IL-6 and IL-8, as well as mucin 5AC (MUC5AC) were measured using qRT-PCR and ELISA, respectively. The results showed that esculetin suppressed histamine-induced expression and secretion of IL-6, IL-8, and MUC5AC in HNEpCs. Furthermore, we examined the effect of esculetin on NF-κB pathway by detecting the expression levels of NF-κB p65, p-p65 and IκBα using western blot analysis. Esculetin treatment suppressed the histamine-induced p-p65 expression and p-IκBα degradation. Inhibiting NF-κB pathway suppressed histamine-induced production of IL-6, IL-8, and MUC5AC in HNEpCs. These findings suggested that esculetin suppressed histamine-induced production of inflammatory cytokines and mucin in HNEpCs, which were partly mediated by the inhibition of NF-κB pathway.

MicroRNA-155 regulates lipopolysaccharide-induced mucin 5AC overproduction via a suppressor of cytokine signaling 1-mediated mechanism in human bronchial epithelial cells

Chronic inflammatory lung diseases accompanied by Gram-negative bacteria infection are characterized by excessive mucin production. Lipopolysaccharide (LPS), the major endotoxin released from Gram-negative bacteria, is a potent inflammatory agonist for mucin overproduction. In this study, we sought to examine whether the toll-like receptor (TLR)-responsive microRNA miR-155 plays a role in LPS-provoked induction of mucin 5AC (MUC5AC) and the potential role of suppressor of cytokine signaling 1 (SOCS1) involved in this process. We found that LPS increased the expression of MUC5AC in association with TLR4-dependent miR-155 induction. The suppression of miR-155 by antagomir led to an excessive production of SOCS1, thereby downregulation of MUC5AC production. Collectively, these data imply that miR-155 is involved in LPS-induced MUC5AC overproduction through a TLR4-dependent manner and thereby the downregulation of SOCS1.

Curcumin suppresses MUC5AC production via interfering with the EGFR signaling pathway.

Excessive mucin production in the airway may contribute to airway inflammatory diseases. Curcumin has been reported to prevent mucin5AC (MUC5AC) production in human airway epithelial cells; however, the molecular targets of curcumin involved in regulating MUC5AC expression have remained elusive. The present study aimed to elucidate the molecular mechanisms by which curcumin regulates MUC5AC production, utilizing the NCI‑H292 human airway epithelial cell line featuring MUC5AC hypersecretion. Curcumin was able to counteract the endothelial growth factor(EGF)‑stimulated mRNA and protein expression of MUC5AC. In addition, curcumin treatment prevented EGF‑induced phosphorylation of EGF receptor(EGFR) as well as the downstream AKT and signal transducer and activator of transcription3(STAT3), while inhibition of PI3K and STAT3 signaling significantly attenuated the expression of MUC5AC that was induced by EGF. Furthermore, EGF‑induced increases in the levels of phosphorylated STAT3 in the nuclear fraction were inhibited by curcumin and PI3K inhibitors. In addition, treatment with curcumin significantly decreased MUC5AC and EGFR expression in a time‑dependent manner under basal conditions. These results demonstrated that curcumin inhibited MUC5AC protein expression in NCI‑H292 cells under basal conditions as well under EGF stimulation. This inhibition was accompanied by decreased activation of the EGFR/AKT/STAT3 pathway and reduced EGFR expression, which indicated that curcumin may have a dual role in interfering with the EGFR signaling pathway and inhibiting mucin expression in human airway epithelial cells.

Bordetella pertussis Adenylate Cyclase Toxin Disrupts Functional Integrity of Bronchial Epithelial Layers.

The airway epithelium restricts the penetration of inhaled pathogens into the underlying tissue and plays a crucial role in the innate immune defense against respiratory infections. The whooping cough agent, Bordetella pertussis, adheres to ciliated cells of the human airway epithelium and subverts its defense functions through the action of secreted toxins and other virulence factors. We examined the impact of B. pertussis infection and of adenylate cyclase toxin-hemolysin (CyaA) action on the functional integrity of human bronchial epithelial cells cultured at the air-liquid interface (ALI). B. pertussis adhesion to the apical surface of polarized pseudostratified VA10 cell layers provoked a disruption of tight junctions and caused a drop in transepithelial electrical resistance (TEER). The reduction of TEER depended on the capacity of the secreted CyaA toxin to elicit cAMP signaling in epithelial cells through its adenylyl cyclase enzyme activity. Both purified CyaA and cAMP-signaling drugs triggered a decrease in the TEER of VA10 cell layers. Toxin-produced cAMP signaling caused actin cytoskeleton rearrangement and induced mucin 5AC production and interleukin-6 (IL-6) secretion, while it inhibited the IL-17A-induced secretion of the IL-8 chemokine and of the antimicrobial peptide beta-defensin 2. These results indicate that CyaA toxin activity compromises the barrier and innate immune functions of Bordetella-infected airway epithelia.

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. Invivo, 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.