This application is a supplement to our recent proposal entitled """"""""lmmunopathogenesis of granulocytic Ehrlichiosis"""""""" (R01 A1041440), in which we proposed to understand the pathogenesis of HGE using both in vitro and in vivo models. Our recently published report demonstrates that A. phagocytophila inhibits the respiratory burst by selectively down-regulating gp91 phox, a major subunit of the NADPH oxidase holoenzyme. The proposed R21 supplement represents an opportunity to use innovative technologies to expand the scope of our current R01 and its pending renewal. We have established a collaboration with Dr. Nancy Berliner's laboratory that offers a unique opportunity to broaden our understanding of the changes in neutrophil biology induced by A. phagocytophila infection. Collaboration with her laboratory provides complementary expertise to our own, since her work has focused on the transcriptional regulatory events governing normal myeloid stem cell maturation. Dr. Nancy Berliner and her colleagues offer expertise in gene expression profiling, chromatin immunoprecipitation, and structure-function analysis of transcription factors that will apply to the proposed studies. In these supplemental studies we will assess and characterize the changes in global gene expression associated with A. phagocytophila infection using a myeloid-specific microarray recently described by Dr Berliner and her colleagues. We will also use chromatin immunoprecipitation (CHIP) and structure-function analyses to further explore the role of CCAAT displacement protein (CDP) in the context of its role in the neutrophil differentiation program and how it is altered by A. phagocytophila infection. As outlined in our R01 proposal, we hypothesize that the repression of gp91 phox by A. phagocytophila is mediated by CDP, a transcriptional repressor of many granulocytic genes. Dr. Berliner and her colleagues have characterized the role of aberrant CDP binding in repressing late neutrophil-specific gene expression in leukemia cell models, and are ideally poised to perform similar studies in concert with our experiments. The proposed studies in combination with those outlined in RO1 A1041440 promise to lead to a greater understanding of HGE and the molecular mechanisms that A. phagocytophila uses to interfere with neutrophil differentiation and function
