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Title: The role of NF-κB p65 and Brd4 in oxidative stress driven inflammation
Author: Khan, Younis
Awarding Body: Imperial College London
Current Institution: Imperial College London
Date of Award: 2013
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Chronic obstructive pulmonary disease (COPD) is caused by prolonged cigarette smoke (CS) exposure. Increased oxidative stress produced by elevated oxidants, derived from cells and CS, or reduced anti-oxidant response is reported in COPD patients. Chronic inflammation is another key characteristic of COPD which persists long after smoking cessation. This suggests that oxidative stress may affect the course of inflammatory gene expression through changes in epigenetic modifications such as DNA and histone methylation and histone acetylation. Hydrogen peroxide (H2O2) is one of many oxidants reported to be elevated in breath condensates of COPD patients and is associated with enhanced inflammation. Inhaled corticosteroids (ICS) are frequently used in the management of inflammation in COPD patients; however, they provide little or no benefits to COPD patients. Understanding the mechanisms underlying this enhanced inflammatory drive and the lack of ICS response may provide novel potential therapeutic targets. Abnormal histone acetylation profiles have been linked to COPD and to relative steroid unresponsiveness. In mononuclear cells, BET bromodomain inhibitors can block acetylation-induced inflammatory responses. H2O2, an oxidant, was used to establish both an acute (2 hours) and a Chronic (low concentration exposure for 5, 10 and 15 days) oxidative stress model. In the acute model, H2O2 (100μM) enhanced TNF-α induced IL-6 and CXCL8 expression in BEAS-2B cells whereas H2O2 alone had no effect on inflammatory gene expression. This confirms that oxidative stress is involved in the enhanced induction of inflammation. Continued exposure of cells to lower concentrations of H2O2 (50μM) for 5 days results in a similar enhancement of TNF-α-induced IL-6 and CXCL8 expression but this effect was lost over 10 and 15 days due to induction of antioxidant genes. We extended the acute model with slight modification by using IL-1β instead of TNF-α as it gave us better IL-6 and CXCL8 induction. Intracellular studies showed that oxidative stress-driven cytokine induction is mediated via activation of the NF-κB pathway and increasing NF-κB p65 promoter binding. This was associated with increased histone H3 acetylation at κB response elements in both the IL-6 and IL-8 promoter regions. Chromatin immunoprecipitation assays also confirmed recruitment of bromodomain protein 4 (Brd4) to the same sites. Inhibition of Brd4 by JQ1 and PFI-1 significantly reduced the expression of IL-6 and CXCL8 cytokines. H2O2 enhanced IL-1β-induced IL-6 and CXCL8 expression by activating the NF-κB pathway. The induction of these cytokines was reduced by the BET bromodomain inhibitors JQ1 and PFI1. Our findings demonstrate that bromodomain inhibitors could potentially be used as new therapeutic agents in inflammatory diseases whereas ICS are ineffective.
Supervisor: Adcock, Ian Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available