ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, July 2006, p. 2420–2427
Copyright © 2006, American Society for Microbiology. All Rights Reserved.
Vol. 50, No. 7
The Antimalarial Artemisinin Synergizes with Antibiotics To
Protect against Lethal Live Escherichia coli Challenge by
Decreasing Proinflammatory Cytokine Release
Jun Wang,1Hong Zhou,1* Jiang Zheng,2* Juan Cheng,1Wei Liu,1Guofu Ding,1Liangxi Wang,1
Ping Luo,1Yongling Lu,1Hongwei Cao,2Shuangjiang Yu,1Bin Li,1and Lezhi Zhang1
Department of Pharmacology, College of Medicine, The Third Military Medical University, Chongqing 400038,
People’s Republic of China,1and Medical Research Center, Southwestern Hospital,
The Third Military Medical University, Chongqing 400038, People’s Republic of China2
Received 16 August 2005 /Returned for modification 29 September 2005/Accepted 14 April 2006
In the present study artemisinin (ART) was found to have potent anti-inflammatory effects in animal models
of sepsis induced by CpG-containing oligodeoxy-nucleotides (CpG ODN), lipopolysaccharide (LPS), heat-
killed Escherichia coli 35218 or live E. coli. Furthermore, we found that ART protected mice from a lethal
challenge by CpG ODN, LPS, or heat-killed E. coli in a dose-dependent manner and that the protection was
related to a reduction in serum tumor necrosis factor alpha (TNF-?). More significantly, the administration
of ART together with ampicillin or unasyn (a complex of ampicillin and sulbactam) decreased mortality from
100 to 66.7% or 33.3%, respectively, in mice subjected to a lethal live E. coli challenge. Together with the
observation that ART alone does not inhibit bacterial growth, this result suggests that ART protection is
achieved as a result of its anti-inflammatory activity rather than an antimicrobial effect. In RAW264.7 cells,
pretreatment with ART potently inhibited TNF-? and interleukin-6 release induced by CpG ODN, LPS, or
heat-killed E. coli in a dose- and time-dependent manner. Experiments utilizing affinity sensor technology
revealed no direct binding of ART with CpG ODN or LPS. Flow cytometry further showed that ART did not
alter binding of CpG ODN to cell surfaces or the internalization of CpG ODN. In addition, upregulated levels
of TLR9 and TLR4 mRNA were not attenuated by ART treatment. ART treatment did, however, block the
NF-?B activation induced by CpG ODN, LPS, or heat-killed E. coli. These findings provide compelling evidence
that ART may be an important potential drug for sepsis treatment.
Sepsis is a potentially lethal condition that results from a
harmful or damaging host response to infection (1, 3, 8, 11).
Sepsis is triggered by bacteria and bacterial components, such
as bacterial DNA (bDNA) and lipopolysaccharide (LPS) (26,
28, 29). Delivery of CpG-containing oligodeoxy-nucleotides
(CpG ODN) can trigger sepsis by mimicking the immunostim-
ulatory effects of bDNA and therefore has provided a useful
animal model of the sepsis condition (21, 23, 26).
Recent surveys conducted in the United States and in
Europe have indicated that approximately 2 to 11% of all
hospital and intensive care unit admissions can be attributed to
severe sepsis. Despite improvements in supportive care and the
increased availability of effective antibacterial agents, hospital
mortality rates from severe sepsis and septic shock (50 to 60%)
have not improved over recent decades (1, 28). Unfortunately,
many experimental inflammatory antagonist-based therapies
have failed in sepsis trials, and currently there is only one
adjuvant therapy in clinical use, e.g., activated protein C, which
targets the coagulation system (28). Thus, it is important to
investigate additional inflammatory antagonist-based treat-
ments with the aim of developing a clinically effective antisep-
Antimalarial drugs such as chloroquine (CQ) and artemisi-
nin (ART) are promising candidates for sepsis treatment. CQ
has been demonstrated to protect mice from CpG ODN and
LPS challenges in vivo via a mechanism that involves a reduc-
tion of proinflammatory cytokine release (14, 18). The protec-
tive effects afforded by CQ may be tightly related to interrupt-
ing endosome maturation (14, 18). The antimalarial drug ART
inhibits the endocytosis of macromolecular tracers by up to
85% in Plasmodium falciparum (15) and may suppress tumor
necrosis factor alpha (TNF-?) and interleukin-6 (IL-6) release
by inhibiting endocytosis of CpG ODN. ART has traditionally
been used to treat malaria (20, 22). It is the active ingredient in
the Chinese herb sweet wormwood, and its derivatives include
dihydroartemisinin, artesunate, artemether, and arteethe. ART
has many applications in addition to treatment of severe malaria,
including use as an antitumor agent (22).
In the present study we examined whether ART provides
protection against animal models of sepsis. Specifically, we
investigated the effects of ART on CpG ODN, LPS, and heat-
killed Escherichia coli- or live E. coli-challenged mice in vitro
and in vivo and further examined the possible molecular mech-
anisms involved in ART inhibition of proinflammatory cyto-
* Corresponding authors. Mailing address for H. Zhou: Department of
Pharmacology, College of Medicine, The Third Military Medical Univer-
sity, Gaotanyan Street 30, Shapingba District, Chongqing 400038, Peo-
ple’s Republic of China. Phone: 86-23-6875-2266. Fax: 86-23-6875-2266.
E-mail: email@example.com. Mailing address for J. Zheng:
Medical Research Center, Southwestern Hospital, The Third Military
Medical University, Gaotanyan Street 30, Shapingba District, Chongqing
400038, People’s Republic of China. Phone: 86-23-6875-4435. Fax: 86-23-
6875-4435. E-mail: firstname.lastname@example.org.
MATERIALS AND METHODS
Materials. LPS from E. coli O111:B4, CQ, D-galactosamine (D-GalN), di-
methyl sulfoxide (DMSO), and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazo-
lium (MTT) were purchased from Sigma (St. Louis, MO). Ampicillin (AMP) and
unasyn (a 2:1 mixture of AMP sodium and sulbactam sodium) were purchased
from the North China Pharmaceutical Group Corp. (Shijiazhuang, China). ART
was purchased from Wulingshan Pharm (Chongqing, China). For in vitro exper-
iments, ART was dissolved in DMSO to make a stock solution of 320 ?g/ml, and
the final concentrations of DMSO were 0.5% (vol/vol) in each cell culture plate
well. For in vivo experiments, ART was ground down to a fine powder and then
added to carboxymethyl cellulose (CMC; China Chengdu Kelong Chemical Re-
agent Factory) solution. The final concentration of CMC used in the experiments
was 0.5% (wt/vol).
