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Stimulation of Toll-Like Receptor 4 by
Lipopolysaccharide During Cellular Invasion
by Live Salmonella typhimurium Is a Critical
But Not Exclusive Event Leading to
Macrophage Responses
Matthew C. J. Royle, Sabine Tötemeyer, Louise C.
Alldridge, Duncan J. Maskell and Clare E. Bryant
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Copyright © 2003 by The American Association of
Immunologists All rights reserved.
Print ISSN: 0022-1767 Online ISSN: 1550-6606.
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J Immunol 2003; 170:5445-5454; ;
doi: 10.4049/jimmunol.170.11.5445
http://www.jimmunol.org/content/170/11/5445
The Journal of Immunology
Stimulation of Toll-Like Receptor 4 by Lipopolysaccharide
During Cellular Invasion by Live Salmonella typhimurium Is a
Critical But Not Exclusive Event Leading to Macrophage
Responses1
Matthew C. J. Royle, Sabine Tötemeyer, Louise C. Alldridge, Duncan J. Maskell, and
Clare E. Bryant2
S
almonella enterica serovars can infect many different hosts
with different outcomes ranging from gastrointestinal diseases through to fully invasive systemic diseases such as
typhoid fever (1). Salmonellae can invade and survive within both
epithelial cells and macrophages, in the process triggering a wide
range of cellular responses that are important in dictating the outcome of infection. Salmonellae possess a range of protein and
nonprotein structures that may be involved in these interactions,
probably by acting as pathogen-associated molecular patterns
(PAMPs),3 which include LPS, lipoproteins, flagellin, peptidoglycan, and bacterial DNA (2). These PAMPs may bind to eukaryotic
cell PAMP receptor proteins, including the family of Toll-like receptors (TLRs), that then signal to the cell and induce a response
(3–5). The investigation of the precise role of the different PAMPs
Department of Clinical Veterinary Medicine, University of Cambridge, Cambridge,
United Kingdom
Received for publication September 23, 2002. Accepted for publication March
25, 2003.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1
M.C.J.R. was funded by a Biotechnology and Biological Sciences Research Council
(BBSRC) studentship. S.T. was funded by BBSRC Project Award 8/9912336. D.J.M.
is the Marks and Spencer Professor of Food Safety. C.E.B. is a Wellcome Trust
Advanced Fellow.
2
Address correspondence and reprint requests to Dr. Clare E. Bryant, Department of
Clinical Veterinary Medicine, University of Cambridge, Madingley Road, Cambridge, CB3 0ES, U.K. E-mail address: [email protected]
3
Abbreviations used in this paper: PAMP, pathogen-associated molecular pattern;
iNOS, inducible NO synthase; JNK, c-Jun N-terminal kinase; LB, Luria-Bertani;
MAPK, mitogen-activated protein kinase; MOI, multiplicity of infection; TLR, Tolllike receptor; TTSS, type III secretion system.
Copyright © 2003 by The American Association of Immunologists, Inc.
in interacting with cells during an infection with live bacteria has
been somewhat neglected in favor of ground-breaking studies with
purified bacterial components that have allowed a comprehensive
understanding of the biology of TLRs and signaling pathways in
cellular responses (6).
LPS is the main component of the outer leaflet of the outer
membrane of salmonellae, and it is one of the most biologically
active of the PAMPs present in these bacteria. Its role in activation
of TLR-4 in modulating responses to infection with salmonellae
has been the subject of considerable speculation, but little direct
experimentation in true infection systems using live bacteria (7–9).
Activation of TLRs initiates complex signal transduction cascades
to activate many signaling proteins, such as the mitogen-activated
protein kinases (MAPKs) and the transcription factor NF-␬B, resulting in transcription of genes encoding proteins such as inducible enzymes and cytokines (10). The importance of LPS in modulating the host response to Salmonella infection is emphasized by
the fact that LPS-hyporesponsive mice (C3H/HeJ), which have a
dominant-negative mutation in the TLR-4 receptor that abrogates
its function, are more susceptible to infections with S. enterica
serovar Typhimurium (hereafter S. typhimurium), an outcome that
may be linked to the failure of macrophages to control bacterial
growth (11, 12). In addition, mutations in salmonellae that led to
changes in the structure of lipid A, the toxic domain of LPS, reduced the toxicity of LPS for macrophages in vitro and reduced
mortality after infection of mice with S. typhimurium (13, 14). In
a recent study using microarrays to compare responses of RAW
macrophage-like cell lines either stimulated with LPS or infected
with high levels of S. typhimurium, it was noted that many similar
genes were activated by both stimuli, for example, inducible NO
0022-1767/03/$02.00
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Invasion of macrophages by salmonellae induces cellular responses, with the bacterial inducers likely to include a number of
pathogen-associated molecular patterns. LPS is one of the prime candidates, but its precise role in the process, especially when
presented as a component of live infecting bacteria, is unclear. We thus investigated this question using the lipid A antagonist
E5531, the macrophage-like cell line RAW 264.7, and primary macrophage cultures from C3H/HeJ and Toll-like receptor 4ⴚ/ⴚ
(TLR-4ⴚ/ⴚ) mice. We show that LPS presented on live salmonellae provides an essential signal, via functional TLR-4, for macrophages to produce NO and TNF-␣. Furthermore, the mitogen-activated protein kinase c-Jun N-terminal kinase and p38 are
activated, and the transcription factor NF-␬B is translocated to the nucleus when RAW 264.7 cells are presented with purified LPS
or live salmonellae. Purified LPS stimulates rapid, transitory mitogen-activated protein kinase activation that is inhibited by
E5531, whereas bacterial invasion stimulates delayed, prolonged activation, unaffected by E5531. Both purified LPS and bacterial
invasion caused translocation of NF-␬B, but whereas E5531 always inhibited activation by purified LPS, activation by bacterial
invasion was only inhibited at later time points. In conclusion, we show for the first time that production of NO and TNF-␣ is
critically dependent on activation of TLR-4 by LPS during invasion of macrophages by salmonellae, but that different patterns
of activation of intracellular signaling pathways are induced by purified LPS vs live salmonellae. The Journal of Immunology,
2003, 170: 5445–5454.
