Abstract

Acute inflammation is an indispensable host response to foreign challenges or tissue injury.

In healthy conditions, inflammatory processes are self-limiting and self-resolving, suggesting
the existence of endogenous mechanisms for the control of inflammation and resolution. A
comprehensive understanding of the cellular and molecular events of a well-orchestrated
inflammatory response is required, and recent studies have uncovered the roles of
endogenous lipid mediators derived from polyunsaturated fatty acids (i.e. lipoxins, resolvins,
protectins) in controlling the resolution of inflammation. This review presents recent
advances in understanding the formation and action of these mediators, especially focusing
on the LC-MS/MS-based lipidomics approach and the emerging roles of eosinophils and
eosinophil-derived lipid mediators in controlling acute inflammation and resolution.
Acute inflammation is an indispensable host response to insult or tissue injury. However, in
many human diseases, uncontrolled inflammation is suspected as a key component of
pathogenesis (1). Excessive or inappropriate inflammatory responses can cause local tissue
damage and remodelling which contribute to a range of chronic diseases. In healthy
individuals, acute inflammation is self-limiting and has an active termination program (2).
Therefore, the identification of such endogenous anti-inflammatory and/or pro-resolution
mechanisms is of wide interest.
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 (LXs), resolvins (Rvs) and protectins (3). The 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 important to know when and where lipid mediators are formed in the
inflammatory sites and which ones are formed. To this end, we developed an LC-ESIMS/MS-based lipidomics system geared to the comprehensive analyses of lipid mediators
with simultaneous and quantitative measurements. Using this system, we revealed a
coordinated class switching of PUFA-derived mediators in the course of acute inflammation,
and a novel role of eosinophils and eosinophil-derived lipid mediators in the resolution of
acute peritonitis.

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 (AA, 20:4 n-6) is a common precursor of many eicosanoids, which
are bioactive lipid mediators that control inflammatory responses. When cells are stimulated,
AA is released from membrane phospholipids by phospholipase A2 (PLA2), which hydrolyzes
the acyl ester bond. This is the first step in the AA 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 PLA 2s (sPLA2s, cPLA2s,
iPLA2s), respectively (4, 5). The different types of PLA2 are regulated differently and are
expressed in different tissues. Unesterified intracellular AA is immediately metabolized by
either COX, LOX or CYP. The COX pathway leads to the formation of PGs and
thromboxanes (TXs), the LOX pathway leads to LTs, LXs and hydroxyeicosatetraenoic acids

(HETE), and the CYP pathway leads to HETEs and epoxyeicosatrienoic acids (EET) (Fig. 1).
As a class, these molecules act as autacoids that are rapidly synthesized in response to before
specific stimuli, act quickly at the immediate locality, and remain active for only a short time
degradation.