Mouse TNF-? and IL-6 enzyme-linked immunosorbent assay (ELISA) kits
were purchased from Biosource International (Camarillo, CA). The RNAeasy
kit was obtained from QIAGEN (Chatsworth, CA). Avian myeloblastosis virus
(AMV) reverse transcriptase and T4 polynucleotide kinase were obtained from
Promega (Madison, WI), and [?-32P]dATP (5,000 Ci/mmol) was purchased from
Furui (Beijing, China). CpG ODN 1826 (5?-TCCATGACGTTCCTGATGCT-3?)
with a nuclease-resistant phosphorothioate backbone, 6-FAM fluorescein-la-
beled CpG ODN (6-FAM CpG ODN), and 5? biotinylated CpG ODN, and all
primers were synthesized by Bioasia Biotechnology, Ltd. (Shanghai, China), and
were determined to be endotoxin negative by Limulus assays.
Bacterial strain and culture. E. coli ATCC 35218 cells maintained in our
laboratory were used for the murine sepsis model. Single colonies from viable,
growing LB agar plates were transferred to sterile liquid LB medium (10 g of
tryptone, 10 g of NaCl, and 5 g of yeast extract per liter) and cultivated in 50-ml
volumes aerobically at 37°C in a heated, shaking environmental chamber for
12 h. These cultures were then transferred to 500 ml of fresh LB medium for
another 12 h. The cells were collected by centrifugation at 9,391 ? g for 5 min at
4°C; the pellet was then washed with sterile saline, and the suspension was
centrifuged (9,391 ? g for 5 min at 4°C). After resuspending the pellet, cell
densities were measured by using UV-visible spectrophotometry (absorbency at
600 nm) and adjusted to optical density values of 0.5 (ca. 2.5 ? 108CFU/ml).
Finally, bacterial suspensions were incubated in a water bath at 100°C for 10 min
in order to inactivate the cells.
MIC determinations. An overnight strain of E. coli ATCC 35218 was tested
against different concentrations of antibiotics and ART in LB medium. The
ranges of concentration assayed for drugs were 0.5 to 512 ?g/ml. MIC was
assayed at 5.0 ? 105CFU/ml by the broth microdilution method according to
procedures outlined by the Clinical and Laboratory Standards Institute (formerly
National Committee for Clinical Laboratory Standards) (25). Inoculated broth
samples in polypropylene 96-well plates (Sigma-Aldrich) were then incubated at
37°C for 24 h. The MIC was taken at the lowest drug concentration at which
observable growth was inhibited.
Cell line and culture. Murine macrophage RAW264.7 cells were purchased
from the American Type Culture Collection (Manassas, VA) and cultured in
Dulbecco modified Eagle medium (DMEM) supplemented with 10% low endo-
toxin fetal calf serum (HyClone, Logan, UT), 2 ?M glutamine, 100 U of peni-
cillin/ml, and 100 ?g of streptomycin/ml in a 37°C humid atmosphere with 5%
CO2. The cells were diluted with 0.4% trypan blue in phosphate-buffered saline
(PBS; 0.1 mM [pH 7.2]), and live cells were counted with a hemacytometer. In
each experiment, 106cells/ml were used, except where otherwise indicated.
Mouse sepsis model and in vivo cytokine assays. BALB/c mice (4 to 8 weeks
old) were obtained from the Experimental Animal Center of The Chongqing
Medical University (Chongqing, People’s Republic of China). All experiments
were conducted in accordance with the National Guidelines for the Care and Use
of Laboratory Animals. Equal numbers of male and female mice were used.
During experiments, D-GalN sensitization is used to reduce the amount of bDNA
or CpG ODN (13). ART (5% [wt/vol]) was administered orally in all animal
experiments; the total volume was 0.4 ml per 20 g of body weight; and 0.2 ml per
20 g of body weight of CpG ODN, LPS, heat-killed E. coli, or live E. coli and
antibiotics was injected intravenously.
In the first series of experiments, mice were challenged with CpG ODN, LPS,
or heat-killed E. coli. A total of 175 mice were weighed on the day of the
experiment, and their average weight was 19.2 ? 2.1 g. For the CpG ODN
challenge, mice were randomly divided into five groups (n ? 10 animals per
group), and all mice except for those in the control group were preinoculated
with D-GalN (intraperitoneally, 600 mg/kg [weight]) 1 h before the experiment.
Mice were divided into the following treatment groups: (i) saline injection only,
(ii) CpG ODN (10 mg/kg), (iii) low dose of ART (50 mg/kg, orally) and imme-
diate subsequent CpG ODN injection, (iv) medium dose of ART (100 mg/kg)
and immediate subsequent CpG ODN injection, and (v) high dose of ART (200
mg/kg) and immediate subsequent CpG ODN injection.
For the LPS challenge, mice were randomly divided into the following five treat-
ment groups (n ? 15 animals per group): (i) saline injection only; (ii) LPS (10
mg/kg); (iii) low dose of ART (50 mg/kg, orally) and immediate subsequent LPS
injection; (iv) medium dose of ART (100 mg/kg) and immediate subsequent LPS in-
jection; and (v) high dose of ART (200 mg/kg) and immediate subsequent LPS
injection. For the heat-killed E. coli challenge, mice were randomly divided into the
following five treatment groups (n ? 10 animals per group): (i) saline injection only;
(ii) heat-killed E. coli (1.5 ? 1011CFU/kg); (iii) low dose of ART (50 mg/kg, orally)
and immediate subsequent injection of heat-killed E. coli; (iv) medium dose of ART
(100 mg/kg) and immediate subsequent injection of heat-killed E. coli; and (5) high
dose of ART (200 mg/kg) and immediate subsequent injection with heat-killed E.
coli. All mice were observed for 7 days posttreatment, and general health and
mortality were noted.
In the second series of experiments, 48 mice were weighed on the day of the
experiment, and the average weight was 18.9 ? 2.0 g. Mice were randomly
divided into the following eight treatment groups (n ? 6 animals per group): (i)
saline injection only; (ii) orally administered ART (200 mg/kg) and saline injec-
tion; (iii) CpG ODN (10 mg/kg); (iv) ART (200 mg/kg) and immediate subse-
quent injection of CpG ODN; (v) LPS (10 mg/kg); (vi) ART (200 mg/kg) and
immediate subsequent LPS injection; (vii) heat-killed E. coli (1.5 ? 1011CFU/
kg); and (viii) ART (200 mg/kg) and immediate subsequent injection of heat-
killed E. coli. At 4 h after stimulator injection, 0.5 ml of blood was collected from
each mouse. The serum was separated and stored at ?80°C for subsequent
TNF-? assay using mouse ELISA kits.
