NAD?-dependent SIRT1 Deacetylase Participates in
Epigenetic Reprogramming during Endotoxin Tolerance*
Tie Fu Liu‡1, Barbara K. Yoza‡§, Mohamed El Gazzar‡, Vidula T. Vachharajani¶, and Charles E. McCall‡?2
North Carolina 27157
Gene-selective epigenetic reprogramming and shifts in cellu-
leukocytes from acute systemic inflammation with sepsis, we
report that energy sensor sirtuin 1 (SIRT1) coordinates the epi-
genetic and bioenergy shifts. After TLR4 signaling, SIRT1 rap-
idly accumulated at the promoters of TNF-? and IL-1?, but not
I?B?; SIRT1 promoter binding was dependent on its co-factor,
NAD?. During this initial process, SIRT1 deacetylated RelA/
termination of NF?B-dependent transcription. SIRT1 then
remained promoter bound and recruited de novo induced RelB,
which directed assembly of the mature transcription repressor
complex that generates endotoxin tolerance. SIRT1 also pro-
moted de novo expression of RelB. During sustained endotoxin
tolerance, nicotinamide phosphoribosyltransferase (Nampt),
the rate-limiting enzyme for endogenous production of NAD?,
and SIRT1 expression increased. The elevation of SIRT1
required protein stabilization and enhanced translation. To
support the coordination of bioenergetics in human sepsis, we
observed elevated NAD?levels concomitant with SIRT1 and
RelB accumulation at the TNF-? promoter of endotoxin toler-
and human sepsis activate pathways that couple NAD?and its
sensor SIRT1 with epigenetic reprogramming.
Two cellular processes predictably accompany TLR-medi-
ated acute systemic inflammation caused by sepsis, a highly
destructive and often lethal process. The first process occurs
when epigenetic alterations reprogram distinct functional sets
of genes to both activate and repress transcription of hundreds
erance requires TLR3receptor signaling of NF?B master regu-
potentially autotoxic proinflammatory TNF-? and IL-1?. To
control the initial recognition and response phases induced by
TLR, gene-specific reprogramming selectively modifies chro-
matin structure and shifts nucleosomes on responsive euchro-
matin to form silent heterochromatin at acute proinflamma-
tory genes; in contrast, genes encoding anti-inflammatory and
antimicrobial mediators maintain responsive euchromatin (4,
relevant phenotypic transition from the hyperinflammatory to
hours, days, or weeks, depending on the strength of the initial
TLR response (4, 5). The physiologic importance of endotoxin
tolerance is still incompletely understood, but likely reflects an
attempt to recover homeostasis (3).
Others and we (6–9) reported how temporal transitions in
epigenetic programming alter the course of acute inflamma-
tion. NF?B master transcription switch directs a phase-shift
between initiating acute inflammation and developing endo-
toxin tolerance. During this sequel, a RelA/p65-dependent
feed-forward loop induces de novo production of NF?B factor
RelB, which directly recruits G9a histone H3K9 methyltrans-
converts structurally responsive euchromatin of acute proin-
heterochromatin, which is maintained until sepsis is resolved.
In contrast, the epigenetic shift generated by the RelB feed-
forward loop also persistently activates euchromatin of genes
encoding anti-inflammatory and antimicrobial mediators (13).
Thus, RelB in innate immunity phagocytes acts as an inducible
dual transcription regulator.
A second predictable feature of sepsis is a shift in cellular
hepatocytes, and muscle cells (14, 15). During this process,
TLR-dependent signaling first increases ATP production by
cytosis. As a by-product, reactive oxygen species injure struc-
Within hours after the initial TLR signaling, mitochondria are
reprogrammed to uncouple oxidative phosphorylation, creat-
ing a state of “relative intracellular hypoxia” (16). If TLR
GrantsRO1AI-065791(toC. E. M.),R01AI-079144(toC. E. M.),andMO-1RR
007122 (Wake Forest University General Clinical Research Center). The
research on human participants was endorsed by Wake Forest University
IRB Protocol BG174.
icine, Section of Molecular Medicine, Wake Forest University School of
Medicine, Medical Center Blvd., Winston-Salem, NC 27157. Tel.: 336-716-
8607; Fax: 336-716-1214; E-mail: firstname.lastname@example.org.
2To whom correspondence may be addressed: Section of Molecular Medi-
cine, Department of Internal Medicine, Wake Forest University School of
Medicine, Medical Center Blvd., Winston-Salem, NC 27157. Tel.: 336-716-
8607; Fax: 336-716-1214; E-mail: email@example.com.
3The abbreviations used are: TLR, Toll-like receptor(s); Nampt, nicotinamide
THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 286, NO. 11, pp. 9856–9864, March 18, 2011
© 2011 by The American Society for Biochemistry and Molecular Biology, Inc.Printed in the U.S.A.
by guest on July 17, 2015
responses are too exuberant, apoptosis kills many cells, and
multiorgan failure occurs. If cells survive, the number of mito-
chondria and ATP levels drop, during which time NAD?/
NADH ratios shift to favor NAD?-dependent deacetylation
processes. During this time, increased glucose uptake provides
ATP from glycolysis. These sequential shifts in bioenergetics
occur in human and animal sepsis and have been linked to a
prosurvival state of cellular “hibernation,” during which time
its apoptosis (17). As sepsis evolves and adaptation continues,
further shifts in gene expression induce mitochondrial biogen-
esis; ultimately, metabolic homeostasis returns. Recent data
lates with sepsis resolution and survival (18).
