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© 2015Nature America, Inc. All rights reserved.
letters
nature medicine ADVANCE ONLINE PUBLICATION 1
The ketone bodies b-hydroxybutyrate (BHB) and acetoacetate
(AcAc) support mammalian survival during states of energy
deficit by serving as alternative sources of ATP1. BHB levels are
elevated by starvation, caloric restriction, high-intensity exercise,
or the low-carbohydrate ketogenic diet2. Prolonged fasting
reduces inflammation; however, the impact that ketones and
other alternative metabolic fuels produced during energy deficits
have on the innate immune response is unknown2–6. We report
that BHB, but neither AcAc nor the structurally related short-
chain fatty acids butyrate and acetate, suppresses activation of
the NLRP3 inflammasome in response to urate crystals, ATP and
lipotoxic fatty acids. BHB did not inhibit caspase-1 activation
in response to pathogens that activate the NLR family, CARD
domain containing 4 (NLRC4) or absent in melanoma 2 (AIM2)
inflammasome and did not affect non-canonical caspase-11,
inflammasome activation. Mechanistically, BHB inhibits the
NLRP3 inflammasome by preventing K+ efflux and reducing
ASC oligomerization and speck formation. The inhibitory effects
of BHB on NLRP3 are not dependent on chirality or starvation-
regulated mechanisms like AMP-activated protein kinase
(AMPK), reactive oxygen species (ROS), autophagy or glycolytic
inhibition. BHB blocks the NLRP3 inflammasome without
undergoing oxidation in the TCA cycle, and independently of
uncoupling protein-2 (UCP2), sirtuin-2 (SIRT2), the
G protein–coupled receptor GPR109A or hydrocaboxylic acid
receptor 2 (HCAR2). BHB reduces NLRP3 inflammasome–
mediated interleukin (IL)-1b and IL-18 production in human
monocytes. In vivo, BHB or a ketogenic diet attenuates caspase-1
activation and IL-1b secretion in mouse models of NLRP3-
mediated diseases such as Muckle–Wells syndrome, familial
cold autoinflammatory syndrome and urate crystal–induced
peritonitis. Our findings suggest that the anti-inflammatory
effects of caloric restriction or ketogenic diets may be linked to
BHB-mediated inhibition of the NLRP3 inflammasome.
The NLRP3 inflammasome controls the activation of caspase-1 and
the release of the pro-inflammatory cytokines IL-1β and IL-18 in
macrophages7–11. It is an important innate immune sensor that is
activated in response to structurally diverse damage-associated
molecular patterns (DAMPs) such as toxins8, ATP8, excess glucose7,
ceramides12, amyloids13,14, urate15 and cholesterol crystals16. Ablation
of NLRP3 attenuates type 2 diabetes12,14,17, atherosclerosis16, multi-
ple sclerosis18, Alzheimer disease14, age-related functional decline19,
bone loss19 and gout15. Thus, identification of endogenous mecha-
nisms that control NLRP3 inflammasome deactivation may provide
insights into the control of several chronic diseases. Although it is
known that immune-metabolic interactions dampen pro-inflamma-
tory responses via glycolysis inhibition6, it is not known whether the
alternative metabolic fuels that are produced during energy deficits,
such as ketones, impact the innate immune response.
The ketone bodies β-hydroxybutyrate (BHB) and acetoacetate
(AcAc) are produced in the liver of mammals and serve as alternative
energy sources for the brain, heart, and skeletal muscles during nutrient
deprivation and adherence to low carbohydrate diets1,2. With pro-
longed fasting, circulating concentrations of BHB can increase to as
much as 6–8 mM as liver glycogen stores get used1,2. These nutritional
states are associated with altered immune cell function, but it is
unknown whether ketone bodies serve as immune effectors.
To test whether BHB affects inflammasome activation, we treated
lipopolysaccharide (LPS)-primed mouse bone marrow–derived
macrophages (BMDMs) with the NLRP3 activator ATP along with
BHB for 60 min and measured caspase-1 activation using a western
blot that detects the enzymatically active p20 subunit of caspase-1.
BHB dose-dependently inhibited both the ATP-induced cleavage
of caspase-1 into p20 and the processing of the biologically active
p17 form of IL-1β. This inhibition occurred at BHB concentrations
similar to the BHB elevations induced by strenuous exercise or 2 d of
fasting1,2 (Fig. 1a and Supplementary Fig. 1a). AcAc and the micro-
biota-derived short-chain fatty acids butyrate and acetate, which
The ketone metabolite β-hydroxybutyrate blocks
NLRP3 inflammasome–mediated inflammatory disease
Yun-Hee Youm1,11, Kim Y Nguyen1,11, Ryan W Grant2, Emily L Goldberg1, Monica Bodogai3, Dongin Kim4,
Dominic D’Agostino5, Noah Planavsky6, Christopher Lupfer7, Thirumala D Kanneganti7, Seokwon Kang8,
Tamas L Horvath1, Tarek M Fahmy4, Peter A Crawford9, Arya Biragyn3, Emad Alnemri8 & Vishwa Deep Dixit1,10
1Section of Comparative Medicine and Program on Integrative Cell Signaling and Neurobiology of Metabolism, Yale School of Medicine, New Haven, Connecticut,
USA. 2Department of Nutrition Sciences, Purdue University, West Lafayette, Indiana. 3Laboratory of Molecular Biology and Immunology, National Institute on
Aging, National Institutes of Health (NIH), Baltimore, Maryland, USA. 4Department of Biomedical Engineering, Yale University, New Haven, Connecticut, USA.
5Department of Molecular Pharmacology and Physiology, University of South Florida, Tampa, Florida, USA. 6Department of Geology and Geophysics, Yale University,
New Haven, Connecticut, USA. 7Department of Immunology, St. Jude Children’s Hospital, Memphis, Tennessee, USA. 8Department of Biochemistry and Molecular
Biology, Thomas Jefferson University, Philadelphia, Pennsylvania, USA. 9Diabetes and Obesity Research Center, Sanford-Burnham Medical Research Institute,
Orlando, Florida, USA. 10Department of Immunobiology, Yale School of Medicine, New Haven, Connecticut, USA. 11These authors contributed equally to this work.
Correspondence should be addressed to V.D.D. (vishwa.dixit@yale.edu).
Received 27 October 2014; accepted 16 January 2015; published online 16 February 2015; doi:10.1038/nm.3804
© 2015Nature America, Inc. All rights reserved.
