MUC5AC Expression Research Articles

Role of notch signaling pathway in Muc5ac secretion induced by atmospheric PM2.5 in rats

BackgroundThe secretion of Muc5ac is closely related to the pathogenesis, treatment and prognosis of bronchial asthma. Atmospheric PM2.5 entered the airway can irritate and corrode the bronchial wall, affecting the expression and secretion of Muc5ac. However, the underlying mechanism is not clear. In this study, we investigated the role of the Notch signaling pathway in mucin section induced by atmospheric PM2.5 in rats. MethodsFifty rats were divided randomly into five groups: the control received physiological saline; the health, health Notch signaling pathway inhibition and asthma, asthma Notch signaling pathway inhibition groups received 7.5 mg/kg PM2.5. PM2.5 or saline was instilled into the trachea at 2-day intervals for two doses. IL-1β, TNF-α and Muc5ac levels were detected by ELISA. The mRNA expression levels of Notch signaling pathway genes were detected by real time PCR. The levels of Notch signaling pathway protein were detected by western blot. ResultsThe levels of Muc5ac in the lungs and TNF-α in serum of asthmatic rats exposed to PM2.5 was the highest, and when Notch signaling pathway was inhibited, the levels of Muc5ac in the lungs and tracheas and TNF-α in serum of asthmatic rats exposed to PM2.5 was significantly decreased. Hes1 mRNA expression level in trachea was the lowest in the asthma inhibition group; and inhibiting the Notch signaling pathway could decrease the mRNA and protein levels of Hes1 in rats’ lung. The mRNA relative levels of Notch3 and Notch4 in rats’ trachea, the protein levels of Notch3 in rats’ lung, and the mRNA relative levels of Jagged1 and Jaggeed2 in rats’ lung were more consist with the changes of Muc5ac, TNF-α and Hes1. ConclusionNotch signaling pathway played an important role in Muc5ac secretion induced by atmospheric PM2.5 of the asthmatic rats’ airways. Jagged1 and Jagged2 interacting with Notch3 and Notch4 regulated the expression of Hes1, further regulated TNF-α in the process of PM2.5 inducing the secretion of Muc5ac.

Epithelial Aryl Hydrocarbon Receptor Protects From Mucus Production by Inhibiting ROS-Triggered NLRP3 Inflammasome in Asthma.

BackgroundDespite long-standing recognition in the significance of mucus overproduction in asthma, its etiology remains poorly understood. Muc5ac is a secretory mucin that has been associated with reduced pulmonary function and asthma exacerbations.ObjectivesWe sought to investigate the immunological pathway that controls Muc5ac expression and allergic airway inflammation in asthma.MethodsCockroach allergen-induced Muc5ac expression and aryl hydrocarbon receptor (AhR) signaling activation was examined in the human bronchial epithelial cells (HBECs) and mouse model of asthma. AhR regulation of Muc5ac expression, mitochondrial ROS (Mito-ROS) generation, and NLRP3 inflammasome was determined by AhR knockdown, the antagonist CH223191, and AhR-/- mice. The role of NLRP3 inflammasome in Muc5ac expression and airway inflammation was also investigated.ResultsCockroach allergen induced Muc5ac overexpression in HBECs and airways of asthma mouse model. Increased expression of AhR and its downstream genes CYP1A1 and CYP1B1 was also observed. Mice with AhR deletion showed increased allergic airway inflammation and MUC5AC expression. Moreover, cockroach allergen induced epithelial NLRP3 inflammasome activation (e.g., NLRP3, Caspase-1, and IL-1β), which was enhanced by AhR knockdown or the antagonist CH223191. Furthermore, AhR deletion in HBECs led to enhanced ROS generation, particularly Mito-ROS, and inhibition of ROS or Mito-ROS subsequently suppressed the inflammasome activation. Importantly, inhibition of the inflammasome with MCC950, a NLRP3-specifc inhibitor, attenuated allergic airway inflammation and Muc5ac expression. IL-1β generated by the activated inflammasomes mediated cockroach allergen-induced Muc5ac expression in HBECs.ConclusionsThese results reveal a previously unidentified functional axis of AhR-ROS-NLRP3 inflammasome in regulating Muc5ac expression and airway inflammation.

CC16 Deficiency in the Context of Early-Life Mycoplasma pneumoniae Infection Results in Augmented Airway Responses in Adult Mice.