As a unifying concept, we hypothesized that NAD?-depen-
dent bioenergetics and epigenetics may combine to influence
the chromatin shifts that generate endotoxin tolerance during
sepsis. To test this, we used the well established THP-1 (the
human promonocytic cell) cell model of endotoxin tolerance
and human sepsis blood leukocytes. Our findings support
that redox sensor SIRT1 and NAD?elevations controlled by
nicotinamide phosphoribosyl transferase (Nampt) coordi-
nate the epigenetic NF?B-dependent p65 and RelB feed-for-
ward loop that regulates gene-selective changes during
Preparation of Human Blood Samples—Blood samples were
failure and healthy controls according to the IRB protocol
approved by Wake Forest University. Leukocytes were sepa-
rated by layering heparinized whole blood over Isolymph (Gal-
lard-Schlesinger Industries, Carle Place, NY) and settling for
1 h. Cells were washed in phosphate-buffered saline, and resid-
ual red blood cells were removed by hypotonic lysis using three
parts distilled H2O for 20 s followed by one part of 3.6% NaCl.
Pelleted leukocytes were subjected to cell culture under the
indicated conditions or NAD?extraction. Cells were ?95%
viable, and because both neutrophils and mononuclear cells
Serum samples were prepared by clotting the blood for 30 min
at room temperature followed by centrifugation for 10 min at
1000 rpm and were stored at ?80 °C.
Type Culture Collection and were maintained in RPMI 1640
medium (Invitrogen) supplemented with 100 units/ml penicil-
lin, 100 ?g/ml streptomycin, 2 mM L-glutamine, and 10% fetal
bovine serum (HyClone, Logan, UT) in a humidified incubator
with 5% CO2at 37 °C. For analysis of TNF-? mRNA, cells were
Ex527 (Tocris Bioscience), or 250 ?M resveratrol (Sigma) fol-
lowed by incubation for the indicated times with 1 ?g/ml of
Gram-negative bacteria LPS (Escherichia coli 0111:B4, Sigma).
For determination of half-life of LPS-induced TNF-? mRNA,
the transcription inhibitor actinomycin D (5 ?g/ml) was added
at the peak time of TNF-? transcription for indicated times.
for 16 h with 1 ?g/ml of LPS. In some experiments, cells were
pretreated with 10 nM FK866 (Cayman Chemical) for 24 h (to
deplete cellular NAD?) or pretreated with 1 ?M cycloheximide
(Sigma) for 15 min (to block protein synthesis).
Real-time RT-PCR—Levels of TNF-?, SIRT1, Nampt, and
Cellular RNA was isolated using the STAT-60 RNA extraction
kit (Tel-Test, Friendswood, TX). One ?g of RNA was reverse-
transcribed to cDNA using murine leukemia reverse transcrip-
tase (Applied Biosystems). The PCR analysis was performed in
an ABI prism 7000 Sequence Detection System (Applied Bio-
systems) in a 25-?l reaction containing 12.5 ?l of 2? TaqMan
universal Master Mix, gene-specific predesigned TaqMan
primer/probe sets, and 1 ?l cDNA. GAPDH mRNA served as
ChIP Assay—DNA-protein interactions at the promoters of
TNF-?, IL-1?, RelB, and I?B? were analyzed by using a ChIP
Chromatin was immunoprecipitated for overnight at 4 °C by
incubation with 5 ?g of antibody against SIRT1, NF-?B p65,
RelB, linker histone H1, I?B? (Santa Cruz Biotechnology),
acetylated p65 at lysine 310 (Abcam), acetylated histone H4 at
lysine 16 (Millipore), or total histone H4 (Cell Signaling). Iso-
type-matched IgG served as a negative control.
For sequential ChIP assay, chromatin was first immunopre-
cipitated with the SIRT1 or H1 antibody. The primary immu-
noprecipitates were then eluted by incubation with 10 mM
dithiothreitol at 37 °C for 30 min, diluted 40 times in immuno-
precipitation buffer, and reimmunoprecipitated with the indi-
cated secondary antibody.
follows: TNF-? ?B1 (at ?598) forward, 5?-CCAAGACTGAA-
ACCAGCAT-3? and reverse, 5?-TAGCAGGGACAAGCCT-
3?; TNF-? ?B2 (at ?216) forward, 5?-GAGGCAATAGGTTT-
TGAGG-3? and reverse, 5?-AAGCATCAAGGATACCCCTC-
3?; TNF-? ?B3 (at ?98) forward, 5?-TACCGCTTCCTCCAG-
ATGAG-3? and reverse, 5?-TGCTGGCTGGGTGTGCCAA-
3?; RelB (forward), 5?-CAGAGCAATGGTCAGCGACG-3?
and reverse, 5?-CACAGT CTGGTGGACGATCG-3? encircl-
ing ?B1 (at ?247) and ?B2 (at ?175) sites; I?B? (forward), 5?-
AGCAGAGGACGAAGCCAGTTCT-3? and reverse, 5?-
GACTGCTGTGGGCTCTGCAG-3? (surrounding ?B1 site at
?96). TNF-? gene-specific primers were obtained from
Applied Biosystems (Hs00174128_m1). Five ?l of immunopre-
cipitated DNA and 1 ?l of input DNA were analyzed in a 25-?l
reaction volume containing 1 ?M of each primer, 2 mM MgCl2,
0.2 ?M dNTP, and 0.04 units/?l AmpliTaq Gold DNA poly-
merase (Applied Biosystems). PCR conditions were set as fol-
lows; one cycle for 10 min at 94 °C, 30 cycles of 30-s each at
94 °C, 58 °C and 72 °C followed by one cycle at 72 °C for 5 min.
and scanned using a typhoon scanner (GE Healthcare). For
SIRT1/p65 ChIP co-immunoprecipitations, DNA-protein
complexes were immunoprecipitated with anti-SIRT1 anti-
body and immunoblotted with anti-p65 antibody.
