![Choline inhibits TNF release (A) and NF-κB activation (B) in endotoxinstimulated RAW-264.7 mouse macrophagelike cells. (A) RAW cells were exposed to the indicated concentration of choline 10 min prior to the addition of endotoxin (4 ng/mL). Culture supernatants were harvested 4 h later and TNF was determined by ELISA. Data represent the mean ± SEM of two representative experiments conducted in duplicate (*P < 0.04 as compared, with vehicle [V] treated controls). (B) RAW cells were exposed to the indicated concentrations of choline 10 min prior to the addition of endotoxin (4 ng/mL) and cells were harvested 2 h after endotoxin challenge for determination of NF-κB activation by EMSA. Autoradiographs were subjected to densitometry by using Quantity One software (Biorad). Data represent the mean ± SEM of three independent experiments (*P < 0.04, **P < 0.006 as compared with the lowest choline concentration tested).](https://www.researchgate.net/profile/Mauricio-Rosas-Ballina/publication/5272185/figure/fig3/AS:667629815615495@1536186760351/Choline-inhibits-TNF-release-A-and-NF-kB-activation-B-in-endotoxinstimulated_Q320.jpg)
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INTRODUCTION
The excessive production of the pro-
inflammatory cytokines TNF, high mo-
bility group box 1 (HMGB1), and other
inflammatory molecules by immune cells
and their subsequent release into the cir-
culation are associated with unrestrained
inflammation: a hallmark of septic shock,
sepsis, and other disorders (1,2). Exacer-
bated release of TNF and other pro-
inflammatory cytokines, and lethality
during endotoxemia and sepsis, can be
controlled by the efferent vagus nerve-
based cholinergic anti-inflammatory
pathway (3–7). Recent research has
demonstrated that the α7 subunit-
containing nicotinic acetylcholine recep-
tor (α7nAChR) is an important compo-
nent of the mechanism underlying the
anti-inflammatory efficacy of the cholin-
ergic anti-inflammatory pathway (4,8).
Activation of this pathway by stimula-
tion of the vagus nerve suppresses serum
TNF levels in endotoxemic animals (3,4),
but fails to cause statistically significant
effects in mice lacking the α7nAChR (4).
Accordingly, α7nAChR agonists, includ-
ing GTS-21, reduce systemic pro-
inflammatory cytokine levels during
murine endotoxemia, sepsis (9), and
other inflammatory conditions (10,11),
and improve survival (9).
Choline is a selective and endogenous
α7nAChR agonist (12–14). Choline also
has other important physiological func-
tions; this essential nutrient is a major
donor of methyl groups, a cell membrane
constituent, and a precursor in the
biosynthesis of the neurotransmitter
acetylcholine (15–17). Although previous
studies have shown protective effects of
choline against endotoxin-induced shock
and organ damage (18–20), the mecha-
nism of the anti-inflammatory action of
MOL MED 14(9-10)567-574, SEPTEMBER-OCTOBER 2008 | PARRISH ET AL. | 567
Modulation of TNF Release by Choline Requires α7 Subunit
Nicotinic Acetylcholine Receptor-Mediated Signaling
William R Parrish,1Mauricio Rosas-Ballina,1Margot Gallowitsch-Puerta,1Mahendar Ochani,1,5
Kanta Ochani,1Li-Hong Yang,1LaQueta Hudson,1Xinchun Lin,2Nirav Patel,1Sarah M Johnson,1
Sangeeta Chavan,1Richard S Goldstein,3Christopher J Czura,1Edmund J Miller,2,5 Yousef Al-Abed,4,5
Kevin J Tracey,1,5 and Valentin A Pavlov1,5
Address correspondence and reprint requests to Valentin A Pavlov, Laboratory of Biomed-
ical Science, The Feinstein Institute for Medical Research, 350 Community Drive, Manhas-
set, NY 11030. Phone: 516-562-2316; Fax: 516-562-2356; Email: vpavlov@nshs.edu.
Submitted June 19, 2008. Accepted for publication June 19, 2008; Epub (www.molmed.
org) ahead of print June 20, 2008.
1Laboratory of Biomedical Science, 2Department of Surgery, 3General Clinical Research Center, 4Laboratory of Medicinal Chemistry,
and 5Center for Immunology and Inflammation, The Feinstein Institute for Medical Research, North Shore-LIJ Health System,
Manhasset, New York, United States of America
The α7 subunit-containing nicotinic acetylcholine receptor (α7nAChR) is an essential component in the vagus nerve-based
cholinergic anti-inflammatory pathway that regulates the levels of TNF, high mobility group box 1 (HMGB1), and other cytokines
during inflammation.Choline is an essential nutrient,a cell membrane constituent, a precursor in the biosynthesis of acetylcholine,
and a selective natural α7nAChR agonist.Here, we studied the anti-inflammatory potential of choline in murine endotoxemia and
sepsis, and the role of the α7nAChR in mediating the suppressive effect of choline on TNF release. Choline (0.1–50 mM) dose-
dependently suppressed TNF release from endotoxin-activated RAW macrophage-like cells, and this effect was associated with
significant inhibition of NF-κB activation. Choline (50 mg/kg, intraperitoneally [i.p.]) treatment prior to endotoxin administration in
mice significantly reduced systemic TNF levels. In contrast to its TNF suppressive effect in wild type mice, choline (50 mg/kg, i.p.)
failed to inhibit systemic TNF levels in α7nAChR knockout mice during endotoxemia. Choline also failed to suppress TNF release
from endotoxin-activated peritoneal macrophages isolated from α7nAChR knockout mice. Choline treatment prior to endotoxin
resulted in a significantly improved survival rate as compared with saline-treated endotoxemic controls. Choline also suppressed
HMGB1 release
in vitro
and
in vivo
, and choline treatment initiated 24 h after cecal ligation and puncture (CLP)-induced polymi-
crobial sepsis significantly improved survival in mice. In addition, choline suppressed TNF release from endotoxin-activated human
whole blood and macrophages. Collectively,these data characterize the anti-inflammatory efficacy of choline and demonstrate
that the modulation of TNF release by choline requires α7nAChR-mediated signaling.
