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Anti-Inflammatory Effects of Omega-3 Fatty Acids in the Brain: Physiological Mechanisms and Relevance to Pharmacology


Abstract and Figures

Classically, polyunsaturated fatty acids (PUFA) were largely thought to be relatively inert structural components of brain, largely important for the formation of cellular membranes. Over the past 10 years, a host of bioactive lipid mediators that are enzymatically derived from arachidonic acid, the main n-6 PUFA, and docosahexaenoic acid, the main n-3 PUFA in the brain, known to regulate peripheral immune function, have been detected in the brain and shown to regulate microglia activation. Recent advances have focused on how PUFA regulate the molecular signaling of microglia, especially in the context of neuroinflammation and behavior. Several active drugs regulate brain lipid signaling and provide proof of concept for targeting the brain. Because brain lipid metabolism relies on a complex integration of diet, peripheral metabolism, including the liver and blood, which supply the brain with PUFAs that can be altered by genetics, sex, and aging, there are many pathways that can be disrupted, leading to altered brain lipid homeostasis. Brain lipid signaling pathways are altered in neurologic disorders and may be viable targets for the development of novel therapeutics. In this study, we discuss in particular how n-3 PUFAs and their metabolites regulate microglia phenotype and function to exert their anti-inflammatory and proresolving activities in the brain.
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PHARMACOLOGICAL REVIEWS Pharmacol Rev 70:1238, January 2018
Copyright © 2017 by The American Society for Pharmacology and Experimental Therapeutics
Anti-Inflammatory Effects of Omega-3 Fatty Acids in the
Brain: Physiological Mechanisms and Relevance to
Sophie Layé, Agnès Nadjar, Corinne Joffre, and Richard P. Bazinet
Institut National pour la Recherche Agronomique and Bordeaux University, Nutrition et Neurobiologie Intégrée, UMR 1286, Bordeaux,
France (S.L., A.N., C.J.); and Department of Nutritional Sciences, University of Toronto, Ontario, Canada (R.P.B.)
Abstract .....................................................................................13
I. Introduction . . ...............................................................................13
II. Definition of PUFAs .........................................................................14
A. PUFA Metabolism .......................................................................14
B. Dietary Origin of PUFAs .................................................................15
C. Accumulation and Regional Distribution in the Brain .....................................15
D. Intrinsic (Age, Sex) and Extrinsic (Diet) Factors Influencing Brain PUFA Content . ........16
III. Mechanisms of Entry of PUFAs into the Brain ............................................... 16
A. Plasma Pools for Brain DHA Supply .....................................................16
B. Mechanisms of DHA Entry into the Brain ................................................17
IV. Anti-Inflammatory Activities of n-3 PUFAs in the Brain ......................................17
A. General Evidence in Humans and Animal Models of Brain Pathologies....................17
B. Overview of DHA Anti-Inflammatory/Proresolving Mechanisms . ..........................18
1. Membrane and Signaling Effects. .....................................................18
2. N-3 LC-PUFA Derivatives.............................................................19
a. Oxylipins. .........................................................................19
b. Endocannabinoids. . . . .............................................................19
V. Microglia as a Target for n-3 PUFAs and SPMs . . . ...........................................20
A. Modulation of Microglial Function by n-3 PUFAs . . . ......................................21
1. In Vitro Evidence. . ...................................................................21
2. In Vivo Evidence. . . ...................................................................22
B. Modulation of Microglial Function by SPMs . . . ...........................................23
1. Lipid Derivatives Target Microglia. . . .................................................23
2. Lipid Derivatives and Their Receptors. ................................................24
C. A Link between n-3 PUFAs and Microglial Extracellular Vesicles? ........................24
D. Sex, Age, and Regional Differences in the Relationship between Microglia and n-3
PUFAs ..................................................................................24
1. Evidence for Sexual Dimorphism of Microglia.. . . ......................................24
2. Evidence for Regionalization of Microglia. . . ...........................................25
3. Evidence for Age Dependence of Microglia. . ........................................... 25
VI. Pharmacological Considerations on the Use of LC-PUFAs or SPMs as Effective Anti-
Inflammatory Drugs in the BrainClinical Use . . . ...........................................26
A. LC-PUFA Dietary Interventions to Limit Neuroinflammation in Humans . . . ..............26
1. LC-PUFA Dietary Intervention and Neuroinflammation in AD Patients. . .............. 26
2. LC-PUFA Dietary Intervention and Neuroinflammation in Patients with Mood
Disorders. . . . .........................................................................27
S.L., C.J., and A.N. are supported by Institut National pour la Recherche Agronomique, Bordeaux University, Foundation for Medical
Research (DRM.20101220441), and the French Foundation (FDF, #00070700). R.P.B. is supported by the Canadian Institutes of Health
Research and the Natural Sciences and Engineering Council of Canada, and holds the Canada Research Chair in Brain Lipid Metabolism.
Address correspondence to: Dr. Sophie Layé, NutriNeuro Institut National pour la Recherche Agronomique and Bordeaux University,
UMR 1286, 146 rue Léo Saignat, 33076 Bordeaux, France. E-mail:
by guest on December 10, 2017Downloaded from
B. The Use of SPMs or n-3 PUFA-Derived Fatty Amides to Target Brain Neuro-
inflammation in Humans.................................................................28
C. Pharmacological Strategy to Promote Endogenous SPM Production in the Brain:
Combination of PUFAs and Anti-Inflammatory Drugs ....................................29
D. The Use of COX or Lipoxygenase Inhibitors to Target Neuroinflammation: a Paradox? . . . . 30
VII. Conclusion . . . ...............................................................................31
Abstract——Classically, polyunsaturated fatty acids
(PUFA) were largely thought to be relatively inert
structural components of brain, largely important for
the formation of cellular membranes. Over the past
10 years, a host of bioactive lipid mediators that are
enzymatically derived from arachidonic acid, the main
n-6 PUFA, and docosahexaenoic acid, the main n-3
PUFA in the brain, known to regulate peripheral immune
function, have been detected in the brain and shown to
regulate microglia activation. Recent advances have
focused on how PUFA regulate the molecular signaling
of microglia, especially in the context of neuroinflamma-
tion and behavior. Several active drugs regulate brain
lipid signaling and provide proof of concept for targeting
the brain. Because brain lipid metabolism relies on a
complex integration of diet, peripheral metabolism,
including the liver and blood, which supply the brain
with PUFAs that can be altered by genetics, sex, and
aging, there are many pathways that can be disrupted,
leading to altered brain lipid homeostasis. Brain lipid
signaling pathways are altered in neurologic disorders
and may be viable targets for the development of novel
therapeutics. In this study, we discuss in particular
how n-3 PUFAs and their metabolites regulate
microglia phenotype and function to exert their anti-
inflammatory and proresolving activities in the brain.
I. Introduction
Polyunsaturated fatty acids (PUFAs) are generally
considered to be essential fatty acids, meaning they are
necessary for maintaining normal physiology, but can-
not be produced by mammals and need to be provided by
the diet (Bazinet and Layé, 2014). There are two main
families of PUFAs, the n-6 and n-3 PUFAs (also referred
as omega 6 and omega 3). Linoleic acid (LA; 18:2n-6) is
the dietary-essential shorter-chain n-6 PUFA precursor
of arachidonic acid (AA), whereas a-linolenic acid (ALA;
18:3n-3) is the dietary-essential shorter chain n-3 PUFA
precursor of eicosapentaenoic acid (EPA) and docosa-
hexaenoic acid (DHA). AA, DHA, and EPA are also
consumed in the diet, although as distinct sources. The
major dietary sources of ALA are green plant tissues,
nuts, flaxseed, and rapeseed oil, whereas oily fish is the
main source of EPA and DHA.
PUFAs from the diet are absorbed from the gut to the
blood and are available for storage (in the adipose
tissue), conversion into longer-chain PUFA (mainly in
the liver), or energy production through b-oxidation. LA
and ALA biosynthetic pathway to AA and EPA and
DHA, respectively, involves a series of desaturation,
elongation occurring in the endoplasmic reticulum
(Fig. 1). The last step in DHA formation involves
b-oxidation, occurring in peroxisomes. As ALA and LA
use the same metabolic pathways to generate long-chain
(LC) PUFA, there is a competition between these two
pathways, with end products generated, at least some-
what, proportional to their precursors.
Generally speaking, LA and ALA poorly accumulate
in tissues, as compared with AA and DHA, which is in
line with their role as precursors to longer-chain PUFA.
The rate of synthesis of ALA into EPA and DHA
occurring mainly in the liver is considered to be low,
with about 8% of ALA being converted to EPA and 1% to
DHA. The enzymes necessary to metabolize ALA are
present in the brain; however, the brains major source
of DHA is coming from the blood, as discussed later.
