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International Immunology Advance Access originally published online on March 30, 2006
International Immunology 2006 18(5):785-795; doi:10.1093/intimm/dxl015
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© The Japanese Society for Immunology. 2006. All rights reserved. For permissions, please e-mail: journals.permissions@oxfordjournals.org

Francisella tularensis LPS induces the production of cytokines in human monocytes and signals via Toll-like receptor 4 with much lower potency than E. coli LPS

Ana I Dueñas1, Mónica Aceves1, Antonio Orduña2, Ramón Díaz3, Mariano Sánchez Crespo1 and Carmen García-Rodríguez1

1 Instituto de Biología y Genética Molecular, Universidad de Valladolid-CSIC, Valladolid, Spain
2 Unidad de Investigación, Hospital Clínico Universitario, Valladolid, Spain
3 Departamento de Microbiología, Universidad de Navarra, Pamplona, Spain

Correspondence to: C. García-Rodríguez; E-mail: cgarcia{at}ibgm.uva.es


   Abstract
Top
 Abstract
Introduction
 Methods
 Results
 Discussion
 References
 
Francisella tularensis is a virulent Gram-negative intracellular pathogen. To address the signaling routes involved in the response of host cells to LPS from F. tularensis live vaccine strain (LVS), experiments were performed in transiently transfected 293 cells. Induction of {kappa}B-driven transcriptional activity by 2.5 µg ml–1 F. tularensis LPS isolated by phenol–water and ether–water extraction, was observed in cells transfected with Toll-like receptor (TLR) 4 and MD-2, although CD14 was required for optimal induction. Conversely, TLR2, TLR2/TLR1 or TLR2/TLR6 transfected cells did not show {kappa}B-driven transcriptional activity in the presence of F. tularensis LPS. In human monocytic cells, F. tularensis LPS activated extracellular signal-regulated kinases and the production of pro-inflammatory proteins. Concentrations of 5–10 µg ml–1 F. tularensis LPS elicited a similar pattern of mRNA and protein induction than 0.1 µg ml–1 E. coli LPS, including the expression of CXC chemokines (IL-8, Gro and IFN-{gamma}-inducible protein-10); CC chemokines (monocyte chemoattractant protein-1 and -2, macrophage-derived chemoattractant, macrophage inflammatory protein-1{alpha} and -1ß and RANTES (regulated upon activation, normal T cell expressed and secreted) and pro-inflammatory cytokines (IL-6 and tumor necrosis factor {alpha}). Altogether, these data indicate that LPS from F. tularensis LVS signals via TLR4 at higher concentrations than those required for E. coli LPS, which may explain the inflammatory reaction and the low endotoxic response associated to vaccination with LVS in humans.

Keywords: bacterial infection, chemokines, LPS, mitogen-activated protein kinase, transcription factors


   Introduction
Top
 Abstract
 Introduction
Methods
 Results
 Discussion
 References
 
The facultative intracellular bacterium Francisella tularensis (order Thiotrichales, family Francisellaceae) is a virulent Gram-negative microorganism that causes tularemia, a severe and often fatal infection of humans and animals (reviewed in 1), the seriousness of which has made it likely its inclusion as a potential agent of bioterrorism (2). F. tularensis is usually classified in two main subspecies: the type A or subspecies tularensis, the most virulent for humans, and the type B or subspecies holartica. The live vaccine strain (LVS), derived from type B, is a relative avirulent strain that remains the only effective tularemia vaccine developed to date. LVS is fully pathogenic in several animals and causes lethal infection (3, 4). F. tularensis LVS has been widely used as a model for studies of immune responses to intracellular bacteria (5), although the molecular mechanisms underlying its biological effects on host cells (reviewed in 6) are not well understood. The structure of the lipid A of LPS in F. tularensis is different from that of Escherichia coli (7), as regards its lack of phosphate groups at the 1- and 4'-positions of the 2-dideoxy, 2-aminoglucose of the disaccharide backbone and the presence of four acyl moieties instead of six chains present in E. coli LPS (Fig. 1A, C).


Figure 1
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  		Fig. 1. Chemical structures of LPS lipids A. The structures of LPS from Escherichia coli (A), Brucella/Ochrobactrum (B) and Francisella tularensis (C) are shown according to the data reported by Zhou et al. (52), Moriyon (53), Velasco et al. (54) and Vinogradov et al. (7). The main structural differences refer to the presence of phosphate groups, the disaccharide backbone and the number and length of the acyl chains. (D) Silver-stained SDS-PAGE pattern of LPS from: lane 1, F. tularensis EW–LPS (2 µg), lane 2, F. tularensis PW–LPS (0.4 µg) and lane 3, E. coli (1 µg).

