Pseudomonas aeruginosa can induce infections that lead to a rapid loss of visual function. The current therapies include antibiotic treatment which reduces the bacterial burden, but fails to control tissue damage that occurs as a result of acute inflammation. The long-term objective is to develop novel therapeutic approaches that harness inflammatory responses during an acute bacterial infection, while maintaining the bactericidal activities of the host. Despite the promising studies conducted during my current R21 funding period which showed that macrophage migration inhibitory factor (MIF) has an unappreciated importance in regulating innate immune responses to P. aeruginosa in the eye, today we know very little of the cellular sources of MIF, mechanisms of MIF release, and MIF-dependent pathways. Our experiments led to the hypothesis that P. aeruginosa-induced infection triggers rapid release of homotrimeric MIF. When the immune response fails to control the infection, the persistence of MIF trimers induces sustained inflammation via CD74-dependent pathways and should be targeted for therapy to reduce tissue-damaging consequences. We propose to pursue this hypothesis by:
Aim 1. Define the cellular types and mechanisms of MIF release during bacterial keratitis. Bone marrow chimeric mice will be infected with invasive or cytotoxic P. aeruginosa strains and the kinetics, magnitude, and oligomeric state of MIF release characterized. We will investigate the molecular mechanisms of MIF protein export by examining the role of the inflammasome assembly in this process.
Aim 2. Define the biological functions of MIF trimers in response to P. aeruginosa infection. MIF deficient mice will be reconstituted with purified rMIF trimers during infection, and disease development will be characterized. The studies will be complemented by analysis of the potency of corneal epithelial responses and PMN bactericidal activities during infection.
Aim 3. Define the contribution of CD74, a cell surface MIF receptor, in regulating sensitivity to P. aeruginosa infection. We will characterize the role of MIF-CD74 interaction in regulating epithelial responses to infection by monitoring the kinetics and distribution of MIF-CD74 complexes in vitro. We will further expand these studies to determine the significance of MIF-CD74 interaction in regulating P. aeruginosa-induced pathology. The expected impact of the proposed project is to open critical opportunities for intervention during an acute P. aeruginosa-induced corneal infection aimed at curbing pathogenic consequences of excessive inflammation, while preserving key bactericidal properties of the host. Regulating the activities of these novel therapeutic targets will result in "controlled" inflammatory responses during infection, limit tissue damage, and facilitate an efficient healing process.
Bacterial keratitis is a sight-threatening complication of contact lens wear and eye trauma and Pseudomonas aeruginosa is a commonly isolated pathogen that can damage the eye integrity during keratitis. We have identified an important molecule-macrophage migration inhibitory factor (MIF)-that is involved in the pathogenesis of P.aeruginosa-induced infection and established that inhibition of MIF is therapeutically beneficial during acute P. aeruginosa-induced keratitis. The project investigates the molecular pathways, cellular types, kinetics for MIF production, and the molecular mechanisms of MIF activities.
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