In the third series of experiments, mice were challenged with live E. coli. A total
of 48 mice were weighed on the day of the experiment, and their average weight was
19.8 ? 1.2 g. Seven groups (n ? 6 animals per group) of mice were challenged with
orally administered ART (100 mg/kg) and immediate subsequent injection of live E.
coli; (iv) 400 mg of AMP/kg and immediate subsequent injection of live E. coli; (v)
ART (100 mg/kg), with immediate subsequent injection of both 400 mg of AMP/kg
and live E. coli; (vi) 600 mg of unasyn/kg (400 mg of AMP/kg) and immediate
injection of live E. coli; and (vii) 600 mg of unasyn/kg and immediate injection of
both live E. coli and ART (100 mg/kg).
In vitro cytokine release assays. In dose-dependent experiments, RAW264.7
cells (106cells/m, 0.4 ml) plated in 48-well plates were preincubated with 5 to 80
?g of ART/ml for 2 h and then stimulated with LPS (0.1 ?g/ml), CpG ODN (10
?g/ml), or heat-killed E. coli (3.5 ? 107CFU/ml) for 4 or 6 h. In time-dependent
experiments, RAW264.7 cells were either preincubated with 40 ?g of ART/ml 2
or 4 h prior to the addition of the specified stimulator, simultaneously treated
with ART and stimulators, or treated with ART 1 or 2 h after the addition of the
specified stimulating agent: LPS (0.1 ?g/ml), CpG ODN (10 ?g/ml), or heat-
killed E. coli (3.5 ? 107CFU/ml). After incubation for another 4 or 6 h, the cells
were centrifuged, and 0.1 ml of supernatant was collected for TNF-? or IL-6
MTT assay. RAW264.7 cells (5 ? 104cells/ml, 0.2 ml) plated in 96 wells were
incubated overnight and then further incubated for 4 h with ART in the range of
0 to 320 ?g/ml. Cells were then washed twice in PBS, and 180 ?l of fresh medium
and 20 ?l of MTT (5 mg/ml) was added to each well, followed by incubation for
another 4 h. The cells were then centrifuged at 138 ? g for 5 min at 4°C, the
supernatant was removed, and 150 ?l of DMSO was added to each well. MTT
crystals were completely solubilized with libration for 10 min. Optical density
values were determined at 490 nm.
Affinity assessment. Lipid A was immobilized on the surface of a hydrophobic
cuvette (Thermo Labsystem) as described previously (17). Biotinylated CpG
ODN was then immobilized on the surface of a streptavidin-coated biotin cuvette
according to the manufacturer’s instructions. Briefly, 5 ?l of biotinylated CpG
ODN and 45 ?l of PBST (PBS containing 0.05% Tween 20 [pH 7.4]) were added
to the cuvette and allowed to bind for about 5 min. The cuvette was then washed
three times with 50 ?l of PBST, and baseline data were collected for approxi-
mately 3 min. After lipid A or CpG ODN molecules were immobilized on the
surface of the hydrophobic cuvette, 5 to 40 ?g of ART/ml was added and a
binding curve was generated. Kdvalues were measured with an Affinity Sensors
IAsys cuvette system. Additional analyses were performed with the FASTplot
and FASTfit software packages (Thermo Labsystem).
Binding and internalization of 6-FAM CpG ODN. Cell surface DNA binding
and internalization experiments were performed as described previously (30).
Briefly, RAW264.7 cells (106cells/ml, 0.5 ml) were preincubated with a range of
concentrations of ART (5 to 40 ?g/ml) or CQ (50 ?g/ml) for 2 h, and 10 ?g of
6-FAM CpG ODN/ml was added. Cells were further cultured in the dark at 4°C
for 0.5 h (for binding) or for 1 h at 37°C (for internalization). After incubation,
VOL. 50, 2006ARTEMISININ PROTECTS AGAINST E. COLI CHALLENGE2421
the cells were washed twice with PBS and resuspended in 500 ?l of PBS. Cells
were analyzed with a FACScan flow cytometer (Becton Dickinson, San Jose,
CA). The fluorescence intensity was analyzed with CELLQuest software (Becton
TLR9 and TLR4 mRNA expressions. RAW264.7 cells (2 ? 106/ml, 2 ml)
plated in 24-well polystyrene plates were incubated in the absence or presence of
40 ?g of ART/ml for 2 h at 37°C. Cells were then recultured for 4 h with CpG
ODN (10 ?g/ml), LPS (0.1 ?g/ml), or heat-killed E. coli (3.5 ? 107CFU/ml).
After removal of the medium and washing the cells once with sterile PBS, total
RNA was purified with an RNAeasy kit according to the manufacturer’s instruc-
tions (QIAGEN, Valencia, CA). After DNase I treatment, 2 ?g of RNA was
reverse transcribed with AMV reverse transcriptase, and 2 ?l of cDNA was
subjected to 33 cycles of PCR. A master mix containing reaction buffer, de-
oxynucleoside triphosphates, Taq polymerase, and 2 ?l of cDNA per 25 ?l of
reaction mixture was distributed into PCR tubes. Forward and reverse primers
corresponding to different individual genes were added to the PCR tubes and
subjected to PCR amplification using primer sets designed against ?-actin,
TLR9, and TLR4. The reactions were run for 34 cycles. Temperatures and times
were 51°C for 30 s for annealing and 94°C for 30 s for denaturing, followed by
extension at 72°C for 20 s. The PCR products were determined by using 1.5%
agarose gel electrophoresis and ethidium bromide staining. Images of the gels
were analyzed by Quantity One software (Bio-Rad, California), which compares
the relative density of objective strap and ?-actin. The PCR primers were de-
signed by Primer Premier 5 software (PREMIER Biosoft International, Califor-
nia) and synthesized by Bioasia Biotechnology, Ltd. (Shanghai, People’s Repub-
lic of China). The primers used were as follows: for TLR4 (285-bp product),
forward (5?-TTTATTCAGAGCCGT TGG-3?) and reverse (5?-TGCCGT TTC
TTG TTC-3?); for TLR9 (287-bp product), forward (5?-TGGACGGGAACT
GCTACT-3?) and reverse (5?-GCCACATTCTATACA GGGATT-3?); and for
mouse ?-actin (455-bp product), forward (5?-CCCTGTATG CCTCTGGTC-3?)
and reverse (5?-TTTACGGATGTCAACG-3?).