letters
2 ADVANCE ONLINE PUBLICATION nature medicine
are structurally related to BHB, did not affect ATP-induced NLRP3
activation (Fig. 1b). We sought to determine whether BHB specifi-
cally targets ATP-induced inflammasome activation or common
signaling mechanisms in response to structurally diverse NLRP3 acti-
vators. BHB, but not butyrate, inhibited monosodium urate (MSU)
crystal– and particulate matter–induced caspase-1 activation (Fig. 1c
and Supplementary Fig. 1b). Furthermore, BHB blocked inflam-
masome activation by five additional NLRP3 activators: nigericin;
silica particles (Supplementary Fig. 1b); and the lipotoxic fatty acids
palmitate, ceramide, and sphingosine (Fig. 1d). BHB also inhibited
f
IL-1β p17
BHB 10 mM
BHB 5 mM
BHB 1 mM
BHB 0 mM
Uninfected
F. tularensis
a
IL-1β p17
LPS
ATP
BHB
Procasp1
p20
49
38
25
17
49
38
25
17
Pro-IL-1β
0
0
– – +
+ + + + + + + +
+ + + + + +
(mM)
–
0 0 1 2 3 4 5 10
1
2
3
Fold change
(casp1 p20)
4
5*
Supernatants (kDa)
b
p20
LPS
ATP
BHB
AcAc
Butyrate
Acetate
Supernatants (kDa)
49
38
25
17
Procasp1
ButyrateAcAcBHB
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
– –
–
–
+
+
+
+
+
++++++
++++
0
20
40
60
Fold change
(casp1 p20)
*
OH O
HO
CH
3
OO
HO
CH
3
O
H
3
C
ONa
d
+ +
+
+ +
+ +
+ +
+
–
– – –
– –
– – –
–LPS
ATP
BHB
Nigericin
0
20
Fold change
(casp1 p20)
40
*
*
Procasp1
49
38
25
17
Supernatants (kDa)
p20
Supernatants (kDa)
+
+
+ +
+
+ +++
+
– –
– ––
–
– – –
–LPS
ATP
BHB
Palmitate
0
20
40
60
Fold change
(casp1 p20)
*
*
p20
49
38
25
17
Procasp1
Supernatants (kDa)
+ +
+
+
+
1 5 10
+
+
+
+
–
– –
– –
–
0
10
20
30
Fold change
(casp1 p20)
*
49
38
25
17 p20
Procasp1
BHB (mM)
Ceramide C6
LPS
p20
S. typhimurium
Procasp1
BHB 10 mM
BHB 5 mM
BHB 1 mM
BHB 0 mM
Uninfected
g
Supernatants (kDa)
c
–00001
1
5
5
10
10
0 0 0 0
+
+
–
–
+ ++ ++ ++
++ ++ +
(mM)
(mM)
+
p20
LPS
MSU
BHB
Butyrate
49
38
25
17
Procasp1
0
2
4
6
8
10
Fold change
(casp1 p20)
*
Supernatants (kDa)
0
10
20
Fold change
(casp1 p20)
*
+ +
+
+
+
1 5 10
+
+
+
+
–
– –
– –
–
BHB (mM)
Sphingosine
LPS
49
38
25
17 p20
Procasp1
0
100
200
300
400
*
Pam3 + ATP
Pam3
0 0 1 5 10
Fold change
(IL-1β p17)
Supernatants (kDa)
e
Lipid A + ATP
0
100
200
300
400
*
Lipid A
BHB(mM) 0 0 1 5 10
49
38
25
17
Fold change
(IL-1β p17)
LTA
IL-1β p17
0
20
40
60
80
*
LTA + ATP
Fold change
(IL-1β p17)
0 0 5 10
Figure 1 BHB specifically inhibits the NLRP3 inflammasome. (a) Representative western blot analysis of caspase-1 (casp1; active subunit p20) and
IL-1β (active p17) in the supernatant of BMDMs primed with LPS for 4 h and stimulated with ATP for 1 h in the presence of various concentrations of
d-BHB. Procasp1, procaspase-1, biologically inactive; Pro-IL-1β, non-secreted biologically inactive form of IL-1β. (b) Western blot analysis of caspase-1
activation in BMDMs stimulated with LPS and ATP and treated with BHB (10 mM), butyrate (10 mM), AcAc (10 mM) or acetate (10 mM). Western blot
analysis of caspase-1 activation in LPS-primed BMDMs stimulated with (c) MSU and treated with butyrate or d-BHB, or (d) nigericin (10 µM) for 1 h,
palmitate (200 µM) for 24 h, C6 ceramide for 6 h (80 µg/ml), and sphingosine (50 µM) for 1 h and treated with BHB. (e) Western blot analysis of IL-1β
activation in BMDMs primed with the TLR ligands lipid A, Pam3-CSK (Pam3) or LTA for 4 h and stimulated with ATP and increasing doses of d-BHB for
1 h. Active IL-1β (p17) was analyzed in supernatants by western blotting. IL-1β (f) or caspase-1 activation (g) in BMDMs infected with (f) F. tularensis
or (g) S. typhimurium and treated with different doses of BHB. Data are expressed as mean ± sem (*P < 0.05) from cells derived from n = 12 (a–d);
n = 6 (e); or n = 3 (f,g) mice with each independent experiment each carried out in triplicate (a–d,e) or in duplicate (f,g). All bar graphs in a–e represent
quantitation of p20 caspase-1 band intensity as fold change by normalizing to inactive p48 procaspase-1, or p17 IL-1β band intensity as fold change
by normalizing to inactive p37 pro-IL-1β. The differences between means and the effects of treatments were determined by one-way analysis of variance
(ANOVA) using Tukey’s test.
© 2015Nature America, Inc. All rights reserved.
letters
nature medicine ADVANCE ONLINE PUBLICATION 3
the processing of IL-1β in response to the Toll-like receptor 4 (TLR4)
pathogen–associated molecular pattern (PAMP) agonist lipid A,
the TLR1/2 ligand Pam3-CSK4 and the TLR2 agonist lipoteichoic
acid (LTA) (Fig. 1e).
We next investigated the specificity of BHB to NLRP3 as com-
pared to other inflammasomes. The BMDMs were infected with
either Francisella tularensis to activate the AIM2 inflammasome or
Salmonella typhimurium to activate the NLRC4 inflammasome. BHB
did not inhibit either AIM2 inflammasome–induced IL-1β activa-
tion (Fig. 1f) or NLRC4-mediated caspase-1 cleavage (Fig. 1g).
Given that the inflammasome can also be activated by LPS through
caspase-11 activation independently of TLR4 (refs. 20,21), we also
evaluated the non-canonical inflammasome pathway. Our data
indicate that neither butyrate nor BHB blocks caspase-11 activa-
tion (Supplementary Fig. 1c). These results suggest that BHB acts
on a central, common signaling pathway specific to the NLRP3
inflammasome in response to PAMPs and a wide array of
pro-inflammatory DAMPs.
Prolonged fasting and subsequent increases in circulating BHB are
linked to a reduction in oxidative stress22, increased AMPK activity23
and autophagy24. All of these mechanisms have also been implicated
in regulating the NLRP3 inflammasome8. Consistent with recent
data25, ROS damage via rotenone or hydrogen peroxide (Fig. 2a;
Supplementary Fig. 2a,b) was not sufficient to induce caspase-
1 cleavage and did not abrogate the suppressive effects of BHB on
ATP-induced NLRP3 inflammasome activation. Caspase-1 activa-
tion was induced by LPS priming alone in macrophages deficient in
the autophagy regulator Atg5 (Fig. 2b and Supplementary Fig. 2a).
