UBC Theses and Dissertations
The role of SHIP1 in IL-10 signalling pathway in primary macrophages So, Yee Wah
The inflammatory response is an important physiological mechanism for hosts’ defense against pathogens and post-assault tissue repair. However, excessive inflammation leads to tissue damage and pathology. Therefore, inflammation is tightly regulated by anti-inflammatory factors such as interleukin-10 (IL-10) in order to maintain homeostasis. IL-10 inhibits macrophages’ activation by suppressing macrophages’ antigen presenting ability and production of pro-inflammatory cytokines such as tumor necrosis factor α (TNFα). The signal transducer and activator of transcription 3 (STAT3) pathway has been regarded as the only downstream pathway of IL-10 for decades. However, our lab has previously shown that IL-10’s early anti-inflammatory action uses the lipid phosphatase SH2 domain containing inositol 5´ phosphatase (SHIP1) but not STAT3. Previous results in our lab suggested that IL-10 activates SHIP1 to inhibit the phosphoinositide 3-kinase (PI3K) pathway; and this accounts for IL-10’s early anti-inflammatory effects. Since the previous results were mostly obtained using cell lines, we sought to verify the results in the more physiological relevant mouse primary cells. We first investigated whether IL-10 activates SHIP1 by assessing the physical interaction of IL-10 receptor (IL-10R) and SHIP1, the localization of SHIP1, and the phosphorylation state of SHIP1 upon IL-10 stimulation. We could not observe any effect of IL-10 on altering the activation state of SHIP1. We next investigated IL-10’s effect on the activation of Akt, a downstream molecule of PI3K, in lipopolysaccharide (LPS) activated macrophages. We demonstrated that IL-10 inhibited phosphorylation of Akt in macrophages from C57BL/6 mice but not macrophages from Balb/C mice. Lastly, we investigated the roles of SHIP1 and STAT3 in IL-10’s inhibition of TNFα protein. We found that the TNFα production profile in SHIP1-/- and STAT3-/- cells were extremely similar. Closer examination showed that SHIP1messenger RNA (mRNA) expression was significantly reduced in STAT3 knock out (-/-) macrophages. Although this work failed to demonstrate some of the observations obtained in cell lines, it shows the significance of genetic background of the cells used in experiments. It also suggests that STAT3-/- macrophages’ unresponsiveness to IL-10 may due to the lower SHIP1 level in these cells, indicating a potentially important role of SHIP1 in IL-10’s anti-inflammatory properties.
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