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Previous Article | Next Article 
Infection and Immunity, January 2004, p. 176-186, Vol. 72, No. 1
0019-9567/04/$08.00+0 DOI: 10.1128/IAI.72.1.176-186.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.
Department of Biochemistry and Immunology, Biological Sciences Institute, Federal University of Minas Gerais,1 Centro de Pesquisas René Rachou, Oswaldo Cruz Foundation, Belo Horizonte,2 EMBRAPA-CNPC, Sobral-CE,3 Department of Parasitology, University of São Paulo, São Paulo, Brazil,4 Department of Animal Health & Biomedical Sciences, University of Wisconsin-Madison, Madison, Wisconsin 53706,5 Department of Basic Medical Science, Medical School, University of Missouri, Kansas City, Missouri 641086
Received 31 July 2003/ Returned for modification 20 September 2003/ Accepted 3 October 2003
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ABSTRACT |
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) and interleukin-12 compared to C3H/HeJ, a TLR4 mutant mouse. The essential role of TLR4 for induction of proinflammatory cytokines was confirmed with diphosphoryl lipid A from Rhodobacter sphaeroides. Furthermore, to determine the contribution of TLR2 and TLR4 in bacterial clearance, numbers of Brucella were monitored in the spleen of C3H/HeJ, C3H/HePas, TLR2 knockout, and wild-type mice at 1, 3, and 6 weeks following B. abortus infection. Interestingly, murine brucellosis was markedly exacerbated at weeks 3 and 6 after infection in animals that lacked functional TLR4 (C3H/HeJ) compared to C3H/HePas that paralleled the reduced gamma interferon production by this mouse strain. Finally, by mass spectrometry analysis we found dramatic differences on the lipid A profiles of R and S strains. In fact, S lipid A was shown to be more active to trigger TLR4 than R lipid A in CHO cells and more effective in inducing dendritic cell maturation. In conclusion, these results indicate that TLR4 plays a role in resistance to B. abortus infection and that S lipid A has potent adjuvant activity. |
INTRODUCTION |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In Drosophila melanogaster, Toll receptors play a key role in antimicrobial host defense (27). Recently, mammalian homologues of Toll, designated Toll-like receptors (TLRs), were also discovered (34, 45, 51). Bacterial cell wall components and lipopolysaccharide (LPS) are recognized by host macrophages and dendritic cells via TLRs (33) that results in activation of professional antigen-presenting cells, initiation of acquired immune responses, and further elimination of the invasive bacteria (53, 54).
Among TLR family members, both TLR2 and TLR4 have been shown to recognize bacterial components. Recent studies have demonstrated that TLR2 is largely required for signaling by numerous ligands from gram-negative and gram-positive bacteria (20, 29, 31, 48, 53, 57), mycobacteria (29, 32, 56), spirochetes (2, 19, 29), and mycoplasmas (29), such as lipoteichoic acid, peptidoglycan, lipoproteins (2, 5, 19, 33), and certain types of LPS (20, 31). In contrast, TLR4 fails to confer responsiveness to gram-positive bacteria and their components and has been postulated to be the main LPS signaling receptor.
LPS is a major constituent of the outer membrane of gram-negative bacteria such as Brucella and is known to activate neutrophils, monocytes, macrophages, and other cell types to upregulate expression of adhesion molecules and produce a number of pro and anti-inflammatory cytokines (37). Recognition of bacterial LPS is mediated by CD14 (17). However, CD14 lacks transmembrane and intracellular domains necessary for signal transduction and thus requires the involvement of molecules belonging to the TLR family. Recently, with fluorescence resonance energy transfer techniques, LPS was shown to trigger a physical association between CD14 and TLR4 (25). B. abortus induces interleukin (IL)-12 production from human monocytes and this effect was blocked by anti-CD14 antibody, suggesting that the Brucella binding and/or signaling to monocytes was mediated via LPS (59). Additionally, Brucella's ability to elicit IL-12 secretion enables it to drive Th0 cells to differentiate into Th1 effector and memory cells that is a central feature of the potential use of B. abortus as a vaccine carrier and adjuvant.
