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
J Immunol June 1, 2003, 170 (11) 5445-5454; DOI: https://doi.org/10.4049/jimmunol.170.11.5445

Abstract

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 4−/− (TLR-4−/−) mice. We show that LPS presented on live salmonellae provides an essential signal, via functional TLR-4, for macrophages to produce NO and TNF-α. Furthermore, the mitogen-activated protein kinase c-Jun N-terminal kinase and p38 are activated, and the transcription factor NF-κB 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-κB, 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-α 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.

Salmonella 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, 4, 5). The investigation of the precise role of the different PAMPs 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, 8, 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-κB, 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 synthase (iNOS) and the receptor for the cytokine TNF-α (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, 19, 20, 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-κB 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-α production is LPS and TLR-4 dependent when macrophage-like cells are invaded by S. typhimurium.

Materials and Methods

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.

Cell culture

RAW 264.7 cells were cultured in DMEM containing 10% FCS supplemented with 2 mM glutamine, 200 U/ml penicillin, and 100 μg/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-CSF-producing 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-α production, following a 2-h incubation, cell supernatants were taken and the cells were washed with PBS and incubated in DMEM containing 50 μg/ml gentamicin for 1 h. Cells were then washed again in PBS and incubated in DMEM containing 10 μg/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 solid-phase 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 β-glycerophosphate, 0.5 mM Na3VO4, 0.2 mM PMSF, and 1 μg/ml leupeptin and aprotinin). The beads were washed once in the lysis buffer and once in kinase buffer containing 20 mM MgCl2, 5 mM β-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 μM ATP and 2 μCi [γ-32P]ATP per reaction for p38 and 25 μM ATP and 1 μCi [γ-32P]ATP per reaction for JNK was added for 30 min. The reaction was stopped by the addition of 10 μl 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 μg/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 μl) 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.).

EMSA

To prepared nuclear pellets, cells (3 × 106) were washed twice with ice-cold 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 μl 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 μg/ml leupeptin, pepstatin, and aprotinin) and put on ice to swell for 15 min. After addition of 25 μl 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 μl 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 μg/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 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-κB binds to a consensus oligonucleotide sequence (5′-AGT TGA GGG GAC TTT CCC AGC C). This oligonucleotide was labeled with [γ-32P]ATP using T4 polynucleotide kinase (Promega, Madison, WI). EMSAs were performed in 10 μl of reaction mixture containing 5 μg 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(dI-dC).poly(dI-dC), and 0.2 ng DNA probe. The reactions were incubated for 20 min at room temperature. After addition of 1 μl 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-α activity

TNF-α 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-α was used. A volume of 100 μl 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.

Reagents

E5531 (in 100 mg/ml lactose, 0448 mg/ml Na2HPO4·7H2O, 0.36 mg/ml NaH2PO4·H2O) was diluted in dH2O to a concentration of 100 μg/ml. The placebo compound contained 100 mg/ml lactose, 0448 mg/ml Na2HPO4·7H2O, and 0.36 mg/ml NaH2PO4·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-κB signaling are activated by LPS stimulation or infection with live S. typhimurium, but bacterial induced signaling is delayed

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. 1⇓B). 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-κB was activated by LPS and S. typhimurium from 30 min and sustained for at least 6 h (Fig. 1⇓, E and F).

FIGURE 1.
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 μg/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) 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 μg/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-κB in response to LPS (1 μg/ml). NF-κB is found in the nucleus after 30-min stimulation with LPS and persists until 6 h. F, Translocation of NF-κB in response to S. typhimurium (MOI = 1). NF-κB is found in the nucleus after 30-min stimulation with Salmonella and persists until 6 h. (Figure legend continues)

NO and TNF-α are produced in RAW 264.7 macrophages by LPS stimulation and by infection with S. typhimurium

NO production (Fig. 2⇓A) 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. 2⇓B) due to the induction of iNOS expression in these cells (Fig. 3⇓B). 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 μg/ml) and live S. typhimurium induced TNF-α release from RAW cells (Fig. 2⇓, C and D).

FIGURE 2.
FIGURE 2.

NO and TNF-α 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 μg/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-α 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 μg/ml), as described in Materials and Methods, and the medium was removed and assayed for TNF-α production. D, TNF-α 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 μg/ml), as described in Materials and Methods, and the medium was removed and assayed for TNF-α production.

FIGURE 3.
FIGURE 3.