The brain is highly enriched in AA and DHA (Bazinet
and Layé, 2014). Both n-3 and n-6 PUFAs are esterified
in the sn-2 position into phospholipids, which are well
known to play critical role in the structures and
functions of brain cell membranes. Brain cell membrane
contains mainly phosphatidylcholine, phosphatidyleth-
anolamine, phosphatidylserine, phosphoinositides, and
plasmalogens with specific PUFA profiles. At the level
of the membrane, PUFAs undergo turnover due to the
activity of phospholipase A2 (PLA2) and acyl-CoA
lysophospholipid transferases. Two distinct groups of
PLA2 are involved in the release of PUFA, namely the
ABBREVIATIONS: AA, arachidonic acid; AD, Alzheimers disease; AEA, anandamide; ALA, a-linolenic acid; AT, aspirin triggered;
Ab, amyloid-b; BBB, blood-brain barrier; CNS, central nervous system; COX, cyclooxygenase; CSF, cerebrospinal fluid; DHA, docosahexaenoic
acid; DHEA, docosahexaenoyl ethanolamide; DPA, docosapentaenoic acid; eCB, endocannabinoid; EFOX, electrophilic oxo-derivatives; EPA,
eicosapentaenoic acid; EPEA, eicosapentaenoyl ethanolamide; ER, estrogen receptor; EV, extracellular vesicle; FABP, fatty acidbinding
protein; FATP, fatty acid transport protein; GPR, G-coupled receptor; IL, interleukin; iNOS, inducible NO synthase; KO, knockout; LA,
linoleic acid; LB, lipid bodies; LC, long chain; LOX, lipoxygenase; LPS, lipopolysaccharide; LT, leukotriene; lysoPC, lysophosphatidylcholine;
LxA4, lipoxin A4; MaR, maresin; MHC, major histocompatibility complex; MMP, matrix metalloproteinase; NF, nuclear factor; NO, nitric
oxide; NPD1, neuroprotectin D1; NSAID, nonsteroidal anti-inflammatory drug; PBMC, peripheral blood mononuclear cell; PG, prostaglandin;
PLA2, phospholipase A2; PPAR, peroxisome proliferator-activated receptor; PUFA, polyunsaturated fatty acid; Rv, resolving; SPM,
specialized proresolving mediator; TLR, Toll-like receptor; TNF, tumor necrosis factor; TX, thromboxane.
New Insights into the Impact of Omega-3 in Microglia 13
group IV cytosolic PLA2, which releases AA, and group
VI calcium-independent phospholipase, which releases
DHA. The free forms of PUFA are metabolized into
specific derivatives [eicosanoids, specialized proresolving
mediators, specialized proresolving mediator (SPMs),
and endocannabinoids (eCBs)], which are key regulators
of inflammation (Lukiw and Bazan, 2000; Buckley et al.,
2013; Serhan, 2014; Calder, 2015; DiMarzo et al., 2015;
Witkamp, 2016). AA derivatives mainly display proin-
flammatory activities, albeit there are some exceptions,
whereas DHA derivatives are anti-inflammatory and
More recently, particular attention has been paid to
these derivatives inthe regulation of neuroinflammation.
Neuroinflammation is a double-edged sword that exerts
both beneficial and detrimental effects on neurons.
Microglia, the brain-resident innate immune cells, are
thought to be protective when properly activated. How-
ever, inadequate activation worsens neuropathological
processes and increases neuronal death, as observed in
neurodegenerative diseases. The complexity of the micro-
glia phenotype and its regulation may account for its
protective and detrimental effects toward neurons, as
discussed elsewhere. An increasing body of evidence
suggests that PUFA and their derivatives may be in-
volved in microglia regulation and the control of neuro-
inflammation (Layé, 2010; Bazinet and Layé, 2014).
Furthermore, because of the high quantity of PUFA in
the brain, specific alterations in PUFA metabolism in the
brain may play an important role in neuroinflammatory
In this review, we will present recent updates on the
metabolism and role of endogenous AA, DHA, and their
bioactive derivatives involved in the resolution of neuro-
inflammation, with a specific focus on microglial cells.
In particular, we will discuss how PUFAs can be used to
target microglia and how drugs targeting PUFA me-
tabolism regulate neuroinflammation. We will high-
light recent controversies and examine adverse events.
II. Definition of PUFAs
Lipids represent 33%40% of the energy intake in
France and the United States (Malvy et al., 1999;
Simopoulos, 2011). They are essentially found (90%95%)
in the form of triacylglycerides, a structure consisting of a
glycerol backbone and three fatty acids. They are also
found in the form of phospholipids, in which the fatty acid
in the three position on the glycerol is replaced by a
phosphorylated functional group. The structure of a
triacylglyceride and a phospholipid is shown in Fig. 2.
Fatty acids have many physiologic roles. They are the
primary source of energy storage for tissues and, as
components of membrane phospholipids, play a structural
role. Fatty acids of phospholipids are also mobilized by the
cells as precursors of lipid mediators, which regulate many
physiologic processes, such as inflammation. There are
three families of fatty acids classified according to the
number of double bonds on their carbon chain they contain:
the saturated ones (no double bond), the monounsaturated
ones (one double bond), and the polyunsaturated ones (two
or more double bonds). In rodents, the brain contains
36%46% saturated fatty acids, 18%33% monounsatu-
rated fatty acids, and 18%28% PUFAs (Joffre et al., 2016).
A. PUFA Metabolism
As previously described, PUFAs are classified into
two main categories, the n-6 PUFAs and the n-3 PUFAs.
LA (18:2 n-6) and ALA (18:3 n-3) are, respectively, the
precursors of these two series. They are called essential
fatty acids because mammals cannot synthesize them.
In vivo, these precursors can be metabolized by series of
elongation, desaturation, and a b-oxidationproducing
PUFA with additional unsaturations and/or carbon
atoms sometimes refered to as LC-PUFA (Fig. 1).
LC-PUFA biosynthesis requires position-specific D6
and D5 desaturases and elongases, and the participa-
tion of both microsomes and peroxisomes (Sprecher,
2000). LC-PUFA biosynthesis takes place predomi-
nately in the liver, despite the brain possessing the
enzymatic equipment necessary for their synthesis.
Both n-6 and n-3 PUFA share the same enzymatic
equipment for the biosynthesis of the LC-PUFAs and
can thus compete (Simopoulos, 2011). The main metab-
olites for the n-6 and n-3 family are AA (20:4 n-6) and
DHA (22:6 n-3), respectively (Kitajka et al., 2004; Joffre
et al., 2016). EPA (20:5 n-3) is also an important n-3
PUFA metabolite, despite its low level in the brain
because of its rapid b-oxidation (Chen and Bazinet,
2015). Docosapentaenoic acid (DPA; 22:5 n-6) for the n-6
Fig. 1. Metabolic pathways of PUFAs. The precursors LA and ALA are
metabolized into LC-PUFAs via several cycles of elongation and
desaturation and one step of b-oxidation within the peroxisome. From
there, LC-PUFAs are released into the bloodstream to reach target
14 Layé et al.
family is also relevant because it replaces DHA during
dietary n-3 PUFA deficiency.
B. Dietary Origin of PUFAs
AA and DHA come mainly from the diet. Although
humans can synthesize them from LA and ALA, re-
spectively, that are found in vegetables, the conversion
efficiency is very low (,1%) even in healthy adults (Kidd,
2007; Plourde and Cunnane, 2007). In the western diet,
there is thought to be an imbalance between n-6 and n-3
PUFAs, leading to a n-3 PUFA consumption 1220 times
lower than n-6 PUFA consumption (Simopoulos, 2002,
2011). This is due to the increased industrialization in
the developed nations accompanied by changes in di-
etary habits. It is particularly characterized by an
increase in LA, abundant in many vegetable oils
(60%65% in sunflower oil for example) (Orsavova et al.,
2015) and AA, found in meats (5%10%) and eggs (15%)
(Taber et al., 1998; Meyer et al., 2003), together with
relatively low intakes of ALA, found in some green
vegetables, rapeseed oil (10%) (Lewinska et al., 2015),
and nuts, and EPA and DHA abundant in fatty fish (18.7%
EPA plus DHA in salmon, 32.9% EPA plus DHA in tuna)
(Strobel et al., 2012). A high intake of LA associated with a
the expression of desaturases and elongases is upregu-
brain (Igarashi et al., 2007). In addition, under dietary n-3
PUFA deficiency, the half-life of brain DHA is increased
by twofold (Demar et al., 2004).
Although not universally accepted, several dietary
recommendations state a ratio LA/ALA close to 45 and
a;500 mg/d supply in EPA and DHA sufficient to meet
the n-3 PUFA needs of the body and to protect against
cardiovascular disease risk (Burdge, 2004; Lucas et al.,
2009). Preclinical and clinical studies indicate that
increasing dietary ALA and reducing LA are beneficial
in increasing n-3 LC-PUFA bioavailability (Blanchard
et al., 2013; Taha et al., 2014). Concerning the bio-
availability of dietary EPA/DHA in the form of phos-
pholipids (krill oil source) or triacylglycerides (fish oil
source), no clear evidence actually identifies a better
source (Salem and Kuratko, 2014; Yurko-Mauro et al.,
2015) to date.
C. Accumulation and Regional Distribution in the Brain
AA and DHA accumulate during brain development,
especially during the perinatal period: in humans be-
tween the beginning of the third trimester and 2 years of
age and in rodents between the 7th and the 21st
postnatal day (Clandinin et al., 1980). These periods
correspond to the rapid neuronal maturation, synapto-
genesis, and gray matter expansion (Morgane et al.,
1993; Giedd et al., 1999).
LC-PUFAs vary across brain regions (Delion et al.,
1994; Carrié et al., 2000; McNamara et al., 2009; Joffre
et al., 2016). For example, in the adult C57BL6/J mice, the
highest level of AA is found in the hippocampus (10.2%),
followed by the prefrontal cortex (9.7%), the hypothala-
mus (8.5%), the cortex (7.7%), the cerebellum (6.5%), and
the brain stem (5.5%) (Joffre et al., 2016). The highest
level of DHA is found in the prefrontal cortex (14.3%) and
in the hippocampus (13.7%), followed by cerebellum
(12.2%), cortex (11.9%), hypothalamus (10.1%), and brain
stem (8.2%) (Joffre et al., 2016). Then the AA/DHA ratio
varies from 0.75 to 0.85 in the hypothalamus and
hippocampus to 0.54 in the cerebellum. These variations
may be due to different LC-PUFA entry mechanisms into
the brain or to different incorporation into membranes of
cells composing the structure considered.
