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Context. Asthma is characterized by chronic symptoms and airway obstruction. Structural changes of the bronchial wall, defined as airway remodelling, contribute to the thickening of the airway wall and to irreversible airflow obstruction (IAO) in a subpopulation of severe asthmatics. These changes are characterized by an abnormal repair of the injured epithelium and by sub-epithelial fibrosis, with increased fibroblast and myofibroblast numbers. Hypothesis. An abnormal epithelial repair in response to injuries by environmental factors such as allergens, eosinophil-derived mediators [cystenyl-leukotrienes (CysLTs)] and by cytotoxic granule proteins, as well as a gradual acquisition of a mesenchymal phenotype by airway epithelial cells (epithelial-mesenchymal transition, EMT), contribute to tissue fibrosis in asthma. Project. 1) Identify specific epithelial abnormalities in relation to sub-epithelial fibrosis in severe asthmatics. Expression of remodelling factors (fibrogenic cytokines, chemokines, growth factors, metalloproteases, extracellular matrix proteins, enzymes involved in the synthesis of CysLTs and their receptors) and of established hallmarks of EMT (junctional molecules, vimentin, -actin and collagen type I) will be assessed by real-time quantitative PCR in laser microdissected bronchial epithelium from endobronchial biopsy samples from controls (n = 10), mild (n = 10) and severe steroid-treated asthmatics without (n = 10) or with (n = 10) IAO. Proteins encoded by selected genes will be detected by immuno-histochemistry. The expression of these markers will be correlated with the number of eosinophils and of fibroblasts/myofibroblasts in the bronchial submucosa. 2) Characterize the contribution of the airway epithelium to subepithelial fibrosis in vitro. Human primary airway epithelial cells will be exposed in vitro to the major antigen of Dermatophagoides ptenonyssimus, the CysLT, LTD4 and the eosinophil-derived cytotoxic proteins. Cells will be subsequently analyzed for the synthesis and release of remodelling factors (see above), and for changes in the expression of markers of EMT. The role of the TGF- signalling molecules, Smads and Snail, as well as of the MAPK pathway, will be investigated in cells undergoing EMT using siRNA-based strategies and pharmacologic inhibitors. 3) Determine the ability of selected stimuli to promote tissue fibrosis in vivo. Eosinophil-derived cationic proteins will be instilled intratracheally to normal mice and features of lung fibrosis will be assessed. In parallel, the expression of EMT markers will be investigated in a model of ovalbumin-immunized and challenged mice. The capability of bone morphogenic protein (BMP)-7, a molecule known to inhibit EMT in vitro, to prevent and reverse lung fibrosis will be assessed. An inducible transgenic system driven by the CC10 promoter and inducing Smads or Snail over-expression in the airway epithelium of naive mice will be also used. The ability of these mice to undergo spontaneously pulmonary fibrosis or to respond with exaggerated fibrogenesis to repeated antigen challenges will be investigated. Conclusion. This project will shed light on the mechanisms underlying sub-epithelial fibrosis, an important feature of airway remodelling in steroid-refractory asthma, lead to the identification of new targets and may help to develop more effective drugs