Pharmacology & Therapeutics 141 (2014) 272282

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Pharmacology & Therapeutics

journal homepage: www.elsevier.com/locate/pharmthera

Associate Editor: M. Madhani

Pharmacology and therapeutics of omega-3 polyunsaturated fatty acids

in chronic inammatory disease
Clara M. Yates a, Philip C. Calder b, G. Ed Rainger a,
a
Centre for Cardiovascular Sciences, School of Clinical and Experimental Medicine, The University of Birmingham, B15 2TT, UK
b
Human Development & Health Academic Unit, Faculty of Medicine, University of Southampton, Southampton SO16 6YD, UK

a r t i c l e i n f o a b s t r a c t

Keywords: Omega-3 (n3) polyunsaturated fatty acids (n3 PUFAs) have well documented anti-inammatory properties,
Omega-3 polyunsaturated fatty acid and consequently therapeutic potential in chronic inammatory diseases. Here we discuss the effects of n3
Eicosapentaenoic acid PUFAs on various inammatory pathways and how this leads to alterations in the function of inammatory
Docosahexaenoic acid cells, most importantly endothelial cells and leukocytes. Strong evidence indicates n3 PUFAs are benecial
Inammation as a dietary supplement in certain diseases such as rheumatoid arthritis; however for other conditions such as
Eicosanoid
asthma, the data are less robust. A clearer understanding of the pharmacology of n3 PUFAs will help to establish
Resolvin
targets to modulate chronic inammatory diseases.
2013 Elsevier Inc. All rights reserved.

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 272
2. n3 polyunsaturated fatty acids and cardiovascular disease . . . . . . . . . . . . . . . . . . . . . . . . 276
3. The use of n3 polyunsaturated fatty acids as a dietary intervention in asthma . . . . . . . . . . . . . . . 277
4. The use of n3 polyunsaturated fatty acids as a dietary intervention in rheumatoid arthritis . . . . . . . . . 278
5. Inammatory bowel diseases and n3 polyunsaturated fatty acids . . . . . . . . . . . . . . . . . . . . . . 278
6. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 279
Conict of interest statement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 280
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 280

1. Introduction adjacent carbon atoms), monounsaturated (one double bond) or poly-

1.1. Omega-3 polyunsaturated fatty acids (n3 PUFAs)
Fatty acids are carboxylic acids with a variable number of carbons
atoms forming a hydrocarbon chain terminated by carboxyl and methyl
groups (Fig. 1). The chains vary in length from two, to greater than thirty
carbon atoms and exist in either saturated (no double bonds between
Abbreviations: AA, Arachidonic acid; ALA, alpha-linolenic acid; CD, Crohn's disease;
COX, cyclooxygensae; CVD, cardiovascular disease; DHA, docosahexaenoic acid; EC, endo-
thelial cells; EPA, eicosapentaenoic acid; FFA, free fatty acids; IBD, inammatory bowel dis-
eases; ICAM-1, intercellular adhesion molecule-1; IL, interleukin; LA, linoleic acid; LOX,
lipoxygenase; LT, leukotrienes; LX, lipoxin; n3 PUFAs, omega-3 polyunsaturated fatty
acids; NPD, neuroprotectin; PBMC, peripheral blood mononuclear cell; PD, protectin; PG,
prostaglandin; PI, phosphatidylinositol; RA, rheumatoid arthritis; RCT, randomised con-
trolled trial; Rv, resolvin; SPM, specialised pro-resolving mediators; TNF, tumour necrosis
factor; UC, ulcerative colitis; VCAM-1, vascular cell adhesion molecule-1.
Corresponding author. Tel.: +44 121 414 4040; fax: +44 121 414 6919.
E-mail address: g.e.rainger@bham.ac.uk (G. Ed Rainger).
unsaturated (more than one double bond) forms. Fatty acids have
both systematic and common names. They are also described by a
nomenclature which includes the number of carbon atoms in the
chain (including the terminal carbons), the number of double bonds
in the chain, and the location of the rst double bond in the chain
from the terminal methyl group, known as the n or carbon. For ex-
ample, arachidonic acid (AA) is shown as 20:4n6 using this nomen-
clature, thus describing a molecule with 20 carbon atoms in the chain,
four double bonds, with the rst double bond at the 6th carbon from
the methyl terminus. This nomenclature enables discrimination of
different families of polyunsaturated fatty acids (PUFAs) based on the
position of the rst double bond in the chain, i.e. n3 and n6 PUFAs
(Ratnayake & Galli, 2009).
In this review we will focus on the biological effects of the so called
marine n3 PUFAs, eicosapentaenoic acid (EPA; 20:5n3) (Fig. 1A)
and docosahexaenoic acid (DHA; 22:6n3) (Fig. 1B). In mammals,
EPA and DHA can be synthesised from the dietary precursor and essen-
tial fatty acid, -linolenic acid (ALA; 18:3n3). Synthesis requires a

