Brucella lipoproteins mimic dendritic cell maturation induced by Brucella abortus

Microbes and Infection 10 (2008) 1346e1354 www.elsevier.com/locate/micinf

Original article

Brucella lipoproteins mimic dendritic cell maturation induced by Brucella abortus Astrid Zwerdling a,b, M. Victoria Delpino a, Paula Barrionuevo a,b, Juliana Cassataro a,b, Karina A. Pasquevich a,b, Clara Garcı´a Samartino a,b, Carlos A. Fossati a, Guillermo H. Giambartolomei a,b,* a

Instituto de Estudios de la Inmunidad Humoral (CONICET), Facultad de Farmacia y Bioquı´mica, Universidad de Buenos Aires (U.B.A.), Buenos Aires, Argentina b Laboratorio de Inmunogene´tica, Hospital de Clı´nicas ‘‘Jose´ de San Martı´n’’, Facultad de Medicina, U.B.A., Buenos Aires, Argentina Received 17 December 2007; accepted 31 July 2008 Available online 12 August 2008

Abstract Infection with Brucella abortus induces a pro-inflammatory response that drives T cell responses toward a Th1 profile. The mechanism by which this bacterium triggers this response is unknown. Dendritic cells (DC) are crucial mediators at the host-pathogen interface and are potent Th1-inducing antigen-presenting cells. Thus, we examined the mechanism whereby B. abortus stimulate human DC maturation. B. abortusinfected DC increased the expression of CD86, CD80, CCR7, CD83, MHCII, MHCI and CD40 and induced the production of TNF-a, IL-6, IL10 and IL-12. Both phenomena were not dependent on bacterial viability since they were also induced by heat-killed B. abortus (HKBA). B. abortus LPS was unable to induce markers up-regulation or cytokine production. We next investigated the capacity of the outer membrane protein 19 (Omp19) as a B. abortus lipoprotein model to induce DC maturation. Lipidated Omp19 (L-Omp19), but not its unlipidated form, increased the expression of cell surface markers and the secretion of cytokines. L-Omp19-matured DC also have decreased endocytic activity and displayed enhanced T cell stimulatory activity in a MLR. Pre-incubation of DC with anti-TLR2 mAb blocked L-Omp19-mediated cytokine production. These results demonstrate that B. abortus lipoproteins can stimulate DC maturation providing a mechanism by which these bacteria generate a Th1-type immune response. Ó 2008 Elsevier Masson SAS. All rights reserved. Keywords: Brucella; Brucellosis; Dendritic cells; Lipoproteins

1. Introduction Brucella abortus has been shown to potently activate the innate as well as the adaptive immune system, leading to a potent pro-inflammatory response that favors a T helper 1 (Th1) profile [1]. This recognized ability to induce a Th1 response, indicates a long lasting recruitment of a proinflammatory mechanism by Brucella-derived products. * Corresponding author. Instituto de Estudios de la Inmunidad Humoral (IDEHU), Facultad de Farmacia y Bioquı´mica, Universidad de Buenos Aires, Junı´n 956 4 Piso, 1113 Buenos Aires, Argentina. Tel.: þ54 11 5950 8755; fax: þ54 11 5950 8758. E-mail address: [email protected] (G.H. Giambartolomei). 1286-4579/$ - see front matter Ó 2008 Elsevier Masson SAS. All rights reserved. doi:10.1016/j.micinf.2008.07.035

Nevertheless, the means whereby B. abortus induces this response have not been completely elucidated. Dendritic cells (DC) play an integral role in host defense in that they are the only antigen (Ag)-presenting cells capable of activating naive lymphocytes, resulting in the initiation of protective immune responses. The efficiency of this process relies on a maturation process in which DC modulate the expression of cell surface molecules and produce immune stimulatory cytokines that will dictate the fate of the immune response [2]. Microbial-induced DC maturation provides a crucial element required for the selective induction of a Th1 response [2] and B. abortus has the ability to infect and multiply inside these cells [3]. Although it is known that microbial products, particularly enterobacterial lipopolysaccharide (LPS), induce