 
Toll-like receptors (TLRs) are pathogen-recognition molecules that activate the immune system and have a central role in the early host response to microbial components (reviewed in 8–11). Members of the TLR family, 11 reported genes in humans to date, are expressed differentially among host cells and specifically respond to diverse conserved molecular structures shared by large groups of microbes. Stimulation of TLRs, with the possible exception of TLR3, triggers a classical signaling cascade involving the association with Toll/IL-1 receptor (TIR) domain containing adaptor molecules such as myeloid differentiation primary response protein 88 (MyD88) and MyD88 adaptor like (also know as TIR domain-containing adaptor protein and TIR domain-containing adaptor molecule-2), which ultimately leads to the activation of the nuclear factor-{kappa}B (NF-{kappa}B) and the expression of pro-inflammatory cytokine genes. In addition, TLR4 and TLR3 activation trigger an alternative signaling pathway that involves adaptors such as TIR domain containing adaptor protein-inducing IFNß (TRIF) and TRIF-related adaptor protein, which leads to the induction of IFN-inducible genes and a late NF-{kappa}B response (reviewed in 10). TLR4 recognizes LPS produced by most Gram-negative bacteria (9), but in view of the structural differences among LPS and their varying ability to produce endotoxic shock, the effect of these molecules from different sources needs to be analyzed in terms of chemical structure versus biological activity. In a previous report, we have demonstrated that LPS from some Gram-negative facultative intracellular bacteria such as Brucella spp. displays a limited capacity to activate TLR4 on host cells, thereby explaining the reduced incidence of endotoxic shock in human brucellosis (12). In addition to NF-{kappa}B activation, LPS induces other intracellular responses like the activation of several members of the mitogen-activated protein kinase (MAPK) family, including extracellular signal-regulated kinases (ERK) 1 and 2 (13), c-Jun amino-terminal kinases (JNKs) (14) and p38 (15).

In contrast to early reports showing that TLR4-defective C3H/HeJ mice were more susceptible to systemic infection with F. tularensis LVS (16), it has recently been reported that TLR4 might play a relative minor role in murine defense against sub-lethal intra-dermal infection of LVS (17), as well as in the model of low-dose aerosol infection with a virulent type of F. tularensis (18). Moreover, LVS suppresses the capability of the murine macrophage-like cell line J774A to respond to E. coli LPS with activation of NF-{kappa}B and c-Jun routes (19), although the molecular analysis of this phenomenon has not been carried out. There have been reported some differences as regards the immunogenic potential of F. tularensis LPS depending on the procedure of purification utilized (20), which might account for changes of the native structure explaining the different biological properties of these LPS. On this basis, we have analyzed the effect of LPS from F. tularensis LVS isolated by two different procedures, ether–water (EW) and phenol–water (PW) in a system of ectopic expression of TLR4 in 293 cells. A weak agonist effect of both EW–LPS and PW–LPS on TLR4 was observed, but optimal induction was observed in the presence of the accessory recognition molecule MD-2 and the co-receptor CD14. We did not find any evidence of the involvement of other TLRs, namely TLR1, TLR2 and TLR6 in F. tularensis LPS signaling. F. tularensis LPS activates ERK1/2 signaling pathways at concentrations of 5–10 µg ml–1. Experiments in monocytic cells showed similar patterns of chemokine mRNA induction and pro-inflammatory protein production elicited by F. tularensis and E. coli LPS, although the induction by F. tularensis LPS needed significantly higher doses than those required for E. coli LPS. These data suggest that the biological actions of LPS from F. tularensis LVS might explain the occurrence of an inflammatory response and a low endotoxic reaction, which makes LVS suitable for use as a vaccine against tularemia.


   Methods
Top
 Abstract
 Introduction
 Methods
Results
 Discussion
 References
 
Expression plasmids
Hemagglutinin-tagged cDNAs of human TLR2 and TLR4, cloned into the expression plasmid pRc/CMV, were provided by Michael Rehli (University of Regensburg, Germany) (21). A pEF-Bos vector encoding human TLR6 tagged with Myc at the C-terminus was provided by Akira and Uematsu (Osaka University, Osaka, Japan). Human CD14 cDNA cloned in pRc/RSV vector and pFLAG-CMV1 human expression vector engineered to introduce human MD-2 were provided by Peter S. Tobias (The Scripps Research Institute, La Jolla, CA, USA) (22). pUNO-TLR1 plasmid and palmitoyl-3-cysteine-serine-lysine-4 (Pam3CSK4) were purchased from InvivoGen (San Diego, CA, USA). RNase, DNase, proteinase K, LPS from E. coli O111:B4, 2-keto-3-deoxyoctulosonic acid (KDO) and 2-deoxi-D-ribose were purchased from Sigma (St Louis, MO, USA). Peptidoglycan was from Fluka (St Louis, MO, USA). Firefly luciferase-linked 5xNF-{kappa}B reporter gene was from Stratagene (La Jolla, CA, USA).