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Cellular NAD?Extraction and Colorimetric NAD?Assay—
EnzyChrom NAD?/NADH Assay kit (BioAssay System)
according to the manufacturer’s instructions. For cellular
NAD?extraction, cells were incubated with NAD?extraction
natants were obtained by spinning the samples down at 14,000
rpm for 5 min. Forty microliters of NAD?standard and sam-
ples were mixed with 80 ?l of working reagent in duplicate in a
96-well plate, and the optical density was read immediately at
subtracted from OD15for concentration analysis. Cellular
NAD?levels of unknown samples were calculated from the
standard curve and analyzed by Prism software (GraphPad
as % of control or ?M/mg proteins.
Transfection—for RNA interference, 60 pmol of a pool of
three target-specific siRNA (Santa Cruz Biotechnology) were
using Amaxa Nucleofector kit V and Amaxa nucleofector II
device (Lonza, Inc.). Twenty four hours after transfection, cells
were stimulated for the indicated times with 1 ?g/ml LPS
before harvest. A pool of scrambled siRNAs was transfected as
a negative control.
RelB plasmid transfection was performed as detailed previ-
HA-RelB plasmid DNA were electronically transfected into
THP-1 cells. Forty-eight hours after transfection, cells were
stimulated for 1 h with 1 ?g/ml LPS in the absence or presence
of 1 ?M Ex527. Cell lysates were subjected to ChIP analysis of
RelB at TNF-? promoter.
Pulse Chase Assay—LPS-responsive or tolerated cells were
starved for 30 min in methionine-free RPMI medium followed
by pulse labeling for 1 h in 5% dialyzed FBS RPMI medium
containing [35S]methionine. After washes, cells were chased in
complete RPMI medium for 0–4 h. The radiolabeled SIRT1
protein was immunoprecipitated with SIRT1 antibody and
resolved onto 9% SDS-PAGE.
Immunoprecipitation—THP-1 cells were stimulated for 24 h
with 1 ?g/ml of LPS. Nuclear extracts were prepared by lysing
mixture) for 5 min in ice followed by spin down for 5 min at
1000 rpm at 4 °C. The supernatants were collected as cytosol
samples. The pellets were washed with wash buffer (10 mM
HEPES (pH 7.9), 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA,
extraction buffer (20 mM HEPES (pH 7.9), 1 M NaCl, 0.2 mM
EDTA, 0.2 mM EGTA, 0.2% IGEPAL, protease inhibitor mix-
ture). Nuclear extracts were collected after centrifugation at
1000 rpm for 5 min and were incubated with anti-SIRT1 anti-
body for overnight at 4 °C. The immunocomplexes were then
precipitated with protein A-Sepharose CL4B beads and ana-
lyzed by Western blot using anti-RelB antibody. IgG immuno-
precipitation severs as negative control.
Immunoblotting—Equal amounts (50 ?g) of cell lysates or
nuclear extracts were separated by SDS-PAGE electrophoresis
and transferred to a polyvinylidene difluoride membrane
(PerkinElmer Life Sciences). Blots were blocked with 5% milk-
PBS for 1 h at room temperature and probed for overnight at
4 °C with 0.4 ?g/ml of primary antibody against SIRT1 or
ing control and probed with 0.04 ?g/ml anti-human ?-actin
monoclonal antibody (Sigma). Protein complexes were
detected by incubation for 1 h at room temperature with sec-
ondary antibody conjugated to horseradish peroxidase (Sigma)
diluted at 1:5000 in blocking buffer and then detected by
Enhanced Chemiluminescence Plus (GE Healthcare).
Statistical Analysis—Data were analyzed with unpaired Stu-
p values of ? 0.05 were considered significant.
TLR4 Stimulation Post-transcriptionally Induces SIRT1 dur-
ing Endotoxin Tolerance—We first determined whether SIRT1
expression changes during endotoxin tolerance. Upon TLR
mRNA, which peaked at ?1 h and quickly decreased to a back-
ground level as endotoxin tolerance developed (Fig. 1A), as
reflected by repression of TNF-? gene expression in response
to LPS restimulation (Fig. 1B). We then assessed SIRT1 mRNA
and protein levels in whole cell extracts of LPS-treated THP-1
after TLR stimulation. In contrast, SIRT1 protein levels
FIGURE 1. SIRT1 increases during TLR4-induced endotoxin tolerance in
tolerance. TNF-? mRNA levels were measured using quantitative real-time
in response to second LPS stimulation. THP-1 cells pretreated with 1 ?g/ml
LPS for indicated times were restimulated with LPS for another 1 h. C, SIRT1
protein expression during the course of endotoxin tolerance. Normal THP-1
were subjected to Western blot analysis for SIRT1 level. ?-Actin serves as
loading control. D, real-time PCR analysis of SIRT1 transcription and densi-
tometry analysis of SIRT1 protein expression in C. med, medium.
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responsible for SIRT1 protein increases. Pulse labeling showed
increases in newly synthesized SIRT1 in tolerant cells over that
of nontolerant normal cells (Fig. 2A, lane 0). We then tested
whether SIRT1, which is degraded in the basal state in the pro-
teasome (19), also is stabilized following TLR stimulation (Fig.
2A, right panel). To do this, THP-1 cells were treated with
idly decreased in normal cells but remained relatively
unchanged in TLR-stimulated tolerant cells (Fig. 2, B and C).
