MUC5B Expression Research Articles

Submandibular Gland Pathogenesis Following SARS-CoV-2 Infection and Implications for Xerostomia.

Although SARS-CoV-2 induces mucin hypersecretion in the respiratory tract, hyposalivation/xerostomia has been reported by COVID-19 patients. We evaluate the submandibular gland (SMGs) pathogenesis in SARS-CoV-2-infected K18-hACE2 mice, focusing on the impact of infection on the mucin production and structural integrity of acini, ductal system, myoepithelial cells (MECs) and telocytes. The spike protein, the nucleocapsid protein, hACE2, actin, EGF, TNF-α and IL-1β were detected by immunofluorescence, and the Egfr and Muc5b expression was evaluated. In the infected animals, significant acinar hypertrophy was observed in contrast to ductal atrophy. Nucleocapsid proteins and/or viral particles were detected in the SMG cells, mainly in the nuclear membrane-derived vesicles, confirming the nuclear role in the viral formation. The acinar cells showed intense TNF-α and IL-1β immunoexpression, and the EGF-EGFR signaling increased, together with Muc5b upregulation. This finding explains mucin hypersecretion and acinar hypertrophy, which compress the ducts. Dying MECs and actin reduction were also observed, indicating failure of contraction and acinar support, favoring acinar hypertrophy. Viral assembly was found in the dying telocytes, pointing to these intercommunicating cells as viral transmitters in SMGs. Therefore, EGF-EGFR-induced mucin hypersecretion was triggered by SARS-CoV-2 in acinar cells, likely mediated by cytokines. The damage to telocytes and MECs may have favored the acinar hypertrophy, leading to ductal obstruction, explaining xerostomia in COVID-19 patients. Thus, acinar cells, telocytes and MECs may be viral targets, which favor replication and cell-to-cell viral transmission in the SMG, corroborating the high viral load in saliva of infected individuals.

The disruptive effects of COPD exacerbation-associated factors on epithelial repair responses.

Exacerbations of chronic obstructive pulmonary disease (COPD) increase mortality risk and can lead to accelerated loss of lung function. The increased inflammatory response during exacerbations contributes to worsening of airflow limitation, but whether it also impacts epithelial repair is unclear. Therefore, we studied the effect of the soluble factor micro-environment during COPD exacerbations on epithelial repair using an exacerbation cocktail (EC), composed of four factors that are increased in COPD lungs during exacerbations (IL-1β, IL-6, IL-8, TNF-α). Mouse organoids (primary CD31-CD45-Epcam+ cells co-cultured with CCL206 fibroblasts) were used to study epithelial progenitor behavior. Mature epithelial cell responses were evaluated using mouse precision cut lung slices (PCLS). The expression of epithelial supportive factors was assessed in CCL206 fibroblasts and primary human lung fibroblasts. EC exposure increased the number and size of organoids formed, and upregulated Lamp3, Muc5ac and Muc5b expression in day 14 organoids. In PCLS, EC imparted no effect on epithelial marker expression. Pre-treatment of CCL206 fibroblasts with EC was sufficient to increase organoid formation. Additionally, the expression of Il33, Tgfa and Areg was increased in CCL206 fibroblasts from EC treated organoids, but these factors individually did not affect organoid formation or size. However, TGF-α downregulated Foxj1 expression and upregulated Aqp5 expression in day 14 organoids. EC exposure stimulates organoid formation and growth, but it alters epithelial differentiation. EC changes the epithelial progenitor support function of fibroblasts which contributes to observed effects on epithelial progenitors.

Pulmonary Fibrosis Ferret Model Demonstrates Sustained Fibrosis, Restrictive Physiology, and Aberrant Repair.

