Endotoxin tolerance in an in vitro model of macrophages co-cultured with epithelial cells

Lung macrophages are the sentinel cells of the respiratory system and have been implicated in a number of chronic inflammatory respiratory disorders. The inflammatory and functional responses of macrophages are governed by signals from their micro-environment. Despite this, they are often studied in isolation in vitro. Recent clinical studies have suggested that respiratory macrophages have impaired functions such as phagocytosis in chronic obstructive pulmonary disease (COPD), and it is hypothesised that endotoxin tolerance (ET) may be involved in the dysfunction of these macrophages. To investigate this, an in vitro system of macrophages co-cultured with respiratory epithelial cells was developed to better represent the normal physiology of respiratory innate immunity. This co-culture system was used to investigate the influence of epithelial cells on complex macrophage biology such as inflammatory responses, induction of ET, and phagocytosis. The epithelial cells were able to regulate the inflammatory responses from single and repeated LPS challenges, and also increase phagocytosis by contributing to the uptake of bacteria and by activating otherwise un-responsive macrophages. In addition, this co-culture system was used to develop and investigate complex protocols of ET induction. It was demonstrated that ET is induced via a “Switch” mechanism that requires the presence of LPS to induce ET but not extended exposure. Furthermore, it was found that the ET inducing stimulus does not need to be removed in order to demonstrate tolerance. In addition, ET suppressed phagocytosis in these models, contrary to what is observed in the literature. After the development of ET models, primary COPD disease state material was used to investigate the implications of ET in COPD. It was demonstrated that gram negative respiratory bacteria can induce ET, and that the ET profiles produced by COPD cells vary compared to healthy controls. These results suggest that the induction of ET in COPD patients may occur too readily, resulting in insufficient clearance of colonised bacteria. However, further investigation is required. In conclusion, this study highlights the importance of studying macrophages in a more physiologically representative system in vitro, and also identifies previously unidentified features of ET, including potential implications in COPD.