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 β-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 β-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-α and IL-6 production as well as NF-κB activation in lipopolysaccharide-induced macrophages. Chemical and enzymatic linkage analyses indicated that GF70-F1 mainly contained the known (1→3),(1→6)-β-d-glucan and a polysaccharide not previously isolated from GF, a nondigestible glucan with a β-(1→4)-linked backbone and β-(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)-β-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)-β-d-glucan in GF70-F1 may contribute to the anti-inflammatory activity via interacting with TLR2 rather than Dectin-1 or CR3 receptors.