The role of MUC5B mucin expression in IPF pathogenesis is unknown. Bleomycin-exposed rodent models do not exhibit sustained fibrosis or airway remodeling. Unlike mice, ferrets have human-like distribution of MUC5B expressing cell types and natively express the risk-conferring variant that induces high MUC5B expression in humans. We hypothesized that ferrets would consequently exhibit aberrant repair to propagate fibrosis similar to human IPF. Bleomycin (5U/kg) or saline-control was micro-sprayed intratracheally then wild-type ferrets were evaluated through 22 wks. Clinical phenotype was assessed with lung function. Fibrosis was assessed with µCT imaging and comparative histology with Ashcroft scoring. Airway remodeling was assessed with histology and quantitative immunofluorescence. Bleomycin ferrets exhibited sustained restrictive physiology including decreased inspiratory capacity, decreased compliance, and shifted Pressure-Volume loops through 22 wks. Volumetric µCT analysis revealed increased opacification of the lung bleomycin-ferrets. Histology showed extensive fibrotic injury that matured over time and MUC5B-positive cystic structures in the distal lung suggestive of honeycombing. Bleomycin ferrets had increased proportion of small airways that were double-positive for CCSP and alpha-tubulin compared to controls, indicating an aberrant 'proximalization' repair phenotype. Notably, this aberrant repair was associated with extent of fibrotic injury at the airway level. Bleomycin-exposed ferrets exhibit sustained fibrosis through 22 wks and have pathologic features of IPF not found in rodents. Ferrets exhibited proximalization of the distal airways and other pathologic features characteristic of human IPF. MUC5B expression through native cell types may play a key role in promoting airway remodeling and lung injury in IPF.

Hyposecretion of cervical MUC5B is related to preterm birth in pregnant women after cervical excisional surgery.

Excisional surgery for cervical intraepithelial neoplasia is a risk factor for preterm birth in subsequent pregnancies. However, the underlying mechanisms of this association remain unclear. We previously showed that cervical MUC5B, a mucin protein, may be a barrier to ascending pathogens during pregnancy. We thus hypothesized that hyposecretion of cervical MUC5B is associated with preterm birth after cervical excisional surgery. This prospective nested case-control study (Study 1) included pregnant women who had previously undergone cervical excisional surgery across 11 hospitals. We used proteomics to compare cervicovaginal fluid at 18-22weeks of gestation between the preterm and term birth groups. In another case-control analysis (Study 2), we compared MUC5B expression in nonpregnant uterine tissues between 15 women with a history of cervical excisional surgery and 26 women without a history of cervical surgery. The abundance of MUC5B in cervicovaginal fluid was significantly decreased in the preterm birth group (fold change=0.41, p=.035). Among the 480 quantified proteins, MUC5B had the second highest positive correlation with gestational age at delivery in the combined preterm and term groups. The cervicovaginal microbiome composition was not significantly different between the two groups. Cervical length was not correlated with gestational age at delivery (r=0.18, p=.079). Histologically, the MUC5B-positive area in the nonpregnant cervix was significantly decreased in women with a history of cervical excisional surgery (0.85-fold, p=.048). The distribution of MUC5B-positive areas in the cervical tissues of 26 women without a history of cervical excisional surgery differed across individuals. This study suggests that the primary mechanism by which cervical excisional surgery causes preterm birth is the hyposecretion of MUC5B due to loss of the cervical glands.

Mucin Expression Profiles in Ulcerative Colitis: New Insights on the Histological Mucosal Healing.

A structural weakness of the mucus barrier (MB) is thought to be a cause of ulcerative colitis (UC). This study aims to investigate the mucin (MUC) composition of MB in normal mucosa and UC. Ileocolonic biopsies were taken at disease onset and after treatment in 40 patients, including 20 with relapsing and 20 with remitting UC. Ileocolonic biopsies from 10 non-IBD patients were included as controls. Gut-specific MUC1, MUC2, MUC4, MUC5B, MUC12, MUC13, MUC15, and MUC17 were evaluated immunohistochemically. The promoters of mucin genes were also examined. Normal mucosa showed MUC2, MUC5B, and MUC13 in terminal ileum and colon, MUC17 in ileum, and MUC1, MUC4, MUC12, and MUC15 in colon. Membranous, cytoplasmic and vacuolar expressions were highlighted. Overall, the mucin expression was abnormal in UC. Derangements in MUC1, MUC4, and MUC5B were detected both at onset and after treatment. MUC2 and MUC13 were unaffected. Sequence analysis revealed glucocorticoid-responsive elements in the MUC1 promoter, retinoic-acid-responsive elements in the MUC4 promoter, and butyrate-responsive elements in the MUC5B promoter. In conclusion, MUCs exhibited distinct expression patterns in the gut. Their expression was disrupted in UC, regardless of the treatment protocols. Abnormal MUC1, MUC4, and MUC5B expression marked the barrier dysfunction in UC.

