טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
M.Sc Thesis
M.Sc StudentSigal Ram
SubjectThe Role of Interferon Regulatory Factors 8 (IRF-8) in
Regulating Innate Resistance to Intraphagosomal
Pathogens
DepartmentDepartment of Biotechnology and Food Engineering
Supervisor Full Professor Levi Ben-Zion
Full Thesis textFull thesis text - English Version


Abstract

Macrophages constitute the first line of defense of the innate immune response against pathogens. Recognition of the pathogen by these cells leads to phagocytosis and the subsequent elimination of the invaders. Among the transcription factors that participate in coordinated macrophage activities against pathogens, is also the Interferon Regulatory Factor-8 (IRF-8). IRF-8 is mainly expressed in hematopoietic cells and its expression is activated by IFN-γ that can be further induced by Toll Like Receptor  ligands. IRF-8 is essential for proper functioning of mature macrophages by playing a key role in the regulation of phagosomal components such as natural resistance-associated protein 1 (Nramp1). Nramp1 is a proton/bivalent cation antiporter, located in late endosomes/lysosomes and is exclusively expressed in monocyte/macrophage cells. Studies from our laboratory demonstrated that the killing efficiency of IRF-8 knockout mouse strain resembled to that of an Nramp1 defective strain and that of a double knockout strain. Therefore, the first aim of this study was to demonstrate a direct genetic link between IRF-8 expression and Nramp1 regulation. For this purpose, bone marrow macrophages derived from IRF-8-/-/Nramp1+/+ mice were complemented by a retroviral vector allowing IRF-8 expression. This rescued expression of IRF-8 led to the recovery of the killing ability of these cells clearly establishing a direct genetic link between IRF-8 expression, Nramp1 regulation and elimination of intraphagosomal pathogens such as Salmonella. The second aim of this study was to generate a Bacterial Artificial Chromosome (BAC) reporter construct for IRF-8 that will allow real time monitoring of IRF-8 expression during macrophage activation. For that purpose, we have swapped the whole coding region of IRF-8 with reporter genes and generated stable IRF-8-BAC reporter clones in RAW264.7 macrophage cell line. Our results showed that this BAC reporter responded only to IFN-γ but not to a combination with TLR ligands or pathogens infection. Furthermore, this BAC reporter exhibited the same expression profile of IRF-8 in NIH3T3 (fibroblast) stable clones and in RAW264.7 stable clones. These results indicate that this BAC reporter does not authentically report on IRF-8 activity due to lack of cell type specific expression element that was lost during the replacement of IRF-8 coding region with the reporter gene. This suggests that the exclusive expression of IRF-8 in hematopoietic cells is probably confined to intronic regions. This is highly supported by bioinformatics and initial results with a new BAC construct in which the whole coding region of IRF-8 was left intact.