Brain DHA levels are comparable in human and mice:
between 12.3% and 15.9% in the prefrontal cortex of rats
and mice (Moriguchi et al., 2001; Xiao et al., 2005; Joffre
et al., 2016) and between 14.1% and 15.9% in post-
mortem frontal cortex in human (Hamazaki et al., 2015,
2016). However, Cortie et al. (2015) reported that mouse
mitochondria contain higher levels of PUFA as com-
pared with those from humans.
Fig. 2. Structure of the two main forms of lipids in food, the
triacylglyceride (A) and phospholipid (B). Triacylglycerides and phospho-
lipids contain a glycerol backbone on which fatty acids are esterified (R1,
R2, and R3 in positions 1, 2, and 3 for the triglyceride and R1 and R2 for
the phospholipid). On the third position of the phospholipid was esterified
a phosphate group associated to a radical, which may be an amino alcohol
or a polyol.
New Insights into the Impact of Omega-3 in Microglia 15
Differences in brain DHA levels depending on brain
structures, dietary intake, gender, and aging may have
consequences on inflammatory processes because n-3
LC-PUFAs have immunomodulatory properties (Layé,
D. Intrinsic (Age, Sex) and Extrinsic (Diet) Factors
Influencing Brain PUFA Content
The brain LC-PUFA levels fluctuate with differential
extrinsic and intrinsic factors. Brain LC-PUFA levels are
modified by the fatty acid composition of the diet (Calder,
2007). Indeed, low consumption in n-3 PUFAs induces a
decrease in brain DHA levels (Connor et al., 1990; Carrié
et al., 2000; Larrieu et al., 2012; Joffre et al., 2016) and an
increase in brain DPA (22:5n-6) and often AA levels
(Connor et al., 1990; Larrieu et al., 2012), whereas
genetic-driven enrichments in n-3 PUFAs induce the
opposite (He et al., 2009; Boudrault et al., 2010; Orr et al.,
2010; Bousquet et al., 2011; Joffre et al., 2016). These
modifications impact all brain structures, but some of
them are more affected than others: the prefrontal cortex
and the hippocampus, which contain the highest DHA
content and are the most sensitive, whereas the hypo-
thalamus, which contains the lowest DHA, is the least
sensitive. These differences may be attributed to the
evolution of brain performance (Crawford et al., 1999;
Broadhurst et al., 2002).
Furthermore, several studies conducted in humans
and rodents suggest that LC-PUFA levels vary with
gender. Indeed, DHA is higher in females than in males,
independently of the status of dietary n-3 PUFA (Lin
et al., 2016). This gender difference is attributed to the
levels of hormones that increase the mRNA expression
of fatty acid desaturase 2, the gene encoding D6
desaturase (Giltay et al., 2004; Magnusardottir et al.,
2009). It was specifically found that DHA was higher in
phosphatidylcholine and phosphatidylethanolamine of
platelets in women (Geppert et al., 2010). Differences
between males and females are also reported in rat liver
and cerebral cortex (Extier et al., 2010).
In addition, age influences brain LC-PUFA levels, as
aging is often characterized by a decrease in LC-PUFAs
(Calderini et al., 1983; Lopez et al., 1995; Zhang et al.,
1996; Favreliere et al., 2003; Little et al., 2007;
McNamara et al., 2008; Labrousse et al., 2012;
Ledesma et al., 2012; Moranis et al., 2012). The decrease
in brain DHA is accentuated in aged animals fed a n-3
PUFA-deficient diet (Joffre et al., 2016).
III. Mechanisms of Entry of PUFAs into the Brain
A. Plasma Pools for Brain DHA Supply
As previously mentioned, the brain is enriched with
PUFAs, particularly DHA and the n-6 PUFA AA.
Although the brain can synthesize saturated and mono-
unsaturated fatty acids, it must rely on uptake of either
the preformed DHA and AA or their dietary precursors,
ALA and LA, respectively, which can be converted to
DHA and AA within the brain. Whereas the brain does
have the capacity to synthesize DHA and AA, their rate
of synthesis relative to uptake from the plasma is low,
suggesting that uptake from plasma and not synthesis
within the brain is the major source (DeMar et al., 2006;
Igarashi et al., 2007). Furthermore, although the liver
can upregulate its ability to synthesize DHA, especially
under conditions of low dietary n-3 PUFA intake, the
brain does not upregulate DHA synthesis under these
conditions, further demonstrating the need for a con-
stant plasma supply to the brain (Igarashi et al., 2007).
Within the blood, DHA can be free (sometimes refered
to as unesterified) or esterified to triacylglycerides,
phospholipids, and cholesteryl esters. Whereas red
blood cells contain esterified DHA, this pool is generally
not thought to contribute DHA, at least not directly, to
the brain. The vast majority of DHA that is esterified in
the blood occurs as circulating lipoproteins, but there
are small pools of free esterified DHA, especially
lysophosphatidylcholine containing DHA (Rapoport
et al., 2001). The plasma pools that contribute DHA to
the brain have been and remain somewhat controver-
sial (for review, see Mitchell and Hatch, 2011), and,
below, we will attempt to highlight potential reasons for
disagreement. Although it was originally hypothesized
that lipoprotein containing DHA was the major source
supplying the brain with DHA, knockout (KO) of either
the low-density or very-low-density lipoprotein recep-
tors does not decrease brain DHA (or AA) concentra-
tions (Chen et al., 2008; Rahman et al., 2010). However,
caution must be taken with interpreting these lifelong
KO studies as compensation via another mechanism
could maintain brain DHA concentrations. However, it
is clear that these lipoprotein receptors are not neces-
sary for maintaining brain DHA levels and other
mechanisms must exist. As an attempt to identify the
major plasma pools for supplying DHA to the brain, we
used a kinetic model in combination with labeled DHA,
in rats, where unesterified DHA is infused i.v. to achieve
a steady state, and calculated the rate of uptake of
unesterified DHA to the brain (Chen et al., 2015). We
then administered radiolabeled DHA by gavage, which
labels multiple plasma pools as well as the brain. We
found that the coefficient of uptake or the rate of uptake
from the unesterified pool, alone, was sufficient to
explain the rate of uptake of all the labeled plasma
pools upon oral administration. Or more, simply put, it
appeared as if the unesterified pool was the major
source, if not the only source, supplying the brain upon
oral administration. We then used another kinetic
model to calculate the rate at which DHA enter the
brain and found the rate of DHA exiting the brain to be
similar to the uptake rate from the plasma unesterified
pool. Because DHA is not accumulating in the adult
rodent brain, this suggested that unesterified DHA is,
again, the major pool supplying the brain. Importantly,
16 Layé et al.
it had been reported that upon acute i.v. administration
of labeled unesterified DHA or DHA esterified to
lysophosphatidylcholine (lysoPC), more radioactivity,
presumably from DHA, was present in the brain of
rodents receiving lysoPC-containing DHA upon several
hours (Thies et al., 1994; Lagarde et al., 2001). Further-
more, evidence that Mfsd2a, a protein that facilitates
the uptake of lysoPC containing DHA into the brain, KO
had lower brain DHA levels compared with wild-type
controls combined with observation of more radiolabel-
ed DHA entering the brain suggested that lysoPC
containing DHA was the major plasma source supplying
the brain (Nguyen et al., 2014). However, upon close
examination, it was determined that i.v. administered
lysoPC containing DHA has a longer plasma half-life
than unesterified DHA; thus, more of it is directed to the
brain, albeit at a much slower rate than unesterified
DHA (Chen et al., 2015). This is of significance as,
although the net rate of plasma lysoPC containing DHA
is lower than unesterified DHA in vivo, i.v. lysoPC
containing DHA would be useful for targeting the brain
with DHA and possibly other lipids (Chauveau et al.,
2011; Lo Van et al., 2016).
B. Mechanisms of DHA Entry into the Brain
Similar to the plasma pools that supply the brain,
there has been considerable debate on the mechanisms
by which DHA is uptaken into the brain. Some of the
confusion may be the result of studies that have failed to
differentiate direct transport from uptake, which is
often coupled to metabolism. Although it is clear that
fatty acids, including DHA, do not need a protein to
cross the cell membrane, several proteins have been
implicated in facilitating the uptake of DHA. These
include members of the fatty acid transport protein
(FATP) family, CD36, Mfsd2a, and fatty acidbinding
proteins (FABP).