B. abortus strain RB51 is a stable rough (R) mutant derived from the standard smooth (S) virulent strain 2308. This rough mutant strain that does not contain the O-antigen (O polysaccharide chain of the smooth LPS) is attenuated in its virulence compared to their smooth virulent parental strain (3). Even though the Brucella RB51 rough strain has been widely used as a live vaccine, it induces lower protection compared to the smooth vaccine strain S19 (56). Similar to most intracellular bacterial infections, cell-mediated immunity plays a major role in acquired resistance to brucellosis, although antibodies to surface antigens, especially to the O-antigen, can confer a certain level of protection against a challenge infection (4). Therefore, it is pivotal to dissect how this difference in protection engendered by these two vaccine strains could be related to the ability of R versus S LPS to signal through TLRs triggering the innate immune system.
In the present study, we investigated the ability of B. abortus R (vaccine) and S (virulent) strains and their purified LPS as well as lipid A to trigger TLR2 and TLR4. Heat-killed bacteria activate both TLR2 and TLR4, whereas purified LPS and lipid A signal through TLR4. Further, we demonstrated that optimal induction of tumor necrosis factor alpha (TNF-) and IL-12 by macrophages exposed to B. abortus as well as efficient bacterial clearance in mouse spleens required functional TLR4. Finally, lipid A from a B. abortus S strain had a stronger immunostimulatory activity than lipid A derived from the R strain. We believe that these findings have important implications in bacterial pathogenesis and vaccine development against brucellosis.
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MATERIALS AND METHODS |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Bacterial strains and LPS and lipid A extraction. Brucella abortus S2308 is the wild-type virulent S strain. The RB51, the current live attenuated U.S. vaccine derived from strain S2308 deficient in O-chain LPS (rough), was a gift from G. G. Schurig (Virginia-Maryland Regional College of Veterinary Medicine, Virginia Polytechnic Institute and State University, Blacksburg, Va.). The R LPS from B. abortus strain RB51 and the S LPS from strain S2308 were purified as detailed by Moreno et al. (35). Final purity of the LPS was determined by both thin-layer chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) on a 4 to 20% gradient gel that was subsequently silver-stained (Pierce Chemical Co., Rockford, Ill.) as previously described (20).
Preliminary dose-response analyses were conducted with heat-killed Brucella and LPS concentrations as described elsewhere (13). The lipid A moiety was isolated from R and S LPS after mild acid hydrolysis with 0.25 N HCl (Pierce) for 1 h at 100°C, followed by Folch partition (12). Lipid A was recovered in the upper phase, dried under N2 and redissolved in chloroform-methanol (1:1, vol/vol). Under the conditions used here, the lipid A species from both Brucella strains were released at high yield and purity. This was inferred from comparative experiments carried out with authentic LPSs from Escherichia coli, Salmonella minnesota, Bordetella pertussis, and Serratia marcescens, strictly under the same conditions. Neither internal lipid A fragments, partially hydrolyzed lipid A species, nor contaminant oligosaccharides were observed on the lipid A preparations when analyzed by electrospray ionization time-of-flight mass spectrometry (ESI-TOF-MS).
Electrospray ionization mass spectrometry. Lipid A species isolated from R and S LPSs were analyzed by electrospray ionization time-of-flight mass spectrometry (ESI-TOF-MS) with a Q-TOF-MS instrument (Ultima, Micromass Ltd., Manchester, United Kingdom). Samples, diluted in chloroform-methanol (1:1, vol/vol), were introduced into the electrospray source by infusion through a 20-µl loop with the assistance of a high-pressure liquid chromatography (HPLC) pump (Shimazu, Japan), at a flow rate of 20 µl/min. Spectra were collected in negative-ion mode over 1 to 2 min at 2 s/scan with an interscan delay of 0.2 s, over a mass range of 1,000 to 2,300. Source, API probe, and desolvation temperatures were set at 80, 100, and 150°C, respectively. Cone voltage and collision energy were 100 and -10 V, respectively. Combined raw spectra were further processed for background subtraction and smoothing (Saviztky-Golay method) with the MassLynx V3.5 software. Multiply charged ion species were deconvoluted to single-ion species with the MaxEnt3 function of the same software. Authentic lipid A preparations from E. coli and S. minnesota (Sigma Chemical Co., St. Louis, Mo.) were used to calibrate the instrument.
