Toll-like receptor 4-MD-2 complex mediates the signal transduction induced by flavolipin, an amino acid-containing lipid unique to Flavobacterium meningosepticum

Flavolipin, an amino acid-containing lipid isolated from Flavobacterium meningosepticum, induces many immune responses. It has been shown that flavolipin does not induce an immune response of macrophages derived from C3H/HeJ mice, which possess a point mutation in Toll-like receptor 4 (TLR4). To determine whether TLR4 or the molecular complex of TLR4 and TLR4 association molecule MD-2 mediates the flavolipin signal, flavolipin responsiveness was examined by measuring NF-kappaB activation in Ba/F3 cells and Ba/F3 transfectants expressing TLR4 or both TLR4 and MD-2. Flavolipin-induced NF-kappaB activation was detected in the cells expressing both TLR4 and MD-2, but not in the other cells. Expression of CD14 in the transfectant expressing both TLR4 and MD-2 increased the sensitivity to flavolipin. Furthermore, flavolipin stereoisomers were chemically synthesized, and their abilities to induce NF-kappaB activation were examined. (R)-Flavolipin, in which the configuration of the lipid moiety is R, induced NF-kappaB activation via the TLR4-MD-2 complex, but (S)-flavolipin did not. In this study, we demonstrated the involvement of TLR4-MD-2 and CD14 in flavolipin signaling and the importance of the (R)-configuration of the flavolipin lipid moiety for the induction of an immune response via TLR4-MD-2.