Acetyl group dictates macrophage immune activation by polysaccharide ME-2 via TLR4-NF-κB/MAPK pathways.

ME-2, a GXG'G''M type polysaccharide with a high O-acetyl group content, was isolated and purified from Auricularia auricula-judae. However, the mechanism underlying its immunomodulatory activity has not been thoroughly investigated. This study demonstrates that ME-2 effectively reverses cyclophosphamide-induced immunosuppression in vivo by normalizing organ indices, increasing serum cytokine levels, and restoring the structure of the spleen tissue. In vitro, ME-2 binds to TLR4 on the surface of RAW264.7 cells. This binding triggered the activation of both the MAPK and NF-κB signaling pathways. Consequently, ME-2 treatment promoted the secretion of immune effector factors, enhanced endocytic activity, and induced a shift toward the M1 phenotype. Subsequently, the above mechanism was reversely validated through TLR4 knockdown and specific signaling pathway inhibitors. These results demonstrate that ME-2 exhibits a potent immune activation effect, laying a foundation for the development of ME-2-based immunomodulators. In addition, experimental results demonstrated that dME-2 with complete de-O-acetylation exhibits no binding affinity for TLR4 and fails to activate its downstream signaling pathways. These findings confirm that O-acetyl groups are indispensable for ME-2-mediated immune activation.