Human milk oligosaccharides and its acid hydrolysate LNT2 show immunomodulatory effects via TLRs in a dose and structure-dependent way

Human milk oligosaccharides (HMOs) have beneficial immune effects but the mechanisms of action are not well understood. Here we study Toll-like receptor (TLR) signaling of the HMOs 2'-FL, 3-FL, 6'-SL, LNT2, and LNnT and their effect on cytokine production in human macrophages. 3-FL activated TLR2 and LNT2 activated all TLRs in a dose-dependent way. In an inhibition assay, 2'-FL, 6'-SL, and LNnT inhibited TLR5 and 7, while 3-FL inhibited TLR5, 7, and 8. 6'-SL showed a synergistic effect on ssRNA40-induced TLR8 activation. In addition, we measured HMO-induced cytokine production in THP1 macrophages. IL-10 and TNF alpha were induced by LNT2, and the effects were NF-kappa B dependent, while the other HMOs had minor effects. The potent effects of LNT2 might be explained by the unique N-acetylglucosamine end that binds to leucine-rich repeats on TLRs. Our data suggest that the effects of HMOs on TLR signaling and immunomodulation of macrophages are HMO-structure dependent.