Asthma is a heterogeneity disease that is mostly managed successfully using bronchodilators and anti-inflammatory drugs. Around 10%-15% of asthmatics however have difficult or severe asthma which is less responsive to treatments. Asthma and in particular severe asthma are now thought of a description of symptoms which may contain possible sub-groups with possible different pathologies which could be useful for targeting different drugs for different sub-groups. However little statistical work has been carried out to determine these sub-phenotypes. Studies have been carried out to partition severe asthma variables in to a number of sub-groups but the algorithms used in these studies are not based on statistical inference and it is difficult to select the number of best fitting sub-groups using such methods. It is also unclear where the clusters or sub-groups returned are actual sub-groups or reflect a bigger non-normal distribution. In the thesis we have developed a statistical model that combines factor analysis, a method used to obtain independent factors to describe processes allowing for variation over variables, and infinite mixture modelling, a process that involves determining the most probable number of mixtures or clusters thus allowing for variation over individuals. This model created is a Dirichlet process normal mixture latent variable model DPNMLVN and it is capable of determining the correct number of mixtures over each factor. The model was tested with simulations and used to analysis two severe asthma datasets and a cancer clinical trial. Sub-groups were found that reflect a high Eosinophilic group and an average eosinophilic group, a late onset older non atopic group and a highly atopic younger early onset group. In the clinical trial data 3 distinct mixtures were found relating to existing biomarkers not used in the mixture analysis.

Traditional animal models of severe asthma do not recapitulate defining asthma characteristics, including persistent airway hyper-responsiveness, and chronic neutrophilic inflammation. This is problematic because moderating airway hyper-responsiveness decreases asthma frequency and severity, making it a paramount pharmacological goal in asthma research. Employing a spontaneous equine asthma model (equine pasture asthma, EPA), we first confirmed reversible airway obstruction in eight diseased horses during asthma exacerbations in response to ß2renergic agonist stimulation. Next, non-specific airway hyper-responsiveness was confirmed using methacholine bronchoprovocation to identify the provocative concentration causing a 40% increase in baseline lung resistance (PC40RL)- a threshold similarly employed in evaluating human asthmatics unable to mount forced expiration. The PC40RL of ten EPA horses was consistently <1mg/ml of methacholine, which is a cutoff that has been used to diagnose severe human asthma. Like non-asthmatic humans, ten control horses did not respond to methacholine doses up to 16 mg/ml. Finally, persistence of AHR was documented during absence of seasonal aeroallergen triggers in five horses that were evaluated between 3 and 31 months following the initial methacholine bronchoprovocation. This unique ability of EPA horses to model AHR attributes that are not addressed by other animal models points to the suitability of EPA horses to decipher the mechanistic basis of airway hyper-responsiveness. Building on knowledge that 2renergic receptor (AR) signaling is required to develop the asthma phenotype in a murine model, differentially expressed genes from serial lung biopsies of two EPA affected and two controls were filtered to identify genes that interact with the 2-AR. Hyperpolarization Activated Cyclic Nucleotide-Gated Potassium Channel 4 (HCN4) was prioritized because of its interactions with the 2-AR. Relative to control horses, HCN4 was constitutively expressed in airway smooth muscle of EPA horses during remission and increased during seasonal disease exacerbation. Agonism of airway contraction by HCN4 was proven using the specific HCN4 antagonist, ivabradine, which caused dose dependent decreases in carbachol induced contractile responses in both EPA and control bronchi in vitro. These findings highlight utility of EPA as a model of severe asthma and HCN4 as a mediator of airway contraction that warrants further investigation in severe human asthma.

