Interaction of endotoxins with Toll-like receptor 4 correlates with their endotoxic potential and may explain the proinflammatory effect of Brucella spp. LPS
Ana I. Dueñas1,
Antonio Orduña1,2,
Mariano Sánchez Crespo3 and
Carmen García-Rodríguez3
1 Unidad de Investigación, Hospital Clínico Universitario, Valladolid, 2 Departamento de Microbiología, Facultad de Medicina, Universidad de Valladolid and 3 Instituto de Biología y Genética Molecular, CSIC-Universidad de Valladolid, Valladolid, Spain
Correspondence to: M. Sánchez Crespo; E-mail: mscres{at}ibgm.uva.es
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Abstract
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Endotoxins displaying differences in the chemical structure of their lipid A were used to induce the expression of chemokines in the human monocytic THP-1 cell line. LPS from two enterobacterial species such as Escherichia coli and Yersinia enterocolitica induced mRNA expression of IFN-
-inducible protein (IP)-10, macrophage-inflammatory protein (MIP)-1
, MIP-1ß, monocyte chemoattractant protein (MCP)-1 and IL-8. LPS from the non-enterobacterial genera Brucella and Ochrobactrum induced the expression of these chemokines to a lower extent. Attempts to address the signaling routes involved in these responses were carried out in transiently transfected HEK293 cells. Induction of
B-driven transcriptional activity by enterobacterial LPS was observed in cells transfected with TLR-4 alone, although co-transfection of TLR-4, MD-2 and CD14 provided optimal induction. The response to Brucella spp. and Ochrobactrum anthropi LPS was only significant at the concentration of 10 µg/ml. These data indicate that LPS from Brucella spp. and O. anthropi, which contain lipid A moieties with structural features different from those of Enterobacteriaceae elicit biochemical signaling via TLR-4 only at high concentrations. Neither TLR-1, TLR-2 and TLR-6 nor heterodimeric combinations of these receptor molecules are involved. Conversely, the ability of LPS to activate the TLR-4 route is a reliable molecular biomarker for endotoxicity.
Keywords: bacterial infection, chemokines, endotoxin shock, lipopolysaccharide, transcription factors
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Introduction
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Members of the genus Brucella are Gram-negative facultative intracellular bacteria that produce clinically relevant infections in a large number of mammals including humans (1). Clinical brucellosis in humans is characterized by either acute or insidious onset, undulating fever, profound weakness, arthralgia and weight loss, but chronic disease with granuloma formation, lymphadenopathy and splenomegaly can also occur (2), thereby indicating a long-lasting recruitment of proinflammatory mechanisms by Brucella-derived products, which agrees with the recognized ability of heat-killed Brucella to activate both the innate and the adaptive immune system, thereby leading to proinflammatory responses (3). In previous studies we have observed upregulation of the inducible isoform of nitric oxide synthase (NOS-2) in rat macrophages (4), as well as induction of cyclooxygenase-2 (COX-2) in the human monocytic cell line THP-1 by Brucella smooth lipopolysaccharide (S-LPS) (5). Both events are transcriptionally regulated, thus suggesting that Brucella S-LPS might trigger signaling events leading to the activation of the transcription factor nuclear factor
B (NF-
B), which displays a central regulatory role in the expression of adhesion molecules, cytokines, chemokines and the aforementioned enzymes NOS-2 and COX-2 [for review, see (6,7)]. Studies in murine models of Brucella infection have also shown a pattern of gene expression induction consistent with the activation of NF-
B (8,9) and the production of cytokines such as TNF-
and IL-12 (1012), thereby indicating that the response to endotoxin might represent a definite component of the host response to Brucella (13). Current views regarding the innate immune response have stressed a role for the Toll-like receptor (TLR) system [for review, see (1418)]. Members of the TLR family are expressed differentially among host cells and specifically respond to different conserved molecular structures shared by large groups of microbes. In this connection, TLR-4 is currently considered as the main element involved in the recognition of LPS. Since the composition of all lipopolysaccharides follows a general principle, which includes a polysaccharide attached to a lipid component termed lipid A through a specific sugar called 2-keto-3-deoxyoctulosonic acid (KDO) (19), it has been assumed that all LPS molecules have identical biological effects. However, this concept has been modified by studies showing great variance in the capacity of several LPS species to produce cytokine synthesis, this effect being related to the structural features of their lipid A portion (20,21). In this connection, the type of disaccharide backbone, the length and number of the acyl moieties, the asymmetry of their distribution and the presence of phosphate groups in both the reducing and non-reducing sugars are of paramount importance to govern the interaction of LPS with TLR (22). According to these principles, the LPS of Brucella and the related species Ochrobactrum anthropi (23,24) possess two phosphate groups and hexaacyl asymmetrical fatty acid moieties, which are also found in the prototypical proinflammatory Escherichia coli LPS. Unlike E. coli LPS, Brucella and Ochrobactrum LPS contain a ß-1,6-linked 2,3-diamino-2,3-dideoxyglucose instead of ß-1,6-linked 2-amino-2-deoxyglucose backbone. Of note, whereas Brucella spp. are highly pathogenic, O. anthropi only produces disease in critically ill patients (25).
