Cellular content plays a crucial role in Non-typeable Haemophilus influenzae infection of preinflamed Junbo mouse middle ear.

Non-typeable Haemophilus influenzae (NTHi) is a major pathogen causing acute otitis media (AOM). The relationship between the cellular content of the middle ear fluid (MEF) during AOM and infection of NTHi is poorly understood. Using the Junbo mouse, a characterised NTHi infection model, we analysed the cellular content of MEF and correlated the data with NTHi titres. The MEF of the Junbo mouse was heterogeneous between ears and was graded from 1 to 5; 1 being highly serous/clear and 5 being heavily viscous/opaque. At seven-day post-intranasal inoculation, NTHi was not found in grade-1 or 2 fluids, and the proportion of MEF that supported NTHi increased with the grade. Analyses by flow cytometry indicated that the cellular content was highest in grade-4 and 5 fluids, with a greater proportion of necrotic cells and a low-live cell count. NTHi infection of the middle ear increased the cell count and led to infiltration of immune cells and changes in the cytokine and chemokine levels. Following NTHi inoculation, high-grade infected MEFs had greater neutrophil infiltration whereas monocyte infiltration was significantly higher in serous noninfected low-grade fluids. These data underline a role for immune cells, specifically monocytes and neutrophils, and cell necrosis in NTHi infection of the Junbo mouse middle ear.