INTRODUCTION: Abnormal ventilation is the functional consequence of airway obstruction. In patients with severe asthma, ventilation patterns visualized by magnetic resonance imaging (MRI) exhibit significant inter-patient heterogeneity. Therefore, our objectives were to identify MRI ventilation phenotypes of severe asthma using an unsupervised clustering approach and examine their associated demographic, clinical, physiologic, and inflammatory characteristics. METHODS: This retrospective analysis included 58 adults with severe asthma who underwent hyperpolarized 129Xe ventilation MRI. Nineteen quantitative variables were extracted from ventilation MRI (including ventilation defect percent (VDP), ventilation defect size, and ventilation texture features) and transformed to principal components for hierarchical clustering. Differences in demographics, clinical characteristics, spirometry, inflammatory biomarkers, and computed tomography (CT) measurements between phenotypes were evaluated using one-way ANOVA or Kruskal-Wallis tests. RESULTS: Three ventilation phenotypes of severe asthma were identified. They were significantly different with respect to their age, prevalence of obesity, spirometry, sputum neutrophil percent, sputum cytokines (interleukin-4, interleukin-6, interleukin-15, B-cell activating factor), total lung capacity, CT air-trapping, and CT mucus score (all p<0.05). They were not different with respect to their asthma control or medication requirement, and ~75% of each phenotype reported uncontrolled asthma (ACQ-5≥1.5). Phenotype 1 had normal ventilation (VDP=1.7±0.9%) and predominantly consisted of young, obese females (88% female, 41±11 years old, 63% obese). They had normal-to-moderately reduced FEV1 (80±15%pred), normal post-bronchodilator FEV1/FVC, and reduced total lung capacity (85%pred [57-108]). 25% had intraluminal inflammation (all eosinophilic) and their sputum interleukin-4 levels were elevated. Phenotype 2 had markedly abnormal ventilation (VDP=6.2±3.8%) and was older than Phenotype 1, but also predominantly consisted of obese females (63% female, 54±13 years old, 59% obese). They had mildly-to-severely reduced FEV1 (61±17%pred) and partially reversible obstructive spirometry (72%, post-bronchodilator FEV1/FVC<0.70). 50% had intraluminal inflammation (28% eosinophilic/13% neutrophilic/9% mixed-granulocytic) and their sputum interleukin-6 levels were elevated. Phenotype 3 had severely abnormal ventilation (VDP=24.8±10.2%) and was also older than Phenotype 1 but was gender-balanced and not obese (50% female, 56±12 years old, 11% obese). They had moderately-to-very severely reduced FEV1 (41±12%pred) and partially reversible obstructive spirometry (89%, post-bronchodilator FEV1/FVC<0.70). 73% had intraluminal inflammation (39% eosinophilic/17% neutrophilic/17% mixed-granulocytic) and their sputum interleukin-15 and B-cell activating factor levels were elevated. They had the highest burden of gas-trapping and mucus on CT. CONCLUSION: Three distinct MRI ventilation phenotypes of severe asthma were identified through unbiased analysis, all of which reported uncontrolled asthma. The discordance in ventilation between phenotypes, and their characteristics, suggest different mechanisms that may be driving severe asthma. / Thesis / Master of Science (MSc) / Severe asthma is an airways disease that is characterized by inflamed, twitchy and obstructed airways. There is remarkable clinical heterogeneity between asthma patients due to the various mechanisms of disease. Abnormal ventilation is the functional consequence of abnormal airway pathology in asthma, which can be directly visualized by hyperpolarized 129Xe magnetic resonance imaging (MRI). Each ventilation pattern is unique and there is significant inter-patient variability. Thus, the goal of the thesis was to extract quantitative information from the 129Xe MRI ventilation patterns of patients with severe asthma, identify novel ventilation phenotypes, and determine their clinical relevance. An unsupervised machine learning approach using quantitative ventilation MRI features identified three unique, clinically relevant ventilation phenotypes of severe asthma with distinct clinical, physiological, and biological characteristics. The discordance in ventilation between phenotypes, and their characteristics, suggest different mechanisms that may be driving severe asthma.

