Oxidized Phospholipid Inhibition of Toll-like Receptor (TLR) Signaling Is Restricted to TLR2 and TLR4: ROLES FOR CD14, LPS-BINDING PROTEIN, AND MD2 AS TARGETS FOR SPECIFICITY OF INHIBITION*

摘要

The generation of reactive oxygen species is a central feature ofinflammation that results in the oxidation of host phospholipids. Oxidizedphospholipids, such as1-palmitoyl-2-arachidonyl-sn-glycero-3-phosphorylcholine (OxPAPC),have been shown to inhibit signaling induced by bacterial lipopeptide orlipopolysac-charide (LPS), yet the mechanisms responsible for the inhibitionof Toll-like receptor (TLR) signaling by OxPAPC remain incompletelyunderstood. Here, we examined the mechanisms by which OxPAPC inhibits TLRsignaling induced by diverse ligands in macrophages, smooth muscle cells, andepithelial cells. OxPAPC inhibited tumor necrosis factor-α production,IκBα degradation, p38 MAPK phosphorylation, andNF-κB-dependent reporter activation induced by stimulants of TLR2 andTLR4 (Pam3CSK4 and LPS) but not by stimulants of otherTLRs (poly(I·C), flagellin, loxoribine, single-stranded RNA, or CpGDNA) in macrophages and HEK-293 cells transfected with respective TLRs andsignificantly reduced inflammatory responses in mice injected subcutaneouslyor intraperitoneally with Pam3CSK4. Serum proteins,including CD14 and LPS-binding protein, were identified as key targets for thespecificity of TLR inhibition as supplementation with excess serum orrecombinant CD14 or LBP reversed TLR2 inhibition by OxPAPC, whereas serumaccessory proteins or expression of membrane CD14 potentiated signaling viaTLR2 and TLR4 but not other TLRs. Binding experiments and functional assaysidentified MD2 as a novel additional target of OxPAPC inhibition of LPSsignaling. Synthetic phospholipid oxidation products1-palmitoyl-2-(5-oxovaleryl)-sn-glycero-3-phosphocholine and1-palmitoyl-2-glutaryl-sn-glycero-3-phosphocholine inhibited TLR2signaling from ∼30 μm. Taken together, these results suggestthat oxidized phospholipid-mediated inhibition of TLR signaling occurs mainlyby competitive interaction with accessory proteins that interact directly withbacterial lipids to promote signaling via TLR2 or TLR4.