11 Mediator Lipidomics in Inflammation Research Makoto Arita, Ryo Iwamoto, and Yosuke Isobe 11.1 Introduction Polyunsaturated fatty acids (PUFAs) exhibit a range of biological effects, many of which are mediated through the formation and actions of lipid mediators such as prostaglandins (PGs), leukotrienes (LTs), lipoxins (LX), resolvins, and protectins. These lipid mediators are potent endogenous regulators of inflammation and related diseases. To better understand the molecular and cellular mechanisms underlying the coordinated processes of inflammation and resolution, it is impor- tant to know when, where, and how much of those lipid mediators are formed in the inflammatory sites. Mediator lipidomics in general, when combined with infor- mation on metabolic pathways such as Kyoto Encyclopedia of Genes and Genomes (KEGG), have broad applications in understanding the role of bioactive lipid media- tors under certain physiological and/or pathological conditions. The potential chal- lenges of lipid mediator analyses are low abundance, labile, and overlapping parent masses and products. In this chapter, we will introduce LC-ESI-MS/MS-based lipi- domics, which is suitable for quantifying lipid mediators in complex samples because this system can provide superior sensitivity, selectivity, and rapid analysis. 11.2 PUFA-Derived Lipid Mediators: Formation and Action PUFA-derived lipid mediators are formed by enzymatic oxidation through the action of cyclooxygenases (COX), lipoxygenases (LOX), and cytochrome P450 monooxygenases (CYP). Arachidonic acid (20:4 n-6) is a common precursor of many eicosanoids, which are bioactive lipid mediators that control inflammatory responses. When cells are stimulated, arachidonic acid is released from membrane phospholipids by phospholipase A 2 (PLA 2 ), which hydrolyzes the acyl ester bond. This is the first step in the arachidonic acid cascade, and is the overall rate- determining step in the generation of eicosanoids. Mammals have three types of PLA 2 , which are classified as secretory, cytoplasmic, and calcium-independent Lipidomics, First Edition. Edited by Kim Ekroos. # 2012 Wiley-VCH Verlag GmbH & Co. KGaA. Published 2012 by Wiley-VCH Verlag GmbH & Co. KGaA. j219
Transcript
11Mediator Lipidomics in Inflammation ResearchMakoto Arita, Ryo Iwamoto, and Yosuke Isobe
11.1Introduction
Polyunsaturated fatty acids (PUFAs) exhibit a range of biological effects, many ofwhich are mediated through the formation and actions of lipid mediators such asprostaglandins (PGs), leukotrienes (LTs), lipoxins (LX), resolvins, and protectins.These lipid mediators are potent endogenous regulators of inflammation andrelated diseases. To better understand the molecular and cellular mechanismsunderlying the coordinated processes of inflammation and resolution, it is impor-tant to know when, where, and how much of those lipid mediators are formed inthe inflammatory sites. Mediator lipidomics in general, when combined with infor-mation on metabolic pathways such as Kyoto Encyclopedia of Genes and Genomes(KEGG), have broad applications in understanding the role of bioactive lipid media-tors under certain physiological and/or pathological conditions. The potential chal-lenges of lipid mediator analyses are low abundance, labile, and overlapping parentmasses and products. In this chapter, we will introduce LC-ESI-MS/MS-based lipi-domics, which is suitable for quantifying lipid mediators in complex samplesbecause this system can provide superior sensitivity, selectivity, and rapid analysis.
11.2PUFA-Derived Lipid Mediators: Formation and Action
PUFA-derived lipid mediators are formed by enzymatic oxidation through theaction of cyclooxygenases (COX), lipoxygenases (LOX), and cytochrome P450monooxygenases (CYP). Arachidonic acid (20:4 n-6) is a common precursor ofmany eicosanoids, which are bioactive lipid mediators that control inflammatoryresponses. When cells are stimulated, arachidonic acid is released from membranephospholipids by phospholipase A2 (PLA2), which hydrolyzes the acyl ester bond.This is the first step in the arachidonic acid cascade, and is the overall rate-determining step in the generation of eicosanoids. Mammals have three types ofPLA2, which are classified as secretory, cytoplasmic, and calcium-independent
Lipidomics, First Edition. Edited by Kim Ekroos.# 2012 Wiley-VCH Verlag GmbH & Co. KGaA. Published 2012 by Wiley-VCH Verlag GmbH & Co. KGaA.
