Macrophages are central to essential physiological processes including the regulation of innate and adaptive immunity, but they are also central to a number of inflammatory disease states. These immune cells also possess remarkable plasticity and display various shades of functionalities based on changes in the surrounding molecular environment. Macrophage biology has defined various phenotypes and roles in inflammation based primarily on cytokine and chemokine profiles of cells in different activation states. Importantly, macrophages are elite producers of eicosanoids and other related lipid mediators during inflammation, but specific roles of these molecules have not generally been incorporated into the larger context of macrophage biology. In this review, we discuss the current classification of macrophage types and their roles in inflammation and disease, along with the practical challenges of studying biologically relevant phenotypes ex vivo. Using the latest advances in eicosanoid lipidomics, we highlight several key studies from our laboratory that provide a comprehensive understanding of how eicosanoid metabolism differs between macrophage phenotypes, along with how this metabolism is altered by changes in membrane fatty acid distribution and varied durations of Toll-like receptor (TLR) priming. In conclusion, we summarize several examples of the benefit of macrophage plasticity to develop accurate cellular mechanisms of lipid metabolism, and insights from lipidomic analyses about the differences in eicosanoid pathway enzyme activity in vitro vs. ex vivo cell studies. Examples of new techniques to further understand the role of macrophage eicosanoid signaling in vivo are also discussed.
Macrophage-mediated inflammation is a major contributor to obesity-associated insulin resistance. The co-repressor NCoR interacts with inflammatory pathway genes in macrophages, suggesting that its removal would result in increased activity of inflammatory responses. Surprisingly, we find that macrophage-specific deletion of NCoR instead results in an anti-inflammatory phenotype along with robust systemic insulin sensitization in obese mice. We present evidence that de-repression of LXRs contributes to this paradoxical anti-inflammatory phenotype by causing increased expression of genes that direct biosynthesis of palmitoleic acid and ω3 fatty acids. Remarkably, the increased ω3 fatty acid levels primarily inhibit NF-κB-dependent inflammatory responses by uncoupling NF-κB binding and enhancer/promoter histone acetylation from subsequent steps required for pro-inflammatory gene activation. This provides a mechanism for the in vivo anti-inflammatory insulin sensitive phenotype observed in mice with macrophage-specific deletion of NCoR. Therapeutic methods to harness this mechanism could lead to a new approach to insulin sensitizing therapies.
nuclear co-repressor; insulin resistance; obesity; macrophage; inflammation
Group VIA calcium-independent phospholipase A2 (GVIA iPLA2) has recently emerged as an important pharmaceutical target. Selective and potent GVIA iPLA2 inhibitors can be used to study its role in various neurological disorders. In the current work, we explore the significance of the introduction of a substituent in previously reported potent GVIA iPLA2 inhibitors. 1,1,1,2,2-Pentafluoro-7-(4-methoxyphenyl)heptan-3-one (GK187) is the most potent and selective GVIA iPLA2 inhibitor ever reported with a XI(50) value of 0.0001, and with no significant inhibition against GIVA cPLA2 or GV sPLA2. We also compare the inhibition of two difluoromethyl ketones on GVIA iPLA2, GIVA cPLA2, and GV sPLA2.
Phospholipase A2; Inhibitor; GVIA iPLA2; Polyfluoroalkyl ketone; Pentafluoroethyl ketones
The lipidome is composed of all of the biomolecules defined as lipids, which encompass compounds of amazing structural diversity and complexity. It has been ∼1 decade since the study of “lipidomics” was begun in earnest, and the technologies and tools for data analysis have advanced considerably over this period. This workshop summarized the scope of the lipidome and technologies for its analysis, lipidomics databases and other online tools, and examples of the application of lipidomics to nutritional research. The slides from the workshop, online lipidomics tools, and databases are available at http://www.lipidmaps.org.