EMSA of NF-?B. RAW264.7 cells (2 ? 106cells/ml, 2 ml) were plated in
six-well plates for 6 h, and then the supernatants were discarded and 2 ml of fresh
DMEM without fetal calf serum was added. The cells were harvested after a
24-h incubation with CpG ODN (10 ?g/ml), LPS (0.1 ?g/ml), or heat-killed
E. coli (3.5 ? 107CFU/ml). Nuclear proteins were then extracted, and an
electrophoretic mobility shift assay (EMSA) was performed as previously
Statistics and presentation of data. The chi-squared test was used to analyze
the significance of mouse mortality differences among groups. Cytokine concen-
trations are expressed as means ? the standard deviation. Each experiment was
repeated a minimum of three times, and each datum point represents the mean
of at least three parallel samples. The Student t test was used to examine the
differences in cytokine concentrations in the cell supernatants and fluorescence
intensity. A P value of ?0.05 was considered significant, and a value of ?0.01 was
considered very significant; values of ?0.05 were considered not significant.
ART protects mice from challenge by CpG ODN, LPS, or
heat-killed E. coli by decreasing proinflammatory cytokine re-
lease. About 80% of the mice challenged with CpG ODN, LPS,
or heat-killed E. coli died within the first 24 h. In contrast, only
30 to 40% of mice in the groups treated with 50 mg of ART/kg
prior to challenge died within 48 h. The larger doses of ART,
100 and 200 mg/kg, provided even as high as 90% protection
from the lethal challenges (Fig. 1, 2, and 3).
The release of a combination of several cytokines acts as a
good indicator of sepsis and SIRS (28, 30). For example,
FIG. 1. Survival of mice challenged with CpG ODN. Mice were
randomly divided into five groups (n ? 10 animals per group) and
preinoculated with D-GalN (i.p., 600 mg/kg [weight]) 1 h before the
experiment. Treatment groups are indicated by symbols as follows: ?,
saline alone; ■, CpG ODN (10 mg/kg); ?, CpG ODN and ART (50
mg/kg); ?, CpG ODN and ART (100 mg/kg); and ‚, CpG ODN and
ART (200 mg/kg). ?, P ? 0.01; ??, P ? 0.05 versus the CpG ODN
FIG. 2. Survival of mice challenged with LPS. The mice were ran-
domly divided into five groups (n ? 15 animals per group): ?, saline
alone; ■, LPS (10 mg/kg); ?, LPS and ART (50 mg/kg); ?, LPS and
ART (100 mg/kg); and ‚, LPS and ART (200 mg/kg). ?, P ? 0.01; ??,
P ? 0.05 versus the LPS group.
FIG. 3. Survival of mice challenged with heat-killed E. coli. The
mice were randomly divided into five groups (n ? 10 animals per
group): ?, saline alone; ■, heat-killed E. coli (1.5 ? 1011CFU/kg); ?,
ART (50 mg/kg) and heat-killed E. coli; ?, ART (100 mg/kg) and
heat-killed E. coli; and ‚, ART (200 mg/kg) and heat-killed E. coli. ?,
P ? 0.01; ??, P ? 0.05 versus heat-killed E. coli.
2422 WANG ET AL.ANTIMICROB. AGENTS CHEMOTHER.
TNF-? is thought to be an early-released cytokine during sep-
sis. Thus, serum levels were tested to determine whether the
protective effects of ART may be related to decreased cytokine
release in mice. We found that TNF-? levels were higher in
serum from mice challenged with CpG ODN, LPS, or heat-
killed E. coli than in the control group. However, pretreatment
with 200 mg of ART/kg significantly reduced TNF-? release
ART with antibiotics has synergistic protective effects on
mice challenged by live E. coli. To further confirm ART’s
apparent ability to protect mice challenged with live bacteria,
mice were injected with live E. coli. In this live bacterial sepsis
model mortality was significantly reduced from 100% to 66.7%
or 33.3% when mice were treated with ART (100 mg/kg) in
combination with AMP or unasyn, respectively. Mice treated
with ART alone or AMP alone did not exhibit a decrease in
mortality after a live E. coli challenge. Unasyn treatment alone
could, however, protect mice challenged with live E. coli, and
mortality in this group was 33.3% (Table 1).
ART has no antibacterial activity in vitro. The MICs of
AMP and unasyn for E. coli were 64 and 8 ?g/ml, respectively.
ART had no antibacterial activity even at the concentration
more than 512 ?g/ml. The combination of ART (0.5 to 512
?g/ml) with AMP or unasyn could not increase the antibacte-
rial activities of either antibacterial agent.
ART reduces cytokine releases in vitro. RAW264.7 cells
were stimulated with CpG ODN, LPS, or heat-killed E. coli,
and the TNF-? and IL-6 levels in the supernatant were mea-
sured. RAW264.7 cells produced abundant amounts of TNF-?
and IL-6 in response to CpG ODN, LPS, and heat-killed E.
coli. When ART was added prior to the stimulators, cytokine
release was potently attenuated in response to the three stim-
ulators in a dose-dependent manner (Tables 2 and 3). Further-
more, when ART was added after the stimulators, cytokine
release was also suppressed, albeit to a lesser degree than when
it was added before stimulation. These data indicate that ART
suppresses CpG ODN- and LPS-induced cytokine release in
vitro in a dose- and time-dependent manner.
ART cannot directly bind to CpG ODN or LPS. ART can
reduce cytokine release induced by CpG ODN and LPS; how-
ever, it remains unclear whether ART’s effects are related to its
binding to CpG ODN or LPS. If binding occurs in vitro, LPS or
CpG ODN are neutralized and cytokine release is inhibited.
Using biosensor technology, we found that ART did not di-
rectly bind CpG ODN or lipid A (data not shown), indicating
that ART-mediated inhibition of proinflammatory cytokine
release is not related to its ability to bind to CpG ODN or LPS.
ART has no influence on CpG ODN binding to the cell
surface or accumulation within RAW264.7 cells. Uptake of
CpG ODN into endosomes is required for TLR9 recognition
in macrophages and monocytes (10, 12, 21). Before TLR9
recognizes CpG ODN, CpG ODN binding and internalization
processes are required (5). Our flow cytometry results showed
that ART had no effect on the binding of 6-FAM CpG ODN
to the cell surface or on accumulation within RAW264.7 cells
(Fig. 5), which indicated that ART-induced inhibition of proin-
flammatory cytokine release was not related to inhibition of
binding and accumulation of CpG ODN.