j219
PLA2s (sPLA2s, cPLA2s, iPLA2s), respectively [1]. The different types of PLA2 areregulated differently and are expressed in different tissues. Unesterified intra-cellular arachidonic acid is immediately metabolized by COX, LOX, or CYP. TheCOX pathway leads to the formation of prostaglandins and thromboxanes (TX); theLOX pathway leads to leukotrienes, lipoxins, and hydroeicosatetraenoic acids(HETE); and the CYP pathway leads to HETEs and epoxyeicosatrienoic acids (EET)(Figure 11.1). As a class, these molecules act as autacoids that are rapidly synthe-sized in response to specific stimuli, act quickly at the immediate locality, andremain active for only a short time before degradation.Arachidonic acid-derived eicosanoids are important in many physiological pro-
cesses. Under nondisease conditions, PGs contribute to the maintenance ofhomeostasis, for example, they have cytoprotective roles in the gastric mucosa,
R : choline, ethanolamine,serine, inositol, and so on
Cleavage site
Phospholipids
Phospholipase A2
Arachidonic acid (AA)
LipoxygenaseCyclooxygenase
Cytochrome P450(LOX)
(COX)(CYP P450)
LeukotrienesLipoxinsHydroxyeicosa-
ProstaglandinsThromboxane
Epoxyeicosa-tetraenoic acids(EETs)
tetraenoic acids(HETEs)
HETEs
Figure 11.1 Eicosanoid production fromarachidonic acid. Phospholipase A2 cleaves theester bond of phospholipids marked by thearrow to release arachidonic acid. Freearachidonic acid is used as a substrate for thecyclooxygenase (COX), lipoxygenase (LOX), andcytochrome P450 monooxygenase (CYP)
pathways. The COX pathway producesprostaglandins and thromboxane. The LOXpathway produces leukotrienes, lipoxins, andhydroxyeicosatetraenoic acids (HETEs). TheCYP pathway produces epoxyeicosatrienoicacids (EETs) and HETEs.
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respiratory tract, and renal parenchyma. PGs are also involved in proinflammatoryprocesses and are responsible for many of the hallmark signs of inflammation suchas heat, redness, swelling, and pain [2]. COX occurs in two isoforms, calledthe constitutive isoform (COX-1) and the inducible isoform (COX-2) [3]. Theseenzymes may contribute to the production of different sets of eicosanoids atdifferent locations at different times. The LOX pathway represents another majorpathway to produce LTs and LXs. Mammals have at least three LOXs, 5-, 12-, and15-LOX present in mammalian systems [4, 5]. 5-LOX-derived LTs (LTB4, cysteinylLTs) are involved in proinflammatory processes such as neutrophil infiltration, increased vascular permeability, and smooth muscle contraction [6]. In contrast, 5-and 15-LOX-derived LXA4 counterregulate the proinflammatory processes and maybe important in the resolution of inflammation [7]. An imbalance in lipoxin–leuko-triene homeostasis may be a key factor in the pathogenesis of inflammatory diseases. The epoxygenation of arachidonic acid by CYP generates EETs, which may haveroles in the regulation of smooth muscle cells and vascular tone [8]. Many of theeicosanoids signal via seven-transmembrane G-protein-coupled receptors [9].Eicosapentaenoic acid (EPA) (20:5 n-3) and docosahexaenoic acid (DHA) (22:6
n-3) are n-3 PUFAs that are abundant in fish oils. EPA-derived mediators include3-series PGs, 5-series LTs and LXs, hydroxyeicosapentaenoic acids (HEPE), andepoxyeicosatetraenoic acids (EpETE) (Figure 11.2). DHA is also converted to
AA
Prostaglandins
LeukotrienesLipoxins
HETEsEETs
EPA
Three-series-Prostaglandins
Five-series-LeukotrienesLipoxins
E-series-Resolvins
DHA
D-series-ResolvinsDocosatrienesProtectinMaresin
Leukotriene B4
Prostaglandin E2 Lipoxin A4
Resolvin E1
10,17-Docosatriene(Protectin D1)
Figure 11.2 Scheme of eicosanoid, docosanoid, and hydroxy-fatty acid production fromarachidonic acid (AA), eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA).