The combination of propylene glycol and vegetable glycerin e-cigarette aerosols induces airway inflammation and mucus hyperconcentration

Despite concerns over their safety, e-cigarettes (e-cigs) remain a popular tobacco product. Although nicotine and flavors found in e-cig liquids (e-liquids) can cause harm in the airways, whether the delivery vehicles propylene glycol (PG) and vegetable glycerin (VG) are innocuous when inhaled remains unclear. Here, we investigated the effects of e-cig aerosols generated from e-liquid containing only PG/VG on airway inflammation and mucociliary function in primary human bronchial epithelial cells (HBEC) and sheep. Primary HBEC were cultured at the air–liquid interface (ALI) and exposed to e-cig aerosols of 50%/50% v/v PG/VG. Ion channel conductance, ciliary beat frequency, and the expression of inflammatory markers, cell type-specific markers, and the major mucins MUC5AC and MUC5B were evaluated after seven days of exposure. Sheep were exposed to e-cig aerosols of PG/VG for five days and mucus concentration and matrix metalloproteinase-9 (MMP-9) activity were measured from airway secretions. Seven-day exposure of HBEC to e-cig aerosols of PG/VG caused a significant reduction in the activities of apical ion channels important for mucus hydration, including the cystic fibrosis transmembrane conductance regulator (CFTR) and large conductance, Ca2+-activated, and voltage-dependent K+ (BK) channels. PG/VG aerosols significantly increased the mRNA expression of the inflammatory markers interleukin-6 (IL6), IL8, and MMP9, as well as MUC5AC. The increase in MUC5AC mRNA expression correlated with increased immunostaining of MUC5AC protein in PG/VG-exposed HBEC. On the other hand, PG/VG aerosols reduced MUC5B expression leading overall to higher MUC5AC/MUC5B ratios in exposed HBEC. Other cell type-specific markers, including forkhead box protein J1 (FOXJ1), keratin 5 (KRT5), and secretoglobin family 1A member 1 (SCGB1A1) mRNAs, as well as overall ciliation, were significantly reduced by PG/VG exposure. Finally, PG/VG aerosols increased MMP-9 activity and caused mucus hyperconcentration in sheep in vivo. E-cig aerosols of PG/VG induce airway inflammation, increase MUC5AC expression, and cause dysfunction of ion channels important for mucus hydration in HBEC in vitro. Furthermore, PG/VG aerosols increase MMP-9 activity and mucus concentration in sheep in vivo. Collectively, these data show that e-cig aerosols containing PG/VG are likely to be harmful in the airways.

Cigarette Smoke Extract Induces MUC5AC Expression Through the ROS/ IP3R/Ca2+ Pathway in Calu-3 Cells.

Chronic obstructive pulmonary disease (COPD) is caused by exposure to noxious external particles, air pollution, and the inhalation of cigarette smoke. Airway mucus hypersecretion particularly mucin5AC (MUC5AC), is a crucial pathological feature of COPD and is associated with its initiation and progression. In this study, we aimed to investigate the effects of cigarette smoke extract (CSE) on MUC5AC expression, particularly the mechanisms by which reactive oxygen species (ROS) induce MUC5AC expression. The effects of CSE on the expression of MUC5AC and mucin5B (MUC5B) were investigated in vitro in Calu-3 cells. MUC5AC and MUC5B expression levels were measured using quantitative reverse transcription-polymerase chain reaction (qRT-PCR), immunofluorescence staining, and enzyme-linked immunosorbent assay (ELISA). Total cellular levels of ROS and Ca2+ were determined using DCFH-DA and Fluo-4 AM. Subsequently, the expression levels of IP3R, IRE1α, p-IRE1α and XBP1s were measured by Western blotting. Gene silencing was achieved by using small-interfering RNAs. Our findings revealed that exposure to CSE increased MUC5AC levels and upregulated ROS, IP3R/Ca2+ and unfolded protein response (UPR)-associated factors. In addition, knockdown of IP3R using siRNA decreased CSE-induced Ca2+ production, UPR-associated factors, and MUC5AC expression. Furthermore, 10 mM N-acetyl-l-cysteine (NAC) treatment suppressed the effects of CSE, including ROS generation, IP3R/ Ca2+, UPR activation, and MUC5AC overexpression. Our results suggest that ROS regulates CSE-induced UPR and MUC5AC overexpression through IP3R/ Ca2+ signaling. Additionally, we identified NAC as a promising therapeutic agent for mitigating CSE-induced MUC5AC overexpression.

