Activation of RAW264.7 macrophage by Exopolysaccharide from Aphanothece halaphytica (EPSAH) and the underlying mechanisms

Exopolysaccharide from Aphanothece halophytica (EPSAH), a potent antitumor agent and immunological adjuvant, was investigated for the activation effect on RAW264.7 macrophages and the underlying mechanisms. EPSAH could significantly enhance macrophage phagocytosis and the secretion of nitric oxide, increase the mRNA expression levels of the pro-inflammatory cytokines (IL-1β, IL-6, IL-12, and TNF-α), anti-inflammatory cytokine IL-10, and chemokines (MCP-1 and MIP-1α). When RAW264.7 cells were treated with EPSAH, the mRNA expression of TLR4 and its downstream molecules TRAF6 and MyD88 were upregulated. When TLR4 was blocked using a TLR4-specific neutralizing antibody, nitric oxide secretion from the macrophages was significantly inhibited. EPSAH was further shown to induce phosphorylation of the mitogen-activated protein kinases (MAPKs) ERK, JNK, and p38, and promote cytoplasmic IκB phosphorylation and increase nuclear NF-κB p65 levels remarkably in RAW264.7 cells. These data demonstrate the capacity of EPSAH to induce macrophage activation possibly via TLR4/MyD88 pathway, which leads to the activation of its main signaling downstream molecules MAPKs and NF-κB.