However, absence of Atg5 did not alter the inhibitory effects of BHB on
the inflammasome (Fig. 2b and Supplementar y Fig. 2a). Consistent
with these findings, the autophagy inhibitor 3-methyladenine (3-MA)
and the proteasome blocker epoxomicin did not abrogate BHB’s
suppressive effects on ATP-induced NLRP3 inflammasome activation
(Fig. 2c and Supplementary Fig. 2a). The activation of AMPK using
AICAR (Fig. 2d) and the inhibition of glycolysis with 2-deoxyglucose
did not mimic the effects of BHB on inhibition of the NLRP3
h
LPS
ATP
(S)-BHB
– –++++++ ++++++
– –– + + + + + – + + + + +
– –– 0 1 5 10 20 – 0 1 5 10 20 (mM)
p20
ProCasp1
(S)-BHB
O
H3C OH
OH
WT Gpr109a–/–
WT Gpr109a–/–
LPS
ATP
BHB
Butyrate
AcAc
++++++++++++++
–++++++–++++++
– – 10 – – – 10 – – 10 – – – 10
– – – 10 – – – – – – 10 – – –
– – – – 5 10 10 – – – – 5 10 10
p20
ProCasp1
g
++++++
– + + – + +
– – + – – +
b
LPS
ATP
BHB
LysM-Cre Atg5fl/fl
Atg5fl/fl
ProCasp1
p20
LPS + ATP
BHB (mM)
Niacin
TSA
WT Gpr109a–/–
IL-1β p17
f–
–
– – – – – – –10 20 10 20
– – – – – – – –++
+++++ +++++–
– – – + – – – – – +
a
LPS
Rotenone
ATP
BHB – – –
– –
–
–
– – –
–
– –+
++++
+++
++ + + + +
+
p20
ProCasp1
49
38
25
17
Cell lysates (kDa)
c
LPS
ATP
BHB
Epoxomicin
3-MA – – – – – – – 10 (mM)
(uM)
(mM)
ProCasp1
p20
49
38
25
17
++++++++
– +
–
––––––1–
– 1 5 10 20 20 20
++++++
Supernatant (kDa)
e
0
20
40
60
80
100
120
140
160
180
200
Proliferation (% change)
LPS + ATP
LPS + ATP
LPS
Control
1 5 10 20 BHB (mM)
*
LPS
ATP
BHB
AICAR
p20
p20
IL-1β p17
Comp. C
ProCasp1
ProCasp1
–++++
– + + + +
– – + – –
– – – – +
– – – + +
49
38
25
17
49
38
25
17
Supernatants (kDa)Cell lysates (kDa)
d
Figure 2 BHB inhibits the NLRP3 inflammasome independently of Gpr109a and
starvation-regulated mechanisms. (a) Western blot analysis of caspase-1 activation
in LPS-primed BMDMs treated with combinations of rotenone (10 µM), ATP (5 µM),
and BHB (10 mM). (b) Western blot analysis of caspase-1 activation in BMDMs
derived from control Atg5fl/fl or LysM-Cre Atg5fl/fl mice primed with LPS and
stimulated with combinations of ATP and BHB (10 mM). (c) Immunoblot analysis
of caspase-1 activation in LPS-primed BMDMs stimulated in the presence of ATP
and different concentrations of BHB and pretreated with either 3-MA or epoxomicin
for 30 min. (d) Western blot analysis of caspase-1 and IL-1β activation in LPS-primed
BMDMs stimulated with ATP and BHB (10 mM) in the presence of an AMPK activator
(AICAR, 2 mM) or AMPK antagonist Compound C (Comp. C; 25 µM). Capase-1
activation was analyzed in both cell supernatants and BMDM cell lysates.
(e) Proliferation of BMDMs in response to increasing concentrations of BHB.
(f,g) Western blot analysis of (f) IL-1β and (g) caspase-1 activation in BMDMs from
control and Gpr109a-deficient mice activated with LPS and ATP and co-incubated with
BHB (10 or 20 mM) and (f) TSA (50 nM) and niacin (1 mM), or (g) butyrate (10 mM)
and AcAc (5 or 10 mM). (h) Western blot analysis of caspase-1 activation in BMDMs of
WT and Gpr109a−/− mice treated with LPS for 4 h and stimulated with ATP in the presence
of (S)-BHB for 1 h. Data are expressed as mean ± sem (*P < 0.05) from cells derived from
n = 6 (a); n = 4 (b,f–h); or n = 10 (c–e) mice with each independent experiment carried out in triplicate. Because of space limitations, the quantitation
of p20 caspase-1 and p17 IL-1β band intensity from each experiment is presented in Supplementary Figure 2a. The differences between means and the
effects of treatments were determined by one-way ANOVA using Tukey’s test.
© 2015Nature America, Inc. All rights reserved.
letters
4 ADVANCE ONLINE PUBLICATION nature medicine
inflammasome (Supplementary Figs. 2c and 3a). Furthermore, inhibi-
tion of AMPK via compound C did not abrogate BHB’s inhibitory effects
on NLRP3-mediated caspase-1 activation. (Fig. 2d and Supplementary
Figs. 2a,c and 3a). BHB also did not impair the viability of
BMDMs, and, at a concentration of 10 mM, it increased cellular
proliferation (Fig. 2e).
h
ASC dimer
BHB 20 mM
BHB 10 mM
BHB 5 mM
BHB 1 mM
LPS + ATP
UnRx
NP40 insoluble ASC
cell pellets
LPS + ATP
ASC monomer
0
2
4
6
Fold change
(Asc dimer)
*
e
0
Unstim
LPS + ATP
LPS + ATP +
BHB 10 mM
20
40
60
80
100
120
K
+
(% change)
*
f
0
20
40
60
80
100
120
LPS + ATP
*
LPS
LPS + ATP
BHB 1 mM
BHB 10 mM
BHB 20 mM
K
+
(% change)
0
20
40
60
80
100
120
*
g
LPS
LPS + MSU
LPS + MSU + BHB
K
+
(% change)
dUcp2–/–
WT
ProCasp1
p17
p20
a b c
i
% of cells containing specks
LPS + ATP (ASC–/–)
LPS + ATP LPS + ATP + BHB
15
10
5
0
LPS + ATP
LPS + ATP + BHB
Oxct
fl/fl
LPS + ATP
BHB –
– –+ + + + + + + + + +
– – –10 20 10 20 –
– –
– – – – – (mM)
AcAc – – – – – – – – – – 10 – – 10 (mM)
LysM-
Cre
Oxct
fl/fl
Oxct
fl/fl
LysM-
Cre
Oxct
fl/fl
ProCasp1 (p48)
p20
SCOT (50kDa)
Actin
LPS + ATP + BHB
+
AGK2
LPS + ATP
LPS + ATP
LPS + ATP + NAD
+
LPS + ATP
LPS + ATP + BHB
p20
ProCasp1
LPS + ATP
+
BHB
LPS + ATP LPS + ATP
LPS
LPS
BHB
(mM)
0 0 1 5 10 0 0 1 5 10
WT Sirt2–/–
ProCasp1
p20
ASC specks
LPS + ATP + BHB
LPS + ATP
LPS
UnRx
UnRx
LPS + ATP + BHB
LPS + ATP
LPS
P = 0.0205
Figure 3 BHB inhibits ASC oligomerization and speck formation without undergoing mitochondrial oxidation. (a) Western blot analysis of caspase-1
activation, SCOT, and actin in BMDMs from Oxctfl/fl and LysM-Cre Oxctfl/fl mice treated with LPS for 4 h and stimulated with ATP in the presence
of BHB and AcAc. p48 indicates the molecular weight of inactive procaspase-1. (b) Western blot analysis of caspase-1 activation in LPS-primed
BMDMs treated with ATP alone and in the presence of BHB (10 mM), Sirt2 antagonist AGK2 (10 µM) or NAD+ (10 µM). (c,d) Western blot analysis
of caspase-1 activation or IL-1β in BMDMs of WT (c,d), Sirt2−/− (c), or Ucp2−/− (d) mice treated with LPS for 4 h and stimulated with ATP alone or in
the presence of BHB (10 mM). UnRx, untreated. (e) Intracellular potassium levels in BMDMs unstimulated (unstim) or stimulated with LPS and ATP in
the presence or absence of BHB (10 mM), as measured by inductively coupled mass spectrometry (ICP-MS). (f,g) Intracellular potassium levels in LPS-
primed BMDMs treated with BHB and (f) ATP or (g) MSU for 1 h as assessed using an APG-1 dye that selectively binds potassium and has an excitation
emission spectrum of 488–540 nm. (h) Representative immunoblot analysis of disuccinimidyl suberate (DSS)–cross-linked ASC in the Nonidet P-
40–insoluble pellet of BMDMs that were primed with LPS (4 h) and stimulated with ATP and BHB for 1 h. The bar graph represents the quantification
of band intensity of the ASC dimer compared to LPS + ATP stimulation. (i) Representative immunofluorescence images of ASC speck formation in
LPS-primed BMDMs stimulated with ATP in the presence or absence of BHB (10 mM). Scale bars, 200 µm in panel and 20 µm in inset. Data are
expressed as mean ± sem (*P < 0.05) from cells derived from n = 5 (a); n = 6 (b,d); n = 8 (e–g); or n = 4 (h) mice with each independent experiment
carried out in triplicate. The differences between means and the effects of treatments were determined by one-way ANOVA using Tukey’s test. (i) Data
are shown as mean ± sem and are representative of two independent experiments. Statistical differences were calculated by Student’s t-test.
