A designed TLR4/MD-2 complex to capture LPS

Abstract

The family of Toll-like receptors (TLRs) is involved in the defense of an organism to microbial attack. TLR4-induced signaling is involved in infectious diseases, chronic inflammatory diseases and sepsis; therefore, we aimed at modulating TLR4-signaling via ligand-binding soluble receptors. Because recognition of microbial structures shows some species-specific traits, we specifically selected the mouse model for later in vivo studies. We first prepared the N-terminally Flag-tagged mouse (m) recombinant (r) soluble (s) fusion proteins mrsTLR4-IgGFc (T4Fc) and mrsMD-2 in Drosophila melanogaster Schneider 2 (S2) cells. The function of these molecules was tested by inhibition of synthesis of pro-inflammatory cytokines after stimulation of mouse macrophage RAW 264.7 cells with purified lipopolysaccharide (LPS). T4Fc alone had no inhibitory activity; however, a T4Fc/MD-2 complex blocked LPS activity. By analogy with `cytokine traps', we then prepared a designer molecule (LPS-Trap) by fusing MD-2 to the C-terminus of soluble TLR4 via a flexible linker. LPS-Trap significantly inhibited TNF production by LPS-stimulated RAW 264.7 cells. Thus, the T4Fc/MD-2 complex as well as the LPS-Trap blocked LPS activity in vitro and might thus represent a new therapeutic option in sepsis by neutralization of TLR4-activating ligands.