ArticlePDF AvailableLiterature Review

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

January 2018
Pharmacological Reviews 70(1):12-38

DOI:10.1124/pr.117.014092

Authors:

Sophie Layé

French National Institute for Agriculture, Food, and Environment (INRAE)

Agnes Nadjar

University of Bordeaux

Corinne Joffre

French National Institute for Agriculture, Food, and Environment (INRAE)

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.

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 organs.

…

Structure of the two main forms of lipids in food, the triacylglyceride (A) and phospholipid (B). Triacylglycerides and phospholipids 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.

…

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 antiinflammatory 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 antiinflammatory depending on the receptor to which they bind.

…

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1521-0081/70/1/12–38$25.00 https://doi.org/10.1124/pr.117.014092

PHARMACOLOGICAL REVIEWS Pharmacol Rev 70:12–38, January 2018

ASSOCIATE EDITOR: ROBERT DANTZER

Anti-Inflammatory Effects of Omega-3 Fatty Acids in the

Brain: Physiological Mechanisms and Relevance to

Pharmacology

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 Brain—Clinical 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: sophie.laye@inra.fr

https://doi.org/10.1124/pr.117.014092.

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

References...................................................................................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 brain’s 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, Alzheimer’s 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 acid–binding

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

proresolving.

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

events.

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-oxidation–producing

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

organs.

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 12–20 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

lowintakeofALAleadstotheaccumulationofn-6PUFA,

includingAA.Inthecaseofseveren-3PUFAdeficiency,

the expression of desaturases and elongases is upregu-

latedinthelivertocompensateandprovideDHAtothe

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 4–5 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,

2010).

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 acid–binding

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

behavior.

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 Alzheimer’sdisease(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).

DHA’s 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

offspring’s 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 protein–coupled 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

-induced

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 oil–rich 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 sac–derived 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

behavior.

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.,

2007).

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), Parkinson’s

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

-mediated

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-g–mediated 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-2–catalyzed 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-seq”database, 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 (40–100 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

(PGE

,PGJ

) (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 sac–originating

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

neuropathologies.

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, microglia–lipid inter-

actions are likely to be extremely diverse.

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

Abstract and Figures