MUC5B Production Research Articles

Chronic airway epithelial hypoxia exacerbates injury in muco-obstructive lung disease through mucus hyperconcentration.

Unlike solid organs, human airway epithelia derive their oxygen from inspired air rather than the vasculature. Many pulmonary diseases are associated with intraluminal airway obstruction caused by aspirated foreign bodies, virus infection, tumors, or mucus plugs intrinsic to airway disease, including cystic fibrosis (CF). Consistent with requirements for luminal O2, airway epithelia surrounding mucus plugs in chronic obstructive pulmonary disease (COPD) lungs are hypoxic. Despite these observations, the effects of chronic hypoxia (CH) on airway epithelial host defense functions relevant to pulmonary disease have not been investigated. Molecular characterization of resected human lungs from individuals with a spectrum of muco-obstructive lung diseases (MOLDs) or COVID-19 identified molecular features of chronic hypoxia, including increased EGLN3 expression, in epithelia lining mucus-obstructed airways. In vitro experiments using cultured chronically hypoxic airway epithelia revealed conversion to a glycolytic metabolic state with maintenance of cellular architecture. Chronically hypoxic airway epithelia unexpectedly exhibited increased MUC5B mucin production and increased transepithelial Na+ and fluid absorption mediated by HIF1α/HIF2α-dependent up-regulation of β and γENaC (epithelial Na+ channel) subunit expression. The combination of increased Na+ absorption and MUC5B production generated hyperconcentrated mucus predicted to perpetuate obstruction. Single-cell and bulk RNA sequencing analyses of chronically hypoxic cultured airway epithelia revealed transcriptional changes involved in airway wall remodeling, destruction, and angiogenesis. These results were confirmed by RNA-in situ hybridization studies of lungs from individuals with MOLD. Our data suggest that chronic airway epithelial hypoxia may be central to the pathogenesis of persistent mucus accumulation in MOLDs and associated airway wall damage.

Hereditary Mucin Deficiency Caused by Biallelic Loss of Function of MUC5B.

Rationale: Mucin homeostasis is fundamental to airway health. Upregulation of airway mucus glycoprotein MUC5B is observed in diverse common lung diseases and represents a potential therapeutic target. In mice, Muc5b is required for mucociliary clearance and for controlling inflammation after microbial exposure. The consequences of its loss in humans are unclear. Objectives: The goal of this study was to identify and characterize a family with congenital absence of MUC5B protein. Methods: We performed whole-genome sequencing in an adult proband with unexplained bronchiectasis, impaired pulmonary function, and repeated Staphylococcus aureus infection. Deep phenotyping over a 12-year period included assessments of pulmonary radioaerosol mucociliary clearance. Genotyping with reverse phenotyping was organized for eight family members. Extensive experiments, including immunofluorescence staining and mass spectrometry for mucins, were performed across accessible sample types. Measurements and Main Results: The proband, and her symptomatic sibling who also had extensive sinus disease with nasal polyps, were homozygous for a novel splicing variant in the MUC5B gene (NM_002458.2: c.1938 + 1G>A). MUC5B was absent from saliva, sputum, and nasal samples. Mucociliary clearance was impaired in the proband, and large numbers of apoptotic macrophages were present in sputum. Three siblings heterozygous for the familial MUC5B variant were asymptomatic but had a shared pattern of mild lung function impairments. Conclusions: Congenital absence of MUC5B defines a new category of genetic respiratory disease. The human phenotype is highly concordant with that of the Muc5b-/- murine model. Further study of individuals with decreased MUC5B production could provide unique mechanistic insights into airway mucus biology.

Influence of silica particles on mucociliary structure and MUC5B expression in airways of C57BL/6 mice

Purpose: Impaired mucociliary clearance is an initial characteristic of recurrent cough, respiratory infection and chronic respiratory diseases. It has been demonstrated that prolonged inhalation of respirable silica particles results in a variety of pulmonary diseases, but whether the mucociliary system is involved in this process is unclear. This study aims to evaluate the effects of silica particles on mucociliary structure and MUC5B production in respiratory tract.Materials and Methods: C57BL/6 mice were administered with 2.5 mg silica particles through a single intratracheal instillation. The changes of mucociliary structure and MUC5B expression in trachea was evaluated by HE and AB-PAS staining, transmission electron microscopy and immunohistochemistry on days 1, 7, 28 and 84 post-exposure.Results: The mucociliary structure of airway epithelium was obviously impaired by silica particles, showing disordered, shortened or partially lost cilia on the surface, increased mucus in mucous layer and submucosal glands from day 7 to day 84. A variety of ultrastructural abnormalities were discovered in silica-exposed airway cilia, including absence of central pair microtubules, disorganized microtubules and clusters of axoneme on day 1 and 7. The numbers of ciliary axonemes and basal bodies in ciliated epithelial cells were significantly decreased, whereas the proportion of abnormal axonemes was gradually increased with exposure to silica particles (P < 0.05). In addition, silica particles significantly decreased MUC5B expression on the surface of airway epithelium on day 28 and 84, but obviously increased its production in submucosal glands from day 1 to day 84 (P < 0.01).Conclusions: Silica particles could lead to ultrastructural defects in airway cilia, mucus hypersecretion and altered MUC5B expression in trachea, indicating that impaired mucociliary structure and altered MUC5B production might participate in the development of silica-related respiratory diseases.

