title: Exploring molecular mechanisms underlying the role of non-typeable Haemophilus influenzae in COPD
Creators: name: KC, R
Publisher: name: University of Tasmania
Year: 2020

description: Chronic obstructive pulmonary disease (COPD) is a severe and progressive condition characterised by persistent respiratory symptoms and airflow limitation. Around 300 million people in the world have COPD. It has emerged as the third leading cause of mortality, claiming 3.2 million lives worldwide in 2017. An acute exacerbation of COPD, a sudden worsening of respiratory symptoms, is a major cause of morbidity and mortality in COPD patients. Various factors, including biomass smoke exposure and infection with bacteria trigger COPD exacerbations. Nearly half of the world’s population uses biomass fuel for cooking and heating and is therefore at risk of exposure to noxious particles released from the combustion of biomass fuel. Among respiratory bacteria, nontypeable Haemophilus influenzae (NTHi) is a key pathogen implicated in colonisation and damage of airways in COPD patients. In this thesis, I assessed the impact of biomass smoke exposure on inflammation and adherence of NTHi to human bronchial epithelial cells. Due to the lack of a standard and easily accessible procedure for the preparation of biomass smoke, I first devised a simple, cost-effective, and reproducible method for the generation of biomass smoke extracts, in particular, cow dung and wood smoke extracts. Using this method, I generated quantifiable batches of biomass smoke extracts that were utilised for the assessment of cellular responses to different types of biomass smoke. I investigated the effect of biomass smoke extracts on human airway epithelial cells with respect to expression of a known receptor of NTHi, platelet-activating factor receptor (PAFR), and the pro-inflammatory cytokines interleukin 6 (IL-6) and IL-8, using quantitative polymerase chain reaction. In addition, I examined the response of bronchial epithelial cells to adherence of NTHi using immunofluorescence microscopy. I observed an increased inflammatory response in cells exposed to biomass smoke, characterised by induction of significant levels of IL-6 and IL-8 mRNA, in comparison to mock exposed cells. I demonstrated a dose-dependent increase in NTHi adhesion to epithelial cells following exposure to biomass smoke extracts. I further established an association between PAFR expression and the adhesion of NTHi in biomass smoke-exposed cells. Pre-treatment with a known PAFR antagonist, WEB-2086 inhibited biomass smoke-induced adherence of NTHi in airway cells in a dose dependent manner. In addition, pre-treatment of biomass smoke-exposed airway epithelial cells with a novel WEB-2086 analogue, C17 reduced NTHi adhesion in a dose-dependent manner. I next assessed the genomes of 568 NTHi isolates, including 40 newly sequenced clinical isolates collected from patients with different diseases, including COPD. Phylogenetic analysis based on polymorphic sites on the core genome did not provide sufficient resolution to separate COPD strains from other clinical phenotypes, suggesting a similar set of core genes are present in all clinical NTHi isolates. I applied discriminant analysis based on the presence or absence profiles of accessory genes and found a clear distinction between COPD and other disease strains. I then applied a pan genome-wide association study approach to identify the accessory genes associated with COPD. I identified a set of accessory genes that regulate metabolic functions, such as the metabolism of organic acids and oxidation-reduction reactions that regulate cellular respiration to be significantly associated with COPD strains. This result suggests that NTHi associated with COPD may exhibit genetically encoded functional variances to isolates collected from other clinical illnesses. In conclusion, this work advances our understanding of how biomass smoke could contribute to the development and progression of COPD and highlights the potential of PAFR as a therapeutic target for reducing the impact of hazardous biomass smoke exposure on respiratory health. Further, this thesis increases our understanding of gene sets shared by NTHi strains that survive and cause disease in the COPD lung.