Azithromycin inhibits MUC5AC production induced by the Pseudomonas aeruginosa autoinducer N-(3-Oxododecanoyl) homoserine lactone in NCI-H292 Cells

Abstract

The features of chronic airway diseases, including chronic bronchitis, cystic fibrosis, bronchiectasis, and diffuse panbronchiolitis, include chronic bacterial infection and airway obstruction by mucus. Pseudomonas aeruginosa is one of the most common pathogens in chronic lung infection, and quorum-sensing systems contribute to the pathogenesis of this disease. The quorum-sensing signal molecule [N-(3-oxododecanoyl) homoserine lactone (3O-C(12)-HSL)] not only regulates bacterial virulence but also is associated with the immune response. In this study, we investigated whether 3O-C(12)-HSL could stimulate the production of a major mucin core protein, MUC5AC. The effect of a macrolide on MUC5AC production was also studied. 3O-C(12)-HSL induced NCI-H292 cells to express MUC5AC at both the mRNA and the protein levels in time- and dose-dependent manners. A 15-membered macrolide, azithromycin, inhibited MUC5AC production that was activated by 3O-C(12)-HSL. 3O-C(12)-HSL induced extracellular signal-regulated kinase (ERK) 1/2 and I-kappa B phosphorylation in cells, and this induction was suppressed by azithromycin. 3O-C(12)-HSL-induced MUC5AC production was blocked by the ERK pathway inhibitor PD98059. Our findings suggest that the P. aeruginosa autoinducer 3O-C(12)-HSL contributes to excessive mucin production in chronic bacterial infection. Azithromycin seems to reduce this mucin production by interfering with intracellular signal transduction.

FIG. 1.

Time-dependent effect of 3O-C₁₂-HSL on MUC5AC synthesis. NCI-H292 cells were stimulated with 100 μM 3O-C₁₂-HSL. (A) Total RNA was isolated, and the MUC5AC mRNA level was analyzed by RT-PCR. (B) MUC5AC protein was measured by an ELISA. Data are expressed as the mean and SEM for three experiments. A dagger indicates a P value of <0.01 for a comparison with culture medium alone.

FIG. 2.

Dose-dependent effect of 3O-C₁₂-HSL on MUC5AC synthesis. Cells were stimulated with 1, 10, or 100 μM 3O-C₁₂-HSL. (A) The mRNA level of MUC5AC expression at 8 h after the addition of 3O-C₁₂-HSL was analyzed by RT-PCR. (B) MUC5AC protein was measured by an ELISA at 12 h after the addition of 3O-C₁₂-HSL. Data are expressed as the mean and SEM for three experiments. An asterisk and a dagger indicate P values of <0.05 and <0.01, respectively, for comparisons with culture medium alone.

FIG. 3.

Effect of azithromycin on MUC5AC production in cells activated by 3O-C₁₂-HSL. Cells were pretreated with 10 or 100 μg of azithromycin/ml for 30 min before exposure to 3O-C₁₂-HSL. (A) MUC5AC mRNA expression at 8 h after the addition of 3O-C₁₂-HSL was determined by RT-PCR. (B) MUC5AC protein was measured by an ELISA at 12 h after the addition of 3O-C₁₂-HSL. Data are expressed as the mean and SEM for three experiments. An asterisk indicates a P value of <0.05 for a comparison with culture medium alone.

FIG. 4.

Phosphorylation of I-κB and ERK1/2 induced by 3O-C₁₂-HSL. Cells were treated with 3O-C₁₂-HSL for 10 min and evaluated by Western blotting. (A) I-κB phosphorylation was induced in 3O-C₁₂-HSL-treated cells but not in untreated cells. (B) ERK1/2 phosphorylation was induced in 3O-C₁₂-HSL-treated cells. This phosphorylation was blocked by the addition of azithromycin. Data are representative of three separate experiments. p, phospho.

FIG. 5.

Effect of PD98059 on MUC5AC production in cells activated by 3O-C₁₂-HSL. Cells were pretreated with 50 μM PD98059 for 30 min prior to 3O-C₁₂-HSL stimulation. (A) MUC5AC mRNA expression was determined by RT-PCR. (B) MUC5AC protein was measured by an ELISA. Data are expressed as the mean and SEM for three experiments. An asterisk indicates a P value of <0.05 for a comparison with culture medium alone.