nd degraded within highly specialized double membrane bound vesicles termed autophagosomes. Over the past decade autophagy has emerged as a central component of the innate and adaptive 2199952 immune responses where it plays roles in antigen presentation Autophagy and P. aeruginosa Infection including cross-presentation, direct and indirect killing of intracellular and extracellular pathogens, generation of bactericidal peptides and the regulation of inflammatory responses. Autophagy has been implicated in P. aeruginosa infection in cultured macrophages in vitro. However, the biological significance of autophagy in P. aeruginosa infection in vivo and its role in mast cell-P. aeruginosa interaction remain undefined. One of the greatest challenges in the treatment of P. aeruginosa infection is the highly antibiotic resistant nature of the bacteria. The recent emergence of multi-drug resistant P. aeruginosa strains leading to increased morbidity and mortality in susceptible populations highlights the need for novel therapeutic strategies for the treatment of P. aeruginosa infections. Recently it has been proposed that P. aeruginosa bacteria have the ability to reside within host cells where they can evade host immune cells, and that the development of intracellular infections may represent a mechanism contributing to antibiotic resistance. Given the well Luteolin 7-O-β-D-glucoside characterized central role of autophagy in the clearance of intracellular pathogens, and the observation that autophagy is impaired in the airways of cystic fibrosis patients, we set out to examine the role of autophagy in host defense against P. aeruginosa in vivo, and explored the therapeutic potential of pharmacological manipulation of the autophagy pathway during P. aeruginosa lung infection. Our results demonstrate that P. aeruginosa infection induces autophagy in mast cells which are abundant in the airways where they play a central role in host defense against P. aeruginosa, as well as in bronchial epithelial cells which have been proposed to act as a reservoir of intracellular bacteria during chronic P. aeruginosa infection. We further demonstrated that inhibition of the autophagy pathway significantly impairs clearance of P. aeruginosa from mast cells and human bronchial epithelial cells, while induction of the process enhances bacterial killing. Finally we demonstrate that pharmacological manipulation of the pathway effectively regulates bacterial clearance in vivo. Thus, induction of autophagy could represent a novel therapeutic approach for the treatment of P. aeruginosa infection. 16104 16HBE14o2 or CFBE41o2 cells were left untreated or pretreated for 1 hour with 20 mM chloroquine diphosphate salt or 2 mM rapamycin. Alternatively, untreated HMC-1 5C6 cells stably expressing Atg5 or Atg7 shRNA were used. Cells were infected in 100 mL of serum free IMDM media with P. aeruginosa strain 8821 at a 1:20 MOI for 3 hours. Extracellular bacteria were then killed with cell impermeable antibiotics, 200 mg/mL gentamycin, 100 mg/mL ceftazidime hydrate and 8402633 100 mg/mL piperacillin sodium salt ) for 10 minutes or 3 hours. Cells were then washed in PBS, and lysed in 100 mL PBS containing 0.2% Triton X-100 and a serial dilution of 10 mL of lysate was streaked in duplicate 
on LB agar plates, and incubated overnight at 37uC. CFUs were counted the next day. Samples containing no cells were used to identify background CFUs which were subtracted from samples at each time point. Bacterial preparation The labo