Tillie-Louise Hackett

Chronic obstructive pulmonary disease (COPD) is an incurable and deadly lung disease, characterized by irreversible airflow limitation and chronic inflammation. Sadly, there has been a disheartening lack of new therapeutic approaches to counteract the progressive decline in lung function, which ultimately leads to disability and death. The majority of COPD patients in primary care have mild and moderate disease, but treatments and clinical trials have historically focused on more severe patients. In this thesis we used a novel approach that combined stereology, multi-resolution computed tomography (CT), and histology to provide the first direct evidence that 41% of terminal and 57% of transitional bronchioles are already lost in mild COPD patients. Additionally, we showed that this loss of small airways occurred in regions of the lung with normal parenchyma, suggesting that airway loss precedes emphysematous tissue destruction. Thus, in the remainder of the thesis we aimed to determine the structural, molecular and cellular alterations that occur in the remaining small airways, and how they might be targeted for therapeutic intervention in COPD.We demonstrate that the remaining terminal bronchioles in COPD patients have thickened walls due to deposition of fragmented fibrillar collagen, and progressively narrowed lumens with disease severity, due to loss of supporting radial tetherings (alveolar attachments).Using a systems biology approach to relate gene expression signatures to disease pathology, we identified a core set of 51 master regulators (transcription factors) that regulate the gene signatures associated with small airways disease and emphysema. Further, we identified mTOR inhibitors as potential novel therapeutics for COPD, and demonstrate the ability of mTOR inhibitors to block pathology associated inflammatory processes at the single cell level in immune cells from COPD patients.Lastly, using highly-multiplexed imaging mass cytometry we created the first single-cell atlas of the terminal bronchiole, demonstrating that the remaining terminal bronchioles of mild and moderate COPD patients have an activated immune response with increased cross-talk between innate and adaptive immune cells.Collectively, our data emphasize that early diagnosis and use of anti-inflammatory treatments early in the disease process will be required to alter small airway loss, and disease outcomes in COPD.

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Asthma and COPD are incurable chronic inflammatory diseases of the lung that involve inflammation and remodeling in the airways. An abnormal interaction between the airway epithelium and fibroblasts in the epithelial mesenchymal trophic unit (EMTU) has been suggested to be a possible disease mechanism in asthma and COPD. The airway epithelium forms the first line of defense in the airways while fibroblasts are the structural cells responsible for producing extracellular matrix (ECM) proteins and maintaining the structural integrity of the lung. In this thesis we developed a co-culture model to study the factors involved in airway epithelial cell and fibroblast interaction and how a dysregulated communication between these two cells contributes to asthma and COPD pathogenesis. We also used multiphoton imaging and 3-dimensional collagen contraction assays to assess the effect of abnormal ECM repair by airway fibroblasts, proposed to cause airway remodeling in asthma and COPD. We found in the co-culture model that the airway epithelium through the release of IL-1α controls the inflammatory and ECM remodeling phenotype of lung fibroblasts. Further, exposing airway epithelial cells to cigarette smoke, the major risk factor for COPD, caused a higher release of IL-1α which subsequently led to the release of higher levels of inflammatory mediators. Again, due to the presence of a single nucleotide polymorphism, rs2910164 (GG allele), COPD-derived lung fibroblasts from our co-culture had a lower induction of miRNA-146a-5p, an anti-inflammatory miRNA that regulates IL-1 signaling, compared to controls. Again, we found asthmatic-derived airway epithelial cells released higher levels of IL-1α and that IL-1 causes the release of inflammatory mediators, the down-regulation of ECM proteins in airway fibroblasts and affects their ability to organize gelatin into fibrillar collagen. In the airways of asthmatics patients, we found that there was disorganization of fibrillar collagen fibers in the lamina propria, due to a defect in the ability of asthmatic-derived airway fibroblasts to remodel collagen fibers compared to controls. Taken together our work sheds new light on how abnormal epithelial-fibroblast communication may contribute to the chronic inflammation and airway remodeling described in asthma and COPD, further it opens new avenues for therapeutic research.