Online address: http://www.molmed.org
doi: 10.2119/2008-00079.Parrish
this compound is not well understood. A
particularly important question is
whether the α7nAChR, which is an es-
sential component of the cholinergic anti-
inflammatory pathway, mediates the
anti-inflammatory action of choline dur-
ing endotoxemia. Another relevant ques-
tion is whether choline suppresses the
pro-inflammatory cytokine response and
affects the survival rate during polymi-
crobial sepsis.
In this study, we provide evidence that
choline functions as an anti-inflammatory
molecule through an α7nAChR-
dependent mechanism. In contrast to its
anti-inflammatory effect in wild type
mice, choline failed to reduce endotoxin-
induced serum TNF levels in α7nAChR
KO mice and TNF release from peri-
toneal macrophages isolated from these
mice. These findings represent the first
direct experimental evidence that the
anti-inflammatory activity of a choliner-
gic agonist is mediated in vivo through
an α7nAChR-dependent mechanism. We
also show that choline suppresses the re-
lease of HMGB1, and choline treatment
initiated within a clinically relevant time
frame significantly improves survival in
mice with severe sepsis.
MATERIALS AND METHODS
Animals
Male mice (BALB/c at 25–28 g
[Taconic], and wild type [WT] or
α7nAChR knockout [KO] C57BL/6 at
8–12 wk old) were used for in vivo stud-
ies, and WT or α7nAChR KO C57BL/
6 female mice at 8–12 wk old were used
in ex vivo peritoneal macrophage stud-
ies. All C57BL/6 animals were bred on
site from heterozygous α7nAChR KO
animals obtained from Jackson Labora-
tories (Bar Harbor, ME, USA). The geno-
type of the α7nAChR locus (CHRNA7)
of all progeny was determined by ge-
nomic PCR using the Extract and Amp
kit (Sigma, St. Louis, MO, USA). Ani-
mals were housed in standard condi-
tions (room temperature 22° C with a
12-h light:dark cycle) with free access to
regular chow and water. Animals were
allowed to acclimate for at least 14 d be-
fore the corresponding experiment. All
animal experiments were performed in
accordance with the National Institutes
of Health Guidelines under protocols
approved by the Institutional Animal
Care and Use Committee of the Fein-
stein Institute for Medical Research,
North Shore-LIJ Health System, Man-
hasset, New York, United States of
America.
RAW Cells, Drug Treatment, TNF, and
HMGB1 Determination
RAW 264.7 cells were purchased from
the American Type Culture Collection
(ATCC TIB-71, Manassas, VA, USA) and
maintained in DMEM supplemented
with 10% heat-inactivated FBS, 2 mM
glutamine (Biowhittaker, Walkersville,
MD, USA) and 100 U/mL penicillin,
100 μg/mL streptomycin (both Gibco,
Carlsbad, CA, USA). Cell cultures were
maintained at 37° C, 5% CO2. Cells were
seeded in 48-well tissue culture plates at
5 ×105cells per well, and were allowed
to adhere for 24 h. Prior to adding com-
pounds, media were removed and re-
placed with serum-free Optimem media
(Gibco). Cells were exposed to lipopoly-
saccharide (LPS; endotoxin) (Escherichia
coli, L4130 0111:B4; Sigma) (4 mg/mL)
in the presence or absence of choline at
the concentrations indicated. Cell cul-
ture media were harvested 4 h after LPS
addition and centrifuged at 800gfor 5
min to sediment cell debris. Secreted
TNF was assayed from the media by
Enzyme-Linked ImmunoSorbent Assay
(ELISA) (R&D Systems, Minneapolis,
MN, USA) according to the manufac-
turer’s recommendations. For HMGB1
determination, cell culture media were
collected 24 h after endotoxin treatment
and centrifuged at 800gfor 5 min to re-
move cellular debris. HMGB1 was ana-
lyzed from cleared media by Western
blot as described previously (9). Briefly,
cleared medium was filtered through
Centricon YM-100 (Millipore, Billerica,
MA, USA) diluted 1:2 with 2 ×Laemmli
sample buffer (BioRad, Hercules, CA,
USA), and subjected to electrophoresis
through 10%–20% Tris-HCl acrylamide
gels (BioRad). Proteins were immobi-
lized onto PVDF membranes (Amer-
sham Pharmacia Biotech, Uppsala,
Sweden) and probed with polyclonal
anti-HMGB1 antibodies. Membranes
were developed using ECL Western
blotting detection reagents (Amersham
Pharmacia Biotech). Autoradiograph
films were scanned and densitometric
analyses performed using Quantity
One Software (BioRad). Standard curves
of human recombinant HMGB1 were
constructed and used to interpolate
HMGB1 levels in the samples. Cell via-
bility was monitored using trypan blue
exclusion.
Nuclear Protein Extraction for NF-κB
Activity Determination and
Electrophoretic Mobility-Shift Assay
(EMSA)
RAW 264.7 macrophages were treated
with the indicated concentrations of
choline followed by LPS (4 ng/mL).
Two h after LPS stimulation, cells were
processed for nuclear protein extraction
as described previously (9). EMSA was
performed using the Nushift NF-κB p65
kit (Active Motif, Carlsbad, CA, USA) ac-
cording to the manufacturer’s instruc-
tions, as described previously (9).