NO and late TNF-α production, but not early TNF-α 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 μg/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 (∗, 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 μg/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-α production from RAW cells after treatment with LPS (0.001–1 μg/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-α production was determined (∗∗, p < 0.01; ∗, p < 0.05 between placebo and E5531 group). D, The effect of E5531 on TNF-α production from RAW cells after treatment with LPS (0.001–1 μg/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-α production was determined (∗∗, p < 0.01 between placebo and E5531 group).

The induction of iNOS and TNF-α production by live S. typhimurium infection is LPS dependent

The lipid A antagonist E5531 inhibited production of both NO and TNF-α 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-α production detected at 9 h after bacterial infection (Fig. 3⇑D), it had no effect on the induction of TNF-α release after 2 h of invasion of RAW macrophages by living S. typhimurium. (Fig. 3⇑C).

The production of TNF-α and NO stimulated by S. typhimurium is through a TLR-4-dependent mechanism

To determine the contribution of TLR-4 to the induction of iNOS and TNF-α, 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-α 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-α release from cells taken from wild-type mice at both 2 and 9 h poststimulation (Fig. 4⇓, B and C). In contrast, TNF-α 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-α 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-α production principally through a TLR-4-dependent mechanism.

FIGURE 4.
FIGURE 4.

The central role of LPS in NO and TNF-α 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-α production at 2 h (Figure legend continues) 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 μg/ml) or S. typhimurium (MOI = 1–10), as described in Materials and Methods, and the medium was removed and assayed for TNF-α production (open bars are macrophages from wild-type mice (MO); filled bars from TLR-4 knockout mice). C, TNF-α 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 μg/ml) or S. typhimurium (MOI = 1–10), as described in Materials and Methods, and the medium was removed and assayed for TNF-α 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 μg/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-α production in macrophages from wild-type (C3H/HeN) and TLR-4 mutant (C3H/HeJ) mice after treatment with LPS (1 μg/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-α production was determined.

MAPK, but not NF-κB, 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-α 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 (wild-type) 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. 5⇓C). Interestingly, the LPS-induced 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 μg/ml) and infection with S. typhimurium (MOI = 1) both stimulated translocation of NF-κB 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-κB 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).

FIGURE 5.
FIGURE 5.

Early signaling events induced by S. typhimurium are unaffected by the presence of E5531, but NF-κB 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 (Figure legend continues) stimulated with LPS (1 or 0.1 μg/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 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 μg/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 μg/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-κB 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-κB were performed using a γ-32P-labeled NF-κB consensus recognition site oligonucleotide. G, The effect of E5531 on NF-κB 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-κB were performed using a γ-32P-labeled NF-κB consensus recognition site oligonucleotide.(Figure legend continues).

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-α 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, 35, 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-α 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 LPS-induced cytokine response than macrophages and may simply reflect differences in the roles of these cells in the host response to bacterial infection.

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-α in vivo during Salmonella infection is protective to the host (44, 45), and we show in this study that in primary macrophages TNF-α 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-α 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-κB 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 LPS-independent early release of TNF-α 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-α production (47, 48, 49).

Activation of the transcription factor NF-κB is necessary for expression of both iNOS and TNF-α in response to TLR-mediated signal transduction, but expression and production of both proteins can be enhanced by other factors (50, 51, 52). For example, TNF-α 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-α production (49). We therefore hypothesized that E5531 was preventing the production of iNOS and TNF-α after macrophage infection with S. typhimurium at least in part by preventing LPS-induced activation of cell signaling pathways leading to NF-κB. 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-κB at 30 min postinfection was also LPS independent, whereas the NF-κB 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-α 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-α and NO production. It is possible that the very early LPS-independent activation of NF-κB during infection is insufficient to stimulate the TNF-α and NO responses seen throughout the infection, that sustained NF-κB 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-κB that is not involved in the regulation of TNF-α and iNOS. The precise roles of the MAPKs JNK and p38 in induction of TNF-α 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 infection-induced 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-α 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 LPS-independent 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-κB and the production of TNF-α 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 these mechanisms, salmonellae may modify macrophage function and thus promote growth and/or dissemination through the host.

Acknowledgments

We thank Eisai Research Institute (Andover, MA) for provision of E5531 and Prof. Akira for provision of mice.

Footnotes

  • ↵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: ceb27{at}cam.ac.uk

  • ↵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, Toll-like receptor; TTSS, type III secretion system.

  • Received September 23, 2002.
  • Accepted March 25, 2003.

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