© 2015Nature America, Inc. All rights reserved.
letters
nature medicine ADVANCE ONLINE PUBLICATION 5
It has been suggested that BHB can act as a signaling molecule by
binding the G protein–coupled receptor GPR109a (ref. 26) or by serv-
ing as a histone deacetylase (HDAC) inhibitor22. Inhibition of HDACs
using trichostatin A (TSA) did not affect inflammasome activation in
LPS-primed and NLRP3 agonist–treated macrophages (Fig. 2f and
Supplementary Fig. 2a) despite induction of H3 acetylation by BHB
in macrophages (Supplementary Fig. 3b). To understand the role of
GPR109a in BHB’s effects on macrophages, we used niacin, a GPR109a
ligand that has been reported to inhibit colonic inflammation27. We
found that, unlike BHB, niacin did not block NLRP3 inflammasome
activation (Fig. 2f and Supplementary Fig. 2a). The effects of BHB on
the inflammasome were not altered in GPR109A-deficient BMDMs
(Fig. 2f,g; Supplementary Fig. 2a), and neither butyrate nor AcAc
altered NLRP3 inflammasome activity in either GPR109A-sufficient
or -deficient cells (Fig. 2g and Supplementary Fig. 2a). BHB is a
chiral compound and its enantiomer, (S)-BHB, cannot enter the TCA
cycle but binds GPR109A with high affinity26. We found that (S)-BHB
exhibited effects on the inflammasome that were similar to -BHB as
it did not require GPR109A to block NLRP3 (Fig. 2h).
Compared to fatty acids, oxidation of BHB is energetically more
efficient as all reducing equivalents generated by ketone oxidation
are delivered through NADH to complex-I within the mitochondrial
electron transport chain2. Furthermore, ketone oxidation increases
the redox span between complex I and complex III by keeping mito-
chondrial ubiquinone oxidized28. We wanted to know whether oxi-
dation, entry into the TCA cycle, or reduced mitochondrial stress
mediate -BHB’s effects on the inflammasome. Macrophages express
ketogenic and ketolytic enzymes encoded by Acat1, Bdh1, Bdh2 and
Hmgcl Supplementary Fig. 4a–c). In terms of macrophage polariza-
tion, classically activated M1 macrophages showed a reduction in
the expression of Acat1, Bdh1, Bdh2 and Hmgcl as compared to M2
macrophages, suggesting that ketones may affect macrophage polari-
zation (Supplementary Fig. 4b). In addition, LPS induced protein
expression of the ketolytic enzyme Scot (succinyl-CoA:3-oxo-acid
CoA-transferase, encoded by Oxct) and the ketogenic enzyme Hmgcl
in BMDMs (Supplementary Fig. 4c). However, the TCA cycle entry–
inhibitor aminoxyacetate did not inhibit the effects of BHB on the
inflammasome (Supplementary Fig. 4d), and the enantiomer (S)-
BHB (which does not enter the TCA cycle) efficiently blocked NLRP3
inflammasome activation (Supplementary Fig. 4e). To directly assess
the role of BHB oxidation, we specifically deleted the ketolytic mito-
chondrial enzyme Scot27 in macrophages (Fig. 3a; Supplementary
Fig. 4f). These experiments confirmed that the TCA intermediates
generated through ketone body oxidation in macrophage mitochon-
dria did not mediate the suppressive effects of BHB on the NLRP3
inflammasome (Fig. 3a; Supplementary Fig. 4f).
b
p20
ProCasp1
49
38
25
17
LPS + ATP
BHB–nLG – – 0.1 1 5 10
– + + + + +
(mM)
Supernatant (kDa)
a
0
1,000
2,000
3,000
4,000
BHB 20 mM
BHB 10 mM
BHB 1 mM
LPS
IL-1β (pg/ml)
LPS
*
*
IL-18 (pg/ml)
0
100
200
300
400
500
600
BHB 20 mM
BHB 10 mM
BHB 1 mM
LPS
LPS
*d e
250
0
50
100
150
200
IL-1β (pg/ml)
* *
nLG
BHB–nLG
nLG + MSU
MSU + BHB–nLG
0
50
100
150
200
250
300
350
400
450
500
nLG
BHB–nLG
nLG + MSU
MSU + BHB–nLG
IL-1β (pg/ml)
* *
c
Gr1
CD45
72.8% 26.1%
0.106%
10
4
10
3
10
2
10
1
10
0
10
0
10
1
10
2
10
3
10
4
21.4% 75.9%
2.59% 0.124%
10
4
10
3
10
2
10
1
10
0
10
0
10
1
10
2
10
3
10
4
37.3% 28.3%
34.1% 0.247%
10
4
10
3
10
2
10
1
10
0
10
0
10
1
10
2
10
3
10
4
f
1 5 10 (nM)
BHB–nLG
LPS
p17
p20
Actin
(MWS)
NLRP3 (A350V) Cre
+
BHB 10
UnRx
g
0.1 1 510
(FCAS)
NLRP3 (L351P) Cre
+
p20
ProCasp1
(mM)
BHB–nLG
LPS
UnRx
(FCAS)
NLRP3 (L351P) Cre
+
IL-1β
p17
BHB–nLG
LPS
0.1 1 510 (mM)
UnRx
h
ASC
dimer
UnRx
BHB 1 mM
BHB 10 mM
BHB–nLG 0.1 mM
BHB–nLG 1 mM
BHB–nLG 5 mM
BHB–nLG 10 mM
ASC
monomer
LPS
40
30
(FCAS)
NLRP3 (L351P) Cre
+
i
Control
diet
1,3-butanediol
KD
Ly6C
+
Ly6G
+
(million)
0
1.0
2.0
3.0
4.0
*
Nanolipogel Nanolipogel + MS U
MSU + BHB
Nanolipogel
0.978%
Figure 4 BHB suppresses NLRP3-mediated inflammatory disease
in vivo and inflammasome activation in human monocytes. (a) Analysis of
IL-1β and IL-18 secretion in culture supernatants of human monocytes
stimulated with vehicle (not shown) or LPS (1 µg/mL) for 4 h in the
presence of increasing concentrations of BHB. n = 6 per treatment;
symbols (circles, triangles, etc.) are data points from individual
subjects. (b) BHB-complexed nanolipogels (nLGs) block NLRP3
inflammasome activation and caspase-1 cleavage (n = 3; repeated
twice). (c) Frequency of CD45+ and Gr1+ immune cells in the
peritoneum of mice treated with MSU (3 mg) and BHB–nLGs
(125 mg/kg of body weight), as assessed by FACS (n = 6 per group).
(d,e) IL-1β secretion from peritoneal cells cultured overnight (d) and
serum IL-1β levels in mice challenged with MSU and treated with BHB–nLGs (n = 6 per group) (e). (f,g) Western blot analysis of caspase-1 and IL-1β
activation in BMDM cells stimulated with LPS in the presence of BHB–nLGs from mice harboring the human (f) MWS NLRP3 (A350V) or (g) FCAS
NLRP3 (L351P) mutation (n = 6; repeated twice). (h) Representative immunoblot analysis of disuccinimidyl suberate (DSS)–cross-linked ASC in
the Nonidet P-40–insoluble pellet of BMDM from FCAS mouse models (n = 6) that were primed with LPS (4 h) and treated with increasing
concentrations of BHB–nLGs. (i) Neutrophil numbers in the peritoneum of FCAS mouse models fed a chow or ketone diester (KD; 1,3-butanediol)
diet for 1 week. (n = 6 per group). Data are expressed as mean ± sem (*P < 0.05) and statistical differences between means and the effects of
treatments were determined by one-way ANOVA using Tukey’s test (a,d,e).
