omega-3 fatty acids prevent inflammation and metabolic ......septic shock (mao et al., 2013; mishra...

Immunity

Article

Omega-3 Fatty Acids Prevent Inflammationand Metabolic Disorder through Inhibitionof NLRP3 Inflammasome ActivationYiqing Yan,1,4 Wei Jiang,1,4 Thibaud Spinetti,2,3,4 Aubry Tardivel,2 Rosa Castillo,2 Carole Bourquin,3 Greta Guarda,2

Zhigang Tian,1,* Jurg Tschopp,2,5 and Rongbin Zhou1,*1Hefei National Laboratory for Physical Sciences at Microscale and School of Life Sciences, University of Science and Technology of China,

Anhui 230027, China2Department of Biochemistry, University of Lausanne, Epalinges 1066, Switzerland3Chair of Pharmacology, Department of Medicine, University of Fribourg, Fribourg 1700, Switzerland4These authors contributed equally to this work5Deceased*Correspondence: [email protected] (Z.T.), [email protected] (R.Z.)

http://dx.doi.org/10.1016/j.immuni.2013.05.015

SUMMARY

Omega-3 fatty acids (u-3 FAs) have potential anti-inflammatory activity in a variety of inflammatoryhuman diseases, but the mechanisms remainpoorly understood. Here we show that stimulationof macrophages with u-3 FAs, including eicosapen-taenoic acid (EPA), docosahexaenoic acid (DHA),and other family members, abolished NLRP3inflammasome activation and inhibited subsequentcaspase-1 activation and IL-1b secretion. In addi-tion, G protein-coupled receptor 120 (GPR120)and GPR40 and their downstream scaffold proteinb-arrestin-2 were shown to be involved in inflamma-some inhibition induced by u-3 FAs. Importantly,u-3 FAs also prevented NLRP3 inflammasome-dependent inflammation and metabolic disorder ina high-fat-diet-induced type 2 diabetes model.Our results reveal a mechanism through which u-3FAs repress inflammation and prevent inflam-mation-driven diseases and suggest the potentialclinical use of u-3 FAs in gout, autoinflammatorysyndromes, or other NLRP3 inflammasome-driveninflammatory diseases.

INTRODUCTION

Omega-3 fatty acids (u-3 FAs) are essential to human health, and

deficiencies can lead to chronic diseases (Zhang and Spite,

2012). In particular, increasing evidences from both human and

animal studies have demonstrated that u-3 FAs, primarily eico-

sapentaenoic acid (EPA) and docosahexaenoic acid (DHA),

can suppress inflammation and have a beneficial role in a variety

of inflammatory human diseases, including diabetes, atheroscle-

rosis, asthma, and arthritis(Fritsche, 2006; Zhang and Spite,

2012). In addition, studies using Fat1 transgenic mice, which

have abundant endogenous u-3 FAs, strongly support the idea

that u-3 FAs are protective in inflammatory pathologies (Hudert

1154 Immunity 38, 1154–1163, June 27, 2013 ª2013 Elsevier Inc.

et al., 2006; Kang et al., 2004). Although the use of u-3 FAs for

treatment of inflammatory disorders in the clinic is promising,

the anti-inflammatory mechanisms of u-3 FAs are poorly

understood.

The inflammasome is a cytosolic protein complex composed

of nucleotide-binding domain and leucine-rich repeat con-

taining proteins (NLRs) or AIM2, ASC and caspase-1, and

is a central regulator of innate immunity and inflammation

(Martinon et al., 2009). The inflammasome is assembled in

response to pathogen infection or ‘‘danger’’ signals and pro-

motes the maturation and release of several proinflammatory

cytokines, including interleukin-1b (IL-1b), IL-18, and IL-33.

Inflammasome is therefore involved in the pathogenesis

of several inflammatory disorders, including diabetes, athero-

sclerosis, and arthritis (Davis et al., 2011; Schroder et al.,

2010; Strowig et al., 2012), suggesting that the activation of

NLRP3 inflammasome needs to be tightly controlled. Type I

interferon has been shown to block NLRP3 inflammasome

activation via Stat1-dependent manner, suggesting that suc-

cess of IFN-b treatment for multiple sclerosis might indeed

rely on suppressing inflammasome-mediated inflammation

(Guarda et al., 2011). Recently, nitric oxide has been identified

as another critical negative regulator of the NLRP3 inflamma-

some activation and can protect mice against LPS-induced

septic shock (Mao et al., 2013; Mishra et al., 2013). However,

the mechanism involved in inflammasome regulation is still

unclear.

u-3 FAs have been shown to inhibit the production of

proinflammatory cytokines, including IL-1b (Endres et al., 1989;

Oh et al., 2010; Robinson et al., 1996), and this prompted us

to investigate whether u-3 FAs exert their anti-inflammatory

activity via inhibition of inflammasome activation. In this study,

we demonstrate that u-3 FAs suppressed inflammation via

inhibition of inflammasome activation. We found that u-3 FAs

inhibited NLRP3 and NLRP1b-dependent caspase-1 activation

and IL-1b secretion. Furthermore, u-3 FAs prevented high-fat-

diet (HFD)-induced metabolic disorder through inhibition of

NLRP3 inflammasome activation in vivo, suggesting that the

inflammasome is an important target for u-3 FAs to exert their

anti-inflammatory activity.

mailto:[email protected]

mailto:[email protected]


http://crossmark.dyndns.org/dialog/?doi=10.1016/j.immuni.2013.05.015&domain=pdf

A

D

B C

E F

Figure 1. u-3 FAs Suppress Caspase-1 Activation and IL-1b Secretion

(A–C), LPS primed-BMDMs were treated with different doses of DHA and then stimulated with nigericin. Medium supernatants (SN) and cell extracts (Input) were

analyzed by immunoblotting as indicated in the text (A). Supernatants were also analyzed by ELISA for IL-1b (B) and IL-18 (C) release.

(D) PMA-differentiated THP-1 cells were treated with different doses of DHA and then stimulated with nigericin.

(E and F) LPS primed-BMDMs were treated with different FAs as indicated in the text at 20 mM and then stimulated with nigericin. Supernatants were analyzed

by ELISA for IL-1b (E) and TNF-a (F) release. *p < 0.05, **p < 0.01. Values are means ± SEM. Data are representative of at least three independent experiments.

See also Figure S1.

Immunity

Omega-3 Fatty Acids Inhibits NLRP3 Inflammasome

RESULTS

u-3 FAs Suppress Caspase-1 Activation and IL-1bSecretionTo assess the effect of u-3 FAs on inflammasome activation and

IL-1b secretion, we first examined whether DHA could inhibit

caspase-1 cleavage and IL-1b secretion. We indeed observed

that pretreatment of LPS-primed bone-marrow-derived macro-

phages (BMDMs) with DHA blocked caspase-1 activation and

IL-1b maturation in a dose-dependent manner (Figures 1A and

1B). Similarly, DHA also inhibited the nigericin-induced secretion

of IL-18, another inflammasome-dependent cytokine (Figure 1C).

