Date of Award

12-24-2008

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Biology

First Advisor

Min Wu

Abstract

Pseudomonas aeruginosa (PA) is a common opportunistic pathogen that invades the susceptible host. Alveolar macrophages (AM) play a pivotal role in defending against invading pathogens in the lungs. The effectiveness of AM in combating PA infection has not been studied in detail. The goal was to identify the key components involved in mounting an effective innate immune response using the murine lung infection as a model to understand the host - pathogen interactions. Our most important finding was the involvement of Lyn tyrosine kinase, a member of Src family tyrosine kinases, in orchestrating the host cell response to PA invasion. Preliminary findings showed that Lyn is constitutively expressed in an epithelial cell type (A549, lung carcinoma derived) and associated with PA infection. PA infection of AEC II cells activates Lyn through innate immune receptor clustering leading to aggregation of membrane lipid raft microdomains. Similarly in macrophage cell type raft mediated Lyn activation plays a crucial role in regulating phagocytosis and respiratory burst activity. Depletion of cholesterol was found to affect raft function and Lyn inhibition by multiple approaches significantly reduced the phagocytosis of PA. The hypothesis proposes that AEC II and AM communicate through secreted factors and raft mediated Lyn activation may play a role in regulating the cell-cell communication. Among the cytokines released by AEC II cells, monocyte chemoattractant protein (MCP-1) plays a major role in recruiting AM to the site of infection. Depleting cholesterol by mevastatin (cholesterol synthesis inhibitor) treatment in macrophages inhibits the raft associated functions and down regulates Lyn activity. Direct Lyn inhibition using siRNA mediated gene silencing had very similar effects and significantly decreased PA phagocytosis. In conclusion, (1) PA infection of AEC II cells induces Lyn kinase activity by lipid raft mediated mechanism; (2) Infected AEC II cells secrete inflammatory cytokines and predominantly MCP-1 that recruits and activates monocytes like alveolar macrophages; (3) Effective phagocytosis of PA by activated AM depends on Lyn function and its downstream signaling mediators. The identification of a novel lipid raft based signaling mechanism that regulates host innate immunity has been demonstrated and holds promise for future therapeutic intervention to combat PA infection.

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