Role for Mucin-5AC in Upper and Lower Airway Pathogenesis in Mice

Generation of Muc5ac Deficient (Muc5ac^−/− ) Mice

Muc5ac ^−/− (B6; 129-Muc5ac^tm1Unc /Ehs) mice were generated by targeted deletion of exons 21 to 31 encoding the MUC5AC protein region responsible for glycosylation. A targeting construct consisting of a neomycin-positive and herpes simplex virus thymidine kinase-negative selection cassette (NeoTKXho) in place of an 8 kilobases (8031 kb) spanning exon 21 to exon 31 of the Muc5ac gene (Supplemental Figure S1-A) was made at the National Institute of Environmental Health Sciences (NIEHS). The construct was introduced into embryonic stem cells (ES-E14TG2a) to generate targeted mice at the Mouse Model Core of the University of North Carolina at Chapel Hill (Supplemental Figure S1-A). The targeted embryonic stem cells were identified by Southern blot analysis and injected into C57BL/6J mice blastocysts which were transplanted into pseudopregnant females. The chimeric offspring obtained were then bred to C57BL/6J mice and the pups screened by Southern blotting. Heterozygous (Muc5ac^+/− ) mice were transferred to the NIEHS animal care facilities and backcrossed into C57BL/6J mice for 8 generations to produce whole body knockout mice and wild-type controls (C57BL/6J-129Ola, Muc5ac^+/+ ). Muc5ac^+/+ and Muc5ac^+/+ mice were identified by Southern blotting (Supplemental Figure S1-B), polymerase chain reaction (PCR; Supplemental Figure S1-C), immunohistochemical analyses (Supplemental Figure S2-A to C), and Western blotting (Supplemental Figure S2-C). Pathogen-free breeding colonies of Muc5ac^+/+ , Muc5ac^−/− , and Muc5ac^+/− mice were maintained in sterilized, filter-topped cages and fed autoclaved food (NIH_31) at the NIEHS animal facilities. All animal use was approved by the NIEHS Animal Care and Use Committee.

Animal Experiments

Male, adult (6-8 weeks old) Muc5ac^+/+ and Muc5ac^−/− mice were used for all studies. Mice were randomly assigned to the experimental groups. For pulmonary fibrosis end points, mice were anesthetized with isoflurane (2% in air), and a single dose (0.5 U/kg body weight [bw]) of bleomycin sulfate (Sigma Chemical Co) or vehicle (PBS) was delivered by oropharyngeal aspiration (intratracheal). Mice were killed at 3, 7, or 14 days postbleomycin. For the RSV disease model, mice were anesthetized with isoflurane and intranasally infected with 10⁶ plaque-forming units of human RSV-19 strain (ViraSource) in 50 µL Hanks balanced salt solution (HBSS). HBSS containing Hep-2 cell lysates (50 μL) was intranasally instilled into control mice as vehicle. Animals were killed at 1, 3, 5, or 7 days after intranasal instillation. To evaluate airway O₃ toxicity, mice were placed in individual stainless-steel wire cages within a whole-body inhalation chamber (Hazelton 1000; Lab Products) with free access to water and foods and exposed continuously to 0.3 parts per million (ppm) O₃ for 48 or 72 hours under constant chamber air temperature (72 °F ± 3 °F) and relative humidity (50% ± 15%) as described in detail previously. ²⁹ Parallel exposure to room air was done in a separate chamber for the same duration. All mice were euthanized by sodium pentobarbital overdose (104 mg/kg bw) for necropsy immediately after the end of designated exposure. Body fat of mice was measured at 6, 9, and 12 months of age using a dual energy X-ray absorptiometry (Lunar PIXImus, GE Lunar Corp).

Bronchoalveolar Lavage Analyses

The right lung of each mouse was lavaged in situ, 4 consecutive times with HBSS (0.5 mL/25 g bw). The bronchoalveolar lavage (BAL) fluid returns were centrifuged (1000g, 10 minutes). An aliquot of the first BAL return supernatants (50 μL) was analyzed for total protein concentration (a marker of edema) in Bradford reagent (Bio-Rad) as indicated in the manufacturer procedure. Cell pellets from all lavage returns were combined and resuspended in 1 mL HBSS for epithelial and inflammatory cell counts as reported previously. ³⁰ To detect cell lysis and toxicity, colorimetric assay for lactate dehydrogenase (LDH) was also performed in aliquots (25 μL) of BAL supernatants by determination of NADH production from added NAD+ (Sigma).

Sandwich Enzyme-Linked Immunosorbent Assay

The BAL supernatants (25 μL) were used to determine neutrophil myeloperoxidase (MPO) using a mouse-specific enzyme-linked immunosorbent assay (ELISA) kit (R&D Systems) according to the manufacturer instructions. Secreted MUC5AC protein was also determined in BAL supernatants following the published method. ²⁹ Briefly, an aliquot of BAL supernatant (20 μL) was loaded in each well of an ELISA plate containing a goat polyclonal anti-MUC5AC capture antibody (1:40 dilution of sc-16903; Santa Cruz Biotechnology Inc) in pH 9.5 bicarbonate–carbonate coating buffer (BD OptEIA Reagent; BD Biosciences). The plate was incubated at 48 °C until the reaction was dry (>5 hours). The wells were washed and blocked overnight with an assay diluent containing 10% fetal bovine serum (BD Opt EIA) at 4 °C. The samples were then incubated with a biotinylated monoclonal anti-MUC5AC detection antibody (1:100 of Clone 45M1, Thermo Scientific) for 1.5 hours at 37 °C. Following incubation with a peroxidase-conjugated secondary antibody (1:2500, goat antimouse-immunoglobulin G [IgG]-horseradish peroxidase), color change was developed by adding the 3,3′,5,5′-tetramethylbenzidine substrate solution (BD Biosciences). Optical density was measured at 450 nm after the stop buffer was added.