It has been reported that DHA can inhibit LPS-induced NF-kB

activation and tumor necrosis factor-a (TNF-a) production

(Oh et al., 2010), we then examined whether DHA had an impact

on LPS-induced priming for inflammasome activation. As shown

in Figure S1, when BMDMs were stimulated with DHA after 3 hr

LPS treatment, DHA had no effect on LPS-induced TNF-a pro-

duction or pro-IL-1b expression (Figures S1A–S1C), suggesting

that DHA didn’t affect LPS-induced priming at this condition. In

contrast, when BMDMswere stimulated with DHA for 3 hr before

LPS treatment, DHA inhibited LPS-induced TNF-a production

and pro-IL-1b expression (Figures S1A–S1C), although the

dose was higher than the dose needed for inflammasome

inhibition. These results suggest that DHA can affect both

LPS-induced priming and inflammasome activation. In order to

clarify the mechanism underlying DHA-induced inflammasome

inhibition, we stimulated BMDMs with DHA after 3 hr LPS treat-

ment in the later experiments. The observed inhibitory effects of

DHA on IL-1b secretion were also confirmed in human THP-1

macrophages (Figure 1D). Furthermore, we found that u-3 FAs

didn’t affect nigericin-induced cell death (Figure S1D), suggest-

ing that the effect of u-3 FAs on IL-1b release is not due to the

inhibition of cell death. We also examined the role of other u-3

FAs, such as EPA and a-Linolenic acid (ALA), and found that

both of EPA and ALA suppressed nigericin-induced IL-1b secre-

tion but had no effect on TNF-a production (Figures 1E and 1F;

Figure S1E). In contrast with u-3 FAs, u-6 FAs—including os-

bond acid (OBA), dihomo-gamma-linolenic acid (DGLA), and

adrenic acid (ADA)—and u-9 FAs—including oleic acid (OA)—

failed to block IL-1b secretion in BMDMs (Figure 1E), suggesting

that only certainu-3 FAs inhibit nigericin-induced NLRP3 inflam-

masome activation. Taken together, these results indicate that

u-3 FAs have the potential to inhibit caspase-1 activation and

IL-1b secretion.

u-3 FAs Inhibit NLRP3 or NLRP1b InflammasomeActivationIn addition to nigericin, NLRP3 inflammasome can be activated

by both pathogen-associated molecular patterns (PAMPs) and

Immunity 38, 1154–1163, June 27, 2013 ª2013 Elsevier Inc. 1155

A

D

B C

E F

Figure 2. u-3 FAs Inhibit NLRP3 or NLRP1b Inflammasome Activation

(A) LPS primed-BMDMs were treated with DHA (20 mM) and then stimulated with MSU, Alum, R837, ATP, and nigericin. Medium supernatants (SN) and cell

extracts (Input) were analyzed by immunoblotting as indicated in the text.

(B–F) LPS primed-BMDMs were treated with FAs (20 mM) and then stimulated with MSU, Lethal Toxin, Salmonella, or poly (dA:dT). Medium supernatants and

cell extracts were analyzed by immunoblotting as indicated in the text (B and E). Supernatants were analyzed by ELISA for IL-1b or IL-18 release (C, D, and F).

**p < 0.01. Values are means ± SEM. Data are representative of at least three independent experiments.

Immunity


danger-associatedmolecular patterns (DAMPs), including R837,

aluminum salts (Alum), ATP and monosodium urate crystals

(MSU) (Davis et al., 2011). To determine whether u-3 FAs only

affect nigericin-induced NLRP3 inflammasome activation, we

examined other NLRP3 agonists. As shown in Figure 2A, we

found that pretreatment with DHA inhibited caspase-1 cleavage

and IL-1b secretion triggered by all examined agonists, including

MSU, Alum, R837, and ATP, similar to nigericin. These results

suggest that u-3 FAs are potent and broad inhibitors of NLRP3

inflammasome activation. To date, four inflammasomes, includ-

ing the NLRP3, the NLRC4, the NLRP1b, and the AIM2 inflam-

masome, have been extensively studied (Davis et al., 2011). We

investigated whether the inhibitory effect ofu-3 FAswas specific

to the NLRP3 inflammasome or more general. We found that

DHA, but not DGLA or OA, blocked caspase-1 activation, IL-1b,

or IL-18 secretion induced by anthrax Lethal Toxin (Lethal Toxin),

an identified NLRP1b activator (Figures 2B–2D). In contrast, DHA

had minimal effect on NLRC4 or AIM2 inflammasome activation,

which were triggered by Salmonella typhimurium (Salmonella)

infection or poly (dA:dT) transfection, respectively (Figures 2E

and 2F). Taken together, these results demonstrate that u-3

FAs can specifically inhibit NLRP3 and NLRP1b inflammasome

activation and subsequent IL-1b production.

u-3 FAs Inhibit Inflammasome Activation Independentof Enzymatic ProductsNext, we investigated the mechanisms underlying the inhibitory

activity ofu-3 FAs on NLRP3 inflammasome activation. In recent


years, some enzymatic oxygenated products generated from

u-3 FAs have been shown to exert anti-inflammatory activity

and contribute functionally to the resolution of inflammation

(Arita et al., 2007; Serhan et al., 2002; Spite et al., 2009). We

thus asked whether u-3 FAs inhibited NLRP3 inflammasome

activation via such modified products. DHA can be converted

to several bioactive compounds, including Protectin D1 (PD1),

Resolvin D1 (RvD1) and aspirin-triggered Resolvin D1 (AT-

RvD1), by lipoxygenases (AlOX5 and AlOX15) and aspirin-acety-

lated COX-2 (Serhan et al., 2000; Zhang and Spite, 2012). As

shown in Figure 3, PD1, RvD1 or AT-RvD1 pretreatment had

no effect on nigericin-induced caspase-1 activation and IL-1b

or IL-18 secretion (Figures 3A–3C). Furthermore, we also found

that Alox5 and Alox15 double deficiency or inhibition of

COX-2 activity by celecoxib had no impact on the inhibitory

activity of DHA on NLRP3 or NLRP1b inflammasome activation

(Figures 3D and 3E; Figure S2). These results suggest that

these enzyme modified enzymatic products are not involved in

u-3 FA-mediated inflammasome inhibition.

Involvement of GPR40, GPR120, and b-Arrestin-2in the Inhibition of NLRP3 InflammasomeActivation Induced by u-3 FAsIt has been previously reported that long-chain FAs can activate

G protein-coupled receptor 120 (GPR120) and GPR40. As

GPR120 is involved in the anti-inflammatory effects of u-3 FAs

(Cintra et al., 2012; Hirasawa et al., 2005; Itoh et al., 2003; Liou

et al., 2011), we examined whether u-3 FAs inhibited NLRP3

A B C

D E

Figure 3. u-3 FA Inhibit Inflammasome Activation Independent of Enzymatic Products

(A–C) LPS primed-BMDMswere treatedwith DHA (20 mM), PD1, RvD1, or AT-RvD1 and then stimulatedwith nigericin.Medum supernatants (SN) and cell extracts

(Input) were analyzed by immunoblotting as indicated in the text (A). Supernatants were also analyzed by ELISA for IL-1b or IL-18 release (B and C).

