Effects of fungi and eosinophils on mucin gene expression in rhinovirus-infected nasal epithelial cells

Abstract

Purpose: Fungi, rhinoviruses (RVs), and eosinophils are associated with upper respiratory diseases. We evaluated the effects of fungal stimulation and eosinophil co-culture on the expression of mucin genes in RV-infected nasal polyp epithelial cells.

Methods: Nasal polyp epithelial cells were obtained from chronic rhinosinusitis patients. Cultured epithelial cells were stimulated with Alternaria and Aspergillus with or without RV-16 infection. The epithelial cells were co-cultured with eosinophils for 16 h. MUC4, MUC5AC, MUC5B, and MUC8 mRNA expressions in the epithelial cells were quantified using real-time RT-PCR. To determine the underlying mechanism, nuclear factor-κB (NF-κB), activator protein-1 (AP-1), and mitogen-activated protein kinase (MAPK) inhibitors were used to inhibit mucin gene expression.

Results: Fungi and RV-16 induced mucin gene expression in nasal polyp epithelial cells. However, there was no synergistic increase in mucin gene expression, with the exception of MUC4 mRNA expression stimulated by 25 µg/mL Aspergillus. When RV-16-infected epithelial cells were stimulated with fungi and then co-cultured with eosinophils, MUC4, MUC5B, and MUC8 mRNA expressions increased. Mucin gene expression was inhibited by NF-κB inhibitors.

Conclusions: RV-16, airborne fungi, and eosinophils may exacerbate the inflammatory process in nasal mucosal diseases by enhancing mucin gene expression.

Keywords: Rhinovirus; eosinophil; fungus; mucin gene; nasal epithelial cell; nuclear factor-κB.

Fig. 1

Quantification of mucin gene expression in nasal polyp epithelial cells stimulated with Alternaria and Aspergillus with or without rhinovirus-16 (RV-16) infection for 24 h. MUC4, MUC5AC, and MUC8 mRNA expression levels were increased significantly by Alternaria; however, only MUC5AC mRNA expression was increased by Aspergillus. RV-16 also induced MUC4, MUC5AC, and MUC8 mRNA expression. When RV-infected epithelial cells were stimulated by fungi, MUC4, MUC 5AC, and MUC8 mRNA expression levels were increased. Values are expressed as means±SD of 6 independent experiments. Alt: Alternaria (µg/mL), Asp: Aspergillus (µg/mL), RV: rhinovirus-16-infected, (-): unstimulated. ^*significantly higher than unstimulated cells, ^†significantly higher than RV-infected cells.

Fig. 2

Quantification of mucin gene expression in nasal polyp epithelial cells co-cultured with eosinophils and stimulated with Alternaria and Aspergillus with or without rhinovirus-16 (RV-16) infection for 16 h. Values are expressed as the means±SD of 6 independent experiments. Alt: Alternaria (µg/mL), Asp: Aspergillus (µg/mL), RV: rhinovirus-16-infected, EO: eosinophils, (-): unstimulated. ^*significantly higher than unstimulated cells, ^†significantly higher than RV-infected cells.

Fig. 3

Effects of curcumin, SB 203580 and BAY 11-7082 on the expression of mucin genes. SB 203580 (MAPK inhibitor) inhibited MUC4 and MUC5AC mRNA expression and BAY 11-7082 (NF-κB inhibitor) inhibited MUC4, MUC5AC, MUC5B, and MUC8 mRNA expression. Values are expressed as the means±SD of 5 independent experiments. Alt: Alternaria (µg/mL), Asp: Aspergillus (µg/mL), V: rhinovirus-16 infected, (-): non-stimulated. ^*P<0.05.