A common polymorphism in the Intelectin-1 gene influences mucus plugging in severe asthma.

By incompletely understood mechanisms, type 2 (T2) inflammation present in the airways of severe asthmatics drives the formation of pathologic mucus which leads to airway mucus plugging. Here we investigate the molecular role and clinical significance of intelectin-1 (ITLN-1) in the development of pathologic airway mucus in asthma. Through analyses of human airway epithelial cells we find that ITLN1 gene expression is highly induced by interleukin-13 (IL-13) in a subset of metaplastic MUC5AC+ mucus secretory cells, and that ITLN-1 protein is a secreted component of IL-13-induced mucus. Additionally, we find ITLN-1 protein binds the C-terminus of the MUC5AC mucin and that its deletion in airway epithelial cells partially reverses IL-13-induced mucostasis. Through analysis of nasal airway epithelial brushings, we find that ITLN1 is highly expressed in T2-high asthmatics, when compared to T2-low children. Furthermore, we demonstrate that both ITLN-1 gene expression and protein levels are significantly reduced by a common genetic variant that is associated with protection from the formation of mucus plugs in T2-high asthma. This work identifies an important biomarker and targetable pathways for the treatment of mucus obstruction in asthma.

Coarse-grained modeling and dynamics tracking of nanoparticles diffusion in human gut mucus.

The gastrointestinal (GI) tract's mucus layer serves as a critical barrier and a mediator in drug nanoparticle delivery. The mucus layer's diverse molecular structures and spatial complexity complicates the mechanistic study of the diffusion dynamics of particulate materials. In response, we developed a bi-component coarse-grained mucus model, specifically tailored for the colorectal cancer environment, that contained the two most abundant glycoproteins in GI mucus: Muc2 and Muc5AC. This model demonstrated the effects of molecular composition and concentration on mucus pore size, a key determinant in the permeability of nanoparticles. Using this computational model, we investigated the diffusion rate of polyethylene glycol (PEG) coated nanoparticles, a widely used muco-penetrating nanoparticle. We validated our model with experimentally characterized mucus pore sizes and the diffusional coefficients of PEG-coated nanoparticles in the mucus collected from cultured human colorectal goblet cells. Machine learning fingerprints were then employed to provide a mechanistic understanding of nanoparticle diffusional behavior. We found that larger nanoparticles tended to be trapped in mucus over longer durations but exhibited more ballistic diffusion over shorter time spans. Through these discoveries, our model provides a promising platform to study pharmacokinetics in the GI mucus layer.

Seihaito, a Kampo medicine, attenuates IL-13-induced mucus production and goblet cell metaplasia.

Goblet cell hyperplasia and increased mucus production are features of airway diseases, including asthma, and excess airway mucus often worsens these conditions. Even steroids are not uniformly effective in mucus production in severe asthma, and new therapeutic options are needed. Seihaito is a Japanese traditional medicine that is used clinically as an antitussive and expectorant. In the present study, we examined the effect of Seihaito on goblet cell differentiation and mucus production. In in vitro studies, using air-liquid interface culture of guinea-pig tracheal epithelial cells, Seihaito inhibited IL-13-induced proliferation of goblet cells and MUC5AC, a major component of mucus production. Seihaito suppressed goblet cell-specific gene expression, without changing ciliary cell-specific genes, suggesting that it inhibits goblet cell differentiation. In addition, Seihaito suppressed MUC5AC expression in cells transfected with SPDEF, a transcription factor activated by IL-13. Furthermore, Seihaito attenuated in vivo goblet cell proliferation and MUC5AC mRNA expression in IL-13-treated mouse lungs. Collectively, these findings demonstrated that Seihaito has an inhibitory effect on goblet cell differentiation and mucus production, which is at least partly due to the inhibition of SPDEF.

Reprogramming of arachidonic acid metabolism using α-terpineol to alleviate asthma: insights from metabolomics.