5446
Materials and Methods
Cell culture
RAW 264.7 cells were cultured in DMEM containing 10% FCS supplemented with 2 mM glutamine, 200 U/ml penicillin, and 100 ␮g/ml streptomycin. Primary bone marrow macrophages were isolated from the femur
and tibia of mice killed by cervical dislocation (28). Briefly, the bone
marrow was flushed out with medium (RPMI ⫹ 10% FCS supplemented
with 2 mM glutamine, 5% horse serum, 1 mM sodium pyruvate) and the
macrophages were seeded into tissue culture flasks. For maintenance of the
bone marrow macrophages in culture, the RPMI medium was supplemented with 20% of supernatant taken from L929 cells (a murine M-CSFproducing cell line (28)). For experiments, cells were plated onto 6-, 24-,
or 96-well plates at a plating density of 2 ⫻ 106, 7 ⫻ 105, or 2 ⫻ 105 per
well, respectively.
LPS or S. typhimurium was added to the cells at the concentrations and
multiplicities of infection (MOI) stated in the text. For signaling assays,
either bacteria or LPS were added to the cells, and at 0 –90 min after
stimulation the cells were washed twice in PBS before lysis. To determine
TNF-␣ production, following a 2-h incubation, cell supernatants were
taken and the cells were washed with PBS and incubated in DMEM containing 50 ␮g/ml gentamicin for 1 h. Cells were then washed again in PBS
and incubated in DMEM containing 10 ␮g/ml gentamicin until the supernatants were taken at 9 h poststimulation. NO and iNOS measurements
were taken following the gentamicin protocol, taking readings at 24 h when
medium was harvested, and the cells were lysed for Western blot analysis.
In vitro kinase assays for MAPK activity
The activity of protein kinases p38 and JNK was assayed using a solidphase kinase reaction, as described by Derijard et al. (29) and McLaughlin
et al. (30), respectively. Protein kinase activity of p38 and JNK was measured in affinity precipitates of the proteins bound to recombinant substrates immobilized on glutathione Sepharose 4B beads (GST-MAPK activated protein kinase-2 for p38 and GST-tagged truncated N terminus of
c-Jun (GST-c-Jun5– 89) for JNK). Cells were lysed on ice in lysis buffer (20
mM Tris-HCl, pH 7.4, 150 mM NaCl, 1% Triton X-100, 10% glycerol, 2 mM
EDTA, 20 mM NaF, 2.5 mM ␤-glycerophosphate, 0.5 mM Na3VO4, 0.2 mM
PMSF, and 1 ␮g/ml leupeptin and aprotinin). The beads were washed once in
the lysis buffer and once in kinase buffer containing 20 mM MgCl2, 5 mM
␤-glycerophosphate, 0.1 mM Na3VO4, 2 mM DTT, and 25 mM HEPES, pH
7.4, for the p38 assay and pH 7.6 for the JNK assay. Kinase buffer with a final
concentration of 50 ␮M ATP and 2 ␮Ci [␥-32P]ATP per reaction for p38 and
25 ␮M ATP and 1 ␮Ci [␥-32P]ATP per reaction for JNK was added for 30
min. The reaction was stopped by the addition of 10 ␮l of 4⫻ Laemmli sample
buffer and boiled for 5 min. Samples were resolved by 11% (w/v) SDS-PAGE,
the gels were dried, and the incorporation of 32P phosphate into GST-MAPK
activated protein kinase-2 or GST-cJun5– 89 was detected using autoradiography. Autoradiographs from four separate experiments were quantified using
Kodak 1D software.
In experiments on primary cell cultures from C3H/HeN and C3H/HeJ,
cells were lysed on ice in buffer containing 10 mM Tris-HCl, pH 7.5, 150
mM NaCl, 1% (v/v) Nonidet P-40, 1 mM EGTA, 1 mM EDTA, 1 mM
Na3V04, 1 mM PMSF, 50 mM NaF, 40 mM Na4P2O7, and 0.5 ␮g/ml
leupeptin and aprotinin. After clearing by centrifugation, p38 MAPK was
immunoprecipitated for 1 h at 4°C, using a polyclonal Ab (Santa Cruz
Biotechnology, Santa Cruz, CA) immobilized on protein G-Sepharose. Immunoprecipitates were washed three times in lysis buffer and once in PBS.
Immunoprecipitates were denatured in Laemmli sample buffer and boiled
for 5 min. Samples (5 ␮l) were resolved by 12% (w/v) SDS-PAGE and
transferred to polyvinylidene difluoride membrane (Millipore, Bedford,
MA) before probing with an Ab to phosphorylated p38 (Santa Cruz Biotechnology). The phosphorylated protein was visualized using ECL (Amersham Pharmacia Biotech, Amersham, Buckinghamshire, U.K.).