Lung and Nasal Airway Histopathology and Immunohistochemistry

The left lung from each mouse was inflated intratracheally in situ with 10% neutral buffered formalin and fixed following procedures published previously. ³¹ The fixed lung lobe was sectioned at proximal (generation 5) and distal (generation 11) levels of the main axial airway. ³² The excised heads of mice from RSV and O₃ studies were flushed retrograde via a cannula through the nasopharyngeal orifice with 10% neutral buffered formalin. The fixed heads were processed for decalcification, and the proximal aspect of the nasal cavity section (ie, at the level of upper incisor teeth, T1) was cut following procedures published previously. ³³ The lung and nasal tissue blocks were embedded in paraffin, and 5 μm-thick section cuts were stained with hematoxylin and eosin (H&E) for histopathological analysis, with Alcian blue (pH 2.5)/periodic acid-Schiff (AB/PAS) reagent for acidic and neutral mucus detection and with Mason trichrome for collagen localization. Severity of histopathologic lung toxicity in the bleomycin study was determined by blinded microscopic evaluation of H&E-stained slides for inflammation and injury and Masson-trichrome-stained slides for collagen associated with areas of inflammation (Experimental Pathology Labs). Tissues were also stained by immunohistochemical methods using mouse monoclonal (IgG₁) anti-MUC5AC (1:100 dilution of Clone 45M1, Thermo Fisher Scientific), rabbit anti-MUC2 (1:667 dilution of GTX100664, GeneTex), goat anti-RSV (1:200 dilution of ab20745, Abcam), and proliferating cell nuclear antigen (PCNA; 1:200 dilution of PCNA; sc-56, Santa Cruz Biotechnology). Briefly, deparaffinized and hydrated tissue sections on microscope slides were treated sequentially with antigen unmasking solution (Vector Laboratories), 0.1% proteinase K, 1 N HCl (1 hour for PCNA only), and endogenous peroxidase quenching solution (5% H₂O₂) before blocking with 1.5% serum (Vectastain ABC kits, Vector Laboratories) at room temperature. Tissue sections were then incubated overnight at 4 °C with each specific primary antibody. After incubation with proper biotinylated secondary antibodies (1:200, Vectastain ABC kits) and avidin/biotin solution, antigens were detected by 3,3′-diaminobenzidine-peroxidase substrate solution (1 minute for PCNA, 5 minutes for RSV, 10 minutes for others, Sigma), and slides were mounted with cover glasses after counterstaining, if necessary, and dehydration. For immunofluorescence staining of RSV, micrograph slides incubated with primary antibody were incubated overnight with fluorochrome-conjugated goat IgG solution (1 μg/mL) for 1 hour and cover-slipped with prolong Gold Antifade reagent with 4′,6-diamidino-2-phenylindole (Thermo Fisher Scientific). Nasal airways and lungs from a subset of male mice (22 Muc5ac^+/+ , 23 Muc5ac^−/− ) at 11 to 12 months were processed for H&E and AB/PAS staining as indicated above. Occurrence of preinvasive alveolar epithelial proliferation was determined by percentage of mice with increased focal cellularity in alveoli including early adenoma on H&E-stained light micrographs. Nodular lymphocytic accumulation was determined by percentage of mice bearing lymphocyte nodules in perivascular-peribronchial pulmonary regions on H&E-stained light micrographs.

Morphometric Analysis of Nasal Airway Mucosubstances and Terminal Bronchial Proliferating Cells

The amount of AB/PAS-stained mucosubstances in nasal airways from RSV and O₃ studies was estimated using computerized image analysis and standard morphometric techniques. ^33,34 Briefly, images of AB/PAS-stained cross-sections from the proximal nasal airway (turbinate section T1) were taken using a light microscope with an attached camera and software (Olympus BX53 and cellSense, Olympus). The AB/PAS-positive areas within the surface epithelium lining mid-septum and ventral septum (from the tip level of nasoturbinate to the bottom of the ventral septum) were calculated from the semiautomatically circumscribed perimeter of the stained material using the NIH Image J 1.52a software (https://imagej.nih.gov/ij/index.html). The length of the basal lamina underlying the surface epithelium was concomitantly calculated from the contour length on digitized images using the same system. Similarly, stored PAS-positive substances in subepithelial glands (mucous glands) in the ventral septum (from the bottom level of maxilloturbinate to the end of the ventral septum) were quantitated and normalized to the length of basal lamina on top of the lamina propria. The volume of intraepithelial mucosubstances per unit surface area (volume density, nL/mm² basal lamina) was determined as previously described in detail. ³⁵ Areas of PAS-positive secretion in the ventral wall region of proximal nasal sections from the O₃ study were also measured as indicated above.

In Situ Hybridization of Airway Mucins

Expression of Muc4 and Muc5b transcripts in lung and nasal tissues was detected by in situ RNA detection techniques in accordance with manufacturer instructions (FFPE assay kit; Advanced Cell Diagnostics). Briefly, formalin-fixed paraffin-embedded tissue sections on charged slides were baked at 60 °C (1 hour for lung, overnight for nasal) and deparaffinized and rehydrated. The sections were pretreated with hydrogen peroxide, incubated in boiling target retrieval solution for 15 minutes, and permeabilized with protease plus solution (40 °C, 30 minutes for lung, 15 minutes for nasal). Tissues were then hybridized with labeled double z RNA probe pairs specific for mouse Muc4 and Muc5b (40 °C for 2 hours). The target messenger RNA (mRNA) signals were sequentially amplified using a horseradish peroxidase-based signal amplification system, and color development was detected with 3,3′-diaminobenzidine. The slides were counterstained with hematoxylin for 10 seconds. Representative light photomicrographs were prepared by Olympus microscope BX53 and cellSense software.