Asthma is an inflammatory airways disease affecting over 339 million people of all ages worldwide. More than 10% of asthmatics have uncontrolled severe disease which is insensitive to high doses of oral corticosteroid treatment. Type 2 innate lymphoid cells (ILC2) are pro-inflammatory lymphomononuclear cells proposed as critical drivers of eosinophilic inflammatory disease of the upper and lower airways. Controlling this activity may provide novel therapies for asthma. This thesis aimed to investigate factors that affect the local activation and expansion of ILC2 in the airways including anti-inflammatory medications such as (i) corticosteroids, (ii) neuro-immune regulation of ILC2, and (iii) effect of locally generated cytokines on ILC phenotypes and the relationship to the ongoing airway inflammatory profile. We firstly investigated the effect of intranasal corticosteroids on activation levels of ILC2 in the upper airway of allergic rhinitics with mild asthma following controlled nasal allergen challenge (Chapter 2). Following pre-treatment with intranasal corticosteroid there was an attenuation in the allergen-induced increase in total ILC2 and IL-5/13+ ILC2 in the nasal mucosa. In addition, HLA-DR expression on ILC2 in the nasal mucosa was down-regulated. Overnight culture with IL-2, TSLP or IFN-γ up-regulated HLA-DR expression on ILC2, in vitro; an effect that is inhibited in the presence of corticosteroids. Attenuation of HLA-DR expression by ILC2 may be an additional mechanism by which corticosteroids modulate adaptive immune responses in the airways. We have previously reported that lung ILC2 are activated within 7h following allergen-inhalation challenge. Since airway mucosal tissue is highly innervated, we investigated whether neuroimmune interactions may trigger early and rapid host immune responses (Chapter 3). In a diluent-controlled allergen-inhalation challenge cross-over study, where mild asthmatics developed early and late bronchoconstrictor responses with sputum eosinophilia (>3%), NMUR1, a receptor for the neuropeptide, neuromedin-U, was up-regulated on sputum ILC2 in 7h post allergen challenge. This was associated with increased expression of IL-5/IL-13 by sputum ILC2 post-allergen and following in vitro culture. ILC2 activation was mediated through a MAPK/PI3 kinase dependent-signaling pathway that was attenuated in the presence of dexamethasone. Co-culture with IL-33 and TSLP, in vitro up-regulated NMUR1 expression on ILC2 at the protein and transcriptomic level which was attenuated by dexamethasone. The close interplay between neuropeptide signalling and tissue-derived alarmin cytokines may be important interactions for rapid ILC2 activation in airway inflammatory responses in asthma. We have reported increased ILC2 with the highest level of IL-5/13+ ILC2 in the airways of severe asthma with uncontrolled eosinophilia (>3%). The prevalence and phenotypic analyses of innate lymphoid cells subsets in severe asthma with neutrophilic or mixed granulocytic airway inflammatory endotypes remains unclear and was investigated in Chapter 4. Sputum ILC3 were most abundant in severe asthma with neutrophilic airway inflammation where IL-17A+ ILC3 correlated with airway neutrophilia. ILC2 were predominant in severe asthma with airway eosinophilia. Importantly, we identified an intermediate ILC2 phenotype displaying ILC3-like markers (c-kit and IL-17A) in severe asthma with neutrophilic and mixed granulocytic airway inflammation. Inflammasome related cytokines, IL-1β and IL-18 were significantly increased in the airways of these patients. At both proteomic and transcriptomic levels, flow sort-purified ILC2 trans-differentiated to the intermediate phenotype when co-cultured with IL-1β+IL-18. Blocking inflammasome-related cytokines may control T2-low severe asthma exacerbations. Collectively, the findings of this thesis highlight the role of corticosteroids, neuropeptides and airway inflammasome related cytokines as modulators of ILC fate and activity in asthma. / Thesis / Doctor of Philosophy (PhD) / Asthma is a disease of the airways that makes breathing difficult. About 10% of asthma patients have uncontrolled severe symptoms despite treatment with high doses of corticosteroids which imposes many unwanted side effects. Investigating processes that worsen the disease will help to discover new treatments for asthma. Type 2 innate lymphoid cells (ILC2) are novel cells that produce large quantities of factors which attract and activate effector cells to the lungs which in turn make breathing difficult. This thesis investigated whether controlling ILC2 activity reduces asthma symptoms by studying i) responsiveness of ILC2 to corticosteroids using a controlled allergen exposure through the nose in people with allergic rhinitis and mild asthma, ii) the role of airway nerves and mediators on ILC2 activation, and iii) the ability of signals produced by the lungs to impact factors released by ILC2 and the relationship to effector cells found in the airways of severe asthma. Overall, ILC2 activation can be modulated by corticosteroids, nerve derived factors and lung tissue derived cytokines, and this is associated with changes in the number and type of effector cells in the lungs.