Whereas earlier work suggested a role of FATPs in
the uptake of fatty acids, and hence their naming, it was
later realized that they possessed acyl CoA synthetase
activity and were, likely, quenching fatty acids and/or
facilitating their metabolism, which led to an increase
in fatty acid uptake, but not transport per se (DiRusso
et al., 2005; Jia et al., 2007; Mashek et al., 2007). An
analogy can be drawn from hexokinase or glucokinase,
which phosphorylates glucose, increasing glucose up-
take, whereas glucose transport is mediated by the
GLUT proteins. Furthermore, it is important to note
that the transport of fatty acids across the membrane
occurs at rates commonly measured in the low msec
range, and studies lasting several seconds, let alone
minutes, do not have the temporal resolution to sepa-
rate transport from uptake and metabolism, especially
in in vivo studies (Hamilton 1998). Nevertheless, it has
become clear that members of the FATP family, espe-
cially FAPT1, are important for DHA uptake (Ochiai
et al., 2017). CD36 has remained more elusive in the
transport of fatty acids, but recent studies suggest that
CD36 is not a fatty acid transporter and, likely, also
facilitates the uptake of fatty acids secondary to
changes in metabolism (Xu et al., 2013a; Jay and
Hamilton 2016). KO of Mfsd2a leads to approximately
50% less neurons and brain DHA as compared with
wild-type controls, and Mfsd2a appears critical for the
brain uptake of LPC, including those esterified with
DHA (Nguyen et al., 2014). However, similar to the
previously mentioned proteins, caution must be taken,
as the studies do not have the temporal resolution to
distinguish between transport and uptake secondary to
changes in metabolism. Members of the FABP family
facilitate the uptake of fatty acid secondary to their
metabolism and are important in the trafficking of fatty
acids, the regulation of eCB signaling among others,
and we refer the reader to several recent reviews
(Moulle et al., 2012; Elsherbiny et al., 2013; Schroeder
et al., 2016). Of particular interest are recent studies on
FAPB-5, which facilitates the uptake of DHA into the
brain, and KO reduces brain DHA by approximately
15% and is associated with impaired working and short-
term memory (Pan et al., 2015, 2016). Although the
precise mechanisms by which DHA is transported and
uptaken into the brain have been and still are of
considerable debate, it is evident that numerous candi-
date mechanisms have been identified that could be
targeted to alter the uptake of DHA into the brain and
ultimately brain levels affecting neuronal survival and
IV. Anti-Inflammatory Activities of n-3 PUFAs in
the Brain
A. General Evidence in Humans and Animal Models
of Brain Pathologies
Inflammation in the brain is beneficial to maintain
organ homeostasis in response to infection. Brain in-
flammation involves microglial cells, the resident mac-
rophages of the central nervous system (CNS) (Aloisi,
2001). When activated, these cells produce pro- and
anti-inflammatory cytokines. However, when the pro-
duction of proinflammatory cytokines is sustained,
these molecules become neurotoxic, leading to neuronal
damage involved in many brain pathologies (Woodroofe
and Cuzner, 1993; Woodroofe, 1995; Blais and Rivest,
2003; Laye, 2010; Solito and Sastre, 2012). Hence,
limiting inflammation is of great importance, and the
identification of mediators able to do that may provide
new targets in brain damage prevention and treatment.
A large number of studies support the hypothesis that
n-3 LC-PUFAs or their products are candidates for
limiting neuroinflammation. Indeed, n-3 LC-PUFAs
downregulate inflammatory gene expression, such as
those of cytokine or enzymes involved in the synthesis of
eicosanoids, while inducing lipid mediators involved in the
resolution of inflammation (Calder, 2006; Serhan, 2014).
New Insights into the Impact of Omega-3 in Microglia 17
In animal models of acute and chronic inflammation,
the effects of n-3 LC-PUFAs and their bioactive
mediators have been demonstrated at the periphery
(Serhan and Chiang, 2013) and in the brain (Orr
and Bazinet, 2008; Rapoport, 2008; Layé, 2010;
Bazinet and Layé, 2014). In humans, higher n-3
LC-PUFA consumption is associated with a lower
risk of inflammation-associated neurologic disorders
(reviewed in Orr and Bazinet, 2008; Layé, 2010;
Bazinet and Layé, 2014). Several epidemiologic and
observational studies report that subjects with higher
n-3 LC-PUFA levels in blood have lower proinflamma-
tory cytokine production (Ferrucci et al., 2006; Kiecolt-
Glaser et al., 2007, 2011; Farzaneh-Far et al., 2009;
Alfano et al., 2012). Moreover, supplementation of
patients diagnosed with Alzheimersdisease(AD)
with a DHA-rich diet led to a reduced release of
proinflammatory cytokines from blood mononuclear
leukocytes (Vedin et al., 2008). In vivo, high levels of
brain DHA are linked to reduced expression of proin-
flammatory cytokines in several rodent models of
acute or chronic neuroinflammation, such as systemic
administration of the bacterial endotoxin lipopolysac-
charide (LPS), brain ischemia-reperfusion, spinal cord
injury, or aging (see Orr et al., 2013b for review). In
addition, a diet rich in EPA attenuates the production
of the proinflammatory cytokine interleukin (IL)-1b
and improves synaptic plasticity impairment in the
hippocampus of old rats (Martin et al., 2002; Lynch
et al., 2007). Aged mice exposed to a diet rich in
EPA/DHA for 2 months express less proinflammatory
cytokine [IL-1b, IL-6, and tumor necrosis factor
(TNF)-acompared with mice fed with a diet with a
ratio of LA/ALA of 5] (Labrousse et al., 2012). Impor-
tantly, the reduction of neuroinflammation linked to
diets enriched in n-3 LC-PUFA is associated with
improvement of spatial memory deficits (Song et al.,
2004; Labrousse et al., 2012). Moreover, increasing
brain DHA by genetic or dietary means is associated
with protection against LPS-induced proinflamma-
tory cytokine production induced by LPS (Mingam
et al., 2008b; Delpech et al., 2015a,b), brain ischemia-
reperfusion (Lalancette-Hebert et al., 2011), or spinal
cord injury (Huang et al., 2007; Lu et al., 2013).
DHAs protective activity on neuroinflammation is
linked to its direct effect on microglia, as suggested
by in vitro studies. For example, DHA decreases the
LPS-induced nuclear factor (NF)kB activation and, as
a consequence, the production of IL-1band TNF-a
(De Smedt-Peyrusse et al., 2008) and chemokines
(Lu et al., 2013) by microglia. In addition, DHA
enhances phagocytosis of AD-related amyloid-b(Ab)
42 by human microglial and decreases inflammatory
markers (Hjorth et al., 2013). Moreover, DHA is able to
normalize the LPS-induced abnormalities in microglia
(Chang et al., 2015). N-3 PUFA activity on microglia is
discussed later.
Conversely, low dietary intake of n-3 PUFA has
deleterious consequences in the brain, especially during
the perinatal period of brain development. For instance,
dietary n-3 PUFA deficiency beginning at the first day
of gestation decreases DHA level, alters microglia
phenotype and motility, and increases brain proinflam-
matory cytokine IL-6 and TNF-aexpression in the
offsprings brain of mice and rats (McNamara et al.,
2010; Madore et al., 2014). In mice, the early-life
exposure to a n-3 PUFA-deficient diet leads to spatial
memory impairment at adulthood (Moranis et al.,
2012), whereas this is not the case in adult mice with
a n-3 PUFA deficiency starting at weaning (Delpech
et al., 2015b). However, adult mice fed a n-3 PUFA-
deficient diet starting at weaning are more vulnerable
to inflammatory insult as spatial memory, synaptic
plasticity, microglia phenotype, and brain cytokine
production is altered in response to LPS (Delpech
et al., 2015b). Altogether these results pinpoint the role
of dietary n-3 PUFA deficiency in regulating brain
proinflammatory cytokine production and microglia
profile in the absence of overt infection (sterile in-
flammation, development, aging) and inflammatory
situation (LPS administration, stroke).
B. Overview of DHA Anti-Inflammatory/
Proresolving Mechanisms
Anti-inflammatory/resolution activities of n-3 PUFAs
are potent with a variety of overlapping and/or additive
mechanisms occurring either directly on the membrane,
via modulation of signaling pathways or control of gene
expression, or indirect through the synthesis of deriva-
tives reviewed elsewhere (Calder, 2011; Serhan, 2017a).
1. Membrane and Signaling Effects. As described in
the introduction, DHA is incorporated in membrane
phospholipids. In direct link with its disordered molec-
ular structure, DHA is believed to adopt a specific
molecular orientation in the membrane likely to modify
membrane domain organization and protein activity
(reviewed in Shaikh, 2012). Notably, formation of DHA-
enriched nanodomains in the membrane or incorpora-
tion of DHA into lipid rafts, a membrane-signaling
platform rich in cholesterol and sphingomyelin, dis-
rupts receptor-signaling interactions. In glial cells,
changes in membrane fluidity due to DHA level have
consequences on several proinflammatory receptor lo-
calization and associated signaling cascades. De Smedt-
Peyrusse et al. (2008) reported that, in microglia, DHA
impairs membrane location of the LPS receptors CD14
and Toll-like receptor (TLR)4, which in turn decreases
proinflammatory activity of LPS. Rockett et al. (2011)
showed that increased n-3 LC-PUFA consumption
disrupts B cell lipid-raft clustering. The membrane
reorganization consequence of DHA increase is in line
with in vitro data showing that DHA modulates the
activation of several proinflammatory transcription
factors (NFkB; mitogen-activated phosphate kinase
18 Layé et al.
p38; c-Jun N-terminal kinases) in microglia (De Smedt-
Peyrusse et al., 2008; Ma et al., 2009; Lu et al., 2013;
Chang et al., 2015). In microglia, DHA upregulates
peroxisome proliferator-activated receptor (PPAR)gnu-
clear translocation, a potent modulator of microglia
(Ajmone-Cat et al., 2010; Corsi et al., 2015).
DHA directly modulates inflammatory gene expres-
sion via surface or intracellular receptors, as reported in
inflammatory cells (Calder, 2011). Notably, DHA binds
to G-coupled receptors (GPR) 120 and 40 and PPARg,
and, in turn, DHA regulates their expression (Im, 2012;
Yamashima, 2012; Calder, 2013, 2015). GPR120
and PPARgmediate DHA anti-inflammatory activity
(Calder, 2011), including in the brain, as described
above. Overall, these results highlight that the anti-
inflammatory activity of DHA is partially attributable
to its effect on cell membrane reorganization and/or
through its effect on specific receptors. Although EPA is
esterified in cell membranes, including those of micro-
glial cells (De Smedt-Peyrusse et al., 2008), its effect on
raft reorganization or GPR has not been extensively
studied in relation to its anti-inflammatory activity
(Williams et al., 2012).