Legionella pneumophila, a Gram-negative facultative intracellular bacteria, is also similar to Brucella as regards both low endotoxicity and chemical structure of its LPS (26). Interestingly, the biological effects of Legionella LPS are independent of the activation of TLR-4 (27) and CD14 (28), and they have been related to its ability to interact with TLR-2 (29). Taking into account the aforementioned structural differences, we have addressed the study of the molecular mechanism underlying the biological effects of different LPS, focusing on Brucella LPS, both because of its distinct structural features and its ability to produce chronic disease in humans. On the basis of previous studies, our working hypothesis was a significant yet weaker than E. coli LPS effect of Brucella LPS on the human TLR-4, thus explaining the lower potency of Brucella S-LPS to induce NOS-2 and COX-2. Our findings show a similar pattern of chemokine induction elicited by Brucella spp. and enterobacterial LPS, although the induction of monocyte chemoattractant protein-1 (MCP-1), macrophage-inflammatory protein (MIP)-1
, MIP-1ß, IFN-
-inducible protein (IP-10) and IL-8, by Brucella spp. LPS was significantly lower than that elicited by enterobacterial LPS. In addition, we have found a weak agonist effect of Brucella LPS on a system of ectopic expression of TLR-4 in HEK293 cells, even in the presence of the accessory recognition molecule MD-2. Moreover, co-transfection of the LPS co-receptor CD14 and the presence of serum were also required. We did not find any evidence of the involvement of other TLR receptors, namely TLR-1, TLR-2 and TLR-6, in Brucella LPS signaling. A corollary to our findings is that the low productive interaction of Brucella LPS with TLR-4 on host cells might explain the low endotoxic effect of this bacterium.
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Methods
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Purification of Brucella LPS and expression plasmids
Brucella melitensis 16M (biotype 1), B. abortus 544 (biotype 1) and US 19 smooth virulent strains and B. melitensis 115 rough strain were grown on Brucella broth (Difco Laboratories, MI). Phenol-inactivated bacteria were harvested by centrifugation and washed twice with saline. LPS were extracted by the phenol/water method modified for Brucella organisms as described by Leong et al. (30), which allows the separation of Brucella LPS in the phenolic phase. Purification of crude LPS isolated in the phenolic phase was performed according to the procedure of Moreno et al. (31), followed by digestion with DNase I, RNase and proteinase K. LPS was recovered by ultracentrifugation (100 000 g for 6 h at 4°C). Heat treatment of Brucella LPS was carried out by boiling for 1 h. The thiobarbituric method was used to measure KDO (32). Under these experimental conditions, B. melitensis 16M purified S-LPS contained 0.79% KDO, B. abortus 544 purified S-LPS 0.70% KDO and E. coli O26:B6 LPS (Sigma Chemical, St Louis, MO) 1.6% KDO. LPS from O. anthropi and Yersinia enterocolitica were provided by Dr Ignacio Moriyón (University of Navarra, Spain). Lipid A from the different bacteria was obtained by hydrolysis of purified LPS with 2% acetic acid at 100°C for 1 h (E. coli) or 5 h (Brucella) (31). Hemagglutinin-tagged cDNAs of human TLR-2, TLR-4 and the dominant-negative mutant of TLR-4(P/H) (33) cloned into the expression plasmid pRc/CMV (Invitrogen Corporation, Carlsbad, CA) were provided by Dr Michael Rehli (University of Regensburg, Germany). Human CD14 cDNA cloned in pRc/RSV vector and pFLAG-CMV1 human expression vector engineered to introduce human MD-2 were provided by Dr Peter S. Tobias (The Scripps Research Institute, La Jolla, CA) (34,35). A pEF-Bos vector encoding human TLR-6 tagged with Myc at the C-terminus was provided by Drs Akira and Uematsu (Osaka University, Osaka, Japan) (36). pUNO hTLR-1 expression vector was from InvivoGen (San Diego, CA). Firefly luciferase-linked 5x NF-
B reporter gene was from Stratagene.
