MUC5AC mRNA Research Articles

Abstract 3714: Muc5ac plays an essential role in promotion of k-ras mutant lung cancer

Abstract Worldwide, lung cancer is the leading cause of cancer mortality, and cigarette smoking (CS) is its principal cause. However, several studies have found that smokers with chronic obstructive pulmonary disease (COPD), an inflammatory disease of the lung, have an increased risk of lung cancer (3 to 10-fold) compared to smokers with comparable cigarette exposure but without COPD. Importantly, among smokers with COPD, even following withdrawal of cigarette smoke, inflammation persists and lung function continues to deteriorate as does the increased risk of lung cancer. These facts suggest a strong link between COPD-related lung inflammation and lung cancer, however, the precise mechanistic link is not known. Mucociliary dysfunction and mucin hyperproduction are important features of COPD with inflammation being the major trigger. Interestingly, lung cancer with mucin overexpression has higher malignancy potential and poor prognosis, which around 76% having mutations in K-ras oncogene, the most frequent oncogenic mutation in lung adenocarcinoma. Taken these together, we hypothesized that mucins contribute to promotion of K-ras mutant lung cancer by inflammation. Here we first investigated whether Muc5ac, predominant airway mucin that plays a primary role in inflammatory lung diseases, is predictive of clinical outcome in KRAS-mutant human lung adenocarcinomas. We determined Muc5ac mRNA expression by array analysis of 150 lung adenocarcinomas from patients that did not received neoadjuvant therapy and we found that, Muc5ac mRNA level was a significant predictor of poor disease-free survival in KRAS-mutant lung adenocarcinomas. We have further found increased mucin and high expression of Muc5ac in lung tumor tissues of the mice with airway specific expression of a mutant form of K-ras (CC-LR mice). Then, we crossed previously developed Muc5ac knockout (KO) mice to CC-LR mice in order to develop a K-ras mutant lung cancer mouse model with lack of Muc5ac (CC-LR/Muc5ac KO mice). This resulted in a significant tumor reduction by ~54% (2.2-fold) in lung of CC-LR/Muc5ac KO mice compared to age and sex matched control CC-LR mice. Lung inflammation was evaluated by analysis of bronchoalveolar lavage fluid and revealed a significant reduction (3-fold) in number of macrophages, and levels of IL-6 and IL-17 in CC-LR/Muc5ac KO mice compared to CC-LR control mice. Immunohistopathological analysis of lung sections confirmed lower inflammation, decreased tumor number and size, less adenomatous lesions, and reduced tumor cell proliferation and angiogenesis in CC-LR mice with lack of Muc5ac compared to control CC-LR mice. Our experimental results suggest that Muc5ac has an essential role in promotion of K-ras mutant lung cancer through autocrine cell intrinsic and paracrine immune cell mediated mechanisms. Citation Format: Misha Umer, Amber M. Cumpian, Nasim Khosravi, Zoulikha Azzegagh, Maede Mohebnasab, Mauricio S. Caetano, Edwin J. Ostrin, Ignacio I. Wistuba, Burton Dickey, Humam Kadara, Christopher M. Evans, Seyed J. Moghaddam. Muc5ac plays an essential role in promotion of k-ras mutant lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3714. doi:10.1158/1538-7445.AM2017-3714

Giardia duodenalis Alters Intestinal Mucin Transcription and Disrupts the Mucus Layer in a Cysteine Protease‐dependent Manner