CHO cell lines.
The CHO reporter cell lines (CHO/CD14 and CHO/CD14/TLR2) (29) were derived from clone 3E10, a CHO/CD14 cell line that has been stably transfected with a reporter construct containing the gene for CD25 under the control of the human E-selectin promoter. This promoter contains an NF-
B binding site, and CD25 expression is completely dependent upon NF-B translocation (10). Cells expressing TLRs were obtained by stable transfection of the CHO/CD14 reporter cell line (which contains endogenous TLR4) with the cDNA for human TLR2 as described (29). In addition to the LPS-responsive cell lines described above, we also tested a CHO/CD14 LPS nonresponder cell line (10) designated clone 7.19 as well as a clonal line derived from this mutant that was transfected with CD14 and TLR2 (7.19/CD14/TLR2). This cell line was derived from 3E10, and reports NF-B activation via the surface expression of CD25, similarly to the other CHO lines described here. The LPS-nonresponsive phenotype of the 7.19 cell lines appears to be due to a mutation in the MD-2 gene, and thus they are defective in signaling via TLR4 (10, 18, 55). All the cell lines were maintained as adherent monolayers in Ham's F-12/Dulbecco's modified Eagle's medium (DMEM) supplemented with 5% fetal bovine serum, at 37°C, 5% CO2, and antibiotics as described (29). Furthermore, the cell lines 7.19, 7.19/CD14/TLR2, CHO/CD14, and CHO/CD14/TLR2 were also termed here as LPS nonresponder control cell lines TLR2, TLR4, and TLR2/TLR4, respectively, according to the TLR that they express.
Flow cytometry analysis. Reporter cells were plated at a density of 105 cells/well in 24-well tissue culture dishes. The next day, either 103 heat-killed bacteria (heat inactivation was performed at 80°C for 1 h)/cell ratio from the S and R strain, 1 µg/ml of purified S LPS and R LPS, and 40, 200, and 1,000 ng of purified S lipid A and R lipid A per ml were added as indicated, in a total volume of 250 µl of medium/well, for 18 h. The cells were harvested with trypsin-EDTA treatment for 50 s, washed once with medium containing 5% serum and again with phosphate-buffered saline, and then 105 cells were stained with phycoerythrin-labeled anti-CD25 (mouse monoclonal antibody to human CD25, PE conjugate; CALTAG Laboratories, Burlingame, Calif.) 1:200 in phosphate-buffered saline, on ice, in the dark, for 45 min. The cells were also harvested physically without trypsin with similar results. After two washes, the cells were resuspended in 1 mM sodium azide in phosphate-buffered saline, and 10,000 cells were examined by flow cytometry (BD Biosciences, San Jose, Calif.). Dead cells were excluded by gating them with the forward scatter and side scatter parameters, and after that the average of 7,641 ± 546 (Fig. 1) and 7,471 ± 265 (Fig. 5A) live cells were analyzed for the expression of surface CD25. Analyses were performed with Cell Quest software (BD Biosciences).
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measurements.
TLR2 knockout (52), C57BL/6 x 129/Ola (wild-type), C3H/HeJ (LPS hyporesponsive), and C3H/HePas mice were inoculated intraperitoneally with 2 ml of 3% thioglycolate and, 4 days later, the elicited peritoneal exudate cells were harvested in cold serum-free DMEM. The medium used in the macrophage cultures (MacMed) consisted of DMEM supplemented with 40 µg of gentamicin/ml and 5% heat-inactivated fetal bovine serum. The macrophages were washed and resuspended in MacMed at 2 x 106/ml, and 100-µl aliquots were dispensed into the wells of a 96-well plate. Cells were allowed to adhere at 37°C and 5% CO2, for 3 h, and were then washed once with serum-free DMEM and 150 µl of MacMed was added to each well, in the presence and absence of 20 U of IFN-
per ml and incubated overnight at 37°C and 5% CO2. The same concentration of Brucella and purified LPS used in flow cytometry was added to the macrophage cultures in a final volume of 200 µl/well at the same time as IFN-. Aliquots of the supernatant were collected after 24 h and 48 h of culture for TNF- and IL-12 (p70) measurements, respectively (8, 9). Levels of TNF- and IL-12 (p70) in the supernatants were measured by a commercially available enzyme-linked immunosorbent assay (ELISA) kit (Duoset; R&D Systems, Minneapolis, Minn.).