Mice
C3H/HeN and C3H/HeJ mice were obtained from Harlan Olac Laboratories (Bichester, U.K.). TLR-4 knockout mice were kindly donated by S.
Akira (Osaka University, Osaka, Japan).
Bacteria and preparation of LPS
S. typhimurium strain C5 (26) was used for all bacterial studies. Live S.
typhimurium C5 was prepared by diluting (1/10) an overnight culture in
fresh Luria-Bertani (LB) broth and incubating for a further 2 h, then washing the bacteria in LB broth and diluting as required in DMEM. S. typhimurium C5 LPS was extracted by hot phenol water purification (27) and
dissolved in distilled water at 1 mg/ml, then sonicated and diluted
in DMEM.
EMSA
To prepared nuclear pellets, cells (3 ⫻ 106) were washed twice with icecold PBS, scraped into a 1.5-ml centrifuge tube, and then pelleted by centrifugation at 13,000 rpm for 1 min. The cell pellet was resuspended in 400
␮l of buffer 1 (10 mM HEPES, pH 7.9; 10 mM KCl; 0.1 mM EDTA; 0.1
mM EGTA; 0.1 mM DTT; 0.5 mM PMSF; and 10 ␮g/ml leupeptin, pepstatin, and aprotinin) and put on ice to swell for 15 min. After addition of
25 ␮l of 10% (w/v) Nonidet P-40, the samples were vortexed for 10 s, then
centrifuged at 13,000 rpm for 20 s. The cell pellets were resuspended in 50
␮l of buffer 2 (20 mM HEPES, pH 7.9; 25% (w/v) glycerol; 0.4 M NaCl;
1 mM EDTA; 1 mM EGTA; 1 mM DTT; 0.5 mM PMSF; 10 ␮g/ml leupeptin, pepstatin, and aprotinin) and, after light vortexing, were extracted
on ice for 15 min. The extract was sonicated on ice for 30 s and then
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synthase (iNOS) and the receptor for the cytokine TNF-␣ (15),
leading to the inference that a considerable part of the host cell
response to live infection by salmonellae was contributed by a
response to LPS. Although this study gives strong circumstantial
evidence for a role for LPS in induction of host cell responses
when delivered as a component of live infecting bacteria, the precise details of how this occurs, and to what extent LPS is required
or interacts with other signals remain unknown.
To investigate these issues further is highly complex, given that
molecular mechanisms are being sought for an interaction between
two living, individually complex cells: the host cell and the infecting bacterium. Live infection studies of the nature required are
further complicated by the fact that salmonellae deliver highly bioactive effector proteins into host cells via a molecular syringe that
comprises a type three secretory system (TTSS), and these themselves have been described as inducing and subverting host cell
responses. For example, roles have been described for SipA and
SipC in modulation of the cellular actin cytoskeleton to permit
bacterial invasion (16, 17); for SptP, SopE, SopE2, and SopB in
interfacing with intracellular signaling pathways (18 –21); and for
SipB in induction of caspase-1-driven cellular death (22). TTSS
effectors are clearly critical in modifying host cell responses, particularly in epithelial cells that lack phagocytic machinery and
have a limited capacity for producing inflammatory mediators such
as cytokines (2). They have also been described as modulating
MAPK function and cellular replication (23). Host macrophage
recognition of, and response to, infection therefore involve the
complex interaction of bacterial TTSS effectors and host PAMP
receptor protein stimulation.
Although it is clear that LPS is an important inducer of macrophage responses to live bacterial invasion with salmonellae, unraveling the relative contributions of LPS, the other PAMPs, and
TTSS effector proteins to stimulating responses in macrophages
remains a key issue. As a first step toward addressing these issues,
we describe in this study experiments using the RAW 264.7 macrophage-like cell line in comparison with primary macrophage cultures from C3H/HeJ and TLR-4 knockout mice (8, 24) to determine the contribution of LPS activation of TLR-4 to the responses
of these cells after invasion by S. typhimurium. We also use the
lipid A antagonist E5531 (25) to investigate the contribution to
stimulation of TLR-4 of lipid A present in live S. typhimurium
during bacterial invasion. We show that invasion by S. typhimurium or stimulation with LPS of RAW cells activates NF-␬B
and the MAPKs c-Jun N-terminal kinase (JNK) and p38. The
time course of S. typhimurium activation of JNK and p38 is
slower than in response to LPS alone. Despite this differential
pattern of signaling activation, NO and TNF-␣ production is
LPS and TLR-4 dependent when macrophage-like cells are invaded by S. typhimurium.
ROLE OF LPS IN Salmonella INFECTIONS
The Journal of Immunology
5447
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FIGURE 1. MAP kinases p38 and JNK are activated by S. typhimurium and its LPS, but with differing profiles. A, Activation profile of p38 following
stimulation with LPS. RAW cells were treated with LPS (1 ␮g/ml) for the time indicated, and the effect on p38 activity was measured using a solid-phase
kinase assay. The intensity of the bands produced on an autoradiograph was determined and related to the control value to produce a fold induction for
each time point. The data presented are the means and SEs from nine experiments. B, Activation profile of p38 following stimulation with live S.
typhimurium. RAW cells were infected with S. typhimurium (MOI ⫽ 1) for the time indicated, and the effect on p38 activity was measured using a
solid-phase kinase assay. The intensity of the bands produced on an autoradiograph was determined and related to the (Figure legend continues)
5448
ROLE OF LPS IN Salmonella INFECTIONS
centrifuged at maximum speed for 15 min at 4°C. The supernatant containing the nuclear fraction was stored at ⫺70°C.