Studies have shown that club cell secretory protein (CC16) plays important protective roles in the lungs, yet its complete biological functions are unclear. We devised a translational mouse model in order to investigate the impact of early life infections, in the context of CC16 deficiency, on lung function in adult mice. CC16 sufficient (WT) and deficient (CC16-/-) mice were infected with Mycoplasma pneumoniae (Mp) as weanlings and assessed as adults (early life infection model; ELIM) and compared to adult mice infected for only 3 days (adult infection model; AIM). CC16-/- Mp-infected mice had significantly increased airway hyperresponsiveness (AHR) in both models compared to WT mice. However, CC16-/- mice infected in early life (ELIM) displayed significantly increased AHR compared to CC16-/- mice infected in adulthood (AIM). In stark contrast, lung function in ELIM WT mice returned to levels similar to saline-treated controls. While WT mice cleared Mp infection in the ELIM, CC16-/- mice remained colonized with Mp throughout the model, which likely contributed to increased airway remodeling and persistence of Muc5ac expression. When CC16-/- mouse tracheal epithelial cells (MTECs) were infected with Mp, increased Mp colonization and collagen gene expression were also detected compared to WT cells, suggesting that CC16 plays a protective role during Mp infection, in part through epithelial-driven host defense mechanisms.

Erythromycin reduces nasal inflammation by inhibiting immunoglobulin production, attenuating mucus secretion, and modulating cytokine expression

Allergic rhinitis (AR) and chronic rhinosinusitis (CRS) share some similar pathological mechanisms. In current study, we intend to investigate the impact of AR on CRS. In addition, we explored the efficacy of erythromycin (EM) treatment on CRS mice with or without AR (CRSwoAR, CRSwAR). Study subjects were divided into control, CRSwoAR, and CRSwAR groups. Experimental mice were divided similarly into control, CRSwoAR, and CRSwAR groups. In addition, CRS mice were treated with EM at 0.75, 7.5, or 75 mg/kg or with dexamethasone (Dex) at 1 mg/kg. In our results, allergy exacerbates inflammation that was evident in nasal histology and cytokine expression both in patients and in mice with CRS. Dex 1 mg/kg, EM 7.5 or 75 mg/kg treatments significantly inhibited serum IgE and IgG2a in CRS mice. EM-treated CRS mice had significantly elevated IL-10 levels and had a reversal of Th-1/Th-2 cytokine expression in nasal-associated lymphoid tissue. MUC5AC expressions were significantly reduced in the 7.5 or 75 mg/kg EM-treated mice compared with untreated mice. EM showed inhibitions on immunoglobulin production and mucus secretion stronger than Dex. We concluded that comorbid AR enhanced inflammation of CRS. EM and Dex treatments showed similar anti-inflammatory effects on CRS but through partly different mechanisms.

PM2.5 Upregulates the Expression of MUC5AC via the EGFR-PI3K Pathway in Human Sinonasal Epithelial Cells

Background: Fine particulate matter (PM) (PM with an aerodynamic diameter <2.5 μm, PM2.5) exposure contributes to respiratory disease development and exacerbation. Objective: We sought to investigate the effect of PM2.5 exposure on mucociliary function in primary human nasal epithelial cells (HNECs) and the underlying mechanism. Methods: HNECs derived from control subjects and patients with chronic rhinosinusitis with nasal polyps were established as air-liquid interface cultures. Confluent cultures were exposed to 100 or 200 μg/mL PM2.5 for 24 h and assessed for expression of specific mucociliary-associated factors, the percentage of β-tubulin IV-positive and MUC5AC-positive cells, expression of epidermal growth factor receptor (EGFR) ligand and activation of phosphoinositide 3-kinase (PI3K)-AKT/ERK. In addition, cultures pretreated for 30 min with AG1478 (an EGFR inhibitor) or LY294002 (a PI3K inhibitor) following PM2.5 exposure were assessed for MUC5AC mRNA and protein expression. Results: PM2.5 exposure at 100 or 200 μg/mL for 24 h did not affect geminin coiled-coil domain containing, multiciliate differentiation and DNA synthesis associated cell cycle protein, FOXJ1, or DNAI2 mRNA expression or the percentage of β-tubulin IV-positive cells. However, 200 μg/mL PM2.5 exposure significantly increased mRNA expression of SAM-pointed domain-containing ETS transcription factor and MUC5AC and the percentage of MUC5AC-positive cells. PM2.5 also increased expression of EGFR ligands, including heparin-binding EGF-like growth factor and amphiregulin. Furthermore, PM2.5induced activation of PI3K, AKT, and ERK, and pretreatment of HNECs with AG1478 or LY294002 attenuated PM2.5-induced MUC5AC mRNA and protein expression. Conclusions and Clinical Relevance: This study demonstrates that short-term PM2.5 exposure increases MUC5AC expression in HNECs. Furthermore, this study shows that PM2.5-induced MUC5AC expression is likely mediated through the EGFR-PI3K pathway.