0163-7258/$ see front matter 2013 Elsevier Inc. All rights reserved.
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 C.M. Yates et al. / Pharmacology & Therapeutics 141 (2014) 272282
A n-3
carbon
H3C COOH
EPA 20:5 n-3
/n-
carbon
B n-3
carbon
COOH
H3C
DHA 22:6 n-3
-/n-
carbon
Fig. 1. Schematic structure of the n3 PUFAs EPA and DHA. A. EPA has 20 carbons and 5
double bonds; B. DHA has 22 carbons and has 6 double bonds. In both fatty acids the
rst double bond occurs at the third carbon from the /n-end of the chain.
number of elongation and desaturation steps and is inefcient in
humans. This makes dietary intake of pre-formed EPA and DHA a more
effective route of assimilation. EPA and DHA in the human diet are de-
rived indirectly from marine algae (higher plants lack the enzymes for
their biosynthesis). Their availability is dramatically increased as they
pass up the food chain, becoming concentrated in the esh of marine
sh. Both EPA and DHA are present in high amounts in the esh of oily
sh e.g. herring, mackerel and salmon, and they are the main PUFAs in
sh oil supplements. Indications of the efcacy of these fatty acids as di-
etary interventions for chronic inammatory disease originated from
epidemiological studies conducted on populations of Greenland Inuits,
native Alaskans and the residents of Okinawa, Japan. These studies uni-
formly showed a relationship between the burden of chronic disease
and life style, with the consumption of high levels of n3 PUFAs being
common to all of the study populations (Dyerberg et al., 1978;
Kromann & Green, 1980; Kagawa et al., 1982; Newman et al., 1993).
Thus, the postulate that dietary marine n3 PUFAs may be protective
against chronic inammatory diseases, in particular cardiovascular dis-
ease (CVD), was engendered. Since these initial studies, much research
has been carried out to conrm this relationship and to investigate the
efcacy of n3 PUFAs, in other disease states with a chronic inamma-
tory component such as rheumatoid arthritis (RA), inammatory bowel
diseases (IBD) and asthma. Here we describe inammation and the
mechanisms by which EPA and DHA can modify inammatory re-
sponses, and the relevance of this to chronic inammatory diseases.
1.2. Inammation
Inammation is a physiological response to infection or injury
characterised by ve classical signs described by Celsus and Galen in
antiquity, i.e. pain, heat, redness, swelling and loss of function. The
overriding purpose of an acute, resolving inammatory response is to
protect the body from invasion and damage and to re-establish physio-
logical homeostasis. Following infection, for example, inammatory
mediators with vasodilatory capacity increase blood ow, thereby caus-
ing redness and heat, and vessels also become more permeable leading
to oedema (swelling). Sensitivity to pain is increased in response to
agents such as bradykinin (Kenji, 2007). A major function of the inam-
matory response is to deliver the molecular and cellular mediators of
immunity to affected tissues. Thus, the concentration of plasma borne
agents such as complement increases due to changes in vascular perme-
ability. Leukocytes are recruited by appropriately activated vascular
endothelial cells (EC) which support a tightly regulated series of events
termed the leukocyte adhesion cascade (Ley et al., 2007) (Fig. 2). In re-
sponse to stimulation by inammatory cytokines, or agents such as
273
histamine, leukocytes are captured from owing blood by specialised
receptors of the selectin and immunoglobulin super families (IgSF).
These molecules also permit a form of dynamic adhesion termed
rolling, during which the velocity of leukocytes is dramatically reduced
in comparison to those being transported in the bulk ow of the blood.
Rolling cells are able to assimilate EC borne signals from agents such as
chemokines, which stabilise adhesion by activating leukocyte 1- and
2-integrins. Integrin-mediated adhesion to specic counter ligands
on the EC surface, in the basement membrane and in stromal tissue sup-
port migration of leukocytes across the vascular barrier and towards the
inammatory locus. Once in tissue, leukocytes have powerful cytotoxic
and tissue remodelling capabilities which must be tightly regulated, not
least because uncontrolled or non-resolving leukocyte recruitment may
be pathogenic. Indeed, it has been postulated that chronic inammation
represents a situation where normal programmes of resolution fail,
resulting in continual inux of leukocytes into tissue, where their exces-
sive activity results in inappropriate tissue remodelling and ultimately
to loss of tissue function (reviewed in McGettrick et al., 2012).
A hallmark of chronic inammatory disease is the continual inltra-
tion of leukocytes from blood, across activated EC and into the affected
tissue. Although the agents that activate EC may show variation in a dis-
ease specic manner, in general many of the receptors and mediators
induced by these agents are common to the leukocyte recruitment
cascade (Fig. 2). It is thus probable that n3 PUFAs can regulate the
magnitude, and possibly the identity, of the leukocytic inltrate, in a
range of inammatory conditions by regulating aspects of EC activation
generic to the inammatory process. It is important to appreciate that
studying the effects of n3 PUFAs on EC function in the context of
leukocyte recruitment is difcult in vivo. Indeed most of the data avail-
able to date have been generated in vitro using cultured cells with the
addition of free fatty acids (FFA) to culture medium as a source of sup-
plementary lipids. However, the signicance of some of these studies
must be questioned due the concentrations of FFA used. Physiologically,
n3 PUFAs are found in the blood plasma as FFA at concentrations less
than 1 M (Cawood et al., 2010) (but at higher concentrations in ester-
ied forms such as triglycerides and phospholipids) and many supple-
mentation regimens do not greatly increase the levels of the FFA. Most
in vitro studies which investigate mechanisms of n3 PUFA action uti-
lise free n3 PUFAs at concentrations 10100 fold higher than this
which may equate to total plasma concentrations of n3 PUFAs achiev-
able with supplementation. However, this does not account for the fact
that the majority of circulating n3 PUFAs are transported in esteried
form within lipoprotein particles which do not have the same prole
of bioavailability as FFA. This is an important point, as in our own
experiments, n3 PUFAs as FFA at concentrations of 10 M or above
induce non-specic calcium transients in cells such as neutrophils
(unpublished observation; Yates & Rainger). Thus, we routinely utilise
free fatty acid at sub-M concentrations and do not normally exceed
5 M, as we feel that this represents the maximal ceiling of free n3
PUFA levels achievable in vivo with oral supplementation.
1.3. Polyunsaturated fatty acid-derived molecules as mediators of the in-
ammatory response
Dietary fatty acids are intimately linked with the inammatory
response, For example the n6 PUFA, AA, is the main precursor of sever-
al important lipid mediators. AA can be derived directly from the diet
(Jonnalagadda et al., 1995) but like the n3 PUFAs, it can also be synthe-
sised through a series of desaturation and elongation reactions from the
essential fatty acid linoleic acid (LA; 18:2-n6). AA from the diet or after
synthesis is stored in membrane phospholipids, e.g. phosphatidylcholine
(PC), phosphatidylethanolamine (PE) and phosphatidylinositol (PI),
mainly at the sn2 position and is liberated under appropriate stimulatory
conditions by the enzyme phospholipase A2 (PLA2). Phospholipase C
(PLC) is also able to release AA specically from PI via a series of reactions
involving the formation of diacylglycerol (DAG) by the enzyme DAG
274 C.M. Yates et al. / Pharmacology & Therapeutics 141 (2014) 272282

Capture Stabilisation

Rolling Arrest Transendothelial

migration

Capture receptors e.g. selectins Chemokines IgSF e.g. ICAM-1 or VCAM-1 integrins

Lipids e.g. PGD2 Junctional molecules e.g. CD31 or JAMs

Fig. 2. The leukocyte adhesion cascade. Leukocytes are captured from owing blood and roll on selectin molecules presented by activated EC. EC derived chemokine and lipid signals are
assimilated by rolling leukocytes, stabilising adhesion and resulting in activation of leukocyte integrins. Integrins bind cognate receptors e.g. ICAM-1 or VCAM-1 on EC and migrate on
the EC surface to intercellular junctions. By engaging junctional adhesion molecules, leukocytes undergo transendothelial migration and enter the underlying tissue. (Figure adapted
from (McGettrick et al., 2012).

lipase (Whatley et al., 1993). Free AA is metabolised by three main
classes of enzymes; (i) the cyclooxygenases (COX), producing prosta-
glandins and thromboxanes, (ii) the lipoxygenases (LOX), generating
leukotrienes (LTs), hydroxyeicosatetraenoic acids (HETEs) and lipoxins
(LX) and (iii) p450 epoxygenases which synthesise epoxyeicosatrienoic
acids (EETs) and HETEs (Fig. 3). The production of these eicosanoids
in vivo can be inuenced by neighbouring cells leading to the process
of transcelluar biosynthesis, where an intermediate generated from
AA in one cell is fully processed into the nal product in a second cell
(Folco & Murphy, 2006).
Alpha-linolenic acid (ALA; 18:3 n3) is the parent n3 PUFA, and
like LA, must be wholly derived from the diet. Although humans have
the enzymes to synthesise EPA and DHA from ALA, the conversion
rate is low, in particular for DHA. Indeed, a diet rich in ALA results in
increased levels of EPA but not DHA in platelet, leukocyte and plasma
phospholipids (Li et al., 1999). In the absence of dietary supplementa-
tion, EPA is present at low levels in the majority of tissues. DHA, however,
comprises up to 50% of all fatty acids in PE and phosphatidylserine
(PS) in the outer cone and rod cells of the retina and is also found at
high levels in the brain (Fliesler & Anderson, 1983) (and reviewed in

Membrane
phospholipids

Arachidonic Acid
(AA)

5-LOX
COX-1,2 P450 15-LOX (leukocytes)
epoxyganase

PGG2/PGH2
Epoxyeicosatrienoic acids 5-HPETE
5,6-EET 5-HPETE
8,9-EET
11,12-EET
14,15-EET LTA4

