Adaptor-Based Quatification of IL-1-induced Cell Activation

• Main Authors: Ko-Wei Liu, 劉克緯
• Other Authors: Nan-Shih Liao
• Format: Others
• Language: en_US
• Published: 2014
• Online Access: http://ndltd.ncl.edu.tw/handle/85641607930677877115

碩士 === 國立陽明大學 === 生命科學系暨基因體科學研究所 === 102 === Interleukin-1 (IL-1) is a proinflammatory cytokine secreted by immune cells that induces further immune responses against pathogen. IL-1 is also induced by endogenous danger signals and results in sterile autoinflammatory and metabolic diseases. Blocking of IL-1 signaling serves as a clinical treatment that significantly ameliorates the disease symptoms. Here, I tried to establish a method to monitor type I IL-1 receptor (IL-1RI) activity for screening IL-1RI modulator. Myeloid differentiation primary response gene (88) (Myd88), an adaptor of Toll like receptor (TLR) and IL-1RI, is recruited to the receptor after ligand binding and displays an aggregation pattern after receptor-mediated endocytosis. I hypothesized that the level of IL-1RI activation can be monitored by Myd88 redistribution after IL-1b induction, and thus generated a HeLa cell line transduced with emerald green fluorescence protein (emGFP)-tagged Myd88 to test this hypothesis. I found that IL-1b induced nuclear translocation of nuclear factor kappa-light-chain-enhancer of activated B cells (NFkB) in the transduced HeLa cells within 20 minutes. However, although the endogenous Myd88 revealed elevated Myd88 aggregation after 5 minutes of IL-1b induction, the transduced cells did not show Myd88-emGFP redistribution prior to NFkB activation. On the other hand, I found that IL-1b up-regulated the level of Myd88 in the transduced HeLa cells, which correlated with Myd88 aggregation at time later than NFkB activation. In summary, the transduced cell line cannot serve to monitor IL-1RI activity due to lack of detectable receptor-mediated Myd88 redistribution upon IL-1b induction, but indicate a correlation between Myd88 expression level and distribution.