However, the prolonged half-life of proteins in tolerant cells is
not a general phenomenon of endotoxin tolerance. We have
reported previously that the anti-inflammatory protein IL-1ra
is stabilized, but the inflammatory protein cox2 is degraded
is also degraded rapidly in tolerance (21).
protein stabilization contribute to the SIRT1 protein incre-
ments during the endotoxin tolerance. This prompted our
determining SIRT1 function during endotoxin tolerance.
tion during Endotoxin Tolerance—We observed that the LPS-
in the presence of the SIRT1-specific inhibitor Ex527 (Fig. 3A).
We then confirmed this effect using RNA interference to
In further support of SIRT1 inhibitory function, pretreatment
ity, markedly depressed TNF-? mRNA synthesis after LPS
stimulation (Fig. 3C).
To exclude the possibility that repression of TNF-? tran-
scription by SIRT1 is due to the increased degradation of
TNF-? mRNA, we compared the half-life of LPS-induced
TNF-? mRNA in the presence or absence of the SIRT1 spe-
cific inhibitor Ex527. Inhibition of SIRT1 activity did not
change TNF-? mRNA degradation rate (Fig. 3D), suggesting
FIGURE 2. THP1 cells translate and stabilize SIRT1 following TLR4 stimu-
labeled SIRT1 protein was immunoprecipitated with SIRT1 antibody and
ant cells were pretreated with 1 ?M cycloheximide for 15 min followed by
was analyzed by Western blot analysis. C, densitometry analysis of the West-
ern blot data shown in B.
transcription. Normal THP-1 cells were pretreated with 1 ?M Ex527 for 1 h followed by stimulation with 1 ?g/ml LPS. TNF-? mRNA was quantified using
real-time PCR. B, SIRT1 knockdown augments LPS-induced TNF-? transcription. Normal THP-1 cells were transfected with SIRT1-specific siRNA for 24 h as
1 h with 1 ?g/ml LPS. D, degradation of LPS-induced TNF-? mRNA. THP-1 cells were stimulated with 1 ?g/ml LPS for 1 h in the presence or absence of Ex527
of control TNF-? mRNA and shown as mean ? S.E. Ctrl, control; KD, knockdown; Ex, Ex527; Res, resveratrol; ETOH, ethyl alcohol.
MARCH18,2011•VOLUME286•NUMBER11 JOURNALOFBIOLOGICALCHEMISTRY 9859
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that SIRT1 regulates TNF-? transcription, instead of its
Because these data addressed only the initial wave of TLR4-
induced transcription, we examined whether SIRT1 also con-
tributed to the hyporesponsive status of fully developed endo-
toxin-tolerance, as assessed at 16 h after the initial TLR4
stimulus. We found that Ex527 treatment of tolerant cells par-
TLR stimulation with LPS (Fig. 3E). Similar results occurred
after knockdown of SIRT1 in tolerant cells (Fig. 3F). Together,
these data suggested that SIRT1 might participate in both the
initiating and sustaining phases of endotoxin tolerance.
SIRT1 Deacetylates RelA/p65 at Lysine 310 at Promoters to
Limit Gene Transcription of Proinflammatory Cytokine Genes
during Initiation of Endotoxin Tolerance—We next reasoned
that SIRT1 might promote the initial shift toward endotoxin
tolerance by inactivating NF?B p65 transcription factor, which
tolerant phenotype (6, 10–12). To test this, we used ChIP
assays. There are three NF?B binding sites at the TNF-? pro-
moter area: the distal ?B1 and ?B2 sites and proximal ?B3 site.
Although ?B1 and ?B2 sites play a minor role in TNF-? tran-
scription, we reported that the proximal site (?B3) plays a cru-
cial role in TNF-? transcription silencing (7). Hence, we per-
formed ChIP assay using primers covering ?B3 site. We found
no SIRT1 bound at the proximal TNF-? promoter in resting
cells, but SIRT1 rapidly accumulated at proximal promoter
after LPS stimulation (Fig. 4A). Further ChIP analysis using a
TNF-? gene specific primer shows SIRT1 exclusively binds to
TNF-? promoter but not to the TNF-? gene coding sequence
(data not shown). As predicted, RelA/p65, a crucial initiator of
TNF-? gene transcription, also rapidly accumulated at proxi-
mal TNF-? promoter, reached its peak after 30 min of LPS
bound to the proximal promoter of acute proinflammatory
? promoter (data not shown). These ChIP standard PCR data
were confirmed by quantitative real-time PCR analysis (data
As SIRT1 contains no DNA-binding domain, it must associ-
ate with partner proteins to affect transcription. One of these,
RelA/p65, can directly bind SIRT1 (22). Accordingly, we tested
whether SIRT1 partners with RelA/p65 during the initiation
phase of endotoxin tolerance. Using ChIP co-immunoprecipi-
tation, we observed that SIRT1 and RelA/p65 formed a com-
plex at the promoter, peaking at 1 h and decreasing by 4 h (Fig.
4B). The kinetics of both p65 promoter binding (Fig. 4A) and
SIRT1-p65 interaction at the promoter (Fig. 4B) parallel the
dynamic changes of nuclear p65 protein, which increases
upon LPS stimulation and decreases after 4 h of LPS stimu-
lation (Fig. 4C).
Lysine 310 on RelA/p65 is a key residue that controls RelA/
p65 transactivation by p300 acetyl transferase; deacetylation at
lysine 310 by SIRT1 can disrupt transactivation by RelA/p65
(22). Accordingly, we tested whether SIRT1 might limit trans-
activation by RelA/p65 by deacetylating lysine 310, while these
two proteins are co-bound in vivo. Although we could not
detect acetylation of RelA/p65 lysine 310 at TNF-? promoter
significantly increased RelA/p65 lysine 310 acetylation (Fig.
