MUC1 deficiency promotes nasal epithelial barrier dysfunction in subjects with allergic rhinitis

The epithelial barrier dysfunction plays a critical role in the pathogenesis of allergic airway inflammation.1Georas S.N. Rezaee F. Epithelial barrier function: at the front line of asthma immunology and allergic airway inflammation.J Allergy Clin Immunol. 2014; 134: 509-520Abstract Full Text Full Text PDF PubMed Scopus (286) Google Scholar Disrupted nasal epithelial barrier is observed in patients with allergic rhinitis (AR) and asthma.2Zhang N. Van Crombruggen K. Gevaert E. Bachert C. Barrier function of the nasal mucosa in health and type-2 biased airway diseases.Allergy. 2016; 71: 295-307Crossref PubMed Scopus (79) Google Scholar For example, Steelant et al3Steelant B. Farre R. Wawrzyniak P. Belmans J. Dekimpe E. Vanheel H. et al.Impaired barrier function in patients with house dust mite-induced allergic rhinitis is accompanied by decreased occludin and zonula occludens-1 expression.J Allergy Clin Immunol. 2016; 137: 1043-1053Abstract Full Text Full Text PDF PubMed Scopus (193) Google Scholar reported decreased epithelial barrier function in patients with AR and found decreased expression of the tight junction proteins occludin and zonula occludens 1. Another study showed that the levels of junction proteins E-cadherin and zonula occludens 1 were reduced in the nasal mucosal tissues of the patient with AR.4Lee H.J. Kim B. Im N.R. Lee D.Y. Kim H.K. Lee S.H. et al.Decreased expression of E-cadherin and ZO-1 in the nasal mucosa of patients with allergic rhinitis: altered regulation of E-cadherin by IL-4, IL-5, and TNF-alpha.Am J Rhinol Allergy. 2016; 30: 173-178Crossref PubMed Scopus (30) Google Scholar However, the etiology of airway epithelial barrier dysfunction remains to be further investigated. It is known that the mucus on the surface of the airway is a protective barrier for the airway.5Ma J. Rubin B.K. Voynow J.A. Mucins, mucus, and goblet cells.Chest. 2018; 154: 169-176Abstract Full Text Full Text PDF PubMed Scopus (179) Google Scholar MUC1 is one of the major components of the mucus, and MUC1 deficiency was found involving the pathogenesis of many inflammatory diseases.6Lu W. Hisatsune A. Koga T. Kato K. Kuwahara I. Lillehoj E.P. et al.Cutting edge: enhanced pulmonary clearance of Pseudomonas aeruginosa by Muc1 knockout mice.J Immunol. 2006; 176: 3890-3894Crossref PubMed Scopus (113) Google Scholar, 7McAuley J.L. Corcilius L. Tan H.X. Payne R.J. McGuckin M.A. Brown L.E. The cell surface mucin MUC1 limits the severity of influenza A virus infection.Muc Immunol. 2017; 10: 1581-1593Crossref PubMed Scopus (76) Google Scholar, 8Milara J. Peiró T. Armengot M. Frias S. Morell A. Serrano A. et al.Mucin 1 downregulation associates with corticosteroid resistance in chronic rhinosinusitis with nasal polyps.J Allergy Clin Immunol. 2015; 135: 470-476Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar However, the role of MUC1 in maintaining the nasal epithelial barrier functions and the role of MUC1 deficiency in the pathogenesis of AR are still unknown. Therefore, this study aimed to elucidate the role of MUC1 in maintaining nasal epithelial barrier functions and the role of MUC1 deficiency in the development of AR. MUC1 expression is decreased in nasal mucosa in both patients with AR and rats with AR. We first evaluated the expression of MUC1 in the nasal mucosa of patients with AR and control subjects. All the patients had typical perennial AR symptoms and positive serum specific IgE (see Table E1 in this article's Online Repository at www.jacionline.org). The results showed that the expression of MUC1 was significantly less in patients with AR than in healthy controls (Fig 1, A-D). An AR rat model was generated (see Fig E1, A, in this article's Online Repository at www.jacionline.org), and the expression of MUC1 in nasal mucosa of rats was