A (1→6)-Branched (1→4)-β-D-Glucan from Grifola frondosa Inhibits Lipopolysaccharide-Induced Cytokine Production in RAW264.7 Macrophages by Binding to TLR2 Rather than Dectin-1 or CR3 Receptors

Mushroom polysaccharides including beta-glucans possess various health-promoting properties and are known to be the major bioactive constituents of Grifola frondosa (GF), which is a popular edible and medicinal mushroom. Dectin-1, a pattern-recognition receptor, is responsible for recognizing beta-glucans. In this study, parental RAW264.7 macrophages and Dectin-1-expressing RAW264.7 macrophages were used to investigate the anti-inflammatory activity and receptor involvement of the water-soluble polysaccharides from GF. Results indicated that the high molecular weight fraction of GF (GF70-F1; 1260 kDa) inhibited TNF-alpha and IL-6 production as well as NF-kappa B activation in lipopolysaccharide-induced macrophages. Chemical and enzymatic linkage analyses indicated that GF70-F1 mainly contained the known (1 -> 3),(1 -> 6)-beta-D-glucan and a polysaccharide not previously isolated from GF, a nondigestible glucan with a beta-(1 -> 4)-linked backbone and beta-(1 -> 6)-linked branches. The ability of GF70-F1 to inhibit cytokine production was not affected by the expression level of Dectin-1 in cells, and a similar inhibitory activity was observed after removing the (1 -> 3),(1 -> 6)-beta-D-glucan from GF70-F1. Blockade of Toll-like receptor 2 (TLR2) but not Dectin-1 or complement receptor 3 (CR3) attenuated the inhibitory activity of GF70-F1. The nondigestible (1 -> 6)-branched (1 -> 4)-beta-D-glucan in GF70-F1 may contribute to the anti-inflammatory activity via interacting with TLR2 rather than Dectin-1 or CR3 receptors.