TXA2 PGE2 PGI2 PGD2 PGF2

5-LOX 12-LOX

LTB4 LTC4
Lipoxin LTD4
15deoxy 12-14PGJ 2 hydrolayses LTE4

Lipoxin A4
Lipoxin B4

Fig. 3. Eicosanoid biosynthesis from AA. Cyclooxygenase (COX)-1,2 metabolise AA to PGH2 by sequential cyclooxygenation and hydroperoxidation reactions. PGH2 is converted via specic
synthases generating thromboxane A2 (TXA2) and the prostaglandins including PGE2, PGI2, PGD2 and PGF2. These products can be non-enzymatically converted into further metabolites,
including 15-deoxy-12,14-PGJ2. 5-lipoxygenase (LOX) binds 5-lipoxygenase activating protein (FLAP) upon cell activation converting AA to 5-HPETE, which is further metabolized to LTA4.
LTA4 can also be converted into LTB4 by LTA4 hydrolase, or the cysteinyl leukotrienes (Cys-LTs) LTC4, LTD4 or LTE4. LTA4 can be metabolised by platelet 12-LOX to lipoxin A4 and lipoxin B4.
15-LOX also metabolises AA to generate 5-HEPTE, which is further converted by 5-LOX and lipoxin hydrolases to lipoxin A4 and lipoxin B4.
 C.M. Yates et al. / Pharmacology & Therapeutics 141 (2014) 272282 275