RNA Sequencing of Whole Blood in Premature Coronary Artery Disease: Identification of Novel Biomarkers and Involvement of T Cell Imbalance.

Serum biomarkers were explored based on the peripheral blood gene expression profiles of premature coronary artery disease (PCAD). RNA sequencing (RNA-Seq) was used to detect PCAD-specific differentially expressed genes (DEGs). Quantitative real-time polymerase chain reaction (RT-PCR) was used to validate the most significant DEGs, and enzyme-linked immunosorbent assay (ELISA) was utilized to quantify the effect on corresponding serum proteins. Fifty-nine PCAD-specific DEGs were identified. Functional analysis showed positive regulation of T cell-mediated cytotoxicity, regulation of T cell-mediated immunity, and the regulation of alpha-beta T cell proliferation which were enriched in PCAD. RT-PCR validated the significant difference in the expression of BAG6, MUC5B, and APOA2 between PCAD and late-onset coronary artery disease (LCAD) patients. ELISA validation showed serum MUC5B increased dramatically in PCAD when compared to LCAD. Our study found T cells contribute to the occurrence of PCAD, and the inflammatory factor MUC5B may be a novel serum marker in PCAD patients.

Proximal and Distal Bronchioles Contribute to the Pathogenesis of Non-Cystic Fibrosis Bronchiectasis.

Rationale: Non-cystic fibrosis bronchiectasis (NCFB) may originate in bronchiolar regions of the lung. Accordingly, there is a need to characterize the morphology and molecular characteristics of NCFB bronchioles. Objectives: Test the hypothesis that NCFB exhibits a major component of bronchiolar disease manifest by mucus plugging and ectasia. Methods: Morphologic criteria and region-specific epithelial gene expression, measured histologically and by RNA in situ hybridization and immunohistochemistry, identified proximal and distal bronchioles in excised NCFB lungs. RNA in situ hybridization and immunohistochemistry assessed bronchiolar mucus accumulation and mucin gene expression. CRISPR-Cas9-mediated IL-1R1 knockout in human bronchial epithelial cultures tested IL-1α and IL-1β contributions to mucin production. Spatial transcriptional profiling characterized NCFB distal bronchiolar gene expression. Measurements and Main Results: Bronchiolar perimeters and lumen areas per section area were increased in proximal, but not distal, bronchioles in NCFB versus control lungs, suggesting proximal bronchiolectasis. In NCFB, mucus plugging was observed in ectatic proximal bronchioles and associated nonectatic distal bronchioles in sections with disease. MUC5AC and MUC5B mucins were upregulated in NCFB proximal bronchioles, whereas MUC5B was selectively upregulated in distal bronchioles. Bronchiolar mucus plugs were populated by IL-1β-expressing macrophages. NCFB sterile sputum supernatants induced human bronchial epithelial MUC5B and MUC5AC expression that was >80% blocked by IL-1R1 ablation. Spatial transcriptional profiling identified upregulation of genes associated with secretory cells, hypoxia, interleukin pathways, and IL-1β-producing macrophages in mucus plugs and downregulation of epithelial ciliogenesis genes. Conclusions: NCFB exhibits distinctive proximal and distal bronchiolar disease. Both bronchiolar regions exhibit bronchiolar secretory cell features and mucus plugging but differ in mucin gene regulation and ectasia.

Effective-compound combination of Bufei Yishen formula III combined with ER suppress airway mucus hypersecretion in COPD rats: via EGFR/MAPK signaling.