ObjectiveGiardia duodenalis is a diarrheagenic protozoan parasite that infects humans and animals across the world. Giardiasis is responsible for both acute and chronic illnesses, including post‐infectious irritable bowel syndrome (PI‐IBS) and extra‐intestinal complications. The mechanisms by which these occur remain unclear. Gastrointestinal mucus is mostly composed of MUC5AC mucin in the stomach and MUC2 in the intestine. We hypothesized that Giardia was able to overcome the protective functions of mucus to facilitate the onset and propagation of disease. Our aim was to assess whether and how Giardia proteases may impair the protective function of mucus.MethodsMuc2−/− (KO‐inf) and C57BL/6 (WT‐inf) mice were gavaged with Giardia trophozoites. Seven days post‐infection, tissues were collected for histology, qPCR, and analysis of bacterial translocation. Human colonic biopsies from healthy donors were exposed to Giardia trophozoites, and amounts of intracellular mucins were quantified. In vitro, Giardia trophozoites were incubated with a human mucus‐producing cell line, LS174T, to assess intracellular mucin content and MUC2 transcription. Trophozoites were incubated with or without E64 inhibitor to assess the role of its cysteine proteases. Lastly, Giardia's secreted products were incubated with purified human MUC2 to assess proteolysis via western blot.ResultsKO‐inf mice had a higher parasitic load compared to WT‐inf mice and lost weight over the period of infection. Compared to non‐infected mice, WT‐inf mice exhibited novel transcription of Muc5ac and decreased transcription of Muc2 in the jejunum. Both Muc2 and Muc5ac mRNA were increased in the colon. Muc5ac transcription was not induced in KO‐inf mice. WT‐inf mice had larger colonic mucus granules but a thinner mucus layer as demonstrated by staining with periodic‐acid Schiff/Alcian blue, wheat germ agglutinin, and fluorescent in situ hybridization for the location of commensal bacteria. Infected mice had bacterial translocation into the liver and spleen. Giardia spent media degraded human MUC2 in vitro. This was attenuated by E64. Human colonic biopsies exposed to Giardia had less intracellular mucin content. Giardia reduced intracellular mucin content in LS174T cells after 20, 40, and 60 minutes post‐infection, whereas it increased mucin content by 3 hours post‐infection. These effects were cysteine protease‐dependent and involved cellular protein kinase C. In vitro, Giardia increased MUC2 transcription after 1 hour post‐infection in a cysteine protease‐dependent manner.ConclusionsMuc2 protects against Giardia accumulation and induced weight loss. Giardia disrupts this mucosal protective barrier by direct proteolysis of the mucus layer and by altering mucin gene transcription‐specifically an increase of Muc5ac and Muc2 in the colon, and a decrease of Muc2 in the jejunum. The resultant loss of the mucus barrier was associated with increased bacterial translocation. Giardia‐induced alterations in mucin transcription, in addition to mucin degradation and depletion, were attenuated by a cysteine protease inhibitor, indicating a potential target to reduce Giardia‐induced pathology. Future research is needed to determine how these effects may contribute to acute and chronic disease.Support or Funding InformationHost‐Parasite Interactions NSERC CREATE and the Canadian Association of Gastroenterology

Neutrophil elastase stimulates MUC5AC expression in human biliary epithelial cells: a possible pathway of PKC/Nox/ROS.

IntroductionBacterial infection and bile flow retardation form a vicious cycle which promotes stone formation and recurrence, and it seems that mucin overexpression plays an important role in this process. However, the mechanism of increased mucus secretion in the biliary tract by bacterial infection and its treatment remain unclear.Material and methodsHuman biliary epithelial cells were induced by neutrophil elastase (NE), and H2O2 production in the cell supernatants was detected by a specific kit, and then cells were pretreated with a H2O2 inhibitor, and expression of MUC5AC was detected by real-time polymerase chain reaction (PCR), Western blot, and immunohistochemistry. Moreover, selective PKC and Nox inhibitors, apocynin and bisindolylmaleimide I, were used to pretreat cells and detect H2O2, MUC5AC mRNA and protein expression. Then, we pretreated cells with selective inhibitors or NE, and detected transforming growth factor α (TGF-α) using an ELISA kit.ResultsH2O2 production increased in an NE dose-dependent manner (p < 0.001), and NE upregulated MUC5AC expression at both mRNA and protein levels, while DMTU, could reduce this high expression (p < 0.01 at mRNA level, p < 0.001 at grey analysis for western blot and p < 0.01 at mean density for immunohistochemical staining at protein level). Moreover, apocynin and bisindolylmaleimide I could reduce the H2O2 production stimulated by NE (p < 0.05), and reduce MUC5AC high expression (p < 0.01 at mRNA level, p < 0.001 at both grey analysis for western blot and mean density for immunohistochemical staining at protein level). In addition, NE induced TGF-α production, and any of the three selective inhibitors could reduce it (p < 0.05).ConclusionsNE-induced reactive oxygen species participated in the upregulation of MUC5AC production. Moreover, protein kinase C and NADPH oxidase (Nox) regulate MUC5AC production in NE-challenged human biliary epithelial cells.

Mometasone Furoate Suppresses PMA-Induced MUC-5AC and MUC-2 Production in Human Airway Epithelial Cells.