(Lauritzen et al., 2001)). Following dietary supplementation, the levels of of eicosanoid production to a less inammatory prole and could be ben-
EPA and DHA in cellular phospholipids increases, sometimes at the ex- ecial in the context of chronic inammatory disease. Interestingly some
pense of AA (Healy et al., 2000). This is important in the context of an in- AA derived mediators do anti-inammatory properties. For example,
ammatory response, as EPA can be metabolised by the same enzymes PGE2 was equally able to inhibit endotoxin-induced TNF- secretion by
as AA to generate alternative series eicosanoids. Although the biological human mononuclear cells as PGE3 (Miles et al., 2002). Additionally, AA
functions of alternative series eicosanoids are poorly described, some derived PGI2 is a potent inhibitor of platelet aggregation and EPA
have been shown to have less pro-inammatory actions than the AA de- derived PGI3 has been show to be as effective in human platelets
rived analogues and some may even have anti-inammatory actions (Kobzar et al., 2001).
(Fig. 4). For example, EPA derived LTB5 is 1030 times less potent as a Competition between the metabolites of EPA with those derived
neutrophil chemoattractant than AA derived LTB4 (Strasser et al., from AA does explain some of the anti-inammatory effects of n3
1985). We have demonstrated that treating EC with EPA results in syn- PUFAs. In addition free DHA and EPA can modulate inammation by
thesis of PGD3 via COX enzymes, which antagonised the neutrophil binding to and activating plasma membrane-bound and cytosolic
PGD2 receptor, inhibiting neutrophil migration (Tull et al., 2009). In- receptors such as GPR120 and the members of the PPAR family of
creasing the ratio of n3:n6 PUFAs may therefore shift the balance transcription factors (Im, 2012). GRP120 is a G-protein coupled receptor
(GPCR), expressed at high levels in macrophages and adipocytes
(Miyauchi et al., 2009), which binds unsaturated fatty acids with a
Membrane
A phospholipids chain length of 1622 carbons (Hirasawa et al., 2005), including EPA
and DHA. In the Raw 264.7 macrophage cell line, DHA inhibited TNF-
and LPS induced cytokine production, with an EC50 of 110 M, an effect
lost in the GPR120 knockdown (although most experiments were per-
Eicosapentaenoic
acid formed with 100 M DHA) (Oh et al., 2010). A similar dependence on
(EPA) GRP120 to mediate anti-inammatory effects of DHA was observed in
Acetylated
COX-1,2 5-LOX COX-2 or the GRP120/ mouse (Oh et al., 2010). These recent ndings suggest
15-LOX an important role for GPR120 in mediating the anti-inammatory
effects of n3 PUFAs. The peroxisome proliferator-activated receptor
(PPAR) family regulate transcription of genes involved in lipid metabo-
PGG3/PGH3
5-HPETE lism and homeostasis by forming heterodimers with retinoid X
18R HPETE receptors (RXR) (Moraes et al., 2006). In addition, PPAR activation
may inhibit pro-inammatory gene transcription, in part by inhibiting
NFB activation (reviewed in Moraes et al., 2006). Of interest both
LTA5 DHA and EPA bind PPARs and in vitro EPA binds PPAR-, and with
TXA3 PGE3 PGI3 PGD3 PGF3 5-LOX IC50 of 14 M (Xu et al., 1999). Binding of free n3 PUFAs to these
receptor classes may represent an alternative mechanism of action.
The concept that there are specic and inducible pathways which
LTB5 LTC5
LTD5 RvE 1,2 support the resolution phase of inammation is attractive, and one
LTE5 that is receiving much attention. Within this eld, specialised pro-
resolving mediators (SPM) derived from EPA and DHA, the resolvins
B Membrane and protectins, have generated signicant interest (Serhan, 2010)
phospholipids
(Fig. 4A and B), and synthetic versions of these molecules have anti-
inammatory and inammation resolving efcacy in vitro, and when
provided exogenously in vivo. The rst pro-resolving mediator to be de-
Docosahexaenoic
acid scribed was resolvin E1 (RvE1), synthesised via the metabolism of EPA
(DHA) by aspirin acetylated COX-2 and transcellular processing by neutrophil
Acetylated
COX-2
15-LOX 5-LOX (Serhan et al., 2000). The precursor of RvE1, 18R-HEPE, can be
synthesised from EPA by microbial cytochrome P450 and is subsequent-
ly metabolised by host 5-LOX to RvE1 in the absence of aspirin (Arita,
Bianchini, et al., 2005, Arita, Clish, et al., 2005). DHA derived resolvins
17R HDHA 17S HDHA were rst described in peritoneal exudates and whole mouse brain
in the presence of aspirin (Serhan et al., 2002), although aspirin is
Epoxygenation not required for the synthesis of D-series resolvins by neutrophil
Enzyme hydrolysis
5-LOX (Hong et al., 2003). Following on from these studies, a series of
5-LOX DHA derived molecules with pro-resolution properties, the protectins,
NPD1 neuroprotectins and maresins, have been identied (Hong et al., 2003;
PD1 Serhan et al., 2009). Recent studies have described the detection of en-
dogenous SPM in murine models of infection (Chiang et al., 2012) and
RvD 1-4
inammation (Oh et al., 2012; Dalli et al., 2013). In a self-resolving
Fig. 4. n3 PUFA derived lipid mediators. A. EPA liberated from membrane phospholipids model of E. coli infection the precursor 17-HDHA and its downstream
is metabolised by COX-1 or 2 to the common precursor PGH3. Subsequent conversion by products, protectin D1 and RvD5, were detected in peritoneal exudates
synthases results in TXA3, PGE3, PGI3, PGD3 and PGF3. 5-LOX also metabolises EPA to
in the ng range (Chiang et al., 2012). In addition, administration of phys-
the 5-series leukotrienes via the formation of 5-HPETE. B. DHA liberated from membrane
phospholipids is metabolised by COX-1 or 2 to the common precursor PGH3. Subsequent iological concentrations of RvD1 or RvD5, in a model of lethal E. coli
conversion by synthases results in TXA3, PGE3, PGI3, PGD3 and PGF3. 5-LOX also infection, led to an increase in macrophage bacterial load and increased
metabolises EPA to the 5-series leukotrienes via the formation of 5-HPETE. EPA is also survival (Chiang et al., 2012). Both RvE2 and the intermediate 18-HEPE
converted by acetylated COX-2 or 15-LOX to 18RHPETE, which is converted via 5-LOX to have been detected in peritoneal exudates from mice with self limiting,
the RvE1 or RvE2. B. DHA is converted by acetylated COX-2 or 15-LOX to 17R HDHA or
17S HDHA respectively. 5-LOX converts both intermediates to the RvD series 14.
zymosan-induced, peritonitis (Oh et al., 2012). Similarly DHA derived
Additionally 17S HDHA can undergo epoxygenation and enzymatic hydrolysis to produce RvD1, 3, 5 and aspirin triggered RvD3 are present in exudates from
PD1, or NPD1 in neural tissue. the same mouse peritonitis model (Dalli et al., 2013).
276 C.M. Yates et al. / Pharmacology & Therapeutics 141 (2014) 272282
The human serum metabolome has been published which, using
multiple methodologies, has produced a comprehensive data set of me-
tabolites in human serum (Psychogios et al., 2011). This includes levels
of DHA (4.7 3.3 M) and EPA (1.1 0.7 M), and the SPMs RvD1
(0.0454 5 0.027 nM) and RvE1 (0.52 0.98 nM) in serum samples
from healthy humans by LC-MS/MS (Psychogios et al., 2011). Addition-
ally, SPM have been detected in plasma and serum of 20 healthy
human subjects following 3 weeks supplementation with 1 g/d DHA
and 1.4 g/d EPA; RvD1 at 24.4 2.5 pg/ml, RvD2 at 26.6 4.7 pg/ml
and 17R-RvD1 at 53.3 6.0 pg/ml (serum measurements) (Mas
et al., 2012). Interestingly, the concentrations reported are within the
range at which SPM have anti-inammatory and pro-resolving proper-
ties in in vivo and in vitro models (Mas et al., 2012). The receptors re-
sponsible for mediating the effects of SPM are starting to be identied.
RVE1 binds and activates the LTB4 receptor, BLT1 on neutrophils and
ChemR23 (Arita, Bianchini, et al., 2005, Arita, Clish, et al., 2005; Arita
et al., 2007), expressed in macrophages and neutrophils. Both cell
types also express the ALX receptor and GPR32 which recognise RvD1
(Krishnamoorthy et al., 2010).
These molecules switch the inammatory environment to one
which promotes tissue homeostasis by inhibiting further neutrophil
recruitment, blocking the production of pro-inammatory PGs, LTs
and cytokines, and promoting the restoration of tissue homeostasis by
inducing macrophage phagocytosis of apoptotic cells (Serhan, 2010).
These molecules represent an opportunity to pharmacologically address
non-resolving chronic diseases by inducing a programme of resolution
by which diseased tissues are returned to a normal. Future studies
are required to assay the levels of SPM in plasma in a larger cohort of
healthy subjects and those with inammatory disease. In addition the
effect of dietary supplementation with n3 PUFAs on SPM levels in
healthy and diseased subjects is of interest and may offer insight into