Asthma is a chronic inflammatory disorder in airways with typical pathologic features of airway inflammation and mucus hypersecretion. α-Terpineol is a monocyclic terpene found in many natural plants and foods. It has been reported to possess a wide range of pharmacological activities including anti-inflammatory and expectorant effects. However, the role of α-terpineol in asthma and its potential protective mechanism have not been well elucidated. This study is designed to investigate the pharmacological effect and mechanism of α-terpineol on asthmatic mice using the metabolomics platform. A murine model of asthma was established using ovalbumin (OVA) sensitization and then challenged for one week. The leukocyte count and inflammatory cytokines in the bronchoalveolar lavage fluid (BALF), lung histopathology, inflammatory  infiltrate and mucus secretion were evaluated. An ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS)-based metabolomics study was performed on lung tissues and serum to explore endogenous small molecule metabolites affected by α-terpineol in asthmatic mice. After α-terpineol treatment, leukocyte count, inflammatory cytokines in the BALF, and peribronchial inflammation infiltration were significantly downregulated. Goblet cell hyperplasia and mucus secretion were attenuated, with the level of Muc5ac in BALF decreased. These results proved the protective effect of α-terpineol against airway inflammation, mucus hypersecretion and Th1/Th2 immune imbalance. To further investigate the underlying mechanisms of α-terpineol in asthma treatment, UPLC-MS/MS-based metabolomics analysis was performed. 26 and 15 identified significant differential metabolites were found in the lung tissues and serum of the control, model and α-terpineol groups, respectively. Based on the above differential metabolites, enrichment analysis showed that arachidonic acid (AA) metabolism was reprogrammed in both mouse lung tissues and serum. 5-Lipoxygenase (5-LOX) and cysteinyl leukotrienes (CysLTs) are the key enzyme and the end product of AA metabolism, respectively. In-depth studies have shown that pretreatment with α-terpineol can alleviate asthma by decreasing the AA level, downregulating the expression of 5-LOX and reducing the accumulation of CysLTs in mouse lung tissues. In summary, this study demonstrates that α-terpineol is a potential agent that can prevent asthma via regulating disordered AA metabolism.

MUC5AC concentrations in lung lavage fluids are associated with acute lung injury after cardiac surgery.

Heart surgery may be complicated by acute lung injury and adult respiratory distress syndrome. Expression and release of mucins MUC5AC and MUC5B in the lungs has been reported to be increased in acute lung injury. The aim of our study was to [1] investigate the perioperative changes of MUC5AC, MUC5B and other biomarkers in mini-bronchoalveolar lavage (minBAL), and [2] relate these to clinical outcomes after cardiac surgery. In this prospective cohort study in 49 adult cardiac surgery patients pre- and post-surgery non-fiberscopic miniBAL fluids were analysed for MUC5AC, MUC5B, IL-8, human neutrophil elastase, and neutrophils. All measured biomarkers increased after surgery. Perioperative MUC5AC-change showed a significant negative association with postoperative P/F ratio (p = 0.018), and a positive association with ICU stay (p = 0.027). In conclusion, development of lung injury after cardiac surgery and prolonged ICU stay are associated with an early increase of MUC5AC as detected in mini-BAL.

The SIRT3 activator ganoderic acid D regulates airway mucin MUC5AC expression via the NRF2/GPX4 pathway.

PURPOSE: The expression of MUC5AC, a highly prevalent airway mucin, is regulated by stimulatory factors such as oxidative stress. Ganoderic acid D (GAD) activates mitochondrial deacetylase SIRT3. SIRT3 regulates mitochondrial function through deacetylation of mitochondrial proteins, thereby playing a significant role in alleviating oxidative stress-related diseases. Therefore, this study aimed to investigate the mechanisms and rationale underlying the regulation of MUC5AC expression by GAD. METHODS: Human airway epithelial cells (NCI-H292) were exposed to pyocyanin (PCN) to establish an in vitro cell model of airway mucus hypersecretion. The expression of SIRT3, MUC5AC, and NRF2 pathway proteins in cells was assessed. Cellular mitochondrial morphology and oxidative stress markers were analyzed. C57BL/6 mice were induced with Pseudomonas aeruginosa (PA) to establish an in vivo mouse model of airway mucus hypersecretion. The expression of SIRT3 and MUC5AC in the airways was examined. In addition, the differential expression of target genes in the airway epithelial tissues of patients with chronic obstructive pulmonary disease (COPD) was analyzed using publicly available databases. RESULTS: The results revealed a significant upregulation of MUC5AC expression and a significant downregulation of SIRT3 expression in relation to airway mucus hypersecretion. GAD inhibited the overexpression of MUC5AC in PCN-induced NCI-H292 cells and PA-induced mouse airways by upregulating SIRT3. GAD activated the NRF2/GPX4 pathway and inhibited PCN-induced oxidative stress and mitochondrial morphological changes in NCI-H292 cells. However, ML385 inhibited the regulatory effects of GAD on MUC5AC expression. CONCLUSION: The SIRT3 activator GAD downregulated MUC5AC expression, potentially through activation of the NRF2/GPX4 pathway. Accordingly, GAD may be a potential treatment approach for airway mucus hypersecretions.