11.2 PUFA-Derived Lipid Mediators: Formation and Action j221
hydroxydocosahexaenoic acids (HDoHE) and epoxy-fatty acids by enzymatic oxida-tion (Figure 11.2). Dietary supplementation of n-3 PUFA has beneficial effects inmany inflammatory disorders, including cardiovascular disease, arthritis, colitis,and metabolic syndrome [10]. Also, elevation in n-3 PUFA levels in n-3 desaturase(fat-1) transgenic mice protected against inflammatory disease models [11]. n-3PUFAs are thought to act via several mechanisms. One role is to serve as an alternative substrate for COX or LOX, resulting in the production of less potent prod-ucts [12]. Another role is to be converted to potent anti-inflammatory and protectivemediators. For example, E-series resolvins are produced from EPA, and D-series resolvins, protectin, and maresin are produced from DHA (Figure 11.2) [13]. Thesemetabolites may have some roles in the beneficial actions of n-3 PUFAs in control-ling inflammation and related diseases.
11.3LC-ESI-MS/MS-Based Lipidomics
A powerful approach for the analysis of mono- and polyhydroxylated fatty acids isliquid chromatography tandem mass spectrometry (LC-ESI-MS/MS). The develop-ment of electrospray ionization (ESI) technology provided an ideal interfacebetween LC and MS, which paved the way for the analysis of hydroxy-fatty acidswithout derivatization and decomposition. ESI is a soft ionization technology usedto form either positive or negative ions through the addition of a proton to form[MþH]þ or through the removal of a proton to form [M�H]�. In case of fattyacid-derived mediators, ESI results in [M�H]� carboxylate ions that can bedetected with relatively high sensitivity. A triple quadrupole mass spectrometer iscapable of carrying out an MS/MS method called multiple reaction monitoring(MRM). A specified precursor ion is selected according to its mass-to-charge ratioin the first quadrupole mass filter and is fragmented into product ions in the sec-ond chamber by collision-induced dissociation (CID). Then, the third quadrupolemass filter is locked on its specified product ion. This MRM mode leads to furtherimprovement of the detection and quantification limits when combined with high-resolution LC separations. We have developed a comprehensive LC-ESI-MS/MSmethod that can simultaneously detect and quantify more than 250 PUFA metabo-lites, including PGs, LTs, LXs, resolvins, protectins, and other AA-, EPA-, DHA-derived products (Figure 11.3).
11.3.1Sample Preparation
Liquid samples are adjusted to 67% methanol (v/v) and kept at �20 �C. Frozen tis-sue samples are homogenized in ice-cold methanol using Precellys 24 biologicalsample grinder (Bertin Technologies). The samples are centrifuged to remove pre-cipitated proteins. The supernatants are diluted with ice-cold water and adjusted to10% (v/v) methanol. Internal standards (1 ng of PGE2-d4, LTB4-d4, 15-HETE-d8,
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and arachidonic acid-d8) are added to each sample, and the clear supernatants acid-ified to pH 4.0 are immediately applied to preconditioned solid-phase extractioncartridges (Sep-Pak C18, Waters) to extract the lipid mediators. Sep-Pak cartridgesare first washed with 20ml water and then with 20ml hexane, and finally the lipidmediators (hydroxy-fatty acids) are eluted with 10ml methyl formate. The lipidextracts are dried under a gentle stream of nitrogen gas, dissolved in 60% metha-nol, and stored at �20 �C before analysis.
11.3.2LC-ESI-MS/MS Analysis
LC-MS/MS-based lipidomic analyses are performed on an Acquity UPLC BEHC18 column (1.0mm� 150mm� 1.7 mm) using an Acquity UltraPerformanceLC system (UPLC; Waters) coupled to an electrospray (ESI) triple quadrupolemass spectrometer (5500 QTRAP; AB SCIEX). Instrument control and dataacquisition are performed using Analyst 1.5.1 (AB SCIEX). Samples are elutedwith a mobile phase consisting of water/acetate (100 : 0.1, v/v) and acetonitri-le/methanol (4 : 1, v/v) (73 : 27) for 5min and ramped to 30 : 70 after 15min, to20 : 80 after 25min and held for 8min, to 0 : 100 after 35min and held for10min with flow rates of 50 ml/min (0–30min), 80 ml/min (30–33min), and100 ml/min (33–45min). MS/MS analyses are conducted in negative ion mode,and fatty acid metabolites are detected and quantified by scheduled MRM withdwell times of �10ms and interchannel delay of 3ms, giving a total cycle timeof 1.3 s. Conditions for the detection of each compound by MRM are repre-sented in Table 11.1. Identification of the target compounds is based on the LCretention time of the analyte compared to that of a standard and on the ratio ofabundance of two specific MRM transitions. For quantification, composite stan-dard solutions ranging from 0.1 to 100 pg/ml are prepared, and calibration linesare calculated by the least-squares linear regression method, as represented inFigure 11.4.