Macrophage cell line and DPLA competition assay.
Rhodobacter sphaeroides diphosphoryl lipid A (DPLA) was purified as described by Qureshi et al. (43). RAW 264.7 murine macrophage-like cells were obtained from the American Type Culture Collection (ATCC) (cell line TIB-71), cultured as previously described (23), except that the medium was RPMI 1640 supplemented with 10% fetal bovine serum, penicillin (50 U/ml) and streptomycin (50 µg/ml). LPS was added to RAW cells 18 h before the culture supernatant was assayed for TNF-. In blocking experiments, DPLA was added 30 min prior to addition of Brucella LPS. TNF- was assayed with duplicate determinations by means of the TNF- Quantikine M Immunoassay (R&D Systems, Minneapolis, Minn.). Each assay was repeated three times.
Infection and Brucella quantitation in spleens. Mice were infected intraperitoneally with 105 CFU of B. abortus strain 2308. To count residual Brucella CFU in the spleens of mice, 10 animals from each group were examined at each sampling period. Spleens from individual animals were homogenized in phosphate-buffered saline, 10-fold serially diluted, and plated on Brucella Broth agar (Difco, Detroit, Mich.). Plates were incubated at 37°C in air with 5% CO2, and the number of CFU was counted after 3 days.
Spleen cultures and IFN- detection assay.
Freshly removed spleens of mice infected with B. abortus strain S2308 were placed in petri dishes (60 x 15 mm) containing 10 ml of phosphate-buffered saline and passed through steel mesh to obtain single-cell suspensions. The splenocyte suspension were then isolated by density gradient centrifugation with Ficoll-Paque (Sigma) for 30 min in a Sorvall H-1000B rotor at 2,000 rpm (800 x g) in 50-ml tubes. Spleen cells floating on top of the high-density solution were isolated by slowly moving the tip of the pipette over the surface of the high-density layer and drawing cells up in a 5-ml pipette and transferred to a sterile 50-ml tube. Cells were then washed twice with sterile phosphate-buffered saline containing penicillin G sodium (100 U/ml), streptomycin sulfate (100 µg/ml), and amphotericin B (250 ng/ml) per ml.
Afterward, splenocytes were cultured in RPMI 1640 medium supplemented with 2 mM L-glutamine, 25 mM HEPES (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid), 50 µM 2-mercaptoethanol, penicillin G sodium (100 U/ml), streptomycin sulfate (100 µg/ml), amphotericin B (250 ng/ml), and 10% heat-inactivated fetal bovine serum (Sigma), then placed at 106 cells per well in 96-well tissue culture plates. Murine splenocytes from infected animals were stimulated with 1 µg of Brucella S LPS and 108 heat-inactivated Brucella abortus S2308 per ml. Unstimulated splenocytes were used as a negative control, and splenocytes stimulated with concanavalin A (5 µg/ml) were a T-cell-activating control. After 72 h at 37°C under 5% CO2, splenocyte culture supernatants were tested for the presence of cytokine with the ELISA Duoset kit (R & D Systems) to mouse IFN-.
Immunization with lipid A-activated dendritic cells and IFN- analysis by ELISPOT.
Immature dendritic cells were obtained by in vitro differentiation of BALB/c mouse bone marrow-derived precursors, as previously reported (7). Differentiated dendritic cells, uninfected and infected 24 h before with AdPyCS, a recombinant adenovirus expressing the CS protein from Plasmodium yoelii, were used to immunize different groups of mice. To determine the effect of LPS on dendritic cell activation, mice received subcutaneously 750,000 dendritic cells/mouse that were treated right after adenoviral infection with B. abortus purified S lipid A and R lipid A (1 µg/ml). Ten days after immunization, mice were sacrificed and the presence of CS-specific IFN--producing CD8+ and CD4+ T cells in their spleens was assayed by ELISPOT with CS-derived CD8+ (SYVPSAEQI) and CD4+ (YNRNIVNRLLGDALNGKPEEK) peptides, as previously described (7).
