A designed TLR4/MD-2 complex to capture LPS.
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.
Brandl
Katharina
K
Department of Internal Medicine I, University of Regensburg, Regensburg, Germany. katharina.brandl@klinik.uni-regensburg.de
Glück
Thomas
T
Hartmann
Pia
P
Salzberger
Bernd
B
Falk
Werner
W
D000818:Animals; D000950:Antigens, Ly / Q000378:metabolism*; D015153:Blotting, Western; D002460:Cell Line; D002478:Cells, Cultured; D004330:Drosophila / Q000166:cytology; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D008070:Lipopolysaccharides / Q000037:antagonists & inhibitors / Q000378:metabolism*; D051218:Lymphocyte Antigen 96; D008264:Macrophages / Q000378:metabolism; D008562:Membrane Glycoproteins / Q000378:metabolism*; D051379:Mice; D011233:Precipitin Tests; D011506:Proteins / Q000032:analysis; D011956:Receptors, Cell Surface / Q000378:metabolism*; D011993:Recombinant Fusion Proteins; D015398:Signal Transduction; D012995:Solubility; D019063:Tenascin / Q000378:metabolism; D051197:Toll-Like Receptor 4; D051193:Toll-Like Receptors; D014409:Tumor Necrosis Factor-alpha / Q000037:antagonists & inhibitors / Q000096:biosynthesis
D000950:Antigens, Ly; D008070:Lipopolysaccharides; C411728:Ly96 protein, mouse; D051218:Lymphocyte Antigen 96; D008562:Membrane Glycoproteins; D011506:Proteins; D011956:Receptors, Cell Surface; D011993:Recombinant Fusion Proteins; C495345:TLR4 protein, human; C074149:TNXA protein, human; D019063:Tenascin; D051197:Toll-Like Receptor 4; D051193:Toll-Like Receptors; D014409:Tumor Necrosis Factor-alpha