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.

ANRIL regulating the secretion of Muc5ac induced by atmospheric PM2.5 via NF-κB pathway in Beas-2B cells.

PM2.5 can cause airway inflammation and promote the excessive secretion of mucin 5ac (Muc5ac), which can further induce many respiratory diseases. Antisense non-coding RNA in the INK4 locus (ANRIL) might regulate the inflammatory responses mediated by nuclear factor kappa-B (NF-κB) signaling pathway. Beas-2B cells were used to clarify the role of ANRIL in the secretion of Muc5ac induced by PM2.5 . The siRNA was used to silence ANRIL expression. Normal and gene silenced Beas-2B cells were respectively exposed to different doses of PM2.5 for 6, 12, and 24 h. The survival rate of Beas-2B cells was detected by methyl thiazolyl tetrazolium (MTT) assay. Tumor necrosis factor-α (TNF-α), interleukin-1ß (IL-1ß) and Muc5ac levels were determined by enzyme linked immunosorbent assay (ELISA). The expression levels of NF-κB family genes and ANRIL were detected by real time polymerase chain reaction (PCR). The levels of NF-κB family proteins and NF-κB family phosphorylated proteins were determined using Western blot. Immunofluorescence experiments were performed to observe the nuclear transposition of RelA. PM2.5 exposure increased the levels of Muc5ac, IL-1ß and TNF-α, and ANRIL gene expression (p < .05). With the dose and time of PM2.5 exposure increasing, the protein levels of inhibitory subunit of nuclear factor kappa-B alpha (IκB-α), RelA, and NF-κB1 decreased, the protein levels of phosphorylated RelA (p-RelA) and phosphorylated NF-κB1 (p-NF-κB1) increased, and RelA nuclear translocation increased, which indicated that the NF-κB signaling pathway was activated (p < .05). Silencing ANRIL could decrease the levels of Muc5ac, IL-1ß, TNF-α, decrease NF-κB family genes expression, inhibit the degradation of IκB-α and the activation of NF-κB pathway (p < .05). ANRIL played a regulatory role in the secretion of Muc5ac and the inflammation induced by atmospheric PM2.5 via NF-κB pathway in Beas-2B cells. ANRIL could be a target for prevention and treatment of the respiratory diseases caused by PM2.5 .

Immunohistochemical detection of MUC5AC and MUC5B mucins in ferrets.

OBJECTIVE: Cystic fibrosis (CF) is a genetic condition that causes abnormal mucus secretions in affected organs. MUC5AC and MUC5B are gel-forming mucins and frequent targets for investigations in CF tissues. Our objective was to qualify MUC5AC and MUC5B immunohistochemical techniques to provide a useful tool to identify, localize and interpret mucin expression in ferret tissues. RESULTS: MUC5AC and MUC5B mucins were detected most commonly in large airways and least in small airways, consistent with reported goblet cell density in airway surface epithelia. We evaluated whether staining method affected the detection of goblet cell mucins in serial sections of bronchial surface epithelia. Significant differences between stains were not observed suggesting common co-expression MUC5AC and MUC5B proteins in goblet cells of airway surface epithelia. Gallbladder and stomach tissues are reported to have differential mucin enrichment, so we tested these tissues in wildtype ferrets. Stomach tissues were enriched in MUC5AC and gallbladder tissues enriched in MUC5B, mucin enrichment similar to human tissues. Mucin immunostaining techniques were further qualified for specificity using lung tissue from recently generated MUC5AC-/- and MUC5B-/- ferrets. Qualified techniques for MUC5AC and MUC5B immunohistochemistry will be useful tools for mucin tissue studies in CF and other ferret models.

