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Activation of Toll-Like Receptor-Mediated NF-κβ by Zymosan-Derived Water-Soluble Fraction: Possible Contribution of Endotoxin-Like Substances

Zymosan is a well-known reagent for the examination of inflammatory response and is prepared from yeast, Saccharomyces cerevisiae. In the activation process, Toll-like receptor (TLR) 2 and TLR6 act as functional receptors for NF-κB activation. Although zymosan is primarily composed of β-glucans, little is known about the active component of zymosan-mediated biological activities. The active moiety of zymosan was fractionated by its solubility in water, and its biological activity on macrophages and TLRs-transfectants examined. The macrophage cell line, RAW264.7, was treated with zymosan-derived preparations, and tumor necrosis factor alpha (TNF-α) produced in the culture supernatant was measured by ELISA. Increased TNF-α production was observed by stimulation with water-soluble (ZWS) or water-insoluble fraction (ZWIS). ZWS showed higher activity in TNF-α production. NF-κB activation via TLR2, TLR1/TLR2, TLR2/TLR6, and TLR4/MD-2/CD14 also was enhanced by stimulation with ZWS and ZWIS. In particular, ZWS showed higher activity via TLR1/TLR2, TLR2/TLR6, and TLR4/MD-2/CD14 than other preparations. ZWS activity was decreased by treatment with polymyxin B, but not with lysozyme and zymolyase. Furthermore, ZWS contained significant more endotoxin than any other preparations. Therefore, we suggest that the active moiety of ZWS for the NF-κB activation has an endotoxin-like substance, that is abundantly observed in Gram-negative bacteria. These results imply that the inflammatory activity of zymosan is induced not only by β-glucans, but also by other endotoxin-like water-soluble substances.