טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
M.Sc Thesis
M.Sc StudentBar-Yosef Hagit
SubjectFlagellin Activation of Tool Like Receptor 5 (TLR5) Directs
Human Monocytes into Distinct Differentiation
towards Dendritic Cells
DepartmentDepartment of Medicine
Supervisor Professor Shimon Pollack
Full Thesis textFull thesis text - English Version


Abstract

Toll-Like Receptors (TLRs) have a crucial role in early host defense against invading pathogens. Some TLRs have also been implicated in the differentiation process of immune cells. Stimulation of TLR5 with its ligand- flagellin, activates a typical MyD88-dependent TLR signaling leading to the translocation of NF-κB to the nucleus. This ultimately leads to the secretion of inflammatory cytokines and/or chemokines.However, whether and how TLR5 plays a role in the differentiation of human monocytes has not been, as yet, clearly elucidated.Monocytes have the capacity to differentiate into macrophages or dendritic cells. This process is regulated both in vivo and in vitro by many immunological factors.  Thus, we aimed to analyze a possible regulatory effect of TLR5 activation on the differentiation process of human monocytes.We used an existing research model in which human monocytes are obtained from peripheral blood of healthy donors and incubated in RPMI medium, supplemented with 20% autologous serum. After 10-12 days non-stimulated human monocytes reach full differentiation into macrophages. We wanted to test whether the addition of flagellin to the culture medium would have an effect on monocyte differentiation. The differentiation of monocytes was analyzed by morphological criteria, by the expression of certain surface markers (CD4, CD64, CD123, CD86, CD304) and by the induced expression of certain cellular proteins (Rel-B). Secretion of cytokines to the culture medium was screened by cytokine array and quantitatively measured by ELISA. Signal transduction pathways in differentiated cells were analyzed by Western blotting.We have found that human monocytes incubated for 10 days with the TLR5 ligand flagellin show morphological characteristics and surface marker expression of dendritic cells. Where as non-stimulated monocytes show morphological characteristics and surface marker expression of macrophages. Flagellin stimulation leads to IRAK-1 phosphorylation in monocytes, indicating TLR5-mediated signal transduction. Increased Rel-b protein expression in flagellin-stimulated monocytes supported the notion of differentiation of monocytes towards dendritic cells.Flagellin stimulation of monocytes was correlated with increased secretion of pro-inflammatory cytokines IL-12, IL-1β and especially IL-6 whereas, in non-stimulated moncytes the secretion of these cytokines was significantly decreased.


In conclusion, we found that flagellin may induce the differentiation of human monocytes into dendritic cells through the activation of TLR5 pathway. The differentiation process was associated by altered cytokine secretion which, in turn, may contribute by itself in an autocrine and/or paracrine manner to the differentiation process towards dendritic cells.