Endotoxemia and Drug Treatment
Endotoxemia was induced by injecting
mice intraperitoneally (i.p.) with 6 mg/kg
endotoxin, which caused ~80% mortality.
Mice were treated i.p. with the indicated
dose of choline or vehicle (sterile saline)
at 6 h, and at 30 min prior to endotoxin
administration. Animals were euthanized
by CO2asphyxiation 1.5 h after endotoxin
injection, and blood was collected by car-
diac puncture. Blood was centrifuged at
1,500gfor 15 min to isolate serum. Sera
were used for TNF analysis by ELISA
(R&D Systems) according to the manu-
facturer’s recommendations. In survival
experiments, mice were treated i.p. with
choline (50 mg/kg or 5 mg/kg) or sterile
saline (controls) at 6 h and at 30 min prior
to endotoxin injection. Survival was mon-
itored for 2 wks.
568 | PARRISH ET AL. | MOL MED 14(9-10)567-574, SEPTEMBER-OCTOBER 2008
CHOLINE ANTI-INFLAMMATORY EFFICACY
Isolation and Treatment of Mouse
Peritoneal Macrophages
WT or α7nAchR KO C57BL/6J female
mice were injected with 2 mL of 9%
thioglycollate broth i.p. to elicit peri-
toneal macrophages. Animals were eu-
thanized by CO2asphyxiation 42–48 h
later, and cells were collected by lavage
of the peritoneal cavity three times with
5 mL ice-cold 11.6% sucrose. Cells were
washed three times with phosphate-
buffered saline (PBS) and once with
RPMI 1640 medium and resuspended
in RPMI 1640 medium supplemented
with 10% heat-inactivated FBS, 2 mM
glutamine (Biowhittaker) and 100 U/mL
penicillin, 100 μg/mL streptomycin
(Gibco). Cells were seeded at 1.5 ×106
per well into 24-well Falcon Primaria
tissue culture dishes and were allowed
to adhere for 2 h at 37° C under 5% CO2.
Then, cells were washed twice with PBS
and supplied with fresh medium as de-
scribed above, returned to the incuba-
tor, and allowed to rest for 18–24 h.
Prior to treatment, media were removed
and replaced with serum free Optimem
media. Cells were incubated with the
indicated concentration of choline for
10 min prior to LPS (100 ng/mL)
exposure. Cell culture media were
harvested 4 h after LPS treatment and
TNF was assayed by ELISA (R&D Sys-
tems) according to the manufacturer’s
recommendations.
Cecal Ligation and Puncture Surgery
and Drug Treatment
Severe polymicrobial sepsis was in-
duced by cecal ligation and puncture
(CLP). Mice were anesthetized using
ketamine (100 mg/kg) and xylazine
(8 mg/kg) administered intramuscularly.
Abdominal access was gained via a mid-
line incision. The cecum was isolated and
ligated with a 6-0 silk ligature below the
ileocecal valve and then punctured once
with a 22 G needle. Stool (approximately
1 mm) was extruded from the hole, and
the cecum placed back into the abdomi-
nal cavity. The abdomen was closed with
two layers of 6-0 Ethilon sutures. An anti-
biotic (Imipenem-Cilastatin, 0.5 mg/kg,
subcutaneously, in a total volume of
0.5 mL/mouse) was administered imme-
diately after CLP as part of the resuscita-
tion fluid. Sham-operated animals had
the cecum isolated and then returned to
the peritoneal cavity without being lig-
ated or punctured. Sham animals also re-
ceived an antibiotic treatment and resus-
citative fluid as described above. For
HMGB1 determination, mice were ran-
domized 24 h after CLP and were in-
jected i.p. with either sterile saline or
choline (25 mg/kg). Mice received addi-
tional treatments at 30 h and 44 h after
CLP. Blood was collected by cardiac
puncture at 45 h after CLP, and serum
HMGB1 levels were determined by
quantitative Western blot analysis as de-
scribed above. For survival studies, 24 h
after CLP, mice were randomized and in-
jected i.p. with either sterile saline or
choline (25 mg/kg or 5 mg/kg). This
treatment was repeated 6 h later (30 h
after CLP) and then twice daily for 2 d
for a total of six treatments. Survival was
routinely monitored for 2 wks.
Isolation and Treatment of Human
Macrophages
Peripheral blood mononuclear cells
(PBMCs) were isolated by density gradi-
ent fractionation from whole blood that
was obtained from anonymous donors
through the Long Island Blood Services
(Westbury, NY, USA), and were differen-
tiated to macrophages in culture as de-
scribed previously (4). Briefly, PBMCs
were harvested from the plasma/Ficol-
Hypaque interface after centrifugation
for 30 min at 550g. Cells were washed
twice with PBS, once with RPMI 1640
medium, and resuspended in RPMI 1640
supplemented with 10% heat-inactivated
human serum, 2 mM glutamine
(Biowhittaker), and 100U/mL penicillin,
100 μg/mL streptomycin (Gibco). Cells
were then seeded at 5 ×107per 10 cm
Falcon Primaria tissue culture plate and
incubated for 2 h at 37° C in 5% CO2to
allow attachment. Plates were then
washed twice with PBS lacking Ca2+ and
Mg2+, and adherent cells were detached
by gentle scraping in PBS lacking Ca2+
and Mg2+ and containing 1mM EDTA.
Cells were washed once and resus-
pended in medium supplemented with
human recombinant macrophage colony-
stimulating factor (hrMCSF), and were
seeded into 24-well Falcon Primaria tis-
sue culture plates at 1 ×106cells per
well. Cultures were incubated for 6 d in
the presence of hrMCSF to promote mac-
rophage differentiation. Macrophages
were rested for 24 h in the absence of
hrMCSF prior to use. Media were re-
moved and replaced with serum free Op-
timem media prior to treatment. Cells
were incubated with the indicated con-
centration of choline for 10 min prior to
exposure to LPS (20 ng/mL). Cell culture
media were harvested 4 h after LPS treat-
ment and secreted TNF was assayed by
ELISA (R&D Systems) as described
above.