The transcription factor NF-␬B binds to a consensus oligonucleotide
sequence (5⬘-AGT TGA GGG GAC TTT CCC AGC C). This oligonucleotide was labeled with [␥-32P]ATP using T4 polynucleotide kinase (Promega, Madison, WI). EMSAs were performed in 10 ␮l of reaction mixture
containing 5 ␮g of nuclear extract, 5% glycerol, 1 mM MgCl2, 0.5 mM EDTA,
0.5 mM DTT, 50 mM NaCl, 10 mM Tris-HCl, pH 7.5, 0.05 mg/ml poly(dIdC).poly(dI-dC), and 0.2 ng DNA probe. The reactions were incubated for 20
min at room temperature. After addition of 1 ␮l of gel-loading buffer (250 mM
Tris-HCl, pH 7.5, 0.2% bromphenol blue, 40% glycerol), the reaction products
were resolved on 4% acrylamide gels containing 2% glycerol pre-electrophoresed with 0.5% Tris-borate-EDTA buffer at 100 V for 30 min. The radioactive
bands were visualized by autoradiography.
iNOS expression and activity
iNOS expression was measured by Western blot analysis of total cell lysates (31). Briefly, cells were lysed in buffer containing 10 mM EDTA and
1% Triton X-100 containing protease inhibitors (1 mM PMSF, 0.05 mM
pepstatin A, and 0.2 mM leupeptin). Western blot analysis for iNOS was
performed, as described previously (31), using rabbit anti-murine iNOS Ab
at a concentration of 1/10,000, and protein bands were visualized by ECL
(Amersham Pharmacia Biotech). As an indicator of iNOS activity, the
supernatants of cultured macrophages were removed 24 h after addition of
LPS and assayed for nitrite accumulation by the Griess reaction (32).
Briefly, an equal volume of Griess reagent (4% sulfanilamide and 0.2%
naphthylethylenediamine dihydrochloride in 10% phosphoric acid) was
added to an equal volume of sample, and the colorimetric difference in OD
at 540 and 620 nm was read immediately. The values obtained were compared with standard concentrations of sodium nitrite dissolved in DMEM,
and the concentration of nitrite in the samples was calculated.
Measurement of TNF-␣ activity
TNF-␣ was detected using a Duoset ELISA development system (R&D
systems, Abingdon, Oxfordshire, U.K.). The materials were all diluted and
stored, according to the manufacturer’s instructions. A seven-point standard curve of 2-fold dilutions from 15.625 to 1000 pg/ml of mouse rTNF-␣
was used. A volume of 100 ␮l of the standards and samples of the appropriate dilution was added to a 96-well microtiter plate. Supernatants taken
at 2 h were diluted 1/4, and those taken at 9 h were diluted 1/10.
control value to produce a fold induction for each time point. The data presented are the means and SEs from seven experiments. C, Activation profile of
JNK following stimulation with LPS. RAW cells were treated with LPS (1 ␮g/ml) for the time indicated, and the effect on JNK activity was measured using
a solid-phase kinase assay. The intensity of the bands produced on an autoradiograph was determined and related to the control value to produce a fold
induction for each time point. The data presented are the means and SEs from 11 experiments. D, Activation profile of JNK following stimulation with
live S. typhimurium. RAW cells were infected with S. typhimurium (MOI ⫽ 1) for the time indicated, and the effect on JNK activity was measured using
a solid-phase kinase assay. The intensity of the bands produced on an autoradiograph was determined and related to the control value to produce a fold
induction for each time point. The data presented are the means and SEs from nine experiments. E, Translocation of NF-␬B in response to LPS (1 ␮g/ml).
NF-␬B is found in the nucleus after 30-min stimulation with LPS and persists until 6 h. F, Translocation of NF-␬B in response to S. typhimurium (MOI ⫽
1). NF-␬B is found in the nucleus after 30-min stimulation with Salmonella and persists until 6 h.
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FIGURE 2. NO and TNF-␣ are produced by macrophages following stimulation with S. typhimurium or its LPS. A, NO production
from RAW macrophages after treatment with LPS. RAW cells were
treated for 24 h with increasing concentrations of S. typhimurium LPS
(0 –10,000 ng/ml), and the medium was removed and assayed for
nitrite concentration as a measure of NO production. B, NO production from RAW cells after treatment with LPS (1 ␮g/ml) or infection
with S. typhimurium (MOI ⫽ 1). RAW cells were treated for 24 h
with S. typhimurium LPS (100 ng/ml) or S. typhimurium (MOI ⫽ 1),
as described in Materials and Methods, and the medium was removed
and assayed for nitrite concentration as a measure of NO production.
C, TNF-␣ production at 2 h postinfection of RAW cells with S. typhimurium. RAW cells were infected for 2 h with S. typhimurium
(MOI ⫽ 10 – 0.01) or treated with LPS (1 ␮g/ml), as described in
Materials and Methods, and the medium was removed and assayed
for TNF-␣ production. D, TNF-␣ production at 9 h postinfection of
RAW cells with S. typhimurium. RAW cells were infected for 9 h
with S. typhimurium (MOI ⫽ 10 – 0.01) or treated with LPS (1 ␮g/
ml), as described in Materials and Methods, and the medium was
removed and assayed for TNF-␣ production.