Bioactive lipid mediators play a crucial role in the induction and resolution of inflammation. To elucidate their involvement during influenza infection, LC/MS lipidomic profiling of 141 lipid species was performed on a mouse influenza model using two viruses of significantly different pathogenicity. Infection by the low pathogenicity strain, X31/H3N2, induced a pro-inflammatory response followed by a distinct anti-inflammatory response; infection by the high pathogenicity strain, PR8/H1N1, resulted in overlapping pro- and anti-inflammatory states. Integration of the large-scale lipid measurements with targeted gene expression data demonstrated that 5 lipoxygenase metabolites correlated with the pathogenic phase of the infection whereas 12/15-lipoxygenase metabolites were associated with the resolution phase. Hydroxylated linoleic acid, specifically the ratio of 13- to 9-HODE, was identified as a potential biomarker for immune status during an active infection. Importantly, some of the findings from the animal model were recapitulated in studies of human nasopharyngeal lavages obtained during the 2009–2011 influenza seasons.
Lipid molecules play an important role in regulating the sensitivity of sensory neurons and enhancing pain perception, and growing evidence indicates that the effect occurs both at the site of injury and in the spinal cord. Using high-throughput mass spectrometry methodology, we sought to determine the contribution of spinal bioactive lipid species to inflammation-induced hyperalgesia in rats. Quantitative analysis of cerebrospinal fluid (CSF) and spinal cord tissue for eicosanoids, ethanolamides and fatty acids revealed the presence of 102 distinct lipid species. After induction of peripheral inflammation by intraplantar injection of carrageenan to the ipsilateral hind paw, lipid changes in cyclooxygenase (COX) and 12-lipoxygenase (12-LOX) signaling pathways peaked at 4 hours in the CSF. In contrast, changes occurred in a temporally disparate manner in the spinal cord with LOX-derived hepoxilins (HX) followed by COX-derived prostaglandin (PG) E2, and subsequently the ethanolamine anandamide (AEA). Systemic treatment with the mu opioid agonist morphine, the COX inhibitor ketorolac, or the LOX inhibitor NDGA significantly reduced tactile allodynia, while their effects on the lipid metabolites were different. Morphine did not alter the lipid profile in the presence or absence of carrageenan inflammation. Ketorolac caused a global reduction in eicosanoid metabolism in naïve animals that remained suppressed following injection of carrageenan. NDGA-treated animals also displayed reduced basal levels of COX and 12-LOX metabolites, but only 12-LOX metabolites remained decreased after carrageenan treatment. These findings suggest that both COX and 12-LOX play an important role in the induction of carrageenan-mediated hyperalgesia through these pathways.
The LIPID MAPS-Nature Lipidomics Gateway is a free, comprehensive online resource providing tutorials and instructional material, experimental data for lipids and genes along with protocols and standards, databases of lipid structures and lipid-associated genes or proteins, and a variety of lipidomics tools.
Graduate Education/Research; Upper-Division Undergraduate; Biochemistry; Interdisciplinary/Multidisciplinary; Computer-Based Learning; Internet/Web-Based Learning; Lipids; Mass Spectrometry
Oxidation of low-density lipoprotein (LDL) is one of the major causative mechanisms in the development of atherosclerosis. In previous studies, we showed that minimally oxidized LDL (mmLDL) induced inflammatory responses in macrophages, macropinocytosis and intracellular lipid accumulation and that oxidized cholesterol esters (OxCEs) were biologically active components of mmLDL. Here we identified a specific OxCE molecule responsible for the biological activity of mmLDL and characterized signaling pathways in macrophages in response to this OxCE. Using liquid chromatography – tandem mass spectrometry and biological assays, we identified an oxidized cholesteryl arachidonate with bicyclic endoperoxide and hydroperoxide groups (BEP-CE) as a specific OxCE that activates macrophages in a TLR4/MD-2-dependent manner. BEP-CE induced TLR4/MD-2 binding and TLR4 dimerization, phosphorylation of SYK, ERK1/2, JNK and c-Jun, cell spreading and uptake of dextran and native LDL by macrophages. The enhanced macropinocytosis resulted in intracellular lipid accumulation and macrophage foam cell formation. Bone marrow-derived macrophages isolated from TLR4 and SYK knockout mice did not respond to BEP-CE. The presence of BEP-CE was demonstrated in human plasma and in the human plaque material captured in distal protection devices during percutaneous intervention. Our results suggest that BEP-CE is an endogenous ligand that activates the TLR4/SYK signaling pathway. Because BEP-CE is present in human plasma and human atherosclerotic lesions, BEP-CE-induced and TLR4/SYK-mediated macrophage responses may contribute to chronic inflammation in human atherosclerosis.