Alterations in p53, Microsatellite Stability and Lack of MUC5AC Expression as Molecular Features of Colorectal Carcinoma Associated with Inflammatory Bowel Disease.

Colitis-associated colorectal carcinoma (CAC) occurs in inflammatory bowel disease (IBD) because of the "chronic inflammation-dysplasia-cancer" carcinogenesis pathway characterized by p53 alterations in the early stages. Recently, gastric metaplasia (GM) has been described as the initial event of the serrated colorectal cancer (CRC) process, resulting from chronic stress on the colon mucosa. The aim of the study is to characterize CAC analyzing p53 alterations and microsatellite instability (MSI) to explore their relationship with GM using a series of CRC and the adjacent intestinal mucosa. Immunohistochemistry was performed to assess p53 alterations, MSI and MUC5AC expression as a surrogate for GM. The p53 mut-pattern was found in more than half of the CAC, most frequently stable (MSS) and MUC5AC negative. Only six tumors were unstable (MSI-H), being with p53 wt-pattern (p = 0.010) and MUC5AC positive (p = 0.005). MUC5AC staining was more frequently observed in intestinal mucosa, inflamed or with chronic changes, than in CAC, especially in those with p53 wt-pattern and MSS. Based on our results, we conclude that, as in the serrated pathway of CRC, in IBD GM occurs in inflamed mucosa, persists in those with chronic changes and disappears with the acquisition of p53 mutations.

Effects of Shiwei Longdanhua formula on LPS induced airway mucus hypersecretion, cough hypersensitivity, oxidative stress and pulmonary inflammation.

Shiwei Longdanhua Granule (SWLDH) is a classic Tibetan medicine (TM) ranking in the top 20 Chinese patent medicines in prescription rate to treat respiratory diseases like pneumonia, acute and chronic tracheobronchitis, acute exacerbation of COPD and bronchial asthma in solution of inflammation, cough and phlegm obstruction in clinical practice. However, its systematic pharmacological mechanisms have not been elucidated yet. Here, we studied the therapeutic efficacy of SWLDH in treatment of acute respiratory diseases in BALB/c mice by comprehensive analysis of airway inflammation, oxidative stress, mucus hypersecretion, cough hypersensitivities and indicators associated with the development of chronic diseases. Our results show that SWLDH might exhibit its inhibitory effects on pulmonary inflammation by interference with arachidonic acid (AA) metabolism pathways. Oxidative stress that highly related to the degree of tissue injury could be alleviated by enhancing the reductive activities of glutathione redox system, thioredoxin system and the catalytic activities of catalase and superoxide dismutase (SOD) after SWLDH treatment. In addition, SWLDH could significantly abrogate the mucus hypersecretion induced bronchiole obstruction by inactivate the globlet cells and decrease the secretion of gel-forming mucins (MUC5AC and MUC5B) under pathological condition, demonstrating its mucoactive potency. SWLDH also showed reversed effects on the release of neuropeptides that are responsible for airway sensory hypersensitivity. Simultaneously observed inhibition of calcium influx, reduction in in vivo biosynthesis of acetylcholine and the recovery of the content of cyclic adenosine monophosphate (cAMP) might collaboratively contribute to cause airway smooth muscle cells (ASMCs) relexation. These findings indicated that SWLDH might exhibited antitussive potency via suppression of the urge to cough and ASMCs contraction. Moreover, SWLDH might affect airway remodeling. We found SWLDH could retard the elevation of TGF-ß1 and α-SMA, which are important indicators for hyperplasia and contraction during the progression of the chronic airway inflammatory diseases like COPD and asthma.

Mucus increases cell iron uptake to impact the release of pro-inflammatory mediators after particle exposure.

We tested the hypothesis that (1) mucus production can be included in the cell response to iron deficiency; (2) mucus binds iron and increases cell metal uptake; and subsequently (3) mucus impacts the inflammatory response to particle exposure. Using quantitative PCR, RNA for both MUC5B and MUC5AC in normal human bronchial epithelial (NHBE) cells decreased following exposures to ferric ammonium citrate (FAC). Incubation of mucus-containing material collected from the apical surface of NHBE cells grown at air-liquid interface (NHBE-MUC) and a commercially available mucin from porcine stomach (PORC-MUC) with iron demonstrated an in vitro capacity to bind metal. Inclusion of either NHBE-MUC or PORC-MUC in incubations of both BEAS-2B cells and THP1 cells increased iron uptake. Exposure to sugar acids (N-acetyl neuraminic acid, sodium alginate, sodium guluronate, and sodium hyaluronate) similarly increased cell iron uptake. Finally, increased metal transport associated with mucus was associated with a decreased release of interleukin-6 and -8, an anti-inflammatory effect, following silica exposure. We conclude that mucus production can be involved in the response to a functional iron deficiency following particle exposure and mucus can bind metal, increase cell uptake to subsequently diminish or reverse a functional iron deficiency and inflammatory response following particle exposure.