the roles of SPM under physiological and pathophysiological conditions.
2. n3 polyunsaturated fatty acids and cardiovascular disease
Following the observations of lower risk of CVD observed in those
consuming traditional diets in Greenland, Alaska and Japan (Dyerberg
et al., 1978; Kromann & Green, 1980; Bjerregaard & Dyerberg, 1988;
Newman et al., 1993), studies in populations consuming a more
western diet were initiated to determine if n3 PUFA or oily sh
consumption affected cardiovascular risk factors and reduced CVD
morbidity or mortality. Varying outcomes were documented in the
studies, which have been discussed in detail elsewhere (Kris-Etherton
et al., 2002; Calder, 2004; von Schacky, 2004; Wang et al., 2006;
London et al., 2007); outcomes studied included coronary heart disease
rate and mortality, cardiovascular mortality, stroke incidence and myo-
cardial infarction.
Crucially, studies involving sh have been the subject of meta-
analyses investigating either coronary heart disease mortality or stroke
incidence. (He et al., 2004a, 2004b) The rst of these (He et al., 2004a)
analysed data from 13 cohorts and 11 independent studies, totalling
222,364 participants where both frequency of sh consumption and rel-
ative risk of coronary heart disease mortality were reported. If the risk of
coronary heart disease in those eating sh less than once a month was
set to 1, those consuming more portions, up to 5 per week, had signi-
cantly reduced (by 38%) risk (He et al., 2004a). In the study investigating
the effect of sh consumption on stroke incidence, 9 cohorts and 8
independent studies comprising 200,575 participants were included
(He et al., 2004b). Similar to the coronary heart disease meta-analysis,
the risk of stroke decreased with greater sh intake. Indeed, in those
who ate 5 portions of sh per week the risk was reduced by 31%
compared to those who never consumed sh (He et al., 2004b).
Large intervention studies have investigated the effect of n3
PUFA supplementation in individuals with existing CVD. The Diet and
Reinfarction Trial (DART), published in 1989, supplemented the diet of
1015 men who had survived a myocardial infarction with two portions
per week of oily sh or with sh oil (Burr et al., 1989). It was the rst
controlled trial which examined the effect of dietary sh (approx.
300 g/week) or sh oils (500900 mg EPA + DHA/d) on the secondary
prevention of myocardial infarction. Overall there was a 29% reduction
in all-cause mortality risk during the rst two years, compared to the
control group (Burr et al., 1989). The GISSI-Prevenzione trial examined
11,324 patients who had recently survived a myocardial infarction
(GISSI-Prevenzione Investigators, 1999). Subjects were randomised to
n3 PUFA (885 mg EPA + DHA/d) and/or vitamin E, or no supplemen-
tation, with up to 3.5 year follow up. The primary combined endpoint
was death, non-fatal myocardial infarction and stroke. n3 PUFAs sig-
nicantly reduced this endpoint (by 15%), almost entirely attributed
to the reduction in cardiovascular death (GISSI-Prevenzione Investiga-
tors, 1999). The Japan EPA Lipid Intervention Study, (JELIS), set out to
test the hypothesis that EPA would be effective as a preventative mea-
sure against a major coronary event in 18,645 hypercholersterolaemic
Japanese patients taking statins (Yokoyama et al., 2007). There was a
19% reduction in the risk in subjects who were randomised to 1.8 g/d
EPA plus 5 mg/d simvastatin or 10 mg/d pravastatin compared to
statins alone (Yokoyama et al., 2007). Effects of n3 PUFAs on mortality
from heart failure was addressed in the GISSI-HF trial (GISSI-HF Investi-
gators, 2004). The trial followed 7000 patients randomised to 850 mg
EPA + DHA/d, or a placebo control, for a median of 3.9 years. All
cause mortality was reduced in the n3 PUFA group by 9% indicating
that n3 PUFAs can confer benet in addition to conventional
treatment.
Although these four studies reported positive outcomes, three re-
cent trials report no effect of n3 PUFA on the same CVD outcomes.
Subjects in the SU.FOL.OM3 trial had a history of myocardial infarction,
ischaemic stroke, or unstable angina (Galan et al., 2010). The study
aimed to test the impact of supplementing 2501 subjects with folate,
B vitamins and/or 600 mg/d EPA + DHA on fatal and non-fatal CVD
events with a median follow up of 4.7 years. No signicant effect of
n3 PUFAs was reported (Galan et al., 2010). The ALPHA-OMEGA
trial gave 4837 myocardial infarction survivors one of four trial marga-
rines, two of which provided 400 mg/d EPA + DHA, for 40 months
(Kromhout et al., 2010). Again, EPA + DHA had no effect on the
rate of major CVD events (Kromhout et al., 2010). Finally, the OMEGA
trial supplemented 3851 survivors of myocardial infarction with
~900 mg/d EPA + DHA and followed them for one year (Rauch et al.,
2010). n3 PUFAs had no affect on sudden cardiac death, total mortal-
ity or major CVD events. These studies have limitations that include a
low dose of EPA + DHA, insufcient sample size, and recruitment of pa-
tients long after the occurrence of a cardiovascular event. Alternatively
the background medication may inuence the efcacy of n3 PUFAs
in secondary prevention of CVD events and mortality.
Despite the conicting results reported in the intervention studies
discussed above, the potential mechanisms of action of n3 PUFAs in
CVD continue to be extensively investigated. CVD encompasses many
conditions, all commonly underlined by the formation of atheromatous
plaques. These plaques have both a lipid and inammatory component
(Libby et al., 2002) and develop as a consequence of EC activation lead-
ing to the inux of leukocytes into the intimal layer of the artery wall.
n3 PUFAs are capable of modulating both EC and leukocyte function
and it has been hypothesised that this may be responsible for the re-
duced CVD risk in populations consuming large amounts of oily sh.
To this end a large number of studies have investigated the effects of
EPA and DHA, either alone or in combination, on endothelial and leuko-
cyte function. These will be discussed in detail below.
One measure of the anti-inammatory properties of n3 PUFAs is
their ability to reduce cytokine induced expression of key EC adhesion
molecules involved in leukocyte recruitment. Several studies have in-
vestigated this possibility using EC of varying origins, as well as different
stimuli, and different concentrations and durations of exposure to n3
PUFAs. Human saphenous vein endothelial cells (HSVEC) treated with
10 M DHA for 24 h, prior to 6 h TNF- stimulation, exhibited reduced
 C.M. Yates et al. / Pharmacology & Therapeutics 141 (2014) 272282
E-selectin protein and reduced VCAM-1 (vascular cell adhesion
molecule-1) protein and mRNA levels compared to TNF- stimulation
alone (De Caterina et al., 1994). 65 M DHA and EPA attenuated IL-1
induced ICAM-1 (intercellular adhesion molecule-1), VCAM-1 and E-
selectin mRNA in HUVEC (Collie-Duguid & Wahle, 1996). Conrming
this data, 20160 M DHA inhibited TNF- induced VCAM-1 and
ICAM-1 protein expression in human aortic endothelial cells (HAEC)
(Wang et al., 2011). Our own studies indicate that 5 M DHA does not
alter mRNA or total cellular levels of E-selectin protein, however the
level of E-selectin expressed on the EC surface was signicantly down-
regulated due to disturbance of intracellular trafcking mechanisms
(Yates et al., 2011). The current evidence suggests a role for DHA in
inhibiting cytokine-induced adhesion molecule expression. Mechanisti-
cally this may be explained by inhibition of NF-B translocation to the
nucleus, for example, resulting in reduced transcription of VCAM-1
(Wang et al., 2011). In addition EPA at a concentration of 25 M has
been reported to inhibit EC surface expression of ICAM-1 (Goua et al.,
2008).
The functional signicance of the modulation of EC expressed adhe-
sion molecules has been investigated in leukocyte adhesion assays. In
vitro adhesion assays carried out in the absence of the hemodynamic
factors imposed by ow demonstrate that DHA inhibits the adhesion
of both THP-1 monocytes (De Caterina et al., 1994; Wang et al., 2011)
and neutrophils (Wang et al., 2003) to EC. Our own ow based adhesion
studies indicate that DHA and EPA modulate different stages of leuko-
cyte recruitment. Thus, DHA inhibited surface expression of E-selectin
in response to TNF-, resulting in reduced recruitment of neutrophils
from ow (Yates et al., 2011). EPA, in contrast, did not affect the number
of neutrophils recruited, but adherent neutrophils were unable to
migrate across the EC efciently. This was achieved by metabolism of
EPA into the anti-inammatory eicosanoid PGD3, using the metabolic
pathways normally utilised to convert the n6 PUFA AA into the pro-
inammatory agent PGD2. PGD3 effectively antagonised the functions
