Identification and characterization of autophagy-inducing compounds in macrophages

• Main Authors: Yi-ju Chou, 周奕如
• Other Authors: Shu-Ling Fu
• Format: Others
• Published: 2013
• Online Access: http://ndltd.ncl.edu.tw/handle/58799790182506293650

碩士 === 國立陽明大學 === 傳統醫藥研究所 === 101 === Activation of Toll-like receptor 4 (TLR4) triggers innate immunity and subsequently leads to induction of adaptive immunity. Recent studies show that induction of TLR4 signaling in macrophages can induce autophagy which is involved in the escape of intracellular pathogens and the enhancement of antigen presentation. Our laboratory has previously developed a synthetic glycolipid, CCL-34, which can activate macrophages through TLR4 signaling pathway and induce TLR4-dependent anticancer immunity in vitro and in vivo. In this study, I used RAW264.7 macrophages as the cell model to examine whether CCL-34 and other herbal compounds can induce autophagy and study its underlying molecular mechanism. Furthermore, whether CCL-34 can function as immune adjuvant was also investigated. The expression of autophagy indicator, LC3-II, was measured using Western blot. Using fluorescent microscope, I detected the percentage of cells with fluorescent punctates in EGFP-LC3-expressing cells to measure the formation of autophgosomes. After CCL-34 treatment, both the expression level of LC3-II and the percentage of cells forming autophagomes were increased. The LC3-II protein was also increased in bone-marrow-derived macrophages (BMDMs) isolated from wild-type (C3H/HeN) mice but not in TLR4-defective (C3H/HeJ) mice upon treatment with CCL-34. Therefore, the LPS- and CCL-34-induced autophagy is via a TLR4-dependent manner. After CCL-34 treatment, the protein levels of activated p38 and pERK, the activity of NF-κB, and the expression level of p62 were also increased in RAW264.7 cells. Inhibition of pERK and NF-κB by PD98059 and Bay117082 also decreased the expression of LC3-II. However, Inhibition of p38 by SB203580 further increased the LC3-II production and the NF-κB activation. These data demonstrate that CCL-34 can induce autophagy in macrophages. Furthermore, the activation of p38-, NF-κB and ERK-signaling pathways as well as the induction of p62 may play a role in the CCL-34-induced autophagy. The adjuvant activity of CCL-34 was also evaluated. Compared with the control group, mice immunized with both CCL-34 and the antigen ovalbumin (OVA) have higher amount of OVA-specific IgG in serum, as measured by ELISA. This data indicate that CCL-34 can also enhance immune response in vivo. In this study, a cell line stably expressing EGFP-LC3 was established and further applied to screen for autophagy-inducing herbal compounds. The fluorescent EGFP-LC3 punctuates was increased in cells treated with cordycepin, piperine and bilobalide. Furthermore, the expression of LC3-II was significantly increased after piperine treatment. How piperine induces autophagy in macrophages remains to be further investigated. In summary, this study demonstrates that CCL-34 can induce autophagy and function as adjuvant in vivo. The possible molecular mechanism of CCL-34-induced autophagy was also studied. The data described in this study will provide crucial information regarding the action mechanisms and the potential application of CCL-34.