Clinicopathological Factors Affecting Mucin 1, Mucin 2, and Mucin 5AC Staining in Patients who Underwent Resection for Colorectal Cancer.

OBJECTIVE: To investigate the clinicopathological factors affecting mucins (MUC 1, MUC 2, and MUC 5AC) staining in patients who underwent resection for colorectal cancer. STUDY DESIGN: An observational study. Place and Duration of the Study: Department of General Surgery and Department of Pathology, Kafkas University Faculty of Medicine, Kars, Turkey, between January 2020 and January 2021. METHODOLOGY: Patients operated on for colorectal adenocarcinoma were included in the study. Patients who underwent colorectal surgery for benign diseases or had a pathological diagnosis other than adenocarcinoma were excluded from the study. Clinicopathological factors affecting MUC1, MUC2, and MUC5AC staining were evaluated with appropriate statistical tests, assuming a significant p-value of less than 0.05. RESULTS: Of the 30 patients who met all study criteria, 18 (60%) were males. The mean age of all patients was 62.83±16.79 (21-88). MUC1 strongly positive staining was observed in 18 (60%) cases, and high expression was detected in pT4 and pT3 cases (p=0.005). In addition, increased expression was also noted in cases with lymph node involvement (p=0.045). MUC2 expression was more than 60% (strongly positive) in 20 (66.7%). The MUC2 expression was increased in moderately differentiated cases (p=0.032). There was no staining (negativity) in 22 (73.3%) cases with MUC5AC, and more than 60% staining (strongly positive) was observed in 3 (10%) cases. In addition, strong expression was noted in rectosigmoid tumours (p=0.001), female patients (p=0.046), and patients with pT3 and pT4 tumours (p=0.05). CONCLUSION: High MUC1 and high MUC5AC staining were observed in advanced colorectal cancer, whereas high MUC2 staining was observed in patients with moderate tumour differentiation. KEY WORDS: Colorectal cancers, Gene expressions, Mucin.

[Dickkopf-1 inhibits the secretion of MUC5AC induced by Mycoplasma pneumoniae P1-C in mouse lung epithelial cells].

Mycoplasma pneumoniae is the most common pathogen of respiratory tract infection in children and adults. Clinical observation shows that M. pneumoniae infection can cause massive mucus secretion in the respiratory tract, which makes the breathing of patients difficult. Studies have shown that M. pneumoniae infection can cause massive secretion of mucin 5AC (MUC5AC). Adhesin P1 plays an important role in the pathogenesis of M. pneumoniae infection by mediating the adhesion of pathogens to host cells, and the C-terminal residues of P1 (P1-C) are immunogenic. This study investigated the molecular mechanism of Wnt/ß-catenin signaling pathway inhibitor Dickkopf-1 (DKK1) in the secretion of MUC5AC in mouse airway epithelial cells (MAECs) induced by P1-C. Scanning electron microscope and hematoxylin-eosin staining were used to observe the effect of P1-C on mucus secretion of MAECs. Protein chip was used to detect the secretion of cytokines and analyse the enrichment of related signaling pathways induced by P1-C in MAECs. Periodic acid schiff stain (PAS) staining, Tunel staining and Masson staining were used to detect the damage of the lungs of mouse exposed to P1-C. Immunohistochemistry was used to detect the secretion of MUC5AC expression, and Western blotting was used to reveal the molecular mechanism of DKK1-regulated secretion of MUC5AC induced by P1-C protein in MACES. The results showed that P1-C induced the massive secretion of mucus and inflammatory factors in MAECs. During P1-C infection, DKK1 down-regulated janus kinase 2 (JAK2), phosphorylation signaling and transcription activator 1 (p-STAT1) and phosphorylation signaling and activator of transcription 3 (p-STAT3) expression. Overexpression of DKK1 significantly up-regulated the expression of MUC5AC repressor transcription factor fork-head box protein A2 (FOXA2). At the same time, the expression of MUC5AC induced by P1-C was inhibited significantly. It is speculated that DKK1 can effectively reduce the secretion of MUC5AC in MAECs induced by P1-C by inhibiting the JAK/STAT1-STAT3 signaling pathway and up-regulating the expression of FOXA2.

