COTL-1 CONTRIBUTES TO EPITHELIAL BARRIER DYSFUNCTION UPON SMOKE AND LYPOLISACCHARIDE EXPOSURE D'Anna Claudia1*, Cigna Diego1*, Di Sano Caterina1, Di Vincenzo Serena1, Dino Paola1, Ferraro Maria1, Bini Luca2, Bianchi Laura2, Gjomarkaj Mark1, Pace Elisabetta1. 1Institute of Biomedicine and Molecular Immunology (IBIM), CNR, Palermo, Italy. 2Laboratory of Functional Proteomics, Life Sciences Department, Università degli Studi di Siena, Siena, Italy

The respiratory epithelium forms a barrier essential for the preservation of the lung respiratory function, and its integrity is crucial for airway homeostasis. Cigarette smoking and recurrent infections cause persistent lung inflammation by inducing structural changes. The underlying mechanisms are not fully understood. The goal of this study was to detect mechanisms involved in barrier dysfunction using a model a bronchial epithelial cell line (16-HBE) stimulated with cigarette smoke extract (CSE) and lipopolysaccharide (LPS), alone and/or in combination, by using two-dimensional electrophoresis (2DE) analysis coupled with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Flow cytometry and immunofluorescence were used to assess F actin polimerization by phalloidin method. The epithelial permeability was examined by measuring fluorescein disulfonic acid flux across a cell monolayer. Fourteen proteins, with significant changes in intensity, were identified in the various experimental points: 6 were up-regulated and 8 were down-regulated. Bioinformatic analysis revealed that most of these proteins are involved in anti-oxidant and immune responses and in cytoskeleton stability. With regard to cytoskeleton stability, we demonstrated for the first time that the co-exposure of CSE and LPS increased COTL-1 expression, an F-actin-binding protein actively involved in actin polymerization in living cells. F-actin levels were measured in cells by flow cytometry from the relatively narrow symmetrical peak revealed by FACS analysis. Both the flow cytometric and microscopic analysis showed that CSE 20%, alone and more in combination with LPS, was able to increase the levels of F-actin in 16HBE cells. Also the values of fluorescein, after 3 h of CSE stimulation, showed a significant increase in epithelial permeability. These results suggest that COTL-1 dysregulation can be regarded as a new mechanism in epithelial barrier dysfunction upon smoke and lypolisaccharide. Barrier dysfunction can contribute to an increased risk of infection and to a progressive lung function deterioration.