Inflammation and macrophage foam cells are characteristic features of atherosclerotic lesions, but the mechanisms linking cholesterol accumulation to inflammation and LXR-dependent response pathways are poorly understood. To investigate this relationship, we utilized lipidomic and transcriptomic methods to evaluate the effect of diet and LDL receptor genotype on macrophage foam cell formation within the peritoneal cavities of mice. Foam cell formation was associated with significant changes in hundreds of lipid species and unexpected suppression, rather than activation, of inflammatory gene expression. We provide evidence that regulated accumulation of desmosterol underlies many of the homeostatic responses observed in macrophage foam cells, including activation of LXR target genes, inhibition of SREBP target genes, selective reprogramming of fatty acid metabolism and suppression of inflammatory response genes. These observations suggest that macrophage activation in atherosclerotic lesions results from extrinsic, pro-inflammatory signals generated within the artery wall that suppress homeostatic and anti-inflammatory functions of desmosterol.
Group VI Ca2+-independent phospholipase A2 (iPLA2) is a water-soluble enzyme that is active when associated with phospholipid membranes. Despite its clear pharmaceutical relevance, no X-ray or NMR structural information is currently available for the iPLA2 or its membrane complex. In this paper, we combine homology modeling with coarse-grained (CG) and all-atom (AA) molecular dynamics (MD) simulations to build structural models of iPLA2 in association with a phospholipid bilayer. CG-MD simulations of the membrane insertion process were employed to provide a starting point for an atomistic description. Six AA-MD simulations were then conducted for 60 ns, starting from different initial CG structures, to refine the membrane complex. The resulting structures are shown to be consistent with each other and with deuterium exchange mass spectrometry (DXMS) experiments, suggesting that our approach is suitable for the modeling of iPLA2 at the membrane surface. The models show that an anchoring region (residues 710–724) forms an amphipathic helix that is stabilized by the membrane. In future studies, the proposed iPLA2 models should provide a structural basis for understanding the mechanisms of lipid extraction and drug-inhibition. In addition, the dual-resolution approach discussed here should provide the means for the future exploration of the impact of lipid diversity and sequence mutations on the activity of iPLA2 and related enzymes.
The Ca2+-independent phospholipase A2 (iPLA2) enzyme is a potential target for the development of medicinal agents against heart and neurological diseases, multiple sclerosis, arthritis, and cancer. However, no structural information is currently available for the iPLA2. The binding of the enzyme to human membranes is driven by favorable electrostatic and non-polar interactions, but the detailed influence of these factors is not well understood. In this paper, we have combined coarse-grained and all-atom simulations of a homology model of the iPLA2. The coarse-grained description allows highly efficient simulations of the protein insertion into a lipid bilayer, while the all-atom simulations are used to refine the structures of the protein–membrane complexes. Finally, the resulting structures are validated experimentally with deuterium exchange experiments. In future works, this approach could be used to build models of other PLA2s. The iPLA2 models presented here open the door to the computational design of new inhibitors with improved potency and selectivity.