© 2015Nature America, Inc. All rights reserved.
letters
6 ADVANCE ONLINE PUBLICATION nature medicine
The NAD-dependent deacetylase SIRT2 regulates acetylation of
α-tubulin, which controls microtubule-driven apposition of NLRP3
and ASC29. Inhibition of SIRT2 by the small molecule AGK2 activates
the NLRP3 inflammasome and supplementation with NAD+ lowers
IL-1β secretion from macrophages29. Inhibition of SIRT2 by AGK2
or ablation of either SIRT2 or uncoupling protein 2 (UCP2), a mito-
chondrial anion transporter did not abrogate the effects of BHB on
the inflammasome, and the addition of NAD+ did not block caspase-1
activation in response to LPS and ATP (Fig. 3b–d and Supplementary
Fig. 4f). These findings rule out a major role for mitochondrial ROS
in the effects of ketone bodies on the inflammasome.
BHB is a strongly anionic endogenous molecule2, and it exerts anti-
epileptic effects by reducing neuronal excitability via regulation of
intracellular potassium cations30. Consistent with recent studies that
demonstrate that K+ efflux triggers NLRP3 inflammasome activa-
tion8,25 , BHB prevented the decline of intracellular K+ in response
to incubation with the NLRP3 activators ATP, MSU and ceramide
(Fig. 3e–g and Supplementar y Fig. 5a). Furthermore, NLRP3-
dependent, ASC nucleation-induced polymerization or oligomeri-
zation are considered to be common mechanisms of NLRP3
inflammasome activation31,32. BHB prevents ATP-induced ASC
oligomerization (Fig. 3h) and speck formation (Fig. 3i). Our data
suggest that BHB blocks NLRP3 inflammasome activation both by
controlling an unknown upstream event that reduces K+ efflux from
macrophages and by inhibiting ASC polymerization, speck formation
and assembly of the inflammasome.
Next we investigated whether delivery of BHB can inhibit the
NLRP3 inflammasome in human monocytes and mouse models of
NLRP3-driven inflammation in vivo. BHB dose-dependently inhib-
ited IL-1β and IL-18 (Fig. 4a) secretion in LPS-stimulated human
monocytes without significantly affecting tumor necrosis factor
(TNF)-α production (Supplementary Fig. 5b). In vivo administra-
tion of BHB is insufficient to sustain high serum concentration owing
to rapid clearance1,2,30. To reduce clearance, BHB was complexed with
nanolipogels (nLGs) to improve its bioavailability33. BHB–nLGs
inhibited NLRP3 inflammasome activation in macrophages
(Fig. 4b). In mice, the Nlrp3 inflammasome was activated follow-
ing intraperitoneal (IP) injection of MSU crystals, resulting in an
influx of neutrophils into the peritoneum and increased secretion
of IL-1β by macrophages 4 h after injection34. Compared to mice
given nLGs alone, BHB–nLGs reduced neutrophil infiltration into the
peritoneum (Fig. 4c and Supplementary Fig. 5c–e) without directly
impairing neutrophil migration (Supplementar y Fig. 5f), suggest-
ing direct effects of BHB in vivo on NLRP3-driven neutrophil influx.
Peritoneal cells from MSU-injected mice treated with BHB–nLGs
produced less IL-1β (Fig. 4d) and the concentration of IL-1β in the
serum was reduced after BHB–nLG treatment (Fig. 4e).
Missense mutations in NLRP3 cause systemic inflammatory diseases
like Muckle–Wells syndrome (MWS) and familial cold autoinflamma-
tory syndrome (FCAS), which are characterized by overproduction of
IL-1β and IL-18 (ref. 35). We tested the efficiency of BHB in mice that
express knocked-in human NLRP3 with gain-of-function mutations
(A350V and L351P) that mimic MWS and FCAS, respectively, render-
ing the inflammasome constitutively active without the requirement
of NLRP3 ligands35. As described before35, in the FCAS mouse model,
tamoxifen-induced, Cre recombinase–mediated excision of a floxed
neomycin cassette induces the expression of a mutant form of NLRP3
(L351P) and subsequent inflammasome activation in macrophages
(Supplementary Fig. 6a).
Treatment of BMDMs from mouse models of MWS (NLRP3
(A350V)) (Fig. 4f and Supplementary Fig. 5g) and FCAS (NLRP3
(L351P)) (Fig. 4g,h; Supplementary Fig. 5g) with BHB–nLGs
dose-dependently inhibited constitutive NLRP3 inflammasome acti-
vation. When complexed with nLGs, -BHB inhibited inflammasome
activation in FCAS macrophages at a lower dose (Supplementary
Fig. 6b,c). BHB reduces ASC oligomerization in macrophages upon
Cre-mediated induction of NLRP (L351P) by tamoxifen treatment
in vitro (Fig. 4h). To model a ketogenic diet and elevate BHB levels
in vivo, the FCAS mice were fed 1,3-butanediol ketone diesters (KD)
for one week before the induction of a missense Nlrp3 mutation.
The KD increased the serum BHB levels in mice to fasting levels of
0.75–1 mM. As reported previously, mouse models of FCAS (NLRP3
(L351P), Cre+) develop severe neutrophila35 in the peritoneum 3 d
after the induction of mutant NLRP3. Compared to the chow-
fed FCAS model, the KD treatment protected these animals from
neutrophilia (Fig. 4i) and hyperglycemia (Supplementar y Fig. 6d),
without any effects on the infiltration of CD11b+F4/80+ perito-
neal macrophages (Supplementary Fig. 6e). Furthermore,the KD
diet did not affect the overall frequency of splenic T cells, macro-
phages or neutrophils (Supplementary Fig. 7a–d). Given that FCAS
is caspase-1 dependent and that IL-1β and IL-18 do not influence
all of the observed pathology35, ketogenic diets that elevate BHB
may improve the therapeutic outcome of patients by inhibiting
the inflammasome.
These findings suggest that the fasting- or exercise- induced
metabolite BHB inhibits the NLRP3 inflammasome in macrophages
independently of binding to surface GPR109A receptors or mito-
chondrial oxidation, which may avoid both competition for receptor
occupancy and requirement for ATP generation. Thus, in states of
extreme energy deficit such as starvation, metabolic signals such
as BHB can dampen innate immune responses, sparing ATP for
the functioning of ketone-dependent organs such as the brain and
heart (Supplementary Fig. 8a). These findings provide insight into
the immunological functions of metabolic signals such as BHB
and suggest that dietary or pharmacological approaches to elevate
BHB—without inducing the generalized starvation response—hold
promise in reducing the severity of NLRP3-mediated chronic
inflammatory diseases.
METHODS
Methods and any associated references are available in the online
version of the paper.
Note: Any Supplementary Information and Source Data files are available in the
online version of the paper.
ACKNOWLEDGMENTS
We thank M. Koch, Y. Kui, P. Chang and D. Albarado for technical assistance, and
V. M. Dixit (Genentech) and R. Medzhitov (Yale School of Medicine) for helpful
discussions and for providing knockout mice. Salmonella typhimurium and
Francisella tularensis (U112) were provided by D. Monack (Stanford University
School of Medicine) and J. Teale (University of Texas at San Antonio). M.B. and
A.B. were supported by the National Institute on Aging–Intramural Research
Program. D.D’A. was supported by the Office of Naval Research (ONR) Grant
N000141310062. V.D.D. was supported in part by the grants from National
Institutes of Health (AG043608, AG31797, DK090556 and AI105097).