4D). We obtained similar results using nicotinamide, a SIRT1-
nonspecific inhibitor (data not shown). Together, these data
supported that SIRT1 deacetylase activity participated in shift-
ing activated to repressed transcription as endotoxin tolerance
SIRT1 Remains Bound to TNF-? Promoter during Endotoxin
and Fig. 1B) have shown that phagocytes become tolerant to a
second stimulation with LPS within 3–6 h after the initial TLR
stimulus. Our finding that either SIRT1 inhibitor Ex527 or
SIRT1-gene specific knockdown in tolerant cells partially
from responsive euchromatin to repressed facultative hetero-
chromatin. In support of this, we found that SIRT1 remained
As RelA/p65 is replaced at the TNF-? promoter in tolerant
THP-1 cells by dimer exchange with RelB (23), we asked
whether SIRT1 interacts with the multicomponent repressor
complex proteins RelB and heterochromatin linker histone H1
(8, 11, 24, 25). Our previous work showed that RelB promoter
recruitment directs facultative heterochromatin formation by
first binding to G9a methyltransferase, which methylates
H3K9; other members of chromatin modifiers include histone
linker H1, heterochromatin protein 1, and DNA CpG methyl-
transferases DNMT 3a/3b (11, 13, 25). Using sequential ChIP
analysis of SIRT1 followed by histone H1 and RelB re-ChIP, we
TNF-? promoter (Fig. 5B). Furthermore, knockdown of SIRT1
This supports that SIRT1 binding preceded that of RelB and
FIGURE 4. SIRT1 deacetylates RelA/p65 at lysine 310 at the TNF-? pro-
moter in the initiation phase of TLR4 response. A, kinetics of SIRT1 and
ChIP analysis using antibodies against SIRT1 or RelA/p65 as described under
“Experimental Procedures.” B, SIRT1 interacts with RelA/p65 at promoters.
THP-1 cells were stimulated for the indicated times, and chromatin-bound
antibody. D, SIRT1-specific inhibitor Ex527 accumulates acetylated RelA/p65
at lysine 310 (p65k310Ac) at proximal TNF-? promoter. THP-1 cells were cul-
tured for 1 h in the absence or presence of 1 ?g/ml LPS, 1 ?M Ex527, or LPS
plus Ex527, respectively. Cell lysates were subjected to ChIP assay using the
indicated antibodies. IB, immunoblotting; EX, Ex527; HC, heavy chain.
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that H1 linker histone binding may be proximal to both SIRT1
Our previous data indicated the RelB is both necessary and
sufficient for promoting transcription repression during endo-
toxin tolerances (12). Our observation here that removal of
SIRT1 decreased RelB promoter binding suggested that SIRT1
of the multicomponent repressor complex. Using immunopre-
cipitation analysis, we further substantiated that SIRT1 and
RelB interacts (Fig. 5D).
We also have found that RelB promoter binding requires
not bind to promoter without TLR signaling.4To investigate
whether SIRT1 is required for RelB loading onto promoter, we
overexpressed RelB in normal THP-1 cells for 48 h (Fig. 5F).
Cells were then stimulated with LPS for 1 h in the presence or
absence of the SIRT1-specific inhibitor Ex527. ChIP analysis
by inhibition of SIRT1 activity (Fig. 5G), supporting SIRT1
ity that RelB may bind to the two distal ?B sites at the TNF-?
promoter without SIRT1 support, we performed RelB ChIP
RelB binds to these two distal kB sites (data not shown).
RelB acts as both a repressor and activator of transcription of
specific genes during endotoxin tolerance and acute systemic
inflammation (5, 10, 13). Reports indicate that SIRT1 also can
activate or repress transcription by deacetylating distinct tran-
scription mediators (26, 27). Accordingly, we asked whether
SIRT1 might promote transcription and de novo synthesis of
RelB. In support of this, we found that SIRT1 accumulated at
bition of SIRT1 deacetylase activity with Ex527 reduced RelB
gene transcription (Fig. 6B) and protein expression (Fig. 6C).
These data support a dual and cooperative function between
SIRT1 and RelB during endotoxin tolerance.
NAMPT Generates NAD?Accumulation during Endotoxin
Tolerance—Because SIRT1 activity is NAD?-dependent, and
because redox states change during acute inflammation, we
tested whether NAD?levels increase following TLR stimula-
tion. We detected NAD?at basal levels in nonstimulated
THP-1 cells and observed that TLR stimulation decreased cel-
lular NAD?to ?20% of its basal level by 15 min to 1 h. There-
(Fig. 7A), which correlated with elevations in SIRT1 protein
(Fig. 1, C and D).
4B. K. Yoza and C. E. McCall, unpublished data.
FIGURE 5. SIRT1 accumulates with RelB and H1 at the TNF-? promoter.
ant cells. LPS-tolerant cells were restimulated for indicated times with 1
?g/ml LPS. Cell lysates were subjected to ChIP analysis using indicated anti-
bodies as detailed under “Experimental Procedures.” B, SIRT1 interacts with
were stimulated for 1 h by LPS, SIRT1/H1, and RelB sequential ChIP analysis
was performed as detailed under “Experiment Procedures.” C, H1/SIRT1 and
RelB sequential ChIP analysis at the TNF-? promoter after SIRT1 knockdown.