Cardiolipin, a major component of mitochondria, is critical for mitochondrial functioning including the regulation of cytochrome c release during apoptosis and proper electron transport. Mitochondrial cardiolipin with its unique bulky amphipathic structure is a potential substrate for phospholipase A2 (PLA2) in vivo. We have developed mass spectrometric methodology for analyzing PLA2 activity toward various cardiolipin forms and demonstrate that cardiolipin is a substrate for sPLA2, cPLA2 and iPLA2, but not for Lp-PLA2. Our results also show that none of these PLA2s have significant PLA1 activities toward dilyso-cardiolipin. To understand the mechanism of cardiolipin hydrolysis by PLA2, we also quantified the release of monolyso-cardiolipin and dilyso-cardiolipin in the PLA2 assays. The sPLA2s caused an accumulation of dilyso-cardiolipin, in contrast to iPLA2 which caused an accumulation of monolyso-cardiolipin. Moreover, cardiolipin inhibits iPLA2 and cPLA2, and activates sPLA2 at low mol fractions in mixed micelles of Triton X-100 with the substrate 1-palmitoyl-2-arachidonyl-sn-phosphtidylcholine. Thus, cardiolipin functions as both a substrate and a regulator of PLA2 activity and the ability to assay the various forms of PLA2 is important in understanding its function.
The Group IVA cytosolic phospholipase A2 (GIVA cPLA2) plays a central role in inflammation. Long chain 2-oxoamides constitute a class of potent GIVA cPLA2 inhibitors that exhibit potent in vivo anti-inflammatory and analgesic activity. We have now gained insight into the binding of 2-oxoamide inhibitors in the GIVA cPLA2 active site through a combination of molecular docking calculations and molecular dynamics simulations. Recently, the location of the 2-oxoamide inhibitor AX007 within the active site of the GIVA cPLA2 was determined using a combination of deuterium exchange mass spectrometry followed by molecular dynamics simulations. After the optimization of the AX007-GIVA cPLA2 complex using the docking algorithm Surflex-Dock, a series of additional 2-oxoamide inhibitors have been docked in the enzyme active site. The calculated binding affinity presents a good statistical correlation with the experimental inhibitory activity (r2 = 0.76, N = 11). A molecular dynamics simulation of the docking complex of the most active compound has revealed persistent interactions of the inhibitor with the enzyme active site and proves the stability of the docking complex and the validity of the binding suggested by the docking calculations. The combination of molecular docking calculations and molecular dynamics simulations is useful in defining the binding of small-molecule inhibitors and provides a valuable tool for the design of new compounds with improved inhibitory activity against GIVA cPLA2.
Persistent pain after resolution of clinically appreciable signs of arthritis poses a therapeutic challenge and immunosuppressive therapies do not meet this medical need. To investigate this conversion to persistent pain, we utilized the K/BxN serum transfer arthritis model, which has persistent mechanical hypersensitivity despite the resolution of visible inflammation. Toll-like receptor (TLR) 4 has been implicated as a potential therapeutic target in neuropathic and other pain models. We compared the relative courses of serum transfer arthritis and mechanical hypersensitivity in wild type (WT) and Tlr4−/− mice. K/BxN serum transfer induced similar joint swelling and inflammation from days 4–22 in WT and Tlr4−/− mice. Unlike WT mice, Tlr4−/− mice displayed a significant reversal in mechanical hypersensitivity and diminished appearance of glial activation markers after resolution of peripheral inflammation. Intrathecal (IT) delivery of a TLR4 antagonist, LPS-RS (10μg), on days 6, 9, and 12 abrogated the transition to persistent mechanical hypersensitivity in WT arthritic mice, while later administration had no impact. We utilized a lipodomics LC/MS/MS methodology to determine spinal cord profiles of bioactive lipid species following early LPS-RS treatment compared to vehicle treated controls. WT arthritic mice had reduced spinal levels of the anti-inflammatory prostaglandin 15d-PGJ2 on day 6, compared to IT LPS-RS treated mice. Direct IT application of 15d-PGJ2 (0.5μg) on day 6 improved mechanical hypersensitivity in arthritic mice within 15 minutes. Hence, TLR4 signaling altered spinal bioactive lipid profiles in the serum transfer model and played a critical role in the transition from acute to chronic post-inflammatory mechanical hypersensitivity.
