טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
Ph.D Thesis
Ph.D StudentAlter-Koltunoff Michal
SubjectThe Role of IRF-8 in Innate Resistance to Intraphagosomal
Pathogens through the Regulation of Nramp1
DepartmentDepartment of Biotechnology and Food Engineering
Supervisor Professor Ben-Zion Levi
Full Thesis textFull thesis text - English Version


Abstract

Macrophages are central arm of innate immune defense against most intracellular pathogens. They internalize microbes into phagosomes where the invaders are subjected to a lethal hit mediated by oxygen and nitrogen reactive species. Amazingly, despite this battery of antimicrobial molecules, some pathogens are able to survive and thrive within macrophages without escaping the phagosomes. These are termed intraphagosomal pathogens among which are Salmonella, Leishmania and Mycobacteria. In mice, a single dominant gene termed Natural Resistance-Associated Macrophage Protein 1 (Nramp1) controls innate resistance to such pathogens. This protein is a proton/divalent cation antiporter exclusively expressed in monocyte/macrophage cells. In humans it is linked to several infectious diseases as well as to autoimmune diseases. We show that the restricted expression of Nramp1 is regulated by a myeloid cell specific transcription factor termed Interferon Regulatory Factor-8 (IRF-8). This factor exerts its activity via protein-protein interaction, which facilitates its binding to target DNA. Using yeast two-hybrid screen we identified Myc Interacting Zinc finger protein 1 (Miz-1) as new interacting partner. This interaction is restricted to immune cells and takes place on the promoter Nramp1 in association with PU.1, a transcription factor essential for myelopoiesis. Consistent with these data, IRF-8 knockout mice are sensitive to a repertoire of intracellular pathogens and express low levels of Nramp1 that can not be induced any further. Thus, our results explain in molecular terms the role of IRF-8 in conferring innate resistance to intracellular pathogens and point to its possible involvement in autoimmune diseases. Induction of Nramp1 expression in activated macrophages is accompanied by a promoter shift from a repression state elicited by c-Myc to an activation state elicited by the induction of IRF-8 in activated macrophages. This transition from repression to activation is facilitated by a competitive protein-protein interaction with the transcription factor Miz-1. Thus, IRF-8 antagonized c-Myc repression of Miz-1. To show that IRF-8 is directly involved in the elimination of intraphagosomal pathogens through the regulation of Nramp1 gene expression, we bred wild type as well as IRF-8 and Nramp1 null mouse strains and examined macrophages derived from bone marrow and peritoneum. Our results clearly show that the absence of IRF-8 and Nramp1 leads to the same phenotype; defective killing of intraphagosomal Salmonella enterica serovar Typhimurium and Mycobacterium Bovis (bacille Calmette-Guerin (BCG). Thus, interplay between repression and activation state of Nramp1 promoter mediated by IRF-8 provides the molecular basis by which macrophages resist intraphagosomal pathogens at early stage after infection.