Guifu Dihuang Pills Ameliorated Mucus Hypersecretion by Suppressing Muc5ac Expression and Inactivating the ERK-SP1 Pathway in Lipopolysaccharide/Cigarette Smoke-Induced Mice.

Mucus hypersecretion is a hallmark of chronic obstructive pulmonary disease (COPD) and is associated with increasing sputum production and declining pulmonary function. Therefore, reducing mucus secretion can be a new therapeutic opportunity for preventing COPD. The Guifu Dihuang pill (GFDHP) is a classical Chinese medicine and has been used as an immunoregulator for treatment of kidney yang deficiency syndrome, including hypothyroidism, adrenocortical hypofunction, chronic bronchitis, and COPD, for more than 2000 years. However, the protective effects and mechanisms of GFDHP against mucus hypersecretion in COPD remain obscure. The aim of the present study was to explore the inhibitory effects of GFDHP on lipopolysaccharide/cigarette smoke- (LPS/CS-) induced Mucin5ac (Muc5ac) overproduction and airway goblet cell hyperplasia in mice. The mice were randomly assigned into 6 groups: control, model, GFDHP-L, GFDHP-M, GFDHP-H, and dexamethasone. The mice were given LPS twice through intranasal inhalation and then exposed to CS daily for 6 weeks. Three doses of GFDHP were orally administered daily during the last 3 weeks of the experiment. Pulmonary function was examined with an EMKA pulmonary system, and pulmonary hyperpermeability and lung damage were evaluated with an in vivo imaging system. Inflammatory cells and cytokines in bronchoalveolar lavage fluid (BALF) were detected with a cell count analyzer and though ELISA analysis, respectively. Lung pathological changes and airway goblet cell hyperplasia were analyzed with hematoxylin and eosin and Alcian blue periodic acid Schiff staining. The protein expression levels of Muc5ac and extracellular signal-regulated kinase (ERK)-specificity protein1 (SP1) signaling pathway were measured with Western blot and immunohistochemistry. The results demonstrated that GFDHP improved pulmonary function and suppressed mouse pulmonary hyperpermeability and edema. GFDHP suppressed inflammatory cell infiltration and cytokine release in BALF, thereby elevating pulmonary function. It ameliorated lung pathological changes and airway goblet cell hyperplasia, and suppressed expression levels of Muc5ac mRNA and protein and phospho-ERK and SP1 levels in the lung tissues of the COPD mice. In conclusion, GFDHP inhibited mucus hypersecretion induced by LPS/CS by suppressing the activation of the ERK-SP1 pathway.

Compatibility characteristics of Bufei Yishen formula III in regulating chronic obstructive pulmonary disease mucus hypersecretion