Extraction of LPS from F. tularensis
Stock cultures of F. tularensis NCTC 10857 (LVS) were prepared in chocolate agar plates. The cultures were incubated for 48 h at 37°C; the bacteria were then collected in distilled water and washed three times by centrifugation (9000 x g). PW–LPS was extracted by Westphal and Jann's hot PW method (23) modified for F. tularensis (24). Briefly, the bacteria from the stock cultures were killed by treatment with 0.5% phenol (final concentration) for 24 h at 37°C. PW–LPS of F. tularensis was collected from the aqueous phase, and precipitated for 24 h at –20°C in four volumes of cold methanol reagent (99 parts of methanol and 1 part of methanol saturated with sodium acetate). The crude PW–LPS was collected by centrifugation at 8000 x g for 20 min, suspended in distilled water, dialyzed and then lyophilized. To obtain EW–LPS (25), 50 ml of diethyl ether was added to 150 ml of bacterial suspension and the mixture was shaken vigorously. The suspension was left overnight at room temperature, and the aqueous phase was then drawn off and centrifuged to eliminate cell residues. The supernatant was precipitated in four volumes of cold methanol reagent for 24 h at –20°C. The crude EW–LPS was collected and lyophilized. After extraction, crude EW–LPS and PW–LPS were treated three times with 0.1 mg ml–1 RNase A and 0.1 mg ml–1 DNase II type V at 37°C for 30 min, and then three times with proteinase K at 1 mg ml–1 at 55°C for 3 h. The purified EW–LPS and PW–LPS were harvested by centrifugation at 100 000 x g for 6 h, and later dialyzed and lyophilized. The KDO content was determined colorimetrically by a modified thiobarbituric acid method (26). KDO was used as standard and 2-deoxy-D-ribose used to correct for deoxysugar interference, and absorbance at 552 and 536 nm was measured. The KDO contents were 0.45 and 0.35% for EW–LPS and PW–LPS, respectively. Purified LPS was boiled for 1 h before use and was characterized by SDS-PAGE followed by oxidation with periodate and silver staining (27).

Cell culture and transient transfection
Human embryonic kidney 293 cells (American Type Tissue Collection, Rockville, MD, USA) were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, 100 U ml–1 penicillin G, 100 µg ml–1 streptomycin and 2 mM L-glutamine. Cells were cultured at 37°C in an atmosphere containing 5% CO2. THP-1 cells were cultured in RPMI 1640 medium supplemented with 100 U ml–1 penicillin, 100 µg ml–1 streptomycin, 2 mM glutamine and 10% fetal bovine serum. Human monocytes were isolated from blood of healthy donors by centrifugation into Ficoll cushions and adherence to plastic dishes. For protein production experiments, monocytes were kept 48 h in ITS (bovine insulin, human transferrin, sodium selenite) supplemented media, and later stimulated in RPMI supplemented with 2% fetal bovine serum for 24 h. 293 cells were transiently transfected using the calcium phosphate method (28). The usual experimental protocol included transfection with different combinations of TLR4, MD-2, CD14, TLR1 and TLR2 expression plasmids together with NF-{kappa}B 5x-Luc reporter plasmid, and empty vector to keep constant the total amount of DNA. Cells were also transfected with pRL-TK (Renilla-luciferase expressing plasmid) control reporter vector (Promega Inc., Madison, WI, USA) to normalize the assays.

Transactivation experiments
Twenty-four hours after transfection, cells were treated with different concentrations of LPS for 12–14 h or with vehicle solution. After these treatments, cells were harvested, lysed and assayed for firefly- and Renilla-luciferase activities following the manufacturer's instructions (Promega Inc.). Quantification of corresponding luminescence signals was performed in a microplate luminometer equipped with a dual injector system (EG&G Berthold, Bad Wildbad, Germany).

SDS whole cell lysates and Western blotting
THP-1 cells (4 x 106) were stimulated with either 10 µg ml–1 F. tularensis or 0.1 µg ml–1 E. coli LPS at 37°C for the indicated times. After incubation, cells were harvested and lysed with 200 µl of lysis buffer (20 mM Tris–HCl, 150 mM NaCl, 5 mM EDTA, 0.01% Nonidet P40, pH 7.4) supplemented with protease inhibitors (2 mM leupeptin, 1 µg ml–1 aprotinin and 1 mM phenylmethylsulfonyl fluoride) and phosphatase inhibitors (1 mM Na3VO4, 50 mM NaF), and incubated for 10 min on ice. Cell lysate was centrifuged at 12 000 r.p.m., and the pellet was discarded. A total of 150 µg protein per lane was analyzed by 10% SDS-PAGE followed by Western blotting. To examine ERK1 and ERK2 phosphorylation, antibodies against either phosphorylated ERK1 and ERK2 (Promega Inc.) or non-phosphorylated ERK2 (Santa Cruz Biotechnology, Santa Cruz, CA, USA) were used. Protein detection was performed using the Amersham (Buckinghamshire, UK) enhanced chemiluminescence's system.

RNA extraction and RNase protection assays (RPA)
Total cellular RNA was extracted by the TRIzol method (Life Technologies, Grand Island, NY, USA) and used to assay the level of expression of RANTES (regulated upon activation, normal T cell expressed and secreted), IFN-{gamma}-inducible protein-10 (IP-10), macrophage inflammatory protein (MIP)-1ß and -1{alpha}, monocyte chemoattractant protein (MCP)-1, IL-8, L32 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) messenger RNAs by RiboQuant RNase protection assay using the hCK-5 multiprobe template set from PharMingen (San Diego, CA, USA). For this purpose, riboprobes were labeled with [{alpha}-32P]uridine 5'-triphosphate in the presence of T7 RNA polymerase and used for overnight hybridization with 3 µg RNA. The hybridized RNA was digested with RNase and proteinase K, and the RNase-protected probes were purified and resolved on denaturing PAGE. Radiolabeled bands on the gel were acquired using the Personal Molecular Imager FX and quantified using Quantity One software (Bio-Rad, Hercules, CA, USA). The housekeeping genes L32 and GAPDH were used to normalize sample loading.