Statistical analysis. Statistical analyses were performed with Student's t test and analysis of variance with computer software package MINITAB (Minitab Inc., State College, Pa.).
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RESULTS |
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B activation was assessed by measuring the expression of CD25 by flow cytometry (10, 29). Increased induction of CD25 expression by 7.19/CD14/TLR2, CHO/CD14, and CHO/CD14/TLR2 cells exposed to R and S B. abortus was observed, indicating that heat-killed Brucella activates both TLR2 and TLR4 pathways (Fig. 1A, top panels). Additionally, Fig. 1B (top panel) showed that the difference in the percentage of CD25 positive CHO cells stimulated with R and S B. abortus was much higher (P < 0.05) on those cells that express TLR2, TLR4, and TLR2/TLR4 compared to the LPS nonresponder control cell line (7.19). In contrast, NF-B activation was detected only in CHO/CD14 and CHO/CD14/TLR2 but not in 7.19/CD14/TLR2 cells treated with S LPS and R LPS (Fig. 1A, bottom panels). Further, the percentage of CD25-positive cells following treatment with S LPS and R LPS was statistically significant on CHO cells that express TLR4 and TLR2/TLR4 compared to those cells that possess TLR2 and the LPS nonresponder control cell line (Fig. 1B, bottom panel). These data demonstrate that LPS derived from either S or R strains of Brucella triggers cell activation through TLR4 but not TLR2.
TLR4 is required for maximal proinflammatory activity of B. abortus and its LPS on murine macrophages.
Previous studies have demonstrated that B. abortus induces high levels of IFN-, TNF-, and IL-12 (38, 39). Here, we tested the involvement of TLR2 and TLR4 on cytokine production by murine inflammatory macrophages stimulated with B. abortus S and R strains and their LPS. C3H/HePas cells treated with Brucella S and R strains and their purified LPS produced significantly higher levels of TNF- and IL-12 compared to cells from C3H/HeJ TLR4 mutant mice (Table 1). No statistically significant differences were observed in TNF- and IL-12 production by macrophages from TLR2 knockout mice compared to the wild-type control group (C57BL/6 x 129/Ola). Additionally, priming inflammatory macrophages with IFN- enhanced TNF- and IL-12 synthesis by these cells of all mouse strains studied, but a significant difference was still observed only on cytokine production by C3H/HePas versus C3H/HeJ cells. Furthermore, IFN--primed macrophages of all mice studied stimulated with S LPS produced much higher levels of IL-12 compared to R LPS. This result corroborates the data demonstrated here showing more effective adjuvant activity of B. abortus S LPS. Overall, these findings suggest that TLR4 has a dominant role in macrophage signaling and production of proinflammatory cytokines by macrophages exposed to R and S strains of B. abortus and their LPS.
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and IL-1ß in both murine and human cells through CD14/TLR4 interaction (24, 30, 41, 42). The DPLA inhibitory effect on the ability of B. abortus LPS to induce the production of TNF- was tested. Addition of 1 µg of DPLA on Brucella LPS-treated RAW 264.7 macrophage-like cells dramatically reduced TNF- production by these cells (Fig. 2). This result confirmed our previous data described here regarding the role of TLR4 on proinflammatory cytokine production induced by Brucella LPS.
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production by spleen cells parallels enhanced susceptibility to Brucella infection in TLR4 mutant mice.
