Multi-Hit Inhibition of Circulating and Cell-Associated Components of the Toll-Like Receptor 4 Pathway by Oxidized Phospholipids

Abstract

Objective-Oxidized phospholipids (OxPLs) that are abundant in atherosclerotic lesions are increasingly recognized as context-dependent lipid mediators demonstrating both pro-and antiinflammatory activities. Molecular mechanisms of their effects are largely unknown. Here we present novel information on the mechanisms whereby OxPLs modulate activation of TLR4 by lipopolysaccharide (LPS). Methods and Results-We show, using several cell types and various inflammatory genes as readouts, that different classes and molecular species of OxPLs do not stimulate TLR4 but exert prominent inhibitory effects on LPS-induced reactions. Our data demonstrate that binding of OxPLs to the LPS-binding protein (LBP) and CD14 prevents recognition of LPS by these proteins, thus impairing activation of TLR4. In addition, OxPLs inhibited LBP-and CD14-independent activation of TLR4 by the synthetic TLR4 agonist E6020 indicating that in parallel with LBP and CD14, OxPLs target cell-associated steps in TLR4 cascade. Conclusions-Our data suggest that OxPLs inhibit action of LPS via a multi-hit mechanism. These results support the notion that OxPLs are endogenous inhibitors of TLR4 produced in response to oxidative stress. O xidized phospholipids (OxPLs) are generated as a result of oxidation of esterified polyunsaturated fatty acids (PUFAs). Oxidized phosphatidylcholines (OxPCs) were identified as proinflammatory components of LDL minimally modified by oxidation (mmLDL) and are increasingly recognized as lipid mediators with a broad spectrum of activities. Apart from OxPCs, oxidation of other classes of PUFA-PLs generates biologically active products that were detected in a number of pathologies including ischemia/reperfusion, radiation injury, cell aging, and apoptosis. [1][2][3][4] OxPCs are especially abundant in human and animal atherosclerotic lesions 5-7 where they are thought to induce inflammatory See accompanying article on page 337 reactions characteristic of atherosclerosis. In particular, OxPCs initiate mononuclear cell recruitment by inducing activation of adhesion molecules and production of MCP-1, IL-8, and other chemokines by endothelial cells (ECs). 8 On the other hand, oxidized LDL and OxPLs demonstrate tissueprotective and antiinflammatory activities including upregulation of antioxidant enzymes or inhibition of inflammation induced by bacterial lipopolysaccharide (LPS, endotoxin) via TLR4. 9,10 The mechanisms of the antiendotoxin effects of OxPLs are only partially understood. In particular, it is not clear which chemical groups are important for the antiendotoxin activity of OxPLs and whether there are strict structural constraints for the activity. Furthermore, the data showing that certain effects of OxPLs are inhibited in TLR4 knockout animals 11,12,13 suggest that OxPLs are not pure inhibitors/antagonists of TLR4 but may possess partial agonistic activity for TLR4. Last but not least, an important open question concerns the molecular targets of the antiendotoxin action of OxPLs. According to one hypothesis, OxPLs ...

Oxidized Phospholipids Are More Potent Antagonists of Lipopolysaccharide than Inducers of Inflammation

“…Previously, we showed that OxPLs inhibited LPS-induced accumulation of both E-selectin mRNA and protein in HUVECs (21). Therefore, only expression of E-selectin mRNA was measured in this work.…”

“…Oxidation was monitored by TLC and electrospray ionization-mass spectrometry (12). Mass-spectra of OxPLs have been published previously (21). 1-Palmitoyl-2-(5-oxovaleroyl)-sn-glycero-3-phosphocholine (POVPC), 1-palmitoyl-2-glutaroyl-sn-glycero-3-phosphocholine (PGPC), a mixture of 1-palmitoyl-2-(12-hydroperoxyeicosa-5,8,10,14-tetraenoyl)-sn-glycero-3-phosphocholine and 1-palmitoyl-2-(15-hydroperoxyeicosa-5,8,11,13-tertraenoyl)-sn-glycero-3-phosphocholine (PAPC-OOH), and 1-palmitoyl-2-(5,6-epoxy isoprostane E2)-sn-glycero-3-phosphocholine (PEIPC)-enriched fraction were prepared as described previously (25).…”

“…1-Palmitoyl-2-(5-oxovaleroyl)-sn-glycero-3-phosphocholine (POVPC), 1-palmitoyl-2-glutaroyl-sn-glycero-3-phosphocholine (PGPC), a mixture of 1-palmitoyl-2-(12-hydroperoxyeicosa-5,8,10,14-tetraenoyl)-sn-glycero-3-phosphocholine and 1-palmitoyl-2-(15-hydroperoxyeicosa-5,8,11,13-tertraenoyl)-sn-glycero-3-phosphocholine (PAPC-OOH), and 1-palmitoyl-2-(5,6-epoxy isoprostane E2)-sn-glycero-3-phosphocholine (PEIPC)-enriched fraction were prepared as described previously (25). Noncleavable analogue of PAPC was synthesized as described previously (21). Concentration of PLs was determined by phosphorus assay (26).…”

“…However, under different biological conditions, OxPLs can also induce anti-inflammatory effects. In particular, various classes and molecular species of OxPLs were shown to inhibit action of LPS and TLR2 ligands in vivo and in vitro (11,(16)(17)(18)(19)(20)(21). Several cell types were shown to be targets of the anti-LPS action of OxPLs, including human ECs (11,16), blood monocytes (21), monocyte-derived macrophages (22), human and mouse macrophage cells lines (17), dendritic cells (23), skin fibroblasts (21), and epithelial cells transfected with TLR4 and TLR2 together with coreceptors (17,21).…”

“…The mechanisms of the anti-LPS effect of OxPLs seem to be complex. Available data suggest that OxPLs inhibit both recognition of LPS by LPS-binding protein, CD14, and MD-2 (17,21), as well as cell-associated events such as coupling of TLR4 to intracellular signaling adapters (22,24).…”