Analysis of cytokine expression levels by protein arrays
THP-1 cells (2 x 106) and human monocytes (3 x 106 cells plated and attached for 48 h) were used for the experiments. Cells were stimulated with LPS for 24 h at 37°C in RPMI supplemented with fetal bovine serum. To analyze multiple cytokine expression, supernatants were then incubated with Human Cytokine Antibody Array III (RayBiotech, Norcross, GA, USA) following the manufacturer's instructions. Briefly, previously blocked membranes were incubated with supernatants for 2 h, later incubated with the primary biotin-conjugated antibody for 2 h, followed by incubation with HRP-conjugated streptavidin antibodies for 1 h. Chemiluminiscence detection was followed with exposure to X-ray film. Densitometry of cytokine spots was analyzed by Image Master 2D platinum software (Amersham, Buckinghamshire, UK).

ELISA of human MIP-1{alpha}, MCP-1 and IL-8 production
MIP-1{alpha}, MCP-1 and IL-8 protein production was assayed in cell culture medium from human monocytes (3 x 106 cells plated and attached for 48 h) stimulated with LPS for 24 h at 37°C. The procedure was conducted with reagents from Amersham/GE Medical. These methods use antibody pre-coated well plates, biotinylated rabbit anti-human antibodies and streptavidin conjugated to HRP. Absorbance was measured using a microplate reader Versamax (Molecular Devices, Sunnyvale, CA, USA).

Statistical analysis
Results are expressed as mean ± SD. Data were analyzed by unpaired t'-test using GraphPad Prism version 4 (GraphPad Prism Software, San Diego, CA, USA). Differences were considered statistically significant for a P < 0.05.


   Results
Top
 Abstract
 Introduction
 Methods
 Results
Discussion
 References
 
Effect of LPS from F. tularensis on the TLR4 system
To address the molecular mechanism involved in the response of host cells to F. tularensis, 293 cells were transfected with a NF-{kappa}B reporter construct and several expression plasmids encoding the proteins involved in LPS signaling. No significant effect on luciferase activity was observed with both EW–LPS and PW–LPS in cells transfected with TLR4 alone (Fig. 2A). A limited effect was observed in cells transfected with TLR4 in combination with MD-2 or CD14 in medium supplemented with serum, whereas optimal productive response was observed with a combination of TLR4, MD-2 and CD14 (Fig. 2A), thus agreeing with previous results with Brucella and Ochrobactrum LPS (12). Incubation with 2.5 µg ml–1 EW–LPS produced a 1.95 ± 0.25-fold induction, and 2.5 µg ml–1 PW–LPS produced a 1.95 ± 0.10-fold induction of luciferase activity as compared with that observed in cells transfected with both the expression and reporter plasmids, but treated with vehicle instead of LPS. A dose-dependent effect was observed with both EW–LPS and PW–LPS, and both LPS showed comparable results (Fig. 2B). The effect of F. tularensis LPS was always below those reported for E. coli LPS at a concentration of 0.1 µg ml–1 under similar conditions (12), thereby agreeing with the low endotoxic potential of LPS from other facultative intracellular bacteria. In keeping with the reduced potency of F. tularensis LPS as compared with E. coli LPS, F. tularensis lipid A appears to be de-glycosylated and de-phosphorylated (Fig. 1D).


Figure 2
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  		Fig. 2. LPS from F. tularensis activates the TLR4 system. (A) Transactivation experiments were conducted in cells transfected with the different elements of the TLR4 signaling route (TLR4, MD-2 and CD14) or pRc/CMV (empty vector) as indicated. Twenty-four hours after transfection, cells were stimulated for an additional period of 12–14 h with 2.5 µg ml–1 of EW–LPS and PW–LPS in the presence of fetal bovine serum, and then collected for the assay of firefly- and Renilla-luciferase activities. (B) Dose-dependence effect of F. tularensis LPS was tested with EW–LPS and PW–LPS at the indicated concentrations. Results are normalized and expressed as fold increase of the activity detected in cells treated only with vehicle used to dissolve the LPS. Data from (A) represent mean ± SD of at least five experiments in duplicate. *Indicates P < 0.05 as compared with transfected cells treated with vehicle. Data from (B) are a representative experiment of three with similar results.

 
Lack of effect of LPS from F. tularensis on the TLR2 system
It has been reported that LPS from some Gram-negative bacteria might elicit its biological effect by engaging other TLRs, in particular TLR2 (29). To study the putative effect of F. tularensis LPS on other TLRs, further experiments were conducted in 293 cells transfected with TLR2 and a combination of TLR1/TLR2 or TLR2/TLR6. The TLR2 ligands peptidoglycan and Pam3CSK4 were used as positive controls. Since it has been described that soluble CD14 interacts with TLR2 and facilitates the binding of TLR2 to peptidoglycan (30), experiments co-transfecting 293 cells with CD14 and TLR2 were performed. A lack of effect was observed in all cases (Fig. 3) even when concentrations of LPS as high as 5–10 µg ml–1 were used, thus suggesting that binding to TLR2, TLR2/CD14, TLR1/TLR2 or TLR2/TLR6 might not explain the pro-inflammatory effects of F. tularensis LPS.


