Investigating the local Th2 cytokine profile and its impact in a sheep model of allergic asthma

Abstract

Asthma is a chronic inflammatory disease affecting the airways in response to certain stimuli. It is characterized by pulmonary inflammation, reversible bronchial constriction, and airway remodelling triggered by exposure to inhaled allergens. Key pathological features include infiltration of the airways by activated eosinophils, lymphocytes and macrophages, damage to the bronchial epithelium, mast cell degranulation, mucous gland hyperplasia and bronchial mucosal thickening by oedema. Infiltrating leukocytes, as well as airway structural cells including smooth muscle, fibroblasts, endothelial and epithelial cells, are all important sources of mediators that drive or exacerbate the asthmatic condition. Extensive work in experimental animal models and studies in the clinic support a central role of allergen-specific T helper type 2 (Th2) lymphocytes in pathophysiological responses. Th2 lymphocytes drive the allergic reaction and activation of inflammatory cells via the production of cytokines crucial to allergic disease, the most important of which are interleukin (IL)-4 and IL-13. These cytokines are known to directly and indirectly drive several aspects of allergic inflammation and have numerous effects on the functional activity of airway structural cells, resulting in subepithelial fibrosis, airway smooth muscle proliferation, and goblet cell hyperplasia. Current asthma therapies are centred on the use of short and long acting bronchodilators, often in combination with corticosteroids, which are largely ineffective or induce resistance in a sub-group of asthmatics. There is clearly a need for more effective drugs for the prevention and treatment of allergic asthma. Hence, IL-4 and IL-13 have been the focus of much research as therapeutic targets that might be beneficial to consider in many asthma phenotypes. The aim of this thesis was to extend our knowledge of the role of the Th2 cytokines, IL-4 and IL-13, in the inflammatory response seen in asthma, using an established sheep experimental model of allergic asthma previously shown to mimic many of the main characteristics of the human condition. An investigation of the kinetics of IL-4 and IL-13 release during the inflammatory response as well as alterations to immune cells due to the effects of IL-4 and IL-13 in the sheep model was the focus of this study. Methods for detecting protein levels of ovine IL-4 and IL-13 in biological fluids by enzyme-linked immunosorbent assay (ELISA) were developed and optimized for use in the present studies. Sandwich ELISAs for the detection of ovine IL-4 and IL-13 were confirmed for specificity and precision, as well as quantitative detection of native proteins. The ELISA assays were then used to investigate temporal changes in the levels of IL-4 and IL-13 in airway fluid (bronchoalveolar lavage or BAL) samples collected during the course of the allergic airway response in the sheep asthma model. In this part of the study, it was found that similar to human asthma, these cytokines are involved and altered during an acute phase of asthmatic inflammation; IL-4 and IL-13 are released by different cellular sources and each exhibit a unique profile of secretion in response to allergen challenge. It is well known that macrophages are amongst the cellular targets of IL-4 and IL-13 and emerging evidence shows that these cells are involved in asthmatic inflammation. Macrophages undergo activation in response to IL-4 and IL-13 and their resultant activities contribute to asthma pathogenesis. The sheep model of asthma is a powerful tool for studying macrophages as it is easy to access large numbers of alveolar macrophages (AMs) at multiple time points. Also, there is evidence showing similarities in the features of activation of human and sheep macrophages. Therefore, taking advantage of the sheep asthma model, the phenotype and activity of AMs were investigated with the aim to better understand IL-4 and IL-13 effects on macrophages, as well as understanding the macrophage characteristics that contribute to asthmatic symptoms. As a result, it was found that AMs from asthmatic sheep undergo a shift in the expression of their markers and activities which may contribute to an altered or compromized immune response in asthma. The research presented in this thesis provides a comprehensive investigation into the complex immunological responses occurring in asthma. Further, this study has strengthened our understanding of the critical elements of asthma disease by further exploring the immunopathological features of the sheep model of allergic asthma. It is hoped that this thesis will contribute towards developing novel and effective targeted therapies for asthma.