BackgroundMucus hypersecretion from airway epithelium is a characteristic feature of airway inflammatory diseases. Tumor necrosis factor α (TNF-α) regulates mucin synthesis. Glucocorticoids including mometasone fuorate (MF) have been used to attenuate airway inflammation. However, effects of MF on mucin production have not been reported.MethodsEffects of MF and budesonide (BUD) on the phorbol-12-myristate-13-acetate (PMA)–induction of mucin and TNF-α in human airway epithelial cells (NCI-H292) were investigated in the present study. Confluent NCI-H292 cells were pretreated with PMA (200 nM) for 2 hours. Subsequently, the cells were stimulated with MF (1–500 ng/mL) or BUD (21.5 ng/mL) for 8 hours. Dexamethasone (1 µg/mL) was used as the positive control. Real-time polymerase chain reaction was used to determine MUC2 and MUC5AC mRNA levels. The level of total mucin, MUC2, MUC5AC, and TNF-α in culture supernatants were measured using enzyme-linked immunosorbent assay.ResultsMF and BUD significantly suppressed MUC2 and MUC5AC gene expression in PMA-stimulated NCI-H292 cells. The inhibitory effects of the two steroid drugs were also observed in the production of total mucin, MUC2 and MUC5AC proteins, and TNF-α.ConclusionOur findings demonstrated that MF and BUD attenuated mucin and TNF-α production in PMA-induced human airway epithelial cells.

Peroxiredoxin 6 suppresses Muc5ac overproduction in LPS-induced airway inflammation through H2O2-EGFR-MAPK signaling pathway

Mucus hypersecretion is a prominent mechanism in airway inflammation. Muc5ac is a major component of mucus and can be activated by reactive oxygen species (ROS). Peroxiredoxin 6 (Prdx6) highly expresses in airway epithelium and protects the airway from oxidative stress. In this study, we investigated the roles of Prdx6 in lipopolysaccharide (LPS)-induced mucin production in mice. We found that the levels of H2O2 and the Muc5ac mRNA were significantly increased in Prdx6 (-/-) mice compared to those in C57BL/6J mice after LPS instillation, which were markedly inhibited by epithelial growth factor receptor (EGFR) inhibitor Elrotinib. In vitro studies showed that mRNA levels of Prdx6 were decreased while H2O2 and Muc5ac were increased in a dose-dependent manner after LPS exposure, with significant increase in Prdx6 knockdown bronchial epithelial cells compared with those in normal epithelial cells. LPS-induced Muc5ac release was significantly inhibited by EGFR inhibitor, p38 inhibitor and JNK inhibitor, but not ERK1/2 inhibitor, indicating that the H2O2-EGFR-MAPK pathway is likely involved in the responses. This study indicated that Prdx6 decreased LPS-induced Muc5ac increase and played important roles in mucin hypersecretion after LPS exposure.

Interleukin-33 induces mucin gene expression and goblet cell hyperplasia in human nasal epithelial cells

We investigated whether IL-33 is involved in mucus overproduction and goblet cell hyperplasia in eosinophilic chronic rhinosinusitis (ECRS). IL-33 mRNA was significantly higher in the eosinophilic CRS group than in the non-eosinophilic CRS group from human nasal polyps. IL-33 induced MUC5AC mRNA and MUC5AC protein, and also goblet cell hyperplasia at air liquid interface culture in human nasal epithelial cells. In addition to that, IL-33 induced MUC5B and FOXA3, and reduces FOXJmRNA.In conclusion, our present study demonstrated that the direct evidence of IL-33 which lead to increase mucin gene and protein expression, as well as goblet cell hyperplasia. This study provides novel insights into the role of IL-33 on mucus overproduction in eosinophilic inflammation of human airways.

Expression of airway mucus-associated proteins in rats with chronic obstructive pulmonary disease with a cold-dryness symptom pattern