Statistical Analysis
Data are expressed as mean ±SEM.
Significant differences were assessed by
using one way analysis of variance
(ANOVA) followed by a Student ttest.
The statistical significance of differences
between groups of animals in survival
experiments was analyzed by the log-
rank test. Differences with P<0.05 were
considered statistically significant.
RESULTS
Choline Inhibits Endotoxin-Induced
TNF Release and NF-κB Activation
The anti-inflammatory efficacy of
cholinergic agonists has been tested pre-
viously by using RAW-264.7 mouse
macrophage-like cells (9), a well-
established cell culture system for in vitro
studies of innate immune cell inflamma-
tory responses. We studied the efficiency
of choline in inhibiting TNF release from
endotoxin-activated RAW cells. The cells
were pre-incubated for 10 min with in-
creasing choline concentrations prior to
endotoxin activation. As shown in Fig-
ure 1A, choline dose-dependently sup-
pressed endotoxin-stimulated TNF re-
lease from RAW cells. NF-κB is a key
transcription factor that is activated in
RESEARCH ARTICLE
MOL MED 14(9-10)567-574, SEPTEMBER-OCTOBER 2008 | PARRISH ET AL. | 569
response to endotoxin for the production
of inflammatory mediators such as TNF.
Therefore, we tested whether the
choline-induced suppression of TNF was
associated with inhibition of NF-κB acti-
vation. RAW cells were incubated for 10
min in the absence or presence of in-
creasing concentrations of choline prior
to exposure to endotoxin. Nuclear ex-
tracts were prepared and electrophoretic
mobility shift assays (EMSA) were con-
ducted to measure activated NF-κB.
Choline dose-dependently suppressed
NF-κB activation in response to endo-
toxin (Figure 1B).
Choline Suppresses Systemic TNF
Levels During Endotoxemia Through
an α7nAChR-Mediated Mechanism
Based on our in vitro data, we next
tested whether choline reduces systemic
TNF during endotoxemia. Choline
(5 mg/kg or 50 mg/kg) or vehicle
(saline) was injected into BALB/c mice at
6 h and at 30 min prior to endotoxin
(6 mg/kg i.p.) administration. This dose
of endotoxin previously was shown to
cause about 80% mortality (9). The
higher choline dose (50 mg/kg) signifi-
cantly suppressed serum TNF levels
(Figure 2A). The lower choline dose
(5 mg/kg) failed to alter serum TNF lev-
els (see Figure 2A). In light of the re-
cently discovered role for the α7nAChR
in mediating the cholinergic suppression
of systemic TNF during endotoxemia (4),
and the fact that choline is a selective
α7nAChR agonist, we tested the efficacy
of choline in suppressing systemic TNF
levels during endotoxemia in WT and
α7nAChR KO mice. WT and age-
matched α7nAChR KO mice were in-
jected i.p. with choline (50 mg/kg) or ve-
hicle (saline) at 6 h and at 30 min prior to
endotoxin (6 mg/kg, i.p.) administration.
Choline significantly suppressed sys-
temic TNF levels in endotoxemic WT
mice as compared with saline-treated en-
dotoxemic controls (Figure 2B, P<0.05).
In contrast, choline administration in
α7nAChR KO mice did not alter sys-
temic TNF levels significantly, as com-
pared with controls (see Figure 2B). Mac-
rophages represent a major source of
TNF during endotoxemia (21,22), and the
α7nAChR expressed on macrophages
plays a critical role in mediating cholin-
ergic anti-inflammatory signaling (4).
Therefore, we reasoned that choline may
suppress macrophage TNF release
through an α7nAChR-dependent signal-
ing mechanism. Accordingly, we exam-
ined the effect of choline on TNF release
from endotoxin-activated peritoneal
macrophages collected from WT mice
and α7nAChR KO mice. Peritoneal mac-
rophages were exposed to the indicated
concentrations of choline 10 min prior to
the addition of endotoxin, and TNF lev-
els were measured in media super-
natants collected 4 h later. As shown in
Figure 2C, choline dose-dependently
suppressed TNF release by macrophages
from WT mice. However, choline treat-
ment did not suppress TNF release by
macrophages isolated from α7nAChR
KO mice (see Figure 2C).
Choline Improves Survival in Lethal
Endotoxemia
We have shown previously that the
α7nAChR agonist GTS-21 significantly
improves survival of BALB/c mice dur-
ing endotoxemia (9). We next studied
whether choline improves survival in
lethal endotoxemia. Choline (5 mg/kg or
50 mg/kg, i.p.) or vehicle (saline, i.p.)
was injected into BALB/c mice at 6 h
and at 30 min prior to the i.p. adminis-
tration of endotoxin (6 mg/kg, i.p.). Ve-
hicle-treated mice showed a 27% survival
rate that was not improved by 5 mg/kg
choline (Figure 3). In contrast, treatment
with 50 mg/kg choline resulted in a sig-
nificantly improved survival rate of 63%
(see Figure 3).