of PGD2 which were necessary to support neutrophil transit across the
EC monolayer.
The incorporation of DHA into membrane phospholipids may alter
the nature of cholesterol rich microdomains which play key roles in
anchoring receptors and signalling molecules (Insel & Patel, 2009). For
example, in EC supplemented with 100 M DHA, cholesterol was
displaced from lipid rafts, resulting in reduced association of src family
kinases with the raft (Chen et al., 2007), indicating that DHA may also
modulate EC signalling. In addition 50 M DHA has been shown to
displace caveolin-1 and endothelial nitric oxide synthase (eNOS) from
caveolae in EC (Li et al., 2007).
The ability of n3 PUFAs to modulate leukocyte function has been
investigated both in vitro using FFA as the supplemental lipids, but
more importantly, ex vivo after supplementing trial participants with
DHA, EPA or a mixture of these, usually in the form of sh oils. Studies
using sh oil are too numerous to examine in detail and only a few
will be discussed here. PBMCs (peripheral blood mononuclear cell) iso-
lated from 9 subjects supplemented with 18 g/d of sh oil concentrate
secreted reduced levels of IL-1 and TNF- in response to stimulation
with LPS (lipopolysaccaride; endotoxin) (Endres et al., 1989). 9.4 g/d
EPA + 5 g/d DHA for 10 weeks resulted in decreased neutrophil che-
motaxis to LTB4 (Sperling et al., 1993). Supplementation of 28 healthy
male volunteers with 1.62 g/d EPA + 1.08 g/d DHA for 4 weeks
reduced secretion of TNF- and IL-1 by PBMCs, in response to LPS com-
pared to secretion prior to dietary supplementation; the reduction in cy-
tokine secretion inversely correlated with the EPA content of
mononuclear cells (Caughey et al., 1996). 2.3 g/d n3 PUFAs for 3 -
months reduced ex vivo IL-1 and TNF- secretion by PBMCs in response
to concanavalin A (ConA) or phytohaemagglutinin (PHA) (Meydani
et al., 1991). Trebble et al. demonstrated that a regime supplementing
16 healthy men with increasing concentrations of EPA + DHA over
12 weeks (0.32.0 g/d) inhibited TNF- and IL-6 production in re-
sponse to LPS (Trebble et al., 2003). 1.8 g/d DHA and EPA for
277
26 weeks in healthy elderly subjects decreased PBMC expression of
genes associated with inammation and atherogenesis including NFB
and eicosanoid synthesis (Bouwens et al., 2010).
In a study designed to compare the effects of DHA vs. EPA on a vari-
ety of leukocyte functions, participants were supplemented with oil rich
in either DHA (4.9 g/d) or EPA (4.7 g/d) for 4 weeks. Neither DHA nor
EPA had any effect on the ability of isolated PBMCs to secrete cytokines,
including TNF- and IL-10, in response to ex vivo stimulation with ConA
or LPS (Kew et al., 2004). In addition there was no effect of supplemen-
tation on the ability of monocytes or neutrophils to phagocytose uo-
rescently labelled E. coli or the expression of a number of monocyte
adhesion molecules, including CD18 and CD11b (Kew et al., 2004).
DHA supplementation did reduce T lymphocyte expression of CD69,
an early marker of activation (Kew et al., 2004). Gorjao et al. investigat-
ed the effect of supplementation with 1.62 g/d DHA in 10 male subjects
for 8 weeks (Gorjo et al., 2006). They reported that DHA increased
lymphocyte proliferation in response to ConA, increased monocyte
and neutrophil phagocytosis of opsonised zymosan and increased neu-
trophil chemotaxis to complement C5a. In vitro, 100 M DHA inhibited
LPS induced TNF- and IL-1 secretion by the THP-1 monocytic cell
line (Zhao et al., 2005), an observation conrmed in subsequent studies
(Weldon et al., 2007). There is also data indicating that EPA can modu-
late NFB activation in primary human monocytes (Zhao et al., 2004). In
addition to modulating cytokine secretion, DHA (6 g/d) reduced LPS
stimulated PGE2 and LTB4 release by mononuclear cells (Kelley et al.,
1999). There is also data indicating that EPA can modulate up-stream
signalling events which control pro-inammatory transcription factors.
EPA inhibited NFB activation in the THP-1 cell line (Mullen et al., 2010)
as well as in human monocytes by preventing phosphorylation and
subsequent degradation of the inhibitory subunit IkappaB-alpha (Zhao
et al., 2004). A daily dose of 4 g n3 PUFA (EPA + DHA) for 9 months
signicantly reduced monocyte chemotaxis towards the bacterial peptide
formyl-methionyl-leucyl-phenylalanine (fMLP) (Schmidt et al., 1991).
3. The use of n3 polyunsaturated fatty acids as a dietary interven-
tion in asthma
Asthma is a chronic inammatory disease of the airways; sufferers
are hypersenstitve to specic allergens and exaggerated Th2-like cellu-
lar responses are considered to be crucial for the initiation and progres-
sion of the disease (Georas et al., 2005). AA derived eicosanoids
including LTB4, LTC4, and LTD4 are thought to be important mediators
of airway inammation and airway obstruction in asthma (Hallstrand
& Henderson, 2010). Indeed, LTB4 is increased in bronchoalveolar lavage
(BAL) uid of asthmatics compared with healthy controls (Wardlaw
et al., 1989) and may be responsible for the inux of neutrophils, eosin-
ophils and mast cells into the lung (Ohnishi et al., 2008). Owing to the
important role of n6 PUFA derived mediators in asthma, the hypoth-
esis that a diet high in these lipids might contribute to the onset of
asthma was postulated (Black & Sharpe, 1997). Moreover, rebalancing
the dietary prole of n3 PUFAs and n6 PUFAs might prevent, or at
the least reduce, morbidity associated with the disease.
Many dietary intervention studies have been conducted, mostly
with sh oils rather than individual n3 PUFAs. Measured endpoints
for efcacy have been retarding disease onset and/or reducing patho-
logical changes occurring in the lung. Epidemiological data indicated
that children who did not eat sh were three times more likely to devel-
op asthma than those who ate oily sh more than once a week (Hodge
et al., 1996). Other studies suggest a positive association of oily sh
intake with lung function and fewer asthma symptoms (Smit et al.,
1999). The data from clinical trials is more varied. Randomised con-
trolled trials (RCTs) have investigated if n3 PUFA supplementation
has any effect on established disease in adults. Twelve asthmatic
patients were randomised to either low dose (0.1 g/d) or high dose
(4.0 g/d) EPA for 8 weeks, and lung function tests carried out before
and during supplementation (Kirsch et al., 1988). The authors reported
278 C.M. Yates et al. / Pharmacology & Therapeutics 141 (2014) 272282
no effect of EPA on clinical status of disease or lung function. Arm et al.
(Arm et al., 1988) investigated the effect of n3 PUFA supplementation
on mild asthma. 12 asthmatics were randomised to receive 3.2 g
EPA + 2.2 g DHA/d and 8 to receive placebo (olive oil) for 10 weeks.
A number of clinical parameters relating to lung health were measured
at baseline and 10 weeks including airway responsiveness to histamine
and exercise, diurnal peak expiratory ow, symptom scores and bron-
chodilator use. In addition neutrophil fatty acid composition was deter-
mined and calcium ionophore induced LTB4 and LTB5 synthesis, as well
as neutrophil chemotaxis towards fMLP, was measured. There was no
effect of n3 PUFAs on any of the clinical measurements of pulmonary
function, despite the fact that there was a signicant increase in neutro-
phil EPA levels, a 50% inhibition of LTB4 + LTB5 synthesis and a signi-
cant reduction in neutrophil chemotaxis. A study comparing 15 to
20 ml/d of evening primrose oil or sh oil with an olive oil placebo for
10 weeks as a supplementary treatment of asthmatic patients reported
no effect of either oil on any clinical readout (Stenius-Aarniala et al.,
1989). 46 patients with mild to moderate asthma were randomised to
receive 50 mg/d of DHA + EPA plus 100 mg/d olive oil or 150 mg/d
olive oil (as a placebo) for 2 months (Emelyanov et al., 2002). Compared
to the placebo group, daytime wheezing was signicantly reduced in
the n3 PUFA group. However there was no difference in the forced
expiratory volume in one second (FEV1), a measure of airway obstruc-
tion, between groups. A smaller study supplemented 7 individuals
with 3 g/d DHA + EPA and reported a signicant decrease in airway re-
sponsiveness following challenge with an allergen. In agreement with
Emelyanov et al. (2002), there was no change in FEV1 following n3
PUFA supplementation. Conversely a study has reported a positive effect
on FEV1 following 9 months of supplementation with 1 g/d DHA + EPA
compared to a control group (Dry & Vincent, 1991). A recent meta-
analysis and systematic review of the use of n3 PUFA containing sup-
plements in asthma, suggested that supplementation with sh oil was
unlikely to be useful in the primary prevention of asthma (or other aller-