2. N-3 LC-PUFA Derivatives.
a. Oxylipins. Some of the anti-inflammatory activi-
ties of n-3 PUFAs are attributed to eicosanoids (a
subclass of oxylipins), the signaling molecule by the
enzymatic and nonenzymatic oxidation of EPA and
DHA (Fig. 3) (De Roos et al., 2009; Arita, 2012). The
main EPA-derived mediators include 3-series prosta-
glandin (PG), 5-series leukotrienes (LT), and 3-series
thromboxane (TX), and are reported to be relatively
nonactive (Fig. 3). DHA is also converted into 3-series
PG (Fig. 3). EPA- and DHA-derived eicosanoids are
biologically less active than the one derived from AA
(Calder, 2002). As an example, PGE3 is much less
effective than PGE2 to induce IL-6 and cyclooxygenase
(COX)-2 expression in macrophages, despite binding to
the same receptor (Bagga et al., 2003). In addition, when
copresent, EPA-derived eicosanoids antagonize those
synthesized from AA. Moreover, EPA counteracts
AA-derived eicosanoid production, as EPA is a compet-
itive inhibitor for the enzymes involved (Calder, 2002).
First, EPA inhibits the activity of D5-desaturase, con-
verting dihomo g-linolenic acid to AA. EPA also inhibits
the activity of PLA2, thus preventing the release of AA;
the activity of COX-2 generating the PG, prostacyclins,
and TX (Needleman et al., 1979; Obata et al., 1999); and
the activity of 5-lipoxygenases (LOX) that generates the
LT (Sperling et al., 1993). Overall, EPA reduces both the
proportion of AA and the production of proinflammatory
eicosanoids derived from AA. DHA also is able to reduce
the production of PGE2 and LTB4 derived from AA in
stimulated peripheral blood mononuclear cells (Kelley
et al., 1999). In addition, eicosanoids synthesized from
AA and EPA act in competition as they share the same
G proteincoupled receptors.
Recently, SPM derived from n-3 LC-PUFAs have
gained much more attention. These lipid mediators have
both anti-inflammatory and proresolving properties with-
out immune suppression (Serhan et al., 2002, 2008, 2014).
They act in physiologic doses around the nanomolar level
as compared with DHA, which acts at micromolar levels.
Among the resolvins, resolving D1 (RvD1, 7S,8R,17S-
trihydroxy-4Z,9E,11E,13Z,15E,19Z-docosahexaenoic acid)
and resolvin E1 (RvE1, 5S,12R,18R-trihydroxy-
6Z,8E,10E,14Z,16E-eicosapentaenoic acid) are of par-
ticular interest in the resolution of inflammation
because they actively turn off the inflammatory re-
sponse (Fig. 3) (Fredman and Serhan, 2011). They are
thought to underlie many of the beneficial effects
attributed to their precursors (Calder, 2013; Serhan
and Chiang, 2013; Bazinet and Laye, 2014; Headland
and Norling, 2015). They act via cell surface G protein
coupled receptors: GPR32 and ALX/FPR2 for RvD1, and
chemoattractant receptor 23 for RvE1 (Zhang and Spite,
2012). They were mainly studied in peripheral immune
cells, both in vitro and in vivo. In vitro, they act on
macrophages to stimulate the clearance of apoptotic cells
and inflammatory debris, inhibit the expression of the
proinflammatory cytokines, and block neutrophil infiltra-
tion (Arita et al., 2005; Schwab et al., 2007; Fredman and
Serhan, 2011; Zhang and Spite, 2012). RvE1 inhibits the
activity of NFkB and the subsequent production of
proinflammatory cytokines (Xu et al., 2013b; Rey et al.,
2016). RvD1 also displays its anti-inflammatory activity
through miRNA modulation (Rey et al., 2015). In vivo,
RvD1 decreases proinflammatory cytokine production in
acute models of kidney injury (Chen et al., 2014) or lung
(Wang et al., 2011a, 2014; Zhou et al., 2013; Yaxin et al.,
2014), and in a model of allergic airways (Rogerio et al.,
2012). RvE1 also modifies cytokine production in exper-
imental models of colitis (Arita et al., 2005) and perito-
nitis (Schwab et al., 2007) and significantly modulates
the inflammatory profile and activation of microglia
(Harrison et al., 2015).
SPMs derived from DHA include neuroprotectin D1
(NPD1) and maresin 1 (MaR1) (Bazan, 2006; Bannenberg
and Serhan, 2010; Bazan et al., 2012). NPD1 protects the
brain toward leukocyte infiltration, COX-2 expression,
cytokine production, and microglia activation (Hong
et al., 2003; Marcheselli et al., 2003, 2010; Lukiw et al.,
2005; Orr et al., 2013). MaR1 reduces cytokine production
in human peripheral blood lymphocytes (Chiurchiu et al.,
2016). It protects against cerebral ischemia/reperfusion
injury through the modulation of the proinflammatory
response (Xian et al., 2016). It downregulates Ab
inflammation in human microglial cells in culture through
the stimulation of Abphagocytosis (Zhu et al., 2016).
b. Endocannabinoids. N-3 LC-PUFAs can also exert
their effect through the modulation of the eCB system,
(Meijerink et al., 2013; Bazinet and Layé, 2014; Kuda,
2017; Nadjar et al., 2017). eCBs are synthesized from
PUFA, the most well-known being anandamide (AEA)
New Insights into the Impact of Omega-3 in Microglia 19
and the 2-arachidonoyl derived from AA. However, n-3
LC-PUFAs are also precursors of DHA- and EPA-
derived eCBs, namely docosahexaenoyl ethanolamide
(DHEA or synaptamide) and eicosapentaenoyl ethano-
lamide (EPEA). The formation of these compounds in
mice is increased in various tissues, including the brain
after consumption of a fish oilrich diet and in the
plasma of volunteers supplemented with DHA and EPA
(see Meijerink et al., 2013 for review). Furthermore, the
level of DHEA in the brain is higher than that of AEA
even in animals fed a control diet (see Meijerink et al.,
2013 for review). The immunomodulatory effect of
DHEA has been demonstrated in the brain and at the
periphery. Indeed, in the brain, Park et al. (2016b)
showed that DHEA is a potent suppressor of LPS-
induced neuroinflammation in mice, by enhancing
cAMP/protein kinase A signaling and inhibiting NFkB
activation. In macrophages, Meijerink et al. (2011,
2015) showed that DHEA modulates inflammation by
reducing monocyte chemoattractant protein-1, nitric
oxide (NO), and eicosanoid production. Moreover,
Rossmeisl et al. (2012) suggested a possible role for
DHEA in modulating inflammation in adipocytes. Im-
portantly, DHEA can be oxidized to form derivatives
with anti-inflammatory properties (Yang et al., 2011;
Shinohara et al., 2012; Kuda, 2017).
eCBs bind mainly to the eCB receptors CB1 and
CB2 (Piomelli and Sasso, 2014) that are expressed in
neurons and glial cells and more specifically in micro-
glial cells (Stella, 2009). In microglial cells, CB2 is more
highly expressed than CB1 (Nunez et al., 2004). It was
already shown that in inflammatory conditions eCBs
act via CB2, with the role of CB1 remaining unclear
(Mecha et al., 2015). The importance of CB2 in regulat-
ing microglia activity was demonstrated in vivo and
in vitro. CB2 knockout (KO) mice microglia lose their
phagocytic activity, and microglia motility is reduced in
presence of a CB2 antagonist (Walter et al., 2003).
All of these studies highlight the central role of n-3
LC-PUFA and their derivatives in the regulation of
inflammation, with emerging data in the brain, espe-
cially through their effect on microglia.
V. Microglia as a Target for n-3 PUFAs and SPMs
Microglia are a glial cell of myeloid origin whose role
is to maintain brain homeostasis in a sex-, age-, and
region-dependent manner (Hanisch and Kettenmann,
2007; Tay et al., 2017). Microglia are a highly plastic and
multitasking cell, important from brain development to
pathologic conditions, via inflammatory and noninflam-
matory responses (Ransohoff and Brown, 2012). Any
situation leading to undesirable microglial activity at
different stages of life could severely impair brain
function. During development, yolk sacderived micro-
glia colonize the brain and spread evenly in the whole
Fig. 3. Conversion of LC-PUFAs into lipid mediators. AA, DHA, and EPA released from the membrane can be metabolized into various classes of
derivatives via COX and LOX activity. AA is always converted into proinflammatory metabolites, whereas DHA and EPA are precursors of anti-
inflammatory mediators. Only in situations in which COX-2 is acetylated (treatment with aspirin), LC-PUFAs (AA, DHA, and EPA) can be converted
into AT mediators that are all anti-inflammatory. AA can also be metabolized into endocannabinoids (2-arachidonoyl and AEA) that are pro- or anti-
inflammatory depending on the receptor to which they bind.
20 Layé et al.
CNS, where they shape neuronal circuits (Paolicelli
et al., 2011; Schafer et al., 2012). In the adult brain,
microglia sense the microenvironment with their
processes in search for nonhomeostatic signals, and
they also regulate neuronal architecture and function
(Davalos et al., 2005; Nimmerjahn et al., 2005; Wake
et al., 2009; Tremblay et al., 2010; Sipe et al., 2016).
