The barrier function of the airway epithelium is central to the prevention of infection; in patients with chronic respiratory disease the integrity of this barrier is compromised leading to an increase in nutrient leakage into the airway surface liquid (ASL). Not only does this provide a nutrient source for bacterial growth but it is also likely to cause a change in the metabolism of the epithelial cells and resident immune cells thereby affecting their ability to control the infection. Healthy human ASL only contains around 0.4 mM glucose – this increases in patients with respiratory diseases such as COPD and cystic fibrosis, with the greatest increase in patients with both respiratory disease and diabetes (2-6 mM). The increase in ASL glucose seen in hyperglycaemia is associated with increased risk of respiratory S.aureus and P.aeruginosa infections and poorer outcomes for the patients. Cystic fibrosis affects more than 10,400 people in the UK with approximately half of adult patients developing hyperglycaemia (CF-related diabetes) and the associated increased risk of pulmonary infection. We propose that regulating the glucose concertation of ASL may provide an antibiotic free therapy for respiratory infections in CF and CFRD. However it is vital to fully investigate the consequences of modulating glucose in terms of infection as glucose is required for the function of phagocytic cells located within the lungs. Consequently the key aim of this project is to develop a disease relevant in vitro co-culture model of airway infections including epithelial cells, immune cells and bacteria to investigate the effect of controlling glucose levels on infection severity. In order to develop this complex in vitro model we need to first determine the metabolic requirements of phagocytic cells. Initial results suggest that neutrophils show no loss of their ability to kill live bacteria across a wide range of glucose concentrations (0-15 mM). Monocyte derived dendritic cell (moDC) maturation and antigen uptake are unaffected by acute changes to glucose concentration. Further work is ongoing to look at moDC cytokine secretion and capacity to stimulate T cells. To generate epithelium for co-culture models human tissue samples were obtained from routine nasal surgeries. Basal epithelial cells isolated from this tissue were grown on Transwell supports at air-liquid-interface (ALI) into fully differentiated airway epithelia. These cell cultures mimic much of the function and complexity of human airways. Preliminary data shows that elevated glucose promotes S.aureus and P.aeruginosa growth in ALI epithelial-bacterial co-cultures and increases their binding to airway epithelial monolayers. The addition of neutrophils to the co-culture reduced apical P.aeruginosa growth under all glucose concentrations, consistent with a limited influence of glucose on neutrophil phagocytic capacity. Next we need to determine optimum conditions for the addition of multiple phagocytic cells (neutrophils, moDCs and monocyte-derived macrophages) in addition to bacteria to the ALI cultures to generate a physiological human in vitro airway infection model to further investigate the influence of glucose, hyperglycaemia and CF on bacterial killing/growth. Development of this co-culture model will be of huge value for modelling human respiratory disease without the need for animal models in accordance with the NC3Rs.