IFN- is a pivotal cytokine involved in the control of murine brucellosis (26). To determine the level of IFN- produced by C3H/HeJ and C3H/HePas mice following infection, we measured this cytokine in the supernatant of splenocytes from these animals after stimulation with B. abortus and its LPS. At 1 week postinfection, no difference was observed in the levels of IFN- produced by TLR4 mutant and C3H/HePas cells treated with B. abortus (Table 2), but a statistically significant difference in IFN- production by C3H/HePas splenocytes was observed compared to C3H/HeJ cells when S LPS was used as the stimulus. Interestingly, at 3 and 6 weeks postinfection, a major difference in IFN- production was detected in C3H/HePas splenocyte supernatants compared to C3H/HeJ when B. abortus was tested as the antigen. These data parallel our in vivo study that shows the decrease in bacterial load in C3H/HePas spleens compared to C3H/HeJ at 3 and 6 weeks postinfection. Together, these results demonstrate that TLR4 is required for inducing a robust adaptive immunity and IFN- synthesis for B. abortus infection and therefore an efficient control of murine brucellosis.
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This structural difference may be the key factor that contributes to the enhanced activity observed when S lipid A is used as the stimulus. Based on previous work by Qureshi et al. (44) and Moryion and Lopez-Goni (36) and our present observations, we tentatively assigned the structure of lipid A series originated from m/z 2065 of R and S strains (Fig. 4C). It is noteworthy to point out that the predicted monoisotopic masses (pMmi) for all tentative structures are very close (in average < 0.200 Da mass error) to the experimental monoisotopic masses (eMmi) obtained in this study. Nevertheless, we are currently carrying out extensive GC-MS, ESI-TOF-MS/MS, and nuclear magnetic resonance analyses of R and S lipid A species in order to confirm the proposed structural assignments and to determine the structure of the species that we could not determine here.
Enhanced TLR4 signaling through purified Brucella smooth strain lipid A. To determine whether the difference in composition of lipid A species from Brucella S and R strains could actually result in different biological activity of these molecules, we measured cell activation through TLR4 signaling. Differently from the data shown on Fig. 1 for whole LPS (1 µg/ml), purified lipid A from Brucella S strain was shown to activate TLR4 in much lower concentrations (40 and 200 ng/ml) compared to lipid A from Brucella R strain as observed by increased CD25 expression on CHO/CD14/TLR2 (TLR2/TLR4) and CHO/CD14 (TLR2) cell surface (Fig. 5, top and bottom panels).
Brucella lipid A activates dendritic cells to trigger IFN- production by malaria peptide-specific CD4+ and CD8+ T cells.
Bruna-Romero and Rodriguez (7) have previously shown that dendritic cells expressing the CS protein from the rodent malaria parasite Plasmodium yoelii with a CS recombinant adenovirus vector were able to present this malaria antigen to the immune system upon maturation. In the present work, we used equivalent adenovirus-transduced immature dendritic cells treated with purified S lipid A and R lipid A from B. abortus to immunize mice to test the dendritic cell maturation ability of these LPS molecules.
BALB/c instead of C57BL/6 mice were used in this experiment because of the H-2d restriction of the CS peptides. Mice vaccinated with adenovirus-transduced dendritic cells treated with S lipid A induced a much greater antigen-specific IFN- production by either CD4+ and CD8+ T cells compared to animals immunized with uninfected dendritic cells and infected dendritic cells not treated or treated with R lipid A (Fig. 5B). Enhanced IFN- production by CD4+ and CD8+ T lymphocytes of mice immunized with S lipid A-treated dendritic cells is probably related to the ability of Brucella S LPS to induce high levels of IL-12 in primed mouse macrophages compared to R LPS (Table 1). These results suggest that S LPS obtained from B. abortus has a great capacity to activate dendritic cells compared to R LPS and, as a consequence, displays a strong adjuvant effect for the induction of an adaptive immune response.
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DISCUSSION |
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First, with CHO reporter cell lines transfected with CD14 alone, TLR2 and CD14, both expressing endogenous TLR4 and MD-2 and the control cell line 7.19 not expressing MD-2 and therefore not possessing functional TLR4, we determined NF-B activation by measuring CD25 expression on the cell surface after exposure to B. abortus R and S strains and their LPS. The data presented here demonstrate that B. abortus triggers TLR2 and TLR4 activation, while its LPS induces cell activation through TLR4. Recent studies have demonstrated that TLR2 confers responsiveness to a wide variety of gram-positive bacterial cell wall components as well as to lipoproteins that are found in gram-positive and gram-negative bacteria (54). Further, Lorenz et al. (31) have demonstrated that LPS from Bacteroides fragilis functions through TLR4-independent mechanisms. But LPS preparations from Escherichia coli and Salmonella spp. lose their ability to induce TLR2-dependent responses after removal of contaminant proteins, suggesting that lipoproteins rather than the LPS are responsible for cellular activation via TLR2 (21).