Chromatin was immunoprecipitated with H1 antibody and reimmunopre-
were stimulated for 24 h with 1 ?g/ml LPS, nuclear extracts were immuno-
precipitated with SIRT1 antibody. Immunoprecipitates were subjected to
Western blot analysis using anti-RelB antibody. E, overexpression of RelB in
pcDNA3-HA vector plasmid DNA or HA-RelB plasmid DNA. Total cell lysates
were subjected to Western blot analysis of RelB expression using anti-RelB
antibody. F, SIRT1 facilitates RelB loading onto the TNF-? promoter. RelB-
presence or absence of Ex527. ChIP analysis of TNF-? promoter bound RelB
was performed as described under “Experimental Procedures.” IP, immuno-
precipitation; IB, immunoblotting; Ctrl, control; KD, knockdown; Med, medium;
tin was immunoprecipitated with SIRT1 antibody. The SIRT1-DNA complex
was analyzed by standard PCR using TNF-? promoter-specific or RelB pro-
control. B and C, inhibition of SIRT1 deacetylase activity decreases LPS-in-
duced RelB gene transcription and protein expression during the develop-
ment of endotoxin tolerance. THP-1 cells were pretreated for 1 h with 1 ?M
Ex527 followed by stimulation with 1 ?g/ml of LPS for indicated times. RelB
using Western blot. RelB mRNA levels are presented as fold changes relative
to unstimulated control. Ctrl, control; med, medium.
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Cellular NAD?homeostasis is partially sustained by recy-
cling NAD?degradation products by the rate-limiting enzyme
Nampt (28). Because Nampt has an NF-?B consensus binding
naling might increase cellular NAD?by inducing Nampt expres-
activity. Western blot analysis showed increased Nampt expres-
of Nampt mRNA (Fig. 7C). As predicted, inhibition of Nampt
activity by FK866, a Nampt-specific inhibitor (29, 30), markedly
Nampt-dependent Up-regulation of NAD?Supports SIRT1
Activation and Epigenetic Gene Reprogramming during Endo-
toxin Tolerance—Given that SIRT1 deacetylase activity is
endotoxin tolerance. To do this, we employed the Nampt-spe-
cific inhibitor FK866 to diminish cellular NAD?before LPS
stimulation (Fig. 8A). We found that predepletion of cellular
NAD?by FK866 treatment enhanced LPS-induced TNF-?
mRNA (Fig. 8B). We then demonstrated that FK866 prevented
NAD?accumulation and disrupted SIRT1 binding to the
TNF-? promoter (Fig. 8C); FK866 also increased acetylation of
histone H4 on lysine 16 at the TNF-? promoter (Fig. 8D).
ulation of Nampt expression positively regulates cellular
NAD?biosynthesis, SIRT1 activation, and SIRT1 promoter
binding during the development of endotoxin tolerance.
ulators Accumulate at TNF-? Promoter of Blood Leukocytes
during Human Sepsis—We previously demonstrated that RelB
is induced in sepsis blood leukocytes during endotoxin toler-
ance, concomitant with the formation of facultative hetero-
chromatin (10, 12). To test whether our unifying concept of
SIRT1 incorporating bio-energy and epigenetics might trans-
sepsis and multiorgan failure and then assessed the binding of
SIRT1 and RelB at the TNF-? promoter. As expected, leuko-
cytes from sepsis patients were endotoxin-tolerant with
repressed TLR4-induced transcription of TNF-? (Fig. 9A).
Coincident with this phenotype, we found SIRT1 and RelB
participants showed increased SIRT1 accumulated at both
TNF-? and IL-1? promoters as determined by real-time RT-
PCR analysis (data not shown). We also detected that cellular
NAD?in sepsis blood leukocytes and extracellular Nampt in
sepsis serum were increased (Fig. 10, C and D).
sis (3), involves interplay between energy sensor SIRT1 and the
NF?B feed-forward loop that shifts TLR4-responsive euchro-
matin state of the TNF-? and IL-1? promoters to silent facul-
tative heterochromatin. This coordinated process uses distinct
pathways (redox sensor SIRT1, Nampt-dependent generation
of NAD?, transcription regulators NF-?B p65 and RelB, and
modifiers of chromatin structure) to shift from inflammation
FIGURE 7. Nampt expression and cellular NAD?increase during TLR4-
course of endotoxin tolerance. Cells were cultured for different times in the
commercial kit as detailed under “Experimental Procedures”. Results are
of Nampt expression at the indicated times after 1 ?g/ml of LPS stimulation.
LPS-inducedNAD?biosynthesisanddepletescellularNAD?.THP-1 cells were
Intracellular NAD?was extracted and analyzed as in A.
FIGURE 8. Cellular NAD?is required for SIRT1 promoter binding and
repression of TNF-? transcription. A, cellular NAD?is diminished by the
Nampt-specific inhibitor FK866. THP-1 cells were pretreated for 24 h with 10
LPS in the presence of 10 nM FK866. B, depletion of cellular NAD?by FK866
augments LPS-induced TNF-? transcription. THP-1 cells were pretreated for
TNF-? mRNA was quantified using real-time PCR. C, depletion of cellular
as in A. Cell lysates were subjected to SIRT1 ChIP assay at the TNF-? proximal
activity enhances accumulation of acetylated histone H4 at lysine 16. Cells
1 mM nicotinamide followed by ChIP analysis with H4K16Acantibody. Med,
medium; Nic, nicotinamide.
9862 JOURNALOFBIOLOGICALCHEMISTRY VOLUME286•NUMBER11•MARCH18,2011
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initiation to the tolerant or adaptive phase of TLR responses.
