The long-term goal of this application is to understand the molecular and cellular responses of corneal epithelial cells to Pseudomonas aeruginosa and develop chemotherapeutic and immunotherapeutic interventions for treatment of this serious eye infection. P. aeruginosa enters corneal epithelial cells via the cystic fibrosis transmembrane conductance regulator (CFTR), and these intracellular bacteria are able to persist in the tissue and avoid host defenses, leading to serious corneal pathology. Goals of aim 1 include exploring the role of the CFTR-P. aeruginosa interactions in corneal pathology as manifest by host chemokine and cytokine responses. Using isogenic human corneal epithelial cell lines expressing either mutant or wild-type (WT) CFTR, P. aeruginosa induced and CFTR-dependent release of cytokines implicated in pathogenesis and resistance to infection, notably IL-1, IL-6, IL-18 and IFN-gamma, will be examined. The role of the identified factors in eye infection and pathology will be tested in appropriate WT and transgenic mice using a corneal scratch-injured eye infection model or in mice in which the factor is neutralized with antibody. P. aeruginosa- CFTR interactions depend on formation of lipid rafts, and preliminary data indicate disruption of rafts prevents corneal pathology and promotes bacterial clearance from infected eyes.
In aim 2, the role of these rafts in pathogenesis will be further evaluated using the WT and CF corneal cell lines as well as mice unable to form lipid rafts due to disruption of the acid sphingomyelinase gene, and in mice treated with cyclodextrin, which disrupts lipid rafts. Cyclodextrin treatment of P. aeruginosa eye infections has the potential to be highly efficacious and become an important component of therapy. Finally, a fully human monoclonal antibody (MAb) has been developed to P. aeruginosa alginate a conserved, cell surface polysaccharide that is expressed at low levels by corneal isolates. Importantly, alginate expression is detected n the corneas of P. aeruginosa infected mice and the MAb is highly protective against P. aeruginosa infection.
In aim 3, the Mab will be evaluated for therapeutic efficacy against 6 different P. aeruginosa strains in the murine corneal infection model with the goal of having a single immunotherapeutic reagent useful against most clinical isolates as an adjunct to current treatment modalities for P. aeruginosa ulcerative keratitis. Overall at the conclusion of these aims, significant advances in the development of therapies for P. aeruginosa keratitis should become apparent based on use of cellular and molecular studies on host-pathogen interactions.
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