OBJECTIVETo reveal the effects on expression of airway mucus-associated proteins in rats with chronic obstructive pulmonary disease (COPD) and a cold-dryness symptom pattern induced by elastase and smoking. METHODSThe COPD model was established with an elastase dose into the trachea combined with exposure to smoking; the COPD model cold-dryness symptom pattern was further developed by exposure to a cold, dry environment. After 90 days, pathologic lung sections, inflammatory cytokine levels (measured by enzyme linked immunosorbent assay), mRNA and protein expression of mucus-associated proteins and aquaporins (measured by real-time polymerase chain reaction and western blots) were examined. RESULTSCytokines interleukin-6 (IL-6), interleukin-8 (IL-8), and tumor necrosis factor-α (TNF-α) in the COPD and the cold-dryness symptom pattern COPD groups were all significantly higher than in controls (each P < 0.01). IL-6 and IL-8 levels were higher in the cold-dryness symptom pattern COPD group than in the COPD group (each P < 0.05). The AQP5 mRNA expression in the cold-dryness symptom pattern COPD and COPD groups was lower than in the control group (P < 0.01), and that in the cold-dryness symptom pattern COPD group was lower than the COPD group (P < 0.05). The expression of MUC5AC and MUC5B mRNAs in the cold-dryness symptom pattern COPD group and COPD group was higher than in the control group (each P < 0.01), and that in the cold-dryness symptom pattern COPD group was higher than the COPD group (P < 0.01, and P < 0.05, respectively). The ratio of MUC5AC mRNA/MUC5B mRNA was COPD group < the cold-dryness symptom pattern COPD group < the control group. AQP4 and AQP5 protein expression in the cold-dryness symptom pattern COPD group was lower than that in the COPD group which was lower again than in the control group. MUC5AC and MUC5B expression in the cold-dryness symptom pattern COPD group was higher than in the COPD group and higher again than in the control group. CONCLUSIONSCold-dryness affects the expression of mucus-associated protein mRNA and its corresponding proteins, reducing the secretion of aquaporins and increasing the secretion of mucins. Imbalance in aquaporins and mucins can affect the function of mucus, increasing airway obstruction.

IFN-γ Blocks Development of an Asthma Phenotype in Rhinovirus-Infected Baby Mice by Inhibiting Type 2 Innate Lymphoid Cells.

Early-life wheezing-associated infections with rhinovirus (RV) have been associated with asthma development in children. We have shown that RV infection of 6-day-old mice induces mucous metaplasia and airways hyperresponsiveness, which is dependent on IL-13, IL-25, and type 2 innate lymphoid cells (ILC2s). Infection of immature mice fails to induce lung IFN-γ expression, in contrast to mature 8-week-old mice with a robust IFN-γ response, consistent with the notion that deficient IFN-γ production in immature mice permits RV-induced type 2 immune responses. We therefore examined the effects of intranasal IFN-γ administration on RV-induced ILC2 expansion and IL-13 expression in 6-day-old BALB/c and IL-13 reporter mice. Airway responses were assessed by histology, immunofluorescence microscopy, quantitative polymerase chain reaction, ELISA, and flow cytometry. Lung ILC2s were also treated with IFN-γ ex vivo. We found that, compared with untreated RV-infected immature mice, IFN-γ treatment attenuated RV-induced IL-13 and Muc5ac mRNA expression and mucous metaplasia. IFN-γ also reduced ILC2 expansion and the percentage of IL-13-secreting ILC2s. IFN-γ had no effect on the mRNA or protein expression of IL-25, IL-33, or thymic stromal lymphoprotein. Finally, IFN-γ treatment of sorted ILC2s reduced IL-5, IL-13, IL-17RB, ST2, and GATA-3 mRNA expression. We conclude that, in immature mice, IFN-γ inhibits ILC2 expansion and IL-13 expression in vivo and ex vivo, thereby attenuating RV-induced mucous metaplasia. These findings demonstrate the antagonistic function of IFN-γ on ILC2 expansion and gene expression, the absence of which may contribute to the development of an asthma-like phenotype after early-life RV infection.

Clarithromycin Suppresses Chloride Channel Accessory 1 and Inhibits Interleukin-13-Induced Goblet Cell Hyperplasia in Human Bronchial Epithelial Cells.

Activation of the interleukin-13 (IL-13) receptor leads to signal transducer and activator of transcription 6 (STAT6) activation and subsequent induction of SAM pointed domain containing ETS transcription factor (SPDEF) and chloride channel accessory 1 (CLCA1), increasing secretion of the gel-forming mucin MUC5AC. Activation of the epidermal growth factor receptor (EGFR) also leads to MUC5AC production via extracellular signal-regulated kinase (ERK1/2). We examined the effect of clarithromycin IL-13 signaling leading to production. Normal human bronchial epithelial (NHBE) cells were grown for 14 days at an air-liquid interface (ALI) with IL-13 and/or clarithromycin. Histochemical analysis was performed using hematoxylin and eosin (HE) staining and MUC5AC immunostaining. MUC5AC, SPDEF, and CLCA1 mRNA expression were evaluated by real-time PCR. Western analysis was used to assess phosphorylation of STAT6 and ERK1/2. Clarithromycin decreased IL-13-induced goblet cell hyperplasia and MUC5AC mRNA expression in a dose-dependent manner. Clarithromycin decreased IL-13-stimulated SPDEF and CLCA1 mRNA expression in a dose-dependent manner, and at 32 μg/ml CLCA1 was profoundly decreased (P < 0.001). Although clarithromycin had no effect on STAT6 phosphorylation induced by IL-13, it decreased constitutive phosphorylation of ERK1/2 (P < 0.05).