Figure 3
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  		Fig. 3. Lack of effect of F. tularensis LPS on the TLR2 system. Transactivation experiments were conducted in cells transfected with TLR2 in the presence or absence of CD14, a combination of TLR2 and TLR1, or a combination of TLR2 and TLR6 as indicated, and stimulated with either 5 µg ml–1 of EW–LPS or PW–LPS. Peptidoglycan (10 µg ml–1) and Pam3CSK4 (100 ng ml–1) were used as positive controls for TLR2 response. Results are normalized and expressed as fold increase of the activity detected in cells treated only with the vehicle used to dissolve the LPS. Data represent mean ± SD of at least three experiments in duplicate. *Indicates P < 0.05 as compared with transfected cells treated with vehicle.

 
F. tularensis LPS induces phosphorylation of ERK1 and ERK2 in monocytic cells
LPS has been shown to activate ERK1 and ERK2 in a CD14-dependent manner (13). On this basis, phosphorylation of the ERK module of MAPK was studied in THP-1 cells by Western blot analysis using phospho-specific antibodies and an antibody against ERK2 to demonstrate equal loading of the samples. F. tularensis EW–LPS and PW–LPS phosphorylated ERK1 and ERK2 (Fig. 4, upper panel, and data not shown) at a concentration of 10 µg ml–1. Similar results were obtained with 0.1 µg ml–1 E. coli LPS. These data indicate that F. tularensis LPS activates ERK signaling pathways in monocytic cells.


Figure 4
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  		Fig. 4. F. tularensis LPS induces phosphorylation of ERK1 and ERK2. THP-1 cells incubated with either 10 µg ml–1 F. tularensis EW–LPS or 0.1 µg ml–1 E. coli LPS were analyzed by Western blot as described in experimental procedures. P-ERK1 appears as the upper band (44 kDa) and P-ERK2 appears as the lower band (42 kDa). Anti-ERK1/2 phospho-specific antibody recognizes the activated forms of ERK1 and ERK2 (upper panel). Anti-ERK2 antibody recognizes ERK2 (lower panel). The depicted data are representative from two to three independent experiments.

 
Chemokine gene expression induction by F. tularensis LPS in monocytic cells
The expression of the chemokine genes induced by F. tularensis LPS was addressed using RPA. A fixed time of 3 h of incubation was selected on the basis of previous studies (12, 31). As shown in Fig. 5A, resting THP-1 cells showed a significant expression of RANTES. Incubation with both EW–LPS and PW–LPS showed a similar pattern of induction characterized by the expression of IP-10, MIP-1ß, MIP-1{alpha}, MCP-1 and IL-8 (Fig. 5A, left panel). EW–LPS seems to be more potent than PW–LPS in THP-1 cells. Even though LPS from F. tularensis showed a similar pattern of expression than 0.1 µg ml–1 LPS from E. coli (Fig. 5A, right panel), concentrations as high as 2.5–5 µg ml–1 were necessary to induce significant effects (Fig. 5A, left panel).


Figure 5
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  		Fig. 5. Effect of F. tularensis LPS on chemokine mRNA induction expression. (A) THP-1 cells and (B) human peripheral blood monocytes were incubated with EW–LPS, PW–LPS (left panel) or E. coli LPS (right panel) at the indicated doses for 3 h. At the end of this period, total RNA was extracted and used for the assay of chemokine mRNA using a multiprobe template, as described in experimental procedures. These are representative experiments of three with similar results. A densitometry quantitation corresponding to data corrected for housekeeping gene expression is shown below each figure. Data are expressed as fold induction of the activity detected in cells treated only with vehicle used to dissolve the LPS and represents mean ± SD of three experiments.

 
Adhered human peripheral blood monocytes showed a different pattern of chemokine mRNA expression than the THP-1 monocyte-like cells. Strong mRNA expression of IL-8 and low mRNA expression of RANTES, IP-10, MIP-1ß, MIP-1{alpha} and MCP-1 were observed (Fig. 5B, left panel). The discrepancy of the results obtained in THP-1 as compared with human monocytes can be explained by previous reports showing that THP-1 cells do not express CD14 in the absence of phorbol ester treatment (32), which is a strong indication of the important role of membrane bound CD14 for full LPS signaling. Incubation with both EW–LPS and PW–LPS triggered the induction of MIP-1ß, MIP-1{alpha}, MCP-1 and IL-8 mRNA expression (Fig. 5B, left panel). RANTES and IP-10, which are TRIF-dependent and MyD88-independent genes (33), showed a low mRNA expression but no induction after 3-h activation with neither E. coli nor F. tularensis LPS was observed (results were more apparent on a more exposed image, data not shown). These results do not rule out a later mRNA induction of RANTES, as it has been described for other stimuli (31). Consistent with results from THP-1, LPS from F. tularensis (EW and PW) showed a similar pattern of expression than LPS from E. coli in monocytes (Fig. 5B, right panel), although significant effects were only observed at concentrations as high as 5–10 µg ml–1 (Fig. 5B, left panel). PW–LPS seems to be more potent than EW–LPS in human monocyte cells, but higher concentrations than those used for THP-1 stimulation were required to elicit significant responses.