(D) LPS primed-BMDMs from Alox5�/�Alox15�/� mice were treated with DHA (20 mM) and then stimulated with nigericin. Medium supernatants and cell extracts

were analyzed by immunoblotting as indicated in the text.

(E) LPS primed-BMDMs were treated with celecoxib (50 mM) for 30min and then treated with DHA (20 mM). After that cells were stimulated with nigericin. Medium

supernatants and cell extracts were analyzed by immunoblotting as indicated in the text. **p < 0.01. Values are means ± SEM. Data are representative of at least

three independent experiments. See also Figure S2.

Immunity


inflammasome activation via GPR120 or GPR40. First, we found

that pretreatment with GW9508, a small-molecular agonist of

GPR120 and GPR40 (Briscoe et al., 2006), markedly repressed

caspase-1 activation and IL-1b or IL-18 secretion induced by

nigericin or Lethal Toxin, similar to DHA (Figure 4A; Figure S3A,

B). To further confirm the role of GPR120 andGPR40 in the inhib-

itory role of u-3 FAs in inflammasome activation, we established

THP-1 cells stably expressing shRNA targeting the mRNA en-

coding either GPR40, GPR120, or both of them (Figure S3C).

We found that, although single deficiency of either GPR40 or

GPR120 only partially impaired the inhibitory effect of DHA on

NLRP3 inflammasome activation (Figures 4B and 4D; Fig-

ure S3D), double deficiency of GPR40 and GPR120 significantly

relieved DHA-mediated inflammasome inhibition (Figures 4C–

4E; Figure S3E). Similar results were observed when THP-1 cells

were pretreated with EPA (Figure 4F). These data suggest that

u-3 FAs can signal through GPR40 and GPR120 to inhibit

NLRP3 inflammasome activation and may explain the enhanced

inflammation observed in adipose tissue of Gpr120�/� mice

(Ichimura et al., 2012).

b-arrestin-2 (ARRB2) is a downstream scaffold protein of

GPR120 (Oh et al., 2010), so to further investigate the molecular

mechanisms mediating the inhibitory effect of u-3 FAs, we

investigated the role of ARRB2. We found that deficiency of

ARRB2 in THP-1 cells abrogated the inhibitory effects of DHA

and EPA on NLRP3 or NLRP1b inflammasome activation (Fig-

ure 5A; Figure S4A–S4C). We further used Arrb2�/� mice to

confirm the results observed in THP-1 cells and found that

deletion of Arrb2 only partially inhibited the activity of DHA (Fig-

ures 5B and 5C), suggesting that another mechanism might be

involved in the inhibition of NLRP3 inflammasome activation

induced by DHA in BMDMs. We next tested the possibility that

ARRB2 interacts with NLRP3 in HEK293T cells. Overexpressed

ARRB2 bound full-length NLRP3 and the leucine-rich repeat

(LRR) and NACHT regions of NLRP3 (Figure 5D). We further

found that ARRB2 interacted with NLRP1, but not with NLRC4

or AIM2. This might explain why DHA can inhibit NLRP3 or

NLRP1b inflammasome activation but has no effect on NLRC4

or AIM2 inflammasome activation (Figure 5E). Importantly, DHA

and EPA treatments induced the endogenous interaction

between NLRP3 and ARRB2 in THP-1 cells via GPR120 and

GPR40, whereas DGLA did not (Figure 5F; Figure S4D). These

results indicate that ARRB2 acts downstream of GPR120 and

GPR40 to inhibit inflammasome activation via binding with

NLRP3 or NLRP1.

u-3 FAs Prevent HFD-Induced Insulin Resistancethrough Inhibition of NLRP3 Inflammasome ActivationType 2 diabetes (T2D) is a leading cause of morbidity and

mortality worldwide, and increasing evidence suggests chronic


C

D E F

BA

Figure 4. u-3 FAs Inhibit NLRP3 Inflammasome Activation via GPR40 and GPR120

(A) LPS primed-BMDMs were treated with DHA (20 mM) or different doses of GW9508 and then stimulated with nigericin. Medium supernatants (SN) and cell

extracts (Input) were analyzed by immunoblotting as indicated in the text.

(B–F) PMA-differentiated THP-1 cells stably expressing shRNA targeting themRNA encodingGPR40 orGPR120 or both of themwere treatedwith DHA (20 mM) or

EPA (20 mM) and then stimulated with nigericin (B–D, and F) or Lethal Toxin (E). Medium supernatants (SN) and cell extracts (Input) were analyzed by immu-

noblotting as indicated in the text (B and C). Supernatants were analyzed by ELISA for IL-1b release (D–F). *p < 0.05, **p < 0.01. Values aremeans ± SEM. Data are

representative of at least three independent experiments. See also Figure S3.

Immunity


inflammation as an important pathogenetic factor in the develop-

ment of insulin resistance and T2D (Donath and Shoelson, 2011).

As we and others have shown that the NLRP3 inflammasome is

implicated in the development of insulin resistance in T2D (Mas-

ters et al., 2010; Vandanmagsar et al., 2011; Wen et al., 2011;

Zhou et al., 2010), we investigated whetheru-3 FAs can improve

insulin sensitivity via inhibition of inflammasome activation. We

fedmice with HFD or normal diet (ND) for 10 weeks. HFD-treated

wild-type (WT) mice exhibited a robust elevation in plasma

insulin accompanied by increased plasma glucose in fasted

states (Figures 6A and 6B; Figures S5A and S5B), indicating

the emergence of insulin resistance. However, HFD-induced

elevations of plasma glucose and insulin were ameliorated by

DHA supplementation, but not by DGLA or OA supplementation,

suggesting that DHA has a beneficial effect on HFD-induced

insulin resistance (Figures 6A and 6B; Figures S5A and S5B).

To confirm these results, we performed glucose tolerance test

(GTT) and insulin tolerance test (ITT) in HFD-treated mice with

or without DHA supplementation. GTT showed that DHA supple-

mented mice were more glucose tolerant than WT mice (Fig-

ure 6C). ITT showed that insulin sensitivity, as measured by the

reduction in plasma glucose after insulin administration, was

markedly improved by DHA in HFD-treated mice (Figure 6E).

Furthermore, phosphorylation of the major marker for insulin

signaling, Akt (on Ser473), was enhanced both in liver and

adipose tissues in DHA-treated mice as compared to WT mice


(Figures S5C and S5D). In contrast with WT mice, the beneficial

effects of DHA, including reduction of fasted glucose concentra-

tions, improved glucose tolerance and better insulin sensitivity,

were all abrogated in Nlrp3�/� mice, suggesting that the benefi-

cial effect of u-3 FAs on metabolic disorder depends on NLRP3

inflammasome inhibition (Figures 6A–6F).

