We have identified a subset of genes that is specifically induced by stimulation of TLR3 or TLR4 but not by TLR2 or TLR9. Further gene expression analyses established that upregulation of several primary response genes was dependent on NF-kappaB, commonly activated by several TLRs, and interferon regulatory factor 3 (IRF3), which was found to confer TLR3/TLR4 specificity. Also identified was a group of secondary response genes which are part of an autocrine/paracrine loop activated by the primary response gene product, interferon beta (IFNbeta). Selective activation of the TLR3/TLR4-IRF3 pathway potently inhibited viral replication. These results suggest that TLR3 and TLR4 have evolutionarily diverged from other TLRs to activate IRF3, which mediates a specific gene program responsible for innate antiviral responses.

The liver X receptors (LXR) alpha and beta are regulators of cholesterol metabolism and determinants of atherosclerosis susceptibility. Viral and bacterial pathogens have long been suspected to be modulators of atherogenesis; however, mechanisms linking innate immunity to cholesterol metabolism are poorly defined. We demonstrate here that pathogens interfere with macrophage cholesterol metabolism through inhibition of the LXR signaling pathway. Activation of Toll-like receptors (TLR) 3 and 4 by microbial ligands blocks the induction of LXR target genes including ABCA1 in cultured macrophages as well as in aortic tissue in vivo. As a consequence of these transcriptional effects, TLR3/4 ligands strongly inhibit cholesterol efflux from macrophages. Crosstalk between LXR and TLR signaling is mediated by IRF3, a specific effector of TLR3/4 that inhibits the transcriptional activity of LXR on its target promoters. These findings highlight a common mechanism whereby bacterial and viral pathogens may modulate macrophage cholesterol metabolism and cardiovascular disease.

It has been proposed that chemically reactive lipids released during lipid peroxidation convert low density lipoprotein (LDL), the major carrier of plasma cholesterol, to an abnormal form and that receptor-mediated clearance of this altered LDL produces cholesteryl ester deposition in macrophage-derived foam cells of atheroma. Immuno-cytochemical analyses now reveal the presence of protein modified by malondialdehyde, a peroxidative end product, which colocalizes with the extracellular deposition of apolipoprotein B-100 protein of LDL in atheroma from Watanabe heritable hyperlipidemic rabbits. These findings provide direct evidence for the existence in vivo of protein modified by a physiological product of lipid peroxidation within arterial lesions.

Cholesterol has a maximum solubility in aqueous solutions of 1.8 jug/ml or 4.7 ,M. It undergoes a thermodynamically reversible self-association with a critical micelle concentration of 25 to 40 nM at 250. The cholesterol micelle is heterogeneous in size, probably rodlike in shape, and stabilized by an unusually high interaction energy between the aggregated monomers.A critical problem in biochemistry today is the elucidation of the molecular organization of lipid and protein components in biological membranes and serum lipoproteins. Not only is a definition of the structure itself essential, but also an explanation in thermodynamic terms for its existence, i.e., free energies of association among the various components. However, any such investigation requires prior knowledge of the physical properties of the components themselves in aqueous solvents.Lipids, for example, are amphiphiles that undergo monomeraggregate equilibria. Just as it was essential to define these equilibria in thermodynamic terms in order to interpret binding of simple detergents to serum albumin (1, 2), so the same information is required to determine the modes of association of biological lipids with membrane proteins and serum lipoproteins. Numerous structural investigations of pure phospholipid bilayers and of the incorporation of cholesterol into these bilayers have been published (3-5). However, only a single phospholipid system, dipalmitoyl phosphatidylcholine, has been studied from the standpoint of monomer-micelle equilibrium (6). Cholesterol-water solutions have been completely neglected despite the ubiquitous nature of this neutral lipid in eukaryotes.We report here the critical micelle concentration of cholesterol in water and the formation of a heterogeneous selfaggregate in which the forces of interaction are stronger than those usually found in micellar systems. (Beckman Instruments). Scintillation fluid containing 2 liters of toluene, 1 liter of Triton X-100, 8.0 g of 2,5-diphenyloxazole, and 0.4 g of 1,4-bis-2-(5 phenyl-oxazolyl)-benzene was used. The aqueous sample was added to 15 ml of scintillation fluid; the mixture was equilibrated for 4 hr in the dark and counted to 1.5% error. The efficiencies of counting over a range of 0-0.1 ml of water were determined by standardization with [3H]toluene of 3.12 X 106 dpm/ml. A sample of 50-100 jl had a counting efficiency of 40%. All counts were corrected for background, efficiency, and quenching before conversion to concentration units.Critical micelle concentrations were determined by rates of dialysis by use of 10 ml of water in a Visking dialysis bag (Union Carbide Corp.) suspended in a glass vessel containing 15 or 25 ml of cholesterol solution. The apparatus was designed so that a closed system existed except at the time of sampling. The solution was mixed by rotation at 250. The dialysis tubing was brought to a boil in water, rinsed extensively, and used within 1 day.Lengths of micellar aggregates were estimated by use of