AUTHOR CONTRIBUTIONS
Y. H.Y. and K.Y.N. designed and conducted the majority of in vitro and all
in vivo experiments, analyzed and interpreted the data, and participated in
writing the manuscript. R.W.G. participated in design and conduct of
inflammasome activation experiments. E.L.G. performed ASC speck and
© 2015Nature America, Inc. All rights reserved.
letters
nature medicine ADVANCE ONLINE PUBLICATION 7
neutrophil assays. M.B. and A.B. performed the human monocytes experiments.
D.K. and T.M.F. synthesized the BHB–nanolipogels and conducted control
experiments to determine the dose response. D.D’A. formulated the ketone diester
diet. N.P. conducted the ICP-MS experiments to determine K+ efflux. C.L. and
T.D.K. conducted the F. tularensis and S. typhimurium infection experiments.
T.L.H. designed the experiments and provided essential reagents for experiments
involving mitochondrial ROS and UCP2. P.A.C. generated the macrophage-
specific, Scot-deficient mice and contributed to experiment design. S.K. and E.A.
designed and conducted the ASC oligomerization experiments. V.D.D. conceived
and supervised the project, interpreted the data, and wrote the manuscript.
COMPETING FINANCIAL INTERESTS
The authors declare no competing financial interests.
Reprints and permissions information is available online at http://www.nature.com/
reprints/index.html.
1. Newman, J.C. & Verdin, E. Ketone bodies as signaling metabolites. Trends
Endocrinol. Metab. 25, 42–52 (2014).
2. Cotter, D.G., Schugar, R.C. & Crawford, P.A. Ketone body metabolism and
cardiovascular disease. Am. J. Physiol. Heart Circ. Physiol. 304, H1060–H1076
(2013).
3. Shido, O., Nagasaka, T. & Watanabe, T. Blunted febrile response to intravenous
endotoxin in starved rats. J. Appl. Physiol. 67, 963–969 (1989).
4. Johnson, J.B. et al. Alternate day calorie restriction improves clinical findings and
reduces markers of oxidative stress and inflammation in overweight adults with
moderate asthma. Free Radic. Biol. Med. 42, 665–674 (2007).
5. Mercken, E.M. et al. Calorie restriction in humans inhibits the PI3K/AKT
pathway and induces a younger transcription profile. Aging Cell 12, 645–651
(2013).
6. McGettrick, A.F. & O’Neill, L.A. How metabolism generates signals during innate
immunity and inflammation. J. Biol. Chem. 288, 22893–22898 (2013).
7. Martinon, F., Mayor, A. & Tschopp, J. The inflammasomes: guardians of the body.
Annu. Rev. Immunol. 27, 229–265 (2009).
8. Lamkanfi, M. & Dixit, V.M. Mechanisms and functions of inflammasomes. Cell 157,
1013–1022 (2014).
9. Wen, H., Miao, E.A. & Ting, J.P. Mechanisms of NOD-like receptor-associated
inflammasome activation. Immunity 39, 432–441 (2013).
10. Latz, E., Xiao, T.S. & Stutz, A. Activation and regulation of the inflammasomes.
Nat. Rev. Immunol. 13, 397–411 (2013).
11. Franchi, L., Eigenbrod, T., Muñoz-Planillo, R. & Nuñez, G. The inflammasome: a
caspase-1-activation platform that regulates immune responses and disease
pathogenesis. Nat. Immunol. 10, 241–247 (2009).
12. Vandanmagsar, B. et al. The NLRP3 inflammasome instigates obesity-induced
inflammation and insulin resistance. Nat. Med. 17, 179–188 (2011).
13. Masters, S.L. et al. Activation of the NLRP3 inflammasome by islet amyloid
polypeptide provides a mechanism for enhanced IL-1β in type 2 diabetes. Nat.
Immunol. 11, 897–904 (2010).
14. Heneka, M.T. et al. NLRP3 is activated in Alzheimer’s disease and contributes to
pathology in APP/PS1 mice. Nature 493, 674–678 (2013).
15. Martinon, F., Pétrilli, V., Mayor, A., Tardivel, A. & Tschopp, J. Gout-associated uric
acid crystals activate the NALP3 inflammasome. Nature 440, 237–241 (2006).
16. Duewell, P. et al. NLRP3 inflammasomes are required for atherogenesis and
activated by cholesterol crystals. Nature 464, 1357–1361 (2010).
17. Wen, H. et al. Fatty acid-induced NLRP3-ASC inflammasome activation interferes
with insulin signaling. Nat. Immunol. 12, 408–415 (2011).
18. Shaw, P.J. et al. Cutting edge: critical role for PYCARD/ASC in the development of
experimental autoimmune encephalomyelitis. J. Immunol. 184, 4610–4614
(2010).
19. Youm, Y.H. et al. Canonical Nlrp3 inflammasome links systemic low-grade
inflammation to functional decline in aging. Cell Metab. 18, 519–532 (2013).
20. Kayagaki, N. et al. Noncanonical inflammasome activation by intracellular LPS
independent of TLR4. Science 341, 1246–1249 (2013).
21. Hagar, J.A., Powell, D.A., Aachoui, Y., Ernst, R.K. & Miao, E.A. Cytoplasmic LPS
activates caspase-11: implications in TLR4-independent endotoxic shock. Science
341, 1250–1253 (2013).
22. Shimazu, T. et al. Suppression of oxidative stress by β-hydroxybutyrate, an
endogenous histone deacetylase inhibitor. Science 339, 211–214 (2013).
23. Laeger, T., Pöhland, R., Metges, C.C. & Kuhla, B. The ketone body β-hydroxybutyric
acid influences agouti-related peptide expression via AMP-activated protein kinase
in hypothalamic GT1–7 cells. J. Endocrinol. 213, 193–203 (2012).
24. Finn, P.F. & Dice, J.F. Ketone bodies stimulate chaperone-mediated autophagy.
J. Biol. Chem. 280, 25864–25870 (2005).
25. Muñoz-Planillo, R. et al. K+ efflux is the common trigger of NLRP3 inflammasome
activation by bacterial toxins and particulate matter. Immunity 38, 1142–1153
(2013).
26. Taggart, A.K. et al. (D)-beta-Hydroxybutyrate inhibits adipocyte lipolysis via the
nicotinic acid receptor PUMA-G. J. Biol. Chem. 280, 26649–26652 (2005).
27. Singh, N. et al. Activation of Gpr109a, receptor for niacin and the commensal
metabolite butyrate, suppresses colonic inflammation and carcinogenesis. Immunity
40, 128–139 (2014).
28. Cotter, D.G., Ercal, B., d’Avignon, D.A., Dietzen, D.J. & Crawford, P.A. Impact of
peripheral ketolytic deficiency on hepatic ketogenesis and gluconeogenesis during
the transition to birth. J. Biol. Chem. 288, 19739–19749 (2013).
29. Misawa, T. et al. Microtubule-driven spatial arrangement of mitochondria promotes
activation of the NLRP3 inflammasome. Nat. Immunol. 14, 454–460 (2013).
30. Lutas, A. & Yellen, G. The ketogenic diet: metabolic influences on brain excitability
and epilepsy. Trends Neurosci. 36, 32–40 (2013).
31. Lu, A. et al. Unified polymerization mechanism for the assembly of ASC-dependent
inflammasomes. Cell. 156, 1193–1206 (2014).
