Polyunsaturated fatty acids are precursors of multiple pro- and anti-inflammatory molecules generated by enzymatic stereospecific and positionally specific insertion of oxygen, which is a prerequisite for recognition of these mediators by cellular receptors. However, nonenzymatically oxidized free and esterified polyunsaturated fatty acids also demonstrate activities relevant to inflammation. In particular, phospholipids containing oxidized fatty acid residues (oxidized phospholipids; OxPLs) were shown to induce proinflammatory changes in endothelial cells but paradoxically also to inhibit inflammation induced via TLR4. In this study, we show that half-maximal inhibition of LPS-induced elevation of E-selectin mRNA in endothelial cells developed at concentrations of oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine (OxPAPC) 10-fold lower than those required to induce proinflammatory response. Similar concentration difference was observed for other classes and molecular species of OxPLs. Upon injection into mice, OxPAPC did not elevate plasma levels of IL-6 and keratinocyte chemoattractant but strongly inhibited LPS-induced upregulation of these inflammatory cytokines. Thus, both in vitro and in vivo, anti-LPS effects of OxPLs are observed at lower concentrations than those required for their proinflammatory action. Quantification of the most abundant oxidized phosphatidylcholines by HPLC/tandem mass spectrometry showed that circulating concentrations of total oxidized phosphatidylcholine species are close to the range where they demonstrate anti-LPS activity but significantly lower than that required for induction of inflammation. We hypothesize that low levels of OxPLs in circulation serve mostly anti-LPS function and protect from excessive systemic response to TLR4 ligands, whereas proinflammatory effects of OxPLs are more likely to develop locally at sites of tissue deposition of OxPLs (e.g., in atherosclerotic vessels).

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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 ...

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