A. Zwerdling et al. / Microbes and Infection 10 (2008) 1346e1354

the maturation of DC [4], LPS from B. abortus is an unlikely candidate to induce DC maturation since we and others have shown that this molecule is a weak cellular activator [5] and a poor inducer of pro-inflammatory cytokines [6,7]. Similarly, DNA from B. abortus has also been shown to be relatively inefficient in eliciting cytokine production in murine spleen cells [6]. On the other hand, bacterial lipoproteins are known to have multiple effects on the immune system in mice and humans [8,9]. In that respect, we have recently shown that B. abortus lipoproteins, and not its LPS, are the molecules responsible for the activation of monocytes/macrophages and the induction of the pro-inflammatory response elicited by heat-killed B. abortus (HKBA) [5]. Considering the relevance of DC in linking innate and adaptive immune responses, and that Brucella-induced DC maturation may provide the crucial element required for the selective induction of a Th1 response, we focused our present work on the interactions between B. abortus and human DC. We first investigated the ability of B. abortus to induce the phenotypic and functional changes associated with DC maturation. In addition, given the notion that other bacterial lipoproteins are able to activate DC [10], together with the finding made in our laboratory indicating that B. abortus lipoproteins can activate cells of the innate immune system [5], we investigated the role of Brucella lipoproteins on Brucella-induced DC maturation. For that purpose, we used purified recombinant outer membrane protein 19 (Omp19) as a B. abortus lipoprotein model. 2. Materials and methods 2.1. Bacteria B. abortus S2308 and Escherichia coli strain 11105 (ATCC) were cultured in tryptose-soy agar supplemented with yeast extract (Merck). Bacterial numbers were determined as described [5]. To obtain Heat-killed B. abortus, bacteria were washed five times for 10 min each in PBS and heat-killed by boiling for 20 min. Absence of B. abortus viability subsequent to heat-killing was verified by the absence of bacterial growth.

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gradient (GE Bio-Sciences). Monocytes were then purified from the PBMC by Percoll gradient (GE Bio-Sciences). Purity of the isolated CD14þ monocytes was more than 90% as determined by flow cytometry. Monocytes were cultured at 2  106 cell/ml under a humidified atmosphere of 5% CO2 at 37  C in complete medium (RPMI 1640, 10% FBS, 1 mM glutamine, 100 U/ml penicillin, 100 mg/ml streptomycin; Invitrogen) supplemented with 50 ng/ml recombinant granulocyte-monocyte colony stimulating factor (GM-CSF) (Gautier laboratories) and 10 ng/ml recombinant interleukin (IL)-4 (Prepotech). At day 6, cells exhibited morphology typical of immature DC and a surface phenotype of CD14, CD1aþ and DC-SIGNþ as assessed by flow cytometry. To induce further maturation, cells were either infected (as indicated below) or re-cultured in fresh medium containing different stimulants for an additional 24 h. The concentrations of the stimuli used were: Escherichia coli LPS (10 ng/ml), B. abortus LPS (1000 ng/ml), HKBA (1  108 and 1  109 bacteria/ml), Pam3Cys (50 ng/ml), L-Omp19 and U-Omp19 (10, 100 and 1000 ng/ml). 2.3.1. Infection of DC DC (1  106/ml) were infected with B. abortus or E. coli at a MOI of 5 for 1 h in medium containing no antibiotics. Cells were extensively washed to remove uninternalized bacteria and infection was maintained for an additional 24 h in the presence of antibiotics (100 mg/ml gentamicin and 50 mg/ml of streptomycin) in order to kill remaining extracellular bacteria. To monitor Brucella intracellular replication, infected cells (5  105/well) were washed and lysed at several intervals post-infection with 0.1% (vol/vol) Triton X-100. The number of intracellular viable bacteria (in CFU/well) was determined by plating serial dilutions onto TSB agar plates (Fig. 1). 2.4. Flow cytometry FITC-conjugated mAbs to CD14 and CD1a and, PEconjugated mAbs to CD86, CD80, CCR7, CD83, MHCII, 107

2.2. Lipoproteins and LPS Log CFU/well

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Lipidated Omp19 (L-Omp19) and unlipidated Omp19 (UOmp19) were obtained as described [5]. Both recombinant proteins contained less than 0.25 endotoxin U/mg of protein as assessed by Limulus amebocyte assay (Associates of Cape Cod). B. abortus 2308 LPS and Escherichia coli O111k58H2 LPS were provided by I. Moriyon. The synthetic lipohexapeptide (tripalmitoyl-S-glyceryl-Cys-Ser-Lys4-OH [Pam3Cys]) was purchased from Boehringer Mannheim. Lipidated B. burgdorferi outer surface protein (L-OspA) was obtained from Dr. John Dunn. 2.3. Generation of monocyte-derived DC

Fig. 1. Intracellular growth of B. abortus in DC. DC were infected with B. abortus at a MOI of 5:1, and the number of live intracellular bacteria was evaluated by determining the number of CFU/well at different times postinfection, as described in Section 2. Data are means  SD of the means of three experiments performed in duplicate.