To further confirm that DHA prevents HFD-induced metabolic

disorder through inhibition of NLRP3 inflammasome activation,

we tested whether DHA supplementation inhibited metabolic

stress-induced inflammasome activation in vivo. Consistent

with above results, liver and adipose tissue from HFD-treated

mice showed higher caspase-1 activation and IL-1b or IL-18

production as compared to ND-treated mice (Figures 7A–7D,

7I). Importantly, DHA administration or Nlrp3 deficiency blocked

HFD-induced IL-1b or IL-18 production and caspase-1

activation in these tissues (Figures 7A–7D, 7I), indicating that

DHA supplementation can suppress metabolic stress-induced

inflammasome activation in HFD-treated mice. Moreover, DHA

also suppressed the upregulation of proinflammatory cytokines,

such as TNF-a and MCP-1, in WT mice, but not in Nlrp3�/� mice

(Figures 7E–7H), suggesting that NLRP3 inflammasome is a key

target throughwhichu-3 FAs exert their broad anti-inflammatory

activity in vivo. Taken together, these results indicate that u-3

FAs can exert beneficial effect on HFD-induced insulin resis-

tance by blocking metabolic stress-induced NLRP3 inflamma-

some activation.

B

DHA

NigericinA

Nigericin +++-+++-+++-+++-

DHA

C MockNigericinDHA+Nigericin

p20

mIL-1β

SN

DHA

EPA

p20

mIL-1β

SN

EPA

- - + - - - + -

+---+---+---+---

- - + - - - + -DHA

3000

4000 ****

g/m

l)

EPA+Nigericin

Pro-casp1

Pro-IL-1β

p 0

Input

Scramble shARRB2

Pro-casp1

Pro-IL-1β

p 0

Input

WT Arrb2-/-0

1000

2000 **

WT Arrb2-/-

IL-1

β(p

g

ED F

Isotype Anti-NLRP3

WT Arrb2

Flag

VSV-ARRB2 + + + + + +

ARRB2

NLRP3

VSV-ARRB2 + + + + + Flag

ARRB2

Fl

ARRB2

IP

Moc

kD

HA

1hD

HA

2hD

HA

3h

Isotype Anti NLRP3

Moc

kD

HA

1hD

HA

2hD

HA

3h

IP

NLRP3NACHTLRR

PYD

ARRB2

ARRB2

Flag

Proteins

FlI t

IP

ARRB2

NLRP3

NLRP3Input

IP

Input

ARRB2

NLRP3NACHTLRR

PYD

Flag

Proteins

Input

PYD

Figure 5. ABBR2 Acts Downstream of GPR120 and GPR40 to Inhibit NLRP3 Inflammasome Activation via Binding with NLRP3

(A–C) PMA-differentiated THP-1 cells stably expressing shRNA targeting the mRNA encoding ARRB2 (A) or LPS-primed BMDMs from Arrb2�/� mice (B and C)

were treated with DHA (20 mM) or EPA (20 mM) and then stimulated with nigericin. Medium supernatants (SN) and cell extracts (Input) were analyzed by

immunoblotting as indicated in the text (A and B). Supernatants were analyzed by ELISA for IL-1b release (C). *p < 0.05, **p < 0.01. Values are means ± SEM.

(D) Flag-tagged NLRP3 constructs and VSV-tagged ARRB2 construct were cotransfected in HEK293T cells. After 24 hr, cell lysates were immunoprecipitated

with anti-Flag antibody and then the samples were analyzed by immunoblotting as indicated in the text. EV, empty vector. IP, immunoprecipitation.

(E) Flag-taggedNLRP3, NLRP1, NLRC4, or AIM2 and VSV-tagged ARRB2were cotransfected in HEK293T cells. After 24 hr, cell lysates were immunoprecipitated

with anti-Flag antibody and then the samples were analyzed by immunoblotting as indicated in the text.

(F) THP-1 cells were treated with DHA for different time points and then the cell lysates were immunoprecipitated with anti-NLRP3 antibody and then the samples

were analyzed by immunoblotting as indicated in the text. Data are representative of at least three independent experiments. See also Figure S4.

Immunity


DISCUSSION

u-3 FAs are attractive candidates for prevention of many inflam-

mation-driven human diseases because of their anti-inflamma-

tory properties. The first evidence of the important role of u-3

FAs in inflammation was derived from epidemiological observa-

tions of the low incidence of inflammatory disorders, such

as psoriasis, diabetes, and multiple sclerosis in Greenland

Eskimos compared with gender and age-matched people living

in Denmark (Dyerberg and Bang, 1979). In the last years, both

human clinical studies and animal experiments have demon-

strated that u-3 FAs have anti-inflammatory activity and

beneficial roles in a variety of inflammatory human diseases,

including diabetes, atherosclerosis, asthma, and arthritis. Thus,

understanding the underlying anti-inflammatory mechanisms

is helpful for an appropriate use of u-3 FAs in the clinic. We

show here that the inflammasome was a key target for u-3

FAs to suppress inflammation and exert their anti-inflammatory

activity in inflammatory disorders. u-3 FAs suppressed both

LPS-induced priming and inflammasome activation (caspase-1

cleavage) in macrophages. The inhibitory effect of u-3 FAs

on inflammasome activation was specific to NLRP3 or NLRP1b

inflammasome, because u-3 FAs did not inhibit NLRC4 or

AIM2 inflammasome activation. Mechanistically, we found that

u-3 FAs repressed NLRP3 inflammasome activation indepen-

dently of their enzyme modified products, including PD1,

RvD1, and AT-RvD1. Furthermore, our data suggest that u-3

FAs inhibited NLRP3 inflammasome activation via GPR40 and

GPR120. Indeed, ARRB2 acted downstream of GPR120 and

GPR40 to inhibit NLRP3 inflammasome activation via binding

to NLRP3, at least partially. Importantly, u-3 FAs supplemen-

tation could suppress HFD-induced NLRP3 inflammasome


A WT B WT CWT

152025

**6

810 ***

*

*

Nlrp3-/-

gluc

ose

(mM

)

se (m

M)

3

4*

***

*

insu

lin (n

g/m

l)

NDHFDHFD+DHA

Nlrp3-/-

0 30 60 9005

10*

024

Fast

ing

bloo

d

Bloo

d gl

uco

0

1

2 *

Fast

ing

bloo

d D FENlrp3-/- WT Nlrp3-/-

) e%)

e%)

Time (min)

ND ND ND

20406080

100

* ** *

510152025

20406080

100

lood

glu

cose

(mM

glu

cose

(bas

elin

glu

cose

(bas

elinHFD

HFD+DHAHFDHFD+DHA

HFDHFD+DHA

0 30 60 900

0 30 60 900

0 30 60 900B

Bloo

d

Bloo

d

Time (min)Time (min)Time (min)

Figure 6. u-3 FAs Prevent NLRP3-Dependent Insulin Resistance-Induced by HFD

WT or Nlrp3�/� mice were fed with normal diet (ND) or high fat diet (HFD) for 10 weeks with or without treatment with DHA. Fasting serum glucose (A), fasting

serum insulin (B), glucose tolerance (C and D), and insulin tolerance (E and F) were tested as indicated. Values are means ± SEM of 6–9 mice per group. *p < 0.05,

**p < 0.01 (nonparametric Mann Whitney test). Data are representative of three independent experiments. See also Figure S5.