Cell culture and transient transfections
Human embryonic kidney (HEK) 293 cells (ATCC, Rockville, MD) were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, 100 U/ml penicillin G, 100 µg/ml streptomycin, 2 mM L-glutamine and 1 mM HEPES pH 7.4. 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 penicillin, 100 µg/ml streptomycin, 2 mM glutamine and 10% fetal bovine serum. HEK293 cells were transiently transfected using the calcium phosphate method (37). The usual experimental protocol included transfection with different combinations of TLR-4, MD-2, CD14, TLR-1, TLR-2 and TLR-6, expression plasmids together with 0.52 µg of reporter plasmid DNA (firefly luciferase-expressing plasmid), or empty vector to keep constant the total amount of transfected DNA. pRL-TK (Renilla-luciferase expressing plasmid) control reporter vector (Promega Inc., Madison, WI) was also employed to normalize the transfection assays. After transfection, cells were maintained in medium with 10% FBS for 24 h. At the end of this period, the medium was removed and replaced with new DMEM medium containing the agonists both in the presence and absence of 10% FBS.
Transactivation experiments
Twenty-four hours after transfection, cells were treated with different concentrations of LPS for 1214 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. Quantification of corresponding luminescence signals was performed in a microplate luminometer equipped with a dual injector system (EG&G Berthold). Data represent mean ± SD of at least three experiments.
RNA extraction and RNase protection assays
Total cellular RNA was extracted by the TRIzol method (Life Technologies, Grand Island, NY) and used to assay the level of expression of RANTES, IP-10, MIP-1
, MIP-1ß, MCP-1, IL-8, L32 and GAPDH messenger RNAs by RiboQuant RNase protection assay using the hCK-5 multiprobe template set from Pharmingen (San Diego, CA). For this purpose, riboprobes were labeled with [
-32P]UTP 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. The identification of the specific chemokine bands was carried out on the basis of their individual migration patterns in comparison with the undigested probes. Radiolabeled bands on the gel were acquired using the Personal Molecular Imager FX and quantitated using Quantity One software (Bio-Rad). Sample loading was normalized by the housekeeping genes L32 and GAPDH.
ELISA assay of human MIP-1
and MIP-1ß production
MIP-1
and MIP-1ß were assayed in cell culture medium with reagents from Endogen (Woburn, MA). This procedure uses antibody precoated well plates, biotinylated rabbit anti-human antibodies, and streptavidin conjugated to horseradish peroxidase. The ELISA were developed using a peroxidase reaction, and the minimum detectable doses of this assay are 5 pg/ml.
Statistical analysis
Results are expressed as mean ± SD. Data were analyzed by unpaired Student's t-test using GraphPad Prism version 4 (GraphPad Prism Software, San Diego, CA). Differences were considered statistically significant for P < 0.05.
