Differential expression of cytokine genes and iNOS induced by nonviable nontypeable Haemophilus influenzae or its LOS mutants during acute otitis media in the rat.

OBJECTIVE: We have previously demonstrated that the disruptions of nontypeable Haemophilus influenzae (NTHi) lipooligosaccharide (LOS) htrB and rfaD genes may play a role in the pathogenesis of otitis media (OM). The purpose of this study was to determine whether NTHi LOS gene disruptions influence the induction of gene expression for proinflammatory mediators in vivo using the rat model of acute OM. METHODS: At 3, 6, 12, 24, 48 and 72 h after transbullar inoculation with nonviable NTHi, expression of genes for the cytokines and chemolines; tumor necrosis factor alpha (TNF-alpha), interleukin-lbeta (IL-1beta), and IL-6, IL-1alpha, IL-8, IL-10, and inducible nitric oxide synthase (iNOS) were quantitated by real-time PCR. Enzyme-linked immunosorbent assay was performed to confirm the gene expression data as determined by real-time PCR. The middle ear inflammatory responses were also evaluated. RESULTS: The NTHi 2019 parent and its isogenic LOS htrB (B29) and rfaD (DK-1) mutant strains induced a significant up-regulation in gene expression for the cytokines examined compared to the sham-inoculated controls at 3, 6 and 12 h post-inoculation (P<0.05 in all cases). However, the NTHi 2019 cohort demonstrated a significant increase in gene expression for TNF-alpha (up to 6 h), IL-1alpha and IL-8 (up to 24 h), IL-1beta and IL-6 (up to 48 h), and IL-10 and iNOS (up to 72 h) relative to the animals inoculated with NTHi B29 (P<0.05, in all cases), Moreover, the concentrations of inflammatory cells in the middle ear lavage fluid samples from the NTHi 2019 cohort were 2.8-5.3-fold higher than those of the B29 cohort. There were no significant differences in mRNA expression of the cytokines between the NTHi 2019 and the DK-1-treated groups. CONCLUSIONS: Data from this study indicate that the disruption of the NTHi htrB gene may impact the temporal mRNA expression of inflammatory mediators and inflammation within the middle ear.

Sortase A contributes to pneumococcal nasopharyngeal colonization in the chinchilla model.

Sortase A (SrtA) is required to anchor neuraminidase, beta-galactosidase, and possibly other LPXTG motif proteins to the pneumococcal cell surface. We examined the role of SrtA in Streptococcus pneumoniae nasopharyngeal (NP) colonization in the chinchilla model. The srtA mutant colonized the nasopharynx at a significantly lower level than the D39 parent strain during the second and third week of the carriage, and was eliminated from nasopharynx one week earlier than the D39 pneumococci. Our data indicate that SrtA contributes to pneumococcal NP colonization in this animal model.

Alteration of gene expression in human middle ear epithelial cells induced by influenza A virus and its implication for the pathogenesis of otitis media.

Influenza A virus infection plays a significant role in the pathogenesis of Streptococcus pneumoniae-induced acute otitis media in children. An understanding of how influenza A virus modulates host cellular responses is critically important in efforts to explore the molecular mechanisms of this synergism. We used microarray technology to characterize the mRNA expression profile in human middle ear epithelial cells induced by influenza A virus. Alterations of mRNA expression in 142 out of approximately 12,600 genes were observed at 24h after virus infection. Of these 142 genes with altered expression, interferon inducible genes, chemokine and cytokine genes, pro- and antiapoptotic genes, signal transduction and transcription factors, cellular immune response, cell cycle and metabolism genes were the most prominent. Our results reveal several previously unknown alterations of host gene expression induced by influenza A virus which may provide new targets for further analysis of its role in this particular host-pathogen interaction.