Investigating the role of insulin in the presence of allergic pulmonary inflammation in diabetic and healthy mice

Abstract

Asthma is a complex disease that simultaneously involves the participation of several cells, cytokines and inflammatory mediators on which the initial phase is completely different from the late stage. In previous work, we used an experimental omdel developen in mice pre-immunized with ovalbumin (OVA) and found that insulin interferes with the production/release of cytokines, cell migration, collagen deposition and mucus secretion in pulmonary remodeling of a murine asthma model. The asthma model proposed in this project uses a booster dose of antigen twelve days after the first immunization and following, the animals are subjected to seven subsequently antigenic challenges. This model is characterized by a specific pattern of inflammation in the airway mucosa, involving eosinophils with a predominantly lymphocytes subpopulation of T helper (Th)2. Furthermore, structural changes occurring in the airways (remodeling) are observed, including increased collagen deposition around the bronchial sub-epithelial fibrosis, hyperplasia and hypertrophy of smooth muscle of the airways, angiogenesis, and increased mucus secretion. With this additional grant, we aim to assess the modulatory role of insulin in pulmonary inflammation. The study performed in healthy mice will be transferred to diabetic mice and molecular basis of inflammation will be targeted. The study will include: a) glucose levels; b) study of airway responsiveness after bronchial provocation by methylcholine (buxco), and assessment of respiratory function (flexiVent) c) study of cells present in bronchoalveolar lavage (BAL), as well as analysis of cell subpopulations by flow cytometry in samples from the bone marrow, lung, thymus and spleen; d) determination of serum levels of: insulin, corticosterone and immunoglobulins (IgE, IgG1 and IgG2a) by ELISA; e) cytokines concentration in BAL supernatant samples by ELISA (IL-4, IL-5, IL-10, IL-13, TNF-± and IFN-³); f) expression of the molecules involved in cell signalling pathways (p38 MAPK, collagen I, Smad 2 and Smad 3) in the lungs and bone marrow by Western blot g) alterations in the m-RNA expression by RT-PCR in the intracellular signaling pathways during the course of allergic inflammation. (AU)