- Title
- Epigenetic mechanisms underlying the development and progression of chronic obstructive pulmonary disease
- Creator
- Jones-Freeman, Bernadette
- Relation
- University of Newcastle Research Higher Degree Thesis
- Resource Type
- thesis
- Date
- 2020
- Description
- Research Doctorate - Doctor of Philosophy (PhD)
- Description
- Chronic obstructive pulmonary disease (COPD) is a leading global cause of morbidity and mortality and is expected to increase in importance as the world population continues to age. It is known that cigarette smokers develop some degree of lung inflammation, however patients with COPD develop a far greater degree of inflammation that progresses rapidly with advanced disease, often accompanied by systemic inflammation. Despite the identification of the inflammatory role within COPD the mechanisms that underpin the development and progression of disease and disease symptoms are yet to be fully elucidated. It is possible that epigenetics, the regulation of gene expression based off environmental stimuli, plays a fundamental role in the development and progression of COPD. The body of work conducted during this PhD candidature, explored the role of epigenetic mechanisms, particularly DNA methylation and histone acetylation in COPD using a combination of our novel short-term cigarette smoke (CS)-induced murine model of COPD and the complementary analyses of some clinical samples. Utilising both cell lines and the murine model of COPD, I assessed the role of the DNA methylation. I show that CS exposure in vitro and in experimental COPD; there are aberrant changes in expression of DNA methylation regulatory enzymes DNA methyltransferases (DNMT)-1, -3a, -3b and ten-eleven translocation (TET)-1, -2 & -3. Furthermore, CS leads to increased amounts modified DNA states that are indicative of DNA de-methylation. These observations led to prophylactic treatment of the experimental model of COPD with DNMT inhibitor 5-azacytidine. Despite no changes to the CS induced inflammation, there was a protection against lung remodelling such as alveolar enlargement (emphysema), and collagen deposition around the airways; these changes in turn led to protection against lung function changes. Thus far, this data suggests that DNA methylation has a role in the development of COPD and that by further analysis may have the potential to lead to novel therapeutic targets. Two key enzyme families, histone acetyltransferases (HATs) which activate gene transcription and histone deacetylases (HDACs) which halt gene transcription, regulate histone acetylation. I established that there is an imbalance of HATs and HDACs associated with a murine model of COPD and furthermore in clinical samples. The change in expression and activity of these enzymes was accompanied by an increase of histone H3 acetylation in early stages of disease development (4wk cigarette smoke exposure), where when disease features in our experimental model have been fully established, we had a shift to an increase of histone H4 acetylation. This could be the pivotal shift that leads to irreversible disease features of COPD, and that histone acetylation could be a key mechanism in the development and progression of COPD. As a number of HATs and HDACs were shown to have altered expression in COPD, I investigated the potential of targeting bromodomain and extra-terminal (BET) proteins. BET proteins possess bromodomain motifs that bind acetylated lysine residues in histones. In this investigation I used a selective, small molecule inhibitor of BET proteins, I-BET151. When administered prophylactically and therapeutically in the experimental model of COPD, I-BET151 was effective with reducing airway inflammation, halting progression of alveolar destruction and restored lung function parameters. I-BET151 also restored elevated levels of tumour necrosis factor (TNF)-alpha and CXC motif chemokine ligand 1 (CXCL1)/(KC). To confirm that these changes were directly related to the use of I-BET151, I ran chromatin immunoprecipitation (ChIP)-PCR pulling down either Brd2 or Brd4; Brd2 has an increased affinity to the promotor region of TNF-alpha in lung tissue from mice with experimental COPD, furthermore and interestingly mice treated with I-BET151 displayed a decreased affinity of Brd2 to the promoter region, whereby Brd4 pulldowns resulted in similar finding for the affinity of CXCL1 promoter region. Hence, I-BET151 displaces both Brd2 and Brd4 from promoter regions of pro-inflammatory genes leading to resolved disease features. Expanding our knowledge on the effects of disease features, I assessed the effects of I-BET151 in a murine model whereby mice are exposed to CS for 8wks (to obtain COPD features) and then infected with a mouse strain of influenza virus. This study showed that the exacerbated and exaggerated immune response to flu in mice previously exposed to CS was substantially reduced in mice treated with I-BET151, this coincided with an increase in viral clearance. This was validated in clinical primary bronchial epithelial cells (pBECs) obtained from patients with COPD. In summary, by targeting the BET proteins, Brd2 and Brd4 downstream with a selective inhibitor I-BET151, we can restore features of COPD experimentally, which do not hinder the subjects’ effect to clear influenza infection. This could lead to a targeted and effective treatment for patients with COPD. From these studies I have shown that both DNA methylation and histone acetylation are imbalanced in response to CS in vitro, in vivo and ex vivo. These data provide the foundation for further epigenetic regulation investigations in COPD development and disease progression. And more importantly, my studies have shown the therapeutic potential of targeting epigenetic mechanisms, by preventing disease prophylactically and halting progression of disease therapeutically. This can potentially lead to more effective therapeutics for patients with COPD whereas now there is no cure of effect treatments that halt the progression of disease.
- Subject
- COPD; epigenetics; DNA methylation; histone acetylation; bromodomains; influenza
- Identifier
- http://hdl.handle.net/1959.13/1424001
- Identifier
- uon:38008
- Rights
- Copyright 2020 Bernadette Jones-Freeman
- Language
- eng
- Full Text
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| Thumbnail | File | Description | Size | Format | |||
|---|---|---|---|---|---|---|---|
| View Details Download | ATTACHMENT01 | Thesis | 3 MB | Adobe Acrobat PDF | View Details Download | ||
| View Details Download | ATTACHMENT02 | Abstract | 515 KB | Adobe Acrobat PDF | View Details Download |