The aim of this study was to explore the combined efficacy ofeffective-component compatibility of Bufei Yishen formula III (ECC-BYF III) and exercise rehabilitation (ER) in inhibiting airway mucus hypersecretion in a chronic obstructive pulmonary disease (COPD) rat model. A total of 48 SD rats were divided into control, model, acetylcysteine (NAC), ECC-BYF III, ER, and ECC-BYF III + ER groups (n=8). COPD rats were exposed to cigarette smoke and bacteria for 8 weeks and administered various treatments over the next eight weeks. Rats were euthanized at week 17 after pulmonary function testing. Pathological examination of lung tissues was performed. IL-6 and IL-10 levels were measured in bronchoalveolar lavage fluid (BALF) and protein levels of MUC5AC, MUC5B, AQP-5, EGFR, ERK, JNK, and p38 were measured in lung tissues. Improved pulmonary function and pathological changes were observed in ECC-BYF III, ECC-BYF III + ER, and NAC groups. ECC-BYF III and ECC-BYF III + ER had greater mean alveolar number (MAN) compared with NAC. Lung inflammation and goblet cell generation were reduced and MUC5AC, MUC5B and AQP-5 expressions were lower in all treatment groups. ECC-BYF III has more significant effect on MUC5AC than ER and NAC. ECC-BYFIII + ER had a greater effect on suppressing IL-6 in BALF compared with other treatments. ECC-BYFIII, ER, and ECC-BYF III + ER reduced EGFR, ERK, JNK, and p38 phosphorylated protein levels. ECC-BYFIII+ER had a greater effect on p-JNK and p-p38 than ECC-BYFIII and NAC. ECC-BYF III, ER, and ECC-BYF III + ER have efficacy in inhibiting airway mucus hypersecretion with improved pulmonary function and pathological changes. ECC-BYF III had a greater effect in improving MAN and MUC5AC in lung tissue. ECC-BYF III+ER had a greater effect in alleviating pulmonary pathology and inflammation. These effects may be mediated by inhibition of the EGFR/MAPK pathway.

Clonal Somatic Mutations in Chronic Lung Diseases Are Associated with Reduced Lung Function.

Rationale: Constantly exposed to the external environment and mutagens such as tobacco smoke, human lungs have one of the highest somatic mutation rates among all human organs. However, the relationship of these mutations to lung disease and function is not known. Objectives: To identify the prevalence and significance of clonal somatic mutations in chronic lung diseases. Methods: We analyzed the clonal somatic mutations from 1,251 samples of normal and diseased noncancerous lung tissue RNA sequencing with paired whole-genome sequencing from the Lung Tissue Research Consortium. We examined the associations of somatic mutations with lung function, disease status, and computationally deconvoluted cell types in two of the most common diseases represented in our dataset, chronic obstructive pulmonary disease (COPD; 29%) and idiopathic pulmonary fibrosis (IPF; 13%). Measurements and Main Results: Clonal somatic mutational burden was associated with reduced lung function in both COPD and IPF. We identified an increased prevalence of clonal somatic mutations in individuals with IPF compared with normal control subjects and individuals with COPD independent of age and smoking status. IPF clonal somatic mutations were enriched in disease-related and airway epithelial-expressed genes such as MUC5B in IPF. Patients who were MUC5B risk variant carriers had increased odds of developing somatic mutations of MUC5B that were explained by increased expression of MUC5B. Conclusions: Our identification of an increased prevalence of clonal somatic mutation in diseased lung that correlates with airway epithelial gene expression and disease severity highlights for the first time the role of somatic mutational processes in lung disease genetics.

235 Reducing MUC5AC or MUC5B expression using antisense oligonucleotides as a therapeutic approach for cystic fibrosis and other muco-obstructive diseases

235 Reducing MUC5AC or MUC5B expression using antisense oligonucleotides as a therapeutic approach for cystic fibrosis and other muco-obstructive diseases

Maintenance of adult stem cells from human minor salivary glands via the Wnt signaling pathway

BackgroundXerostomia is a salivary gland dysfunction that negatively impacts the life quality of patients; however, there is no effective treatment for xerostomia. Bioengineered organs, generated using stem cells obtained from newborn salivary glands and ligated injury models, are a new organ transplantation strategy that could be feasible for xerostomia treatment. Reconstruction of salivary gland organoids by seed cells obtained from human minor salivary glands will offer theoretical fundaments and technology support for clinical application and organ regeneration research. Herein, we aimed to propose a new method for culturing and enriching adult human minor salivary gland stem cells in vitro in a three-dimensional (3D) environment via Wnt signaling activation.MethodsObtained and characterized human minor salivary gland stem cells (hMSGSCs) with self-organization ability were 3D-cultured to generate organoids. We examined hMSGSCs proliferation and colony formation using MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assays. Telomerase reverse transcriptase staining, flow cytometry, immunofluorescence assay, RNA isolation, RT-PCR, and qPCR were performed to assess hMSGSCs structure and the function of reconstructive organoids in vitro.ResultshMSGSCs showed typical epithelial-like characteristics, such as positive for CD49f and cell KRT expression. hMSGSCs served as adult stem cells in salivary glands and could differentiate into acinar and duct cells. Upon the addition of Noggin, CHIR99021, and Wnt3A to the 3D culture system, hMSGSCs showed higher LGR5 expression and decreased AMY1B and MUC5B expression. Therefore, the Wnt and bone morphogenetic protein (BMP) pathways are important in regulating hMSGSCs self-organization and differentiation.ConclusionsWe showed that the stem cell properties of hMSGSCs in a 3D culture system can be maintained by activating the Wnt signaling pathway and inhibiting the BMP signaling pathway. Our findings contribute new insights on salivary gland organoid generation in vitro.