Lung RNA Isolation and PCR

Total lung RNAs were isolated from lung homogenates (RNeasy Mini Kit) and reverse transcribed into complementary DNAs (cDNAs). Aliquots of cDNA (40 ng) were subjected to quantitative PCR with mouse-specific primers for Muc5ac (amplifying nucleotide 3547 to 3667 of NM_010844; 5′-TGCATGCGTACCTGCCAGAA-3′, 5′-CACACTGCATTGTGCCCTCA-3′), Muc5b, ³⁶ Muc4 (RealTimePrimers), and Muc2, ³⁶ and 18s ribosomal RNA, an internal control (amplifying nucleotide 324-507 of K1364; 5′-TACCTGGTTGATCCTGCCAG-3′, 5′-CCGTCGGCATGTATTAGCTC-3′) using CFX Connect Realtime System (Bio-Rad). Copy number of RSV nucleoprotein (N) was determined by droplet digital PCR. Briefly, an aliquot of lung cDNA (2 ng) in PCR mixture containing a SYBR Green dye (Bio-Rad) and 125 nM of RSV N gene primers (amplifying nucleotide 1137-1220 of M11486; 5′-AGATCAACTTCTGTCATCCAGCAA-3′, 5′-TTCTGCACATCATAATTAGGAGTATCAAT-3′) was partitioned into approximately 20,000 individual nanoliter-sized water-in-oil droplets by Bio-Rad QX200 Automated Droplet Generator followed by PCR reaction (C1000 Touch Thermal Cycler, Bio-Rad). The droplets were then read one-by-one in QX200 Droplet Reader (Bio-Rad) and assigned as positive (1) or negative (0). The copy number of the N gene per µg RNA was calculated by the positive droplets.

Western Blot Analyses

Protein level of epithelial cadherin (E-cadherin, 135 kDa for precursor and 120/80 kDa for matured), an intercellular adherens junction protein in epithelial barrier, was determined. Total lung proteins were isolated from individual mouse lung homogenate in radioimmunoprecipitation assay buffer (n = 4/group), and equal amounts of the lung proteins from 2 mice were pooled. Aliquots of pooled proteins (70-150 μg) were separated on Tri-HCl sodium dodecyl sulfate polyacrylamide gel electrophoresis genes (7.5% or 10%-20%, Bio-Rad) and analyzed by routine Western blotting using specific antibodies (Santa Cruz Biotechnology) against E-cadherin (sc-59778, 1:200 dilution) and pan-actin (sc-1616, 1:500 dilution). Blot images were scanned using the FluorChem HD2 System (ProteinSimple) and quantified by densitometry using Image J Gel analysis software (https://imagej.nih.gov).

Sircol Assay for Lung Collagen Quantitation

Total soluble collagen content in right lungs was determined using the Sircol collagen-dye binding assay kit (Biocolor). Briefly, whole right lung lobes from each mouse were homogenized in 1 mL of acetic acid solution (0.5 N with protease inhibitors) and incubated on a gentle shaker overnight at room temperature. The acid extracts were centrifuged (15,000g for 30 minutes), and aliquots of the supernatants (200 μL) containing acid-soluble collagens were mixed with 1 mL Sirius Red dye reagent for 30 minutes on a shaker (800 rpm). The pellets (bead-dye-collagen complex) acquired by centrifugation (10,000g for 10 minutes) were dissolved in 1 mL alkali reagent (0.5 M NaOH). Absorbance of alkali reagent in which collagen-dye complex was dissolved was measured at 550 nm. The amount of collagen was quantitated using a standard curve prepared from collagen standard.

Serum IgE Detection

From each mouse, an aliquot of serum (1 μL) was isolated from blood samples collected by cardiac puncture and was diluted in an assay diluent (1:100). The immunoglobulin E (IgE) levels were determined using a mouse-specific sandwich ELISA kit (BD Opt EIA) and similarly prepared serial dilution of IgE standards (BD Biosciences).

Statistics

Data were expressed as the group mean ± standard error of the mean. Two-way analysis of variance (ANOVA) was used to evaluate the effects of genotype (Muc5ac^+/+ vs Muc5ac^−/− ) and exposure (RSV vs vehicle, bleomycin vs vehicle, O₃ vs air) at each time point. One-way ANOVA was used to assess MUC5AC secretion or Muc5ac mRNA expression in Muc5ac^+/+ mice. Kruskal-Wallis ANOVA on Ranks test was applied to the histology score data. The Student-Newman-Keuls test was used for a posteriori comparisons of means for all multiple comparisons (P < .05). All statistical analyses were performed using SigmaPlot 13.0 program (Systat Software).

Enhanced Responsivity of Muc5ac^−/− Mice to Bleomycin-Induced Pulmonary Injury and Fibrosis

The bw gain at 14 days after bleomycin treatment was significantly lower in Muc5ac^−/− mice compared to Muc5ac^+/+ mice (Figure 1A). Relative to respective controls, bleomycin significantly increased the BAL protein concentration (a marker of protein edema), LDH activity (a marker of cell death), and the numbers of total cells and leukocytes (neutrophils and lymphocytes) in Muc5ac^+/+ and Muc5ac^−/− mice (Figure 1B-F). However, bleomycin-induced protein edema and lung cell death (7-14 days) as well as numbers of lung leukocytes (3-14 days) in Muc5ac^−/− mice were significantly greater than those in Muc5ac^+/+ mice (Figures 1B-F).

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Figure 1.