analyzed by immunohistochemical staining, Western blotting, and real-time quantitative PCR. The results showed that the expression of MUC1 was also decreased in rats with AR compared with control rats (Fig 1, E-H). The results suggest that MUC1 may be involved in the pathogenesis of AR. To explore the role of MUC1 in AR, we developed an AR model with MUC1 knock-out (KO) rats and wild-type (WT) rats with ovalbumin as the specific antigen. We observed that the nasal epithelial permeability was higher in WT rats with AR than in naive control rats, but was significantly increased in MUC1 KO rats with AR (Fig 2, A). The results indicate that MUC1 deficiency can aggravate nasal epithelial permeability of rats with AR. The nasal mucosal tissues were collected; the expression of epithelial connection proteins was analyzed by Western blotting, immunohistochemistry, and real-time quantitative PCR. The results showed that the expression of occludin, claudin-1, and E-cadherin significantly decreased in MUC1 KO rats compared with WT rats. The results suggest that MUC1 deficiency may downregulate the expression of claudin-1, occludin, and E-cadherin. The results also showed that the expression of occludin, claudin-1, and E-cadherin was significantly decreased in WT rats with AR than in naive control rats, and was further decreased in MUC1 KO rats with AR. The results demonstrate that MUC1 deficiency aggravates the reduction of nasal epithelial connection proteins in rats with AR (Fig 2). MUC1 deficiency aggravates experimental AR. After the final intranasal ovalbumin challenge, as compared with naive control rats, the number of sneezing and nasal scratching was increased, the number of neutrophils and eosinophils was increased, and the levels of IL-4, IL-5, IL-13, IL-17a, and TNF-α were increased in nasal lavage of rats with AR (see Fig E1, E-K), and all these were further increased in MUC1 KO rats with AR (Fig E1, E, F, and I). The results indicate that MUC1 deficiency aggravates AR. We knocked down MUC1 expression in BEAS-2B cells by transfecting small interfering RNA (see Fig E2, A-C, in this article's Online Repository at www.jacionline.org). Western blotting showed that occludin and claudin-1 protein levels were significantly decreased after knocking down MUC1 expression (Fig E2, D-F). Confocal images showed that the positive staining of occludin and claudin-1 was also significantly reduced after knocking down MUC1 expression (Fig E2, G and H). The results indicate that MUC1 inhibition reduces occludin and claudin-1 protein production in BEAS-2B cells (Fig E2). In summary, the present data show that MUC1 is significantly less in the nasal epithelium of patients with AR and rats with AR. MUC1 deficiency aggravates AR symptoms and AR-related nasal epithelial barrier functions. Depletion of MUC1 suppresses the expression of epithelial cell connection protein. Our findings suggest that MUC1 deficiency is associated with the pathogenesis of AR, which may be considered as a promising therapeutic target in patients with AR. The inferior turbinates were collected from patients with AR (n = 20) and healthy controls (n = 11). Control subjects did not report any nasal symptoms of AR, and did not have a history of AR or rhinosinusitis. All the patients had typical perennial AR symptoms and positive serum specific IgE (sIgE). Serum sIgE level against 31 common allergens was measured using the Pharmacia Immuno-CAP system according to the manufacturer's instructions (Pharmacia Diagnostics, Clayton, NC). The analysis for the presence of a sIgE was defined as positive when the serum level of sIgE was greater than or equal to 0.35 kU/L. Patient information can be found in Table E1. This study was approved by the Medical Ethics Committee of the First Affiliated Hospital of Guangzhou Medical University. We obtained written informed consent from all participants. The BEAS-2B cell