The response of human bronchial epithelial cells to outdoor air pollution: Interventions to protect the diseased lung against diesel emission exposure

Abstract

Introduction Prolonged exposure to diesel particulate matter (PM) has been linked to inflammation and oxidative stress, which can lead to the development and progression of chronic obstructive pulmonary disease (COPD). A common strategy to reduce diesel PM is the substitution of diesel fuel with biodiesel or a fuel additive. The use of biodiesels or fuel additives effectively lower diesel particulate emissions. However, very little is known about the health effects of these alternative diesel emissions. Furthermore, antioxidant treatment may be a potential therapeutic intervention to protect the lungs by maintaining oxidative balance and preventing oxidative stress after diesel emission exposure; however, this has not previously been comprehensively tested. Understanding the cell response within the lungs after air pollution exposure and how air pollution interacts with the diseased lungs of COPD patients would facilitate development of preventive strategies for COPD.  Aim The main objectives of this PhD program were to investigate adverse effects of diesel emission with varied physical and chemical compositions on human bronchial epithelial cells, to test the effectiveness of antioxidant therapy to protect against adverse effects, and to identify potential novel targets in the COPD lung transcriptome for interventions aimed at protecting vulnerable lungs from air pollution exposure.  Methods This project incorporates three different study designs:1.      Diesel and biodiesel toxicity studies characterized human bronchial epithelial cells (HBECs) responses to diesel and biodiesel emissions with varied chemical and physical compositions. HBECs were cultured and differentiated at an air-liquid interface (ALI) for 28 days. After exposure, bioassays measured cell metabolism, cell death and cytokine secretion. Gene expression assays measured biomarkers for apoptosis, anti-apoptosis, xenobiotic metabolism and oxidative stress responses. 2.      An antioxidant intervention was tested for attenuation of HBECs responses to diesel emissions. Cell response testing measured cell proliferation, cell death, oxidative stress, inflammation and xenobiotic metabolism. The effect of the diesel emissions with or without intervention on transcriptomic changes was assessed using Nanostring GX Intracellular Signalling and Immunology panels.3.      Transcriptomic profiling of the COPD lung was assessed using RNAseq using the Ion Torrent technology. Comparative data analysis was used to isolate potential targets for preventative cell response intervention. Results This PhD program identified 6 key findings:1.      Both organic content and residual components of diesel emissions were important in determining HBEC responses in vitro.2.      Biodiesel blends worsened primary HBEC response to diesel emission exposure.3.      Diesel fuel substitution with raw coconut oil reduced the adverse effects of diesel emission on 16HBE cells when percentage substitution is controlled.4.      NAC intervention potentiated, rather than reduced, adverse cellular responses of immortalised 16HBE cells to diesel emission exposure. 5.      NAC intervention may be effective intervention against the adverse effects of diesel emission exposure in primary HBECs from a COPD cohort.6.      Transcriptomic profiling of the COPD lung identified 8 differentially expressed genes, when compared to the non-COPD lung, which may be potential targets for intervention against diesel emission exposure and COPD progression. Conclusions This thesis has investigated two potential strategies to reduce the adverse effects of diesel emissions on HBECs; the addition of coconut oil in diesel fuel and the administration of NAC as a therapeutic antioxidant. These coconut oil derived alternative diesel emissions showed highly differential effects on HBEC responses, despite the similarities in diesel composition. By studying the effects of diesel emissions with varied physical and chemical compositions, this thesis has improved current understanding of how HBECs respond to diesel, biodiesel and alternative diesel emissions. Moreover, an antioxidant treatment with 5 mM NAC had differential effects on HBECs after diesel emission exposure. NAC intervention was toxic to immortalized 16HBE cells after diesel emission exposure. When tested on primary HBECs from a COPD cohort, NAC did not significantly reduce the adverse effects of diesel emission exposure, however there was a trend for attenuation.                   This thesis has highlighted the importance of toxicity testing for novel diesel alternatives, as minor differences in composition can alter HBEC responses and the potential for negative health impacts. Furthermore, intervention studies showed no significant improvement of HBEC responses with the administration of an antioxidant therapy, despite oxidative stress being a known driver of diesel-induced injury. This thesis has identified a limitation in our understanding of diesel emission toxicity and how to effectively intervene and protect the airways from diesel emission exposure.   The COPD lung transcriptome displayed potential novel targets for future intervention studies. Future research will look to validate these potential targets and assess their potential for intervention. Future diesel toxicity studies should continue to explore alternative diesel blends that reduce diesel pollution and the adverse effects of diesel emission exposure.            Intervention studies should investigate the toxicity of NAC on immortalised cell lines and the potential to protect against pre-malignant cells after diesel emission exposure. These findings provide a scientific basis for the development of strategies to protect vulnerable populations from diesel emissions.