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The airway epithelium is the interface between the environment and the submucosa of the lung and thus is the first line of defense against inhaled exogenous agents. In addition to maintaining a structural barrier through homeostasis and repair, the airway epithelium is involved in co-ordination of the mucosal immune response to the inhaled environment. In asthma it is now understood that the airway epithelium is abnormal with dysregulation of genes integral to differentiation, proliferation, and inflammation. Alteration of the chromatin architecture through epigenetic modifications including DNA methylation and histone modifications is reactive to the environment and can establish chromatin states which are permissive or repressive to gene expression. Epigenetic regulation of gene expression is cell specific, as such, it is important to understand epigenetic regulation in cells that are thought to play a central role in asthma.As epigenetic processes are critical to cellular specificity and disease susceptibility the overarching hypothesis of this thesis is that alterations to the epigenome of asthmatic airway epithelial cells contribute to the dysregulation of genes involved in key epithelial functions. To investigate this hypothesis, we performed analysis of DNA methylation, expression of epigenetic modifying genes, and histone acetylation and methylation in airway epithelial cells, airway fibroblasts, and peripheral blood mononuclear cells from asthmatic and healthy subjects.We identified unique signatures of both DNA methylation and expression of epigenetic modifying enzymes in airway epithelial cells and showed that epithelial cells were epigenetically distinct from other cell types. Furthermore, we found that airway epithelial cells from asthmatic subjects displayed changes in DNA methylation, expression of histone kinases, acetylation of lysine 18 on histone 3, and occupancy of this histone modification at genes important to epithelial functions. Therefore, epigenetic differences between tissue types were more evident and plentiful than within cell types highlighting the importance of the epigenome to cell specificity, yet subtle differences within each tissue were determined and may play a role in disease pathogenesis. Thus, these findings enhance our understanding of the unique epigenetic landscape which may contribute to the airway epithelial phenotype in health and disease.

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Asthma is characterized by reversible airflow limitation, airway inflammation and remodeling, which includes increased smooth muscle mass, sub-epithelial fibrosis, goblet cell metaplasia and loss of columnar epithelial cells. Basal cells are progenitor cells of the pseudostratified airway epithelium that undergo distinct phenotypic transitions to maintain epithelial homeostasis following damage. We hypothesized that differentiation of epithelial basal cells is defective in asthma, leading to impaired repair. We used three distinct in vitro models of human airway epithelial basal cell plasticity – epithelial-mesenchymal transition (EMT), repair of mechanical scratch wounds, and differentiation at air-liquid interface – which together provide a complete overview of basal cell function in epithelial repair. In Chapter 3 we assessed the ability of transforming growth factor (TGF)-β₁ to induce molecular reprogramming indicative of EMT and found that basal cells from asthmatic and non-asthmatic patients undergo TGFβ₁-induced EMT. However, an expanded population of basal cells in differentiated epithelial cultures from asthmatic donors led to an increased EMT response. In Chapter 4 we found that inhibition of ΔNp63α impaired restitution of scratch wounds in monolayer culture, due to decreased proliferation. Additionally, ΔNp63α regulated several genes known to be involved in epithelial repair, including β-catenin, epidermal growth factor receptor, and jagged1. In Chapter 5 we found that basal epithelial cells from asthmatic donors with (+) and without (-) exercise-induced bronchoconstriction (EIB) were impaired in the transition to a ciliated but not goblet cell phenotype in an air-liquid interface (ALI) model of mucociliary differentiation. EIB(-) asthmatics also had an expansion of the basal cell population and shorter cilia. In Chapter 6, we used an unbiased RNA sequencing approach to identify aberrant expression of pathways involved in actin cytoskeleton dynamics and cellular metabolism that were distinctly different in EIB(-) and EIB(+) asthma during mucociliary differentiation. We also identified a miRNA-mRNA network that regulates the epithelial transition from proliferation to differentiation. This thesis provides compelling evidence that lineage commitment and molecular reprogramming in basal cells are skewed in asthma to favour epithelial plasticity in response to TGFβ₁, rather than mucociliary differentiation, and that epithelial remodeling is more pronounced in the EIB(-) phenotype of asthma.

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