The Journal of Immunology
Reagents
E5531 (in 100 mg/ml lactose, 0448 mg/ml Na2HPO4 䡠 7H2O, 0.36 mg/ml
NaH2PO4 䡠 H2O) was diluted in dH2O to a concentration of 100 ␮g/ml. The
placebo compound contained 100 mg/ml lactose, 0448 mg/ml
Na2HPO4 䡠 7H2O, and 0.36 mg/ml NaH2PO4 䡠 H2O dissolved in water, pH
7.5. Aliquots were made and stored at ⫺20°C for no more than 1 mo.
Unless otherwise stated, other reagents were obtained from Sigma-Aldrich
(Poole, Dorset, U.K.). LB broth and L-agar constituents were purchased
from Difco (Detroit, MI) and Oxoid (Basingstoke, U.K.).
Results
MAPK and NF-␬B signaling are activated by LPS stimulation
or infection with live S. typhimurium, but bacterial induced
signaling is delayed
NO and TNF-␣ are produced in RAW 264.7 macrophages by
LPS stimulation and by infection with S. typhimurium
NO production (Fig. 2A) and iNOS expression (data not shown)
from RAW cells were stimulated by concentrations of LPS greater
than 1 ng/ml and were dose dependent. Similarly, cells stimulated
by infection with S. typhimurium produced NO (Fig. 2B) due to the
induction of iNOS expression in these cells (Fig. 3B). An MOI of
1 stimulated a substantial NO response, whereas the responses
seen with an MOI of either 0.1 or 10 were reduced, the latter
probably due to macrophage mortality (data not shown). Increasing doses of LPS (1 ␮g/ml) and live S. typhimurium induced
TNF-␣ release from RAW cells (Fig. 2, C and D).
The induction of iNOS and TNF-␣ production by live
S. typhimurium infection is LPS dependent
The lipid A antagonist E5531 inhibited production of both NO and
TNF-␣ induced by purified LPS in all experiments (Fig. 3, A–D).
Similarly, when used to treat RAW cells before infection with S.
typhimurium, E5531 inhibited the production of NO in all cases
(Fig. 3, A and B). In contrast, although E5531 inhibited TNF-␣
production detected at 9 h after bacterial infection (Fig. 3D), it had
no effect on the induction of TNF-␣ release after 2 h of invasion
of RAW macrophages by living S. typhimurium. (Fig. 3C).
FIGURE 3. NO and late TNF-␣ production, but not early TNF-␣ production, by macrophages following infection with live S. typhimurium is inhibited
by the presence of E5531. A, The effect of E5531 on NO production from
RAW cells after treatment with LPS (0.001–1 ␮g/ml) or infection with S.
typhimurium (MOI of 1 or 0.1). RAW cells were simultaneously treated with
either E5531 or its placebo as well as three concentrations of S. typhimurium
LPS or 2 MOI of live bacteria, as described. Medium was removed and nitrite
release was determined as a measure of NO production (ⴱ, p ⬍ 0.05 between
placebo and E5531 group). B, The effect of E5531 on NO synthase induction
in RAW cells after treatment with LPS (1 ␮g/ml) or infection with S. typhimurium (MOI of 1). RAW cells were simultaneously treated with either E5531
or its placebo as well as S. typhimurium LPS or live bacteria, as described for
9 h. Medium was removed, and the cells were lysed and analyzed by Western
blot to measure iNOS protein expression. C, The effect of E5531 on TNF-␣
production from RAW cells after treatment with LPS (0.001–1 ␮g/ml) or infection with S. typhimurium (MOI of 1 or 0.1) for 2 h. RAW cells were simultaneously treated with either E5531 or its placebo as well as three concentrations of S. typhimurium LPS or 2 MOIs of live bacteria (0.1 and 1), as
described, for 2 h. Medium was removed and TNF-␣ production was determined (ⴱⴱ, p ⬍ 0.01; ⴱ, p ⬍ 0.05 between placebo and E5531 group). D, The
effect of E5531 on TNF-␣ production from RAW cells after treatment with
LPS (0.001–1 ␮g/ml) or infection with S. typhimurium (MOI of 1 or 0.1) for
9 h. RAW cells were simultaneously treated with either E5531 or its placebo
and either three concentrations of S. typhimurium LPS or 2 MOI of live bacteria, as described, for 9 h. Medium was removed and TNF-␣ production was
determined (ⴱⴱ, p ⬍ 0.01 between placebo and E5531 group).
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LPS produces a transient activation of both p38 and JNK (Fig. 1,
A and C). Compared with LPS-induced activation, S. typhimurium
induces a more delayed profile of activation of p38, which peaks
at 90 min (Fig. 1B). The magnitude of the p38 response to both
LPS and bacterial invasion was similar. The magnitude of JNK
activation by S. typhimurium was reduced in comparison with that
induced by LPS. In addition, the time course of activation of JNK
was delayed in response to infection with S. typhimurium with no
obvious peak (Fig. 1, C and D). NF-␬B was activated by LPS and
S. typhimurium from 30 min and sustained for at least 6 h (Fig. 1,
E and F).
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ROLE OF LPS IN Salmonella INFECTIONS
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FIGURE 4. The central role of LPS in NO and TNF-␣ production is confirmed using primary macrophages from TLR-4 knockout mice and C3H/HeJ
mice. A, NO production after treatment or infection of macrophages from wild-type and TLR-4 knockout mice with LPS or S. typhimurium, respectively.