Human peripheral blood mononuclear cells (PBMC) from healthy donors were isolated from a Ficoll-Paque density

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MHCI, CD40 and DC-SIGN were obtained from BD Biosciences. After staining, cells were washed and fixed in 1% paraformaldehyde before analysis on a FacsCa1ibur Flow CytometerÒ. Gating was on large granular cells, and 10,000 gated events were collected from each sample. Data were processed using the CellQuest software (BD Biosciences). Histograms were draw from and mean fluorescence intensity (MFI) values were determined on the gated population. 2.5. Cytokine ELISA IL-6, IL-10, IL-12p40 and tumor necrosis factor (TNF)-a in culture supernatants were quantified by ELISA (BD Bioscience). 2.6. Endocytic activity Endocytic activity was measured by the uptake of FITCconjugated ovalbumin (OVA-FITC) (kindly gifted by Dr. Vermeulen) as previously described [11]. Briefly, DC were incubated in complete medium plus 100 mg/ml OVA-FITC for 1 h at 4  C to measure non-specific binding, or at 37  C to measure specific uptake. Cells were then washed extensively and analyzed by flow cytometry. 2.7. MLR L-Omp19, U-Omp19, HKBA or E. coli LPS-stimulated DC were treated with 30 mg/ml of mitomycin C (Sigma) at 37  C for 30 min, extensively washed and incubated in fresh medium in 96-wells round bottom plates (Costar) for 5 days with PBMC (2  105/well) from an unrelated donor to obtained final DC:PBMC ratios of 1:20; 1:50; 1:100. For measuring lymphocyte proliferation 1.0 mCi/well of [3H] thymidine (ICN Pharmaceuticals Inc.) was added and the culture was incubated for another 18 h. The assay was then harvested and the radioactive incorporation was measured in a liquid scintillation counter (Beckman Instruments). 2.8. Blocking of TLRs DC (1  106/ml) were incubated with 20 mg/ml of antihTLR2 (clone TL2.1), anti-hTLR4 (clone HTA125) or IgG2a isotype control (eBioscience) for 30 min at 4  C and then incubated with E. coli LPS, L-OspA, HKBA or L-Omp19 to reach a final concentration of 10 ng/ml of E. coli LPS, 500 ng/ ml of L-OspA, 1  108 bacteria/ml of HKBA or 500 ng/ml of L-Omp19 in a final volume of 0.4 ml. Culture were incubated for 24 h and supernatants were assayed for cytokine production as described. 2.9. Statistical analysis Results were logarithmically transformed and analyzed by one-way analysis of variance followed by post-hoc Bonferroni analysis (InStat; GraphPad).

3. Results 3.1. B. abortus induces DC maturation We first evaluated the ability of B. abortus to induce human DC maturation. DC phenotype was confirmed by the expression of CD1a and DC-SIGN; and the absence of CD14. E. coli infection was used as positive control for DC maturation. B. abortus infection induced DC maturation as evidenced by the up-regulated expression of CD86, CD80, CCR7, CD83, MHCII, MHCI and CD40 (Fig. 2A,B). Additionally, the culture supernatants were analyzed for cytokine production by ELISA. As with the up-regulation of cell surface markers, B. abortus infection also induced a significant (P < 0.05) production of TNF-a, IL-6, IL-10 and IL-12 (Fig. 2C). To test whether viable bacteria were necessary to induce DC maturation, the ability of HKBA to up-regulate the expression of co-stimulatory molecules and induce the secretion of cytokines was examined. E. coli infection also induced up-regulation of cell surface markers and cytokine production (Fig. 2). As observed with live B. abortus, HKBA-induced the upregulation of CD86, CD80, CCR7, CD83, MHCII, MHCI and CD40. The level of expression of these markers was dependent on the amount of bacteria present in the culture (Fig. 3A,B). HKBA also induced a significant (P < 0.05) production of TNF-a, IL-6, IL-10 and IL-12 in a dose-dependent fashion (Fig. 3C). E. coli LPS, a known maturation stimulus used as a positive control, also induced up-regulation of cell surface markers and cytokine production (Fig. 3). These results demonstrate the ability of B. abortus to induce human DC maturation. The fact that HKBA also induces DC maturation, suggest that this phenomenon is mediated by a structural component of B. abortus. 3.2. Omp19 induces DC maturation When we analyzed the contribution of B. abortus LPS on HKBA-induced DC maturation, we found that highly purified B. abortus LPS was unable to induce DC maturation, at concentrations comparable to the ones estimated to be present in the concentration of bacteria used [5] (data not shown). As B. abortus LPS is not involved in the Brucella-induced DC maturation we examined if B. abortus lipoproteins were able to induce the phenotypic and functional changes associated with DC maturation using L-Omp19 as a Brucella lipoprotein model. Immature DC cultured with L-Omp19 increased the cell surface expression of CD86, CD80, CCR7, CD83, MHCII, MHCI and CD40 in a dose-dependent fashion (Fig. 4A). DC maturation induced by Brucella lipoproteins was dependent on the lipid moiety since unlipidated Omp19 (U-Omp19) induced only small or none increase in the levels of CD80, CCR7, CD83, MHCII, MHCI and CD40 at all the concentrations tested. The requirement for lipidation was further supported by the fact that Pam3Cys, a lipohexapeptide with an irrelevant peptide sequence, also increased the expression of the molecules investigated (Fig. 4A). Additionally, L-Omp19-stimulated DC secreted TNF-a, IL-6, IL-10 and IL-12. Cytokine production

