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A4700 - The Defenders of the Alveolus: A Study of ATII Simple Cell and Complex Cultures Using Ex Vivo Precision Cut Lung Slices from Human and Mouse
Author Block: D. Yan1, C. Mwase2, Y. Rai3, S. Daya2, K. Feldman2, T. Chen2, M. Dowling2, C. Bauer2; 1Physiology and Pharmacology, University of Calgary, Calgary, AB, Canada, 2Respiratory Disease, Novartis Institutes for BioMedical Research INC, Cambridge, MA, United States, 3School of Medicine, University of Tokyo, Tokyo, Japan.
INTRODUCTION: Chronic obstructive pulmonary disease (COPD) is projected to be the third leading cause of death worldwide with a large and increasing unmet medical need for novel modalities of treatment. Individuals with COPD are susceptible to acute worsening of symptoms, often accompanied by positive cultures for gram-negative bacterial species in the lower airways. Alveolar epithelial type II (ATII) cells are surfactant producing progenitor cells responsible for epithelial repair. In this study, using a short culturing condition of isolated ATII cells and a complex tissue slice model, response to LPS stimulation was explored. Understanding the contribution of ATII cells during innate host defense could provide novel therapeutic targets for the treatment of COPD exacerbation. METHODS AND RESULTS: Mouse and human ATII cells are isolated and cultured on 2D transwell plates for 24 hours prior to stimulation with LPS. Increased concentrations of surfactant protein A (SPA) were detected in culture media in a time dependent manner from mouse and human cultures. In addition, inflammatory cytokines, IL-6 and IL-8, were detected following LPS stimulation. Interestingly, LPS-induced SPA secretion is inhibited in COPD-derived ATII cells while IL-8 secretion is enhanced. To translate ATII cell in vitro observations, Precision Cut Lung Slices (PCLS) are generated from BALB/c mice following agarose inflation. Slices can be cultured for up to 7 days with minimal loss of viability. Following total RNA isolation from lung slices, TaqMan PCR for SFTPC, FOXA3, MUC5B, ABCA3, AQP5 and other genetic markers indicate retention of variable known cell types. Using live cell confocal imaging, ATP + PMA-dependent laminar body release from ATII cells are visualized. These studies indicate that PCLS can be used to study functional responses of ATII cells in a complex culture setting. SUMMARY: ATII cells serve as defenders of the alveolus through their ability to secrete immune mediators in response to infectious insults. These data suggest a reduced host defense mechanism in COPD patients which may contribute to acute bacterial exacerbations. By exploring the mechanisms of altered ATII cell function in COPD, using novel techniques such as PCLS, we may identify therapeutic targets for the prevention and treatment of infection-induced exacerbations.