Macrophages were isolated and characterized, as described. Cells were treated for 24 h with S. typhimurium LPS (0 –100 ng/ml) or S. typhimurium (MOI ⫽
1), as described in Materials and Methods, and the medium was removed and assayed for nitrite concentration as a measure of NO production (open bars
are macrophages from wild-type mice (MO); filled bars from TLR-4 knockout mice). B, TNF-␣ production at 2 h (Figure legend continues)
The Journal of Immunology
The production of TNF-␣ and NO stimulated by S. typhimurium
is through a TLR-4-dependent mechanism
MAPK, but not NF-␬B, signaling stimulated by S. typhimurium
is LPS independent
Our data to date show that despite LPS and S. typhimurium showing differential patterns of cell signaling activation, the release of
both NO and TNF-␣ is LPS dependent. We therefore investigated
to what extent the delayed activation of signaling pathways seen
during S. typhimurium infection was dependent on LPS. LPS-induced activation of both p38 and JNK was inhibited by E5531
(Fig. 5, A and D). In contrast, E5531 had no effect on the activation
of these MAPKs in response to S. typhimurium infection (Fig. 5, B
and E). To confirm that our signaling results were not a cell line
artifact, we assayed p38 MAPK activity in LPS-treated and S.
typhimurium-infected bone marrow cells from C3H/HeN (wildtype) and C3H/HeJ (TLR-4 mutant) mice. Activation of p38 was
seen in cells from C3H/HeN mice in response to both LPS and
bacterial infection, the latter having a later time course of action
than that seen with LPS. S. typhimurium infection activated p38 in
cells from C3H/HeJ mice, showing a late time course of activation,
confirming the presence of TLR-4-independent signaling by the
bacteria in macrophage-like cells (Fig. 5C). Interestingly, the LPSinduced signaling activity of p38 MAPK was not fully inhibited in
the C3H/HeJ cells, although the time course of activation was different from that seen in the LPS-activated C3H/HeN cells. In
RAW macrophages, LPS (1 ␮g/ml) and infection with S. typhimurium (MOI ⫽ 1) both stimulated translocation of NF-␬B to the
nucleus at the 30-min and 4-h time points (Fig. 5, F and G). Incubation of the RAW cells with E5531 inhibited NF-␬B translocation by both LPS and bacterial invasion at 4 h, but had no detectable effect on the 30-min time point after S. typhimurium
infection (see Fig. 5, F and G).
Discussion
In this work, data are presented that strongly support the view that
LPS presented by living S. typhimurium, through activation of TLR-4,
is vital to stimulating innate immune responses in macrophages. Most
researchers in the field have assumed this to be the case, and some
have inferred this conclusion from experiments using purified bacterial components, but these are the first experiments to provide direct
experimental evidence supporting this view.
In gene array studies, altered expression of a number of genes
was observed upon stimulation of RAW 264.7 macrophages with
either LPS or a high MOI of S. typhimurium, many of which were
the same for both stimuli, including TNF-␣ and iNOS (15). The
changes in gene expression were generally greater in the cells
treated with LPS than in those infected by bacteria. The fact that
the expression of a similar set of genes was affected by both stimuli
may infer that LPS is more important as a stimulator of cytokine
production than TTSS effector proteins produced by S. typhimurium in macrophages (15).
Our in vitro studies compared responses from macrophages
from mice of the C3H/HeJ and C3H/HeN lineages, as well as from
the TLR-4 knockout mice, and combined these studies with the use
of the lipid A antagonist E5531. Synthetic lipid A analogs such as
E5531 inhibit in vivo and in vitro responses stimulated by LPS (25,
34 –36) principally by acting as an antagonist at TLR-4 (37, 38).
Our combined experimental approach has enabled the determination of the importance of LPS as a component of live S. typhimurium in inducing the macrophage response to infection.
Our data provide direct evidence to support the idea that LPS is
a centrally important stimulator of the expression of genes encoding proteins such as TNF-␣ and iNOS in response to live bacterial
infection, as inhibition of the effects of S. typhimurium LPS by
E5531 or infection of TLR-4-defective macrophages prevents production of both these proteins. Studies showing a primary role for
TTSS bacterial proteins in stimulating host cytokine production
have been in epithelial cells (2), which have a more limited LPSinduced cytokine response than macrophages and may simply reflect differences in the roles of these cells in the host response to
bacterial infection.
postinfection of macrophages from wild-type and TLR-4 KO mice with S. typhimurium. Macrophages were isolated and characterized, as described. Cells
were treated for 2 h with S. typhimurium LPS (1 ␮g/ml) or S. typhimurium (MOI ⫽ 1–10), as described in Materials and Methods, and the medium was
removed and assayed for TNF-␣ production (open bars are macrophages from wild-type mice (MO); filled bars from TLR-4 knockout mice). C, TNF-␣
production at 9 h postinfection of macrophages from wild-type and TLR-4 KO mice with S. typhimurium. Macrophages were isolated and characterized,
as described. Cells were treated for 9 h with S. typhimurium LPS (0.1–1 ␮g/ml) or S. typhimurium (MOI ⫽ 1–10), as described in Materials and Methods,
and the medium was removed and assayed for TNF-␣ production (open bars are macrophages from wild-type mice; filled bars from TLR-4 knockout mice).
