Mouse macrophage lipids: All systems go

By combining lipidomic with transcriptomic methods, researchers have uncovered a whole-system picture of immunologically- and pharmacologically-induced lipid perturbations in mouse macrophages.

The ultimate goal of the LIPID MAPS consortium is to improve understanding of lipid metabolism and the active role that lipids play in human disease, which could aid the development of new therapies. The first step is to define the lipidome of the mouse macrophage — a cell with important roles in innate and adaptive immunity, and cardiovascular and inflammatory disease.

Now, Edward A. Dennis and colleagues from the consortium, writing in the Journal of Biological Chemistry, reveal the most complete lipids “parts list” to date for the mouse macrophage. By harnessing advanced mass spectrometry techniques to accurately measure the levels of lipid molecules, and coupling these with gene-transcription analysis, they present snapshots through time of the lipidomic response to immune-system and pharmacological perturbations. The research uncovers both expected and unexpected changes, including evidence of cross-talk between different lipid categories.

To analyse the effects of an immune-system perturbation on cellular lipid levels, the researchers used Kdo2 Lipid A (KLA), the active component of an inflammatory lipopolysaccharide. This ligand mimics aspects of bacterial infection by specifically acting on Toll-like receptor 4. The team also tested compactin — a drug which inhibits cholesterol biosysnthesis — both with and without KLA stimulation.

Over 400 lipid species were analysed by six research teams using liquid chromatography-mass spectrometry techniques, applying a different protocol for each major lipid category. In addition, to investigate how and why lipid levels might change, the researchers also measured the cellular mRNA of over 20,000 genes, including those that encode lipid-synthesizing proteins. Both mRNA and lipid measurements were taken at time intervals following perturbation by KLA and compactin.

Using statistical techniques, the researchers calculated correlations between lipids and genes over time and were able to distinguish between the effects of KLA and compactin. Many genes coding for lipid biosynthetic proteins were differently expressed, correlating with associated lipid concentrations.

One pathway significantly altered by KLA was the arachidonate oxidation pathway, in which cyclooxygenase 2 activity, prostaglandins and secondary metabolites increased. However, unsaturated fatty acid levels decreased, correlating with down-regulated elongase and desaturase genes. Also decreased were acyl CoAs, which have a key position in the lipid synthetic system, and their associated genes.

Glycerolipids, glycerophospholipids, sphingolipids and sterol esters were all remodelled by KLA. In particular, the authors found evidence for de novo synthesis of sphingolipids and for sterol synthesis via acetyl CoA. Saturated and monounsaturated cholesteryl esters and their associated enzymes also increased, suggesting that these species might accumulate due to TLR4 activation. Because oxysterols promote sphingomyelin biosynthesis, the authors suggest that elevated sphingomyelins might be due to cross-talk between the two pathways. Galactoceramides, which promote phagocytosis by macrophages, were also elevated. Interestingly, increases in saturated and monounsaturated phosphatidic acid and phosphatidylinositol lipid species were seen as late as 24 hours following KLA stimulation.

As expected, compactin, which inhibits the conversion of HMG-CoA to mevalonic acid, blocked the biosynthesis of cholesterol, but did not prevent accumulation of cholesterol owing to lipoprotein particle degradation. It also slowed the KLA-induced increase in CoQ. Unexpectedly, however, compactin appeared to increase two inflammatory eicosanoids — PGD₂ and PGE₂ — and their synthetic enzymes. To explain this finding, the authors propose cross-talk between sterols and eicosanoids via a new, as yet undiscovered, pathway.

Overall, the paper highlights the growing maturity of the lipidomics field, showing how developments in mass spectrometry are allowing the accurate measurement of an ever-growing number of lipid molecules, and that such data can reveal not just concentrations of individual lipids, but processes involving the whole system.

Lipidomics Gateway (27 October 2010) [doi:10.1038/lipidmaps.2010.32]