Enhanced bleomycin-induced lung injury in mucin-5AC-deficient (Muc5ac^−/− ) mice: Body weight change (A) and bronchoalveolar lavage (BAL) analysis for protein concentration (B) and lactate dehydrogenase concentration (C) as well as the number of total cells (D), neutrophils (E), and lymphocytes (F) were determined in wild-type (Muc5ac^+/+ ) and Muc5ac^−/− mice after vehicle or bleomycin instillation. Data presented as mean ± standard error of the mean (n = 3-7 mice/group for body weight, 6-8 mice/group for vehicle-BAL, n = 3/group for 3-day bleomycin-BAL, n = 5-7 for 7- and 14-day bleomycin-BAL). Graph symbols: *P < .05 versus genotype-matched vehicle control mice; †P < .05 versus exposure-matched Muc5ac^+/+ mice.

Histopathologic analysis found that bleomycin caused sporadic fibrotic patches in parenchyma with predominant fibrogenesis in perivascular–peribronchial regions from 7 days and more widespread fibrosis at 14 days postbleomycin in both genotypes (Figure 2). Relative to Muc5ac^+/+ mice, the magnitude of the bleomycin-induced fibrosis was more severe and widespread in Muc5ac^−/− mice at 7 to 14 days (Figure 2). Histopathologic scores of inflammation and fibrosis as well as lung soluble collagen contents (collagens type I-V) were significantly higher in Muc5ac^−/− mice than in Muc5ac^+/+ mice at 14 days (Figure 3A). In Muc5ac^+/+ lung, Muc5ac mRNA abundance (3 days) and airway secretion (7 days) were significantly enhanced by bleomycin (Figure 3B). The E-cadherin levels determined as an intercellular junction marker were significantly lower in vehicle (PBS)-treated Muc5ac^−/− mice than in vehicle-treated Muc5ac^+/+ mice (Figure 3C). Bleomycin treatment significantly decreased amounts of mature lung E-cadherin (80kDa) in both genotypes of mice but more markedly in Muc5ac^−/− mice than in Muc5ac^+/+ mice at 3 and 14 days (Figure 3C).

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Figure 2.

Augmented bleomycin-induced pulmonary histopathology in mucin-5AC-deficient (Muc5ac^−/− ) mice. Masson trichrome staining exhibits differential progress and severity of pulmonary fibrosis and collagen accumulation between wild-type (Muc5ac^+/+ ) and Muc5ac^−/− mice at 7 and 14 days postbleomycin or at 14 days post vehicle. Representative light photomicrographs of lung sections (n = 6-7/group) were presented. Bar = 100 μm. AV indicates alveoli; BR, bronchi or bronchiole; PA, pulmonary artery.

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Figure 3.

Bleomycin-induced changes in fibrotic lung injury, mucin-5AC (MUC5AC), and epithelial barrier expression in mucin-5AC-sufficient (Muc5ac^+/+ ) and -deficient (Muc5ac^−/− ) mice. A, Severity of histopathologic lung inflammation and fibrosis at 14 days postbleomycin was scored by microscopic evaluation of H&E and Masson trichrome-stained slides (n = 6-7/group). Total soluble collagen contents were determined in whole right lung lysates using a colorimetric Sirius Red dye-collagen binding assay. Mean ± SEM presented (n = 4/group). B, Pulmonary Muc5ac mRNA expression was determined by qRT-PCR using a mouse specific Muc5ac primer set in Muc5ac^+/+ mice (n = 3/group). Secreted Muc5ac level was determined in the aliquots of bronchoalveolar lavage supernatants from Muc5ac^+/+ mice using a sandwich enzyme-linked immunosorbent assay (n = 3-4/group). Mean ± SEM presented. C, Protein level of E-cadherin in aliquots of lung homogenates was determined by Western blot analysis using actin as an internal control (n = 2 pooled samples/group). Representative digitized images from duplicate assays are presented. Intensity of blot images was quantified by densitometry (n = 2/group, mean ± SEM presented). Graph symbols: *P < .05 versus genotype-matched vehicle control mice, †P < .05 versus exposure-matched Muc5ac^+/+ mice. H&E indicates hematoxylin and eosin; mRNA, messenger RNA; qRT-PCR, quantitative real-time polymerase chain reaction; SEM, standard error of the mean.

Bleomycin caused mucous (goblet) cell metaplasia (MCM) in Muc5ac^+/+ and Muc5ac^−/− mice at 7 and 14 days (Figure 4A). Microscopic analysis indicated that MUC5AC proteins were localized in goblet cells of bronchi and bronchioles after vehicle or bleomycin in Muc5ac^+/+ mice and were not detected in Muc5ac^−/− mice (Figure 4A, insets). Although basal level of AB/PAS-stained bronchial intraepithelial mucus was relatively scarce in Muc5ac^−/− lungs compared to Muc5ac^+/+ lungs, the overall magnitude of bleomycin-induced MCM was greater in Muc5ac^−/− lungs than in Muc5a^+/+ lungs as MCM was more widely extended to smaller airways adjacent to fibrotic regions (at 14 days) in Muc5ac^−/− mice compared to Muc5a^+/+ mice (Figure 4A), suggesting a compensatory increase of non-MUC5AC mucin in Muc5ac^−/− airways. Expression of MUC2 protein, Muc5b mRNA, and Muc4 mRNA were determined as these mucins were known to increase in pathologic airways ^{37 –39} and compensate for Muc5ac deficiency. ^15,36,40,41 In situ hybridization (ISH) detected Muc4 and Muc5b mRNAs in vehicle-treated bronchial epithelium of both genotypes (Figure 4A). Bleomycin increased Muc4 and Muc5b transcripts in bronchial-to-bronchiolar epithelium of fibrogenic regions, and their increase was colocalized with MCM throughout the conducting airways (Figure 4A). High Muc4 expression was detected in honeycombing alveoli of Muc5ac^−/− mice at 14 days postbleomycin and it was minimal throughout epithelium at 7 days postbleomycin (Figure 4A). Induced expression of Muc4 and Muc5b mRNA markedly in proximal-to-distal conducting airway epithelium was higher in Muc5ac^−/− mice than in Muc5ac^+/+ mice at 7 to 14 days, indicating compensatory overexpression (Figure 4A). Immunohistochemical staining found that MUC2 proteins were constitutively expressed in AB/PAS-positive bronchial-to-bronchiolar epithelium of both strains and it was increased by bleomycin in Muc5ac^−/− mice at 7 days (Figure 4A). Quantitative PCR detected significantly greater expression of Muc5b (14 days), Muc4 (7 and 14 days), and Muc2 (3 days) messages in Muc5ac^−/− mice than in Muc5ac^+/+ mice after bleomycin treatment (Figure 4B). Upregulation of Muc2 mRNA by bleomycin treatment preceded (at 3 days) the increase of MUC2 proteins (at 7 days) in Muc5ac^−/− mice (Figure 4A and B). Overall, these results indicated that MUC4 and MUC5B together with MUC2 contribute to bleomycin-induced MCM in Muc5ac^−/− mice.