A 3D "In Vitro" Model to Study Hyaluronan Effect in Nasal Epithelial Cell Line Exposed to Double-Stranded RNA Poly(I:C).

Environmental agents, including viral and bacterial infectious agents, are involved in the alteration of physicochemical and biological parameters in the nasal epithelium. Hyaluronan (HA) has an important role in the regulation of tissue healing properties. High molecular weight HA (HMW-HA) shows greater anti-inflammatory responses than medium molecular weight HA (MMW-HA) and low molecular weight HA (LMW-HA). We investigated the effect of HMW-HA, MMW-HA and LMW-HA on the regulation of physicochemical and biological parameters in an “in vitro” model that might mimic viral infections of the nasal epithelium. Human nasal epithelial cell line RPMI2650 was stimulated with double-stranded RNA (dsRNA) Poly(I:C) for 5 days in air-liquid-interface (ALI) culture (3D model of airway tissue). dsRNA Poly(I:C) treatment significantly decreased transepithelial electrical resistance (TEER) in the stratified nasal epithelium of RPMI2650 and increased pH values, rheological parameters (elastic G’ and viscous G’’), and Muc5AC and Muc5B production in the apical wash of ALI culture of RPMI2650 in comparison to untreated cells. RPMI2650 treated with dsRNA Poly(I:C) in the presence of HMW-HA showed lower pH values, Muc5AC and Muc5B production, and rheological parameters, as well as increased TEER values in ALI culture, compared to cells treated with Poly(I:C) alone or pretreated with LMW-HA and MMW-HA. Our 3D “in vitro” model of epithelium suggests that HMW-HA might be a coadjuvant in the pharmacological treatment of viral infections, allowing for the control of some physicochemical and biological properties affecting the epithelial barrier of the nose during infection.

Can PBDEs affect the pathophysiologic complex of epithelium in lung diseases?

Brominated flame-retardant (BFRs) exposure promotes multiple adverse health outcomes involved in oxidative stress, inflammation, and tissues damage. We investigated BFR effects, known as polybrominated diphenyl ethers (PBDEs) (47, 99 and 209) in an air-liquid-interface (ALI) airway tissue derived from A549 cell line, and compared with ALI culture of primary human bronchial epithelial cells (pHBEC). The cells, exposed to PBDEs (47, 99 and 209) (0.01-1 μM) for 24 h, were studied for IL-8, Muc5AC and Muc5B (mRNAs and proteins) production, as well as NOX-4 (mRNA) expression. Furthermore, we evaluated tight junction (TJ) integrity by Trans-Epithelial Electrical Resistance (TEER) measurements, and zonula occludens-1 (ZO-1) expression in the cells, and pH variations and rheological properties (elastic G', and viscous G″, moduli) in apical washes of ALI cultures. N-acetylcysteine (NAC) (10 mM) effects were tested in our experimental model of A549 cells. PBDEs (47, 99 and 209) exposure decreased TEER, ZO-1 and pH values, and increased IL-8, Muc5AC, Muc5B (mRNAs and proteins), NOX-4 (mRNA), and rheological parameters (G', G″) in ALI cultures of A549 cell line and pHBEC. NAC inhibited PBDE effects in A549 cells. PBDE inhalation might impairs human health of the lungs inducing oxidative stress, inflammatory response, loss of barrier integrity, unchecked mucus production, as well as altered physicochemical and biological properties of the fluids in airway epithelium. The treatment with anti-oxidants restored the negative effects of PBDEs in epithelial cells.

Primary infection by Pneumocystis induces Notch-independent Clara cell mucin production in rat distal airways.