Thus, B. abortus NF-B activation via TLR2 in 7.19/CD14/TLR2 cells is probably due to lipoproteins and other cell wall components from this organism, since LPS from R and S strains did not trigger TLR2. Regarding TLR4 signaling, B. abortus and its LPS from R and S strains activate NF-B in the CHO/CD14 cell line that does not possess TLR2 on the surface. Thus, TLR2 expression is not required for mediating responses to LPS from B. abortus strains used here. The importance of TLR4 for responses to LPS and gram-negative bacteria has been confirmed by the observation that targeted disruption of the tlr4 gene renders mice unresponsive to LPS and that this defect can be reversed in macrophages derived from TLR4-deficient mice by reintroducing a wild-type copy of the gene (22).
As with other intracellular microbial pathogens, IL-12-derived IFN- production, and TNF- are critical components of the immune system to control murine brucellosis (38, 39). Our findings revealed that TLR4 is required for B. abortus LPS signaling to produce TNF- and IL-12, while TLR2 is not. Additionally, when whole B. abortus was used as the stimulus, we observed a marked difference on inflammatory macrophage derived IL-12 and TNF- production from C3H/HePas mice via TLR4 activation compared to C3H/HeJ mouse cells. Nevertheless, C3H/HeJ cells still produced TNF- and IL-12 implying the involvement of additional TLRs besides TLR4 in B. abortus signaling.
To confirm our hypothesis that TLR4 itself is sufficient to confer the ability to recognize Brucella LPS, we took advantage of the species-specific pharmacology of diphosphoryl lipid A from Rhodobacter sphaeroides (DPLA). This molecule acts as an antagonist of LPS in mouse cells (30). Macrophage cell line RAW 264.7 treated with DPLA dramatically reduced TNF- production induced by B. abortus LPS. The most likely explanation for the mechanism of LPS inhibition by DPLA is that this antagonist competes with the lipid A portion of LPS for a common binding site on TLR4, since DPLA blocks E. coli LPS-induced signaling in a TLR4-dependent fashion (30). In contrast, DPLA fails to block Porphyromonas gingivalis LPS-induced TNF- production because P. gingivalis LPS signals via TLR2 (20).
To determine the role of TLR4 and TLR2 in Brucella infection, we infected C3H/HeJ, C3H/HePas, TLR2 knockout, and wild-type mice with the virulent B. abortus strain S2308. TLR4 mutant mice (C3H/HeJ) demonstrated an enhanced susceptibility to B. abortus infection at 3 and 6 weeks postinfection compared to C3H/HePas mice. Recently, TLR4 mutant mice were apparently highly effective in controlling the primary Mycobacterium tuberculosis infection, but host immune protection to M. tuberculosis was insufficient for long-term persistence in vivo (1). Regarding TLR2-deficient mice, these animals are more susceptible to infection by Staphylococcus aureus (50). But, in the case of infection with B. abortus we found that TLR2 knockout mice controlled infection as efficiently as wild-type mice. These in vivo data confirmed the dominant role of TLR4 versus TLR2 in triggering innate immunity in murine brucellosis and therefore helping the host to control this bacterial infection. Additionally, Brucella-primed splenocytes from C3H/HeJ exposed to heat-killed bacteria produced much lower levels of IFN- at 3 and 6 weeks postinfection time when the number of bacteria was elevated compared to C3H/HePas animals.
A recent study with IFN--deficient mice has demonstrated that at 3 weeks postinfection with B. abortus these animals had twice the spleen weights, a threefold increase in the total number of splenic leukocytes, and a higher number of bacteria than the control mice (39). A major mechanism to explain enhanced C3H/HeJ susceptibility to infection is based on limited IFN- production. This drop in IFN- synthesis probably occurred due to decreased IL-12 and TNF- production by C3H/HeJ inflammatory macrophages exposed to Brucella. Zhan and Cheers (61) previously demonstrated that TNF- produced by Brucella acts as an autocrine factor to up-regulate IL-12 and consequently IFN- production.