arthritis; allodynia; astrocytes; microglia; inflammation; Toll-like receptor 4 (TLR4)
Historically considered to be simple membrane components serving as structural elements and energy storing entities, fatty acids are now increasingly recognized as potent signaling molecules involved in many metabolic processes. Quantitative determination of fatty acids and exploration of fatty acid profiles have become common place in lipid analysis. We present here a reliable and sensitive method for comprehensive analysis of free fatty acids and fatty acid composition of complex lipids in biological material. The separation and quantitation of fatty acids is achieved by capillary gas chromatography. The analytical method uses pentafluorobenzyl bromide derivatization and negative chemical ionization gas chromatography-mass spectrometry. The chromatographic procedure provides base line separation between saturated and unsaturated fatty acids of different chain lengths as well as between most positional isomers. Fatty acids are extracted in the presence of isotope-labeled internal standards for high quantitation accuracy. Mass spectrometer conditions are optimized for broad detection capacity and sensitivity capable of measuring trace amounts of fatty acids in complex biological samples.
Lipidomics, a major part of metabolomics, constitutes the detailed analysis and global characterization, both spatial and temporal, of the structure and function of lipids (the lipidome) within a living system. As with proteomics, mass spectrometry has earned a central analytical role in lipidomics, and this role will continue to grow with technological developments. Currently, there exist two mass spectrometry-based lipidomics approaches, one based on a division of lipids into categories and classes prior to analysis, the “comprehensive lipidomics analysis by separation simplification” (CLASS), and the other in which all lipid species are analyzed together without prior separation, shotgun. In exploring the lipidome of various living systems, novel lipids are being discovered, and mass spectrometry is helping characterize their chemical structure. Deuterium exchange mass spectrometry (DXMS) is being used to investigate the association of lipids and membranes with proteins and enzymes, and imaging mass spectrometry (IMS) is being applied to the in situ analysis of lipids in tissues.
CLASS; DXMS; imaging mass spectrometry; lipidomics; novel lipids; shotgun
Maternal immunization with oxidized low-density-lipoprotein prior to pregnancy prevents pathogenic in utero programming by gestational hypercholesterolemia, but it is unknown whether gestational hypercholesterolemia and maternal immunization affect similar pathways.
A lipidomic approach was used for unbiased plasma eicosanoid profiling in adult offspring of immunized and non-immunized normo- or hypercholesterolemic rabbit mothers.
Gestational hypercholesterolemia was associated with increased levels of some eicosanoids formed by the cyclooxygenase and 12-lipoxygenase pathways only (including TXB2, PGF2α, PGE2, and PGD2). Immunization of hypercholesterolemic or normocholesterolemic mothers reduced 9 of 14 eicosanoids of the cyclooxygenase pathway, 21 of 23 eicosanoids of the 5- and 12-lipoxygenase pathways (e.g. 5-HETE, HXB3, 12-HETE), 8 of 19 eicosanoids of the cytochrome P-450 pathway and all metabolites of the nonenzymatic pathway.
Maternal immunization not only counteracts in utero programming by gestational hypercholesterolemia but reduces a broad range of eicosanoid modulators of immunity and inflammation in offspring.
Cardiovascular disease; developmental programming; eicosanoids; immunization; inflammation; rabbits
Lipoprotein-associated phospholipase A2 (Lp-PLA2) plays important roles in both the inhibition and promotion of inflammation in human disease. It catalyzes the hydrolytic inactivation of plasma platelet activating factor (PAF) and is also known as PAF acetylhydrolase. High levels of PAF are implicated in a variety of inflammatory diseases such as asthma, necrotizing enterocolitis and sepsis. Lp-PLA2 also associates with lipoproteins in human plasma where it hydrolyzes oxidized phospholipids to produce pro-inflammatory lipid mediators that can promote inflammation and the development of atherosclerosis. Lp-PLA2 plasma levels have recently been identified as a biomarker of vascular inflammation, atherosclerotic vulnerability, and future cardiovascular events. The enzyme is thus a prominent target for the development of inflammation and atherosclerosis-modulating therapeutics. While the crystallographically-determined structure of the enzyme is known, the enzyme's mechanism of interaction with PAF and the function-modulating lipids in lipoproteins is unknown. We have employed peptide amide hydrogen-deuterium exchange mass spectrometry (DXMS) to characterize the association of Lp-PLA2 with dimyristoyl phosphatidylcholine (DMPC) vesicles, and found that specific residues 113-120 in one of the enzyme's surface-disposed hydrophobic α-helices likely mediate liposome binding.