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Figure 4.

Bleomycin-induced compensatory mucin overexpression in mucin-5AC-deficient (Muc5ac^−/− ) mouse lungs. A, Mucus in conducting airways was visualized by AB/PAS-stained proximal aspect of left lung sections from vehicle (14 days) and bleomycin (7 and 14 days)-treated mice. Expression and localization of single mucin were determined by immunohistochemical staining (IHC) using specific antibodies for MUC5AC (insets in AB/PAS) and MUC2 or in situ hybridization (ISH) using specific probes for Muc5b and Muc4 on the serial cuts of the same tissue blocks. Arrow heads indicate MUC5AC proteins localized in bronchial epithelium of Muc5ac^+/+ mice. Arrows indicate more markedly increased Muc4 and Muc5b message or MUC2 protein in Muc5ac^−/− mice compared to those in Muc5ac^+/+ mice. Representative light micrographs presented. Bars = 100 μm. AV = alveoli, BR = bronchi or bronchiole, PA = pulmonary artery, TB = terminal bronchiole. (B, Pulmonary Muc5b, Muc4, and Muc2 mRNA expressions were determined by qRT-PCR using mouse-specific primer sets (n = 3/group). Mean ± SEM presented. Graph symbols: *P < .05 versus genotype-matched vehicle control mice; †P < .05 versus exposure-matched Muc5ac^+/+ mice. AB/PAS indicates Alcian blue/periodic acid-Schiff; mRNA, messenger RNA; qRT-PCR, quantitative real-time polymerase chain reaction; SEM, standard error of the mean.

Enhanced Sensitivity of Muc5ac^−/− Mice to RSV

Lung injury by RSV

Respiratory syncytial virus infection caused significant airway inflammation and bronchial and bronchiolar epithelial hyperplasia and hyperproliferation in Muc5ac^+/+ and Muc5ac^−/− mice (Figure 5A). Large syncytia (a multinucleated cell resulted from fusions of uninuclear cells by infected RSV) formation was more evident in lungs from Muc5ac^−/− mice (Figure 5A, arrow heads). Compared to Muc5ac^+/+ lungs, RSV-induced increases in the numbers of BAL epithelial cells and the concentration of neutrophilic MPO were significantly greater in Muc5ac^−/− lungs at 1-day post-RSV (Figure 5B). Consistent with more severe airway epithelial hypertrophy/hyperplasia and smooth muscle thickening (Figure 5A, arrows), serum IgE level was significantly higher in Muc5ac^−/− mice than in Muc5ac^+/+ mice at 5 days post-RSV (Figure 5C).

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Figure 5.

Exacerbated respiratory syncytial virus (RSV)-induced pulmonary injury and airway narrowing in mucin-5AC-deficient (Muc5ac^−/− ) mice. A, Differential severe bronchial/bronchiolar hyperplasia and hypertrophy and smooth muscle thickening in Muc5ac^−/− mice than in Muc5ac^+/+ mice demonstrated by H&E staining of proximal aspect of left lung after 3 to 5 days post-RSV infection or 3 days post vehicle. Large syncytia (multinucleated cells) formation and lymphocytic inflammation in alveoli were detected more markedly in Muc5ac^−/− mice at 5 days post-RSV. Representative light photomicrographs presented (n = 5-8/group). Arrows indicate perivascular-peribronchiolar edema and smooth muscle hyperplasia. Arrow heads indicate syncytia. AV = alveoli, BR = bronchi or bronchiole. Bar = 100 μm. B, Significantly higher number of epithelial cells and concentration of neutrophil myeloperoxidase (MPO) in bronchoalveolar lavage (BAL) fluids from Muc5ac^−/− mice compared to those in Muc5ac^+/+ after 1-day post-RSV infection. Mean ± SEM (n = 4-8/group for epithelial cells, n = 4-5/group for MPO) presented. C, Immunoglobulin E (IgE) level determined in aliquots of serum (1 μL) using a colorimetric enzyme-linked immunosorbent assay kit. Mean ± SEM (n = 3/group) presented. Graph symbols: *P < .05 versus genotype-matched vehicle control mice; ^† P < .05 versus exposure-matched Muc5ac^+/+ mice. H&E indicates hematoxylin and eosin; SEM, standard error of the mean.

