The Role of Neutrophils in Pseudomonas aeruginosa Chronic Suppurative Otitis Media

Abstract

Abstract Chronic suppurative otitis media is largely due to biofilm-forming bacteria, of which a common pathogen is Pseudomonas aeruginosa. Neutrophils are well-known to have various contributions in both promoting and restricting bacterial growth in other biofilm-mediated diseases, as they are one of the first cells recruited to sites of infection. Herein we propose that 1) fluorescent P. aeruginosa develop into biofilms and form chronic infections in the middle ear of a mouse model of Eustachian tube obstruction and acute tympanic membrane wounds; 2) this biofilm reduces functional capacity of professional phagocytes; 3) biofilm initiates apoptosis of responding neutrophils; and 4) neutrophils contribute to disease burden. We also determine neutrophil subsets with a leading role in the establishment of bacterial biofilm-mediated disease, and test the capacity of polymorphonuclear leukocytes to suppress planktonic infection. Neutrophil function is quantified as a measure of phagocytosis and the production of extracellular traps; we also measure cell survival and clearance via efferocytosis to determine host-mediated barriers to this recalcitrant infection. Using flow cytometry coupled with fluorescent-tagged P. aeruginosa, we are able to assess middle ear effusion post-inoculation and develop a timeline of response to chronic infection in our novel CSOM model. Importantly, this study establishes a model system for further immunological and translational studies, including testing of topical therapeutics. The results of this work will also inform potential efforts of neutrophils as predictors for disease exacerbation and guide additional therapeutic development for an illness affecting millions of children globally.