Darren S. Dumlao

High-Throughput Lipidomic Analysis of Fatty Acid Derived Eicosanoids and N-Acylethanolamines

Fatty acid-derived eicosanoids and N-acylethanolamines (NAE) are important bioactive lipid mediators involved in numerous biological processes including cell signaling and disease progression. To facilitate research on these lipid mediators, we have developed a targeted high-throughput mass spectrometric based methodology to monitor and quantitate both eicosanoids and NAEs, and can be analyzed separately or together in series. Each methodology utilizes scheduled multiple reaction monitoring (sMRM) pairs in conjunction with a 25 min reverse-phase HPLC separation. The eicosanoid methodology monitors 141 unique metabolites and quantitative amounts can be determined for over 100 of these metabolites against standards. The analysis covers eicosanoids generated from cycloxygenase, lipoxygenase, cytochrome P450 enzymes, and those generated from non-enzymatic pathways. The NAE analysis monitors 36 metabolites and quantitative amounts can be determined for 33 of these metabolites against standards. The NAE method contains metabolites derived from saturated fatty acids, unsaturated fatty acids, and eicosanoids. The lower limit of detection for eicosanoids ranges from 0.1pg to 1pg, while NAEs ranges from 0.1pg to 1000pg. The rationale and design of the methodology is discussed.

Spinal 12-lipoxygenase-derived hepoxilin A3 contributes to inflammatory hyperalgesia via activation of TRPV1 and TRPA1 receptors

Peripheral inflammation initiates changes in spinal nociceptive processing leading to hyperalgesia. Previously, we demonstrated that among 102 lipid species detected by LC-MS/MS analysis in rat spinal cord, the most notable increases that occur after intraplantar carrageenan are metabolites of 12-lipoxygenases (12-LOX), particularly hepoxilins (HXA3 and HXB3). Thus, we examined involvement of spinal LOX enzymes in inflammatory hyperalgesia. In the current work, we found that intrathecal (IT) delivery of the LOX inhibitor nordihydroguaiaretic acid prevented the carrageenan-evoked increase in spinal HXB3 at doses that attenuated the associated hyperalgesia. Furthermore, IT delivery of inhibitors targeting 12-LOX (CDC, Baicalein), but not 5-LOX (Zileuton) dose-dependently attenuated tactile allodynia. Similarly, IT delivery of 12-LOX metabolites of arachidonic acid 12(S)-HpETE, 12(S)-HETE, HXA3, or HXB3 evoked profound, persistent tactile allodynia, but 12(S)-HpETE and HXA3 produced relatively modest, transient heat hyperalgesia. The pronociceptive effect of HXA3 correlated with enhanced release of Substance P from primary sensory afferents. Importantly, HXA3 triggered sustained mobilization of calcium in cells stably overexpressing TRPV1 or TRPA1 receptors and in acutely dissociated rodent sensory neurons. Constitutive deletion or antagonists of TRPV1 (AMG9810) or TRPA1 (HC030031) attenuated this action. Furthermore, pretreatment with antihyperalgesic doses of AMG9810 or HC030031 reduced spinal HXA3-evoked allodynia. These data indicate that spinal HXA3 is increased by peripheral inflammation and promotes initiation of facilitated nociceptive processing through direct activation of TRPV1 and TRPA1 at central terminals.

Spinal TLR4 mediates the transition to a persistent mechanical hypersensitivity after the resolution of inflammation in serum-transferred arthritis

Summary Neither Tlr4−/− K/BxN serum transfer arthritic mice nor those administered spinal TLR4 pharmacological blockade display persistent mechanical hypersensitivity, in contrast to WT mice. ABSTRACT 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, lipopolysaccharide Rhodobacter sphaeroides (LPS‐RS; 10 μg), 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 lipidomics liquid chromatography tandem mass spectrometry methodology to determine spinal cord profiles of bioactive lipid species after early LPS‐RS treatment compared to vehicle‐treated control animals. WT arthritic mice had reduced spinal levels of the anti‐inflammatory prostaglandin 15‐deoxy‐Δ12,14‐PGJ2 (15d‐PGJ2) on day 6, compared to IT LPS‐RS‐treated mice. Direct IT application of 15d‐PGJ2 (0.5 μg) on day 6 improved mechanical hypersensitivity in arthritic mice within 15 min. 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 postinflammatory mechanical hypersensitivity.

Pharmacological correction of a defect in PPAR-[gamma] signaling ameliorates disease severity in Cftr-deficient mice

Cystic fibrosis is caused by mutations in the cystic fibrosis transmembrane conductance regulator (encoded by Cftr) that impair its role as an apical chloride channel that supports bicarbonate transport. Individuals with cystic fibrosis show retained, thickened mucus that plugs airways and obstructs luminal organs as well as numerous other abnormalities that include inflammation of affected organs, alterations in lipid metabolism and insulin resistance. Here we show that colonic epithelial cells and whole lung tissue from Cftr-deficient mice show a defect in peroxisome proliferator–activated receptor-γ (PPAR-γ, encoded by Pparg) function that contributes to a pathological program of gene expression. Lipidomic analysis of colonic epithelial cells suggests that this defect results in part from reduced amounts of the endogenous PPAR-γ ligand 15-keto-prostaglandin E2 (15-keto-PGE2). Treatment of Cftr-deficient mice with the synthetic PPAR-γ ligand rosiglitazone partially normalizes the altered gene expression pattern associated with Cftr deficiency and reduces disease severity. Rosiglitazone has no effect on chloride secretion in the colon, but it increases expression of the genes encoding carbonic anhydrases 4 and 2 (Car4 and Car2), increases bicarbonate secretion and reduces mucus retention. These studies reveal a reversible defect in PPAR-γ signaling in Cftr-deficient cells that can be pharmacologically corrected to ameliorate the severity of the cystic fibrosis phenotype in mice.