Immunohistologic localization of RSV indicated that viral proteins were predominantly in pleura, conducting airway epithelium, and blood vessels at 1-day post-RSV and more severe viral pleuritis was found in Muc5ac^−/− mice than in Muc5ac^+/+ mice (Figure 6A). By 3 days post-RSV, virus diffused throughout the lung parenchyma in both genotypes, but infection diminished by 5 days (Figure 6A). There was little difference in the magnitude of lung RSV localization between 2 genotypes at 3 and 5 days (Figure 6A), suggesting that Muc5ac may contribute to a barrier against RSV in early stages of viral entry and spread. Corresponding to RSV immunohistochemistry, viral nucleoprotein (N) gene copy number was significantly higher in Muc5ac^−/− lung than in Muc5ac^+/+ lung at 1-day post-RSV but not at later times (Figure 6B).

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Figure 6.

Differential respiratory syncytial virus (RSV) load and epithelial barrier expression in mucin-5AC-sufficient (Muc5ac^+/+ ) and -deficient (Muc5ac^−/− ) mouse lungs. A, RSV localization in lung sections detected by immunohistochemical (DAB) and immunofluorescent (FITC) methods using an anti-RSV antibody. Arrows indicate virus enrichment in pleurae (pleuritis) at 1-day post-RSV infection detected by DAB. Confocal images display more diffused RSV visualized with fluorescence (green, top) in Muc5ac^−/− mouse lung than in Muc5ac^+/+ lung. Nuclei were stained with DAPI (blue, bottom). Bars = 200 μm. AV = alveoli, BR = bronchi or bronchiole, PL = pleurae (pleural cavity), BV = blood vessel. Representative light photomicrographs presented (n = 5-8/group).

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Figure 6.

B, RSV infectivity was determined by quantification of viral nucleoprotein (N) gene expression using a digitized droplet PCR (ddPCR) method. Mean ± SEM (n = 3/group) presented. C, Protein level of E-cadherin in aliquots of lung homogenates was determined by Western blot analysis using actin as an internal control (n = 2 pooled samples/group). Representative digitized images from duplicate assays are presented. Graph symbols: *P < .05 versus genotype-matched vehicle control mice; †P < .05 versus exposure-matched Muc5ac^+/+ mice. DAB indicates 3,3’-diaminobenzidine; FITC, fluorescein isothiocyanate; DAPI, 4′,6-diamidino-2-phenylindole; PCR, polymerase chain reaction; SEM, standard error of the mean.

The amount of mature E-cadherin protein (80 kDa) was basally lower in vehicle-treated Muc5ac^−/− mice than in vehicle-treated Muc5ac^+/+ mice (Figure 6C). After 1 and 3 days post-RSV, 120 kDa (mature)/135 kDa (precursor) E-cadherin levels were detected in Muc5ac^+/+ lungs, which was declined at 5 days post-RSV in these mice (Figure 6C). E-cadherin was barely detected in Muc5ac^−/− lungs after RSV infection (Figure 6C).

Nasal airway injury by RSV

Immunohistochemical localization in cross-sections of the proximal nasal turbinate (T1) indicated that RSV was mainly in surface epithelium lining septum, maxilloturbinate, blood vessels, and subepithelial glands in septum and walls, and walls at 1-day post-RSV (Figure 7). As found in the lung, RSV was most extensively detected at 3 days when nasal airway injury peaked, and virus propagation was relatively resolved at 5 days (Figure 7). Respiratory syncytial virus was more intensely and diffusively localized in Muc5ac^−/− mice than in Muc5ac^+/+ mice at 1 day, and nasal airway injury indicated by epithelial sloughing and hyperplastic changes in medial meatus region (medial wall, nasoturbinate, maxilloturbinate) was more prevalent in Muc5ac^−/− mice than in Muc5ac^+/+ mice at 1 to 5 days post-RSV (Figure 7).

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Figure 7.

Heightened respiratory syncytial virus (RSV) load in nasal airway of Muc5ac-deficient (Muc5ac^−/− ) mice. RSV detected by immunohistochemical (DAB) and immunofluorescent (Alexa Fluor 647) methods in proximal nasal airways of Muc5ac^+/+ and Muc5ac^−/− mice treated with vehicle (3 days) or RSV (1, 3, 5 days) using an anti-RSV antibody. Confocal images display more diffused virus visualized with fluorescence (left, red) in Muc5ac^−/− turbinate than in Muc5ac^+/+ turbinate at 1-day post-RSV. Nuclei were stained with DAPI (right, blue). Arrows indicate virus enriched in submucosal glands underneath septum and maxilloturbinate (MT). e = surface epithelium lining nasal mucosa, VS = ventral septum, NT = nasoturbinate. Bars = 200 μm. DAB indicates 3,3’-diaminobenzidine; DAPI, 4′,6-diamidino-2-phenylindole.