Immunohistological profiling confirms salivary gland-like nature of the tubarial glands and suggests closest resemblance to the palatal salivary glands

Background and purposeHigh label uptake in 68 Ga-PSMA-11 PET/CT recently identified a bilateral nasopharyngeal structure as a salivary gland (SG)-like additional ‘area of interest’, to be considered in conditions affecting SGs. These structures were termed ‘tubarial glands’. We aimed to further characterize their histological and immunohistochemical position compared to established SGs. MethodsTubarial gland tissue was compared with parotid, submandibular, sublingual, palatal and labial SGs tissue using immunohistological techniques. ResultsExpression of acinar cell-associated aquaporin-5 (AQP5) was detected in tubarial glands, in an apical location associated in control, established SGs with polarized, secretory acinar cells. Keratin14 (KRT14) expression in cells peripheral to AQP5+ clusters also suggested presence of myoepithelial cells. α-amylase, prolactin-induced protein, proline rich protein Haelll subfamily 2, and Muc5B expression suggests mucous acinar cell presence, and presence of muco-serous acinar cells peripheral to putative mucous acinar cells. Expression of adrenergic receptor-β1 by acinar-like cells of the tubarial gland suggests ability to transduce sympathetic neuronal signaling. In terms of ductal architecture, tubarial glands contained large excretory-like ducts (similar to all other SGs), and squamous ducts, comprised of intermingled KRT14+ and KRT7+ cells. These squamous ducts were also observed in palatal, sublingual and labial SGs. No striated or intercalated ducts were observed, similar to palatal SGs. ConclusionBased on histological and immunohistochemical analyses, the tubarial glands resemble SGs. They most convincingly echo characteristics of the palatal SGs in terms of ductal cells, and both the palatal and labial SGs when considering acinar cells.

TNF-α and MMPs mediated mucus hypersecretion induced by cigarette smoke: An in vitro study

Cigarette smoke is one of the leading causes of oxidative stress due to high levels of free radicals, which in turn leads to the degradation of alveolar cell walls and development of emphysema. Cigarette smoking has been linked to chronic bronchitis, Chronic Obstructive Pulmonary Disease (COPD) and lung cancer as well. The aim of the present study was to observe the effect of cigarette smoke extract (CSE) on TNF-α and MMPs mediated mucus hypersecretion in A549 cell line. The MTT experiments showed that CSE caused a dose-dependent decline in the level of viability of A549 cells. In addition, AO/PI and Mitotracker Red staining assays demonstrated that CSE caused the A549 cells to undergo apoptosis. This was determined by observing the reduction in mitochondrial membrane potential. CSE was found to be responsible for the formation of intracellular ROS, which was observed by DCFDA staining through fluorescence microscopy. Approximately 65% migration rate was decreased in 20% CSE exposed cells. CSE exposure led to the significantly increased mRNA levels of TNF-α, MMP-7, and MMP-12, in comparison to the control cells. Additionally, the expression of MUC5AC and MUC5B was provoked by CSE as well. Human epithelial cells are stimulated by TNF-α and MMPs secreted mucus, as shown by expression of MUC5AC and MUC5B. CSE could induce mucus in lungs through TNF-α and MMPs mediated pathways.

Bioinformatic Analysis of Key Regulatory Genes in Adult Asthma and Prediction of Potential Drug Candidates.

