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.The "omics" revolution has provided significant insight into the genes, mRNAs, and proteins of mammalian cells, biological systems, and disease (1-3). An important function of these macromolecular classes is the production of metabolites that in turn are used by cells for replication and function. Lipids comprise major structural and metabolic components of cells and have essential functions in the formation of membranes, energy production, and intracellular signaling. Despite the central role of lipids in mammalian cell function, there has been no systematic effort to define the lipid "parts list" of a mammalian cell or the changes in these lipids associated with cellular function and disease. Many biochemical pathways leading to the synthesis and degradation of major lipid categories are known, but how these pathways interact under normal and pathological conditions remains unexplored. Recent advances in mass spectrometry have made it possible to qualitatively and quantitatively analyze a majority of cellular lipids (4 -8). We report here a comprehensive systems-level analysis of a mammalian cell lipidome through temporal measurements.We characterized lipidomic responses of RAW264.7 (RAW) macrophages to a highly specific ligand for Toll-like receptor 4 (TLR4) 4 that mimics aspects of bacterial infection. This model is of particular interest because of the essential roles that alterations in macrophage lipid metabolism play in innate and adaptive immune responses and the development of chronic inflammatory and cardiovascular diseases. Recent studies further suggest that TLR signaling in macrophages is not only required for innate immunity against viral and bacterial pathogens but also contributes to the pathogenesis of atherosclerosis, diabetes, arthritis, and other inflammatory diseases (9). Although TLR4 signaling is known to exert profound effects on the macrophage transcriptome (10), proteome (11), and selected lipid species that...

Palmitate and Lipopolysaccharide Trigger Synergistic Ceramide Production in Primary Macrophages

“…The effects of lipids and inflammatory signaling on ceramide production have been studied in established cell lines, and our data show that these stimuli regulate ceramide production differently in primary macrophages. In cultured RAW 264.7 macrophages, TLR activation has been shown to increase sphingolipid and ceramide content (15,16). As with other cell types, palmitate treatment of RAW cells has also been shown to increase total cellular ceramide (21).…”

“…In addition to cytokine production, another consequence of TLR4-mediated macrophage activation is the remodeling of cellular lipids. Recent lipidomic data has revealed that TLR4 activation of RAW 264.7 cells increases total cellular sphingolipids, including ceramides (15,16). These findings are of particular interest because ceramides have been implicated in a wide variety of cell stress responses including cell death, proliferation, inflammation, autophagy, and insulin resistance (17,18).…”

Membrane lipidome of an epithelial cell line

“…Although the current lipidomic methodologies have succeeded in elucidating the molecular composition of major GP classes, complex GSPs were neglected within common lipidomic routines (14,(23)(24)(25). These lipid classes, however, are both biologically important and abundant (19,26,27).…”

“…By combining high mass resolution MS, an improved extraction procedure that increases the recovery of polar lipids (13), and the use of internal standard mixtures for absolute quantification, we expanded the lipid repertoire covered by shotgun lipidomics to glycosphingolipids (GSPs). Importantly, the analysis of the GSPs did not require the hydrolysis of glycerophospholipids (GPs) (14), and this simplification allowed us to determine the membrane lipidome with unprecedented comprehensiveness (more than 300 lipid species from 14 different lipid classes) from a single sample of 10 5 cells.…”

Liberating Chiral Lipid Mediators, Inflammatory Enzymes, and LIPID MAPS from Biological Grease

“…Together, we developed novel liquid chromatographic-mass spectrometric-based lipidomics techniques termed "CLASS" (138) using a quantitative approach based on over 500 LIPID MAPS synthetic standards and specific agonists such as Kdo 2 -Lipid A (154). This approach was applied to the overall omics analysis of some 500 lipid species in immunologically activated macrophages integrating transcriptomics, proteomics, and metabolomics of lipid metabolites (155). Novel methods for separating and assessing the purity of subcellular organelles (156) were developed and employed to determine the subcellular localization of many individual lipid molecular species and their changes during macrophage activation (157).…”