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Results
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Chemokine gene expression induction by LPS from different bacteria
Incubation of THP-1 monocytes with LPS obtained from different species of bacteria showed the induction of the expression of the mRNA of several chemokines, even though these cells do not exhibit surface display of CD14 (38). A fixed time of 3 h of incubation was selected to address the response to LPS on the basis of previous studies, which suggested that time as the most adequate one to address the pattern of induction of the different chemokines elicited by proinflammatory stimuli, including E. coli LPS (39). As shown in Fig. 1(A), resting THP-1 cells showed a significant expression of RANTES, but not of other chemokines. Incubation of THP-1 cells with LPS of both Y. enterocolitica and E. coli showed a pattern of induction characterized by the expression of IP-10, MIP-1ß, MIP-1
, MCP-1 and IL-8, whereas LPS from Brucella spp. and O. anthropi mainly induced the expression of MIP-1
, MCP-1 and IL-8, but to a lower extent than the effect produced by equivalent concentrations of enterobacterial LPS (Fig. 1A). Incubation with the lipid A moieties showed a similar pattern of expression, thereby suggesting that the effects elicited by the different LPS may be best explained by differences in the chemical structures of their cognate lipids A (Fig. 1B and E). Attempts to compare the dose-dependency of the mRNA induction showed that near maximum effect of E. coli and Y. enterocolitica LPS could be detected with concentrations as low as 0.1 µg/ml (Fig. 1C and Fig. 2, left panel). In contrast, LPS from Brucella spp. and O. anthropi had to be tested at 1020 µg/ml to induce significant effects (Fig. 1D and Fig. 2, right panel). To confirm that the changes in the chemokine mRNA expression levels correlated with the expression of protein, MIP-1
and MIP-1ß proteins were assayed in the culture media. MIP-1
protein was < 5 pg/ml in resting cells and reached a concentration of 748 ± 87 pg/ml, 16 h after addition of E. coli LPS, versus 53 ± 4 pg/ml in the case of B. melitensis LPS and 33 ± 1 pg/ml with O. antropi LPS. In the case of MIP-1ß, E. coli LPS also showed maximum effect, but B. melitensis and O. anthropi LPS significantly enhanced the expression of the protein (Table 1).

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Fig. 1. Effect of LPS and lipid A from different bacteria on chemokine mRNA induction expression. THP-1 monocytes were incubated with 1 µg/ml of LPS (A) or lipid A (B) from the indicated bacteria for 3 h. After this time, total RNA was extracted and used for the assay of chemokine mRNA using the hCK-5 multiprobe template from Pharmingen. These are representative experiments of three with similar results. Effect of different concentrations of E. coli LPS on chemokine mRNA induction (C). The effect of different concentrations of LPS from E. coli and Brucella spp. is shown in (D). (E) Comparison of the effect of 10 µg/ml of lipid A from Brucella melitensis and 1 µg/ml of lipid A from E. coli.
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Fig. 2. Effect of LPS from Y. enterocolitica and O. anthropi on chemokine mRNA induction expression: dose-dependency. THP-1 monocytes were incubated with different concentrations of LPS for 3 h as indicated. After this time, total RNA was extracted and used for the assay of chemokine mRNA. These are representative experiments of three with similar results.