Asthma is a common chronic disease that is characterized by respiratory symptoms including cough, wheeze, shortness of breath, and chest tightness. The underlying mechanisms of this disease are not fully elucidated, so more research is needed to identify better therapeutic compounds and biomarkers to improve disease outcomes. In this present study, we used bioinformatics to analyze the gene expression of adult asthma in publicly available microarray datasets to identify putative therapeutic molecules for this disease. We first compared gene expression in healthy volunteers and adult asthma patients to obtain differentially expressed genes (DEGs) for further analysis. A final gene expression signature of 49 genes, including 34 upregulated and 15 downregulated genes, was obtained. Protein-protein interaction and hub analyses showed that 10 genes, including POSTN, CPA3, CCL26, SERPINB2, CLCA1, TPSAB1, TPSB2, MUC5B, BPIFA1, and CST1, may be hub genes. Then, the L1000CDS2 search engine was used for drug repurposing studies. The top approved drug candidate predicted to reverse the asthma gene signature was lovastatin. Clustergram results showed that lovastatin may perturb MUC5B expression. Moreover, molecular docking, molecular dynamics simulation, and computational alanine scanning results supported the notion that lovastatin may interact with MUC5B via key residues such as Thr80, Thr91, Leu93, and Gln105. In summary, by analyzing gene expression signatures, hub genes, and therapeutic perturbation, we show that lovastatin is an approved drug candidate that may have potential for treating adult asthma.

The association between MUC5AC and MUC5B genes expression and remodeling progression in severe neutrophilic asthma: A direct relationship

BackgroundMUC5 dysregulation is a hallmark of severe neutrophilic asthmatic patients. This study investigates the expression of MUC5AC and MUC5B at mRNA levels on asthma severity and airway wall thickness in severe neutrophilic asthmatic patients. MethodIn this case-control clinical trial, twenty-five severe neutrophilic asthmatic patients and ten control subjects were enrolled. Subjects underwent ACT, pulmonary functions tests, and fractional exhaled nitric oxide (FENO). Also, induced sputum has been obtained to assess the expression of MUC5AC and MUC5B by the real-time PCR. In addition, the thickness of the airway wall was assessed by high-resolution computed tomography (HRCT), and bioinformatic analysis was implemented to approve the selection of the appropriate genes and for further investigations. ResultA significant difference was observed between the asthmatic and control in MUC5AC and MUC5B mRNA expression. Meanwhile, the expression of MUC5AC increased remarkably by asthma severity; also, it is associated with airway wall thickness (WT) (both P-value <0.05). The expression of MUC5B in asthmatic patients was lower than in control. There is no significant correlation between MUC5B mRNA level and WT and asthma severity. Notably, MUC5AC transcription level was correlated to sputum neutrophil percentage, while MUC5B transcription level had a positive correlation with sputum macrophages and a negative one with sputum neutrophils. ConclusionIn severe neutrophilic asthma, airway wall thickness increases with MUC5AC mRNA overexpression, which is probably related to asthma severity and the formation of mucus plugs. However, the expression of MUC5B was decreased, resulting in poor mucociliary clearance in the airways. Trial registrationIR.IAU.MSHD.REC.1400.124

Abstract 1479: FOXA2 induces invasive mucinous adenocarcinoma of the lung in the presence of mutant KRAS

Abstract Introduction: The role of the endoderm lineage transcription factor FOXA2 in autochthonous lung tumors has been elusive though the expression of FOXA2 has been reported in human lung cancers. Here, using GEMM (Genetically Engineered Mouse Models) and human lung cancer cell lines, we delineate the role of FOXA2 in lung cancers. Experimental Procedures: GEMM that carry mutant KRAS or mutant EGFR along with FOXA2 (CCSP-rtTA; tetO-Foxa2; tetO-Kras.G12D or tetO-EGFR.L858R) were developed allowing for lung tumor induction by doxycycline administration. The growth of lung tumors affected by FOXA2 was assessed by microCT and pathologically. FOXA2 downstream genes were also evaluated in vivo by immunohistochemistry of mouse lung sections and in vitro by gain (ectopic lentivirus transduction) and loss (siRNA) of function approaches with human lung cancer cell lines using TaqMan gene expression, RNA-seq, ChIP-seq and CRISPRi analyses. FOXA2 protein expression in human lung cancer specimens was also assessed. Data: FOXA2 suppressed the growth of EGFR-mutant lung tumors while promoting that of KRAS-mutant lung tumors in GEMM. Notably, half of the KRAS-mutant lung tumors presented invasive mucinous adenocarcinoma of the lung (IMA) with the expression of a master mucous transcription factor SPDEF and the two major IMA mucin genes MUC5AC and MUC5B. In vitro, FOXA2 induced the expression of SPDEF, MUC5AC and MUC5B by directly binding to their loci. H3K27ac was introduced at the enhancer region of MUC5AC (most highly expressed IMA gene) by FOXA2 and the expression of MUC5AC initiated by FOXA2 was enhanced by mutant KRAS. FOXA2 was highly expressed in human IMA compared to normal lung. Conclusions: FOXA2 induces IMA in the presence of mutant KRAS by directly regulating the expression of mucous genes. Citation Format: Yutaka Maeda, Koichi Tomoshige, William D. Stuart, Iris M. Fink-Baldauf, Masaoki Ito, Tomoshi Tsuchiya, Takeshi Nagayasu, Tomoki Yamatsuji, Morihito Okada, Takuya Fukazawa, Minzhe Guo. FOXA2 induces invasive mucinous adenocarcinoma of the lung in the presence of mutant KRAS [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1479.