ART doesn’t reduce the expression of TLR9 and TLR4
mRNA. Signaling by TLR family members is required for CpG
ODN and LPS to induce cytokine release (13, 16). In macro-
phages and monocytes, TLR9 is a pattern recognition receptor
for CpG ODN (13, 27), whereas TLR4 is a pattern recognition
receptor for LPS (16). Because several lines of evidence sug-
gest that the molecular recognition of CpG ODN occurs within
the cell, we investigated the mRNA expression of TLR9 and
TLR4 in ART-treated cells. In agreement with previous results
(14), TLR9 or TLR4 mRNA in nonstimulated RAW264.7 cells
were expressed at low levels. CpG ODN and LPS elevated
TLR9 and TLR4 mRNA expression significantly, and pretreat-
ment of the cells with ART did not change the expression
levels. In addition, heat-killed E. coli stimulated TLR4 and
TLR9 expression levels, and these were not attenuated mark-
edly by pretreatment with ART (Fig. 6).
ART blocks NF-?B activation. By EMSA, we found that
CpG ODN, LPS, and heat-killed E. coli activated NF-?B (Fig.
FIG. 4. ART decreased the serum TNF-? release (pg/ml) after
CpG ODN, LPS, and heat-killed E. coli challenge. Mice were intrave-
nously injected with CpG ODN (10 mg/kg), LPS (10 mg/kg), or heat-
killed E. coli (1.5 ? 1011CFU/kg) in the absence or presence of 200 mg
of ART/kg orally administered, and 0.5 ml of blood per mouse was
drawn after challenge for 4 h. The serum was stored at ?80°C for
subsequent TNF-? assays using mouse ELISA kits. ?, P ? 0.05 versus
groups receiving no ART treatment. The datum points are presented
as means ? the standard deviation. The experiment was repeated
TABLE 1. Survival of mice challenged with live E. colia
No. of animals
AMP ? E. coli
ART ? E. coli
ART ? E. coli ? AMP
Unasyn ? E. coli
ART ? E. coli ? unasyn
aIn a concurrent experiment seven groups of mice (n ? 6 animals per group)
were challenged with live E. coli as follows: (i) saline control; (ii) live E. coli
(2.5 ? 108CFU/kg, intravenously); (iii) ART (100 mg/kg) and live E. coli; (iv)
400 mg of AMP/kg and live E. coli; (v) 400 mg of AMP/kg, ART at 100 mg/kg,
and live E. coli; (vi) 600 mg of unasyn/kg and live E. coli; or (vii) 600 mg of
unasyn/kg, live E. coli, and ART at 100 mg/kg. The total injection volume was 0.2
ml per 20 g of body weight.
b?, P ? 0.05 versus E. coli; †, P ? 0.05 versus unasyn plus E. coli.
VOL. 50, 2006ARTEMISININ PROTECTS AGAINST E. COLI CHALLENGE2423
7), suggesting that the nuclear transcription factor is involved
in the observed cytokine release. Pretreatment with ART uni-
formly blocked activation of NF-?B induced by CpG ODN,
LPS, or heat-killed E. coli. These results suggest that the ob-
served inhibition of proinflammatory cytokine release may be
associated with the ART-induced block of NF-?B.
ART has little cellular toxicity in vitro. MTT assay revealed
that the concentration of DMSO (0.5% [vol/vol]) in which
ART was dissolved did not affect the morphological features or
growth of RAW264.7 cells. Although in a concentration range
of 5 to 80 ?g/ml and incubated for 6 h or 24 h, ART did not
influence cell viability, at more than 80 ?g of ART/ml and with
a 24-h incubation ART may be cytotoxic (Fig. 8). The concen-
tration of ART used in our in vitro experiments was less than
80 ?g/ml, and the results suggest there is no correlation be-
tween the ART-induced inhibition of cytokine release and
We have shown here for the first time, to our knowledge,
that ART can act in synergy with antibiotics to protect against
a lethal live E. coli challenge by decreasing the release of
In the present study, mice were challenged by pure LPS or
CpG ODN or by heat-killed E. coli. Heat-killed E. coli lacks
viability, but bDNA and LPS still exist in the cells. Therefore,
the sepsis model made using heat-killed E. coli represents the
ability of E. coli to induce sepsis. We found that mice pre-
treated with ART had a higher survival rate no matter whether
they were challenged with LPS or CpG ODN or heat-killed E.
coli. This protection is correlated with the ability of ART to
inhibit the release of proinflammatory cytokines, such as serum
TNF-?, which are known to be involved in sepsis.
Indeed, since sepsis is mostly caused by live bacteria, a sepsis
model using live E. coli is a better approximation of the septic
patient in the clinic. In the present study, E. coli ATCC 35218
was used. E. coli ATCC 35218 was well known for a ?-lacta-
mase-producing isolate (6). In vitro, the MICs of AMP and
unasyn for the strain were 32 and 8 ?g/ml, respectively. The
difference in antibacterial activity between AMP and unasyn
might result from the inactivation of AMP by ?-lactamase
from E. coli. In vivo, although AMP or ART alone did not
protect mice challenged with live E. coli, the combination of
ART and AMP did lead to a decrease in acute sepsis mortality.
Unasyn alone could protect mice challenged with live E. coli
because of its strong bactericidal activity, but ART also could
increase the protection of unasyn. The findings that ART did
not inhibit bacterial growth at an even higher concentration
(?512 ?g/ml), even in combination with AMP or unasyn, sug-
TABLE 2. Dose-dependent ART effects on TNF-? and IL-6 release from RAW264.7 cells induced by CpG ODN, LPS, or heat-killed E. colia
Mean cytokine release (pg/ml) ? SD
CpG ODN LPS Heat-killed E. coli
3,363.1 ? 192.3
1,948.2 ? 110.8*
1,905.6 ? 140.5**
1,884.6 ? 60.1*
1,554.6 ? 161.6**
907.5 ? 140.4**
1,630.7 ? 15.2
1,040.5 ? 75.5*
950.2 ? 26.5**
890.7 ? 68.2**
810.1 ? 105.6**
710.1 ? 150.1**
1,770.0 ? 87.0
1,709.0 ? 35.1
1,469.7 ? 109.4**
1,279.6 ? 98.3**
928.0 ? 102.5**
829.5 ? 31.8**
1,528.7 ? 55.8
820.4 ? 100.8*
530.9 ? 140.1**
411.9 ? 110.7**
370.3 ? 130.5**
358.0 ? 14.1**
3,951.0 ? 60.5
2,801.8 ? 268.1*
2,345.3 ? 183.4**
2,203.9 ? 45.1**
2,037.0 ? 48.9**
1,578.4 ? 122.5**
1,054.8 ? 72.1
488.5 ? 56.5*
295.4 ? 45.5**
293.0 ? 48.2**
230.7 ? 24.5**
184.8 ? 13.8**
Medium only40.2 ? 32.074.2 ? 10.142.6 ? 6.4 46.6 ? 3.1 377 ? 29.0 24.6 ? 3.9
aRAW264.7 cells (106/ml) were pretreated for 2 h with the indicated concentrations of ART and then incubated with 10 ?g of CpG ODN/ml or LPS (0.1 ?g/ml)
and heat-killed E. coli (3.5 ? 107CFU/ml) or medium only for another 2 or 4 h. The relative concentrations of TNF-? and IL-6 in the cell-free supernatants were
determined by a quantitative ELISA assay. The experiment was repeated three times. ?, P ? 0.05; ??, P ? 0.01 versus ART ? 0.