To evaluate the compatibility laws of effective-component compatibility of Bufei Yishen formula III (ECC-BYF III) in regulating mucus hypersecretion of chronic obstructive pulmonary disease (COPD). According to the efficacy of the original Chinese medicine, the components of ECC-BYF III were divided into four categories: Buqi (Ginsenoside Rh1+Astragaloside), Bushen (Icariin), Huatan (Nobiletin), and Huoxue (Paeonol). The four categories were divided into 14 groups based on the method of mathematical permutation. (1) The rats were divided into control group, model group, ECC-BYF III, and different components compatibility groups according to the random number table, totaling 17 groups. COPD rat model in stable phase was established by cigarette smoke exposure combined with repeated bacterial infections. The corresponding drugs were given by gavage at the 9th week of modeling, and the samples were collected at the end of the 16th week. The levels of matrix metalloproteinase-9 (MMP-9) and tissue inhibitors of metalloproteinase 1 (TIMP-1) in serum and bronchoalveolar lavage fluid (BALF), and the levels of mucin (MUC) 5AC in lung tissue and BALF were detected by enzyme linked immunosorbent assay (ELISA). (2) Human lung epithelial cells BEAS-2B were divided into blank group, model group, and different components compatibility groups. Hypoxia-induced mucus hypersecretion model of human lung epithelial cells BEAS-2B was established 4 hours after corresponding drug pretreatment. The mRNA expressions of MUC5AC, MUC5B, and MUC1 were detected by quantitative polymerase chain reaction (PCR). The mucus secretion indexes of rats and BEAS-2B cells were evaluated by Region (R) value comprehensive evaluation method. (1) Compared with the control group, MMP-9 in serum and BALF from the model group were significantly increased, the level of TIMP-1 was significantly decreased, and MUC5AC in lung tissue and BALF were significantly increased. The results of R value comprehensive evaluation showed that except for the Buqi and Bushen groups, ECC-BYF III and other components compatibility groups significantly corrected mucus hypersecretion in COPD rats, ECC-BYF III, Bushen Quxie, Fuzheng Huatan, and Quxie groups were much better (R values were 2.15±0.42, 2.11±0.23, 2.16±0.23 and 2.16±0.55, respectively), compared with the model group (R value: 3.00±0.00), the differences were statistically significant (all P < 0.05). (2) Compared with the blank group, the mRNA expressions of MUC5AC, MUC5B, and MUC1 increased in the model group. But different components compatibility groups had no significant effects on the mucus secretion of BEAS-2B cells. (3) The comprehensive evaluation results of R value about each in vivo and in vitro index showed that ECC-BYF III, Huoxue, Quxie, Bushen Huoxue, Fuzheng Huatan, Buqi Quxie groups significantly corrected the mucus hypersecretion (R values were 2.30±0.43, 2.33±0.44, 2.12±0.68, 2.27±0.64, 2.24±0.27 and 2.29±0.47, respectively), compared with the model group (R value: 3.00±0.00), the difference was statistically significant (all P < 0.01). The order was: Quxie > Fuzheng Huatan > Bushen Huoxue > Buqi Quxie > ECC-BYF III > Huoxue. Different components compatibility of ECC-BYF III had different effects on COPD mucus secretion. The components containing Huatan (Nobiletin) or Huoxue (Paeonol) showed a better inhibitory effect on mucus secretion.

The therapeutic promises of Lianhuaqingke in the mice model of coronavirus pneumonia (HCoV-229E and SARS-CoV-2)

BackgroundLianhuaqingke (LHQK) has been approved for the treatment of acute tracheobronchitis and exerts a broad-spectrum antiviral effect in our previous study.MethodsAcute pneumonia caused by HCoV-229E was modeled in BALB/c mice. The anti-viral effect of LHQK was assessed by measuring the lung index and virus titer of lung tissues. The expression levels of pro-inflammatory cytokines in lung tissues and peripheral blood were measured by ELISA. The morphological changes of lung tissues were observed by H&E staining. The subsets of Th cells were assayed by the flow cytometry, including Th0, Th1, Th2, Treg, and Th17. The expression level of MUC5AC in 16HBE cells treated with TNFα was measured by ELISA. Immunofluorescence staining for β-IV tubulin was used to identify the airway epithelial ciliary in the condition-cultured RTE cells treated with TNFα. The direct antiviral effect of LHQK was assessed in vitro in Vero E6 infected by SARS-CoV-2, validated in vivo in the COVID-19 model of hACE2 transgenic mouse by detecting the lung index, the SARS-CoV-2 virus load, and the morphological changes of lung tissues.ResultsLHQK reduced the weight loss and the lung index by inhibiting the HCoV-229E replication and reducing the expression of pro-inflammatory cytokines in lung tissues. An assay for the Th cell subsets in peripheral blood revealed that LHQK could reduce the ratio of Th1/Th2 and increase the Treg/Th17 ratio in a dose-dependent way, which indicated that LHQK could coordinate the Th-mediated immune responses against the virus. In in vitro injury by TNFα, LHQK inhibited MUC5AC expression in 16HBE cells and increased the number of β-IV tubulin positive staining cells in the condition-cultured RTE cells. In the SARS-CoV-2-infected mice, LHQK could reduce weight loss, inhibit viral replication, and alleviate lung tissue damage.ConclusionsOur results demonstrate that LHQK exerts therapeutic effects on pneumonia caused by HCoVs (HCoV-229E and SARS-CoV-2) in mice, and that the anti-HCoV effects might depend on its immunomodulatory capacities. All these results suggest that LHQK serves as a potential adjuvant for anti-HCoV therapies.

Whole-genome copy number and immunohistochemical analyses on surgically resected intracholecystic papillary neoplasms.