Immunity


activation and prevent NLRP3 inflammasome-dependent insulin

resistance in vivo. Taken together, our results demonstrate a

previously unrecognized mechanism through which u-3 FAs

repress inflammation and prevent inflammation-driven diseases.

T2D is a leading cause of morbidity and mortality worldwide

and its etiology remains unclear. Oxidative stress, endoplasmic

reticulum stress, amyloid deposition in the pancreas, lipotoxicity,

and glucotoxicity are the most popular mechanisms being used

to explain insulin resistance and islet b-cell dysfunction in T2D

(Harding and Ron, 2002; Prentki and Nolan, 2006; Robertson

et al., 2004; Weir and Bonner-Weir, 2004), although it has been

difficult to determine which one is the most important. It is note-

worthy that each of these cellular stresses is also thought to

elicit or associate with inflammatory response (Hotamisligil and

Erbay, 2008; Masters et al., 2010). Indeed, increasing evidence

suggests chronic inflammation as an important pathogenetic

factor in the development of insulin resistance and T2D (Donath

and Shoelson, 2011). Proinflammatory cytokines, such as TNF-a

and IL-1b, are significantly elevated in T2D (Herder et al., 2009;

Spranger et al., 2003). u-3 FAs have been reported to enhance

systemic insulin sensitivity via inhibition of TNF-a signaling

pathway (Oh et al., 2010), however, some animal studies and

several clinical trials using TNF-a blockade have failed to

improve insulin resistance (Dominguez et al., 2005; Lo et al.,

2007; Ofei et al., 1996; Rosenvinge et al., 2007), suggesting

that TNF-a may be not the primary target of u-3 FAs to exert

beneficial effect in T2D. Recently, we and others have shown

that the NLRP3 inflammasome activation and subsequent


IL-1b production is implicated in the development of insulin

resistance in T2D (Masters et al., 2010; Vandanmagsar et al.,

2011; Wen et al., 2011; Zhou et al., 2010). Here we present

that u-3 FAs suppress NLRP3 inflammasome-dependent IL-1b

production in macrophages, and more importantly, u-3 FAs

prevent HFD-induced insulin resistance in WT mice, but have lit-

tle effect on Nlrp3�/� mice. Although some reports propose

other targets for u-3 FAs, such as TNF-a and PPAR-g (Moraes

et al., 2006; Oh et al., 2010), our data support a key role for the

inhibition of the NLRP3 inflammasome underlying the beneficial

effect of u-3 FAs in inflammatory disorders, at least in the model

of T2D we explored.

The inflammasome is a multiprotein complex that is

composed of a sensor protein, the adaptor protein ASC, and

the inflammatory protease caspase-1(Davis et al., 2011; Marti-

non et al., 2009). Although several inflammasomes have been

described, the NLRP3 inflammasome is the most extensively

studied but also the most elusive. K+ efflux, elevated ROS

levels, mitochondria damage, and lysosomal rupture have

been proposed to participate in NLRP3 inflammasome activation

(Gross et al., 2011; Zhou et al., 2011), but we still know very little

about the precise mechanisms that can positively or negatively

regulate NLRP3 inflammasome activation. Because NLRP3

inflammasome is clearly involved in host defense and autoin-

flammatory disorders, understanding the networks that control

NLRP3 inflammasome activation and identifying its potential

inhibitors will likely be extremely useful in developing new treat-

ments for inflammatory disorders. Here we demonstrateu-3 FAs

WAT Li WAT

400

600**

**NS

75

100 ***NS

WAT Liver

A B

100

150 ****NS

WAT

C

ml/m

g)

/ml/m

g)

/ml/m

g) NDHFDHFD+DHA

NDHFDHFD+DHA

NDHFDHFD+DHA

0

200

400

NS

0

25

50 NS

0

50 NS

WT Nlrp3-/-

IL-1

β(p

g/m

IL-1

β(p

g/

IL-1

8 (p

g/

WT Nlrp3-/- WT Nlrp3-/-

1000

1500

***NS

600

800**

***

*

TAWTAW

300

400**

**NSD

Liver

/mg)

ml/m

g)

ml/m

g)NDHFDHFD+DHA

NDHFDHFD+DHA

NDHFDHFD+DHA

0

500

1000

*NS

0

200

400 * NS*

0

100

200*

IL-1

8 (p

g/m

l/

TNF-

α(p

g/m

MC

P-1

(pg/

mHFD DHA

WT Nlrp3-/- WT Nlrp3-/- WT Nlrp3-/-

I

200250 ***

*300

****

reviLreviL

E F

G H

HFD

HFD

+DH

AN

D

HFD

HFD

+DH

AN

D

WT Nlrp3-/-

/mg)

/mg)ND

HFDHFD DHA

NDHFDHFD+DHA

WT Nlrp3 WT Nlrp3 WT Nlrp3

050

100150200

** NS

0

100

200

* NS*

Actin

p20

Pro-caspase-1

H H N H H N

TNF-

α(p

g/m

l/

MC

P-1

(pg/

ml/HFD+DHA HFD+DHA

/ /WT Nlrp3-/- WT Nlrp3-/-

Figure 7. u-3 FAs Suppress HFD-Induced NLRP3 Inflammasome Activation In Vivo

WTorNlrp3�/�micewere fedwith ND orHFD for 10weekswith or without treatment with DHA. Adipose tissue (WAT) (A, C, E, and F) and liver (B, D, G, andH) were

isolated and cultured for 24 hr, and supernatants were analyzed by ELISA for IL-1b (A and B), IL-18 (C andD), TNF-a (E andG) andMCP-1 (F andH) release. Values

aremeans ± SEMof 6–9mice per group. Caspase-1 activation inWATwas analyzed by immunoblotting as indicated (I). *p < 0.05, **p < 0.01, NS p> 0.05. Data are

representative of three independent experiments.

Immunity


as inhibitors for NLRP3 inflammasome activation and suggest

the potential clinic use of u-3 FAs in NLRP3 inflammasome-

driven diseases. We also demonstrate that u-3 FAs inhibit

NLRP3 inflammasome activation via GPR40 and GPR120-

dependent pathway, at least partially, suggesting that GPR40

and GPR120 downstream signaling pathway are involved in

the signaling network that controls NLRP3 inflammasome

activation, although the precise mechanisms need to be further

investigated. The role of GPR40 and GPR120 signaling in

NLRP3 inflammasome inhibition may also explain the enhanced

inflammation observed in adipose tissue of Gpr120�/� mice

(Ichimura et al., 2012).

