D-086. Haemophilus Influenzae as Predator and Prey: Evaluation of a Model to Explain Heme Source Availability to Nontypeable Haemophilus Influenzae in the Chinchilla Model of Otitis Media

T. W. Seale, D. J. Morton, W. A. Kaserer, V. Awasthi, P. W. Whitby, T. M. VanWagoner, T. L. Stull;
Univ. Oklahoma Hlth. Sci. Ctr., Oklahoma City, OK.

Haemophilus influenzae (HI) has an absolute growth requirement for heme and possesses multiple mechanisms for acquiring this essential nutrient. Potential heme sources are normally intracellular within host cells and their extracellular concentrations restricted by innate immunity mechanisms. How HI acquires heme at concentrations sufficient to permit rapid growth to high titers in vivo is unclear. NTHI are now the most frequent cause of otitis media in children. We hypothesized the following scenario to explain how heme sources become available to NTHI in the middle ear. NTHI entry into the middle ear initially alters epithelial cell function and elicits entry of high numbers of neutrophils, and perhaps monocytes, into the bullar space. Phagocytosis of a fraction of the NTHI cell population activates apoptotic pathways in these host cells and their death liberates intracellular heme sources at concentrations sufficient to support rapid proliferation of the remaining NTHI cells. We used a chinchilla model of otitis media and the well characterized NTHI strain 86-028NP to initiate the evaluation of this hypothesis. Our methodological approaches included imaging (e.g. positron emission tomography, confocal microscopy and transmission electron microscopy), biochemical and molecular analyses (e.g. gel electrophoresis, Western blots, Q-RT-PCR analysis of NTHI gene expression) and quantitative bacteriology. The following summarize our current results. Proteins in middle ear effusions originate from both host serum and cellular sources. Specific effusion hemoproteins have yet to be identified; catalase, an HI heme source, is a good candidate. An influx of neutrophils occurs early after challenge and outnumbers monocytes. Apoptotic neutrophils and neutrophil “nets” become prominent within a few days of challenge and increase with time post challenge. Our Q-RT-PCR analyses suggest that once maximal NTHI titers (~109 cfu/ml effusion) are achieved, heme source availability is low enough to fully derepress iron/heme-repressible gene expression. We conclude that a neutrophil/epithelial apoptotic model for provision of heme sources to NTHI invading the middle ear deserves further study.