Under pathologic conditions, unusual/danger signals
trigger microglial response, including release of inflam-
matory factors and/or redirection of its phagocytic
activity to the clearance of hazardous factors
(Ransohoff and Brown, 2012; Sierra et al., 2014; Tay
et al., 2017). Understanding how n-3 PUFAs modulate
microglial phenotypes and functions is a major chal-
lenge for future development of innovative lipid-based
therapies with positive effects on physiology and
A. Modulation of Microglial Function by n-3 PUFAs
1. In Vitro Evidence. N-3 PUFAs are potent modu-
lators of microglial functions (Nadjar et al., 2017). The
first evidence came in 2007 from an in vitro study in
which microglial cells (BV2 cell line) were incubated
with or without EPA for 60 minutes prior to LPS
application, and their inflammatory response was ana-
lyzed (Moon et al., 2007). EPA dose-dependently
inhibited the expression of the two inflammatory en-
zymes, inducible NO synthase (iNOS) and COX-2, as
well as the subsequent production of NO and PGE
BV2 cells. EPA also dampened the production of proin-
flammatory cytokines (IL-1b, IL-6, and TNF-a). This
report was followed by another study in which primary
cultures of rat microglia were incubated simultaneously
with LPS and n-3 PUFAs (Liuzzi et al., 2007). The
authors measured the LPS-mediated induction of the
matrix metalloproteinase (MMP)9 in presence or in
absence of a mixture of EPA and DHA. MMP9 is an
endopeptidase that degrades the extracellular matrix,
and as such plays a role in inflammation by regulating
processes such as cell motility, blood-brain barrier
(BBB) disruption, cell infiltration, etc. Application of
n-3 PUFAs concomitantly to LPS on microglial cells was
sufficient to significantly reduce MMP9 expression and
activity, in a dose-dependent manner (Liuzzi et al.,
Since then, a total of 16 publications on various
in vitro models brought converging evidence on the
anti-inflammatory action of n-3 PUFAs on microglia
(Nadjar et al., 2017). They also revealed new regulatory
roles of n-3 PUFAs on these cells, such as modulation on
microglial phenotype, migration, phagocytosis, auto-
phagy, or lipid bodies accumulation, as well as some of
the molecular mechanisms implicated (extensively
reviewed in Nadjar et al., 2017). Briefly, whatever the
inflammatory challenge applied on these cells (TLR3-4
or 7 agonists, Ab, interferon-g, hypoxia, or myelin), all
studies reported a dose-dependent decrease in the
production of proinflammatory factors (cytokines and/or
chemokines) when treated with n-3 PUFAs (De Smedt-
Peyrusse et al., 2008). N-3 PUFAs also inhibit the
production and activity of the enzymes COX-2, iNOS,
and the production of NO and reactive oxygen species
(Moon et al., 2007; Lu et al., 2010; Antonietta Ajmone-
Cat et al., 2012; Pettit et al., 2013; Chen et al., 2014;
Corsi et al., 2015; Zendedel et al., 2015). This is
correlated with a switch in microglial marker expres-
sion, from a proinflammatory to an anti-inflammatory
phenotype (decreased CD40 and CD86, increased
CD206) (Ebert et al., 2009; Chhor et al., 2013; Hjorth
et al., 2013; Chen et al., 2014). The phagocytic capacity
of microglia is also modulated by n-3 PUFAs in vitro.
Using flow cytometry on the human microglial cell line
CHME3, Hjorth et al. (2013) assessed the effects of
DHA and EPA on microglial phagocytosis of the AD
pathogen Ab
. They showed that both DHA and EPA
exacerbate Ab
engulfment by microglia in a dose- and
time-dependent manner. Microglia employ a wide
repertoire of mechanisms to phagocytose various types
of cellular elements/debris (Sierra et al., 2013; Brown
and Neher, 2014). Yet, the prophagocytic effects of n-3
PUFAs are likely to be generalizable to all stimuli, as
1 year later Chen et al. (2014) showed a significant
increase in myelin engulfment by DHA- or EPA-
treated microglia primary cultures. N-3 PUFAs also
modulate microglia migration capacities. Using a
Transwell migration assay, Ebert et al. (2009) showed
that DHA dose-dependently inhibits LPS-activated
microglial migration, whereas it does not affect the
migratory abilities of BV2 cells in basal conditions.
Very recently, by measuring the classic autophagy
index LC3-I/LC3-II ratio (for the lipidated form of the
microtubule-associated protein 1 light chain 3, LC3-II,
and nonlipidated LC3, LC3-I), Inoue et al. (2017)
showed that application of EPA plus DHA increases
autophagy in MG6 microglial cells. Autophagy is an
essential process for immune cell homeostasis that
leads to dampening of inflammatory processes (Levine
et al., 2011). Inoue et al. (2017) highlight it as a new
process by which n-3 PUFAs modulate microglial
inflammatory response. Finally, lipid bodies (LBs)
are functionally active organelles that are formed
within immune cells, such as macrophages, in re-
sponse to different inflammatory stimuli and are sites
for synthesis and storage of inflammatory factors
(Melo et al., 2011). Ebert et al. (2009) were the first
to demonstrate that DHA significantly reduces the
accumulation of LBs that is usually observed in LPS-
treated microglia. These data were confirmed and
extended a few years later by the group of Maysinger.
Chang et al. (2015) showed in N9 cell line that DHA
normalizes LPS-induced abnormalities in microglia,
by promoting small LB formation and LB interaction
with mitochondria, and by restoring mitochondrial
function (Chang et al., 2015; Tremblay et al., 2016).
New Insights into the Impact of Omega-3 in Microglia 21
Importantly, none of these studies reported detrimental
effects of PUFAs on microglia viability, except at very high
doses (Moon et al., 2007; Antonietta Ajmone-Cat et al.,
2012; Nadjar et al., 2017). Moreover, although EPA and
DHA are both efficient almost to the same extent when
applied separately, their effects are most often potentiated
when combined (Zhang et al., 2010; Hjorth et al., 2013;
Chen et al., 2014; Kurtys et al., 2016; Inoue et al., 2017).
Some of the studies presented above explored the
molecular mechanisms by which n-3 PUFAs modulate
microglial functions. Our group demonstrated that
DHA significantly downregulates the cell surface ex-
pression of CD14 and TLR4, the two coreceptors that
bind LPS, in LPS-stimulated BV2 cells (De Smedt-
Peyrusse et al., 2008). Beyond the membrane effects of
n-3 PUFAs, many studies have reported converging
evidence on the modulatory role of n-3 PUFAs on
signaling pathways. Indeed, the beneficial effects of
n-3 PUFAs on inflammatory processes are attributable
in part to their inhibitory action on inflammatory
signaling pathways such as NFkB (De Smedt-
Peyrusse et al., 2008; Zhang et al., 2010; Wang et al.,
2015b; Inoue et al., 2017), mitogen-activated protein
kinases (P38, c-Jun N-terminal kinases, or extracellular
signal-regulated kinase 1/2) (Liuzzi et al., 2007;
Antonietta Ajmone-Cat et al., 2012; Chang et al.,
2015), or Akt (Liuzzi et al., 2007).
Finally, it was reported that DHA and EPA are
natural ligands for several nuclear receptors, including
peroxisome proliferator-activated receptors (PPARs)
that are highly expressed in microglial cells (Xu et al.,
1999; Zhang et al., 2014). These latter play an important
role in the general transcriptional control of numerous
cellular processes, including lipid homeostasis and in-
flammation (Clark, 2002). Several in vitro studies
showed that DHA and EPA activate PPARgin micro-
glial cells as well, hence significantly decreasing the
expression of inflammatory factors (Ebert et al., 2009;
Antonietta Ajmone-Cat et al., 2012; Corsi et al., 2015;
Wang et al., 2015; Kurtys et al., 2016).
Overall, the plethora of in vitro studies rather con-
vincingly demonstrated the anti-inflammatory role of n-3
PUFAs. However, although they are very convenient to
study molecular mechanisms, the relevance of in vitro
models to study microglial function has been recently
questioned (Hickman et al., 2013; Butovsky et al., 2014).
By comparing gene expression in cultured microglia with
in situ microglia and other myeloid cells, several groups
showed that in vitro microglia do not express the same
molecular signature as brain microglia and have a
transcriptome signature that is closer to macrophages,
putting into question the relevance of data presented
above to the brain (Hickman et al., 2013; Butovsky et al.,
2014). To evaluate the validity of in vitro studies, we will
now review reports that assessed the effects of PUFAs on
neuroinflammation in various physiologic and pathologic
contexts. The data relating to their anti-inflammatory
actions have been presented in the previous section. We
will in this work focus on the evidence regarding micro-
glial cells, as microglial activation was studied as a
secondary outcome in most of these studies.
2. In Vivo Evidence. The first in vivo study showing
a relationship between n-3 PUFAs and microglia was
performed on aged rats supplemented with 125 mg/d
EPA-containing chow for 4 weeks (Lynch et al., 2007).
EPA supplementation was able to significantly reduce
microglial activation marker expression [major histo-
compatibility complex (MHC)II, CD40] and microglia-
mediated production of IL-1b. This was paralleled by an
increase in the anti-inflammatory cytokine IL-4 expres-
sion, the complete inhibition of aging-induced synaptic
plasticity impairment, and a decreased vulnerability
to Abstimulus (Lynch et al., 2007). Concomitantly,
Connor et al. (2007) demonstrated, in a model of retinal
degeneration, that supplementation with n-6 PUFAs
increases microglial production of TNF-ain the retina,
as a plausible cause for vascular growth and pathology.
These effects were prevented by increasing dietary n-3
PUFA intake (Connor et al., 2007).
Many studies have reported a close relationship
between microglial function and n-3 PUFAs since then.
N-3 PUFA supplementation reduces microglial activa-
tion and/or phenotype alteration in models of brain
development (Kuperstein et al., 2008; Madore et al.,
2014; Abiega et al., 2016), respiratory system develop-
ment (Tenorio-Lopes et al., 2017), healthy aging
(Grundy et al., 2014), ischemia (Zhang et al., 2010;
Belayev et al., 2011; Okabe et al., 2011; Eady et al.,
2012a,b, 2014; Chang et al., 2013; Zendedel et al., 2015;
Jiang et al., 2016), spinal cord injury (Huang et al.,
2007; Lim et al., 2013a,b; Paterniti et al., 2014;
Tremoleda et al., 2016; Xu et al., 2016), Parkinsons
disease (Muntane et al., 2010; Ji et al., 2012; Tian et al.,
2015; Delattre et al., 2017; Mori et al., 2017), AD (Lynch
et al., 2007; Hopperton et al., 2016; Serini and Calviello,
2016; Wen et al., 2016), systemic inflammation (Delpech
et al., 2015b), traumatic brain injury (Pu et al., 2013;
Harrison et al., 2015; Harvey et al., 2015; Desai et al.,
2016), neuropathic pain model (Xu et al., 2013b;
Manzhulo et al., 2015; Huang and Tsai, 2016), aging
(Labrousse et al., 2012), demyelination (Chen et al.,
2014), amyotrophic lateral sclerosis (Yip et al., 2013),
retinal degeneration (Ebert et al., 2009; Mirza et al.,
2013), or experimental autoimmune uveoretinitis
(Saraswathy et al., 2006). In all these studies, n-3
PUFAs were provided under the form of DHA or EPA
supplementation, a combination of EPA plus DHA,
largely as fish oil, or the precursors of LC-PUFAs.
