Effect of corticosteroids and long-acting ß2- agonists in a human cell culture based "in vitro" model of airway inflammation and tissue remodeling

Abstract

Asthma and chronic obstructive pulmonary disease (COPD) are characterized by chronic airway inflammation and remodeling. In non-pathological conditions tissue homeostasis is maintained by the balance between extracellular matrix (ECM) synthesis and degradation. However, this equilibrium is altered in asthma and COPD leading to structural changes such as increased ECM deposition. Fibroblasts and airway smooth muscle cells (ASMC) are the main source of collagen and glycosaminoglycans (GAGs) and transforming growth factor-b1 (TGF-b1) is the most potent ECM stimulator in vitro. In turn, the ECM influences cell proliferation, migration, differentiation, and secretion of cytokines and growth factors. Thus, ECM deposition may perpetuate the inflammatory and remodeling processes, contributing to progression and severity of diseases. Inhaled corticosteroids and long acting b2-agonists (LABA) are the current therapy for asthma and COPD but their effect on airway remodeling is not clear. Therefore, the purpose of this thesis was to investigate the effect of corticosteroids and LABA on airway inflammation and tissue remodeling using a human cell based in vitro model. In the present thesis, primary human lung fibroblasts and ASMC were cultured under serumfree condition to reflect a non-inflammatory environment or with 5% fetal calf serum (FCS) and/or TGF-b1 to mimic inflammation. This experimental design allowed us to assess the effect of corticosteroids and LABA on total ECM, collagen and GAGs deposition, cell proliferation, cytokines release, collagen and ECM mediators mRNA expression, and gelatinolytic activity under both non-inflammatory and inflammatory conditions. Concerning the effect of corticosteroids and LABA on ECM deposition by fibroblasts, we showed that 5% FCS and TGF-b1 increased total ECM and collagen deposition. Under serumfree condition corticosteroids reduced ECM deposition and the effect was partly mediated by the glucocorticoid receptor and collagen de novo synthesis. Interestingly, in the presence of 5% FCS corticosteroids had the opposite effect. LABA reduced total ECM and collagen deposition under both conditions. Combined drugs further decreased ECM deposition under serum-free condition whereas they counteracted each other in 5% FCS, independently of TGF-b1. Furthermore, we showed that 5% FCS and TGF-b1 increased GAGs secretion and deposition. Under serum-free condition corticosteroids inhibited GAGs secretion and deposition whereas LABA alone had no effect but partly reversed the effect of corticosteroids. On the contrary, in 5% FCS corticosteroids and LABA increased GAGs deposition. Upon TGF-b1 stimulation, similar effects were obtained except in 5% FCS, where both drugs decreased the TGF-b1-induced GAGs secretion and deposition. Together, these findings indicate that the effect of corticosteroids but not of LABA, on ECM deposition by fibroblasts is altered by ongoing inflammation. The effects of corticosteroids and LABA on GAGs, matrix metalloproteinases (MMPs) and total ECM/collagen deposition by ASMC from healthy, asthma and COPD patients were also investigated. First, no difference in GAGs secretion and deposition was observed between ASMC from healthy, asthma and COPD patients. Under serum-free condition corticosteroids inhibited GAGs secretion and deposition while LABA had no clear effect nor modulated the effect of corticosteroids. In 5% FCS corticosteroids decreased GAGs secretion to a greater extent than LABA and their combination resulted in the same effect as corticosteroids alone. None of the drugs had any effect on GAGs deposition. Second, we assessed the effect of the drugs on ASMC-derived MMPs. In ASMC only proMMP-2 gelatinolytic activity was detected, which was decreased in asthma and COPD patients compared to healthy controls. However, corticosteroids and/or LABA had no effect on proMMP-2 activity. Lastly, we demonstrated that corticosteroids and LABA generated similar effects on total ECM and collagen deposition by ASMC from healthy subjects as observed with fibroblasts. In conclusion, our novel findings suggest that the action of corticosteroids and LABA on ECM deposition differs under non-inflammatory and inflammatory conditions in lung fibroblasts and ASMC. In the presence of acute inflammation with vessel leakage, increased ECM deposition may be regarded as an attempt of stromal cells to block further serum and cells infiltration into the tissue and this effect would be supported by corticosteroids. The addition of LABA would counteract the corticosteroid-induced ECM deposition and therefore their combination with corticosteroids may depend on the inflammatory status of the patient. Thus, when airway inflammation is resolved, combination therapy may beneficially reduce pathological tissue remodeling. Thus, our findings might have implications for the short- and long-term treatment strategies in regard to airway remodeling in asthma and COPD.