Alcian blue/periodic acid-Schiff staining of nasal airway epithelium revealed mucous cell hyperplasia in the mid-septum of Muc5ac^+/+ mice from 1-day post-RSV (Figure 8A and B). Relative to vehicle control, RSV infection increased MUC5AC protein expression mostly in AB/PAS-positive nasal respiratory epithelium (mid- and ventral septum) and some in Bowman glands in Muc5ac^+/+ mice (Supplemental Figure S3-A). The H&E staining found that RSV infection caused most morphological changes including epithelial sloughing and hypertrophy at 3 days postinfection largely in mid-septum, maxilloturbinate, and lateral meatus wall, and the injury was greater in Muc5ac^+/+ mice than in Muc5ac^−/− mice (Figure 8A, mid-septum shown). Although basal levels of AB/PAS-positive goblet cells were relatively fewer in Muc5ac^−/− mice, RSV also caused significant goblet cell hyperplasia in Muc5ac^−/− mice at later (7 days) postinfection days (Figure 8A and B). Interestingly, increase in PAS-positive substances in submucosal glands (mostly mucous glands) was more prevalent in Muc5ac^−/− mice than in Muc5ac^+/+ mice after RSV infection (Figure 8A). Respiratory syncytial virus-induced cellular proliferation and injury indicated by PCNA immunostaining were greater in Muc5ac^−/− turbinates than in Muc5ac^+/+ turbinates (Supplemental Figure S3-B). Consistent with the subepithelial gland hyperplasia (increased grand volume), PCNA localization in RSV-infected Muc5ac^−/− turbinates was predominant in subepithelial gland cells in ventral septum (Figure S3-B). Immunohistochemical staining of MUC2 (Supplemental Figure S3-C) and ISH of Muc5b mRNA (Supplemental Figure S3-D) found that overall nasal expression of MUC5B and MUC2 may be increased by RSV infection similarly in both genotypes. Muc5b mRNA was detected mainly in hyperplastic/metaplastic respiratory epithelium (goblet cells and other cells) lining septum, nasoturbinate, and ventral wall and less frequently in Bowman glands (Supplemental Figure S3-D). It was, however, seldom detected in subepithelial glands. In contrast, MUC2 protein was increased in seromucosal glands in septum and lateral wall and in Bowmans glands with minor localization in septal surface epithelium (Supplemental Figure S3-C). Muc4 mRNA was detected only in cells surrounding maxillary sinus of the nasal airways (data not included). Therefore, compensatory septal goblet cell hyperplasia and submucosal gland hyperplasia manifest in Muc5ac^−/− nasal airways may be at least in part contributed by upregulated MUC5B and MUC2, respectively.

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Figure 8.

Differential changes of respiratory syncytial virus (RSV)-induced nasal airway mucosubstances in mucin-5AC-sufficient (Muc5ac^+/+ ) and -deficient (Muc5ac^−/− ) mice. A, Changes of intraepithelial and submucosal glandular mucosubstances in mid-septum (MS) of vehicle (1 day) and RSV (1 and 7 days)-treated mice. Representative light photomicrographs presented (n = 3-5/group). Arrow heads indicate stored intraepithelial mucus increased early (1 day) in Muc5ac^+/+ mice and later (7 day) in Muc5ac^+/+ and Muc5ac^−/− vehicle after RSV infection. Arrow indicates PAS-positive mucus in hyperplastic mucosal glands. Asterisks indicate AB/PAS-stained mucus in newly developed glands (potentially submandibular) of Muc5ac^−/− mouse septum. sg = serous gland; mg = mucous gland, sm = seromucous (submandibular) gland, d = gland duct, v = blood vessel, c = cartilage. Bar = 50 μm. B, RSV-induced increase in volume density (Vs) of AB/PAS-stained stored intraepithelial mucus in MS was morphometrically measured by Image J software. Mean ± SEM (n = 3/group) presented. Graph symbols: *P < .05 versus genotype-matched vehicle control mice; †P < .05 versus exposure-matched Muc5ac^+/+ mice.

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Figure 8.

C, Transmission electron micrographs of nasal septum from vehicle-treated Muc5ac^+/+ tissue with an intact periciliary layer, ciliated respiratory epithelium, and goblet cell vesicles filled with electron-dense materials (secretory mucins), while goblet cells in vehicle-treated Muc5ac^−/− septum had relatively reduced granule storage. RSV-infected nasal epithelial cell surface with loss of cilia in both genotypes of mice. Goblet cells in Muc5ac^+/+ mice had more frequent evidence of vacuoles (1 day), viral infection-associated debris trapped in fused granules (1 day), and granule exocytosis (3 days) against RSV indicating active antiviral activity. These goblet cell changes were relatively scarce in RSV-infected Muc5ac^−/− mice and their secretory granule storage was decreased until 3 days post-RSV. In contrast, subepithelial glands in Muc5ac^−/− septum contained uniformly stained granules connected with rich rough endoplasmic reticulum (rer) while subepithelial granules in Muc5ac^+/+ septum had particularly electron-lucent outer rim (3 day). g = surface goblet cell with granules, c = cilia. Bar = 5 μm. AB/PAS indicates Alcian blue/periodic acid-Schiff; SEM, standard error of the mean.

Analyses of electron micrographs of the vehicle control epithelium from T1 found reduced mucus storage in goblet cell vesicles of Muc5ac^−/− mice as indicated by relatively empty granules and electron-dense central core compared with the granule features in Muc5ac^+/+ mice (Figure 8C). After 1 to 3 days of RSV infection, swollen granules, exocytosis, and formation of vacuoles were evident in goblet cells, and trapping of virus infection-associated debris in fused granules undergoing exocytosis were more commonly detected in wild-type mice (Figure 8C). However, Muc5ac^−/− septum was less marked in these goblet cell activities after virus infection (Figure 8C). In contrast to the surface goblet cells, subepithelial glands in septum of Muc5ac^−/− mice presented uniformly stained and electron-dense granules connected with rich rough endoplasmic reticulum at 3 days post-RSV, while RSV-infected glandular vesicles in Muc5ac^+/+ septum had particularly electron-lucent outer rim (Figure 8C).