Hypoxic conditions promote a proliferative, poorly differentiated phenotype in COPD lung tissue progenitor cells in vitro.

PURPOSE: Chronic obstructive pulmonary disease (COPD) patients experience hypoxemia and lung tissue hypoxia, causing vasoconstriction, and at its most severe Cor pulmonale. However, minimal attention has been given to the effects of hypoxia at the cellular level. We hypothesize that a persistent progenitor cell population undergoes an aberrant differentiation process, influenced by changes in oxygen. METHODS: Distal lung progenitor cells from two emphysematous donors were cultured in 21% and 2% oxygen. Proliferation was determined on collagen-coated plastic and in 3T3-J2 co-culture. Epithelial (E-cadherin, pan-cytokeratin) and progenitor (TP63, cytokeratin 5) marker expressions were examined. Cells were differentiated at air-liquid interface, and ciliated, mucus-producing, and club cell populations identified by immunofluorescence. MUC5AC, MUC5B, CC10, and TP63 expression were determined using qRT-PCR, mucin5AC, and mucin5B protein levels by ELISA, and secreted mucin by periodic acid biotin hydrazide assay. RESULTS: Cells were positive for epithelial and progenitor markers at isolation and passage 5. Passage 5 cells in hypoxia increased the proportion of TP63 by 10% from 51.6 ± 1.2% to 62.6 ± 2.3% (p ≤ 0.01). Proliferative capacity was greater on 3T3J2 cells and in 2% oxygen, supporting the emergence of a proliferation unrestricted population with limited differentiation capacity. Differentiation resulted in ßIV tubulin positive-ciliated cells, mucin5AC, mucin5B, and CC10 positive secretory cells. Epithelial barrier formation was reduced (p ≤ 0.0001) in hypoxia-expanded cells. qRT-PCR showed higher mucin expression in 2% cells, significantly so with MUC5B (p ≤ 0.05). Although overall mucin5AC and mucin5B content was greater in 21% cells, normalization of secreted mucin to DNA showed a trend for increased mucin by low oxygen cells. CONCLUSIONS: These results demonstrate that hypoxia promotes a proliferative phenotype while affecting subsequent progenitor cell differentiation capacity. Furthermore, the retained differentiation potential becomes skewed to a more secretory phenotype, demonstrating that hypoxia may be contributing to disease symptoms and severity in COPD patients.

Nebulized Menthol Impairs Mucociliary Clearance via TRPM8 and MUC5AC/MUC5B in Primary Airway Epithelial Cells.

Flavorings enhance the palatability of e-cigarettes (e-cigs), with menthol remaining a popular choice among e-cig users. Menthol flavor remains one of the only flavors approved by the United States FDA for use in commercially available, pod-based e-cigs. However, the safety of inhaled menthol at the high concentrations used in e-cigs remains unclear. Here, we tested the effects of menthol on parameters of mucociliary clearance (MCC) in air-liquid interface (ALI) cultures of primary airway epithelial cells. ALI cultures treated with basolateral menthol (1 mM) showed a significant decrease in ciliary beat frequency (CBF) and airway surface liquid (ASL) volumes after 24 h. Menthol nebulized onto the surface of ALI cultures similarly reduced CBF and increased mucus concentrations, resulting in decreased rates of mucociliary transport. Nebulized menthol further increased the expression of mucin 5AC (MUC5AC) and mRNA expression of the inflammatory cytokines IL1B and TNFA. Menthol activated TRPM8, and the effects of menthol on MCC and inflammation could be blocked by a specific TRPM8 antagonist. These data provide further evidence that menthol at the concentrations used in e-cigs could cause harm to the airways.

