The long-term goal of this project is to perform a careful kinetic analysis of the immune response to Francisella tularensis and the resulting pathology. The underlying hypothesis is that the innate immune response and/or early adaptive immune responses to pneumonic tularemia caused by F. tularensis subsp. tularensis (Schu4) is delayed or different compared to less virulent subspecies or attenuated mutants. The rationale is that comparing immune responses between virulent and less virulent strains will provide new insights into protective responses concentrating on the less virulent strains known to modulate the immune response.
Three aims are proposed to compare Schu4 with targeted attenuated mutants. First, bacterial trafficking will be compared using labeled organisms followed by imaging in real time with the microPET and biodistribution of radioisotope and viable organisms. This will help determine whether differences in bacterial migration correlate with virulence as well as to identify the most important organs/tissues for study. Secondly, mice will be infected with Francisella strains/mutants (aerosol chamber) and sacrificed at various times post infection for removal of blood and relevant tissues defined in Aim 1. Initially, gene profiling will be done on isolated RNA from infected tissues by targeted macroarrays, thus focusing on immune related molecules important in the infection. Subsequently, in situ immunofluoresence approaches will be used, so that the pathogen, immune cells, and mediators can be identified at the same time. In addition, the extent of pathology will be assessed with time. Based upon our preliminary data, the third aim will concentrate on chemokines and the role of infiltrating neutrophils in disease progression. In collaboration with Project 2, we will also analyze TLR expression in situ. Information regarding virulence/protective mechanisms obtained in Projects 1-3 will interface with the 'humanized' transgenic model from Project 4. The data from this subproject, using real time imaging and in situ protocols, will provide a foundation for the disease processes associated with pneumonic tularemia. This project is therefore an integral part of the overall goal of this Program Project to understand the pathogenesis and host response to tularemia. (Cores C, B, and A required.)
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