Introduction: The pathogenesis and treatment of allergic bronchopulmonary aspergillosis (ABPA), severe asthma-non fungal sensitised (SANFS) and severe asthma with fungal sensitization (SAFS) is poorly understood. IL-17A, IgE and microbiome may be associated with pathogenesis of asthma, but their role in fungal-associated asthma is uncertain. Further, the efficacy of voriconazole, posaconazole and nebulised amphotericin B (NAB) in ABPA and SAFS has not been fully studied. Aims and objectives: The aim of this PhD thesis was to evaluate the role of IL-17A, IgE and lung microbiome in patients with SANFS, SAFS and ABPA. We also studied the efficacy and safety of NAB, voriconazole and posaconazole. Methods: Airway lymphocytes and peripheral blood mononuclear cells (PBMC) from patients with ABPA (n=16), SAFS (n=15), SANFS (n=11), mild asthma (MA) (n=6) and NH (n=11) were characterized by flow cytometric analysis (FACS) to determine the % of CD (+) IL-17A expressing cells. We also evaluated microbiome population using culture and PCR plus sequencing from BAL of these patients. In chapter 3, we analysed total and specific IgE in blood from adult cohorts of SAFS (n=34) and ABPA (n=48) using ImmunoCAP 100. In chapter 5 we studied the efficacy of voriconazole and posaconazole and in chapter 6; we studied the efficacy of NAB.Results: %CD4+IL-17A expressing cells were significantly higher in patients with severe asthma and correlated positively with serum neutrophil and presence of fungi in the airways. ABPA, SAFS and SANFS were similar but all were significantly higher than MA and NH. There were no differences in IL-17A expression between blood and the lung. Fungi were more frequently associated with severe asthma and low FEV1. Steroid treatment significantly increased airway fungal load. IgE against staphylococcal aureus (SE-IgE) correlated positively with FEV1 and OCS dose. Voriconazole and posaconazole improved asthma severity and radiological abnormalities. NAB was associated bronchospasm, but was extrely effective in the few patients (n=3) that took treatment for >12 months. These responders had unique characteristics. Conclusions: IL-17A, SE-IgE, and lung microbiome are associated with asthma severity. Steroid use in these patients may increase airway fungal load. Whereas voriconazole and posaconazole are efficacious, the use of NAB is associated with significant bronchospasm. SE-IgE -high asthma patients may be a distinct asthma phenotype. Larger studies are needed.

L’asthme sévère de l’enfant est une pathologie respiratoire chronique dont le traitement est difficile. L’épithélium bronchique à l’interface de l’organisme et de l’environnement, est un pivot central dans la maladie asthmatique. Le but de ce travail a donc été d’étudier l’épithélium bronchique de l’enfant asthmatique sévère.Dans un premier temps, j’ai développé un modèle de reproduction d’épithélium bronchique in vitro obtenu à partir de cellules épithéliales bronchiques issues de biopsies bronchiques d’enfants asthmatiques sévères. J’ai comparé ces épithélia obtenus chez l’enfant à ceux obtenus chez l’adulte. Nous avons spécifiquement étudié un marqueur inflammatoire important dans l’asthme sévère, le TSLP. J'ai montré 2 isoformes de TSLP ayant un rôle contraire, anti inflammatoire et proinflammatoire. Dans un deuxième temps, j’ai mis au point un modèle d’exacerbation d’asthme viro-induite. J’ai utilisé le modèle de culture et soumis les cellules à une stimulation Poly I:C. Les différentes cytokines sécrétées lors d’une exacerbation asthmatique virale ont été retrouvées augmentées dans notre modèle : CXCL8, CXCL10, CCL2, CXCL9 et RANTES. Enfin, j’ai étudié le propionate de fluticasone dans le modèle d’exacerbation asthmatique. Nous avons montré que l’effet de la fluticasone est différent entre les cellules épithéliales bronchiques issues de témoins et celles issues d’asthme sévère. Le modèle de stimulation viro-induite a permis d’étudier l’effet des corticoïdes inhalés sur l’épithélium bronchique et va permettre d’étudier les voies mécanistiques en jeu dans les exacerbations viro-induites, de tester d’autres molécules et proposer d’autres pistes thérapeutiques. / Severe childhood asthma is a chronic respiratory disease difficult to control despite treatment. Bronchial epithelium, at the interface between the body and the environment, is a central pivot in the asthmatic disease. The aim of this study was to examine the bronchial epithelium of severe asthmatic children. Initially, I developed a bronchial epithelium in vitro reproduction model obtained from bronchial epithelial cells from bronchial biopsies of severe asthmatic children. I compared these epithelia obtained in children with those obtained in adults. We specifically studied an important inflammatory marker in severe asthma,TSLP. I have highlighted the presence of two isoforms of TSLP which have an opposite role, anti-inflammatory for the short form and the long form for proinflammatory.Secondly, I developed an asthma exacerbation model of virus-induced. I used the culture model and challenged bronchial cells with poly I:C to. Different cytokines secreted upon viral asthma exacerbation were found increased in our model: CXCL8, CXCL 10, CCL2, RANTES and CXCL9.Finally, I have studied fluticasone propionate in the exacerbation asthmatic model. I studied cells from asthmatic and from non asthmatic children. Interestingly, we have shown that the effect of fluticasone is different between bronchial epithelial cells from non asthmatic children and those from severe asthma.The model of virus-induced stimulation was used to study the effect of inhaled corticosteroids on bronchial epithelium and will allow studying the mechanistic pathways involved in virus-induced exacerbations, testing other molecules and propose other therapeutic approaches.