Administration was made s.c., i.v., i.p., by gavage or
via dietary approaches, acutely or chronically. Micro-
glial response was evaluated by quantifying the number
of cells or by measuring expression of some phenotype
markers, including Iba-1, Arg1, Ym1/2, CD16, CD32,
CD40, CD36, CD68, CD86, CD206, CD11b, and MHCII.
22 Layé et al.
Of all these studies, 90% found a significant decrease of
microglial density and/or activation after exposure to
n-3 PUFAs, as a criterion for decreased neuroinflam-
mation, whereas 10% could not find any effect (Orr
et al., 2013; Vauzour et al., 2015; Trepanier et al., 2016;
Nadjar et al., 2017). Moreover, we showed that the
sourcing of PUFAs (from plants or dairy products) has a
differential impact on LPS-modulating microglial phe-
notype (Dinel et al., 2016). However, none of these
studies ever addressed the intimate relationship be-
tween brain PUFA contents and microglial function.
Notably, our group demonstrated that low dietary
consumption of n-3 PUFA precursors over the perinatal
period not only impairs microglia phenotype but also its
morphologic dynamic (assessed by two-photon micros-
copy of microglial processes motility) in the postnatal
developing brain (Madore et al., 2014). On the same
model of developmental n-3 PUFA deficiency, we also
showed that the phagocytic activity of microglia was
enhanced in the offspring, as a consequence of an
increased density in apoptotic cells (Abiega et al.,
2016). It is important to highlight that these data are
not in accordance with in vitro studies that show higher
phagocytic activity in n-3 PUFA-treated cells (Hjorth
et al., 2013; Chen et al., 2014), emphasizing even more
the need for thorough in vivo work. To improve our
knowledge of how PUFA specifically modulate microglia
in vivo, new technological tools are required. The recent
development of new tools to study these cells, such as
CX3CR1-Cre mice, will surely provide informative re-
sults in the coming years (Wieghofer and Prinz, 2016).
B. Modulation of Microglial Function by SPMs
1. Lipid Derivatives Target Microglia. Hundreds of
biologically active metabolites of DHA and EPA have
been described in the literature (see details in previous
section) (Dyall, 2015; Kuda, 2017) (Fig. 3). Briefly, DHA
derivatives can be divided into oxygenated metabolites
[SPMs, epoxides, electrophilic oxo-derivatives (EFOX),
and neuroprostanes] and conjugates of DHA (ethanol-
amines, acylglycerols, docosahexaenoyl amides of
amino acids or neurotransmitters, and branched DHA
esters of hydroxy fatty acids). EPA can also be metab-
olized via oxygenation, hydroxylation, or peroxidation
processes that lead to the production of eicosanoids.
Some of them have been studies in relation to microglial
functions, and data are summarized hereafter.
The EPA-derived RvE1 inhibits LPS-induced micro-
gliosis and proinflammatory cytokine release [IL-6, TNF-a,
and IL-1bin microglial cell culture (primary cultures
and BV2 cell line)], by inhibiting NFkB pathway (Rey
et al., 2016), and inhibits spinal cord microglial activa-
tion following peripheral nerve injury (Xu et al., 2013b).
DHA-derived lipid mediators (resolvins of the D-series,
MaR, and neuroprotectins) also modify microglial func-
tions. RvD1 promotes anti-inflammatory phenotype in
BV2 cells, enhancing Arg1 and Ym1 expression, IL-4
synthesis, and subsequent NFkB and PPARgactivation
and decreasing CD11b expression (Li et al., 2014; Zhu
et al., 2015), via the regulation of miRNA expression
(Rey et al., 2016). In vivo, RvE1 and the aspirin-
triggered (AT) 17R-epimer of RvD1 (AT-RvD1) signifi-
cantly modify microglial morphology in a model of
traumatic brain injury, decreasing the proportion of
rod/activated microglia at the expense of ramified
microglia (Harrison et al., 2015). The DHA derivative
MaR1 also modulates microglial response to Ab
application in vitro, downregulating Ab
phenotype alterations (CD40 and CD11b expression)
in CHME3 cells (Zhu et al., 2016). MaR1 also promotes
phagocytosis by microglial cells in culture (Zhu
et al., 2016). Finally, they provide the demonstration
that microglia can produce SPMs [e.g., PD1, lipoxin A4
(LxA4), and RvD1] (Zhu et al., 2015). RvD2 is also a
modulator of microglial cells in vitro, as shown by Tian
et al. (2015). In this study, they incubated microglia
with LPS and increasing doses of RvD2 for 24 hours.
Using Western blot, they showed that RvD2 inhibits
LPS-mediated activation of TLR4 and its downstream
signaling pathway NFkB (Tian et al., 2015). Finally,
NPD1 signaling induces an increase of microglial
ramification size typical of nonactivated phenotype
and coincident with attenuation of retina structural
alterations (Sheets et al., 2013), whereas the AT-NPD1
significantly reduces the number of ED1-positive cells
(microglia/macrophages) in a model of cerebral ische-
mia (Bazan et al., 2012).
Even though this review focuses on the anti-
inflammatory effects of n-3 PUFAs in the brain and the
complexity of the lipid-dependent inflammatory response,
one should mention in this work that some AA-derived
lipid mediators also display anti-inflammatory activity
via the modulation of microglial function (Fig. 3). LxA4 for
instance inhibits interferon-gmediated inflammatory
response (TNF-arelease and P38 mitogen-activated
phosphate kinase activation) in primary cultures of micro-
glia (Martini et al., 2016). It also dampens microglial
proliferation, TNF-aupregulation, and the expression of
microglial markers such as P2Y12, in a model of spinal
cord injury (Martini et al., 2016). In a transgenic model of
AD (3xTg-AD mice), the AT-LxA4 significantly decreases
the number of CD11b, Iba-1, and CD45-positive cells
(presumably microglia) around the plaques (Medeiros
et al., 2013; Dunn et al., 2015). In vitro experiments on
LPS-treated BV2 cells revealed that AT-LxA4 inhibits
NFkB signaling pathway and NADPH oxidase activity,
hence reducing proinflammatory cytokines and reactive
oxygen species production, respectively, as potential
mechanisms to explain its anti-inflammatory action
(Wang et al., 2011; Wu et al., 2012).
Another class of DHA-derived oxygenated metabolites
has been linked to inflammation. DHA can be converted
into EFOX by a COX-2catalyzed mechanism (Groeger
et al., 2010) (Fig. 3). Whereas EFOX have been shown to
New Insights into the Impact of Omega-3 in Microglia 23
modulate macrophage inflammatory activity, no data are
yet available on microglia. However, EFOX are natural
ligands for PPARgand modulate the Nrf2 and NFkB
inflammatory pathways (Groeger et al., 2010), all these
molecules being highly expressed in microglial cells
(Zhang et al., 2014). Hence, EFOX also look like good
candidates to explain modulatory activity of DHA on
microglial cells, claiming for more studies on these lipids.
Besides oxidation, DHA can be conjugated with
alcohols and amines to form esters and amides, re-
spectively (Kuda, 2017). Among all DHA conjugates, the
amine conjugate N-docosahexaenoyl dopamine has
been shown to modulate microglial function in vitro
(BV2 cells) (Wang et al., 2017). N-docosahexaenoyl
dopamine dose-dependently (1 or 2 mM) inhibits LPS-
induced IL-6 and CCL20 production in BV2 cells,
whereas neither DHA nor dopamine alone (at the same,
low concentrations, 1 or 2 mM) is able to produce the
same effects. This was paralleled by a decreased pro-
duction of PGE
, whereas COX-2 gene expression
remained stable (Wang et al., 2017).
2. Lipid Derivatives and Their Receptors. Only few
receptors for DHA and EPA derivatives have been
determined to date. These include ALX/FPR2 and
GPR32, both receptors for LxA4 and RvD1; ChemR23,
receptor for RvE1; and LTB4R (or BLT1), receptor for
LTB4 and RvE1 (Serhan et al., 2011). Most of these
receptors have been found on microglial cells in vitro
(Rey et al., 2016; Zhu et al., 2016). Data mining in the
Barres brain RNA-seqdatabase, which provides in-
formation on the transcriptome of glial cells (including
microglia) and neurons sorted from adult cortex, pro-
vides the information that microglia in vivo also express
the genes cmklr1 (for ChemR23), ltb4r1 (for LTB4R or
BLT1), and fpr2 (for ALX/FPR2) at very high levels.
Even more interestingly, they express these genes at
much higher concentration than any other cell type
(Zhang et al., 2014). Beyond receptors, some of these
lipid mediators target PPARs, also highly expressed by
microglia (Forman et al., 1997; Zhang et al., 2014).
Hence, based on this evidence, it is highly likely that
microglia are a target for EPA and DHA derivatives
within the brain.