gic diseases including eczema, allergic rhinitis and food allergies)
(Anandan et al., 2009). The conclusions drawn about efcacy in asthma
were based on data from four studies which assessed disease incidence
in high and low risk cohorts (Marks et al., 2006, Dunstan et al., 2003;
Lauritzen et al., 2005, Olsen et al., 2008). A comprehensive report from
a United States governmental agency also concluded there was not
enough consistent evidence from such studies to state denitively that
n3 PUFAs were or were not effective in improving respiratory out-
comes in adults or children (Schachter et al., 2004).
4. The use of n3 polyunsaturated fatty acids as a
dietary intervention in rheumatoid arthritis
Rheumatoid arthritis (RA) is a disease characterised by chronic in-
ammation of the joints. It can be an extremely painful and debilitating
condition which affects any peripheral joint, but most commonly the
feet, knees and hands (Smolen et al., 1995). The aetiological agent re-
sponsible for triggering the onset of RA (if such an agent exists) is un-
known. However, it is hypothesised that infection or traumatic insult
may induce the initial inammatory response in the synovial lining of
the affected joint(s) (Firestein et al., 1987). Healthy joints do not contain
leukocytes; however in RA, neutrophils, B lymphocytes, T lymphocytes
and monocyte-derived macrophages are abundant (Firestein et al.,
1987). As the disease develops, the cells of the synovial lining prolifer-
ate, forming an invasive pannus which ultimately results in loss of
cartilage, bone destruction and dramatic loss of function (Wong &
Lord, 2004). Cytokines and chemokines, including IL-1, IL-6 and TNF-
, have been detected in the synovial uid of patients with RA and
have been implicated in the development of the disease (Brennan &
McInnes, 2008). Indeed, all of these mediators are therapeutic targets
in RA, with anti-TNF- treatment proving highly effective at controlling
disease progression in a signicant proportion of patients (Moreland
et al., 1997). Importantly, the AA derived lipid mediators of
inammation, PGE2 and LTB4 have been detected in synovial uid
(Sperling, 1995) and are thought to contribute to the inltration of leu-
kocytes into the diseased tissue, both being powerful chemo-attractants
for cells such as neutrophils.
In view of the role for AA derived lipid mediators in RA, the utility of
supplementing the diet with n3 PUFAs to reduce the level of AA de-
rived eicosanoids and to promote formation of n3 PUFA derived
agents with putative anti-inammatory activity, has been tested . One
of the rst studies to address this hypothesis supplemented the
diet of patients with 1.8 g EPA for 12 weeks and reported both a re-
duction in joint stiffness in the morning and in the number of tender
joints (Kremer et al., 1985). Since then numerous trials have been
reported with the supplemental levels of DHA and EPA varying
from 1.6 g/d to 7.1 g/d (Kremer et al., 1995, Remans et al., 2004,
Calder, 2006). An overview of placebo controlled studies using long
chain n3 fatty acids and published prior to 2006, concluded that
there is sound evidence suggesting that long-chain n3 PUFAs
have some clinical benets in rheumatoid arthritis (Calder, 2006).
Similarly a meta-analysis published in 2007 set out to provide infor-
mation concerning the efcacy of n3 PUFA supplementation on pain
management, primarily in RA, but also in inammatory bowel disease
(IBD) and dysmenorrhea (Goldberg & Katz, 2007). 17 studies were
included which had supplemented over 800 patients. The dosage of n
3 PUFA ranged from 1.7 to 9.6 g/d in the following combinations:
EPA or ALA alone; a combination of DHA and EPA; a combination of
DHA, EPA and ALA; krill oil (n3 content not reported); DHA and
docosapentaenoic acid (DPA; 22:5n3) and EPA. Pain was quantied
using six different measures: patient assessed, physician assessed, dura-
tion of morning joint stiffness, number of painful joints, joint tenderness
(Ritchie articular index (Ritchie et al., 1968)) and NSAID consumption.
The meta-analysis concluded that n3 PUFAs reduce the intensity of pa-
tient assessed joint pain, joint stiffness in the morning, number of painful
and/or tender joints and NSAID consumption. The authors suggest that in
order to maximise therapeutic effects future trials should supplement
with N2.7 g/d EPA + DHA and should last for a minimum of 3 months.
Overall n3 PUFAs appear to reduce pain and therefore have an
NSAID sparing effect in patients with RA. This is potentially important
as prolonged NSAID use is associated with an increased risk of develop-
ing gastrointestinal ulcers and gastrointestinal bleeding (Traversa et al.,
1995), therefore any intervention which may reduce NSAID use could
have signicant benet to patient welfare.
5. Inammatory bowel diseases and n3 polyunsaturated fatty acids
Inammatory bowel diseases (IBD) encompass two main subtypes
of disease, ulcerative colitis (UC) and Crohn's disease (CD) (Abraham
& Cho, 2009). Both are chronic inammatory diseases; UC mainly affects
the colon, whereas CD can occur in any part of the gastrointestinal (GI)
tract. The precise causative agent of IBD is unknown, however mounting
evidence indicates that inappropriate activation of the immune system
by intestinal bacteria is key (Cho, 2008). Interestingly, in the absence of
enteric bacteria in a rat model of colitis, there was no evidence of
inammation (Sellon et al., 1998). Familial and twin studies have dem-
onstrated the role of genetics in disease, indeed polymorphisms in
genes associated with microbial defence and intestinal immunity are as-
sociated with increased disease susceptibility (Cho, 2008). Environmen-
tal factors, including smoking, the composition of the diet and hygiene,
have been linked with the development of IBD (Koutroubakis et al.,
1996). The gut mucosa of healthy individuals ordinarily contains macro-
phages, T cells and dendritic cells which secrete both pro- and anti-
inammatory cytokines. There is a balance maintained which works
to both restrict excessive levels of enteric microbiota and protect against
infections. In IBD this balance between the immune system and gut ora
is disrupted due to greater intestinal permeability and the breakdown of
epithelial tight junctions (Abraham & Cho, 2009). This leads to increased
inltration of neutrophils and T cells and enhanced levels of pro-
 C.M. Yates et al. / Pharmacology & Therapeutics 141 (2014) 272282
inammatory cytokines including TNF- (Sands & Kaplan, 2007). Here
we discuss whether n3 PUFA as a dietary supplementation may be a
useful tool in the primary prevention or management of the disease.
The European prospective cohort study investigated the risk of de-
veloping UC with dietary fatty acid intake in 139 patients compared
with age and sex matched controls (Hart et al., 2008). There was no
correlation between saturated or monounsaturated intake and UC,
however there was a slight increase in disease risk with total PUFA in-
take (Hart et al., 2008). Further analysis of the study revealed that the
high intake of the n6 PUFA LA was responsible for the increased UC
risk, and there was no association with DHA or EPA intake (The IBD in
EPIC Study Investigators, 2009). A prospective cohort study of 25,639
patients between the ages of 4574 years used 7 d food diaries to inves-
tigate the hypothesis that n3 PUFA intake is protective in UC (John
et al., 2010). Total dietary n3 PUFAs, in particular DHA, were associat-
ed with protection from UC in this cohort (John et al., 2010). Overall
these data suggest that n3 PUFA intake is associated with a decreased
risk, and higher n6 intake with an increased risk of developing UC.
This protective effect of n3 PUFAs may be due to modulation of
inammatory events which take place within the GI environment
(Vilaseca et al., 1990).
Rat colitis models demonstrate that inammation in the colonic
mucosa results in an increase in the levels of AA derived LTB4 and
PGE2 (Hirata et al., 2001, Nieto et al., 2002) and mucosal LTB4 is also el-
evated in humans with IBD (Sharon & Stenson, 1984). LTB4 is a potent
leukocyte chemoattractant and plays a role in mediating the inamma-
tion associated with IBD. Inhibition of the LT biosynthetic pathway
in colitic rats reduced mucosal injury and decreased levels of LTB4
(Empey et al., 1992). Interestingly, this reduction in LTB4 was accompa-
nied by an increase in PGE2, suggesting a shift towards PG synthesis
(Empey et al., 1992). PGE2 has reported anti-inammatory actions, in-
cluding inhibition of 5-LO, resulting in reduced 4-series LT synthesis
(Levy et al., 2001).
IBD clearly has an inammatory component, consequently a number
of animal studies have taken place to assess the efcacy of n3 PUFAs in
IBD. Many of these studies used chemically induced models of colitis.