Group VIA calcium-independent phospholipase A2 (GVIA iPLA2) has recently emerged as a novel pharmaceutical target. We have now explored the structure-activity relationship between fluoroketones and GVIA iPLA2 inhibition. The presence of a naphthyl group proved to be of paramount importance. 1,1,1-Trifluoro-6-(naphthalen-2-yl)hexan-2-one (FKGK18) is the most potent inhibitor of GVIA iPLA2 (XI(50) 0.0002) ever reported. Being 195 and >455 times more potent for GVIA iPLA2 than for GIVA cPLA2 and GV sPLA2, respectively, makes it a valuable tool to explore the role of GVIA iPLA2 in cells and in vivo models. 1,1,1,2,2,3,3-Heptafluoro-8-(naphthalene-2-yl) octan-4-one inhibited GVIA iPLA2 with a XI(50) value of 0.001, while inhibiting the other intracellular GIVA cPLA2 and GV sPLA2 at least 90-times less potently. Hexa- and octa-fluoro ketones were also found to be potent inhibitors of GVIA iPLA2; however they are not selective.
heptafluoropropyl ketones; inhibitors; polyfluoro ketones; phospholipase A2
The cyclooxygenase (COX) enzymes are known modulators of innate immune cell function; however, their contributions to adaptive immunity are relatively unknown. We investigated the roles of COX-1 and COX-2 in the humoral immune response to infection with the Lyme disease pathogen, Borrelia burgdorferi. We report that in vitro, murine B cells constitutively expressed COX-1 and up-regulated expression of both COX-1 and COX-2 as well as their products PGE2, PGF2α and TXB2 and their receptors following stimulation with B. burgdorferi or anti-CD40. In vitro inhibition of COX-1 and/or COX-2 in murine B cells resulted in decreased eicosanoid production, and altered antibody production. Importantly, infection of mice lacking COX-1, but not COX-2 activity resulted in a defect in immunoglobulin class-switching and a lack of Borrelia-specific IgG production. This defect correlated with decreased germinal center formation and IL-6 and IL-17 production, and could be partially recovered by restoration of IL-6, but fully recovered by IL-17. Furthermore, sera from COX-1 inhibitor-treated mice were dramatically less effective in killing B. burgdorferi, but borreliacidal activity was restored in COX-1 inhibitor-treated mice administered IL-17. We conclude that IL-17 plays a role in antibody production and immunoglobulin class-switching in response to infection and that COX-1 is a critical, previously unrecognized regulator of this response.
Group IVA phospholipase A2 (GIVA PLA2) catalyzes the release of arachidonic acid (AA) from the sn-2 position of glycerophospholipids. AA is then further metabolized into terminal signaling molecules including numerous prostaglandins. We have now demonstrated the involvement of phosphatidic acid phosphohydrolase 1 (PAP-1) and protein kinase C (PKC) in the Toll-like receptor-4 (TLR-4) activation of GIVA PLA2. We also studied the effect of PAP-1 and PKC on Ca+2 induced and synergy enhanced GIVA PLA2 activation. We observed that the AA release induced by exposure of RAW 264.7 macrophages to the TLR-4 specific agonist Kdo2-Lipid A is blocked by the PAP-1 inhibitors bromoenol lactone (BEL) and propranolol as well as the PKC inhibitor Ro 31-8220; however these inhibitors did not reduce AA release stimulated by Ca+2 influx induced by the P2X7 purinergic receptor agonist ATP. Additionally, stimulation of cells with diacylglycerol (DAG), the product of PAP-1 mediated hydrolysis, initiated AA release from unstimulated cells as well as restored normal AA release from cells treated with PAP-1 inhibitors. Finally, neither PAP-1 nor PKC inhibition reduced GIVA PLA2 synergistic activation by stimulation with Kdo2–Lipid A and ATP.