Choline Suppresses HMGB1 Release
and Improves Survival in Mice with
Severe Sepsis
HMGB1 is a late pro-inflammatory cy-
tokine mediator of inflammation during
experimental sepsis and an important
therapeutic target in the treatment of this
disorder (23–25). We studied whether
choline suppresses HMGB1 release in
570 | PARRISH ET AL. | MOL MED 14(9-10)567-574, SEPTEMBER-OCTOBER 2008
CHOLINE ANTI-INFLAMMATORY EFFICACY
Figure 1. Choline inhibits TNF release (A)
and NF-κB activation (B) in endotoxin-
stimulated RAW-264.7 mouse macrophage-
like cells. (A) RAW cells were exposed to
the indicated concentration of choline
10 min prior to the addition of endotoxin
(4 ng/mL). Culture supernatants were
harvested 4 h later and TNF was deter-
mined by ELISA. Data represent the
mean ±SEM of two representative ex-
periments conducted in duplicate
(*
P
< 0.04 as compared, with vehicle [V]
treated controls). (B) RAW cells were ex-
posed to the indicated concentrations
of choline 10 min prior to the addition of
endotoxin (4 ng/mL) and cells were har-
vested 2 h after endotoxin challenge for
determination of NF-κB activation by
EMSA. Autoradiographs were subjected
to densitometry by using Quantity One
software (Biorad). Data represent the
mean ±SEM of three independent ex-
periments (*
P
< 0.04, **
P
< 0.006 as com-
pared with the lowest choline concen-
tration tested).
vitro and in vivo and improves survival
of mice with polymicrobial sepsis. Based
on our data that choline suppresses TNF
release and NF-κB activation in RAW
cells (see Figure 1A,1B), we tested
whether choline also attenuates HMGB1
release from RAW cells. Choline dose-
dependently reduced HMGB1 release
from endotoxin-stimulated RAW cells
(Figure 4A). We next studied whether
choline suppresses serum levels of
HMGB1 in mice with CLP-induced sep-
sis. Choline (25 mg/kg) or saline were in-
jected i.p. to septic mice 24 h after the
CLP surgery. Mice received additional
treatments 30 h and 44 h after CLP, and
serum HMGB1 levels were determined in
blood obtained at 45 h after CLP. Serum
HMGB1 levels were reduced by 85% in
the animals receiving choline as com-
pared with the saline-administered con-
trols (Figure 4B). It is noteworthy that the
mortality rate at the 45-h time point was
higher in saline-treated mice (5/12) com-
pared with the choline-treated group
(1/12) (data not shown). We then specifi-
cally tested whether choline improves
survival when therapeutically adminis-
tered to septic mice. Choline (25 mg/kg,
or 5 mg/kg) or vehicle (saline) was ad-
ministered i.p. to mice with CLP-induced
sepsis 24 h after surgery. This treatment
was repeated 6 h later (30 h after surgery)
and twice daily for 2 d more. The sur-
vival rate for choline-treated (25 mg/kg)
mice (64%) was improved significantly
when compared with control animals
(23%) (Figure 4C). The 42% survival rate
of septic mice treated with the lower
choline dose (5 mg/kg) was not signifi-
cantly different as compared with control
mice (see Figure 4C). These data show
that therapeutically administered choline
attenuates systemic HMGB1 levels and
improves survival in polymicrobial sepsis.
Choline Suppresses TNF Production
from Endotoxin-Stimulated Human
Whole Blood and Cultured
Macrophages
To examine the anti-inflammatory effi-
ciency of choline in human cells, we
RESEARCH ARTICLE
MOL MED 14(9-10)567-574, SEPTEMBER-OCTOBER 2008 | PARRISH ET AL. | 571
Figure 2. Choline suppresses systemic TNF levels during endotoxemia through an
α7nAChR-dependent mechanism. (A) Choline or vehicle (V, saline) was injected i.p. in
BALB/c mice (n = 10 per group) at 6 h, and at 30 min, prior to endotoxin (6 mg/kg, i.p.)
administration. Serum TNF was analyzed by ELISA in blood obtained 90 min after endo-
toxin administration. Results show the mean ±SEM for each group (*
P
< 0.05 as compared
with vehicle (V) administered controls). (B) Choline (50 mg/kg, i.p.) or saline was injected
i.p. in age-matched WT and α7nAchR KO mice (WT n = 8–9/group,α7nAChR KO n =
7–9/group) at 6 h, and at 30 min, prior to endotoxin (6 mg/kg, i.p.) administration. Serum
TNF was analyzed by ELISA in blood obtained 90 min after endotoxin administration.Re-
sults show the mean ±SEM for each treatment group (*
P
< 0.001 as compared with saline
administered controls). (C) Peritoneal macrophages from age-matched WT and α7nAChR
KO mice were incubated with the indicated concentrations of choline or vehicle (V) for
10 min prior to exposure to endotoxin (100 ng/mL). TNF in cell culture media was deter-
mined by ELISA 4 h after endotoxin addition. Results represent the mean ±SEM of three in-
dependent experiments conducted in duplicate (*
P
< 0.04, **
P
< 0.02, ***
P
< 0.002 as
compared with lowest choline concentration tested).
Figure 3. Choline improves survival in lethal
endotoxemia. BALB/c mice (n = 30/ group)
were injected i.p. with either vehicle (saline)
or choline at 6 h, and at 30 min, prior to en-
dotoxin (6 mg/kg, i.p.) administration. Sur-
vival was monitored for 14 d (*
P
< 0.002).
tested whether choline suppressed TNF
release from endotoxin-activated human
whole blood and cultured human macro-
phages. Blood was collected from
healthy volunteers and blood samples
were exposed to increasing concentra-
tions of choline or vehicle for 10 min
prior to the addition of endotoxin. As
shown in Figure 5A, choline (50 mM)
significantly suppressed endotoxin-
induced TNF release from human whole
blood. In a parallel set of experiments,
human macrophages that express the
α7nAChR (4) were differentiated from
peripheral blood mononuclear cells
(PBMCs) and treated with various
choline concentrations 10 min prior to
the addition of endotoxin. As shown in
Figure 5B, choline (1 mM) significantly
reduced TNF release from endotoxin-
stimulated human macrophages. The
level of TNF suppression did not in-
crease with higher concentrations of
choline (up to 50mM) (data not shown).