Intracholecystic papillary neoplasms are newly defined precancerous lesions. According to Classification of the World Health Organization, they have four histological morphologies, which are biliary, gastric, intestinal, and oncocytic. This study evaluated 17 patients with resected intracholecystic papillary neoplasms in terms of histological, immunohistochemical, and copy number variation (CNV). The histological subtypes included 5 cases of low-grade (5 gastric) and 12 cases of high-grade (6 gastric and 6 biliary) neoplasms. Most cases showed high expression of MUC1, MUC5AC, and CK7, moderate expression of MUC6 and Ki-67, and low expression of CK20, MUC2, and CDX2. The CNV profile identified gain of 7q in 12%, and loss of 1p (18%), 5q (29%), 9p (35%), 12p (17%), 17p (24%), and 19p (18%). No CNVs were observed in low-grade neoplasms, whereas high-grade ones had increasing abnormalities. β-catenin was often expressed in the nucleus of neoplasms with gastric morphology, suggesting the involvement of the Wnt/β-catenin pathway. However, it was not expressed among those with biliary morphology, which instead exhibited high p53 expression. Neoplasms with biliary morphology showed more CNV changes (9p, 17p, 19p losses). Distinct immunological and CNV patterns were seen in both morphologies, suggesting differences in their pathogenesis. More CNVs accumulated with tumor progression.

Lactobacillus casei T1 from kurut against Helicobacter pylori-induced inflammation and the gut microbial disorder

• L. casei T1 inhabited H. pylori growth. • L. casei T1 suppressed H. pylori adhesion. • L. casei T1 alleviated gastric inflammation caused by H. pylori -infection. • L. casei T1 pretreatment prevent the gut microbial disorder caused by H. pylori . Probiotics have the functions of inhibiting pathogen infection and maintaining gastrointestinal homeostasis. The aim of this study was to investigate the preventive effect of Lactobacillus casei T1 ( L. casei T1) isolated from kurut against the gastric inflammation and gut microbiota alteration induced by Helicobacter pylori (H. pylori ) infection. The results showed that L. casei T1 and its supernatant had a potent inhibitory effect on H. pylori growth in vitro . L. casei T1 adhered to GES-1 cells, down-regulated the expression of MUC5AC, IL-6, IL-8 and TNF-α in GES-1 cells induced by H. pylori . This followed by in vivo studies demonstrated that L. casei T1 pretreatment alleviated gastric inflammation and prevent gut microbiota alteration caused by H. pylori infection. These results demonstrated that L. casei T1 have a preventive effect against H. pylori infection and it can be served as a further development and utilization of probiotic.

APC mutations are common in adenomas but infrequent in adenocarcinomas of the non-ampullary duodenum.

Recent studies highlighted the clinicopathological heterogeneity of non-ampullary duodenal adenomas and adenocarcinomas, but the detailed process of the malignant transformation remains unclear. We analyzed 144 adenomas and 54 adenocarcinomas of the non-ampullary duodenum for immunohistochemical phenotypes, genetic alterations, and mismatch repair (MMR) status to probe their histogenetic relationship. The median ages of patients with adenoma and adenocarcinoma were the same (66years). Adenomas were histologically classified as intestinal-type adenoma (n = 124), pyloric gland adenoma (PGA, n = 10), gastric-type adenoma, not otherwise specified (n = 9), and foveolar-type adenoma (n = 1). Protein-truncating APC mutations were highly frequent in adenomas (85%), with the highest prevalence in intestinal-type adenomas (89%), but rare in adenocarcinomas (9%; P = 2.1 × 10-23). Close associations between phenotypic marker expression and genetic alterations were observed in adenomas, but not in adenocarcinomas, excluding the common association between GNAS mutations and MUC5AC expression. MMR deficiency was more frequent in adenocarcinomas (20%) than in adenomas (1%; P = 2.6 × 10-6). One MMR-deficient adenoma and three MMR-deficient adenocarcinomas occurred in patients with Lynch syndrome. Additionally, three other patients with an MMR-deficient adenocarcinoma fulfilled the revised Bethesda criteria. The discrepant APC mutation frequency between adenomas and adenocarcinomas suggests that APC-mutated adenomas, which constitute the large majority of non-ampullary duodenal adenomas, are less prone to malignant transformation. Non-ampullary duodenal adenocarcinomas frequently exhibit MMR deficiency and should be subject to MMR testing to determine appropriate clinical management, including the identification of patients with Lynch syndrome.