Specificity of eicosanoid production depends on the TLR-4-stimulated macrophage phenotype

Eicosanoid metabolism differs in profile and quantity between macrophages of different tissue origin and method of elicitation, as well as between primary and immortalized macrophages after activation with inflammatory stimuli. Using a lipidomic approach, we comprehensively analyzed the eicosanoids made by murine RPMs, TGEMs, BMDM, and the macrophage‐like cell line RAW after stimulation with the TLR‐4‐specific agonist KLA. Direct correlation among total COX metabolites, COX side‐products (11‐HETE, 15‐HETE), COX‐2 mRNA, and protein at 8 h was found when comparing each cell type. Comprehensive qPCR analysis was used to compare relative transcript levels between the terminal prostanoid synthases themselves as well as between each cell type. Levels of PGE2, PGD2, and TxB2 generally correlated with enzyme transcript expression of PGES, PGDS, and TBXS, providing evidence of comparable enzyme activities. PGIS transcript was expressed only in RPM and TGEM macrophages and at an exceptionally low level, despite high metabolite production compared with other synthases. Presence of PGIS in RPM and TGEM also lowered the production of PGE2 versus PGD2 by approximately tenfold relative to BMDM and RAW cells, which lacked this enzyme. Our results demonstrate that delayed PG production depends on the maximal level of COX‐2 expression in different macrophages after TLR‐4 stimulation. Also, the same enzymes in each cell largely dictate the profile of eicosanoids produced depending on the ratios of expression between them, with the exception of PGIS, which appears to have much greater synthetic capacity and competes selectively with mPGES‐1.

Inflammatory hyperalgesia induces essential bioactive lipid production in the spinal cord.

J. Neurochem. (2010) 114, 981–993.

Lipidomics Analysis of Essential Fatty Acids in Macrophages

The Lipid Metabolites and Pathway Strategy (LIPID MAPS) Consortium is a nationwide initiative that has taken on the task of employing lipidomics to advance our understanding of lipid metabolism at the molecular and mechanistic level in living organisms. An important step toward this goal is to craft enabling analytical procedures to comprehensively measure all lipid species, to establish the precise structural identity of the lipid molecules analyzed, and to generate accurate quantitative information. The LIPID MAPS Consortium has succeeded in the implementation of a complete infrastructure that now provides tools for analysis of the global lipidome in cultured and primary cells. Here we illustrate the advancement of a gas chromatography mass spectrometry (GC/MS) procedure for the analysis of essential fatty acids in RAW 264.7 cells. Our method allows for the specific identification and quantification of over 30 fatty acids present in cells in their free form in a single analytical GC/MS run. Free fatty acids are selectively extracted in the presence of deuterated internal standards, which permit subsequent estimation of extraction efficiencies and quantification with high accuracy. Mass spectrometer conditions were optimized for single-ion monitoring, which provides an extremely sensitive technology to measure fatty acids from biological samples in trace amounts. These methods will be presented in the context of our broader effort to analyze all fatty acids as well as their metabolites in inflammatory cells.

Assessing Phospholipase A2 Activity toward Cardiolipin by Mass Spectrometry

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.

Polyoxygenated Cholesterol Ester Hydroperoxide Activates TLR4 and SYK Dependent Signaling in Macrophages

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.

Systematic analysis of rat 12/15-lipoxygenase enzymes reveals critical role for spinal eLOX3 hepoxilin synthase activity in inflammatory hyperalgesia

Previously, we observed significant increases in spinal 12‐lipoxygenase (LOX) metabolites, in particular, hepoxilins, which contribute to peripheral inflammation‐induced tactile allodynia. However, the enzymatic sources of hepoxilin synthase (HXS) activity in rats remain elusive. Therefore, we overexpressed each of the 6 rat 12/15‐LOX enzymes in HEK‐293T cells and measured by LC‐MS/MS the formation of HXB3, 12‐HETE, 8‐HETE, and 15‐HETE from arachidonic acid (AA) at baseline and in the presence of LOX inhibitors (NDGA, AA‐861, CDC, baicalein, and PD146176) vs. vehicle‐treated and mock‐transfected controls. We detected the following primary intrinsic activities: 12‐LOX (Alox12, Alox15), 15‐LOX (Alox15b), and HXS (Alox12, Alox15). Similar to human and mouse orthologs, proteins encoded by rat Alox12b and Alox12e possessed minimal 12‐LOX activity with AA as substrate, while eLOX3 (encoded by Aloxe3) exhibited HXS without 12‐LOX activity when coexpressed with Alox12b or supplemented with 12‐HpETE. CDC potently inhibited HXS and 12‐LOX activity in vitro (relative IC50s: CDC, ~0.5 and 0.8 μM, respectively) and carrageenan‐evoked tactile allodynia in vivo. Notably, peripheral inflammation significantly increased spinal eLOX3; intrathecal pretreatment with either siRNA targeting Aloxe3 or an eLOX3‐selective antibody attenuated the associated allodynia. These findings implicate spinal eLOX3‐mediated hepoxilin synthesis in inflammatory hyperesthesia and underscore the importance of developing more selective 12‐LOX/HXS inhibitors.—Gregus, A. M., Dumlao, D. S., Wei, S. C., Norris, P. C., Catella, L. C., Meyerstein, F. G., Buczynski, M. W., Steinauer, J. J., Fitzsimmons, B. L., Yaksh, T. L., Dennis, E. A. Systematic analysis of rat 12/15‐lipoxygenase enzymes reveals critical role for spinal eLOX3 hepoxilin synthase activity in inflammatory hyperalgesia. FASEB J. 27, 1939–1949 (2013). www.fasebj.org