Profile of MUC5AC and MUC5B mucins expression in asthma patients under cold exposure

Introduction. Cold airway hyperresponsiveness (CAH) is a common condition in patients with asthma, which worsens the clinical course of the disease and the patients’ quality of life. MUC5AC and MUC5B are the main secreted mucins in the respiratory tract, which are involved in normal mucociliary clearance, but also capable of provoking the development of pathological changes in case of dysregulation of their balanced production.Aim. The aim of this study was to determine the dynamics of MUC5AC and MUC5B expression during experimental cooling in patients with asthma depending on the status of CAH.Materials and methods. The study enrolled 98 subjects including 26 patients with chronic non-obstructive bronchitis without exacerbation (control group) and 72 patients with asthma. The expression of MUC5AC, MUC5B and TRPM8 was determined in the upper respiratory tract by quantitative reverse transcription PCR. The production of MUC5AC and MUC5B was also measured in sputum by ELISA. All patients underwent a bronchoprovocation test with isocapnic cold air hyperventilation to detect CAH, and a similar nasal challenge was performed to assess the effect of cooling on the expression of the studied genes.Results. Patients with asthma had 4.22-fold increase in the expression of MUC5AC (p=0.02) in the nasal epithelium as compared with the control group. CAH was associated with an initial 7.33-fold upregulation of MUC5AC (p=0.008) as well as with further increase in MUC5AC expression but a decrease in MUC5B in response to cooling, which was not observed in asthma patients without CAH. Basal TRPM8 expression was associated with baseline level of MUC5AC (ρ=0.41, p=0.04), MUC5B (ρ=0.55, p&lt;0.001) and amount of sputum produced after the cold bronchoprovocation.Conclusion. Asthma patients with CAH demonstrate a more pronounced imbalance in the production of mucins, which is aggravated by cold exposure. This, in turn, can lead to a number of pathological disorders associated with a more severe course of the disease.

Clinical Significance and Levels of NLRP3, MUC5AC, and MUC5B in Asthma

Objective: To explore the role of NLRP3 in mucus hypersecretion in asthmatic patients. Methods: From January 2020 to June 2022, 90 patients with asthma and 60 healthy patients under the Department of Pulmonary and Critical Care Medicine of the First Affiliated Hospital of Xi’an Medical University were selected. Immunohistochemistry and enzyme-linked immunosorbent assay were performed. NLRP3 inflammasome and mucins MUC5AC and MUC5B levels in lung tissue and sputum were detected. Results: Compared to the healthy control group, the asthma group had significantly higher sputum MUC5A (20.12 ± 5 .07 versus 36.21 ± 6.13) and NLRP3 (72.31 ± 15.13 versus 119.21 ± 31.21) levels (P &lt; 0.05) but lower MUC5B levels (1.35 ± 0.12 versus 0.53 ± 0.11, P &lt; 0.05). Immunohistochemistry showed that NLRP3, MUC5AC, and MUC5B expressions were consistent with the sputum results. Conclusion: NLRP3 and MUC5AC levels are significantly increased in asthmatic patients, whereas MUC5B levels are reduced in these patients. They can be used as targets for the diagnosis and treatment of asthma.