Increased O₃-Induced Injury in Nasal Turbinate and Centriacinar Region of Muc5ac^−/− Mice

Exposure to 0.3-ppm O₃ caused epithelial exfoliation in mid- and dorsal-septum, mucous cell hyperplasia throughout the septal respiratory epithelium, mucoserous gland proliferation and hyperplasia, and blood vessel congestion in nasal airways of both genotypes from 48 hours (Figure 9A). Subepithelial gland expansion and congestion accompanying hypertrophy of basal lamina beneath septum, nasal wall, and turbinate were greater in Muc5ac^−/− mice than in Muc5ac^+/+ mice at 48-hour exposure (Figure 9A). Although O₃ significantly increased AB/PAS-positive intraepithelial substances in mid-septum only in Muc5ac^+/+ mice, PAS-positive stored mucoserous gland substances were significantly higher in Muc5ac^−/− mice than in Muc5ac^+/+ mice basally and after O₃ (Figure 9B). In addition, these changes corresponded with the O₃-induced secretion of PAS-positive mucosubstances in ventral wall detected only in Muc5ac^−/− mice (Figure 9B). Using ISH, we found that O₃ markedly increased Muc5b mRNA (peak at 48 hours) mainly in respiratory epithelium undergoing goblet cell hyperplasia (lining septum, nasoturbinate, and ventral wall) and less frequently in Bowman glands in both genotypes of mice (Figure 9C). However, Muc5b was seldom expressed in subepithelial glands, indicating other mucin genes/proteins (eg, Muc2, Muc7) may involve in PAS-positive subepithelial gland hyperplasia and secretion in Muc5ac^−/− mice.

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Figure 9.

Exacerbation of nasal airway responses to ozone (O₃) exposure in mucin-5AC-deficient (Muc5ac^−/− ) mice. A, Increase in AB/PAS-stained septal intraepithelial mucosubstances (arrow heads) and PAS-stained septal and lateral wall subepithelial gland mucosubstances (arrows) in Muc5ac^+/+ and Muc5ac^−/− nasal airways after O₃ exposure (48 hours). PAS-positive mucus-like secretion seen only in O₃-exposed Muc5ac^−/− terminates was indicated by an asterisk. S = septum, MT = maxilloturbinate, NT = nasoturbinate, LM = lateral meatus, VM = ventral meatus. Bar = 200 μm. B, Morphometric analyses identified O₃-induced significant increase in AB/PAS-stained intraepithelial mucosubstances in Muc5ac^+/+ turbinate (mid-septum) and PAS-stained subepithelial gland (mid-ventral septum) and PAS-positive mucus-like secretion (ventral meatus) in Muc5ac^−/− turbinate. Mean ± SEM (n = 3/air group, n = 4/O₃ group) presented. Graph symbols: *P < .05 versus genotype-matched air control mice; †P < .05 versus exposure-matched Muc5ac^+/+ mice.

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Figure 9.

C, Nasal airway Muc5b mRNA expression (brown dots) determined by in situ hybridization (ISH) was markedly increased by O₃ in respiratory epithelium throughout the septum and nasoturbinate undergoing mucous cell hyperplasia/metaplasia. Muc5b mRNA was also detected marginally after O₃ exposure in Bowman glands. O₃-induced Muc5b expression was higher in Muc5ac^+/+ mice than in Muc5ac^−/− mice, concurrent with AB/PAS-positive mucus increases (peak at 2 days) by O₃ exposure. S = septum, NT = nasoturbinate, re = respiratory epithelium, oe = olfactory epithelium, BG = bowman glands. Bar = 100 μm. AB/PAS indicates Alcian blue/periodic acid-Schiff; mRNA, messenger RNA; SEM, standard error of the mean.

Mucous cell metaplasia developed in bronchial epithelium after O₃ exposure (peaked at 48 hours) was predominant in Muc5ac^+/+ mice relative to that in Muc5ac^−/− mice (Figure 10A). In contrast, O₃-induced injury and proliferation in the centriacinar region (junction of terminal bronchiole and alveoli) as determined by the abundance of PCNA-positive S phase cells were markedly higher in Muc5ac^−/− mice than in wild-type mice at 48 hours (Figure 10B). Total protein concentration and total cells (Figure 10C) as well as inflammatory (macrophages, neutrophils, lymphocytes) and epithelial cell numbers in BAL were, however, not significantly different between 2 genotypes of mice after 48- to 72-hour exposure to O₃. Lung E-cadherin amount detected at 120 (mature)/135 (precursor) kDa was basally lower in air-exposed Muc5ac^−/− mice than in air-exposed Muc5ac^+/+ mice (Figure 10D). O₃ exposure decreased E-cadherin proteins in Muc5ac^+/+ mice at 72 hours, and E-cadherin levels in Muc5ac^−/− mice remained lower than in Muc5ac^+/+ mice after O₃ exposure (Figure 10D).

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Figure 10.

Exacerbation of pulmonary airway responses to ozone (O₃) exposure in mucin-5AC-deficient (Muc5ac^−/− ) mice. A, AB/PAS staining determined O₃-induced bronchial epithelial mucous cell metaplasia in Muc5ac^+/+ lung (peaked at 48 hours) but not in Muc5ac^−/− lung. AV = alveoli. Bar = 100 μm. B, Epithelial cell proliferation in terminal bronchiole of centriacinal region, a marker of pulmonary O₃ sensitivity, was determined by immunohistochemical staining of proliferating cell nuclear antigen (PCNA). Brown dots = PCNA. AV = alveoli, BR = bronchiole, TB = terminal bronchiole, BV = blood vessel. Bar = 100 μm.

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Figure 10.

C, Bronchoalveolar lavage (BAL) analysis found no significant differences in lung protein concentration and cellular injury between Muc5ac^+/+ and Muc5ac^−/− mice after subacute exposure to 0.3 parts per million O₃ exposure. Data presented as mean ± SEM (n = 4 for air groups, n = 6 for O₃ groups). D, Protein level of E-cadherin in aliquots of lung homogenates was determined by Western blot analysis using actin as an internal control (n = 2 pooled samples/group). Representative digitized images from duplicate assays are presented. Intensity of blot images was quantified by densitometry (n = 2/group, mean ± SEM presented). Graph symbols: *P < .05 versus genotype-matched air control mice; †P < .05 versus exposure-matched Muc5ac^+/+ mice.