TABLE 3. Time-dependent ART effects on TNF-? and IL-6 release from RAW264.7 cells induced by CpG ODN, LPS, or heat-killed E. colia
Treatment Time (h)
Mean cytokine release (pg/ml) ? SDb
CpG ODN LPSHeat-killed E. coli
1,231.5 ? 272.1**
1,648.1 ? 17.2**
1,450.6 ? 61.9**
1,802.6 ? 21.2*
1,848.8 ? 222.3
233.5 ? 29.5**
349.5 ? 39.5**
429.6 ? 11.5**
584.7 ? 49.6**
873.2 ? 92.8*
1,314.5 ? 212.4**
1,096.6 ? 117.9**
1,288.9 ? 104.3**
959.8 ? 71.3*
1,657.9 ? 23.0
100.6 ? 19.9**
93.4 ? 15.9**
104.8 ? 7.2**
96.4 ? 12.3*
155.5 ? 43.8
1,376.9 ? 24.1**
847.0 ? 134.9**
2,020.1 ? 57.0**
2,004.0 ? 76.5*
2,734.4 ? 41.5*
258.8 ? 26.8**
292.7 ? 74.2**
893.2 ? 75.7*
877.2 ? 86.7*
925.3 ? 32.5
No ART 2,491.4 ? 141.5861.5 ? 90.5 2,136.2 ? 23.4 379.5 ? 55.72,562.9 ? 155.0942.1 ? 11.0
Medium77.6 ? 31.5241.5 ? 29.653.3 ? 10.6 90.7 ? 6.8 49.1 ? 11.5 240.8 ? 28.2
aRAW264.7 cells (106/ml) were preincubated with 40 ?g of ART/ml 2 or 4 h prior to, coincident with (0 h), or 1 or 2 h after the addition of the stimulator LPS (0.1
?g/ml), CpG ODN (10 ?g/ml), or heat-killed E. coli (3.5 ? 107CFU/ml). After incubation for another 2 or 4 h, the cells were centrifuged, and 0.1 ml of supernatant
was collected for TNF-? or IL-6 assay with the corresponding ELISA kits. The experiment was repeated three times. ?, P ? 0.05; ??, P ? 0.01 versus no ART.
2424WANG ET AL.ANTIMICROB. AGENTS CHEMOTHER.
gest that the synergistic protection for sepsis by ART combi-
nation with antibiotics is more likely to be closely related to the
anti-inflammatory effects of ART rather than an antibacterial
In all experiments, the prestimulation with oral administra-
tion of ART provided some level of protection from the chal-
FIG. 5. Binding (A) and internalization (B) of CpG ODN 1826 to RAW264.7 cells treated with ART. RAW264.7 cells (2 ? 106/ml) were
pretreated for 2 h with ART (10, 20, or 40 ?g/ml) or 50 ?g of CQ/ml in 12-well plates and then incubated with 10 ?g of CpG ODN 1826/ml in
the dark at 4°C for 30 min (A) or at 37°C for 1 h (B). Cells left untreated served as controls (medium). After incubation, the cells were washed
twice with ice-cold PBS and then resuspended in PBS for assay. Fluorescence intensity (FI) was analyzed by FACScan. ‡, P ? 0.05; ??, P ? 0.05
versus the CpG ODN. Error bars indicate the mean ? the standard deviation. Each experiment was repeated at least three times.
FIG. 6. Effect of ART on TLR4 and TLR9 mRNA expression in
stimulated RAW264.7 cells. RAW264.7 cells (106/ml) were pretreated for
2 h with 40 ?g of ART/ml and then incubated with 10 ?g of CpG ODN
1826/ml (A), 0.1 ?g of LPS/ml (B), or 3.5 ? 107CFU of heat-killed E.
coli/ml (C and D). Total RNA was prepared with the RNAeasy kit. After
DNase I treatment, 2 ?g of RNA was reverse transcribed with AMV
with suitable primers; ?-actin, TLR9, and TLR4 were amplified. Molec-
ular weight markers (M) were 2,000, 1,000, 750, 500, and 250 bp from
upper to lower.
FIG. 7. Inhibition of ART on NF-?B activation induced by CpG
ODN, LPS, and heat-killed E. coli. RAW264.7 cells (106/ml) were left
untreated (medium only [lane A]), incubated with 40 ?g of ART/ml for
2 h (ART [lane B]), or pretreated with the indicated concentration of
ART and then incubated with 10 ?g of CpG ODN 1826/ml (lane C),
0.1 ?g of LPS/ml (lane E), or 3.5 ? 107CFU/ml of heat-killed E. coli
(lane G) or else treated with only CpG ODN 1826 (lane D), 0.1 ?g of
LPS/ml (lane F), or 3.5 ? 107CFU of heat-killed E. coli/ml (lane H)
for another 4 h. The cells were collected, and nuclear protein extracts
were examined for NF-?B p65 activation by EMSA.
VOL. 50, 2006ARTEMISININ PROTECTS AGAINST E. COLI CHALLENGE 2425
lenges. When administered after stimulation, however, the
drug’s potency was not manifest (data not shown). Obviously,
in the clinic the occurrence of infection cannot be predicted. In
the present study we only examined a single postinfection ad-
ministration protocol; however, if ART was administered
many times it may work well postinfection. Indeed, our pre-
liminary results (data not shown) of ongoing follow-up studies
examining postinfection protocols are encouraging.
Previous studies have shown that ART shares with other
sesquiterpene lactones the ability to inhibit nitric oxide synthe-
sis in cytokine-stimulated human astrocytoma T67 cells (1).
However, there have been no prior reports on the effects of
ART on proinflammatory cytokines. As demonstrated in our in
vitro experiments, ART exhibits powerful inhibition potency
on proinflammatory cytokines in a dose-dependent manner in
murine macrophage RAW264.7 cells. In the present study, the
dose was selected according to studies on cancer and malaria
(7, 19). Although the lowest dose of ART (5 ?g/ml) is higher
than the peak concentration of ART in plasma (402 ng/ml
from mice in our lab and 500 ng/ml from humans in a previous
study ), we think there is a difference between the in vitro
and in vivo results. The results from in vitro studies cannot
completely represent the in vivo truth. The cells in the human
body may be more sensitive to the drug.