Clara cells are the main airway secretory cells able to regenerate epithelium in the distal airways through transdifferentiating into goblet cells, a process under negative regulation of the Notch pathway. Pneumocystis is a highly prevalent fungus in humans occurring between 2 and 5 months of age, a period when airways are still developing and respiratory morbidity typically increases. Pneumocystis induces mucus hyperproduction in immunocompetent host airways and whether it can stimulate Clara cells is unknown. Markers of Clara cell secretion and Notch1 activation were investigated in lungs of immunocompetent rats at 40, 60, and 80 days of age during Pneumocystis primary infection with and without Valproic acid (VPA), a Notch inducer. The proportion of rats expressing mucin increased in Pneumocystis-infected rats respect to controls at 60 and 80 days of age. Frequency of distal airways Clara cells was maintained while mRNA levels for the mucin-encoding genes Muc5B and Muc5ac in lung homogenates increased 1.9 and 3.9 times at 60 days of infection (P. = 0.1609 and P. = 0.0001, respectively) and protein levels of the Clara cell marker CC10 decreased in the Pneumocystis-infected rats at 60 and 80 days of age (P. = 0.0118 & P. = 0.0388). CC10 and Muc5b co-localized in distal airway epithelium of Pneumocystis-infected rats at day 60. Co-localization of Muc5b and Ki67 as marker of mitosis in distal airways was not observed suggesting that Muc5b production by Clara cells was independent of mitosis. Notch levels remained similar and no transnucleation of activated Notch associated to Pneumocystis infection was detected. Unexpectedly, mucus was greatly increased at day 80 in Pneumocystis-infected rats receiving VPA suggesting that a Notch-independent mechanism was triggered. Overall, data suggests a Clara to goblet cell transdifferentiation mechanism induced by Pneumocystis and independent of Notch.

Transgenic Mouse Reporter to Study Muc5b In Vivo.

Dysregulation of gel-forming mucins is associated with many airway diseases. Better knowledge of the pathophysiological mechanisms linking mucins and respiratory diseases will advance the understanding of their pathogenesis and should provide opportunities to develop new therapeutic compounds for treatment. MUC5B and MUC5AC are the two main gel-forming mucins in the respiratory tract. The organization in domains and the expression profile of mouse Muc5b are very similar to those in humans, which makes the mouse a relevant model for studies of the translational activities of human mucins. To assess the in vivo biological functions of Muc5b, a mouse reporter tagged in frame with the green fluorescent protein marker has been engineered by homologous recombination. The proof of concept that this reporter model may be informative for translational studies was confirmed by the finding that interleukin-13 administration in living mice upregulated Muc5b production.

In vivo imaging of the Muc5b gel-forming mucin

Gel-forming mucins are macromolecules produced by goblet cells and responsible for the mucus gel formation. Changes in goblet cell density and in gel-forming mucin production have emerged as sensitive indicators for mucosal diseases. A Muc5b-GFP tagged reporter mouse was used to assess Muc5b production in mouse tissues by immunofluorescence microscopy and fluorescent activity using stereromicroscopy and probe-based confocal laser endomicroscopy. Muc5b production was followed longitudinally by recording the fluorescent activity in vagina and in embryonic lung explants under stimulation by interleukin 13. We show that the GFP is easily visualized in the mouse adult ear, nose, trachea, gallbladder, and cervix. Live Muc5b is also easily monitored in the nasal cavity, trachea and vagina where its production varies during the estrus cycle with a peak at the proestrus phase and in pregnant mice. Explant culture of reporter mouse embryonic whole lung shows that interleukin 13 stimulates Muc5b production. The transgenic Muc5b-GFP mouse is unique and suitable to study the mechanisms that regulate Muc5b production/secretion and mucous cell differentiation by live imaging and can be applied to test drug efficacy in mucosal disease models.

MUC5B Production Is Unaffected by Akt Inhibition in Human Lung Epithelial NCI-H292 Cells

In the human airway, the gelforming mucin subtypes MUC5B and MUC5AC play important roles in biophylaxis. However, the regulation of MUC5B production is less clear than that of MUC5AC. Therefore, the regulation of MUC5B production by cell attachment and Akt was investigated in human lung epithelial NCI-H292 cells. We found that low cell attachment to culture plates induced the upregulated production of both MUC5B and MUC5AC. Cell attachment induces the activation of Akt, a serine/threonine kinase. Cell treatment with Akt inhibitor I decreased Akt phosphorylation and activation. However, MUC5B production was unaffected by Akt inhibition, whereas MUC5AC production was upregulated. MUC5B production was also unaffected by Akt inhibition in cells cultured on type IV collagen or fibronectin. These results suggest that the production of both MUC5B and MUC5AC is regulated by cell attachment. However, the regulation of MUC5B is unaffected by Akt inhibition, in contrast to that of MUC5AC.