was cultured in DMEM-F12 media with supplements, including 10% FBS, 100 U/mL penicillin, 0.1 mg/mL streptomycin, and 2 mM glutamine, at 37°C with 5% CO2 in humidified air. The culture medium was replaced every 48 hours. MUC1 KO Sprague Dawley rats were produced by targeted gene mutation at Cyagen Biosciences, Inc (Guangzhou, China). The deletion was made by microinjection of TALENs and located in the exon 1 of rat MUC1 gene (Gen Bank: NM_012602.1). Genotyping was performed by PCR of tail DNA using primers specific for the rat MUC1 gene with forward primer 5′-CTAGCAAGCCTAAAAGGTGAGAGGT-3′ and reverse primer 5′-ACGAAGAGCATTTGCCTACTC-3′, followed by DNA sequencing analysis. PCR protocols included 95°C for 2 minutes and 30 cycles at 95°C for 15 seconds, 57°C for 30 seconds, 72°C for 45 seconds, and 72°C for 10 minutes. PCR product (250bp) was analyzed on 1.5% agarose gel. Adult female Sprague Dawley rats (250 ± 20 g) were purchased from the Guangdong Medical Laboratory Animal Center (Guangdong, China). This study was approved by the Ethics Committee of Guangzhou Medical University, and all procedures were conducted in accordance with the experimental animal guidelines of Guangzhou Medical University (Guangzhou, China). Rats were housed in the SPF Experimental Animal Center of Guangzhou Medical University. All rats had free access to standard food and water and allowed to be acclimated for 7 days before experiment. AR rat model was developed with ovalbumin (OVA) as the specific antigen following published proceduresE1Nishijima H. Kondo K. Toma-Hirano M. Iwasaki S. Kikuta S. Fujimoto C. et al.Denervation of nasal mucosa induced by posterior nasal neurectomy suppresses nasal secretion, not hypersensitivity, in an allergic rhinitis rat model.Lab Invest. 2016; 96: 981-993Crossref PubMed Scopus (15) Google Scholar (Fig E1). Briefly, rats were sensitized with an intraperitoneal injection of 1000 μg of OVA (grade V; Sigma, St Louis, Mo) and 20 mg of aluminum hydroxide gel (Sigma Aldrich) on days 1, 8, and 15. One week after the last intraperitoneal injection, rats were intranasally challenged with OVA diluted in sterile normal saline (300 μg OVA/20 μL per nostril) on days 22 to 36. The control (normal) group was given saline. After the final intranasal OVA challenge, the AR was confirmed using a symptom score calculated from the total number of sneezing and nasal scratching during a 30-minute period. Cytokine levels were determined by ELISA with commercial reagent kits following the manufacturer instructions (Thermo Scientific, Waltham, Mass). On day 36 (after the final antigen challenge), rats were deeply anesthetized and sacrificed. Nasal mucosa samples were excised and fixed in 4% paraformaldehyde (Biosharp, Hefei, Anhui, China) for 24 hours, and then embedded in paraffin. Paraffin-embedded tissue samples were cut into 3-μm thick sections and stained with hematoxylin and eosin. Histopathologic changes were evaluated and photographed using an orthorhombic optical photomicroscope (Eclipse Ci, Leica, Germany). For immunohistochemistry, sections were deparaffinized and then submerged in citrate buffer (pH 6.0) for antigen retrieval. Samples were treated with H2O2 for 15 minutes to block endogenous peroxidase, and then incubated overnight at 4°C in recommended dilutions of antioccludin, anti–E-cadherin and anti–claudin-1 antibodies (Invitrogen, Carlsbad, Calif). After washing with PBS, sections were incubated with a secondary antibody for 30 minutes at room temperature. Signals were visualized with a diaminobenzidine peroxidase substrate kit (Zhong shan Jin qiao, Beijing, China). For immunofluorescence staining, sections were incubated with appropriate Alexa Fluor–conjugated secondary antibodies and cell nuclei were stained with 4′-6-diamidino-2-phenylindole, dihydrochloride (Invitrogen). Tissue samples and BEAS-2B cell were lysed in RIPA buffer (Beyotime