Fig. 10 models this temporal coupling of energy and epigenet-
ics. First, TLR induces rapid promoter binding of constitutive
SIRT1, which uses available nuclear NAD?to support its pro-
moter binding and deactivate RelA/p65 through lysine 310
deacetylation, thus limiting transcription of acute proinflam-
matory genes. SIRT1 then remains promoter bound, and TLR-
dependent responses increase the expression of Nampt, which
sustains NAD?elevation by recycling nicotinamide. Increases
lation, supports recruitment of RelB and its directly facilitating
formation of locus-specific facultative heterochromatin to
silence acute proinflammatory genes, or activate other genes
with distinct physiologic effects. Unexpectedly, we also
observed that SIRT1 also participates in inducing RelB tran-
there is an interplay between feed-forward loops that involve
transcription, translation, and post-translation programming.
A product of this sequel is the development of endotoxin toler-
ance through epigenetic reprogramming.
We (in humans) and others (in mice) (7–11) showed that
epigenetic reprogramming regulates inflammation by both
repressing acute proinflammatory genes and activating anti-
inflammatory, antimicrobial, and other functionally distinct
gene sets, including those that regulate cellular energy and
mitochondrial biogenesis. In this study, we further find, like
inflammatory IkB? promoter, it represses transcription of
TNF-? and IL-1?, and it activates RelB transcription. Thus,
SIRT1 is part of the master switch that coordinates epigenetic
reprogramming and shifts functional phenotypes during acute
TLR4 responses and sepsis inflammation.
Although the THP-1 cell model of endotoxin tolerance has
been faithful in predicting what happens in human and animal
To support application of the cell model to the epigenetics of
human acute systemic inflammation, we previously demon-
strated gene-specific formation of facultative heterochromatin
at the TNF-? and IL-1? promoters in both THP-1 cells and in
(9) have shown gene-specific heterochromatin formation in
murine macrophages stimulated with endotoxin ex vivo and in
peritoneal macrophages and splenocytes from murine sepsis.5
Here, we provide support of concept for energy and epigenetic
NAD?and concomitant SIRT1 and RelB accumulation at the
TNF-? promoter of blood leukocytes obtained during the
endotoxin tolerant state of septic humans. This leukocyte pop-
5M. El Gazzar and C. E. McCall, unpublished data.
FIGURE 9. Increases in cellular NAD?and extracellular Nampt and accu-
mulation of SIRT1 and RelB at TNF-? promoter in septic leukocytes.
A, TNF-? transcription in blood leukocytes in response to LPS stimulation.
Blood leukocytes were isolated from normal and septic subjects and stimu-
lated with LPS for 1 h. RNA was isolated and analyzed for TNF-? mRNA by
real-time PCR. B, ChIP analysis of TNF-? promoter-bound SIRT1 and RelB in
normal and septic leukocytes. C, intracellular NAD?levels in normal (n ? 3)
and septic blood leukocytes (n ? 12). D, extracellular Nampt levels in normal
(n ? 3) and septic serum (n ? 12). Data in C and D are shown as mean ? S.E.
N, normal control; P, septic patient.
FIGURE 10. A model for SIRT1 in bridging bioenergetics and epigenetics
during endotoxin tolerance and sepsis. TLR induces rapid promoter bind-
ing of constitutive SIRT1, which uses available nuclear NAD?to support its
thus limiting transcription of acute proinflammatory genes. SIRT1 then
sion of Nampt, which sustains NAD?elevation by recycling nicotinamide.
protein and enhancing translation. The combined availability of nuclear
sion and supports recruitment of RelB to direct formation of locus-specific
facultative heterochromatin to silence acute proinflammatory genes or its
activating gene sets with distinct functions.
by guest on July 17, 2015
macrophages, both of which develop the endotoxin-tolerant
phenotype (13, 25). A recent study supports the concept that
SIRT1 may regulate inflammation after endotoxin administra-
tion to humans and mice (31). Further investigations are
needed to determine whether this paradigm extends to other
cells or compartments, such as skeletal muscle, liver, lung, and
in many cellular functions, including stem cell development,
cell intermediary metabolism, and cell senescence (33).
Although this study focused on Sir2 homologue SIRT1, other
NAD?-dependent sirtuin family members might also coordi-
nate energy transitions in many cells: in immunocytes or other
as an energy source, although the functions of SIRT3 in innate
immunity cells are unclear (34). SIRT6, like SIRT1, can bind to
and inactivate RelA/p65, which modifies development of new-
born mice (35). SIRT6 also promotes translation of TNF-? and
deacetylates histone H3 lysine 9 to augment heterochromatin
formation in some tissues (36). We have demonstrated that
SIRT6 also represses TNF-? transcription in our THP-1 cell
model of acute inflammation6and thus may have redundant
functions with SIRT1. Finally, SIRT1 itself has diverse non-
nuclear functions that may impact inflammation regulation
ment, modifies circadian rhythm, enhances antioxidant pro-
duction, and promotes mitochondrial biogenesis, all of which
are prominent features of endotoxin tolerance (3). Taken
together, this study and others support that the sirtuin family
may link energy and inflammation by distinct spatial and tem-
poral processes among diverse cell types.
and gene-specific epigenetic programming converge through
SIRT1 to influence the course of TLR-induced endotoxin tol-
erance as an indicator of the epigenome of acute systemic
inflammatory responses such as sepsis. When this occurs, four
highly conserved biologic processes interact: TLR sensing,
NAD?-directed deacetylation, NF-?B, and epigenetic modifi-
cations of germ line DNA. Our findings may inform new ways
Acknowledgments—We thank Sue Cousart, Jean Hu, and Ashley
Lyles for critical discussion.