Fig. 1
Eicosanoid production from AA. PLA2 cleaves the ester bond of phospholipids to release
AA. Free AA is used as a substrate for the LOX, COX and CYP pathways. The COX pathway
produces PGs and TX. The LOX pathway produces LTs, LXs and HETEs. The CYP pathway
produces EETs and HETEs.
AA-derived eicosanoids are important in many physiological processes. Under non-disease
conditions, PGs contribute to the maintenance of homeostasis, e.g. they have cytoprotective
roles in the gastric mucosa, respiratory tract and renal parenchyma. PGs are also involved in
pro-inflammatory processes, and are responsible for many of the hallmark signs of
inflammation such as heat, redness, swelling and pain (6). COX occurs in two isoforms,
called the constitutive isoform (COX-1) and the inducible isoform (COX-2) (7). These
enzymes may contribute to the production of different sets of eicosanoids at different
locations at different times. The LOX pathway represents another major pathway to produce
LTs and LXs. Mammals have at least three LOXs, 5 -, 12 - and 15-LOX present in
mammalian systems (8, 9). 5-LOX-derived LTs (LTB4, cysteinyl LTs) are involved in proinflammatory processes such as neutrophil infiltration, increased vascular permeability and
smooth muscle contraction (10). In contrast, 5 - and 15-LOX-derived LXA 4 counter-regulates
the pro-inflammatory processes and may be important in the resolution of inflammation (11).
An imbalance in lipoxin-leukotriene homeostasis may be a key factor in the pathogenesis of
inflammatory diseases. The epoxygenation of AA by CYP generates EETs, which may have
roles in regulation of smooth muscle cells and vascular tone (12). Many of the eicosanoids
signal via seven-transmembrane G protein-coupled receptors (GPCRs) (6, 13).
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 include 3-series PGs, 5-series
LTs and LXs, hydroxyeicosapentaenoic acids (HEPE), and epoxy-eicosatetraenoic acids
(EpETE) (Fig. 2). DHA is also converted to hydroxydocosahexaenoic acids (HDoHE) and
epoxy-fatty acids by enzymatic oxidation. Dietary supplementation of n-3 PUFA has
beneficial effects in many inflammatory disorders, including cardiovascular disease, arthritis,
colitis and metabolic syndrome (14). Also, elevation in n-3 PUFA levels in n-3 desaturase
(fat-1) transgenic mice protected against inflammatory disease models (15). N-3 PUFAs 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 products (16). Another role is to be
converted to potent anti-inflammatory and protective mediators. For example, E-series Rvs
are produced from EPA, and D-series Rvs, protectin and maresin are produced from DHA
[reviewed in (17)] (Fig. 2). Rvs, protectin and maresin are distinct families of local mediators
identified in the resolving exudates of acute inflammation. They were initially identified
using a systems approach with LC-MS/MS-based lipidomics and then complete structural
elucidation of the bioactive mediators was achieved. RvE1 (5 S,12 R,18 R-trihydroxy-6 Z,8
E,10 E,14 Z,16 E-EPE) and RvE2 (5 S,18 R-dihydroxy-6 E,8 Z,11 Z,14 Z,16 E-EPE) are
biosynthesized via the 5-LOX pathway from a precursor, 18-hydroxyeicosapentaenoic acid
(18-HEPE) (1821). From DHA, Protectin D1 (10 R,17 S-dihydroxy-docosa-4 zZ,7 Z,11 E,13
E,15 Z,19 Z-hexaenoic acid; PD1) is formed via the 15-LOX pathway (22), and maresin 1 (7
S,14 S-hydroxy-docosa-4 Z,8 E,10 E,12 Z,16 Z,19 Z-hexaenoic acid; MaR1) is formed via the
12-LOX pathway (23). D-series Rvs such as RvD1 (7 S, 8 R, 17 S-trihydroxy-docosa-4 Z,9
E,11 E,13 Z,15 E,19 Z-hexaenoic acid) and RvD2 (7 S,16 R,17 S-trihydroxy-docosa-4 Z,8
E,10 Z,12 E,14 E,19 Z-hexaenoic acid) are formed via combination of 5 - and 15-LOX
pathways from DHA (24, 25). These mediators, not only act as potent anti-inflammatory lipid
mediators limiting neutrophil influx, but also promote resolution of inflammation by
stimulating the clearance of apoptotic cells and inflammatory debris by macrophages (25
27). These mediators possess stereoselective properties and display potencies in the low
nanomolar range in many mammalian systems. As part of their molecular mechanism, these
mediators exert their potent actions via cell-surface receptors such as GPCRs and transient
receptor potential (TRP) ion channels (2123). For example, Rvs differentially interact with
distinct GPCRs (e.g. GPR32, ALX/FPRL1, ChemR23, BLT1, etc.) to prevent excessive
inflammation and elicit pro-resolving signals by promoting macrophage phagocytic activity
and clearance of apoptotic cells and inflammatory debris (28, 29). Also specific Rvs inhibit
inflammatory and neuropathic pain in mice and suppress distinct TRP channels (e.g. TRPA1,
V1, V3, V4) to control selective pain modalities (30, 31). To date, receptor candidates for
PD1 or MaR1 have not been reported. Neither RvE1 nor RvD1 directly activated nuclear
receptors such as peroxisome proliferator-activated receptor (PPAR)-, and , or retinoid X
receptor (RXR) compared to known nuclear receptor agonists (29).

Fig. 2
Eicosanoid, docosanoid and hydroxy-fatty acid production from AA, EPA and DHA.