Changes of Gastric Juice Microenvironment in Patients with Fundic Gland Polyp and Hyperplastic Polyp

Introduction: The aim of this study was to study the relationship between the formation of gastric fundic gland polyp and gastric hyperplastic polyp (HP) and the changes of gastric juice microenvironment. Methods: The proton-pump inhibitor (PPI) applications to patients were recorded. Gastric juices and biopsy polyps were collected for pathological examination, H. pylori tests, biomarkers, and MUC1, MUC2, MUC5AC expression measurement. Results: Among 34,892 patients, the detection rate of gastric fundic gland polyps was significantly higher than that of gastric HPs (p < 0.01). The incidence rate of gastric fundic gland polyp and gastric HP in PPI users (n = 3,886) was higher than that of non-PPI users (p < 0.01). The occurrence of polyp was positively related to the duration of PPI application and the H. pylori-positive rate. The bile reflux rate between fundic gland polys group (17.61%) and HPs (28.67%) was significantly different (p < 0.01). The levels of gastric juice Gastrin-17, epidermal growth factor (EGF) and MUC₂ from patients with gastric fundic gland polyps and gastric HPs were higher than those in the control group (p < 0.01). However, patients with gastric fundic gland polyps and HPs had significantly lower gastric juice PGE₂ and MUC5AC (p < 0.01). Conclusion: PPI application, H. pylori infection, and bile reflux are the potential risk factors for formation of fundic gland polyps and HPs. The potential mechanism of polyps’ formation can be related to the levels of Gastrin-17, EGF, MUC₂, PGE₂, and MUC5AC in gastric juice.

Peroxiredoxin 2 Inhibits Lipopolysaccharide Induced Mucin Expression and Reactive Oxygen Species Production in Human Airway Epithelial Cells

Background and Objectives Peroxiredoxin (Prx) is an antioxidant enzyme involved in signaling pathway. Prx2 is the most abundant in mammalian gray matter neurons and has protective role under oxidative stress. MUC5AC and MUC5B are typical mucin genes in human airway epithelial cells. Even if free radicals play a key role in chronic respiratory inflammatory diseases, the effects of the Prx2 on mucin expression and oxidative stress are not clearly known. The purpose of this study is to investigate the effect of Prx2 on lipopolysaccharide (LPS)-induced MUC5AC/5B expression and reactive oxygen species (ROS) in human airway epithelial cells.Subjects and Method In NCI-H292 cells and human nasal epithelial cells, the effects of Prx2 on LPS-induced MUC5AC/5B expression and ROS production were investigated using reverse transcriptase-polymerase chain reaction, real-time polymerase chain reaction, enzyme linked immunosorbent assay (ELISA) and flow cytometry analysis.Results MUC5AC, MUC5B mRNA expression and protein production were increased by LPS. ROS production was also increased by LPS. Prx2 suppressed the LPS-induced MUC5AC mRNA expression and protein production as well as ROS production. However, Prx2 did not inhibit MUC5B mRNA expression and protein production. N-acetylcysteine, diphenyleneiodonium, and apocynin also inhibited LPS-induced ROS production.Conclusion These results may show that Prx2 suppresses LPS-induced MUC5AC expression via ROS in human airway epithelial cells.

Deficient inflammasome activation permits an exaggerated asthma phenotype in rhinovirus C-infected immature mice.

Compared to other RV species, RV-C has been associated with more severe respiratory illness and is more likely to occur in children with a history of asthma or who develop asthma. We therefore inoculated six-day-old mice with sham, RV-A1B, or RV-C15. Inflammasome priming and activation were assessed, and selected mice treated with recombinant IL-1β. Compared to RV-A1B infection, RV-C15 infection induced an exaggerated asthma phenotype, with increased mRNA expression of Il5, Il13, Il25, Il33, Muc5ac, Muc5b and Clca1, increased lung lineage-negative CD25+CD127+ST2+ ILC2s, increased mucous metaplasia, and increased airway responsiveness. Lung vRNA, induction of pro-inflammatory type 1 cytokines, and inflammasome priming (pro-IL-1β and NLRP3) were not different between the two viruses. However, inflammasome activation (mature IL-1β and caspase-1 p12) was reduced in RV-C15-infected mice compared to RV-A1B-infected mice. A similar deficiency was found in cultured macrophages. Finally, IL-1β treatment decreased RV-C-induced type 2 cytokine and mucus-related gene expression, ILC2s, mucous metaplasia and airway responsiveness, but not lung vRNA level. We conclude that RV-C induces an enhanced asthma phenotype in immature mice. Compared to RV-A, RV-C-induced macrophage inflammasome activation and IL-1β are deficient, permitting exaggerated type 2 inflammation and mucous metaplasia.