Effect of F. tularensis LPS on pro-inflammatory protein production in monocytes
To address the effect of F. tularensis LPS on the production of pro-inflammatory proteins, supernatants from cells activated for 24 h were incubated with antibody arrays. Since no apparent differences were observed between EW–LPS and PW–LPS in preliminary experiments, only EW–LPS was used for a more detailed scrutiny. Resting THP-1 cells, in the presence of serum, showed a low expression level of IL-8, IL-10, tumor necrosis factor {alpha} (TNF-{alpha}), macrophage colony-stimulating factor, oncostatin M, MIP-1{delta} and RANTES (Fig. 6A). A total of 0.1 µg ml–1 E. coli LPS, used as a positive control, induced the expression of IL-8, MCP-1, RANTES and IL-10 (Fig. 6A). F. tularensis LPS showed a similar pattern of induction of cytokines, although to a lower extent than LPS from E. coli, even when concentrations as high as 5 µg ml–1 were used. Resting human monocytes in the presence of serum, showed a different pattern of expression than THP-1 cells. Monocytes showed basal production of IL-8 and macrophage-derived chemoattractant (MDC) (Fig. 6B). Strong induction of IL-6, IL-8, MCP-1, Gro ({alpha}, ß, {gamma}) and low induction of MCP-2, RANTES, TNF-{alpha} and MDC were observed under stimulation with 0.1 µg ml–1 LPS from E. coli and 10 µg ml–1 F. tularensis EW–LPS (Fig. 6B). Consistent with RPA experiments and arrays in THP-1 cells, F. tularensis LPS behaved as a low–moderate activator, as compared with E. coli LPS.


Figure 6
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  		Fig. 6. Effect of F. tularensis LPS on pro-inflammatory protein production. Protein arrays were used to analyze multiple cytokine production in THP-1 cells (A) and human peripheral blood monocytes (B) attached to plastic dishes for 48 h after isolation. Cells were incubated with vehicle, 5–10 µg ml–1 EW–LPS or 0.1 µg ml–1 E. coli LPS for 24 h. Supernatants were then incubated with Human Cytokine Antibody Array III as indicated in experimental procedures. Array includes in duplicate: epithelial-neutrophil activating peptide-78, G-CSF, GM-CSF, growth regulated oncogene (Gro), Gro-{alpha}, I-309, IL1-{alpha}, IL-1ß, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-12p40p70, IL-13, IL-15, IFN-{gamma}, MCP-1, MCP-2, MCP-3, M-CSF, MDC, monokine induced by gamma-interferon, MIP-1{delta}, RANTES, stem cell factor, stromal cell-derived factor-1, thymus and activation-regulated chemokine, TGF-ß1, TNF-{alpha}, TNF-ß, epidermal growth factor, insulin-like growth factor-1, angiogenin, oncostatin M, thrombopoietin, vascular endothelial cell growth factor, BB, leptin and IgG as positive control. Protein production was detected by chemiluminiscence followed by exposure to X-ray film. Positive control was used to normalize results from different membranes. Squares indicate positive controls. Arrows indicate cytokines produced under basal conditions. Ovals indicate the cytokines unambiguously induced upon stimulation. Images are representative of two independent experiments. M-CSF, macrophage colony-stimulating factor.

 
To further validate these data ELISA were carried out for MIP-1{alpha}, MCP-1 and IL-8 (Fig. 7A–C). A dose response was performed with 0.25, 2.5 and 5 µg ml–1 F. tularensis EW–LPS and 0.01, 0.1 and 1 µg ml–1 LPS from E. coli in human monocytes. Consistent with RPA and cytokine arrays, F. tularensis LPS behaved as a low–moderate gene expression activator of chemoattractants for mononuclear cells (MIP-1{alpha}, MCP-1), as compared with E. coli LPS. However, F. tularensis LPS behaved as a moderate stimulus for the production of the PMN chemoattractant IL-8 as compared with E. coli LPS.


Figure 7
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  		Fig. 7. Effect of F. tularensis LPS on pro-inflammatory protein production. Human monocytes were incubated in the presence (black bars) or absence (white bars) of corresponding LPS. A dose response was performed with 0.25, 2.5 and 5 µg ml–1 F. tularensis EW–LPS and 0.01, 0.1 and 1µg ml–1 LPS from E. coli in human monocytes. Cell supernatants were collected after 24 h and used for the assay of MIP-1{alpha} (A), MCP-1 (B) and IL-8 (C) as described. Data represent mean and range of two experiments.