In addition, although ART added after the stimulators in-
hibited less cytokine release than that added prior to the stim-
ulators, our results indicate that ART suppresses CpG ODN-
and LPS-induced cytokine release in vitro in a time-dependent
manner. Although there are several stimulators in the suspen-
sion of heat-killed E. coli, ART also inhibits TNF-? and IL-6
releases induced by heat-killed E. coli. Thus, our results sug-
gest that ART is a strong inhibitor of many stimulators and
may provide an important means for treating sepsis.
Endocytosis or internalization is a fundamental process of
eukaryotic cells and fulfills numerous functions. Malaria par-
asites invade red blood cells and during their intracellular
development endocytose large amounts of host cytoplasm for
digestion in a specialized lysosomal compartment called the
food vacuole. Heinrich et al. demonstrated that ART inhibits
endocytosis of macromolecular tracers by up to 85% in Plas-
modium falciparum (15).
Uptake of CpG ODN into endosomes is required for the
recognition of TLR9. TLR9 recognizes CpG ODN in the ma-
ture endosomes of macrophages and monocytes, and CpG
ODN binding and internalization processes are required for
cell activation (14). Increased cell surface DNA binding and
internalization could increase RAW264.7 macrophage cyto-
kine release (24) and vice versa. Therefore, it is possible that
ART-induced suppression of TNF-? and IL-6 release might be
related to the inhibition of cell surface DNA binding and the
internalization of CpG ODN. However, in contrast to the
findings in P. falciparum, our results showed that ART does
not influence binding and internalization of CpG ODN in
CpG ODN activates the TLR9-mediated signal transduction
pathway to regulate the release of cytokines. Although previ-
ous studies showed a correlation among cytokine release, the
uptake of CpG ODN, and TLR9 expression (30), our data
indicate that decreased cytokine release by ART is not asso-
ciated with decreased CpG ODN uptake and TLR9 expres-
sion. LPS-induced signal transduction in macrophages is me-
diated by TLR4 on the cell surface (16). Our data demonstrate
that ART can inhibit LPS-induced proinflammatory cytokine
release; however, ART cannot block LPS-induced TLR4
NF-?B is an important downstream regulator of the expres-
sion of various proinflammatory cytokines induced by CpG
ODN and LPS (1, 2, 9, 11, 31). In 2003, Aldieri et al. reported
that ART blocks a mix containing LPS and cytokine-induced
activation of NF-?B in human astrocytoma T67 cells (1). By
EMSA, we found that CpG ODN, LPS, and heat-killed E. coli
activated NF-?B (Fig. 7), whereas little NF-?B was activated in
the absence of stimulators, suggesting that a nuclear transcrip-
tion factor is involved in the observed cytokine release. Pre-
treatment with ART uniformly blocked NF-?B activation in-
duced by CpG ODN, LPS, or heat-killed E. coli. These results
suggest that inhibition of proinflammatory cytokine release
may be associated with the ART-induced blockade of NF-?B
in RAW264.7 cells.
The inhibitory effects of ART on CpG ODN- and LPS-
induced cytokine release are unlikely due to its nonspecific
cellular toxicity. ART treatment did not affect RAW264.7 cell
viability as measured by MTT assay. More importantly, in mice
treated with ART, we did not observe any side effects, such as
liver or kidney dysfunction (data not shown). We have shown
in our laboratory that the concentration of ART in plasma at
1 h (402 ng/ml after a single dose of ART treatment [100
mg/kg]) is similar to that (500 ng/ml) reported in healthy adult
males given ART (4). Thus, the doses of ART used in our
study are comparable to that used for malaria treatment.
Therefore, ART should be considered a safe putative candi-
date for development into a sepsis treatment.
FIG. 8. Cytotoxicity of ART on RAW264.7 cells. After overnight
culture in a 96-well culture plate, cells (104cells/well in DMEM) were
washed and incubated with various concentrations of ART for 6 or
24 h. Subsequently, 20 ?l of MTT solution (5 mg/ml in PBS) were
added in a total volume of 200 ?l of medium. Cells were incubated for
4 h at 37°C and 5% CO2; the supernatant was then removed, and 150
?l of DMSO was added to each well to dissolve the produced formazan
crystals. The extinction was measured at 490 nm by using a microplate
reader. ?, P ? 0.05; ??, P ? 0.01 versus medium only group. The datum
points are presented as means ? the standard deviation. The experi-
ment was repeated three times. OD450, optical density at 450 nm.
2426 WANG ET AL.ANTIMICROB. AGENTS CHEMOTHER.
This study was supported by grant 30572365 from the National
Natural Science Foundation of China and a grant from Sci-Tech of
1. Aldieri, E., D. Atragene, L. Bergandi, C. Riganti, C. Costamagna, A. Bosia,
and D. Ghigo. 2003. Artemisinin inhibits inducible nitric oxide synthase and
nuclear factor NF-?B activation. FEBS Lett. 552:141–144.
2. An, H., H. Xu, Y. Yu, M. Zhang, R. Qi, X. Yan, S. Liu, W. Wang, Z. Guo, Z.
Qin, and X. Cao. 2002. Up-regulation of TLR9 gene expression by LPS in
mouse macrophages via activation of NF-?B, ERK and p38 MAPK signal
pathways. Immunol. Lett. 81:165–169.
3. Angus, D. C., W. T. Linde-Zwirble, J. Lidicker, G. Clermont, J. Carcillo, and
M. R. Pinsky. 2001. Epidemiology of severe sepsis in the United States:
analysis of incidence, outcome, and associated costs of care. Crit. Care Med.
4. Ashton, M., T. Gordi, N. H. Trinh, V. H. Nguyen, D. S. Nguyen, T. N. Nguyen,
X. H. Dinh, M. Johansson, and D. C. Le. 1998. Artemisinin pharmacokinet-
ics in healthy adults after 250, 500, and 1000 mg single oral doses. Biopharm.
Drug Dispos. 19:245–250.
5. Bennett, R. M., G. T. Gabor, and M. M. Merritt. 1985. DNA binding to
human leukocytes: evidence for a receptor-mediated association, internal-
ization, and degradation of DNA. J. Clin. Investig. 76:2182–2190.
6. Butler, D. L., C. J. Jakielaszek, L. A. Miller, and J. A. Poupard. 1999.
Escherichia coli ATCC 35218 as a quality control isolate for susceptibility
testing of Haemophilus influenzae with haemophilus test medium. Antimi-
crob. Agents Chemother. 43:283–286.
