A mouse macrophage lipidome

Abstract

We report the lipidomic response of the murine macrophage RAW cell line to Kdo(2)-lipid A, the active component of an inflammatory lipopolysaccharide functioning as a selective TLR4 agonist and compactin, a statin inhibitor of cholesterol biosynthesis. Analyses of lipid molecular species by dynamic quantitative mass spectrometry and concomitant transcriptomic measurements define the lipidome and demonstrate immediate responses in fatty acid metabolism represented by increases in eicosanoid synthesis and delayed responses characterized by sphingolipid and sterol biosynthesis. Lipid remodeling of glycerolipids, glycerophospholipids, and prenols also take place, indicating that activation of the innate immune system by inflammatory mediators leads to alterations in a majority of mammalian lipid categories, including unanticipated effects of a statin drug. Our studies provide a systems-level view of lipid metabolism and reveal significant connections between lipid and cell signaling and biochemical pathways that contribute to innate immune responses and to pharmacological perturbations.

FIGURE 1.

Interrelationships between cellular metabolic pathways. Red arrows denote multistep transformations among the major lipid categories and acetyl CoA. Black arrows from fatty acids to sterol esters indicate transfer of fatty acids (in the form of acyl-CoAs) to sterol cores. Values in orange ovals represent the number of analytes within each lipid category that were quantified by mass spectrometry in RAW cells in response to stimuli (see under “Results”). The panel at the lower left shows a temporal profile of quantitative changes that occur for some lipid species in the various lipid categories in these cells over a 24-h period upon treatment with KLA. The horizontal arrows denote the time periods during which statistically significant changes with KLA treatment were observed.

FIGURE 2.

Changes in eicosanoids in cell media and transcripts in response to KLA treatment of RAW cells. Green boxes represent genes coding for enzymes that catalyze the indicated conversions, whereas yellow boxes represent lipid metabolites. The heat maps (see color key) below each lipid and gene represent the KLA/control (KLA/Ctrl) ratios at 0, 0.5, 1, 2, 4, 8, 12, and 24 h of treatment. Multiple gene isoforms are shown as heat maps that are stacked with the first isoform at the top. KLA/control ratio measurements for mRNAs and lipids were generated from averaging triplicate analyses of three separate RAW cell preparations. 5-HETE, 5-hydroxyeicosatetraenoic acid; 11(R)HETE, 11(R)-hydroxyeicosatetraenoicacid.

FIGURE 3.

Changes in sterols and transcripts in response to KLA treatment in RAW cells. Green boxes represent genes coding for enzymes that catalyze the indicated conversion, whereas yellow boxes represent lipid metabolites. The heat maps below each lipid and gene represent the KLA/control (KLA/Ctrl) ratios at 0, 0.5, 1, 2, 4, 8, 12, and 24 h of treatment. Lipid and mRNA measurements were obtained as described in the legend for Fig. 2. Chol. esters, cholesteryl esters.

FIGURE 4.

Time courses for selected lipids in RAW cells or media treated with KLA and/or compactin. The legend is as follows: blue diamond, control; red box, KLA; green triangle, compactin; purple X, KLA + compactin. KLA/control ratio measurements for lipids were generated from the average analyses of three separate RAW cell preparations and are shown with standard errors.