Knowing that the lipid A moiety is the major molecule responsible for the proinflammatory properties of LPS, we decided to define the Brucella S and R strains lipid A profile by mass spectrometry. The mass spectra revealed very distinct profiles between R and S lipid A (Fig. 4). These differences seem to be a result of variations in the number and size of the fatty acid chains of the lipid A species from both strains, and perhaps it could account for some of the significant differences in their biological activity observed here. Goldstein et al. (16) demonstrated that Brucella abortus lipid A has diaminoglucoses instead of glucosamines and long-chain fatty acids, and therefore it is less pyrogenic than enterobacterial LPS. To confirm if this difference in lipid A structure between S and R strains could be responsible for different biological activity of these molecules, we measured cell activation through TLR4 signaling. Our results demonstrated that at the same concentration S lipid A had a greater ability to induce NF-B activation trough TLR4 than R lipid A.
Immature dendritic cells are unable to present antigens and induce immune responses in the absence of inflammatory stimuli (like TNF- and IL-1), which, acting immediately after antigen uptake, induce dendritic cells to mature. We wanted to gain more insights into the ability of Brucella S LPS and R LPS to activate dendritic cells and, as a consequence, to induce specific adaptive immune responses. Mice immunized with adenovirus-transduced immature dendritic cells expressing the CS protein from the rodent malaria parasite Plasmodium yoelii and treated with purified Brucella S lipid A, induced an enhanced IFN- production by murine CD4+ and CD8+ T cells specific to CS-derived peptides compared to untreated dendritic cells and treated with R lipid A. These data confirm that Brucella S LPS is a more powerful adjuvant compared to R LPS not only to trigger innate but also adaptive immune responses. Recently, B. abortus LPS has been shown to induce relevant quantities of ß-chemokines known to bind to the human immunodeficiency virus type 1 coreceptor CCR5 and block virus entry (60). Therefore, heat-inactivated B. abortus and its LPS have been proposed as carriers for therapeutic vaccines for individuals with human immunodeficiency virus (15). LPS from B. abortus differs structurally from the LPS of E. coli and S. enterica serovar Typhimurium, is much less pyrogenic in rabbits and mice, and induces less TNF- from human monocytes (16). Thus, these properties make B. abortus LPS a safe adjuvant to be used in vaccine production.
The activation of macrophages represents one of the first events in the innate resistance to intracellular infection. Intracellular pathogens such as Brucella, phagocytosed by macrophages and acting through surface molecules like LPS that signal through TLR4 activate these cells to produce a series of cytokines that further contribute to bacterial clearance. In this study, we have shown that TLR4 has a dominant effect compared to TLR2 on Brucella macrophage activation and host control of infection. In addition to improve our understanding of Brucella pathogenesis our findings have an important implication in vaccine development. At similar concentrations, the Brucella S vaccine strain (S19) induces higher protection than the R vaccine strain (RB51) (46). The lack of protective antibodies to the O side chain of the LPS in animals immunized with B. abortus RB51 may explain in part why the R strain induces lower protection against infection (49). Herein, we demonstrated that structural difference between lipid A moiety of LPS from S and R strains are responsible for enhanced cell activation through TLR4 and cytokine production. Therefore, we suggest that the difference in protection induced by the S versus R vaccine strains may also be dependent on activation of the innate immune system and proinflammatory cytokine production via TLR4.
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ACKNOWLEDGMENTS |
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This work was supported by the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), PADCT/CNPq, CBAB/CNPq, FAPEMIG, FIOCRUZ (PAPES III and PDTIS) and National Institutes of Health grant RO1 AI48490. M.A.C. and O.B.R. are visiting scientists from CNPq/FIOCRUZ, G.M.S.R. is a Postdoctoral Fellow from CNPq, and R.T.G., I.C.A., and S.C.O. are Research Fellows from CNPq. I.C.A. is supported by a grant from the Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP).
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FOOTNOTES |
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