7. Chen, H. H., H. J. Zhou, and X. Fang. 2003. Inhibition of human cancer cell
line growth and human umbilical vein endothelial cell angiogenesis by arte-
misinin derivatives in vitro. Pharmacol. Res. 48:231–236.
8. Cohen, J. 2002. The immunopathogenesis of sepsis. Nature 420:885–891.
9. Hacker, H. 2000. Signal transduction pathways activated by CpG-DNA.
Curr. Top. Microbiol. Immunol. 247:77–92.
10. Hacker, H., H. Mischak, T. Miethke, S. Liptay, R. Schmid, T. Sparwasser, K.
Heeg, G. B. Lipford, and H. Wagner. 1998. CpG-DNA-specific activation of
antigen-presenting cells requires stress kinase activity and is preceded by
nonspecific endocytosis and endosomal maturation. EMBO J. 17:6230–6240.
11. Hacker, H., R. M. Vabulas, O. Takeuchi, K. Hoshino, S. Akira, and H.
Wagner. 2000. Immune cell activation by bacterial CpG-DNA through my-
eloid differentiation marker 88 and tumor necrosis factor receptor-associ-
ated factor (TRAF)6. J. Exp. Med. 192:595–600.
12. He, H., and M. H. Kogut. 2003. CpG-ODN-induced nitric oxide production
is mediated through clathrin-dependent endocytosis, endosomal maturation,
and activation of PKC, MEK1/2 and p38 MAPK, and NF-?B pathways in
avian macrophage cells (HD11). Cell Signal 15:911–917.
13. Hemmi, H., O. Takeuchi, T. Kawai, T. Kaisho, S. Sato, H. Sanjo, M.
Matsumoto, K. Hoshino, H. Wagner, K. Takeda, and S. Akira. 2000. A
Toll-like receptor recognizes bacterial DNA. Nature 408:740–745.
14. Hong, Z., Z. Jiang, W. Liangxi, D. Guofu, L. Ping, L. Yongling, P. Wendong,
and W. Minghai. 2004. Chloroquine protects mice from challenge with CpG
ODN and LPS by decreasing proinflammatory cytokine release. Int. Immu-
15. Hoppe, H. C., D. A. van Schalkwyk, U. I. Wiehart, S. A. Meredith, J. Egan,
and B. W. Weber. 2004. Antimalarial quinolines and artemisinin inhibit
endocytosis in Plasmodium falciparum. Antimicrob. Agents Chemother. 48:
16. Hoshino, K., O. Takeuchi, T. Kawai, H. Sanjo, T. Ogawa, Y. Takeda, K.
Takeda, and S. Akira. 1999. Cutting edge: Toll-like receptor 4 (TLR4)-
deficient mice are hyporesponsive to lipopolysaccharide: evidence for TLR4
as the Lps gene product. J. Immunol. 162:3749–3752.
17. Jiang, Z., Z. Hong, W. Guo, G. Xiaoyun, L. Gengfa, L. Yongning, and X.
Guangxia. 2004. A synthetic peptide derived from bactericidal/permeability-
increasing protein neutralizes endotoxin in vitro and in vivo. Int. Immuno-
18. Karres, I., J. P. Kremer, I. Dietl, U. Steckholzer, M. Jochum, and W. Ertel.
1998. Chloroquine inhibits proinflammatory cytokine release into human
whole blood. Am. J. Physiol. 274:R1058–R1064.
19. Kim, J. T., J. Y. Park, H. S. Seo, H. G. Oh, J. W. Noh, J. H. Kim, D. Y. Kim,
and H. J. Youn. 2002. In vitro antiprotozoal effects of artemisinin on Neo-
spora caninum. Vet. Parasitol. 103:53–63.
20. Klayman, D. L. 1985. Qinghaosu (artemisinin): an antimalarial drug from
China. Science 228:1049–1055.
21. Krieg, A. M. 2002. CpG motifs in bacterial DNA and their immune effects.
Annu. Rev. Immunol. 20:709–760.
22. Li, Y., and Y. L. Wu. 2003. An over four millennium story behind qinghaosu
(artemisinin): a fantastic antimalarial drug from a traditional chinese herb.
Curr. Med. Chem. 10:2197–2230.
23. Lipford, G. B., T. Sparwasser, M. Bauer, S. Zimmermann, E. S. Koch, K.
Heeg, and H. Wagner. 1997. Immunostimulatory DNA: sequence-dependent
production of potentially harmful or useful cytokines. Eur. J. Immunol.
24. McCoy, S. L., S. E. Kurtz, F. A. Hausman, D. R. Trune, R. M. Bennett, and
S. H. Hefeneider. 2004. Activation of RAW264.7 macrophages by bacterial
DNA and lipopolysaccharide increases cell surface DNA binding and inter-
nalization. J. Biol. Chem. 279:17217–17223.
25. National Committee for Clinical Laboratory Standards. 2003. Methods for
dilution antimicrobial susceptibility tests for bacteria that grow aerobically,
6th ed. Approved standard. NCCLS document M7–A6. National Committee
for Clinical Laboratory Standards, Wayne, Pa.
26. Sparwasser, T., T. Miethke, G. Lipford, K. Borschert, H. Hacker, K. Heeg,
and H. Wagner. 1997. Bacterial DNA causes septic shock. Nature 386:336–
27. Takeshita, F., C. A. Leifer, I. Gursel, K. J. Ishii, S. Takeshita, M. Gursel, and
D. M. Klinman. 2001. Cutting edge: role of Toll-like receptor 9 in CpG
DNA-induced activation of human cells. J. Immunol. 167:3555–3558.
28. Vincent, J. L., Q. Sun, and M. J. Dubois. 2002. Clinical trials of immuno-
modulatory therapies in severe sepsis and septic shock. Clin. Infect. Dis.
29. Yao, Y. M., H. Redl, S. Bahrami, and G. Schlag. 1998. The inflammatory
basis of trauma/shock-associated multiple organ failure. Inflamm. Res. 47:
30. Yi, A. K., R. Tuetken, T. Redford, M. Waldschmidt, J. Kirsch, and A. M.
Krieg. 1998. CpG motifs in bacterial DNA activate leukocytes through the
pH-dependent generation of reactive oxygen species. J. Immunol. 160:4755–
31. Yi, A. K., J. G. Yoon, S. C. Hong, T. W. Redford, and A. M. Krieg. 2001.
Lipopolysaccharide and CpG DNA synergize for tumor necrosis factor-alpha
production through activation of NF-?B. Int. Immunol. 13:1391–1404.
VOL. 50, 2006ARTEMISININ PROTECTS AGAINST E. COLI CHALLENGE2427