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Effect of LPS from different bacteria on the TLR system
To address the molecular elements involved in the response of host cells to Brucella spp. and O. anthropi LPS, as well as the mechanisms that could explain the distinct effects of enterobacterial and non-enterobacterial LPS, HEK293 cells were transfected with a NF-
B reporter construct and different expression plasmids encoding the proteins involved in LPS signaling. Transfection of TLR-4 cDNA alone increased 2.36 ± 0.7-fold the relative luciferase activity produced by E. coli LPS as compared to that observed in cells transfected with both the expression and reporter plasmids, but treated with vehicle instead of LPS (Fig. 3A). Interestingly, the response was further enhanced by co-transfection with MD-2 (5.94 ± 0.8-fold increase), whereas the isolated transfection of CD14, MD-2, or CD14 and MD-2 did not increase the response above that observed in cells transfected with an empty vector. In contrast, the combined transfection of TLR-4, MD-2 and CD14 showed the optimal productive response, thus indicating that CD14 enhances the response to E. coli LPS by the TLR-4 route, but can not replace the effect of MD-2 (Fig. 3A). Of note, a dominant-negative mutant TLR-4(P/H) (33) inhibited E. coli LPS action, and the inhibitory effect was dose-dependent (data not shown), thus suggesting the strict dependence of the observed effect on TLR-4. A similar pattern of response was observed with Y. enterocolitica LPS (Fig. 3B), thereby indicating that a similar assembly of a multi-receptor complex is involved in the triggering of the TLR-4 route by enterobacterial LPS. Lipid A from E. coli also enhanced TLR-4-mediated
B-driven transcriptional activity (Fig. 3C). However, this effect was not observed in cells transfected with TLR-4 alone, and the co-transfection of MD-2 was needed. Since serum is a source of LPS-binding protein (LBP) as well as of other proteins, namely CD14 (40) and MD-2 (41), that can facilitate LPS signaling, the dose-dependence of enterobacterial LPS was assayed both in the presence and absence of serum. As shown in Fig. 4, serum enhanced the effect of LPS; however, this effect was only observed when the cells were co-transfected with TLR-4 plus MD-2, or TLR-4 plus MD-2 and CD14. Moreover, both E. coli and B. melitensis 16M lipid A also enhanced
B-driven transcriptional activity in cells transfected with TLR-4 and MD-2 in the absence of serum (5.2-fold increase, for 1 µg/ml E. coli LPS and 2.5-fold increase for 10 µg/ml Brucella LPS, n = 2), thus indicating that 10% FCS does not provide a comparable source of MD-2 as that provided by the expression of this protein (42), and also that serum effect should be explained by another mechanism, most likely as a source of LBP. Since LPS from Brucella and O. anthropi at the dose of 1 µg/ml did not enhance
B-driven transcriptional activity in cells transfected with several combinations of elements of the TLR-4 route, under conditions fully responsive to both Enterobacteriaceae LPS and the proinflammatory cytokine TNF-
(Fig. 5A), cells transfected with TLR-4 in combination with MD-2 and/or CD14 in medium supplemented with serum were used as a reference system for the screening of the effect of LPS from these bacteria at the dose of 10 µg/ml. As shown in Fig. 5(B), significant increases of luciferase activity in response to Brucella and Ochrobactrum LPS were only observed in cells transfected with TLR-4, MD-2 and CD14, in the presence of serum. The effect of Brucella LPS resisted 1 h boiling, but it was not observed after polymyxin B treatment (Fig. 5B). However, these responses were always below those produced by LPS from E. coli and Y. enterocolitica, even when these LPS were tested at a concentration of 0.1 µg/ml or lower (Fig. 4C and D).

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Fig. 3. Induction of B-driven transcriptional activity by E. coli O26:B6 and Y. enterocolitica O:9 LPS, and E. coli O26:B6 lipid A in HEK293 cells transfected with different proteins of the TLR-4 route. Twenty-four hours after transfection, cells were stimulated for an additional period of 1214 h with 1 µg/ml LPS in the presence of fetal bovine serum, and then collected for the assay of firefly- and Renilla-luciferase activities. Results are normalized and expressed as fold-increase of the activity detected in cells treated only with the vehicle used to convey the different LPS. Background firefly luciferase activity ranged between 50 000 and 100 000 relative luciferase units in control cells. (A) HEK293 cells stimulated with E. coli O26:B6 LPS. (B) Cells stimulated with Y. enterocolitica O:9 LPS. (C) Cells stimulated with 1 µg/ml of E. coli O26:B6 lipid A. Results are expressed as fold induction of the activity measured in cells transfected with both expression and reporter plasmids and treated with vehicle. Data represent mean ± SD of 35 experiments in duplicate. *P < 0.05 as compared to cells treated with vehicle.
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Fig. 4. Induction of B-driven transcriptional activity by E. coli O26:B6 and Y. enterocolitica O:9 LPS: dose-dependence and effect of serum. HEK293 cells were incubated for 1214 h with different concentrations of LPS in the absence (A and B) or presence (C and D) of fetal bovine serum. Results are normalized and expressed as fold-increase of the activity detected in cells transfected and treated with LPS as compared to cells treated with the vehicle used to convey the LPS. Data represent mean ± SD of 35 experiments in duplicate. *P < 0.05 as compared to cells treated under the same conditions in the absence of serum.