<p>Various studies have shown an association between neutrophilic airway inflammation and severe asthma, but have failed to establish a causal relationship. In these studies airway neutrophilia could be due to high steroid doses, an airway infection, an epiphenomenon of severe asthma or a combination of these. We have examined the role of neutrophils in severe asthma in patients on optimum steroid doses with controlled eosinophilic airway inflammation and chemotactic activity of IL-17 as one potential mechanism of neutrophil recruitment to the airway.</p> <p>We examined the number, viability and activity of neutrophils in blood and sputum of three groups of asthma subjects divided on the basis of asthma severity. We also compared direct migration of blood neutrophils towards IL-17 between non-asthmatics and severe asthma subjects.</p> <p>Viability and survival at 24 hours was measured by examining apoptotic and non-apoptotic cells. Activation was examined by measuring the production of hydrogen peroxide and the expression of primary and secondary granule proteins. In migration study, migration of neutrophils towards IL-17 was measured.</p> <p>Blood neutrophils were increased in severe asthma subjects as compared to moderate and mild asthma subjects. There was no difference in sputum neutrophil numbers. There was no difference in viability, although blood neutrophil 24 hour survival was increased in severe asthma subjects as compared to moderate asthma subjects. There was no difference in the level of activation amongst the three groups.</p> <p>IL-17 was not a chemotactic stimulus for neutrophils. The study results show that sputum neutrophil numbers and activation are not increased in severe asthma as compared to less severe asthma. Therefore, the study results do not support a causal relationship between airway neutrophilia and severe asthma.</p> <p>Airway neutrophilia observed in previous studies might be due to airway infections or high doses of steroids taken by study subjects.</p> / Master of Health Sciences (MSc)

The present study explored the interaction between paediatric asthma and family functioning, within the context of family systems theory. Eight families, each with an asthmatic child between the ages of 8 and 15, were included in this study. In each case, the parents were interviewed and completed a quantitative measure, the Family Assessment Device, The results indicated that each family has a unique way of functioning and of integrating the child's asthma into its lifestyle. Factors found to be involved in the mutual impact of paediatric asthma and family functioning were the size of the family, the severity of the child's condition, the ways in which the family copes with the stress of asthma, communication patterns between parents, compliance with family rules and boundaries, and the affective responsiveness and involvement of family members. / Psychology / M.A. (Psychology)

The present study explored the interaction between paediatric asthma and family functioning, within the context of family systems theory. Eight families, each with an asthmatic child between the ages of 8 and 15, were included in this study. In each case, the parents were interviewed and completed a quantitative measure, the Family Assessment Device, The results indicated that each family has a unique way of functioning and of integrating the child's asthma into its lifestyle. Factors found to be involved in the mutual impact of paediatric asthma and family functioning were the size of the family, the severity of the child's condition, the ways in which the family copes with the stress of asthma, communication patterns between parents, compliance with family rules and boundaries, and the affective responsiveness and involvement of family members. / Psychology / M.A. (Psychology)