32. Yu, J.W. et al. Cryopyrin and pyrin activate caspase-1, but not NF-κB, via ASC
oligomerization. Cell Death Differ. 13, 236–249 (2006).
33. Demento, S.L., Siefert, A.L., Bandyopadhyay, A., Sharp, F.A. & Fahmy, T.M.
Pathogen-associated molecular patterns on biomaterials: a paradigm for engineering
new vaccines. Trends Biotechnol. 29, 294–306 (2011).
34. Martinon, F., Pétrilli, V., Mayor, A., Tardivel, A. & Tschopp, J. Gout-associated uric
acid crystals activate the NALP3 inflammasome. Nature 440, 237–241 (2006).
35. Brydges, S.D. et al. Inflammasome-mediated disease animal models reveal roles
for innate but not adaptive immunity. Immunity 30, 875–887 (2009).
© 2015Nature America, Inc. All rights reserved.
nature medicine doi:10.1038/nm.3804
ONLINE METHODS
Mice and animal care. The global Nlrp3−/−, Gpr109a−/−, Ucp2−/− and Sirt2−/−
knockout mice have been described before12 ,19,20. Oxct1fl /fl28 and Atg5 fl/f l
mice were crossed with LysM-Cre (B6.129P2-Lyz2tm1(cre)Ifo/J) animals for
macrophage specific gene ablations. NLRP3L351P gain of function familial
cold autoinflammatory syndrome (FCAS) and NLRP3A350V Muckle–Wells
syndrome (MWS) knock-in mutations have been described before32 ,33.
Briefly, the Nlrp3 (L351PneoR/+) and Nlrp3 (A350VPneoR/+) mutation was
conditionally activated by breeding these animals with tamoxifen-inducible
Cre mice (B6.Cg-Tg(CAG-cre/Esr1*)5Amc/J) or in vitro by treating cells with
4-hydroxy tamoxifen. Mice were fed 1,3-butanediol ketone diesters (KD)
(B84785, Sigma-Aldrich) for 1 week after weaning and injected with tamoxifen
for 3 d and analyzed. The WT littermates and mutant cohorts were housed
with a 12 h–light/12 hour–dark cycle at 22 °C. The mice were multi-housed
and were either fed ad libitum normal chow diet consisting of 4.5% fat (5002;
LabDiet) or ad libitum normal chow diet mixed in with 20% 1,3-butanediol
ketone diesters and aged in the specific-pathogen free barrier facility in
ventilated cage racks that deliver HEPA-filtered air to each cage with free access
to sterile water through a Hydropac system. Sentinel mice in our animal rooms
were negative for currently tested standard murine pathogens (Ectromelia,
EDIM, LCMV, Mycoplasma pulmonis, MHV, MNV, MPV, MVM, PVM,
REO3, TMEV and Sendai virus) at various times while the studies were
performed (Research Animal Diagnostic Laboratory, Columbia, MO). All
experiments and animal use were conducted in compliance with the National
Institutes of Health Guide for the Care and Use of Laboratory Animals
and were approved by the Institutional Animal Care and Use Committee at
Yale and Washington University.
Human monoc ytes. CD14+ monoc ytes were sorted from cryopreserved
peripheral blood mononuclear cells using the Human Monocyte Isolation
Kit II from Miltenyi (130-091-153). A total of 6 healthy subjects (three females
aged 67, 31 and 44 years old and three males aged 67, 31 and 35 years old)
were used for monocyte isolations. Monocytes were seeded in six-well plates
at a concentration of 3 million cells per ml of RPMI1640 media (11875119;
Life Technologies) with 10% FCS (10437028; Life Technologies) and antibiotic/
antimycotic mixture (10378-016; Life Technologies) and stimulated with LPS
(62325; Sigma-Aldrich) for 4 h. The supernatants were used to measure IL-1β,
IL-18 and TNF-α using the Human IL-1β, IL-18 and TNF-α Platinum ELISA
from eBioscience (BMS 267/2, BMS2034, BMS224). Human peripheral blood
was collected by the Health Apheresis Unit and the Clinical Core Laboratory,
both part of the National Institute on Aging-Intramural Research Program,
under Human Subject Protocol no. 2003054 and Tissue Procurement Protocol
no. 2003-071; consent was obtained.
Cell culture. All steps were performed using sterile technique. Femurs were
collected in RPMI (22400105; Life Technologies) + 10% FBS (R10; Omega
Scientific). Both ends of the femur were then cut and the femur was flushed with
R10. The bone marrow was centrifuged at 450 g for 5 min, the supernatant was
decanted and red blood cells were lysed using ACK lysis buffer (118-156-101;
Quality Biological). After neutralization with R10, bone marrow cells were
centrifuged, resuspended in 10 ml of R10 and placed into a six-well plate.
Non-adherent cells were collected the following morning. The non-adherent
cells were resuspended at 4 × 106 cells per ml in medium consisting of 10 ml
supernatant of non-adherent cells, 7.2 ml L929 conditioned medium, 6.8 ml
R10 and MCSF (10 ng/ml; 416-ML; R&D Systems). An additional 2 ml of fresh
medium was added 4 d after isolation. Non-adherent cells were collected on
day seven, separated by density gradient separation using Fico/Lite (I40650;
Atlanta Biologicals) and mononuclear cells were collected. Cells were rinsed
twice with Dulbecco’s PBS + 2% FBS, and resuspended at 1 × 10 6 cells per
ml. Cells were treated with ultrapure LPS (L6529-1mg, Sigma-Aldrich) alone
or in combination with 5 mM ATP (1A7699-1G; Sigma-Aldrich) or 200 µM
palmitate-BSA (P9767; Sigma-Aldrich). The BMDMs were also primed with
ultrapure lipid A (10 ug/ml; tlrl-mpla; InvivoGen), lipoteichoic acid (10 ug/ml;
tlrl-pslta; InvivoGen), or Pam3-CSK4 (10 ug/ml; tlrl-pms; InvivoGen) for 4 h
and stimulated with various NLRP3 activators (ATP, 5 mM (1 h); MSU,
250 µg/ml (5 h) (tlrl-msu, InvivoGen); silica, 200 µg/ml (5 h) (tlrl-sio,
InvivoGen); palmitate-BSA, 200 µM (24 h) (P9767, Sigma-Aldrich); Ceramide
C6, 80 µg/ml (6 h) (62525-10, Cayman); and sphingosine, 40 µM (1 h) (62570,
Cayman); together with -BHB or (S)-BHB (54920, 54925, Sigma-Aldrich) at
indicated concentration and time. The cell supernatants and cell lysates were
collected after BHB treatment and analyzed for caspase-1(1:250; Genentech)
and IL-1β (1:1,000; GTX74034; Genetex).
Salmonella typhimurium were grown overnight and then subcultured to mid-
log phase as described previously36. BMDMs were infected with 1 multiplicity
of infection of S. typhimurium and Francisella tularensis and treated 1 h after
infection with 0, 1, 5 or 10 mM of BHB. Cell supernatants and cell lysates were
collected 3 h after treatment (4 h after infection).
ASC oligomerization and ASC speck formation. ASC oligomerization
blotting was performed as described previously32. BMDMs were plated on
chamber slides and allowed to attach overnight. The following day cells were
primed with LPS and treated with ATP in the presence or absence of BHB
(10 mM). Cells were fixed with 4% paraformaldehyde followed by ASC
(ADI-905-173-100; Enzo Lifesciences) and DAPI (D9542; Sigma-Aldrich)
staining. ASC specks were quantified using ImageJ software. At least five
distinct fields were analyzed and a minimum of 550 cells from each treatment
condition were quantified.