Collectively, our findings demonstrate that u-3 FAs sup-

pressed inflammation by reducing NLRP3 inflammasome acti-

vation in macrophages and provide a new anti-inflammatory

mechanism for u-3 FAs. Our results also show that the inhibitory

activity of u-3 FAs on inflammasome activation was important

for their beneficial effects in T2D. Considering that NLRP3

inflammasome is involved in the development of a broad range

of inflammatory diseases, u-3 FAs might have potential clinic

use in gout, autoinflammatory syndromes, or other NLRP3-

driven diseases.

EXPERIMENTAL PROCEDURES

Mice

Nlrp3�/� andArrb2�/�mice were described previously (Bohn et al., 1999; Mar-

tinon et al., 2006).Alox5�/� and Alox15�/�mice were from Jackson laboratory.

All mice are in C57BL/6 background except that the cells used for the Lethal

Toxin stimulation were from BALB/c mice. All animal experiments were

approved by a local ethics committee (The Ethics Committee of University of

Science and Technology of China; Service Veterinaire Cantonal, Lausanne,

Switzerland).

Reagents

Nigericin, MSU, ATP, PMA, GW9508, poly (dA:dT), celecoxib, insulin, and

glucose were purchased from Sigma. R837 and ultrapure LPS were obtained

from Invivogen. Docosahexaenoic acid, eicosapentaenoic acid, a-Linolenic

acid, osbond acid, dihomo-gamma-linolenic acid, adrenic acid, oleic acid,

protectin D1, resolvin D1, and aspirin-triggered Resolvin D1 were from

Cayman chemical. Lethal Toxin, consisting of protective antigen and lethal

factor, was from List Biological Laboratories. Salmonella is a gift from R.V.

Bruggen. Imject-Alum was from Pierce Biochemicals. Anti-Flag (M2) antibody

and anti-VSV antibody were from Sigma. Anti-human cleaved IL-1b (D116),

anti-human caspase-1, anti-AKT, and anti-pAKT were purchased from Cell

Signaling. Anti-human Pro-IL-1b was from Proteintech. The antibody against

mouse IL-1b was from R&D. Anti-mouse caspase-1 (p20) and anti-NLRP3

were from Adipogen. Anti-b-actin was from Abmart. Anti-ARRB2, anti-GPR40,


Immunity


and anti-GPR120 were from Abcam. All tissue culture reagents were bought

from Invitrogen.

Generation of THP-1 Cells Expressing shRNA

The shRNA targeting the mRNA encoding ARRB2, GPR40, and GPR120 were

from Sigma. The protocol generating THP-1 cells stably expressing shRNA

was described previously (Papin et al., 2007).

Cell Preparation and Stimulation

Human THP-1 cells were grown in RPMI 1640 medium, supplemented with

10% FBS and 50 mM 2-mercaptoethanol. THP-1 cell were differentiated for

3 hr with 100 nM phorbol-12-myristate-13-acetate (PMA). Bone marrow

macrophages were derived from tibia and femoral bone marrow cells as

described elsewhere and cultured in DMEM complemented with 10% FBS,

1 mM sodium pyruvate, and 2 mM L-glutamine(Didierlaurent et al., 2006).

For inducing IL-1b, 106 macrophages were plated in 12-well plate overnight

and the medium was changed to opti-MEM in the following morning, and then

the cells were primedwith ultrapure LPS (100 ng/ml) for 3 hr. After that,u-3 FAs

were added into the culture for another 1 hr and then the cells were stimulated

with MSU (150 mg/ml), R837 (15 mg/ml), and Alum (300 mg/ml) for 6 hr, or u-3

FAs were added into the culture for another 3 hr and then the cells were

stimulated with ATP (1 mM) and nigericin (5 mM) for 45 min. For poly(dA:dT)

transfection, poly(dA:dT) (0.5 mg/ml) was transfected by using Lipofectamine

(1 ml/ml) as the manufacturer’s protocol (Invitrogen). Cell extracts and precip-

itated supernatants were analyzed by immunoblotting.

ELISA

Supernatants from cell culture or tissue culture were assayed for mouse IL-1b

(R&D), human IL-1b (BD biosciences), mouse IL-18 (eBioscience), and mouse

MCP-1 (R&D) according to manufacturer’s instructions.

Tissue Culture

Adipose or liver tissues were isolated and then washed in cold PBS supple-

mented with penicillin and streptomycin (Hyclone). These tissues were

cultured in 12-well plates in opti-MEM medium supplemented with penicillin

and streptomycin. After 24 hr, supernatants were collected and stored at

�20�C until analyzed.

Metabolic Studies

At 6 weeks of age, mice were placed on ND, HFD (D12331, Researchdiets), or

HFD withu-3 FAs supplementation for 10 weeks.u-3 FAs (100 mg/kg, twice a

week) were administrated by gavage for 6 weeks after mice were fed with HFD

for 4 weeks. For testing fasting blood glucose or insulin, the mice were fasted

for 8 hr and the samples were collected for glucose measurements by using a

Glucometer (Roche) or for insulin measurements by using ELISA. For GTT,

mice fasted for 8 hr and were injected intraperitoneally with glucose at a

dose of 1.5 mg/g body weight. Blood samples were obtained at different

time points for glucose measurements by using a Glucometer. For ITT, mice

were injected with 1.5 IU/kg body weight of recombinant human insulin

after 2 hr fasting, and blood glucose concentration was determined with the

Glucometer.

Salmonella Infection

Salmonellawas precultured on day 1. On day 2, BMDMs were infected for 1 hr

with the Salmonella culture (1:100) and then incubated for 3 hr in the presence

of gentamycin (Invitrogen).

Transfection and Immunoprecipitation

Constructs were transfected into HEK293T cells by using polyethylenimine.

After 18 hr, cells were collected and resuspended in lysis buffer (50 mM

Tris, pH 7.8, 50 mM NaCl, 0.1% [vol/vol] Nonidet-P40, 5 mM EDTA and 10%

[vol/vol] glycerol). Extracts were immunoprecipitated with anti-Flag antibody

and beads and then were assessed by immunoblotting. THP-1 cells (1 3

107) were differentiated with PMA and then were treated with DHA and

EPA for different time points. THP-1 cells were then resuspended in lysis

buffer and proteins were immunoprecipitated from extracts with anti-NLRP3

antibody.


Statistical Analyses

All values are expressed as the mean SEM of individual samples. Samples

were analyzed by using Student’s t test unless indicated in the figure legends.

SUPPLEMENTAL INFORMATION

Supplemental Information includes five figures and can be found with this

article online at http://dx.doi.org/10.1016/j.immuni.2013.05.015.

ACKNOWLEDGMENTS

We thank Gang Pei for providing the Arrb2�/� mice. This work was supported

by NSFC (81222040, 81273318, 31200659, 91029303), National Basic

Research Program of China (2013CB944904), One Hundred Person Project

(R.Z.), and CAS President’s fund (R.Z.) from The Chinese Academy of

Sciences, the Doctoral Fund of Ministry of Education of China

(20123402120001, 20123402110010), the Program for New Century Excellent

Talents in University, the Fundamental Research Funds for the Central Univer-

sities (R.Z. and W.J.), and the start-up funds (R.Z. and W.J.) from University of

Science and Technology of China. J.T. was supported by grants from the

Swiss National Science Foundation, the NCCR Molecular Oncology, the

Foundation Louis-Jeantet and the EU-FP7 program APO-SYS, and the Insti-

tute for Arthritis Research. G.G. was supported by the Swiss National Science

Foundation (PP00P3_139094).

