title: Suppression of Toll-like receptor 4 activation by caffeic acid phenethyl ester is mediated by interference of LPS binding to MD2.
abstract: OBJECTIVE
  Toll-like receptors (TLRs) play a crucial role in recognizing invading pathogens and endogenous danger signal to induce immune and inflammatory responses. Since dysregulation of TLRs enhances the risk of immune disorders and chronic inflammatory diseases, modulation of TLR activity by phytochemicals could be useful therapeutically. We investigated the effect of caffeic acid phenethyl ester (CAPE) on TLR-mediated inflammation and the underlying regulatory mechanism.
  
  
  METHODS
  Inhibitory effects of CAPE on TLR4 activation were assessed with in vivo murine skin inflammation model and in vitro production of inflammatory mediators in macrophages. In vitro binding assay, cell-based immunoprecipitation study and liquid chromatography-tandem mass spectrometry analysis were performed to determine lipopolysaccharide (LPS) binding to MD2 and to identify the direct binding site of CAPE in MD2.
  
  
  RESULTS
  Topical application of CAPE attenuated dermal inflammation and oedema induced by intradermal injection of LPS (a TLR4 agonist). CAPE suppressed production of inflammatory mediators and activation of NFκB and interferon-regulatory factor 3 (IRF3) in macrophages stimulated with LPS. CAPE interrupted LPS binding to MD2 through formation of adduct specifically with Cys133 located in hydrophobic pocket of MD2. The inhibitory effect on LPS-induced IRF3 activation by CAPE was not observed when 293T cells were reconstituted with MD2 (C133S) mutant.
  
  
  CONCLUSIONS
  Our results show a novel mechanism for anti-inflammatory activity of CAPE to prevent TLR4 activation by interfering with interaction between ligand (LPS) and receptor complex (TLR4/MD2). These further provide beneficial information for the development of therapeutic strategies to prevent chronic inflammatory diseases.
authors.affiliation: School of Life Sciences, Gwangju Institute of Science and Technology, Gwangju, Korea.
authors.forename: So Young
authors.initials: SY
authors.lastname: Kim
authors.forename: Jung Eun
authors.initials: JE
authors.lastname: Koo
authors.forename: Yun Jee
authors.initials: YJ
authors.lastname: Seo
authors.forename: Nisha
authors.initials: N
authors.lastname: Tyagi
authors.forename: Eunshil
authors.initials: E
authors.lastname: Jeong
authors.forename: Jaeyoung
authors.initials: J
authors.lastname: Choi
authors.forename: Kyung-Min
authors.initials: KM
authors.lastname: Lim
authors.forename: Zee-Yong
authors.initials: ZY
authors.lastname: Park
authors.forename: Joo Young
authors.initials: JY
authors.lastname: Lee
mesh_terms: D000818:Animals; D000893:Anti-Inflammatory Agents / Q000494:pharmacology*; D002109:Caffeic Acids / Q000494:pharmacology*; D002460:Cell Line; D002849:Chromatography, Gas; D004195:Disease Models, Animal; D005786:Gene Expression Regulation; D057809:HEK293 Cells; D006801:Humans; D007249:Inflammation / Q000139:chemically induced; D050838:Interferon Regulatory Factor-3 / Q000378:metabolism; D008070:Lipopolysaccharides / Q000008:administration & dosage / Q000378:metabolism*; D051218:Lymphocyte Antigen 96 / Q000378:metabolism*; D051379:Mice; D008807:Mice, Inbred BALB C; D008810:Mice, Inbred C57BL; D016328:NF-kappa B / Q000378:metabolism; D010626:Phenylethyl Alcohol / Q000031:analogs & derivatives* / Q000494:pharmacology; D015398:Signal Transduction / Q000187:drug effects; D053719:Tandem Mass Spectrometry; D051197:Toll-Like Receptor 4 / Q000819:agonists* / Q000378:metabolism*
chemical_list: D000893:Anti-Inflammatory Agents; D002109:Caffeic Acids; D050838:Interferon Regulatory Factor-3; C495377:LY96 protein, human; D008070:Lipopolysaccharides; C411728:Ly96 protein, mouse; D051218:Lymphocyte Antigen 96; D016328:NF-kappa B; C495345:TLR4 protein, human; C493487:Tlr4 protein, mouse; D051197:Toll-Like Receptor 4; C055494:caffeic acid phenethyl ester; D010626:Phenylethyl Alcohol