LC-ESI-MS/MS-Based Lipidomics
A powerful method for the analysis of mono- and polyhydroxylated fatty acids is liquid
chromatography tandem mass spectrometry (LC-ESI-MS/MS). Electrospray ionization (ESI)
is a soft ionization technology used to form either positive or negative ions without

derivatization and decomposition. In the case of fatty acid-derived mediators, ESI results in
the removal of a proton to form [M-H] carboxylate ions. A triple quadrupole mass
spectrometer is capable of carrying out an MS/MS method called multiple reaction
monitoring (MRM). A specified precursor ion is selected according to its mass-to-charge ratio
(m/z) in the first quadrupole mass filter, and is fragmented into product ions in the second
chamber by collision-induced dissociation (CID). Then the third quadrupole mass filter is
locked on its specified product ion. We developed a comprehensive LC-ESI-MS/MS method
that can simultaneously detect and quantify more than 250 PUFA metabolites including PGs,
LTs, LXs, Rvs, protectins and other AA-, EPA-, DHA-derived products (36, 37).
Representative MRM chromatograms are depicted in Fig. 3. The LC-MS/MS system using
MRM mode provides structure-specific signal detection and further improves the
quantification limits when combined with high-resolution LC separations.

Fig. 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 and
quantified by MRM using triple quadrupole MS/MS. Representative MRM chromatograms
of fatty acid metabolites are depicted.

Mediator Lipidomics
Resolution

in

Acute

Inflammation

and

The local inflammatory response is characterized by a sequential release of mediators and the
recruitment of different types of leukocytes (32). Acute inflammation is characterized by
edema formation and the initial recruitment of neutrophils followed by the recruitment of
monocytes that differentiate into macrophages. Lipid mediators such as PGs and LTs, and
cytokines and chemokines coordinatedly regulate the initial events of acute inflammation.
Also the resolution of acute inflammation is an active process that is controlled by
endogenous pro-resolving mediators, thereby leading to the efficient clearance of
inflammatory leukocytes and exudates, and restoration of the inflamed tissue to homeostasis
(2). In contained inflammatory exudates, coordinated lipid mediator class switching occurs in
the course of acute inflammation and resolution (3335). To better understand the molecular
and cellular mechanisms underlying the coordinated processes of inflammation and
resolution, we applied LC-ESI-MS/MS-based mediator lipidomics to the self-resolving acute
inflammation model, namely murine zymosan-induced peritonitis (36).
Temporal and quantitative differences in the lipid mediator profiles were observed in the
course of acute inflammation and resolution. The maximum levels of 5-LOX products such
as LTB4 were observed in initiation phase, and subsequently subsided during the resolution.

In contrast, the levels of 12/15-LOX products such as DHA-derived mediator PD1 were low
at the initiation, then gradually increased during the resolution phase (Fig. 4A). PD1 not only
acts as potent anti-inflammatory lipid mediators limiting neutrophil influx but also promotes
the resolution of inflammation by stimulating macrophage ingestion of apoptotic cells and
inflammatory debris, and by increasing the movement of phagocytes into draining lymph
nodes (26). Thus, the endogenous cellular source of PD1 in the resolution phase was of
interest. Recently, eosinophils were found to be the major source of PD1 biosynthesis in the
early phase of resolution (36). The predominant population expressing 12/15-LOX in the
early resolution phase was eosinophils, and isolated eosinophils produced a significant
amount of PD1 when stimulated ex vivo. Also, in vivo depletion of eosinophils by antiinterleukin(IL)-5 antibody increased the number of neutrophils in inflammatory exudates, and
reduced the number of phagocytes moving from the inflamed peritoneum to the draining
lymph nodes, both of which showed a resolution deficit. LC-MS/MS-based lipidomics
analysis revealed that the amounts of 12/15-LOX-derived mediators dramatically decreased
in eosinophil-depleted mice, whereas the amounts of COX and 5-LOX-derived products did
not differ between the two groups. Furthermore, the resolution deficit caused by eosinophil
depletion was rescued by eosinophil restoration or local administration of PD1, and
eosinophils deficient in 12/15-LOX were unable to rescue the resolution phenotype.
Collectively, eosinophils are recruited to the inflamed loci in the early resolution phase,
where they locally produce resolution mediators including PD1 via a 12/15-LOX-initiated
biosynthetic route, and play roles in promoting resolution of acute inflammation (36) (Fig.
4B).

Fig. 4
Emerging roles of eosinophils and eosinophil-derived lipid mediators in the resolution of
acute inflammation. (A) Temporal and quantitative differences of lipid mediator profiles in
the course of acute inflammation and resolution. 5-LOX products such as LTB 4 are present in
the initiation phase, and 12/15-LOX products such as PD1 are increased during the resolution
phase. (B) Eosinophils are recruited in the early resolution phase, where they locally produce
resolution mediators such as PD1 via a 12/15-LOX-initiated biosynthetic route, and play
roles leading to the efficient clearance of inflammatory leukocytes and exudates (36).

