Objectives/HypothesisChronic otitis media (COM) develops after sustained inflammation and is characterized by secretory middle ear epithelial metaplasia and effusion, most frequently mucoid. Nontypeable Haemophilus influenzae (NTHi), the most common acute otitis media (OM) pathogen, is known to activate inflammation and mucin expression in vitro and in animal models of OM. The goals of this study were to examine histopathological and expression profiling epithelial effects of NTHi challenge in murine middle ears.Study DesignIn vitro and in vivo murine model of OM.MethodsWeekly transtympanic inoculation of Balb/c mice with 300 μg/ml of NTHi lysates versus saline was performed. Histopathologic analysis was carried out at 4 weeks. Expression microarray analysis was performed at 1 and 7 days. Microarray findings were validated in independent animal samples and in a cultured murine middle ear epithelial cell (mMEEC) line.ResultsHistopathologic analyses revealed middle ear mucosal thickening after NTHi exposure. Microarray analyses of inflammatory response genes which changed significantly demonstrated that the chemokine Cxcl2 had the largest fold‐change, with significantly increased expression at 1 and 7 days after NTHi injection compared to either saline or no‐injection (P <0.01). Validation by real‐time qPCR revealed similar significantly increased relative mRNA levels for Cxcl2. NTHi lysates were also found to significantly upregulate the transcription of Cxcl2 in mMEEC in a time‐ and dose‐dependent manner (P <0.05).ConclusionsMiddle ear NTHi challenge in mice leads to chronic epithelial mucosal metaplasia and overexpression of inflammatory mediators, most notably Cxcl2. This finding is parallel to NTHi‐mediated pulmonary mucosal metaplasia where Cxcl2 has been identified as an important inflammatory mediator.Level of EvidenceN/A. Laryngoscope, 123:E66–E71, 2013