| Popis: |
Chronic otitis media (OM) is a common cause of deafness in children. Two novel murine models of chronic OM, Junbo and Jeff, generated by chemical mutagenesis with ethyl nitrosurea (ENU), have been developed at MRC Harwell. Junbo heterozygote mice (Jbo/+) mice have a mutation in transcription factor Evi-1 and Jeff heterozygote mice (Jf/+) mice have a mutation in gene for Fbxo protein Fbxo11. Pathologic hypoxia has been characterized as a common feature of the inflamed middle ear in both models. HIF-VEGF pathways dysregulation has been indicated in both models previously. However, the mechanisms involved in pathogenesis of OM in these two models are yet to be elucidated. \ud In this thesis I have described an in-vitro model system to study cellular processes in OM to study and identify pathways involved in OM pathogenesis. This model system involves culturing BMDM and exposing them to various treatments under standard defined conditions. \ud Results from this thesis reveal that Evi-1 is expressed at comparable levels in both WT and Jbo/+ genotypes and that Evi-1A2288T mutation is a loss of function mutation which results in dysregulated expression of Smad responsive and hypoxia responsive genes such as Vegf and Glut-1 under prolonged hypoxic conditions. Although Jbo/+ BMDM are hyper-responsive to LPS in normoxia, as was indicated by higher Vegf and IL-6 levels, macrophage phagocytic function is potentially attenuated in Jbo/+ under hypoxic conditions as was indicated by lower levels of Tnf-α and IL-1β in Jbo/+ BMDM in LPS and hypoxia combination treatment. A perturbed resolution of IL-6 and IL-1β in hypoxia was also observed. \ud Studies performed in this thesis revealed dysregulation in TGF-β pathway in both Jbo/+ and Jf/+ BMDM in normoxia which suggests a common role of TGF-β signalling in OM pathogenesis. Differential expression of cytokines was observed in Jbo/+ BMDM after prolonged treatment with TGF-β and hypoxia indicating a pro-angiogenic and pro-inflammatory phenotype. Levels of pro-inflammatory cytokines such as IL-1β, C5a, IL-17, IL-23, and Ccl5 were higher in Jbo/+ BMDM and levels of anti-inflammatory cytokines such as IL-1ra, IL-10, and IL-4 were lower in Jbo/+ BMDM after prolonged hypoxia and TGF-B treatment which further validates the role of dysregulated HIF and TGF-β signalling pathways in OM in Jbo/+ mice. The differential cytokine expression observed in Jbo/+ BMDM is favourable for Th17 cell differentiation and may indicate a role of cross-talk of innate immunity and cell mediated immunity in OM chronicity in hypoxia. \ud To conclude, HIF signalling and TGF-β signalling in Jbo/+ BMDM is dysregulated. Pro-angiogenic function, impaired resolution of inflammation and predisposition to Th17 cell differentiation by Jbo/+ BMDM under hypoxia contribute to the prolonged and chronic inflammation in Jbo/+ mice and may provide some evidence of pathways that could be affected in OM patients. |