Novel Bioactive Products Derived from Eosinophils

Since eosinophils function to promote resolution by locally producing pro-resolving
mediators such as PD1, we questioned whether other mediator(s) with potent antiinflammatory properties could be formed by eosinophils. To this end, isolated leukocytes
were incubated with fatty acid precursors and the lipids were analysed by LC-MS/MS.

Several hydroxylated products were identified using MRM with established or predicted
precursor-product ion pairs.
RvE1 and RvE2 are biosynthesized by human polymorphonuclear leukocytes (PMNs) via the
5-LOX pathway from a common precursor 18-HEPE (1821). 18-HEPE formation in vivo is
related to dietary intake of EPA (19), and a recent study demonstrated two parallel
stereospecific pathways, 18 R- and 18 S-, in the biosynthesis of E series Rvs both in human
sera and murine exudates (21). Side-by-side MRM chromatograms of products from human
PMN and eosinophil incubations with 18-HEPE demonstrated that human PMNs converted
18-HEPE into RvE1 and RvE2, and in comparison, eosinophils converted 18-HEPE into
novel 8,18-dihydroxy-EPE (8,18-diHEPE), 11,18-diHEPE, 12,18-diHEPE and 17,18-diHEPE
via the 12/15-LOX pathway (Fig. 5). Among them, two stereoisomers of 17,18-diHEPE,
collectively termed resolvin E3 (RvE3), displayed a potent anti-inflammatory action by
limiting neutrophil infiltration in zymosan-induced peritonitis. The basic structures of these
bioactive products were identified as 17,18 R/S-dihydroxy-5 Z,8 Z,11 Z,13 E,15 E-EPE,
denoted 18 R-RvE3 and 18 S-RvE3, respectively. Both 18 R- and 18 S-RvE3 inhibited
neutrophil chemotaxis in vitro at low nanomolar concentrations (37).

Fig. 5
Biosynthesis of a novel eosinophil-derived mediator RvE3. EPA-derived 18-HEPE is
converted via the sequential actions of LOX, which leads to formation of E series Rvs. 5LOX expressed in PMNs converts 18-HEPE into RvE1 and RvE2 (1921). 18-HEPE is also
converted by eosinophils via the 12/15-LOX pathway into potent anti-inflammatory mediator
RvE3 (17,18-diHEPE) together with other novel metabolites 8,18-diHEPE, 11,18-diHEPE
and 12,18-diHEPE (37).

Perspectives
Determining the contributions of eosinophils to the biosynthesis of RvE3 and other 12/15LOX-derived mediators such as PD1 is of interest because such mediators have roles in
controlling acute inflammation and resolution. Eosinophils are thought to primarily assist in
the host defense against parasitic infection through the release of cytotoxic products such as
major basic protein and eosinophil-derived peroxidase (38). The release of these products
also induces tissue damage and dysfunction, especially under allergic conditions. It is of
interest whether eosinophils in the resolution phase represent a distinct subset. Besides
eosinophils, 12/15-LOX is expressed in tissue-resident macrophages, dendritic cells, mast
cells and airway epithelial cells (9). Also, the expression level of 12/15-LOX is upregulated
in various cell types by Th2 cytokines including interleukin(IL)-4 and IL-13 (9). Cells

expressing 12/15-LOX might be involved in regulating inflammatory responses by locally

producing anti-inflammatory lipid mediators such as PD1 and RvE3. This is consistent with
the finding of exacerbated inflammatory responses in 12/15-LOX-deficient mice in several
disease models (3942).
Mediator lipidomics concerns the simultaneous and quantitative analysis of bioactive lipid
mediators in biological systems. When combined with proteomic, transcriptomic and
genomic profiles (multi-omics profiling), it can greatly assist in understanding the role of
lipid mediators in certain biological and/or pathological conditions. This technology could
potentially identify the metabolic fingerprint of a disease for clinical diagnosis and treatment.
Moreover, the recently identified Rv and protectin families and the LX class of eicosanoids
constitute a new group of endogenous mediators with potent anti-inflammatory and tissueprotective actions. They could lead to the development of resolution agonist-based
therapeutics for many human diseases when sustained inflammation and impaired resolution
are suspected as key components of pathogenesis.

Funding
This work was supported by Japan Science and Technology Agency Precursory Research for
Embryonic Science and Technology, and a grant-in-aid for scientific research from the
Ministry of Education, Culture, Sports, Science and Technology of Japan.

http://jb.oxfordjournals.org/content/152/4/313.full