 

   Discussion
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
References
 
Invasion of macrophages by facultative intracellular bacteria is of prime importance for the occurrence of disease, and this results from a complex balance between host anti-microbial defense mechanisms and the ability of bacteria to evade those mechanisms. In the particular case of tularemia, F. tularensis LPS possesses an immunogenic potential, which makes this component suitable to use for vaccine preparation. Noteworthy, F. tularensis displays a strong ability to inhibit TLR4 mediated activation of murine macrophages (19), whereas F. tularensis elicits cytokine production in human cells (34), therefore pointing to the existence of species-specific mechanisms relevant to the survival of F. tularensis as an intracellular pathogen.

The paradigm of defense stimulation of monocytic cells by LPS has evolved along recent years from a concept involving LBP, CD14, TLR4 and MD-2 to a more complex model of heterotypic receptor associations linked to the formation of activation clusters, which may include additional elements such as CD11b/CD18, CD81 and CXCR4 at a variable array of stoichiometric ratios, thereby allowing the tailoring of the immune responses against the particular pathogen (reviewed in 35). In this context, the chemical structure of LPS and the proportion of rough and smooth LPS chemotypes represent another group of variables explaining the occurrence of different responses. In keeping with this concept, tetra-acyl lipid A has been found to produce less recruitment of TLR4 and MD-2 molecules and a lower activation of the NF-{kappa}B system than archetypical hexa-acyl LPS, whereas they efficiently activate MAPK signaling cascades (36). In the present study, we have observed that LPS from F. tularensis LVS shows a potency slightly lower than that of Brucella LPS to activate the TLR4 system. This seems of interest, since both Francisella and Brucella are facultative intracellular pathogens and their lipid A moieties show different structural characteristics, which provides some hints regarding the relationship between the chemical structures of their lipids A and the biological effects of their LPS. In fact, F. tularensis LPS possesses four fatty acid moieties somewhat longer than those of E. coli LPS, and lacks phosphate groups, whereas Brucella spp. contain six asymmetrical fatty acid moieties (Fig. 1A–C), a disaccharide backbone including 2,3-dideoxy, 2,3-diamino-D-glucose and two phosphate groups. The phosphate groups have been associated with endotoxic potential, since they allow interaction with cationic amino acid side chains on LPS-binding proteins and receptors (37–39). However, the presence of phosphates is not sufficient to endow the molecule with high endotoxic potential, in view of the results obtained with Brucella LPS. On the other hand, F. tularensis LPS contains a 2-deoxy, 2-amino-D-glucose disaccharide backbone analogous to that of E. coli LPS, suggesting again that this structural feature can not explain the reduced endotoxic potential, and pointing to the fatty acid moieties as the structural elements responsible for the low endotoxic potential. Noteworthy, the role of the acyl moieties in the endotoxic potential of lipid A has been the subject of intense scrutiny and some controversy still exists. In fact, tetra-acyldisaccharide lipid A from E. coli, and penta-acyl lipid A have been reported to act as LPS antagonists on human monocytic cells because of their cylindrical shapes (40–43), whereas they exert pro-inflammatory effects on rodent macrophages, thus exhibiting species-specific pharmacology. On the other hand, triacyl lipid A displays pro-inflammatory potential as judged from its ability to stimulate the production of IL-6 from human monocytes (44). Interestingly, a detailed study with LPS from Yersinia pestis has disclosed a decrease of the endotoxic potential as a function of the number of fatty acid substituents. However, the tetra-acyl lipid A obtained by culture of the bacterium at 37°C exhibits similar potency as their hexa-acyl lipid A counterpart when tested at a concentration of ~1 µg ml–1 in human macrophages of the U937 cell line (45).

To ascertain the cell host elements involved in the recognition of F. tularensis LPS, experiments were carried out in a system of ectopic expression of the different elements of the TLR system in 293 cells. These experiments disclosed a moderate response of the TLR4 route to F. tularensis LPS in cells expressing TLR4, MD-2 and CD14, which was in sharp contrast to the robust response to enterobacterial LPS, which occurred in the presence of lower concentrations of LPS, and even in the absence of the ectopic expression of the LPS co-receptor CD14. The expression of CD14 and the presence of serum at the time of stimulation were absolute requisites for a significant induction of {kappa}B-dependent transactivation in response to LPS from F. tularensis LVS, which points to the need for a concomitant presence of LBP. Consistent with a weak activation of the TLR4 route, it has recently been reported that TLR4-defective C3H/HeJ mice were not more susceptible than TLR4+/+ C3H/HeOuJ mice to a moderate sub-lethal intra-dermal infection with F. tularensis LVS, but were more susceptible to abnormally large inocula of the pathogen (17).

LPS triggers cellular responses through signaling cascades that include members of the MAPK family. In the case of the ERK module in mononuclear phagocytes, this involves the co-receptor CD14 (13). A recent study in murine macrophages has reported ERK1 and ERK2 phosphorylation by F. tularensis LVS, as well as the involvement of MAPK pathways in infection-induced apoptosis (46). Our data show that LPS from F. tularensis LVS produces ERK phosphorylation in human monocyte-like cells, although higher doses than those of LPS from E. coli are required, in agreement with lower TLR4 activation. Consistent with a weak activation of MAPK pathways, LPS from other facultative intracellular bacteria such as Brucella abortus activate ERK1/2 and JNK in macrophage-like cells at a lower extent than E. coli LPS (47).