MUC5B mucin production is upregulated by fibronectin and laminin in human lung epithelial cells via the integrin and ERK dependent pathway.

MUC5B mucin is a principal component of airway mucus and plays a key role in biodefense. We investigated the regulation of MUC5B production using the signals from extracellular matrix (ECM) components in NCI-H292 human lung epithelial cells. We found that MUC5B production in NCI-H292 cells cultured on fibronectin or laminin increased by 4-5-fold, with the increase occurring in a dose- and time-dependent manner. In contrast, MUC5B production was unchanged on type-IV collagen. Inhibition of integrin β1 induced upregulation of MUC5B and MUC5AC; however, inhibition of p38 MAPK did not show any remarkable change in overproduced MUC5B. Inhibition of extracellular signal-regulated kinase (ERK) pathway or the transcription factor NF-κB induced the recovery of overproduced MUC5B on fibronectin and laminin. These results suggest that MUC5B production can be regulated by ECM components and that MUC5B is upregulated by fibronectin and laminin via the integrin, ERK, and NF-κB dependent pathway.

Muc5b is required for airway defence

Respiratory surfaces are exposed to billions of particulates and pathogens daily. A protective mucus barrier traps and eliminates them through mucociliary clearance (MCC). However, excessive mucus contributes to transient respiratory infections and to the pathogenesis of numerous respiratory diseases. MUC5AC and MUC5B are evolutionarily conserved genes that encode structurally related mucin glycoproteins, the principal macromolecules in airway mucus. Genetic variants are linked to diverse lung diseases, but specific roles for MUC5AC and MUC5B in MCC, and the lasting effects of their inhibition, are unknown. Here we show that mouse Muc5b (but not Muc5ac) is required for MCC, for controlling infections in the airways and middle ear, and for maintaining immune homeostasis in mouse lungs, whereas Muc5ac is dispensable. Muc5b deficiency caused materials to accumulate in upper and lower airways. This defect led to chronic infection by multiple bacterial species, including Staphylococcus aureus, and to inflammation that failed to resolve normally. Apoptotic macrophages accumulated, phagocytosis was impaired, and interleukin-23 (IL-23) production was reduced in Muc5b(-/-) mice. By contrast, in mice that transgenically overexpress Muc5b, macrophage functions improved. Existing dogma defines mucous phenotypes in asthma and chronic obstructive pulmonary disease (COPD) as driven by increased MUC5AC, with MUC5B levels either unaffected or increased in expectorated sputum. However, in many patients, MUC5B production at airway surfaces decreases by as much as 90%. By distinguishing a specific role for Muc5b in MCC, and by determining its impact on bacterial infections and inflammation in mice, our results provide a refined framework for designing targeted therapies to control mucin secretion and restore MCC.

TGF-β2 decreases baseline and IL-13-stimulated mucin production by primary human bronchial epithelial cells

ABSTRACTIntroduction: Mucus hypersecretion is a major contributor to asthma pathology and occurs as part of a spectrum of structural changes termed airway wall remodeling. Transforming growth factor (TGF)-β is proposed to play a key role in regulating airway matrix remodeling although less is known about the specific action of TGF-β isoforms in regulating mucus production. Methods: Primary human bronchial epithelial (HBE) cells cultured at air–liquid interface were treated with exogenous TGF-β1, TGF-β2, and/or a Th2 cytokine, interleukin (IL)-13. Expression and production of respiratory mucins, MUC5AC and MUC5B, were analyzed by real-time PCR, agarose gel electrophoresis, and western blotting. A murine-transformed Clara cell line (mtCC1-2) transfected with a luciferase reporter driven by the Muc5ac promoter containing Smad4 site-mutated cis sequences was used to determine whether exogenous TGF-β2 affects Muc5ac promoter function. Results: Surprisingly, TGF-β1 showed no measurable effect on MUC5AC or MUC5B production by HBE cells whereas TGF-β2 caused a decrease in both MUC5AC and MUC5B mRNA and protein. Dual treatment with TGF-β2 and IL-13 partially attenuated the increase in mucin production found with IL-13 alone. This effect was confirmed by using mtCC1-2 cells where addition of TGF-β2 reduced the ability of IL-13/EGF to induce Muc5ac promoter expression in wild-type cells; however, this decrease was absent in mutant promoter-transfected cells. Discussion and conclusion: Findings suggest that normal regulation of MUC5AC and MUC5B production by HBE cells is TGF-β isoform-specific and that TGF-β2 downregulates both MUC5AC and MUC5B. Furthermore, TGF-β2 controls baseline and IL-13-driven Muc5ac promoter function in murine Clara cells via an endogenous Smad4 recognition motif.