S-allylmercapto-l-cysteine modulates MUC5AC and AQP5 secretions in a COPD model via NF-кB signaling pathway

Garlic has shown versatile medicinal activities in the prevention and treatment of diseases such as chronic obstructive pulmonary disease (COPD). However, no individual garlic bioactive components have yet been determined in the COPD treatment effects. In this work, S-allylmercapto-l-cysteine (SAMC) identified in the aged garlic was selected as a model compound to determine its COPD therapeutic potential. The COPD model was established by using lipopolysaccharides (LPS) to stimulate the human airway submucosal gland cell line SPC-A1. Previous studies show that both MUC5AC up-regulation and AQP5 down-regulation play an important role in viscous COPD mucus secretions. The modulation effects of SAMC on LPS-induced MUC5AC and AQP5 productions in SPC-A1 cells were then evaluated. Pretreatment of the SPC-A1 cells with SAMC attenuated MUC5AC secretion and increased AQP5 expression in a dose-dependent manner in the non-cytotoxic concentration range of 20 to 100μM. Mechanistic studies suggested that SAMC could suppress the accumulation of MUC5AC mRNA and inhibit IкBα degradation and NF-кB p65 translocation. These results suggest that SAMC could be a promising candidate in the prevention and treatment of MUC5AC-associated disorders such as COPD.

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

BackgroundExcessive 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. MethodsHuman 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. ResultsTransforming 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. ConclusionsThese results suggest that IFN-γ represses MUC5AC expression, disturbing binding of Sp1 to its target sequences.

Effectiveness of atorvastatin in suppressing MUC5AC gene expression in human airway epithelial cells.

We recently reported that chronic cholesterol depletion in NCI-H292 cells by lovastatin suppresses interleukin (IL)-1β-induced MUC5AC gene expression. However, as there are numerous statins affect MUC5AC expression, we sought to determine which statin is most effective in reducing MUC5AC expression, and whether this activity of statins is related to IL-1 receptor (IL-1RI) and mitogen-activated protein kinase (MAPK) activity. Four statins, namely atorvastatin, fluvastatin, lovastatin, and simvastatin, were tested. Cholesterol depletion was measured via modified microenzymatic fluorescence assay and filipin staining. NCI-H292 cells were pretreated with 10 μM of each statin for 1 hour, 10 ng/mL of IL-1β was added, and cells were then co-incubated with statin and IL-1β for 24 hours. MUC5AC mRNA expression was measured via real-time polymerase chain reaction (PCR). The phosphorylation levels were assayed by Western blot. Cholesterol in the plasma membrane was markedly decreased by all 4 statins, of which atorvastatin was the most potent. IL-1β-induced MUC5AC messenger RNA (mRNA) expression was most significantly decreased by 10 μM atorvastatin, to 1.4 ± 0.2-fold of the level of the untreated control group, as opposed to an increase to 4.7 ± 0.5-fold for IL-1β alone, and this suppression of MUC5AC expression was dose-dependent. This decrease in MUC5AC expression by atorvastatin was mediated via the IL-1 receptor and the MAPK pathway, including both phospho-p38 MAPK and phospho-extracellular signal-regulated kinase (phospho-ERK). These results suggest that atorvastatin is the most potent of the assayed statins with respect to suppression of IL-1β-induced MUC5AC mRNA expression, and may be considered as an anti-hypersecretory agent.