SampleSolid-phaseextraction HPLC ESI Triple quadrupole
MS/MS
Multiple reaction monitoring (MRM)
Figure 11.3 Flow chart depicting the system of LC-ESI-MS/MS-based lipidomics. After solid-phase extraction, samples are separated by HPLC and fatty acid metabolites are detected andquantified by multiple reaction monitoring (MRM) using triple quadrupole MS/MS.
11.3 LC-ESI-MS/MS-Based Lipidomics j223
Table 11.1 Multiple reaction monitoring (MRM) transitions and LC retention times of lipidmediators and hydroxy-fatty acids.
Compound MRM transition (m/z) Retention time (min)
Representative chromatograms of mono-HEPEs are depicted in Figure 11.5.Among HEPE isomers, 8-HEPE and 12-HEPE did not resolve well using a C18 col-umn (retention time of 21.2min). However, the choice of structure-specific productions allowed their differentiation (8-HEPE m/z 317> 155 and 12-HEPE m/z317> 179). Also, the MRM transition used for 18-HEPE (m/z 317> 215) showedcross-reactivity with 17,18-EpETE, but these hydroxy- and epoxy-fatty acids wereresolved well by C18 column chromatography (retention times of 20.4 and22.5min, respectively). Therefore, the LC-MS/MS system using MRM mode pro-vides structure-specific signal detection and further improves the quantificationlimits when combined with high-resolution LC separations. New MS instrumentssuch as QTRAP 5500 can conduct hundreds of MRM analyses, and high-resolutionLCs such as UPLC can improve quantification by optimal peak separation.
11.4Mediator Lipidomics in Inflammation and Resolution
In many human diseases, uncontrolled inflammation is suspected as a key compo-nent of pathogenesis [14]. Acute inflammation is an indispensable host response toinsult or tissue injury. However, excessive or inappropriate inflammatory responsescan cause local tissue damage and remodeling, which contribute to a range ofchronic diseases. In healthy individuals, acute inflammation is self-limiting andhas an active termination program [15]. Therefore, the mechanisms by which acuteinflammation is resolved are of interest.
Inte
nsity
15-HEPEy = 2562.1x + 16131
R² = 0.99915
1.0E+06
2.0E+06
3.0E+065-HEPEy = 3619.5x + 15783
R² = 0.99956
1.0E+06
2.0E+06
3.0E+06
4.0E+06
0.0E+0010005000
LTB4y = 3651.7x + 32044
R² = 0.99760
2.0E+06
3.0E+06
4.0E+06
0.0E+0010005000
LXA4y = 1607.7x + 1515.4
R² = 0.99875
1.0E+06
2.0E+06
Inte
nsity
/ 10µlpg
0.0E+00
1.0E+06
100050000.0E+00
10005000
/ 10µlpg
Figure 11.4 Representative standard curves for hydroxy-fatty acids.
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The local inflammatory response is characterized by a sequential release of medi-ators and the recruitment of different types of leukocytes that become activated atthe inflamed site [15, 16]. To better understand the molecular and cellular mecha-nisms underlying the coordinated processes of inflammation and resolution, weapplied LC-ESI-MS/MS-based mediator lipidomics to the self-resolving acuteinflammation model, namely, murine zymosan-induced peritonitis [17]. As shownin Figure 11.6, temporal and quantitative differences in the lipid mediator profileswere observed in the course of acute inflammation and resolution. COX pathway
5-HEPE5-HEPE
21.821.8
m/z 317 > 115317 > 11521.221.2
8-HEPE8-HEPE
12-HEPE12-HEPE
21.221.2
20.820.8 m/zm/z 317 > 179317 > 179
m/zm/z 317 > 155317 > 155
15-HEPE15-HEPE
20.420.4
22.522.514.9
18-HEPE18-HEPE 17,18-EpETE17,18-EpETE
m/zm/z 317 > 219317 > 219
m/zm/z 317 > 259317 > 25915.315.3
PGEPGE2
Rel
ativ
e A
bund
ance
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ativ
e A
bund
ance
18.018.0 m/zm/z 351 > 217351 > 217
LXA4
15.915.9 m/zm/z 351 > 271351 > 271
PGDPGD2
LTBLTB4
PD1
18.518.5 m/zm/z 359 > 153359 > 153
m/zm/z 335 > 195335 > 195
TICTIC
Retention Time ( min )Retention Time ( min )10.0 40.040.0
Figure 11.5 Representative MRM chromatograms for hydroxy-fatty acids.
