Omega-3 Fatty Acids Prevent Inflammation and Metabolic ......septic shock (Mao et al., 2013; Mishra...
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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.
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
Omega-3 Fatty Acids Inhibits NLRP3 Inflammasome
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
1156 Immunity 38, 1154–1163, June 27, 2013 ª2013 Elsevier Inc.
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.
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Omega-3 Fatty Acids Inhibits NLRP3 Inflammasome
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
Immunity 38, 1154–1163, June 27, 2013 ª2013 Elsevier Inc. 1157
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
Omega-3 Fatty Acids Inhibits NLRP3 Inflammasome
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
1158 Immunity 38, 1154–1163, June 27, 2013 ª2013 Elsevier Inc.
(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.
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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
Omega-3 Fatty Acids Inhibits NLRP3 Inflammasome
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
Immunity 38, 1154–1163, June 27, 2013 ª2013 Elsevier Inc. 1159
A WT B WT CWT
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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
Omega-3 Fatty Acids Inhibits NLRP3 Inflammasome
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
1160 Immunity 38, 1154–1163, June 27, 2013 ª2013 Elsevier Inc.
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
Omega-3 Fatty Acids Inhibits NLRP3 Inflammasome
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 38, 1154–1163, June 27, 2013 ª2013 Elsevier Inc. 1161
Immunity
Omega-3 Fatty Acids Inhibits NLRP3 Inflammasome
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.
1162 Immunity 38, 1154–1163, June 27, 2013 ª2013 Elsevier Inc.
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
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