D, The effect of E5531 on NO production in macrophages from wild-type (C3H/HeN) and TLR-4 mutant (C3H/HeJ) mice after treatment with LPS (1
␮g/ml) or infection with S. typhimurium (MOI ⫽ 1). Macrophages were isolated and characterized, as described. Cells were simultaneously treated with
either E5531 or its placebo and either S. typhimurium LPS (1000 ng/ml) or infected with S. typhimurium (MOI ⫽ 1), as described. Medium was removed
after 24 h, and nitrite release was determined as a measure of NO production. E, The effect of E5531 on TNF-␣ production in macrophages from wild-type
(C3H/HeN) and TLR-4 mutant (C3H/HeJ) mice after treatment with LPS (1 ␮g/ml) or infection with S. typhimurium (MOI ⫽ 1) for 9 h. Macrophages were
isolated and characterized, as described. Cells were simultaneously treated with either E5531 or its placebo and either S. typhimurium LPS or infected with
S. typhimurium, as described. Medium was removed after 9 h, and TNF-␣ production was determined.
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To determine the contribution of TLR-4 to the induction of iNOS
and TNF-␣, bone marrow macrophages were isolated and cultured
from C3H/HeJ mice, with a dominant-negative mutation in TLR-4
(8) and compared with cells from the C3H/HeN mouse strain that
expresses wild-type TLR-4. Cells were also isolated from TLR-4
knockout mice (24, 33) and compared with cells from wild-type
mice. NO and TNF-␣ production was assayed in all of these primary cell cultures in response to stimulation by LPS or live S.
typhimurium infection. In these experiments, NO production in
response to either stimulus was observed in macrophages from
C3H/HeN or wild-type animals, but not from the C3H/HeJ and
TLR-4 knockout animals (Fig. 4, A and D). This is strong evidence
supporting the view that iNOS induction is mediated through a
TLR-4-dependent mechanism, which is similar whether live bacteria or LPS is used to stimulate the cells. LPS and S. typhimurium
were both able to induce TNF-␣ release from cells taken from
wild-type mice at both 2 and 9 h poststimulation (Fig. 4, B and C).
In contrast, TNF-␣ release was inhibited in macrophages from
both C3H/HeJ and TLR-4 knockout mice in response to both LPS
and S. typhimurium infection. In macrophages from wild-type
C3H/HeN mice, the induction of both NO and TNF-␣ release was
inhibited by E5531 (Fig. 4, D and E) at all time points tested. This
suggests that both LPS treatment and infection with S. typhimurium induce NO and TNF-␣ production principally through a
TLR-4-dependent mechanism.
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ROLE OF LPS IN Salmonella INFECTIONS
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FIGURE 5. Early signaling events induced by S. typhimurium are unaffected by the presence of E5531, but NF-␬B translocation at 4 h is blocked by
the antagonist. A, E5531 blocks LPS-induced activation of p38. RAW cells were treated with E5531 or placebo and stimulated with LPS (1 or 0.1 ␮g/ml)
for the time indicated. Activity of p38 was measured by a solid-phase kinase assay. The autoradiograph shown is representative of three repeats. B, E5531
has no effect on the activation of p38 induced by S. typhimurium. RAW cells were treated with E5531 or placebo and (Figure legend continues)
The Journal of Immunology
venting LPS-induced activation of cell signaling pathways leading
to NF-␬B. Our study showed LPS-independent activation of the
MAPKs JNK and p38 throughout the course of infection. Similarly, TLR-4-independent signaling activity was seen in S. typhimurium-infected primary bone marrow macrophage cells isolated
from C3H/HeJ mice, confirming these observations were not an
artifact of the RAW cell line. Activation of NF-␬B at 30 min
postinfection was also LPS independent, whereas the NF-␬B response at 4 h postinfection was LPS dependent. Despite these differences in signaling activation in RAW macrophages in response
to LPS or S. typhimurium infection, we could not detect major
differences in TNF-␣ or NO responses to either stimulus.
There are several possible explanations for why we see differences in signaling between LPS- and infection-stimulated cells,
and yet both stimuli lead to TNF-␣ and NO production. It is possible that the very early LPS-independent activation of NF-␬B during infection is insufficient to stimulate the TNF-␣ and NO responses seen throughout the infection, that sustained NF-␬B
activation is required for these responses, and that this is LPS dependent. It is also possible that the early LPS-independent event leads to
activation of an isoform of NF-␬B that is not involved in the regulation of TNF-␣ and iNOS. The precise roles of the MAPKs JNK and
p38 in induction of TNF-␣ and iNOS are unclear, but many studies
have suggested a number of roles for these kinases in full responsiveness. The fact that there are very different profiles of MAPK activation
when cells are stimulated by LPS or infection, and that the infectioninduced events are not LPS dependent raises fascinating questions
concerning precisely how the encounter between macrophages and
infecting salmonellae leads to the responses observed. It will be intriguing to discover how important cellular responses other than
TNF-␣ and iNOS production differ between cells stimulated by purified LPS or live infection and whether these correlate with the differences that we have seen in MAPK stimulation. Bacteria of the
Salmonella family produce a number of specialized effector proteins
that can modify host cell signaling (2), so this might explain the LPSindependent signal transduction activation seen in our cells. The functional consequences of this LPS/TLR-4-independent signaling are
currently unclear, but a number of macrophage functions may be influenced, such as phagocytosis or cellular survival rather than the
production of inflammatory cytokines.