The procedures used for the purification of F. tularensis LPS have been reported as a factor influencing the biological properties, that is, PW versus EW extraction; however, we have not observed significant differences related to the purification procedure. A possible reason for this reported difference in some systems might be the presence of contaminants acting on pathogen-recognition receptors other than TLR4, since the PW procedure yields LPS containing glucans, which are not recognized by TLR4, and only interact with TLR2 after recognition by the C-type lectin receptor dectin-1 in some cell systems (48, 49), but we can rule out that any of those extraction procedures could significantly damage the TLR4-activating structure of the LPS.

As regards the cytokines induced by F. tularensis LPS in human monocytic cells, our data show a pattern of response similar to that elicited by E. coli and Brucella spp. LPS, which is characterized by the induction of CXC chemokines such as IL-8 (CXCL8), Gro (CXCL1, 2, 3) and IP-10 (CXCL10); CC chemokines such as MCP-1 (CCL2), MCP-2 (CCL8), MDC (CCL22), MIP (CCL3, 4, 15) and RANTES (CCL5) and pro-inflammatory cytokines such as IL-6 and TNF-{alpha}. These findings agree with previous studies showing the release of pro-inflammatory cytokines by human monocytes in response to the LVS of F. tularensis (34), and point to F. tularensis LPS as an inductor of the cytopathogenic response produced by the bacterium. Interestingly, F. tularensis LPS shows strong induction of IL-6 production, an effect shared by E. coli LPS, but what we have not observed in response to stimuli of the adaptive immune response such as IgG-containing immune complexes (50). In view of the ability of this pro-inflammatory cytokine to stimulate and to differentiate B and T cells, it seems likely that this effect could account for the immunogenic effect of F. tularensis LVS.

As to the mechanisms of evasion from host cell anti-microbial defense that have been associated to F. tularensis, our data do not support a role for its low activity on the TLR4 system, and agree with reports on other Gram-negative bacteria. In fact, LPS from Enterobacteriaceae showing a strong activating activity on the TLR4 system, for instance Yersinia enterocolitica LPS (12), are robust activators of the TLR4 route, whereas Yersinia infection are associated to a state of anergy to LPS because of the concurrent action of the virulence factors Yersinia outer proteins on the route of NF-{kappa}B activation (51). This is reminiscent of the effect of a 23-kDa protein of F. tularensis LVS (19), which suppresses the ability of macrophages to respond to LPS, thus allowing evasion from the anti-microbial mechanisms.

Taken together, our data show a limited ability of F. tularensis LPS to activate the TLR4 system and the ERK route in human monocytic cells. This effect is not due to an antagonistic effect of this LPS on the human receptors. This property might depend on some structural properties of the lipid A moiety, most likely related to the number and length of the acyl chain substituents and the absence of phosphate moieties. Moreover, the inhibition of TLR4 signaling associated to Francisella infection (19) cannot be explained by an effect on the TLR4 system by the LPS itself. In other words, the inhibition of cytopathogenic response associated to the stimulation of the TLR system by some Gram-negative bacteria might rely on the biological effects of bacterial components other than their LPS.


   Acknowledgements
 
Edurne San Vicente and Cristina Gómez are thanked for their technical assistance. Michel Rehli is thanked for the gift of expression plasmids of human TLR2 and TLR4. Peter S. Tobias is thanked for CD14 and MD-2 cDNA. Shizuo Akira and Satoshi Uematsu are thanked for providing a pEF-Bos vector encoding human TLR6. This work was supported by a grant from Plan Nacional de Salud y Farmacia (grant SAF2004-01232), Red de Brucelosis, Red Respira, Red Recava and a grant (FIS 03/1489) from Ministerio de Sanidad. A.I.D. was a recipient of a grant from the Spanish Instituto de Salud Carlos III. C.G.R. is a researcher from the ‘Ramon y Cajal Program’ of the Spanish Ministerio de Ciencia y Tecnología.


   Abbreviations
 
ERK, extracellular signal-regulated kinases
EW, ether–water
GAPDH, glyceraldehyde-3-phosphate dehydrogenase
IP-10, IFN-{gamma}-inducible protein 10
JNK, c-Jun amino-terminal kinase
KDO, 2-keto-3-deoxyoctulosonic acid
LVS, live vaccine strain
MAPK, mitogen-activated protein kinase
MCP, monocyte chemoattractant protein
MDC, macrophage-derived chemoattractant
MIP, macrophage inflammatory protein
MyD88, myeloid differentiation primary response protein 88
NF-{kappa}B, nuclear factor-{kappa}B
Pam3CSK4, palmitoyl-3-cysteine-serine-lysine-4
PW, phenol–water
RANTES, regulated upon activation, normal T cell expressed and secreted
RPA, RNase protection assay
TIR, Toll/IL-1 receptor
TLR, Toll-like receptor
TNF, tumor necrosis factor
TRIF, TIR domain containing adaptor protein-inducing IFNß

   Notes
 
Transmitting editor: Giorgio Trinchieri

Received 2 September 2005, accepted 20 February 2006.


   References
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

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