Young, R. A. (2006) Proc. Natl. Acad. Sci. U.S.A. 103, 5899–5904
2. Medzhitov, R., and Horng, T. (2009) Nat. Rev. Immunol. 9, 692–703
3. McCall, C. E., Yoza, B., Liu, T., and El Gazzar, M. (2010) J. Innate Immun.
4. Hotchkiss, R. S., and Karl, I. E. (2003) N. Engl. J. Med. 348, 138–150
5. McCall, C. E., and Yoza, B. K. (2007) Am. J. Respir. Crit. Care Med. 175,
6. Chan, C., Li, L., McCall, C. E., and Yoza, B. K. (2005) J. Immunol. 175,
J. Biol. Chem. 282, 26857–26864
8. El Gazzar, M., Liu, T., Yoza, B. K., and McCall, C. E. (2010) J. Biol. Chem.
9. Foster, S. L., Hargreaves, D. C., and Medzhitov, R. (2007) Nature 447,
11. El Gazzar, M., Yoza, B. K., Chen, X., Hu, J., Hawkins, G. A., and McCall,
C. E. (2008) J. Biol. Chem. 283, 32198–32208
J. Immunol. 177, 4080–4085
13. Chen, X., Yoza, B. K., El Gazzar, M., Hu, J. Y., Cousart, S. L., and McCall,
C. E. (2009) Clin. Vaccine Immunol. 16, 104–110
14. Suliman, H. B., Welty-Wolf, K. E., Carraway, M. S., Schwartz, D. A., Hol-
lingsworth, J. W., and Piantadosi, C. A. (2005) FASEB J. 19, 1531–1533
15. Suliman, H. B., Carraway, M. S., Welty-Wolf, K. E., Whorton, A. R., and
Piantadosi, C. A. (2003) J. Biol. Chem. 278, 41510–41518
16. Haden, D. W., Suliman, H. B., Carraway, M. S., Welty-Wolf, K. E., Ali,
Crit. Care Med. 176, 768–777
17. Singer, M. (2008) Clin. Chest Med. 29, 655–660
18. Carre ´,J.E.,Orban,J.C.,Re,L.,Felsmann,K.,Iffert,W.,Bauer,M.,Suliman,
(2010) Am. J. Respir. Crit. Care Med. 182, 745–751
19. Sasaki, T., Kim, H. J., Kobayashi, M., Kitamura, Y. I., Yokota-Hashimoto,
20. Learn, C. A., Mizel, S. B., and McCall, C. E. (2000) J. Biol. Chem. 275,
21. Yoza, B. K., and McCall, C. E. (2011) Cytokine 53, 145–152
22. Yeung, F., Hoberg, J. E., Ramsey, C. S., Keller, M. D., Jones, D. R., Frye,
R. A., and Mayo, M. W. (2004) EMBO J. 23, 2369–2380
23. Saccani, S., Pantano, S., and Natoli, G. (2003) Mol. Cell 11, 1563–1574
24. Agwu, D. E., McPhail, L. C., Sozzani, S., Bass, D. A., and McCall, C. E.
(1991) J. Clin. Invest. 88, 531–539
25. El, Gazzar, M., Yoza, B. K., Chen, X., Garcia, B. A., Young, N. L., and
McCall, C. E. (2009) Mol. Cell. Biol. 29, 1959–1971
26. Blander, G., and Guarente, L. (2004) Annu. Rev. Biochem. 73, 417–435
27. Guarente, L. (2007) Cold Spring Harb. Symp. Quant. Biol. 72, 483–488
28. Garten, A., Petzold, S., Ko ¨rner, A., Imai, S., and Kiess, W. (2009) Trends
Endocrinol. Metab. 20, 130–138
29. Nakahata, Y., Sahar, S., Astarita, G., Kaluzova, M., and Sassone-Corsi, P.
(2009) Science 324, 654–657
30. Hasmann, M., and Schemainda, I. (2003) Cancer Res. 63, 7436–7442
31. Zhang, Z., Lowry, S. F., Guarente, L., and Haimovich, B. (2010) J. Biol.
Chem. 285, 41391–41401
32. Irizar, A., Yu, Y., Reed, S. H., Louis, E. J., and Waters, R. (2010) Nucleic
Acids Res. 38, 4675–4686
33. Finkel, T., Deng, C. X., and Mostoslavsky, R. (2009) Nature 460, 587–591
35. Kawahara, T. L., Michishita, E., Adler, A. S., Damian, M., Berber, E., Lin,
M., McCord, R. A., Ongaigui, K. C., Boxer, L. D., Chang, H. Y., and Chua,
K. F. (2009) Cell 136, 62–74
36. Van Gool, F., Gallí, M., Gueydan, C., Kruys, V., Prevot, P. P., Bedalov, A.,
Mostoslavsky, R., Alt, F. W., De Smedt, T., and Leo, O. (2009) Nat. Med.
37. Imai, S., Johnson, F. B., Marciniak, R. A., McVey, M., Park, P. U., and
Guarente, L. (2000) Cold Spring Harb. Symp. Quant. Biol. 65, 297–302
38. Imai, S. (2009) Cell Biochem. Biophys. 53, 65–74
6T. F. Liu and C. E. McCall, unpublished data.
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E. McCall Download full-text
J. Biol. Chem.
Gazzar, Vidula T. Vachharajani and Charles
Tie Fu Liu, Barbara K. Yoza, Mohamed El
during Endotoxin Tolerance
Participates in Epigenetic Reprogramming
-dependent SIRT1 Deacetylase
doi: 10.1074/jbc.M110.196790 originally published online January 18, 2011
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