Effect of methyleugenol on expression of MUC5AC in nasal mucosa of rats with allergic rhinitis

Objective: To investigate the effect of methyleugenol on expression of MUC5AC in nasal mucosa of rats with allergic rhinitis (AR). Methods: Seventy-two Wistar rats were randomly divided into 6 groups:normal control group, AR group, loratadine group, low-dose methyleugenol group, middle-dose methyleugenol group and high-dose methyleugenol group with 12 rats in each group. AR was induced by intraperitoneal injection of ovalbumin in latter 5 groups. 10 mg loratadine q.d was given to rats in loratadine group by gavage; and 10 mg/kg, 20 mg/kg and 40 mg/kg methyleugenol were given by gavege q.d to rats in low-, middle-and high-dose methyleugenol groups, respectively. Nasal mucosa samples were obtained from rats at 1, 2, 4 and 6 weeks after drug intervention. The expression of MUC5AC protein and mRNA in nasal mucosa was detected by immunohistochemistry and real-time fluorescence quota PCR (RT-PCR), respectively. Results: Compared with AR, the percentage of cells staining positively for MUC5AC protein and the relative quantity of MUC5AC mRNA in middle-and high-dose methyleugenol groups were significantly decreased after 2 and 4 weeks of drug intervention (P<0.05), but no such decrease was observed in low-dose methyleugenol group at all time points (P>0.05). The percentage of cells with positive expression of MUC5AC protein and mRNA in loratadine group were significantly decreased after 1 week of administration (P<0.05). The percentage of cells with positive MUC5AC protein in middle-dose methyleugenol group was higher than that in loratadine group (P<0.05) after 6 week of drug intervention, but the difference was not seen in high-dose group (P>0.05). There was no significant difference in relative quantities of MUC5AC mRNA after 4 weeks of administration between high-and middle-dose methyeugenol groups and loratadine group (P>0.05). Conclusion: Methyleugenol can attenuate AR through inhibiting the expression of MUC5AC mRNA and protein in nasal mucosa of AR rats.

Cold-inducible RNA binding protein regulates mucin expression induced by cold temperatures in human airway epithelial cells

Mucus overproduction is an important manifestation of chronic airway inflammatory diseases, however, the mechanisms underlying the association between cold air and mucus overproduction remain unknown. We found that the expression of the cold-inducible RNA binding protein (CIRP) was increased in patients with chronic obstructive pulmonary disease (COPD). In the present study, we tested whether CIRP was involved in inflammatory factors and mucin5AC (MUC5AC) expression after cold stimulation and investigated the potential signaling pathways involved in this process. We found that CIRP was highly expressed in the bronchi of COPD patients. The expression of CIRP, interleukin-1β (IL-1β) and tumor necrosis factor α (TNF-α) were increased, and the CIRP was localized in cytoplasm after cold stimulation. MUC5AC mRNA and protein expression levels were elevated in a temperature- and time-dependent manner after cold stimulation and were associated with the phosphorylation of ERK and NF-κB, which reflected their activation. These responses were suppressed by knockdown of CIRP with a specific siRNA or the ERK and NF-κB inhibitors. These results demonstrated that CIRP was expressed in the bronchi of human COPD patients and was involved in inflammatory factors and MUC5AC expression after cold stimulation through the ERK and NF-κB pathways.

The role of type 2 innate lymphoid cells in RSV-induced asthma exacerbation.

Abstract Respiratory viral infections cause devastating airway inflammation and remodeling in asthmatic children and adults. Type 2 innate lymphoid cells (ILC2) have been characterized individually in both asthma and viral infections. We hypothesize that ILC2 are activated by IL-33 in our model, and thereby enhance the asthmatic phenotype in the lung. Asthma induction was performed in eight week old, female BALB/c mice using ovalbumin (OVA) sensitization and airway challenge. After the last airway challenge, mice were infected with RSV (3,250 TCID50 U/mouse). Lungs were collected for ILC2 sorting, immune cell quantitation, and analysis of IL-4, IL-13, IL-33 and IL-25 on days 1–4 of infection. A subset of lungs were collected and fixed for histological analysis. Results show that mucus production (PAS+ cells) and lymphocytic recruitment were enhanced with RSV infection and asthma. The expression of MUC5AC, CLCA3, IL-25 and IL-33 mRNA levels were increased at least two-fold in the lungs of OVA+RSV mice compared to RSV-infected alone. We detected comparable numbers of ILC2 in BAL fluid from both OVA+ and OVA+RSV mice, yet an evaluation of total lung lymphocytes yielded more ILC2 in the OVA+RSV mice as compared to OVA alone. ILC2 from OVA+RSV mice were sorted and treated with IL-33 for 24 hours. These cells produced greater IL-4 mRNA than cells isolated from OVA only mice. Our results describe a potential role for ILC2 in the exacerbation of asthma with RSV infection. Future studies are planned to determine the exact contribution of ILC2 to type 2 immunity and pulmonary function in our asthma-viral infection model.