11.4 Mediator Lipidomics in Inflammation and Resolution j227
products such as PGE2 and PGD2 appeared upon zymosan stimulation and werepresent in both the initiation (4 h) and the early resolution (24 h) phases. In con-trast, 5-LOX pathway products such as LTB4 appeared only in the initiation phase.Of interest, the 12/15-LOX pathway products, including DHA-derived mediatorprotectin D1 (PD1), were present in the lavage of naive mice. Upon zymosan chal-lenge, the concentrations of the 12/15-LOX products decreased in the initiationphase and recovered in the early resolution phase. Especially, the 5-LOX and 12/15-LOX pathway products displayed a reciprocal pattern.Next we examined the temporal and differential profiles of inflammatory cells
(leukocytes in exudates) in the course of acute peritonitis. Acute inflammatoryresponse is characterized by edema formation and neutrophil infiltration followedby monocyte/macrophage accumulation [15]. In zymosan-induced peritonitis, wefound that eosinophils are recruited to the inflamed loci during the resolutionphase of acute inflammation [17]. In vivo depletion of eosinophils by injecting anti-IL-5 monoclonal antibody caused a resolution deficit, and the differential display oflipid mediators using LC-MS/MS revealed that locally activated eosinophils in theearly resolution phase produced 12/15-LOX-derived mediators, including DHA-derived anti-inflammatory/proresolving lipid mediator PD1 (Figure 11.7) [13].Indeed, the predominant population expressing 12/15-LOX in the early resolutionphase was eosinophils, and isolated eosinophils produced a significant amount of12/15-LOX-derived mediators. In the naive peritoneal cavity, resident macrophages
COX 5-LOX 12/15-LOX
200300400500
pg
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ng
LTB4
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Figure 11.6 Temporal profile of lipid mediators in zymosan-induced mouse peritonitis. Timecourse of COX products PGE2 and PGD2, 5-LOX products LTB4 and LTB5, and 12/15-LOXproducts 12-HETE and PD1 in the peritoneal exudates of zymosan-induced mouse peritonitis.
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are the predominant cell population expressing relatively high levels of 12/15-LOX,and are likely responsible for the generation of 12/15-LOX products in the lavage ofnaive mice. The resolution deficit caused by eosinophil depletion was rescued byeosinophil restoration or local administration of PD1, and eosinophils deficient in12/15-LOX were unable to rescue the resolution phenotype. These results indicate
TxA2
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Figure 11.7 Differential display of LC-MS/MS-based mediator lipidomics. Peritoneal lavagecells in the resolution phase from control IgG-treated (black bars) or anti-IL-5 monoclonalantibody-treated mice (white bars) werestimulated ex vivo with calcium ionophore.Arachidonic acid (AA)-, eicosapentaenoic acid
(EPA)-, and docosahexaenoic acid (DHA)-derived products were quantified by LC-MS/MS.These results indicate that eosinophils in theresolution phase produce 12/15-LOX products,including DHA-derived lipid mediator PD1.12/15-LOX products are boxed. Reprinted fromRef. [17].
11.4 Mediator Lipidomics in Inflammation and Resolution j229
that eosinophils and eosinophil-derived lipid mediators (i.e., 12/15-LOX-derivedmediators) have a role in promoting the resolution of acute inflammation [17].
11.5Conclusion and Future Perspective
The comprehensive lipidomic method described in this chapter has a number ofattractive applications. One of them, as shown above, is a coordinated class switch-ing of lipid mediators in the course of acute inflammation and resolution. Thistechnology could potentially identify the metabolic fingerprint of a disease for clini-cal diagnosis and treatment. Mediator lipidomics concerns the simultaneous andquantitative analysis of bioactive lipid mediators in biological systems. When com-bined with proteomic, transcriptomic, and genomic profiles (multiomics profiling),it can greatly assist in understanding the role of lipid mediators in certain biologicaland/or pathological conditions.
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