Thioredoxin in Neutrophil-Airway Epithelial Interactions

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DESCRIPTION (provided by applicant): The primary objective of this research is to determine the importance of thioredoxin in neutrophil-airway epithelial cell interactions after oxidant injury. Neutrophils play a beneficial role by removing oxidant-injured airway epithelial cells and enhancing subsequent epithelial repair after ambient ozone exposure. The central hypothesis is that neutrophil-dependent removal of oxidant-injured airway epithelial cells requires thioredoxin production by the injured cells that then enhances neutrophil co-localization, removal of injured cells and subsequent epithelial repair. The objectives of this proposal will be addressed in four parts. First, the investigators will determine the molecular interaction between thioredoxin and the cell membrane of oxidant-injured airway epithelial cells. Secondly, they will investigate the importance of thioredoxin in neutrophil co-localization to airway epithelial cells, their removal after injury and enhancing subsequent epithelial repair. Immortalized human bronchial epithelial cells will be stably transfected with a thioredoxin expression construct and the investigators will quantify neutrophil co-localization to these cells, neutrophil-dependent removal of oxidant injured cells and epithelial repair using BrdU. To assess the importance of thioredoxin structure in these functions, the investigators will repeat the experiments using monolayers transfected with thioredoxin expression constructs mutated in key cysteine amino acids. Third, they will show thioredoxin is chemotactic for mouse neutrophils. This will lead to the fourth aim of developing a transgenic mouse over-expressing wild type and mutant thioredoxin in airway epithelium and demonstrating the role of thioredoxin in neutrophil emigration and removal of injured cells in vivo. The primary candidate's long-term research goals are to investigate mechanisms of pulmonary inflammation and repair with special emphasis on the interaction between neutrophils and airway epithelial cells. She hopes to use a career research award to gain additional knowledge and training in molecular biology while transitioning to an independent researcher who will be competitive for R01 funding.