Evaluation of MUC1, MUC2, MUC5AC, and MUC6 Expression Differences in Lung Adenocarcinoma Subtypes by Using a Final Immunoreactivity Score (FIRS).

OBJECTIVE: Lung adenocarcinomas are divided into acinar, lepidic, papillary, micropapillary, and solid predominant subtypes according to the current World Health Organization (WHO) classification. We designed this retrospective study to demonstrate profiles of MUC expression (MUC1, MUC2, MUC5AC, and MUC6) of different histologic patterns within the same tumor among pulmonary adenocarcinomas and investigate correlations of MUC expression with clinicopathologic features. MATERIAL AND METHOD: We analyzed the expression of mucins (MUC1, MUC2, MUC5AC, and MUC6) in a series of 99 resected lung adenocarcinomas, which included a total of 193 patterns (71 acinar, 30 lepidic, 25 papillary, 20 micropapillary, 34 solid and 13 mucinous) and calculated a final immune reactivity score (FIRS) per tumor. RESULTS: MUC1 IRS scores were significantly higher in lepidic and solid patterns compared with mucinous patterns (p=0.013). MUC2 expression was seen only in three cases (1 acinar, 2 mucinous). MUC5AC and MUC2 expression was more common in mucinous patterns (p < 0.001 and p=0.028, respectively). MUC6 expression was only detected in seven patterns and the expression was weak. No significant difference was seen among histologic patterns for the staining scores of MUC6. Mucinous adenocarcinoma differed from other histologic subtypes regarding MUC1 and MUC5AC expression. Mucinous adenocarcinoma showed less MUC1 expression with lower IRS scores and higher MUC5AC expression. Tumor size (p=0.006), lymphatic invasion (p=0.018), vascular invasion (p=0.025), perineural invasion (p=0.019), MUC1 IRS scores (p=0.018), and MUC1 IRS scores > 8.5 (p=0.018) were significant predictors for lymph node metastasis. CONCLUSION: An alternative scoring for MUC1 can be used as a predictor for lymph node metastasis regardless of the histologic subtype.

Tumor Microenvironment Prognostic Risk and Its Association With MUC5AC in Ampullary Carcinoma.

CONTEXT.­: The tumor-host interaction in the tumor microenvironment (TME) affects the prognosis of patients with malignant tumors. TME assessed via tumor budding (BD) and tumor-infiltrating lymphocyte (TIL) had a prognostic impact in patients with nonampullary small intestinal and colorectal carcinomas. In ampullary carcinoma (AC), MUC5AC was recently revealed as a significant prognosticator, but studies about the TME have not been conducted. OBJECTIVE.­: To assess TME-based prognostic risk in AC. DESIGN.­: We generated a collective TME risk index based on high-grade BD at the invasive front (BD3) and high density of stromal-TIL (>5%) in 64 surgically resected ACs. We evaluated its predictive values for overall survival (OS) and recurrence-free survival (RFS). We also investigated the relationship of TME to MUC5AC expression. RESULTS.­: TME prognostic risk index was classified into low-risk (BDLow/TILHigh; 26 of 64; 41%), intermediate-risk (BDLow/TILLow or BDHigh/TILHigh; 23; 36%), and high-risk (BDHigh/TILLow; 15; 23%) groups. Higher TME prognostic risk was associated with higher tumor grade (P = .03), lymphovascular invasion (P = .05), and MUC5AC immunopositivity (P = .02). TME prognostic risk index displayed better predictive ability for both OS (53.9 versus 46.1 versus 42.2) and RFS (24.8 versus 16.9 versus 15.3) than BD or TIL alone. In multivariate analysis, TME prognostic risk index was an independent prognosticator for OS (P = .003) and RFS (P = .03). CONCLUSIONS.­: TME risk index in combination with BD and TIL was a stronger predictor of prognostic risk stratification than either BD or TIL alone for both OS and RFS in patients with AC. MUC5AC may modulate the interaction between tumor cells and immunity toward enhancing invasiveness in TME.

The Regulation Mechanism of MUC5AC Secretion in Airway of Obese Asthma.