Biotechnology Institute, Beijing, China) and lysates subjected to SDS-PAGE and Western blotting for detection of the following antigens: MUC1 (Abcam, Cambridge, Mass), occludin (Invitrogen), claudin-1 (Invitrogen), E-cadherin (Invitrogen), and β-catenin (Cell Signaling Technology, Danvers, Mass). After incubation with a secondary antibody (Cell Signaling Technology), immunoreactive bands were captured on the Gel DOC XR Gel Imaging System (BIO-RAD, Hercules, Calif) and quantified with image lab v5.2 software (Bio-Rad Laboratories, Herts, United Kingdom). Total RNA was isolated from the nasal mucosa and BEAS-2B by using a Total RNA Extraction Kit (Takara, Tokyo, Japan). RNA samples were then reverse transcribed into first-strand cDNA using the PrimeScript RT reagent kit from Takara. Real-time quantitative PCR was performed on an Applied Biosystems 7500 Real-Time PCR System (Life Technologies, Carlsbad, Calif). The amplification protocol was set as follows: 95°C denaturation for 30 seconds followed by 40 cycles of 5-second denaturation at 95°C, 30 s of annealing/extension, and data collection at 60°C. The primer sequences of MUC1, occludin, claudin-1, E-cadherin, and GAPDH (internal reference) are listed in Table E2. A MUC1 small interfering RNA (siRNA) kit was purchased from Huiyuan Biotech (Guangzhou, China). BEAS-2B cells were transfected with siRNA in serum and antibiotic-free medium. After 8 hours, the medium was aspirated and replaced with medium containing serum for a further 48 hours. Lipofectamine-2000 (Invitrogen, Paisley, United Kingdom) was used at a final concentration of 2 μg/mL for the transfection. The results of the 4 experiments were analyzed using SPSS for Windows version 23.0 (SPSS, Chicago, Ill) and expressed as mean ± SEM. Between-groups comparisons were performed using 1-way ANOVA and analyzed by Least—Significant Difference t test (when equal variances are assumed) or Tamhane T2 test (nonparametric test; when equal variances are not assumed), and P values of less than .05 indicated statistical significance.Fig E2MUC1 inhibition reduces occludin and claudin-1 protein production in BEAS-2B cell. We knocked down MUC1 expression in BEAS-2B cells by transfecting siRNA. BEAS-2B cells were transfected with nonsilencing siRNA (control-siRNA) and MUC1-silencing siRNA (MUC1-siRNA). A, MUC1 mRNA levels in BEAS-2B cells. B, MUC1 protein levels in BEAS-2B cells. C, MUC1 protein levels as determined by densitometry normalized to GAPDH. D, Immunoblots show levels of occludin and claudin-1 proteins in BEAS-2B cells. E and F, Densitometric analysis of the blots of Fig E2, D. G, Representative confocal images of the tight junction protein occludin in BEAS-2B cells. Scale bars: 25 μm. H, Representative confocal images of the tight junction protein occludin in BEAS-2B cells. Scale bars: 25 μm. DAPI, 4′-6-Diamidino-2-phenylindole, dihydrochloride. Data of bars are presented as mean ± SEM. *P < .05 vs control-siRNA (n ≥ 3).View Large Image Figure ViewerDownload Hi-res image Download (PPT)Table E1Patient informationGroupAllergy (n = 20)Normal (n = 11)Average age (y) (range)51.1 (22-63)57.8 (32-69)Male:female3:18:3Symptom of AR200Rhinitis duration (y), mean ± SD7.2 ± 7.80sIgE positive200Serum sIgE was defined as positive when the serum level of sIgE was ≥0.35 kU/L. Open table in a new tab Table E2Primer sequencesGeneSpeciesForwardReverseMUC1Rat5-CATTCACATTGACTTCCGAGA-35-TAGCATCGACTGTGATTTTCC-3OccludingRat5- CGTGTAGTCGGTTTCATAGTG-35-GGAGATTCCTCTGACCTTGTC-3E-cadherinRat5-CTGGCTCAAATCAAAGTCCTG-35-TCCTGCTCCTACTGTTTCTACG-3Claudin-1Rat5-ACATGCCTCCAATGCCGTTC-35-AAGATGCAAATCCAGGTCTACCC-3GapdhRat5-CAACGGGAAACCCATCACCA-35-ACGCCAGTAGACTCCACGACAT-3MUC1Human5-TGCCGCCGAAAGAACTACG-35-TGGGGTACTCGCTCATAGGAT-3GapdhHuman5-ACAACTTTGGTATCGTGGAAGG-35-GCCATCACGCCACAGTTTC-3 Open table in a new tab Serum sIgE was defined as positive when the serum level of sIgE was ≥0.35 kU/L.