Middle Ear Response of Muc5ac and Muc5b Mucins to Nontypeable Haemophilus influenzae.

IMPORTANCE Chronic otitis media with effusion is characterized by middle ear secretion of mucin glycoproteins, predominantly MUC5B; MUC5AC, the other secretory mucin studied frequently, has also been identified in the middle ear. Emerging evidence suggests a dichotomous role for these mucins in innate immune responses. We hypothesized that MUC5AC is an acute responder and MUC5B is expressed at later time points, reflecting a chronic situation. OBJECTIVE To determine middle ear regulation of MUC5B and MUC5AC following in vitro bacterial and cytokine exposure. DESIGN, SETTING, AND SAMPLES An in vitro cell-based model of mucin gene regulation was conducted in a basic science laboratory at a tertiary pediatric hospital. The study was conducted from July 1, 2014, to June 30, 2015; data analysis was performed in July 2015. INTERVENTIONS Nontypeable Haemophilus influenzae (NTHi) lysates were generated and used to stimulate mouse middle ear epithelial cells (mMEECs) for 2 hours during 3 weeks. MAIN OUTCOMES AND MEASURES Real-time quantitative polymerase chain reaction, luciferase assays, Western blot assay, and immunofluorescence techniques were performed to determine Muc5ac and Muc5b expression over time, Cxcl2 chemokine response, and nuclear factor-κB activation. Luciferase reporter assays were performed to evaluate specific promoter responses after NTHi exposure. RESULTS Nontypeable H influenzae lysates (200 μg/mL) drove differential mucin gene activation, with Muc5ac being induced up to 2.04 fold at 24 hours and 2.79 fold at 96 hours (P < .05) and Muc5b being induced only at more long-term points: 1.61 fold at 96 hours, 1.41 fold at 1 week, and 1.53 fold at 3 weeks (P < .05). Although NTHi lysates induced robust, early nuclear factor-κB nuclear translocation with nuclear factor-κB-dependent induction of Cxlc2 expression, the lysates had minimal to no effect on Muc5ac and Muc5b promoter activity. However, in contrast to NTHi lysates, CXCL2 induced significant transcription of both Muc5b and Muc5ac as early as 24 hours. CONCLUSIONS AND RELEVANCE Nontypeable H influenzae lysates activate differential mucin gene activation in mMEECs. Although Muc5ac is an early response mucin gene, Muc5b appears to react as a chronic response mucin.