The purpose of this study was to establish a rat asthma model and extract MUC5AC to explore the mechanism of mucin 5AC (MUC5AC) signaling pathway regulating the function of asthmatic airway smooth muscle cells (ASMC) and participating in asthmatic airway remodeling. Western blot was used to detect ß-catenin (ß-catenin), glycogen synthase kinase-3ß (GSK-3ß), proto-oncogene MUC5AC and cyclin D1 (cyclin D1) in MUC5AC of asthmatic and normal groups. After inhibiting the interaction between ß-catenin and transcription cofactor p300 / CBP in ASMC of the asthma group and control group, the cell viability and cycle changes of ASMC were detected by the CCK-8 method and flow cytometry. After inhibiting the activity of P38 mitogen-activated protein kinase (MAPK), the protein expression changes of c-Myc and cyclin D1 were detected by Western blot. Results showed that comprehensive HE staining results of lung tissue sections indicate that the experimental rat model of asthma airway remodeling was successfully established. Compared with the control group, 100 fxmol and L1 Efaroxan promoted insulin secretion (P <0.01), and administration of the MUC5AC antagonist KU14R significantly inhibited the effect of MUC5AC.Western blot showed that the protein expression levels of ß-catenin, c-Myc and cyclin D1 in ASMC of the obese asthma group were significantly higher than those of the control group (P <0.05), while the protein expression level of GSK-3ß was lower than Control group (P <0.05). After inhibiting the interaction between ß-catenin and p300 / CBP, the decrease in cell viability and the degree of cell cycle change of ASMC in the asthma group were more obvious than those in the control group (P <0.05). After inhibiting the activity of P38 MAPK, the expressions of the target proteins c-Myc and cyclin D1 in the MUC5AC signaling pathway in ASMC model rats and control rats were down-regulated, and the difference was statistically significant (P <0.05). The conclusion was that the Wnt/ß-catenin signaling pathway can regulate the proliferation and differentiation of ASMC by up-regulating the expression level of cMyc. Cyclin D1 interacts with the MAPK signaling pathway, thereby affecting the function of ASMC and participating in asthma airway remodeling.

Autocrine TGF-alpha is associated with Benzo(a)pyrene-induced mucus production and MUC5AC expression during allergic asthma.

OBJECTS: Benzo(a)pyrene (BaP), an environmental pollutant, is present in high concentrations in urban smog and cigarette smoke and has been reported to promote high mucin 5AC (MUC5AC) expression. Epithelium-derived inflammatory cytokines are considered an important modulator of mucus oversecretion and MUC5AC overexpression. Here, we investigated whether the effect of BaP on MUC5AC overexpression was associated with cytokine autocrine activity in vivo and in vitro. METHODS: In vivo, BALB/c mice were treated with ovalbumin (OVA) in the presence or absence of BaP. Allergy-induced mucus production was assessed by Alcian Blue Periodic acid Schiff (AB-PAS) staining. The human airway epithelial cell line NCI-H292 was used in vitro. MUC5AC and transforming growth factor (TGF)-α mRNA levels were assessed with real-time quantitative PCR. The concentration of cytokines was measured by ELISA. The MUC5AC, p-ERK, ERK, p-EGFR and EGFR proteins were detected by Western blotting in cells or by immunohistochemistry in mouse lungs. Small-interfering RNAs were used for gene silencing. RESULTS: TGF-α was overproduced in the supernatant of NCI-H292 cells treated with BaP. Knockdown of TGF-α expression inhibited the BaP-induced increase in MUC5AC expression and subsequent activation of the EGFR-ERK signalling pathway. Knocking down aryl hydrocarbon receptor (AhR) expression or treatment with an ROS inhibitor (N-acetyl-L-cysteine) could relieve the TGF-α secretion induced by BaP in epithelial cells. In an animal study, coexposure to BaP with OVA increased mucus production, MUC5AC expression and ROS-EGFR-ERK activation in the lung as well as TGF-α levels in bronchoalveolar lavage fluid (BALF). Furthermore, the concentration of TGF-α in BALF was correlated with MUC5AC mRNA levels. Additionally, TGF-α expression was found to be positively correlated with MUC5AC expression in the airway epithelial cells of smokers. Compared with non-smoker asthma patients, TGF-α serum levels were also elevated in smoker asthma patients. CONCLUSION: Autocrine TGF-α was associated with BaP-induced MUC5AC expression in vitro and in vivo. BaP induced TGF-α secretion by activating AhR and producing ROS, which led to activation of the EGFR-ERK pathway.