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It has recently been shown that LPS from Legionella pneumophila, which shows a chemical structure similar to that of Brucella LPS, elicits its biological effect by engaging TLR-2 (29). On this basis, additional experiments were conducted in HEK293 cells transfected with TLR-2, TLR-2/TLR-1, TLR-2/TLR-6 and TLR-2/MD-2/CD14. In contrast to the robust response produced by the prototypical TLR-2 agonist proteoglycan, no effect of Brucella spp. and O. anthropi LPS was observed in all cases (Fig 5C, and data not shown) even at LPS concentrations as high as 10 µg/ml, thus suggesting that binding to neither TLR-2, TLR-2/TLR-1 nor TLR-2/TLR-6 dimers may explain the proinflammatory effects of Brucella spp. LPS.
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Discussion
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The current paradigm of stimulation of mononuclear phagocytes by LPS stems on a unitary concept involving serum LBP, CD14, TLR-4 and the extracellular molecule MD-2, which physically associates with TLR-4 on the cell surface. However, the structural diversity of the different LPS and the distinct pathophysiological effects elicited by LPS from different bacteria, which might apparently challenge the unitary notion, could be explained by the different abilities of distinct LPS to interact with the TLR system, as well as by the concomitant engagement of other receptors of the innate immunity by particulate or aggregate bacterial-derived products. Based on previous studies in rat peritoneal macrophages and THP-1 monocytes, where it was shown that Brucella S-LPS is less active than E. coli LPS, yet produced the same pattern of responses (3,4,43), we predicted that Brucella LPS effect could be mediated by the common sensing mechanism for LPS through TLR-4. In support of this view was the dependence of these responses on the activation of NF-
B, which is the main signaling pathway associated to the TLR route. Our data confirmed previous findings as to the induction of the expression of the mRNA of several chemokines by Brucella LPS in THP-1 cells, i.e. a cell line showing a reduced surface display of CD14 (38), even though it expresses a wide array of receptors involved in the innate immune response and may respond to LPS by recruiting soluble CD14 from serum. To address the full appraisal of molecular elements involved in the response to Brucella LPS, further experiments were carried out by producing ectopic expression of the different elements of the TLR system in HEK cells. These experiments disclosed a moderate response of the TLR-4 route to Brucella LPS in cells expressing TLR-4, MD-2 and CD14, in the presence of serum, 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, although the expression of MD-2 was of major importance to enhance the response. Noteworthy, both the expression of CD14 and the presence of serum at the time of stimulation were absolute requisites for the induction
B-dependent transactivation in response to Brucella LPS, thus pointing to a requirement for the concomitant presence of LBP. Since the LPS of O. anthropi, which is structurally similar to that of Brucella, elicits a similar response, and comparable results have been reported for L. pneumophila LPS (2730), it seems at first glance likely that the lesser response via TLR-4 to Brucella-related LPS is a common property of those LPS displaying a definite set of structural features of their lipids A, namely the six asymmetrical fatty acid moieties, the disaccharide backbone including 2,3-diamino-2,3-dideoxy-D-glucose, instead of D-glucosamine, and two phosphate groups.
The experiments herein presented help delineate the specific biological effects of the different components of Brucella, in that B. abortus activates the innate and the adaptive immune systems to a state that favors the differentiation of T cells towards a Th1 profile (3,44), and makes this bacterium a potentially useful carrier for antigen targeting (45). However, most studies have been conducted with heat-killed B. abortus (11,13), which does not allow an unambigous appraisal of the effects of LPS, since there is a tight association between B. abortus LPS and lipoproteins on the cell surface which stearically hinders interaction of the LPS with TLR-4 (31,46), and this could explain the triggering of distinct pathophysiological events depending on whether whole bacteria or LPS interact with the receptors of innate immunity. To overcome this possible pitfall, the present study has been conducted with Brucella LPS purified by a procedure including proteinase K treatment, and further controls with heat-treated Brucella LPS to denature protein components. Noteworthy, these treatments yielded Brucella LPS preparations retaining effects on the TLR-4 system, which in turn were abrogated by polymyxin B treatment. In this connection, even though heat-killed bacteria have been found able to activate TLR-2 in vitro, TLR-2 knockout mice showed no statistically significant difference in bacterial load as compared to control animals (47). By contrast, brucellosis in mice that lack functional TLR-4 was associated with an increased bacterial load and a reduced production of
-interferon (47). In the context of cooperation of different bacterial products in bacterial infections, it should be taken into account that even though Y. enterocolitica LPS may be a robust activator of the TLR-4 route; as shown in this study, Yersinia infection may lead to a state of anergy to LPS because of the concurrent action of the virulence factors Yersinia outer proteins (Yops) on the route of NF-
B activation (48), and the development of changes in lipid A acylation pattern under the pressure of environmental stimuli (49).
