טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
M.Sc Thesis
M.Sc StudentKhalfin Yana
SubjectThe Role of PML in the Differentiation and Function of Bone
Marrow Macrophages
DepartmentDepartment of Biotechnology and Food Engineering
Supervisor Professor Ben-Zion Levi
Full Thesis textFull thesis text - English Version


Abstract

Macrophages belong to the myeloid cells family arising from multipotential hematopoietic stem cells (HSCs). The formation of myeloid cells is orchestrated by a relatively small number of transcription factors, among them are Interferon Regulatory Factor-8 (IRF-8). IRF-8 drives monopoiesis of bipotential progenitor cells, while inhibiting at the same time granulopoiesis. Recently, we have identified target genes regulated by IRF-8 in mature activated macrophages, among them was the Promyelocytic Leukemia (PML) gene. PML is a transcriptional regulator localized in sub-nuclear structures called PML-Nuclear Bodies (NBs), which have been recently implicated in a variety of cellular processes, in particular myeloid cell differentiation. These data led us to the hypothesis that the pro-differentiating activities of IRF-8 may be delegated through the regulation of its downstream target gene, PML. Thus, our goal in this study was to gain better insights into the involvement of PML in monopoiesis and to study the role of PML in the function of mature macrophages. For this purpose, PML expression was silenced in bone marrow cells undergoing differentiation toward macrophages, by RNA interference method. To study the effect of PML on macrophage differentiation and function we have analyzed various characteristics of macrophages. Studying the myeloid-specific genes expression profile revealed aberrant expression of granulocyte and macrophage specific genes, such as SR-A, c-fms, MPO and G-CSFR; as well as alteration in the expression level of myeloid-specific transcription factors, like PU.1, IRF-8, Egr-1. Moreover, the expression profile of PML silenced BMM was similar to the profile of the granulocyte monocyte biopotential progenitors (GMPs), implying an arrest in BMM differentiation. Examining the colony forming potential in methylcellulose of PML silenced myeloid precursors indicated a significant reduction in both the number of the generated colonies and their average size. Thus, PML silencing disrupts myeloid precursors proliferative potential. Next, we studied the effect of PML silencing on essential functions of activated macrophages. PML silenced BMM exhibited reduced phagocytosis efficiency, reduced secretion of nitric oxide and impaired mRNA expression of major inflammatory cytokines, like IL-18, IL-6 and IL-1β.

Together, the great significance that emerges from our results points to the important role of PML in myeloid cells differentiation toward macrophages, as well as in the proper function of activated macrophages, clearly strengthening the known data implying the involvement of PML in myelopoiesis. PML knockdown BMM exhibits defects in central features of the innate immune response and possibly other important biological processes mediated by macrophages are impaired.