Overall, all of these reports highlight the modulatory
activity of DHA and EPA derivatives on microglial
functions. However, this promising field is still in its
infancy and requires more studies to unravel the
molecular mechanisms involved and validate the activ-
ity of these lipids in vivo.
C. A Link between n-3 PUFAs and Microglial
Extracellular Vesicles?
Extracellular vesicles (EVs) transport represents a
fundamental mechanism of communication in the CNS.
EVs are released from almost all cell brain types,
including microglia, into the microenvironment and
are involved in cell-to-cell communication (Potolicchio
et al., 2005; Subra et al., 2010; Turola et al., 2012). EVs
include exosomes, which are small vesicles (40100 nm
in diameter) derived from the endosomal multivesicular
bodies that fuse with the plasma membrane via exo-
cytosis. They serve as shuttles for intercellular delivery
of cargo, including specific lipids (Turola et al., 2012).
Microglia-derived exosomes were discovered in 2005 in
N9 microgial cell lines (Bianco et al., 2005; Potolicchio
et al., 2005). Several studies have further shown that
microglia-derived exosomes can deliver a proinflamma-
tory signal on neighboring cells (Bianco et al., 2005;
Verderio et al., 2012; Prada et al., 2013). A recent study
elegantly showed that eCBs such as AEA can also be
secreted through extracellular membrane vesicles pro-
duced by microglial cells and hence inhibit presynaptic
transmission in target GABAergic neurons in a CB1-
dependent manner (Gabrielli et al., 2015).
Interestingly, lipidomic and proteomic analyses of
exosomes from various cell types have shown that these
EVs are not randomly filled with cellular content but
rather display a highly specific pattern in terms of lipid
and protein expression (Subra et al., 2010; Connolly et al.,
2015; Haraszti et al., 2016). Specifically, exosomes contain
free fatty acids, including AA and DHA (Haraszti et al.,
2016), but also AA derivatives such as prostaglandins
) (Subra et al., 2010). Exosomes also express
specific proteins, including enzymes, such as PLA
FABP (Subra et al., 2010). Computer-based analyses of
proteomic data also revealed that they specifically contain
proteins involved in functions such as immune response,
cell adhesion, or integrin signaling (Haraszti et al., 2016).
Overall, exosomes are considered as signalosome carriers
to neighboring cells (Subra et al., 2010).
Based on the currently available evidence, studies are
needed to decipher the lipid and protein composition of
microglial exosomes under various dietary situations.
One might speculate that, depending on the amount of
n-3 PUFAs, exosome composition might change, leading
to differential effects in neighboring cells.
D. Sex, Age, and Regional Differences in the
Relationship between Microglia and n-3 PUFAs
As already exposed in previous sections, n-3 PUFA
distribution varies according to sex, age, and structure,
and so do microglial functions. Hence, microglia/n-3
PUFA interactions could vary according to these fac-
tors, which would have to be considered to understand
whether and how n-3 PUFAs modulate CNS homeosta-
sis and neuroinflammation.
1. Evidence for Sexual Dimorphism of Microglia.
The colonization of the brain by yolk sacoriginating
microglia takes place very early during development,
even before formation of neurons, astrocytes, or oligo-
dendrocytes. Once in the CNS, microglia then proliferate
and spread evenly in all structures all along their
development (Tay et al., 2017). Although entry of micro-
glia in the CNS is likely to be sex-independent, postnatal
24 Layé et al.
microglial colonization and proliferation are sex-
dependent (McCarthy et al., 2015; Nelson and Lenz,
2017b). Indeed, male and female rats do not show sex
differences in microglial number before the testosterone
surge at embryonic day E17 (Schwarz et al., 2012). At
the time of testicular androgen secretion onset, sex
differences in microglia begin to emerge. At P0, females
have a transient increase in the number of ameboid
microglia and microglia with stout processes relative to
males, in the CA3 region of the hippocampus, para-
ventricular nucleus of the hypothalamus, and amygdala
(Schwarz et al., 2012). By P4, the situation is reversed
with males showing greater numbers of ameboid micro-
glia and microglia with stout or thick processes relative
to females, in the amygdala, hippocampus, and cortex
(Schwarz et al., 2012). These sex differences remain
until adolescence period with females displaying more
microglia with thick processes than males (Schwarz
et al., 2012). Slighter sex differences have been reported
in the cerebellum, with males having less ramified
microglia than females, but no overall differences in
ameboid microglia or total microglia during the first
3 weeks of life (Perez-Pouchoulen et al., 2015). At
adulthood, the number of microglia is significantly
higher in females than in males (as quantified at 3,
14, or 24 months old), at least in the dentate gyrus of the
hippocampus (Mouton et al., 2002).
Microglia are also essential for CNS masculinization
by standing at the interface of both endocrine and
nervous systems during development (Lenz et al.,
2013). In the preoptic area, an essential structure for
brain masculinization, male neonates have 30% more
microglia and twofold more ameboid microglia than
females (Lenz et al., 2013). At the time of testicular
surge, estradiol aromatized from testosterone promotes
microglia-mediated PGE
synthesis, a critical step in
masculinization of neurons and behavior (Lenz et al.,
2013). Using repeated central injections of liposomal
clodronate (from P0 to P4) to selectively deplete micro-
glia in the developing brain alters behavior, including
sexual behavior, in male pups, juveniles, and adulthood
(decreased number of and increased latency for mounts
and intromissions in adults), whereas female behavior
is unchanged (VanRyzin et al., 2016).
Besides cellular density and morphology, microglia
exhibit a sexual dimorphism in gene expression profile
(Crain et al., 2009, 2013; Crain and Watters, 2015).
Microglia freshly isolated from males and females differ-
entially express P2X and P2Y purinergic receptors (Crain
et al., 2009). The functional significance of this differen-
tial expression is still unknown, yet purinergic receptors
are involved in functional modal switch of microglia, i.e.,
transition from one phenotype/activity to another pheno-
type/activity (Koizumi et al., 2013). Female microglia also
display higher expression of pro- and anti-inflammatory
cytokines (IL-1b, TNF-a, IL-6, and IL-10) relative to
males (Crain et al., 2013). This sexual dimorphism is
structure-dependent as microglia from cerebral cortex of
females expresses more iNOS mRNA than those of males,
whereas no such difference is observed in the brainstem
(Crain and Watters, 2015). Finally, microglia also display
sexual dimorphisms in function. In female neonates, a
higher number of microglia exhibit phagocytic phenotype
as compared with males (Nelson and Lenz, 2017a,b).
However, the molecular and cellular mechanisms of
sexual dimorphism of microglia and consequences on
brain function are still to be unraveled.
The dynamics of microglia across the developmental
period suggest that gonadal hormones regulate sex dif-
ferences in microglia. Microglia specifically express the
anti-inflammatory estrogen receptor (ER)a,asERbwas
never found on these cells (Vegeto et al., 2003; Sierra et al.,
2008; Crain et al., 2013; Crain and Watters, 2015).
However, ERais expressed to the same extent in males
and females at all ages, suggesting that sexual dimor-
phism of microglia is ER-independent (Crain et al., 2013).
2. Evidence for Regionalization of Microglia.
Microglia are essential in monitoring the environment
and sense variations, and react accordingly to maintain
CNS homeostasis (Kettenmann et al., 2011). Hence, the
local environment, such as BBB permeability or neuro-
nal activity, is key in driving microglial phenotype.
Lawson et al. (1990) described regional variations in
microglial morphology and density as the first evidence
for heterogeneity of these cells. More recently, using
ex vivo flow cytometric analysis surface expression of
CD11b, CD40, CD45, CD80, CD86, F4/80, TREM-2b,
MHCII, CXCR3, CCR9, and CCR7, de Haas et al. (2008)
showed that most of these immunoregulatory markers
displayed region-specific differences in expression lev-
els, confirming other studies (Mittelbronn et al., 2001;
Buschmann et al., 2012; Doorn et al., 2015). Using
genome-wide transcriptional profiling of adult micro-
glia from various brain regions, Grabert et al., 2016)
thoroughly addressed the question of regional hetero-
geneity of microglia and found that the mouse
microglial transcriptome is regionally heterogeneous.
Moreover, regional microglial heterogeneity in immu-
nophenotype suggests differences in immune vigilance
(Grabert et al., 2016). Overall, these data reveal micro-
glia as highly diverse cells under steady-state condi-
tions. In addition, only recently has it become clear that
microglial sensitivity to stimuli may be region-specific
(Lucin and Wyss-Coray, 2009; Ransohoff and Perry,
2009; Olah et al., 2011; Doorn et al., 2015), as a potential
substrate for differential and evolving pattern of
3. Evidence for Age Dependence of Microglia.
Many groups have examined age-dependent modula-
tion of microglial morphology, density, phenotype, and
function (Tay et al., 2017). This literature is too vast
to be described in an exhaustive way in this work. In
substance, microglial activity is continuously evolv-
ing, in a structure and age-dependent manner, from
New Insights into the Impact of Omega-3 in Microglia 25
guidance of axons or phagocytosis of neuronal elements
during development (Paolicelli et al., 2011; Schafer
et al., 2012; Squarzoni et al., 2014), to fine remodeling
of neuronal circuits at adolescence and adulthood
(Tremblay et al., 2010; Parkhurst et al., 2013) or control
of neuroinflammation in normal and pathologic ageing
(Kettenmann et al., 2011; Tay et al., 2017). This list is
far from being exhaustive but shows that microglia are
versatile cells, constantly adapting to the environment
to maintain homeostasis.
In conclusion, age-, sex-, and region-specific variances
in microglial function may allow differential responses
to the same stimulus at different ages, perhaps contrib-
uting to altered CNS vulnerabilities and/or disease
courses. Combined to age, sex, and region dependence
of n-3 PUFA brain composition, microglialipid inter-
actions are likely to be extremely diverse.