Since it has been reported that atypical LPS might exert their biological effects through the TLR-2 route, for instance, L. pneumophila and Porphyromonas gingivalis (50), we carefully addressed this possibility for Brucella LPS; however, our results challenged this concept since we have been unable to show any effect of Brucella LPS on the TLR-2 and the TLR-2/TLR-1 and TLR-2/TLR-6 routes at concentrations as high as 20 µg/ml, even in the presence of MD-2 and CD14. Noteworthy, most recent reports agree with these findings in that the effect of P. gingivalis lipid A (51) and the structurally related Prevotella intermedia lipid A (52) has also been associated to the TLR-4 route, in spite of early reports on the involvement of the TLR-2 pathway. Since the number of receptors mediating the response to LPS has recently been enlarged, for instance members of the NOD family, a corollary to these findings is that the effect of some LPS might be mediated by members of this family or by another sensing molecule(s) as yet undiscovered. In this connection, further evidence of redundancy in the inflammatory response to bacteria has been provided by the isolated occurrence of infections produced by extracellular pyogenic bacteria associated to loss-of-function mutations in central elements of the Toll/IL-1 receptor (TIR) domain (53), thereby suggesting that protective immunity to facultative intracellular bacteria may occur in the absence of a functional TLR-4 route. In summary, we show that LPS from Brucella spp. fail to elicit productive binding to TLR-2, whereas they induce responses on the TLR-4 route at concentrations
10 µg/ml in the presence of MD-2, the LPS co-receptor CD14 and serum. A corollary to these findings is that productive binding to TLR-4 at low concentrations of LPS seems to be a reliable molecular predictor for LPS endotoxicity.
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Acknowledgements
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Edurne San Vicente and Cristina Gómez are thanked for their technical assistance. Dr Ignacio Moriyón is thanked for LPS from O. anthropi and Y. enterocolitica. Dr Michel Rehli is thanked for the gift of expression plasmids of human TLR-2, TLR-4 and TLR-4(P/H). Dr Peter S. Tobias is thanked for CD14 and MD-2 cDNA. Drs Shizuo Akira and Satoshi Uematsu are thanked for providing a pEF-Bos vector encoding human TLR-6. This work was supported by grants from Plan Nacional de Salud y Farmacia (grant SAF2001-0506) and Red Brucella from Instituto de Salud Carlos III. A.I.D. is a recipient of a grant from the Spanish Instituto de Salud Carlos III. C.G.R. is a recipient of a Ramón y Cajal Program of the Spanish Ministerio de Ciencia y Tecnología.
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Abbreviations
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COX-2 | cyclooxygenase-2 |
IP-10 | IFN- -inducible protein |
KDO | 2-keto-3-deoxyoctulosonic acid |
LBP | lipopolysaccharide-binding protein |
MD-2 | myeloid differentiation protein-2 |
MIP-1 | macrophage-inflammatory protein-1 |
MIP-1ß | macrophage-inflammatory protein-1ß |
NOS-2 | nitric oxide synthase-2 |
NF- B | nuclear factor B |
S-LPS | Brucella smooth LPS |
RANTES | regulated upon activation, normal T cell expressed and secreted |
TLR | Toll-like receptor |
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Notes
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Transmitting editor: G. Trinchieri
Received 24 February 2004,
accepted 22 July 2004.
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