Received: August 17, 2012

Accepted: February 22, 2013

Published: June 27, 2013

REFERENCES

Arita, M., Ohira, T., Sun, Y.P., Elangovan, S., Chiang, N., and Serhan, C.N.

(2007). Resolvin E1 selectively interacts with leukotriene B4 receptor BLT1

and ChemR23 to regulate inflammation. J. Immunol. 178, 3912–3917.

Bohn, L.M., Lefkowitz, R.J., Gainetdinov, R.R., Peppel, K., Caron, M.G., and

Lin, F.T. (1999). Enhanced morphine analgesia in mice lacking beta-arrestin

2. Science 286, 2495–2498.

Briscoe, C.P., Peat, A.J., McKeown, S.C., Corbett, D.F., Goetz, A.S., Littleton,

T.R., McCoy, D.C., Kenakin, T.P., Andrews, J.L., Ammala, C., et al. (2006).

Pharmacological regulation of insulin secretion in MIN6 cells through the fatty

acid receptor GPR40: identification of agonist and antagonist small molecules.

Br. J. Pharmacol. 148, 619–628.

Cintra, D.E., Ropelle, E.R., Moraes, J.C., Pauli, J.R., Morari, J., Souza, C.T.,

Grimaldi, R., Stahl, M., Carvalheira, J.B., Saad, M.J., and Velloso, L.A.

(2012). Unsaturated fatty acids revert diet-induced hypothalamic inflammation

in obesity. PLoS ONE 7, e30571.

Davis, B.K., Wen, H., and Ting, J.P. (2011). The inflammasome NLRs in

immunity, inflammation, and associated diseases. Annu. Rev. Immunol. 29,

707–735.

Didierlaurent, A., Brissoni, B., Velin, D., Aebi, N., Tardivel, A., Kaslin, E., Sirard,

J.C., Angelov, G., Tschopp, J., andBurns, K. (2006). Tollip regulates proinflam-

matory responses to interleukin-1 and lipopolysaccharide. Mol. Cell. Biol. 26,

735–742.

Dominguez, H., Storgaard, H., Rask-Madsen, C., Steffen Hermann, T.,

Ihlemann, N., Baunbjerg Nielsen, D., Spohr, C., Kober, L., Vaag, A., and

Torp-Pedersen, C. (2005). Metabolic and vascular effects of tumor necrosis

factor-alpha blockade with etanercept in obese patients with type 2 diabetes.

J. Vasc. Res. 42, 517–525.

Donath, M.Y., and Shoelson, S.E. (2011). Type 2 diabetes as an inflammatory

disease. Nat. Rev. Immunol. 11, 98–107.

Dyerberg, J., and Bang, H.O. (1979). Haemostatic function and platelet poly-

unsaturated fatty acids in Eskimos. Lancet 2, 433–435.

Endres, S., Ghorbani, R., Kelley, V.E., Georgilis, K., Lonnemann, G., van der

Meer, J.W., Cannon, J.G., Rogers, T.S., Klempner, M.S., Weber, P.C., et al.

(1989). The effect of dietary supplementation with n-3 polyunsaturated fatty


Immunity


acids on the synthesis of interleukin-1 and tumor necrosis factor by mononu-

clear cells. N. Engl. J. Med. 320, 265–271.

Fritsche, K. (2006). Fatty acids as modulators of the immune response. Annu.

Rev. Nutr. 26, 45–73.

Gross, O., Thomas, C.J., Guarda, G., and Tschopp, J. (2011). The inflamma-

some: an integrated view. Immunol. Rev. 243, 136–151.

Guarda, G., Braun, M., Staehli, F., Tardivel, A., Mattmann, C., Forster, I., Farlik,

M., Decker, T., Du Pasquier, R.A., Romero, P., and Tschopp, J. (2011). Type I

interferon inhibits interleukin-1 production and inflammasome activation.

Immunity 34, 213–223.

Harding, H.P., and Ron, D. (2002). Endoplasmic reticulum stress and the

development of diabetes: a review. Diabetes 51(Suppl 3 ), S455–S461.

Herder, C., Brunner, E.J., Rathmann, W., Strassburger, K., Tabak, A.G.,

Schloot, N.C., and Witte, D.R. (2009). Elevated levels of the anti-inflammatory

interleukin-1 receptor antagonist precede the onset of type 2 diabetes: the

Whitehall II study. Diabetes Care 32, 421–423.

Hirasawa, A., Tsumaya, K., Awaji, T., Katsuma, S., Adachi, T., Yamada, M.,

Sugimoto, Y., Miyazaki, S., and Tsujimoto, G. (2005). Free fatty acids regulate

gut incretin glucagon-like peptide-1 secretion through GPR120. Nat. Med. 11,

90–94.

Hotamisligil, G.S., and Erbay, E. (2008). Nutrient sensing and inflammation in

metabolic diseases. Nat. Rev. Immunol. 8, 923–934.

Hudert, C.A., Weylandt, K.H., Lu, Y., Wang, J., Hong, S., Dignass, A., Serhan,

C.N., and Kang, J.X. (2006). Transgenic mice rich in endogenous omega-3

fatty acids are protected from colitis. Proc. Natl. Acad. Sci. USA 103,

11276–11281.

Ichimura, A., Hirasawa, A., Poulain-Godefroy, O., Bonnefond, A., Hara, T.,

Yengo, L., Kimura, I., Leloire, A., Liu, N., Iida, K., et al. (2012). Dysfunction of

lipid sensor GPR120 leads to obesity in both mouse and human. Nature

483, 350–354.

Itoh, Y., Kawamata, Y., Harada, M., Kobayashi, M., Fujii, R., Fukusumi, S., Ogi,

K., Hosoya, M., Tanaka, Y., Uejima, H., et al. (2003). Free fatty acids regulate

insulin secretion from pancreatic beta cells through GPR40. Nature 422,

173–176.

Kang, J.X., Wang, J., Wu, L., and Kang, Z.B. (2004). Transgenic mice: fat-1

mice convert n-6 to n-3 fatty acids. Nature 427, 504.

Liou, A.P., Lu, X., Sei, Y., Zhao, X., Pechhold, S., Carrero, R.J., Raybould, H.E.,

and Wank, S. (2011). The G-protein-coupled receptor GPR40 directly

mediates long-chain fatty acid-induced secretion of cholecystokinin.

Gastroenterology 140, 903–912.

Lo, J., Bernstein, L.E., Canavan, B., Torriani, M., Jackson, M.B., Ahima, R.S.,

and Grinspoon, S.K. (2007). Effects of TNF-alpha neutralization on adipocyto-

kines and skeletal muscle adiposity in the metabolic syndrome. Am. J. Physiol.

Endocrinol. Metab. 293, E102–E109.