In conclusion, our study clearly demonstrates that the LPS
present in live S. typhimurium stimulates TLR-4, leading to the
nuclear translocation of NF-␬B and the production of TNF-␣ and
iNOS. It is possible that LPS activation of macrophages during
invasion may modify, through initiation of complex signaling
pathways, the effects of TTSS effector proteins on the cell. Using
stimulated with S. typhimurium (MOI ⫽ 1) for the time indicated. Activity of p38 was measured by a solid-phase kinase assay. The intensity of the bands produced
on an autoradiograph was determined and related to the control value to produce a fold induction for each time point. The data presented are the means and SEs
from six experiments. C, Activation of p38 MAPK induced by S. typhimurium infection is independent of TLR-4. Bone marrow-derived cells from wild-type
(C3H/HeN) and TLR-4 mutant (C3H/HeJ) mice were either stimulated with LPS (1 ␮g/ml) or infected with S. typhimurium (C5; MOI ⫽ 1) for the time indicated.
Activity of p38 was measured by immunoprecipitation of the kinase, followed by an in vitro kinase reaction (pP38, activated p38; IgH, H chain IgG; IgL, L chain
IgG; P38, total p38 MAPK protein). D, E5531 blocks LPS-induced activation of JNK. RAW cells were treated with E5531 or placebo and stimulated with LPS
(1 or 0.1 ␮g/ml) for the time indicated. Activity of JNK was measured by a solid-phase kinase assay. The autoradiograph shown is representative of three repeats.
E, E5531 has no effect on the activation of JNK induced by S. typhimurium. RAW cells were treated with E5531 or placebo and stimulated with S. typhimurium
(MOI ⫽ 1) for the time indicated. Activity of JNK was measured by a solid-phase kinase assay. The intensity of the bands produced on an autoradiograph was
determined and related to the control value to produce a fold induction for each time point. The data presented are the means and SEs from five experiments. F,
The effect of E5531 on NF-␬B translocation in RAW cells after treatment with LPS (100 ng/ml) or infection with S. typhimurium (MOI ⫽ 1) for 30 min. RAW
cells were simultaneously treated with either E5531 or its placebo and either S. typhimurium LPS or live bacteria, as described, for 30 min. After washing with
PBS, the nuclear fraction was extracted, and EMSAs to measure nuclear translocation of NF-␬B were performed using a ␥-32P-labeled NF-␬B consensus
recognition site oligonucleotide. G, The effect of E5531 on NF-␬B translocation RAW cells after treatment with LPS (100 ng/ml) or infection with S. typhimurium
(MOI ⫽ 1) for 4 h. RAW cells were simultaneously treated with either E5531 or its placebo and either S. typhimurium LPS (100 ng/ml) or live bacteria (0.1 and
1), as described, for 4 h. After washing with PBS, the nuclear fraction was extracted, and EMSAs to measure nuclear translocation of NF-␬B were performed using
a ␥-32P-labeled NF-␬B consensus recognition site oligonucleotide.
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The role of macrophage stimulation with the consequent production of mediators, such as NO, in response to infection with S.
typhimurium has been speculated upon for some time (12). Enhanced production of NO, in association with oxygen radicals, is
proposed to have both bactericidal and bacteristatic effects, and
hence may be important in inhibiting bacterial growth (39, 40).
Mice that lack iNOS are more susceptible to salmonellae and are
unable to limit bacterial growth in the later stages of Salmonella
infection (41, 42). Experiments with macrophages from these animals indicate that iNOS may contribute to both early and late
limitation of bacterial growth by enhancing the early oxygen radical killing of S. typhimurium and by a later NO-dependent bacteristatic effect (43). We show in this study that live S. typhimurium infection stimulates the production of NO from
macrophages by an LPS- and TLR-4-dependent mechanism.
The production of the cytokine TNF-␣ in vivo during Salmonella infection is protective to the host (44, 45), and we show in
this study that in primary macrophages TNF-␣ release in response
to live infection is again dependent on LPS and TLR-4. Unexpectedly, given the results with primary cells, we observed that E5531
was ineffective at inhibiting the early release of TNF-␣ from the
RAW macrophage-like cell line. As an immortalized cell line, the
RAW cells may have cellular responses that differ from those seen
in primary macrophages (46). Immortalization may result in alterations in signal transduction pathways possibly involving NF-␬B
and MAPKs, which could influence how these cells respond to
external stimuli. Alternatively, the presence of other TLR ligands,
such as bacterial proteins, during bacterial infection may be sufficient to activate TLRs other than TLR-4 in RAWs, but not primary cells, to cause the early release of this cytokine. The LPSindependent early release of TNF-␣ from RAW cells may be by a
mechanism that modulates translation stimulated either by bacterial
invasion of the RAW cell or by production of specific bacterial components that activate the MAPK pathways known to be associated
with posttranscriptional regulation of TNF-␣ production (47– 49).
Activation of the transcription factor NF-␬B is necessary for
expression of both iNOS and TNF-␣ in response to TLR-mediated
signal transduction, but expression and production of both proteins
can be enhanced by other factors (50 –52). For example, TNF-␣
production within the cell is controlled at both the transcriptional
and posttranscriptional levels, and both need to be fully activated
for maximum production of this cytokine (44, 53, 54). The
MAPKs, particularly JNK, are important in regulating posttranscriptional TNF-␣ production (49). We therefore hypothesized that
E5531 was preventing the production of iNOS and TNF-␣ after
macrophage infection with S. typhimurium at least in part by pre-
5453
5454
these mechanisms, salmonellae may modify macrophage function
and thus promote growth and/or dissemination through the host.
Acknowledgments
ROLE OF LPS IN Salmonella INFECTIONS
29.
We thank Eisai Research Institute (Andover, MA) for provision of E5531
and Prof. Akira for provision of mice.
30.
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