A novel dissociative steroid VBP15 reduces MUC5AC gene expression in airway epithelial cells but lacks the GRE mediated transcriptional properties of dexamethasone

Overproduction of secretory mucins contributes to morbidity/mortality in inflammatory lung diseases. Inflammatory mediators directly increase expression of mucin genes, but few drugs have been shown to directly repress mucin gene expression. IL-1β upregulates the MUC5AC mucin gene in part via the transcription factors NFκB while the glucocorticoid Dexamethasone (Dex) transcriptionally represses MUC5AC expression by Dex-activated GR binding to two GRE cis-sites in the MUC5AC promoter in lung epithelial cells. VBP compounds (ReveraGen BioPharma) maintain anti-inflammatory activity through inhibition of NFκB but exhibit reduced GRE-mediated transcriptional properties associated with adverse side-effects and thus have potential to minimize harmful side effects of long-term steroid therapy in inflammatory lung diseases. We investigated VBP15 efficacy as an anti-mucin agent in two types of airway epithelial cells and analyzed the transcription factor activity and promoter binding associated with VBP15-induced MUC5AC repression. VBP15 reduced MUC5AC mRNA abundance in a dose- and time-dependent manner similar to Dex in the presence or absence of IL-1β in A549 and differentiated human bronchial epithelial cells. Repression was abrogated in the presence of RU486, demonstrating a requirement for GR in the VBP15-induced repression of MUC5AC. Inhibition of NFκB activity resulted in reduced baseline expression of MUC5AC indicating that constitutive activity maintains MUC5AC production. Chromatin immunoprecipitation analysis demonstrated lack of GR and of p65 (NFκB) binding to composite GRE domains in the MUC5AC promoter following VBP15 exposure of cells, in contrast to Dex. These data demonstrate that VBP15 is a novel anti-mucin agent that mediates the reduction of MUC5AC gene expression differently than the classical glucocorticoid, Dex.

Asian Sand Dust Enhances the Inflammatory Response and Mucin Gene Expression in the Middle Ear.

Objectives.Asia sand dust (ASD) is known to cause various human diseases including respiratory infection. The aim of this study was to examine the effect of ASD on inflammatory response in human middle ear epithelial cells (HMEECs) in vitro and in vivo.Methods.Cell viability was assessed using the cell counting kit-8 assay. The mRNA levels of various genes including COX-2, TNF-a, MUC 5AC, MUC 5B, TP53, BAX, BCL-2, NOX4, and SOD1 were analyzed using semiquantitative realtime polymerase chain reaction. COX-2 protein levels were determined by western blot analysis. Sprague Dawley rats were used for in vivo investigations of inflammatory reactions in the middle ear epithelium as a result of ASD injection.Results.We observed dose-dependent decrease in HMEEC viability. ASD exposure significantly increased COX-2, TNF-a, MUC5AC, and MUC5B mRNA expression. Also, ASD affected the mRNA levels of apoptosis- and oxidative stress-related genes. Western blot analysis revealed a dose-dependent increase in COX-2 production. Animal studies also demonstrated an ASD-induced inflammatory response in the middle ear epithelium.Conclusion.Environmental ASD exposure can result in the development of otitis media.

Role of IFN-γ, IL-13, and IL-17 on mucociliary differentiation of nasal epithelial cells in chronic rhinosinusitis with nasal polyps.