Muc5ac Production Inhibited by Decreased lncRNA H19 via PI3K/Akt/NF-kB in Asthma.

IntroductionLncRNAs play important roles in multiple diseases including asthma, while there are a few reports on the role of lncRNA H19 about asthma. This study aimed to investigate the roles and mechanisms of lncRNA H19 in asthma.MethodsWe detected lncRNA H19 and Muc5ac mRNA by establishing a murine asthma model and an in vitro inflammation model. Regulatory roles of lncRNA H19 in asthma were explored by lncRNA H19 overexpression or knockdown in vitro. To study its mechanisms, we detect p-NF-κB and p-Akt expression, and treated 16-HBE cells with inhibitors of PI3K. To study regulatory effects of miR-675-3p on Muc5ac, miR-675-3p mimics and inhibitors were respectively transfected into 16-HBE cells.ResultsFirstly, we established a murine asthma model and an in vitro inflammation model. We found that lncRNA H19 expression was decreased, while Muc5ac mRNA was increased in lung tissues of murine asthma model and in the in vitro inflammation model. lncRNA H19 overexpression increased Muc5ac mRNA expression and lncRNA H19 knockdown decreased Muc5ac mRNA expression in 16-HBE cells. Moreover, lncRNA H19 overexpression further increased Muc5ac expression in TNFα-induced in vitro inflammation model. lncRNA H19 knockdown decreased p-Akt and p-NF-κB expression. Inhibitors of PI3K abolished Muc5ac induced by lncRNA H19 overexpression. Although miR-675-3p was increased by lncRNA H19 overexpression, it had no regulatory effects on Muc5ac expression.DiscussionThese results demonstrated that lncRNA H19 positively regulates Muc5ac expression through PI3K/Akt /NF-κB pathway in the in vitro inflammation model. Therefore, this study indicated that decreased lncRNA H19 in asthma might play a protective role relieving mucus overproduction, and lncRNA H19 might be a potential target for asthma treatment.

TGFβ Signaling Activated by Cancer-Associated Fibroblasts Determines the Histological Signature of Lung Adenocarcinoma.

Invasive lung adenocarcinoma (LADC) can be classified histologically as lepidic, acinar, papillary, micropapillary, or solid. Most LADC tumors manifest several of these histological subtypes, with heterogeneity being related to therapeutic resistance. We report here that in immunodeficient mice, human LADC cells form tumors with distinct histological features, MUC5AC-expressing solid-type or cytokeratin 7 (CK7)-expressing acinar-type tumors, depending on the site of development, and that a solid-to-acinar transition (SAT) could be induced by the tumor microenvironment. The TGFβ-Smad signaling pathway was activated in both tumor and stromal cells of acinar-type tumors. Immortalized cancer-associated fibroblasts (CAF) derived from acinar-type tumors induced SAT in 3D cocultures with LADC cells. Exogenous TGFβ1 or overexpression of an active form of TGFβ1 increased CK7 expression and reduced MUC5AC expression in LADC cells, and knockdown of Tgfb1 mRNA in CAFs attenuated SAT induction. RNA-sequencing analysis suggested that angiogenesis and neutrophil recruitment are associated with SAT in vivo. Our data indicate that CAF-mediated paracrine TGFβ signaling induces remodeling of tumor tissue and determines the histological pattern of LADC, thereby contributing to tumor heterogeneity. SIGNIFICANCE: CAFs secrete TGFβ to induce a solid-to-acinar transition in lung cancer cells, demonstrating how the tumor microenvironment influences histological patterns and tumor heterogeneity in lung adenocarcinoma.

Quantification of increased MUC5AC expression in airway mucus of smoker using an automated image-based approach.

Microscopic analysis of mucus quantity and composition is crucial in research and diagnostics on muco-obstructive diseases. Currently used image-based methods are unable to extract concrete numeric values of mucosal proteins, especially on the expression of the key mucosal proteins MUC5AC and MUC5B. Since their levels increase under pathologic conditions such as extensive exposure to cigarette smoke, it is imperative to quantify them to improve treatment strategies of pulmonary diseases. This study presents a simple, image-based, and high-processing computational method that allows determining the ratio of MUC5AC and MUC5B within the overall airway mucus while providing information on their spatial distribution. The presented pipeline was optimized for automated downstream analysis using a combination of bright field and immunofluorescence imaging suitable for tracheal and bronchial tissue samples, and air-liquid interface (ALI) cell cultures. To validate our approach, we compared tracheal tissue and ALI cell cultures of isolated primary normal human bronchial epithelial cells derived from smokers and nonsmokers. Our data indicated 18-fold higher levels of MUC5AC in submucosal glands of smokers covering about 8% of mucosal areas compared to <1% in nonsmoking individuals, confirming results of previous studies. We further identified a subpopulation of nonsmokers with slightly elevated glandular MUC5AC levels suggesting moderate exposure to second-hand smoke or fine particulate air pollution. Overall, this study demonstrates a novel, user-friendly and freely available tool for digital pathology and the analysis of therapeutic interventions tested in ALI cell cultures.