In a UC model, induced using trinitrobenzene sulphonic acid (TNBS),
rats supplemented with cod liver oil had reduced colonic damage and
inammation compared to the sunower oil group (Vilaseca et al.,
1990). Using the same TNBS model, sh oil supplementation resulted
in lower colonic inammation and a decrease in LTB4 in colonic tissue
(Yuceyar et al., 1999). Recently in two mouse models of colitis, systemic
treatment with aspirin triggered resolvin D1 (RvD1), the precursor
(17(R)-hydroxy docosahexaenoic acid [17R-HDHA]) or resolvin D2
(RvD2) resulted in an improved disease activity score, reduced colonic
damage and a decrease in neutrophil inltration (Bento et al., 2011).
Many human supplementation studies in IBD do not assess the
ability of n3 PUFAs to modulate cellular readouts of inammation
but report the effect on clinical outcomes. However, it has been reported
that n3 PUFAs are incorporated into the colonic mucosa of patients
with IBD and that sh oil reduces LTB4 secretion from patient's neutro-
phils (McCall et al., 1989, Hawthorne et al., 1992; Shimizu et al., 2003).
Randomised, placebo-controlled, double-blind studies of sh oil in IBD
have been published. One of the rst studies supplemented both UC
and CD patients with 1.8 g/d EPA + 1.3 g/d DHA for 7 months
(Lorenz et al., 1989). Clinical activity was unchanged in CD patients
with supplementation but fell in UC patients, although this did not
reach signicance. In a 12 month RCT, 87 patients received 4.5 g/d
EPA and 1.1 g/d DHA (Hawthorne et al., 1992) Supplementation in-
creased the rectal mucosa EPA to 3.2% of total fatty acids compared to
0.63% in the placebo olive oil group. There was limited clinical benet;
patients who started the trial in relapse reported signicantly reduced
corticosteroid use after one and two months of n3 PUFA, but there
was no reduction in the rate of relapse (Hawthorne et al., 1992). Simi-
larly both Lorenz-Meyer et al. (1996) and Feagan et al. (2008) reported
no effect on relapse with n3 PUFA supplementation in UC and CD
279
respectively. Despite the lack of effect on relapse, there are reports of
decreased gut mucosal disease activity and improved histological scores
following supplementation (Stenson et al., 1992, Almallah et al., 1998,
Almallah et al., 2000).
Interestingly although there appears to be some benet of n3
PUFA in IBD, meta-analyses have failed to conrm this. MacLean et al.
(2004) published a meta-analysis of 13 studies of sh oil supplementa-
tion in IBD and concluded there was sufcient data to perform meta-
analysis for relapse in UC only. The analysis concluded that n3 fatty
acids have no effect on relative risk of relapse in ulcerative colitis and
there was a statistically non-signicant reduction in requirement for
corticosteroids for n3 fatty acids relative to placebo in two studies
(MacLean et al., 2004). The most recent meta-analysis set out to review
the efcacy and safety of n3 PUFA in the maintenance of remission in
IBD in three trials of UC and six of CD (Turner et al., 2011). Having exam-
ined the pooled data the authors found a higher rate of diarrhoea and
symptoms of the GI tract in the n3 PUFA treatment group. Overall
the conclusion was there are insufcient data to recommend the use
of omega 3 fatty acids for maintenance of remission in CD and UC. It
therefore appears that although there is some benet reported in ani-
mal models of IBD, in particular UC, there is limited data to suggest
that this translates into benet for human patients.
6. Conclusion
The marine n3 PUFAs EPA and DHA exert a range of anti-
inammatory effects including reduced eicosanoid, cytokine and adhe-
sion molecule production, enhanced SPM production and decreased
leukocyte-EC adhesive interactions. These effects suggest that n3
PUFAs would be useful as therapies in patients with inammatory con-
ditions. However, data from patient or laboratory based studies can be
difcult to interpret due to variations in dosage, treatment length,
confounding medication, or translating evidence from the laboratory
to the clinic. Nevertheless, supplementation with n3 PUFAs may be
benecial in some settings such as RA, although high doses are required.
The lack of consistent data in other diseases such as asthma and IBD sug-
gests that n3 PUFAs may not be benecial in all settings, despite the
presence of common cells and mediators in these different conditions.
One explanation for this could be that n3 PUFAs may not have the
same effects on inammation in healthy, compared with diseased indi-
viduals. There is the possibility that long term supplementation of
healthy individuals may reset the inammatory threshold of the indi-
vidual, providing a degree of protection from cumulative inammatory
injury. However, at least in the context of advanced arterial disease,
short term supplements may not be effective at reversing systemic in-
ammation associated with the disease process. This has been reviewed
(Sijben & Calder, 2007), and current data suggests that supplementation
of healthy subjects with n3 PUFAs may be effective in preventing the
onset of chronic inammatory diseases such as CVD, whereas those
with established disease may receive less benet from dietary supple-
mentation with n3 PUFAs. CVD and a number of other chronic inam-
matory diseases are associated with ageing, and it is possible that ageing
itself may inuence the effectiveness of supplementation. Although
there are data on the effect of n3 PUFA in the elderly in regards to
cognitive function (Denis et al., 2013), this idea has not been thoroughly
investigated in chronic inammatory disease.
Underlying genetic factors controlling the processing of dietary
lipids may offer a further explanation as to why supplementation has
varying effects in individuals (Madden et al., 2011). Recent genome
wide association study (GWAS) studies have identied new loci in-
volved in lipid metabolism (Kim et al., 2011; Teslovich et al., 2010). Of
interest is sirtuin 1 (SIRT1) a member of NAD+ dependant histone
deacetylase family, which can target nuclear receptors/transcription
factors related to lipid metabolism (Finkel et al., 2009). A recent
Japanese study of N1400 individuals linked SITR1 haplotype 2 with
low circulating levels of LDL and high circulating HDL in those who
280 C.M. Yates et al. / Pharmacology & Therapeutics 141 (2014) 272282
consumed a diet with a low n6/n3 ratio(Inamori et al., 2013). In
addition a variant of the n3 lipid sensing GPR120 gene, R270H, has
been identied (Ichimura et al., 2012). Expression of this polymorphism
in a European population is strongly associated with obesity in humans
and inhibits signalling via GPR120 (Ichimura et al., 2012). Whether this
gene mutation affects the ability of people to respond to dietary n3
PUFA supplementation has not been addressed, however these data
raise interesting questions about the role of genetics in lipid metabolism.
Genetic factors may direct the way individuals respond to dietary lipid in-
take and could indicate why there is inter-individual heterogeneity in re-
sponse to n3 PUFA supplementation. Further studies are required to
determine if interactions between genetic and modiable environmental
factors occur and, if so, whether they could exert protective functions.
The current literature indicates that dietary supplementation may
be benecial in some chronic inammatory diseases. However, there
is now an opportunity to identify novel approaches to preventing and
reversing/resolving chronic inammatory disease based upon the
downstream metabolic products of EPA and DHA and their mechanisms
of action or the interaction of these n3 PUFAs with free fatty acid
receptors.
Conict of interest statement
CMY and GER have no conict of interest to report. PCC serves on
Scientic Advisory Boards of the Danone Research Centre in Specialised
Nutrition, Aker Biomarine, Pronova Biopharma and Smartsh. He acts as
a consultant to Mead Johnson Nutritionals, Vifor Pharma and Amarin
Corporation. He has received speaking honoraria from Solvay Healthcare,
Solvay Pharmaceuticals, Pronova Biopharma, Fresenius Kabi, B. Braun,
Abbott Nutrition, Baxter Healthcare, Nestle, Unilever and DSM. He cur-
rently receives research funding from Vifor Pharma. He is immediate
Past-President of the International Society for the Study of Fatty Acids
and Lipids, an organisation that is partly supported by corporate
membership fees, mainly the food and supplements industries. He is a
member of the Board of Directors of ILSI Europe, the Board of Directors
of the European Nutraceutical Association, and the Council of the British
Nutrition Foundation; these organisations are each supported in part by
the food and supplements industries.
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