572 | PARRISH ET AL. | MOL MED 14(9-10)567-574, SEPTEMBER-OCTOBER 2008
CHOLINE ANTI-INFLAMMATORY EFFICACY
Figure 4. Choline treatment suppresses HMGB1 release and improves survival in severe
sepsis. (A) Choline suppresses HMGB1 release from endotoxin-stimulated RAW cells. RAW
cells were exposed to the indicated concentration of choline or vehicle 10 min prior to
LPS (100 ng/mL) addition for 24 h. Culture supernatants were harvested, and secreted
HMGB1 was detected by Western blot analysis. HMGB1 was not detected in the super-
natant from cells that were not treated with LPS. Data represent the mean ±SEM of four
experiments conducted in duplicate (*
P
< 0.05, **
P
< 0.02, ***
P
< 0.001 as compared with
lowest choline concentration tested). (B) Mice (n = 12) were administered i.p.with either
saline or choline (25 mg/kg) 24 h after CLP.Mice received additional treatments at 30 h
and 44 h after CLP.Serum HMGB1 levels were determined in surviving mice (n = 11 for
choline treatment, n = 7 for control treatment) in blood obtained at 45 h after CLP (*
P
<
0.0008). (C) Mice (n = 26 per group) were subjected to CLP surgery. 24 h after CLP, mice
were randomized and injected i.p. with either saline or choline (25 mg/kg). This treatment
was repeated 6 h later (30 h after CLP) and twice daily for 2 d more for a total of six treat-
ments, and survival was monitored for 2 wks (*
P
< 0.002).
Figure 5. Choline suppresses TNF release
from endotoxin-stimulated human
whole blood (A) and human macro-
phages (B). (A) Whole blood was
treated for 10 min with the indicated
concentrations of choline prior to endo-
toxin (10 ng/mL) challenge at 37° C.
Plasma TNF was determined by ELISA
4 h later. Data represent the mean ±
SEM of duplicate determinations from
five donors (*
P
< 0.0001) as compared
with the lowest choline concentration
tested). (B) Peripheral blood mononu-
clear cells (PBMCs) were isolated from
adult donors and were differentiated
into macrophages. Macrophages were
treated with the indicated concentra-
tion of choline 10 min prior to the addi-
tion of endotoxin (20 ng/mL). Culture su-
pernatants were harvested 4 h later
and the level of TNF secreted into the
media was determined by ELISA. Data
represent the mean ±SEM of at least
three experiments conducted in dupli-
cate from independent donors (*
P
<
0.02) as compared with the lowest
choline concentration tested.
DISCUSSION
In this study, we show that choline sup-
presses serum TNF levels in endotoxemic
mice and this anti-inflammatory effect of
choline is dependent on an α7nAChR-
mediated signaling. In addition, we dem-
onstrate the anti-inflammatory efficacy of
choline in experimental polymicrobial
sepsis and in human cells.
Choline suppressed systemic TNF lev-
els in endotoxemic mice, but failed to
reduce TNF levels in mice lacking the
α7nAchR (α7nAchR KO mice) and these
findings clearly indicate the α7nAChR
dependence of this anti-inflammatory in
vivo effect of choline. In contrast to its
suppressive effect on TNF release from
endotoxin-stimulated WT peritoneal
macrophages, choline did not suppress
TNF release from α7nAChR KO cells.
These results strengthen the concept
that α7nAChR expressed on macro-
phages and other immune cells plays a
critical role in controlling inflammatory
responses (4,26).
Choline, a byproduct of acetylcholine
degradation, is a stable, natural, and se-
lective agonist on α7nAChR. Earlier
studies indicated that acetylcholine sup-
presses TNF release from peritoneal
mouse macrophages and human macro-
phages (3,4), but it was unknown previ-
ously whether choline also can regulate
TNF in these cells. Interestingly, our
data show that choline concentrations
that suppress TNF release from these
endotoxin-stimulated immune cells are
significantly higher than acetylcholine
concentrations (in the presence of an
acetylcholinesterase inhibitor) that exert
similar suppressive effects (3,4). These
observations are in line with studies
showing a lower agonistic efficacy of
choline on neuronal α7nAChRs as com-
pared with acetylcholine (14,27,28) and
have implications for signaling after
acetylcholine release, because choline can
persist after acetylcholine degradation.
The expression of the α7nAChR in sev-
eral non-neuronal cells, including macro-
phages, monocytes, and dendritic cells,
has been documented (8,29,30). How-
ever, knowledge about the receptor func-
tion and pharmacological characteristics
related to cytokine production is very
limited. Previously, agonistic properties
of choline have been studied on neuronal
α7nAChRs and it has been shown that
choline is a full agonist on the α7nAChR
with an EC50 of 1.6 mM (12). These data
were suggestive for the drug concentra-
tions we used to study the effects of
choline on TNF release, NF-κB activa-
tion, and HMGB1 release in response to
endotoxin. Our results show that choline
concentrations required to cause statisti-
cally significant suppression of TNF re-
lease and NF-κB activation in vitro are
generally higher than those that sup-
press HMGB1 release. This observation
may indicate that the cellular mecha-
nisms governing the release of HMGB1
could be more sensitive to choline-
stimulated α7nAChR signaling than
those controlling the release of TNF. This
in vitro difference also was extrapolated
to the in vivo studies. While a choline
dose of 50 mg/kg was required to sup-
press serum TNF significantly and to im-
prove survival during endotoxemia, a
lower choline dose of 25 mg/kg signifi-
cantly inhibited serum HMGB1 levels
and improved survival in mice with
polymicrobial sepsis. A possible explana-
tion could be related to differences be-
tween the underlying inflammatory
mechanisms of endotoxemia and CLP-
sepsis. The survival-improving effect of
choline, administered i.p. 6 h and 30 min
prior to endotoxin is in line with a previ-
ous study, demonstrating that a choline-
rich diet improves survival in endotox-
emic rats (18). Moreover, our results that
choline treatment, initiated within a clin-
ically relevant time frame, improves sur-
vival in polymicrobial sepsis indicate the
potential for clinical development of
choline.