Small-Airway Dysfunction is Involved in the Pathogenesis of Asthma: Evidence from Two Mouse Models.

BackgroundThere has been growing evidence of small-airway dysfunction in patients with asthma. Few studies have evaluated the mechanism of small-airway dysfunction in mouse models of asthma.PurposeWe explored the correlation between small-airway spirometric variables and large-airway function or inflammation in different endotypes of asthma.MethodsOvalbumin (OVA) sensitization/challenge was used to produce a type 2 (T2)-high asthma model, and OVA combined with ozone exposure (OVA + ozone) was used for the T2-low asthma model with increased neutrophils. Spirometry, airway responsiveness, cytokine levels in bronchoalveolar lavage fluid (BALF), and pathological analyses of lung slices stained with hematoxylin-eosin, periodic acid–Schiff, and Masson’s trichrome stain were all determined. Muc5ac expression in lung tissue was evaluated by the reverse transcription-polymerase chain reaction (RT-PCR), and alpha-smooth muscle actin was measured by immunohistochemistry.ResultsInflammatory cells infiltrated the lung tissue and inflammatory cytokines were increased in the BALF of both the OVA and OVA + ozone groups, compared with the control group. Peribronchial hypersecretion and collagen deposition were evident in the models. The OVA + ozone group showed greater neutrophilic infiltration and peribronchial smooth muscle proliferation than the OVA group. Large-airway obstruction, small-airway dysfunction, and airway hyperresponsiveness were confirmed in both models. Small-airway functional variables, such as MMEF (mean midexpiratory flow, average flow from 25 to 75% forced vital capacity [FVC]) and FEF50 (forced expiratory flow at 50% of FVC), were positively correlated with large-airway function and had a stronger negative correlation with airway inflammation, mucus secretion, and responsiveness than large-airway function.ConclusionSmall-airway dysfunction was evident in the two endotypes of asthma and was correlated with severe airway inflammation, mucus hypersecretion, and airway hyperresponsiveness. The small airways may be an important target in asthma treatment, and further research in the role of small-airway variables in the pathogenesis of asthma is warranted.

Development of a standardized in vitro approach to evaluate microphysical, chemical, and toxicological properties of combustion-derived fine and ultrafine particles

Ultrafine particles represent a growing concern in the public health community but their precise role in many illnesses is still unknown. This lack of knowledge is related to the experimental difficulty in linking their biological effects to their multiple properties, which are important determinants of toxicity. Our aim is to propose an interdisciplinary approach to study fine (FP) and ultrafine (UFP) particles, generated in a controlled manner using a miniCAST (Combustion Aerosol Standard) soot generator used with two different operating conditions (CAST1 and CAST3). The chemical characterization was performed by an untargeted analysis using ultra-high resolution mass spectrometry. In conjunction with this approach, subsequent analysis by gas chromatography–mass spectrometry (GC–MS) was performed to identify polycyclic aromatic hydrocarbons (PAH). CAST1 enabled the generation of FP with a predominance of small PAH molecules, and CAST3 enabled the generation of UFP, which presented higher numbers of carbon atoms corresponding to larger PAH molecules. Healthy normal human bronchial epithelial (NHBE) cells differentiated at the air-liquid interface (ALI) were directly exposed to these freshly emitted FP and UFP. Expression of MUC5AC, FOXJ1, OCLN and ZOI as well as microscopic observation confirmed the ciliated pseudostratified epithelial phenotype. Study of the mass deposition efficiency revealed a difference between the two operating conditions, probably due to the morphological differences between the two categories of particles. We demonstrated that only NHBE cells exposed to CAST3 particles induced upregulation in the gene expression of IL-8 and NQO1. This approach offers new perspectives to study FP and UFP with stable and controlled properties.

MUC5AC expression is linked to mucinous/endometroid subtype and absence of nodal metastasis in ovarian cancer

MUC5AC expression is linked to mucinous/endometroid subtype and absence of nodal metastasis in ovarian cancer