Neutrophil chemotaxis assay. Neutrophils were isolated from mouse bone
marrow (19762, Stemcell Technologies) and 2 × 105 cells were plated in 3-µm
Transwell 96-well plates. Untreated cells had just RPMI (+ 10% FBS) in the
bottom chamber. To induce chemotaxis, LPS + ATP-stimulated macrophage-
conditioned medium (CM) was diluted 1:1 with RPMI with or without BHB
(10 mM) as indicated. Cells were incubated at 37 °C for 90 min and cells that
passed through the membrane to the lower chamber were counted with a
hemocytometer.
Western blot analysis. The BMDM cell lysates were prepared using RIPA buffer
and immediately snap frozen in liquid nitrogen. Samples were vortexed every
10 min for 1 h. Samples were then centrifuged at 14,000 g for 15 min, the super-
natant was collected and the protein concentration was determined using the DC
Protein Assay (Bio-RAD). The immunoblot analysis was performed as described
previously12. Antibodies to caspase-1 (1:250; 4B4.2.1; Genentech), IL-1β (1:500,
GTX74034; Genetex), caspase-11 (1:1,000 NB120-10454; Clone 17D9; Novus Bio),
Scot (1:500, 12175-1-AP; Proteintech), Asc (1:500, ADI-905-173-100; Enzo Life
Science), acetylated histone H3 (1:500, 06-942; LYS9; EMD Millipore), HMGCL
(1:500, NBP1-58026; Novus Bio), and β-actin (1:1,000 4967L; Cell Signaling)
were used at the dilutions specified by the manufacturer. The immune complexes
were visualized by incubation with horseradish peroxidase-conjugated anti-rat
(PI31470; Pierce) or anti-rabbit secondary antibody (PI31460). Immunoreactive
bands were visualized by enhanced chemiluminescence (PI32209; Pierce).
Densitometry analysis was performed using the ImageJ Gel Analysis tool, where
gel background was also removed individually for each band.
Gene expression analysis. Total RNA was extracted using the Trizol method
and transferred to the Qiagen RNeasy mini kit and purified according to
the manufacturer’s instructions. On the columns, DNA digestion was per-
formed to remove DNA by following the RNase-Free DNase Set’s manufac-
turer’s instructions (79254; Qiagen). Synthesis of cDNA and Q-PCR was
performed as described previously15. The primer pairs used for real-time
PCR:Bdh1 (Forward: 5′-GCTTCCTTGTATTTGCTGGC-3′, Reverse: 5′-TT
CTCCACCTCTTCACTGTTG, Probe: 5′-TGGATGGTTCTCAGTCGGTCAC
TCT-3′), Bdh2 (Forward: 5′-TCTCAATGAATCTCAACGTCCG-3′, Reverse:
5′-ATCTGTTCTCCACCCCTTTG-3′, Probe: 5′-ATCAACATGTCGTCTGT
GGCCTCC-3′), Acat1 (for ward: 5′-GGCTGCTGCAGGAAGTAAGA-3′,
Reverse: 5′-ATCCCTGCCTTCTCAATGGC-3′), Hmgcl (Forward: 5′-CAGGT
GAAGATCGTGGAAGTC-3′, Reverse: 5′-TGGGAGAAACAAAGCTGGTG-3′
and Gapdh (Forward: 5′-TCAACAGCAACTCCCACTCTTCCA-3′, Reverse:
5′-ACCCTGTTGCTGTAGCCGTA TCA-3′).
BHB–nanolipogel (nLG) generation and treatment. nLG is a nanoparticle
that combines the advantages of both liposomes and polymer-based particles
© 2015Nature America, Inc. All rights reserved.
nature medicine
doi:10.1038/nm.3804
and it can provide means for delivery of two or more pharmaceutical agents
at different rates, especially agents with different chemical properties and or
molecular weights. nLGs were fabricated by remotely loading liposomes with
BHB and cross-linkable polyethylene glycol (PEG) oligomers. To prepare
liposomes, a molar ratio mixture of 2:1:0.1 phosphatidylcholine:cholesterol:
DSPE-PEG(2000)-COOH (8400151P, 700000P, 880125P; Avanti Polar Lipids)
in chloroform was evaporated under a nitrogen gas stream and then lyophi-
lized after extrusion. Lyophilized liposomes were rehydrated with the aque-
ous BHB-cyclodextrin-Irgacure-PEG mixture. Vigorous mixing was applied
for 30 min. The liposomes were then cross-linked under a 430 W UV lamp
with UVA light (315–400 nm transmission filter) for 8 min on ice to form the
nLGs, rinsed with PBS, and pelleted by ultracentrifugation. nLGs were stored
at −20 °C until use.
Mice were treated with BHB–nLG (i.p. 125 mg/kg of body weight). After
24 h, mice were challenged with MSU (i.p., 3 mg in 200 µl) and given a second
treatment of BHB nLG (i.p., 125 mg/kg of body weight). 4 h after MSU chal-
lenge, mice were killed. We isolated peritoneum-infiltrating cells by performing
peritoneal lavage with PBS. Lavage fluid containing cells was centrifuged at 1,500
rpm for 5 min. The supernatant was decanted, and the pellet was resuspended
in ACK lysis buffer to lyse the red blood cells. We neutralized the reaction with
RPMI (22400105; Life Technologies) + 10% FBS (R10; Omega Scientific) and
filtered the cells through a 100 micron filter. We pelleted the cells again by cen-
trifuging at 1,500 rpm for 5 min and resuspended cells in 1 ml RMPI to count
with a hemocytometer. Cells were stained for CD45 (clone 30-F11; eBioscience),
Ly6C (clone AL-21; eBioscience), and Gr1 (clone RB6-8C5; eBioscience) and
analyzed using FACS Calibur. All the FACS data was analyzed by post collection
compensation using FlowJO (Tree Star, Inc.) software.
BMDM cell proliferation. Proliferation of BMDMs in response to BHB
treatment was analyzed using the MT T assay (CGD1-1KT; Sigma-Aldrich)
according to manufacturer’s instructions.
Intracellular K+ measurement. BMDM were incubated with asante potas-
sium green 1 (APG-1) (3604, TEFLABS) which is a fluorescent indicator
with a Kd for measuring cytosolic K+ concentration. Its non-ratiometric large
fluorescence dynamic range allows sensing of small changes in the concen-
tration of K+. Optimal excitation was recorded at 517 nm (SpectraMay M5;
Molecular Devices). K+ was also measured in BMDMs using an ElementXR or
Agilent 7700 inductively coupled mass spectrometry (ICP-MS) as described
previously25. For error analysis samples and standard solutions were run
in duplicate.
Enzyme-linked immunosorbent assay (ELISA). Peritoneal cells were
incubated overnight in RPMI (22400105; Life Technologies) + 10% FBS
(R10; Omega Scientific) + antibiotic (15240062; Life Technologies) mixture.
The supernatants were analyzed for IL-1β by the Ebioscience Mouse IL-1β
Platinum ELISA kit (BMS6002TWO). The sera from mice were collected and
stored at −80 °C and used to quantify the concentration of IL-1β in serum
using the eBioscience Mouse IL-1β Platinum ELISA kit (BMS6002TWO).
ELISAs were read on the Tecan Infinite M200 i-control.
Statistical analyses. A two-tailed Student’s t-test was used to examine
differences between genotypes or treatments with a P < 0.05 considered
statistically significant. The results are expressed as the mean ± sem. The
differences between means and the effects of treatments were determined by
one-way ANOVA using Tukey’s test using SigmaStat software, which protects
the significance (P < 0.05) of all pair combinations.
36. Anand, P.K. et al. NLRP6 negatively regulates innate immunity and host defence
against bacterial pathogens. Nature 488, 389–393 (2012).
