title: <i>In Vitro</i> Modelling of Cellular Processes in OM-BMDM Studies in Junbo Mice Reveal Defects in HIF and TGF-β Signalling
Creators: name: Bali, Sulzhan
Publisher: name: The Open University
Year: 2011

description: Chronic otitis media (OM) is a common cause of deafness in children. Two novel murine models of chronic OM, <i>Junbo</i> and <i>Jeff</i>, generated by chemical mutagenesis with ethyl nitrosurea (ENU), have been developed at MRC Harwell. <i>Junbo</i> heterozygote mice (<i>Jbo/+</i>) mice have a mutation in transcription factor Evi-1 and <i>Jeff</i> heterozygote mice (<i>Jf/+</i>) mice have a mutation in gene for Fbxo protein <i>Fbxo11</i>. 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. <br><br>In this thesis I have described an <i>in-vitro</i> 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. <br><br>Results from this thesis reveal that Evi-1 is expressed at comparable levels in both WT and <i>Jbo/+</i> genotypes and that Evi-1<sup>A2288T</sup> mutation is a loss of function mutation which results in dysregulated expression of Smad responsive and hypoxia responsive genes such as <i>Vegf</i> and <i>Glut-1</i> under prolonged hypoxic conditions. Although <i>Jbo/+</i> BMDM are hyper-responsive to LPS in normoxia, as was indicated by higher <i>Vegf</i> and <i>IL-6</i> levels, macrophage phagocytic function is potentially attenuated in <i>Jbo/+</i> under hypoxic conditions as was indicated by lower levels of <i>Tnf-α</i> and <i>IL-1β</i> in <i>Jbo/+</i> BMDM in LPS and hypoxia combination treatment. A perturbed resolution of <i>IL-6</i> and <i>IL-1β</i> in hypoxia was also observed. <br><br>Studies performed in this thesis revealed dysregulation in TGF-β pathway in both <i>Jbo/+</i> and <i>Jf/+</i> BMDM in normoxia which suggests a common role of TGF-β signalling in OM pathogenesis. Differential expression of cytokines was observed in <i>Jbo/+</i> 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 <i>Jbo/+</i> BMDM and levels of anti-inflammatory cytokines such as IL-1ra, IL-10, and IL-4 were lower in <i>Jbo/+</i> BMDM after prolonged hypoxia and TGF-B treatment which further validates the role of dysregulated HIF and TGF-β signalling pathways in OM in <i>Jbo/+</i> mice. The differential cytokine expression observed in <i>Jbo/+</i> 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. <br><br>To conclude, HIF signalling and TGF-β signalling in <i>Jbo/+</i> BMDM is dysregulated. Pro-angiogenic function, impaired resolution of inflammation and predisposition to Th17 cell differentiation by <i>Jbo/+</i> BMDM under hypoxia contribute to the prolonged and chronic inflammation in <i>Jbo/+</i> mice and may provide some evidence of pathways that could be affected in OM patients.