Mao, K., Chen, S., Chen, M., Ma, Y., Wang, Y., Huang, B., He, Z., Zeng, Y.,

Hu, Y., Sun, S., et al. (2013). Nitric oxide suppresses NLRP3 inflammasome

activation and protects against LPS-induced septic shock. Cell Res. 23,

201–212.

Martinon, F., Petrilli, V., Mayor, A., Tardivel, A., and Tschopp, J. (2006). Gout-

associated uric acid crystals activate the NALP3 inflammasome. Nature 440,

237–241.

Martinon, F., Mayor, A., and Tschopp, J. (2009). The inflammasomes: guard-

ians of the body. Annu. Rev. Immunol. 27, 229–265.

Masters, S.L., Dunne, A., Subramanian, S.L., Hull, R.L., Tannahill, G.M., Sharp,

F.A., Becker, C., Franchi, L., Yoshihara, E., Chen, Z., et al. (2010). Activation of

the NLRP3 inflammasome by islet amyloid polypeptide provides a mechanism

for enhanced IL-1b in type 2 diabetes. Nat. Immunol. 11, 897–904.

Mishra, B.B., Rathinam, V.A., Martens, G.W., Martinot, A.J., Kornfeld, H.,

Fitzgerald, K.A., and Sassetti, C.M. (2013). Nitric oxide controls the immuno-

pathology of tuberculosis by inhibiting NLRP3 inflammasome-dependent

processing of IL-1b. Nat. Immunol. 14, 52–60.

Moraes, L.A., Piqueras, L., and Bishop-Bailey, D. (2006). Peroxisome

proliferator-activated receptors and inflammation. Pharmacol. Ther. 110,

371–385.

Ofei, F., Hurel, S., Newkirk, J., Sopwith, M., and Taylor, R. (1996). Effects of an

engineered human anti-TNF-alpha antibody (CDP571) on insulin sensitivity

and glycemic control in patients with NIDDM. Diabetes 45, 881–885.

Oh, D.Y., Talukdar, S., Bae, E.J., Imamura, T., Morinaga, H., Fan, W., Li, P., Lu,

W.J., Watkins, S.M., and Olefsky, J.M. (2010). GPR120 is an omega-3 fatty

acid receptor mediating potent anti-inflammatory and insulin-sensitizing

effects. Cell 142, 687–698.

Papin, S., Cuenin, S., Agostini, L., Martinon, F., Werner, S., Beer, H.D.,

Grutter, C., Grutter, M., and Tschopp, J. (2007). The SPRY domain of

Pyrin, mutated in familial Mediterranean fever patients, interacts with inflam-

masome components and inhibits proIL-1beta processing. Cell Death Differ.

14, 1457–1466.

Prentki, M., and Nolan, C.J. (2006). Islet beta cell failure in type 2 diabetes.

J. Clin. Invest. 116, 1802–1812.

Robertson, R.P., Harmon, J., Tran, P.O., and Poitout, V. (2004). Beta-cell

glucose toxicity, lipotoxicity, and chronic oxidative stress in type 2 diabetes.

Diabetes 53(Suppl 1 ), S119–S124.

Robinson, D.R., Urakaze, M., Huang, R., Taki, H., Sugiyama, E., Knoell, C.T.,

Xu, L., Yeh, E.T., and Auron, P.E. (1996). Dietary marine lipids suppress contin-

uous expression of interleukin-1 beta gene transcription. Lipids 31(Suppl ),

S23–S31.

Rosenvinge, A., Krogh-Madsen, R., Baslund, B., and Pedersen, B.K. (2007).

Insulin resistance in patients with rheumatoid arthritis: effect of anti-

TNFalpha therapy. Scand. J. Rheumatol. 36, 91–96.

Schroder, K., Zhou, R., and Tschopp, J. (2010). The NLRP3 inflammasome: a

sensor for metabolic danger? Science 327, 296–300.

Serhan, C.N., Clish, C.B., Brannon, J., Colgan, S.P., Chiang, N., and Gronert,

K. (2000). Novel functional sets of lipid-derived mediators with antiinflamma-

tory actions generated from omega-3 fatty acids via cyclooxygenase

2-nonsteroidal antiinflammatory drugs and transcellular processing. J. Exp.

Med. 192, 1197–1204.

Serhan, C.N., Hong, S., Gronert, K., Colgan, S.P., Devchand, P.R., Mirick, G.,

and Moussignac, R.L. (2002). Resolvins: a family of bioactive products of

omega-3 fatty acid transformation circuits initiated by aspirin treatment that

counter proinflammation signals. J. Exp. Med. 196, 1025–1037.

Spite, M., Norling, L.V., Summers, L., Yang, R., Cooper, D., Petasis, N.A.,

Flower, R.J., Perretti, M., and Serhan, C.N. (2009). Resolvin D2 is a potent

regulator of leukocytes and controls microbial sepsis. Nature 461, 1287–

1291.

Spranger, J., Kroke, A., Mohlig, M., Hoffmann, K., Bergmann, M.M., Ristow,

M., Boeing, H., and Pfeiffer, A.F. (2003). Inflammatory cytokines and the risk

to develop type 2 diabetes: results of the prospective population-based

European Prospective Investigation into Cancer and Nutrition (EPIC)-

Potsdam Study. Diabetes 52, 812–817.

Strowig, T., Henao-Mejia, J., Elinav, E., and Flavell, R. (2012). Inflammasomes

in health and disease. Nature 481, 278–286.

Vandanmagsar, B., Youm, Y.H., Ravussin, A., Galgani, J.E., Stadler, K.,

Mynatt, R.L., Ravussin, E., Stephens, J.M., and Dixit, V.D. (2011). The

NLRP3 inflammasome instigates obesity-induced inflammation and insulin

resistance. Nat. Med. 17, 179–188.

Weir, G.C., and Bonner-Weir, S. (2004). Five stages of evolving beta-

cell dysfunction during progression to diabetes. Diabetes 53(Suppl 3 ),

S16–S21.

Wen, H., Gris, D., Lei, Y., Jha, S., Zhang, L., Huang, M.T., Brickey, W.J., and

Ting, J.P. (2011). Fatty acid-induced NLRP3-ASC inflammasome activation

interferes with insulin signaling. Nat. Immunol. 12, 408–415.

Zhang, M.J., and Spite, M. (2012). Resolvins: Anti-Inflammatory and

Proresolving Mediators Derived from Omega-3 Polyunsaturated Fatty Acids.

Annual review of nutrition 32, 203–227.

Zhou, R., Tardivel, A., Thorens, B., Choi, I., and Tschopp, J. (2010).

Thioredoxin-interacting protein links oxidative stress to inflammasome activa-

tion. Nat. Immunol. 11, 136–140.

Zhou, R., Yazdi, A.S., Menu, P., and Tschopp, J. (2011). A role formitochondria

in NLRP3 inflammasome activation. Nature 469, 221–225.


omega-3 fatty acids prevent inflammation and metabolic ......septic shock (mao et al., 2013; mishra...

Documents