Choline deficiency has been shown
previously to induce liver injury in hu-
mans and in rodents, which is exacer-
bated upon endotoxin administration
(31,32). In contrast, choline (20 mg/kg,
intravenous [i.v.]) administration sup-
presses TNF release and attenuates in-
flammation during endotoxemia in dogs
(19,20). Moreover, choline (60 mg/kg,
i.v.) attenuates acid-induced lung injury
in mice (33). The effective doses of
choline used in the present study (25–50
mg/kg, i.p.) are within the dose range
used in these other studies. It is impor-
tant to note that we did not observe any
adverse neurobehavioral effects of these
choline doses, which are comparable
with the recommended tolerable upper
limit of dietary choline intake in humans
(34). Unlike other synthetic α7nAChR
agonists, choline is an endogenous mole-
cule with important physiological func-
tions, including its vital roles in main-
taining the structural integrity of cell
membranes and providing methyl
groups for the synthesis of betaine,
thus participating in methionine, folate,
and homocysteine metabolism (34).
While some of these metabolic functions
have been linked previously to anti-
inflammatory effects of choline (18,35)
our data clearly show that α7nAChR
signaling is required for the anti-
inflammatory efficacy of this compound
during endotoxemic shock. Our results
(data not shown) also indicate that
α7nAChR plays a role in mediating the
anti-inflammatory efficacy of choline in
polymicrobial sepsis. Therefore, our find-
ings bring new light to these previous
studies and suggest that endogenous
choline may act on the α7nAChR and
play an important role in regulating in-
nate immune responses to maintain ho-
meostasis. Choline also is a precursor
for the synthesis of acetylcholine, which
is the principle neurotransmitter of the
efferent vagus nerve (36). It is possible
that a portion of the exogenous choline is
metabolized as a substrate for acetyl-
choline biosynthesis, which may con-
tribute to anti-inflammatory effects in
vivo. In the cholinergic synapse, acetyl-
choline is degraded rapidly by acetyl-
cholinesterases into acetate and choline.
Choline generated in this mode may act
to prolong α7nAChR activation selec-
tively (36). While indicating a critical
anti-inflammatory role for choline as an
α7nAChR agonist, we cannot entirely ex-
clude the contribution of other effects of
RESEARCH ARTICLE
MOL MED 14(9-10)567-574, SEPTEMBER-OCTOBER 2008 | PARRISH ET AL. | 573
choline to its anti-inflammatory activity
in vivo, including the stimulation of
cholinergic signaling in the central nerv-
ous system (CNS), which has been
shown recently to play a role in control-
ling inflammation during endotoxemia
(37). However, it is possible that these al-
ternative pathways also culminate in
α7nAChR-mediated signaling.
Choline also suppressed TNF release
from endotoxin-activated human whole
blood and macrophages effectively, dem-
onstrating the anti-inflammatory efficacy
of this compound in human cells. In
conclusion, our data provide experimen-
tal evidence that the cholinergic agonist
choline suppresses TNF release through
an α7nAChR-dependent mechanism and
has therapeutic potential in the treat-
ment of sepsis and other inflammatory
diseases.
ACKNOWLEDGMENTS
This study was supported by a North
Shore-LIJ Health System Research Award
grant (to VAP), MO1 R018535, and
NIGMS R01 GM0557226-08A1 (to KJT).
REFERENCES
1. Tracey KJ, et al. (1986) Shock and tissue injury in-
duced by recombinant human cachectin. Science
234:470–4.
2. Tracey KJ. (2002) The inflammatory reflex. Nature
420:853–9.
3. Borovikova LV, et al. (2000) Vagus nerve stimula-
tion attenuates the systemic inflammatory re-
sponse to endotoxin. Nature 405:458–62.
4. Wang H, et al. (2003) Nicotinic acetylcholine re-
ceptor alpha7 subunit is an essential regulator of
inflammation. Nature 421:384–8.
5. Huston JM, et al. (2007) Transcutaneous vagus
nerve stimulation reduces serum high mobility
group box 1 levels and improves survival in
murine sepsis. Crit. Care Med. 35:2762–8.
6. Pavlov VA, Wang H, Czura CJ, Friedman SG,
and Tracey KJ. (2003) The cholinergic anti-
inflammatory pathway: a missing link in neuro-
immunomodulation. Mol. Med. 9:125–34.
7. Tracey KJ. (2007) Physiology and immunology of
the cholinergic antiinflammatory pathway. J. Clin.
Invest. 117:289–96.
8. Gallowitsch-Puerta M and Pavlov VA. (2007)
Neuro-immune interactions via the cholinergic
anti-inflammatory pathway. Life Sci. 80:2325–9.
9. Pavlov VA, et al. (2007) Selective alpha7-nicotinic
acetylcholine receptor agonist GTS-21 improves
survival in murine endotoxemia and severe sep-
sis. Crit. Care Med. 35:1139–44.
10. van Westerloo DJ, et al. (2006) The vagus nerve
and nicotinic receptors modulate experimental
pancreatitis severity in mice. Gastroenterology
130:1822–30.
11. Yeboah MM, et al. (2008) Cholinergic agonists at-
tenuate renal ischemia-reperfusion injury in rats.