Mucociliary dysfunction is a prominent pathophysiological feature of chronic rhinosinusitis with nasal polyps (CRSwNP); however, the precise mechanisms underlying mucociliary dysfunction are still unclear. The aim of this study was therefore to evaluate the effects of IFN-γ, IL-13, and IL-17 on human nasal mucociliary differentiation and ciliary beat frequency (CBF) in patients with CRSwNP. Human nasal epithelial cells from tissue of patients with CRSwNP and control subjects were established as air-liquid interface (ALI) primary cultures. Confluent cultures were incubated with10 ng/mL each of IFN-γ, IL-13, or IL-17 for 14 days and assessed for expression of specific morphological markers and factors associated with mucociliary differentiation, the percentage of ciliated and goblet cells, and CBF. In comparison with control subjects, percentage of ciliated cells and CBF were decreased; while percentage of goblet cells, FOXJ1, and MUC5AC mRNA expression were increased in nasal polyp-derived epithelial cultures. Treatment with IFN-γ and IL-13 significantly decreased the expression of β-tubulin IV (specific cilia marker), ciliated cell number, and expression of FOXJ1 and DNAI2, in epithelial cultures derived from both CRSwNP patients and control subjects. Furthermore, while both IFN-γ and IL-13 treatment significantly decreased the CBF of cells from both CRSwNP patients and control subjects, IL-13 additionally significantly increased goblet cell number and the expression of MUC5AC and CLCA1, in these cultures. IL-17 treatment did not significantly affect ciliated or goblet cell differentiation, CBF, nor MUC5AC and CLCA1 expression, but increased both MUC5B mRNA and protein expression in these cultures. The demonstration that IFN-γ and IL-13 both significantly reduce ciliated cell differentiation and CBF in CRSwNP patients, and IL-13 additionally induces significant goblet cell hyperplasia and MUC5AC mucin expression, as well as IL-17 significantly increases MUC5B mucin expression, suggests that these inflammatory cytokines may be potential therapeutic targets in the management of CRSwNP.

Human neutrophil elastase induces MUC5AC overexpression in chronic rhinosinusitis through tumour necrosis factor-α converting enzyme

Conclusion The HNE–TACE signalling pathway has an important role in the process of MUC5AC overexpression in chronic rhinosinusitis (CRS). Objectives To provide evidence of HNE-induced MUC5AC overexpression in CRS via TACE. Method HE and PAS staining were used to assess the pathological changes in sinus mucosa samples from CRS or normal control. HNE, TACE, and MUC5AC expression in the sinonasal mucosa was determined using immunohistochemistry (IHC) and real-time polymerase chain reaction (qRT-PCR). In addition, the MUC5AC and TACE expression was determined in a primary culture of human nasal mucosa epithelial cells in vitro. Results On HE staining, the main pathological feature in the sinus mucosa of CRS patients was hyperplasia of goblet cells, inflammatory cells, and submucosal glands. Mucosa from the two experimental groups also showed strong expression on PAS staining. IHC and qRT-PCR demonstrated that HNE, TACE, and MUC5AC expression was significantly higher in the CRS patients compared with control samples (p < 0.05). MUC5AC mRNA expression was higher in cells stimulated by HNE than in untreated cells (p < 0.05). MUC5AC mRNA expression was significantly reduced in cells pre-treated with the TACE inhibitor TAPI-1 prior to HNE stimulation, compared with untreated and HNE-stimulated cells (p < 0.01).

Mediated mechanism of NDRG2 in the regulation of neutrophil nuclear transcription factor-κB signaling pathway through the deubiquitylating enzyme of Zc3h12d

To explore the role NDRG2/Zc3h12d in the regulation of neutrophil nuclear transcription factor-κB signaling pathway and the underlying mechanisms. Sixteen HBE cells were incubated with lipopolysaccharide (LPS) to establish airway mucus hypersecretion model, which was transfected with NDRG2 or Zc3h12d siRNA. The cells were divided into 5 groups: a LPS+NDRG2 siRNA (Group A), a LPS+ NDRG2 and Zc3h12d siRNAs (Group B), a LPS+Zc3h12d siRNA (Group C), a LPS+ empty plasmid (Group D), and a negative control group (Group E). The reverse transcription-polymerase chain reaction (RT-PCR) was used to measure the expression level of mucin (MUC) 5AC mRNA. The levels of MUC5AC and the inflammatory factors were examined by the enzyme-linked immunosorbent assay (ELISA). The NF-κB p65 and the Zc3h12d protein levels were measured by Western blot. The MUC5C expression was further examined by laser confocal method. Compared with Group B, the levels of MUC5AC mRNA and protein in Group A were decreased (P<0.05), there was no significant difference in the MUC5AC mRNA and MUC5AC protein levels between the Group B and the Group C (P>0.05). Compared with Group D, the MUC5AC (mRNA and protein) and inflammatory factor levels in the Group A were significantly decreased (P<0.05); compared with Group E, after incubation with LPS, the levels of MUC5AC (mRNA and protein) and inflammatory factors in the Group D was increased, with significantly difference (P<0.05). NDRG2 can regulate the function of NF-κB signaling pathway through the deubiquitylating enzyme of Zc3h12d.