Mechanistic study of Toll-like receptor 4 (TLR4) activation by a alpha-galactosylcerimide analog (CCL-34)

• Main Authors: Po-Hsiung Huang, 黃柏雄
• Other Authors: Shu-Ling Fu
• Format: Others
• Language: zh-TW
• Published: 2009
• Online Access: http://ndltd.ncl.edu.tw/handle/g3w8xv

碩士 === 國立陽明大學 === 傳統醫藥學研究所 === 97 === Toll-like receptor 4 (TLR4) is commonly expressed in innate immune cells, especially dendritic cells and macrophages. Activation of TLR4 triggers innate immune responses and subsequently induces adaptive immunity. TLR4 agonists are known to function as immunostimulators, and some TLR4 agonists (such as OK-432, BCG-CWS) have been clinically demonstrated to inhibit tumor growth. Our laboratory has previously identified a novel synthetic glycolipid (designated as CCL-34) that can induce murine macrophage activation via TLR4-dependent signaling pathways. In the present study, I carried out the structure-and-activity relationship analysis (SAR) of CCL-34 and its related derivatives (78 compounds with modification in fatty acid moiety, lipid amine moiety, sugar moiety, or glycosidic groups) in human 293 cells constitutively expressing hTLR4, MD2 and CD14(HEK293-TLR4/MD2/ CD14). Based on the NF-kB activity and IL-8 cytokine expression, I conclude that (1) substitution of fatty acid moiety in CCL-34 by bulky structures completely diminishes its activity. (2) length of both lipld amine chain and fatty acid moiety chain are crucial for its activity; compounds with 10~12-carbon lipid amine and 12~13-carbon fatty acid chain are optimal for TLR4 activation and CCL-34 exhibits highest activity among these compounds. (3) Substitution of galactose in CCL-34 by glucose or fucose does not affect its activity; Two CCL-34 sugar analogs (CCL-34-S5 and CCL-34-S6) induce NF-kB activity and IL-8 production to a level comparable with CCL-34. (4) the substitution of the fifth hydroxyl group (-OH) at sugar moiety in CCL-34 with amine group (-NH2) or bulky group reduces or even abolishes its activity. I next examined the TLR4-activating ability of CCL-34, CCL-34-S5 and CCL-34-S6 in human THP-1 monocytes. Treatments with these compounds significantly activated TLR4-downstream MyD88-dependent pathway: both the activity of mitogen-activated protein kinases (Erk1/2, JNK1/2 and p38) and the cytokine production (IL-8 and TNF-��) are greatly enhanced. Furthermore, treatment with a TLR4-neutralizing antibody blocked TNF-�� production induced by CCL34, CCL-34-S5 and CCL-34-S6, indicating that these compounds act in a TLR4-dependent manner. Interestingly, CCL-34 and CCL-34-S6 also stimulated MyD88-independent pathway: the activity of IFN-b promoter and IFN-b gene expression are both increased. Using human 293 cells transfected with various combinations of TLR4, MD2, and CD14 genes, we found that MD2 (but not CD14) is required for CCL-34-mediated TLR4 activation. Our data suggest that CCL-34 and CCL-34-S6 both activated TLR4 receptor via MyD88-dependent and MyD88-independent pathways. Notably, CCL-34-6 is more easily to be chemically synthesized and produced with much higher yield than CCL-34, representing a promising new TLR4 activator. Based on gene expression profiles obtained from microarray analysis, CCL-34- and CCL-34-S6-treated THP-1 cells induced expression of many M1 (anti-cancer) macrophage-associated genes but not M2 (pro-cancer) macrophage-associated genes, suggesting these cells may exhibit anti-cancer features. Certainly, the adjuvant and anti-cancer activities of CCL-34 and CCL-34-S6 in cellular and in animal model merit further evaluation.