Kidney Int. 74:62–9.
12. Alkondon M, Pereira EF, Cortes WS, Maelicke A,
and Albuquerque EX. (1997) Choline is a selec-
tive agonist of alpha7 nicotinic acetylcholine re-
ceptors in the rat brain neurons. Eur. J. Neurosci.
9:2734–42.
13. Mandelzys A, De Koninck P, and Cooper E.
(1995) Agonist and toxin sensitivities of ACh-
evoked currents on neurons expressing multiple
nicotinic ACh receptor subunits. J. Neurophysiol.
74:1212–21.
14. Papke RL, Bencherif M, and Lippiello P. (1996)
An evaluation of neuronal nicotinic acetylcholine
receptor activation by quaternary nitrogen com-
pounds indicates that choline is selective for the
alpha 7 subtype. Neurosci. Lett. 213:201–4.
15. Blusztajn JK. (1998) Choline, a vital amine. Sci-
ence 281:794–5.
16. Zeisel SH. (2000) Choline: an essential nutrient
for humans. Nutrition 16:669–71.
17. Canty DJ and Zeisel SH. (1994) Lecithin and
choline in human health and disease. Nutr. Rev.
52:327–39.
18. Rivera CA, Wheeler MD, Enomoto N, and Thur-
man RG. (1998) A choline-rich diet improves sur-
vival in a rat model of endotoxin shock. Am. J.
Physiol. 275:G862–7.
19. Ilcol YO, Yilmaz Z, and Ulus IH. (2005) Endo-
toxin alters serum-free choline and phospho-
lipid-bound choline concentrations, and choline
administration attenuates endotoxin-induced
organ injury in dogs. Shock 24:288–93.
20. Yilmaz Z, Ilcol YO, Torun S, and Ulus IH. (2006)
Intravenous administration of choline or cdp-
choline improves platelet count and platelet clo-
sure times in endotoxin-treated dogs. Shock
25:73–9.
21. Chensue SW, Terebuh PD, Remick DG, Scales
WE, and Kunkel SL. (1991) In vivo biologic and
immunohistochemical analysis of interleukin-1
alpha, beta and tumor necrosis factor during ex-
perimental endotoxemia. Kinetics, Kupffer cell
expression, and glucocorticoid effects. Am. J.
Pathol. 138:395–402.
22. Grivennikov SI, et al. (2005) Distinct and non-
redundant in vivo functions of TNF produced by
t cells and macrophages/neutrophils: protective
and deleterious effects. Immunity 22:93–104.
23. Wang H, Yang H, and Tracey KJ. (2004) Extracel-
lular role of HMGB1 in inflammation and sepsis.
J. Intern. Med. 255:320–31.
24. Lotze MT and Tracey KJ. (2005) High-mobility
group box 1 protein (HMGB1): nuclear weapon
in the immune arsenal. Nat. Rev. Immunol.
5:331–42.
25. Parrish W and Ulloa L. (2007) High-mobility
group box-1 isoforms as potential therapeutic
targets in sepsis. Methods Mol. Biol. 361:145–62.
26. Saeed RW, et al. (2005) Cholinergic stimulation
blocks endothelial cell activation and leukocyte
recruitment during inflammation. J. Exp. Med.
201:1113–23.
27. Papke RL, et al. (2005) Rhesus monkey alpha7
nicotinic acetylcholine receptors: comparisons to
human alpha7 receptors expressed in Xenopus
oocytes. Eur. J. Pharmacol. 524:11–8.
28. Papke RL and Porter Papke JK. (2002) Compara-
tive pharmacology of rat and human alpha7
nAChR conducted with net charge analysis. Br. J.
Pharmacol. 137:49–61.
29. Cloez-Tayarani I and Changeux JP. (2007) Nico-
tine and serotonin in immune regulation and in-
flammatory processes: a perspective. J. Leukoc.
Biol. 81:599–606.
30. Kawashima K, Yoshikawa K, Fujii YX, Moriwaki
Y, and Misawa H. (2007) Expression and function
of genes encoding cholinergic components in
murine immune cells. Life Sci. 80:2314–9.
31. Nolan JP and Vilayat M. (1968) Endotoxin and
the liver. I. Toxicity in rats with choline deficient
fatty livers. Proc. Soc. Exp. Biol. Med. 129:29–31.
32. Eastin CE, McClain CJ, Lee EY, Bagby GJ, and
Chawla RK. (1997) Choline deficiency augments
and antibody to tumor necrosis factor-alpha at-
tenuates endotoxin-induced hepatic injury. Alco-
hol Clin. Exp. Res. 21:1037–41.
33. Su X, et al. (2007) Activation of the alpha7
nAChR reduces acid-induced acute lung injury
in mice and rats. Am. J. Respir. Cell Mol. Biol.
37:186–92.
34. Zeisel SH. (2006) Choline: critical role during
fetal development and dietary requirements in
adults. Annu. Rev. Nutr. 26:229–50.
35. Kim SK and Kim YC. (2002) Attenuation of bac-
terial lipopolysaccharide-induced hepatotoxicity
by betaine or taurine in rats. Food Chem. Toxicol.
40:545–9.
36. Pavlov VA and Tracey KJ. (2005) The cholinergic
anti-inflammatory pathway. Brain Behav. Immun.
19:493–9.
37. Pavlov VA. et al. (2006) Central muscarinic
cholinergic regulation of the systemic inflamma-
tory response during endotoxemia. Proc. Natl.
Acad. Sci. U. S. A. 103:5219–23.
574 | PARRISH ET AL. | MOL MED 14(9-10)567-574, SEPTEMBER-OCTOBER 2008
CHOLINE ANTI-INFLAMMATORY EFFICACY