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Fig. 2. B. abortus induces DC maturation. Immature DC were infected with B. abortus (B. a) or E. coli (E. c) and after 24 h they were analyzed for the expression of the indicated cell surface markers by flow cytometry (A and B), or TNF-a, IL-6, IL-10 and IL-12 were quantified by ELISA in the culture supernatants (C). Histograms correspond to one representative of five independent experiments. Bars show MFI  SEM of five experiments. ELISA results are expressed as the mean (pg/ml)  SEM. These experiments were performed five times in duplicate. ***P < 0.001, **P < 0.01, * P < 0.05 vs. N.I (not infected).

was a function of the amount of lipoprotein present in the culture. Conversely, U-Omp19 induced little or no cytokine production (Fig. 4B). Upon maturation and concomitant with an increase in Ag presenting function, DC have a reduced capacity for Ag capture via endocytic activity. To determine whether the mechanisms of Ag capture were also modulated by B. abortus lipoproteins, endocytic activity was measured in L-Omp19-treated DC by measuring the uptake of OVA-FITC by flow cytometry. Similar to E. coli LPS-stimulated cells, L-Omp19-treated DC took up lower levels of OVA-FITC in comparison to immature DC (Fig. 5A). This finding provides further evidence that B. abortus lipoproteins can drive DC maturation.

We observed that L-Omp19-matured DC as well as HKBA-matured DC expressed increased levels of Ag-presenting and co-stimulatory molecules. To determine whether, as a result of these phenotypic changes, Omp19 and HKBAmatured DC also had enhanced functional properties, we compared the ability of immature and Omp19-matured or HKBA-matured DC to stimulate T cells in a MLR. DC were treated with L-Omp19, U-Omp19, or HKBA for 24 h before co-cultured with PBMC from an unrelated donor. HKBAmatured DC were more efficient than untreated cells in stimulating a MLR, as observed by an increase in T cell proliferative responses (Fig. 5B). A similar response was obtained for L-Omp19-matured DC. Again, treatment of DC with U-Omp19 did not result in enhanced T cell

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Fig. 3. HKBA-induces DC maturation. Immature DC were incubated with complete medium (RPMI), E. coli LPS (EcLPS) (10 ng/ml) or different concentrations of HKBA and after 24 h were analyzed for the expression of the indicated cell surface markers by flow cytometry (A and B), or TNF-a, IL-6, IL-10 and IL-12 were quantified by ELISA in the culture supernatants (C). Bars show MFI  SEM of five experiments. ELISA results are expressed as the mean (pg/ml)  SEM. These experiments were performed five times in duplicate. ***P < 0.001, **P < 0.01, *P < 0.05 vs. RPMI.

proliferation. The greater efficiency of L-Omp19 and HKBAmatured DC than untreated cells in stimulating MLR correlates with the capacity of these stimuli to up-regulate the expression of both MHCII and MHCI. Taken together, these results indicate that B. abortus and its lipoproteins are able to induce not only the phenotypic but also the functional changes necessary for DC maturation. They also indicate that L-Omp19-matured DC have enhanced T cell stimulatory activity at the same level of HKBAmatured DC.

3.3. HKBA-induced cytokine production is dependent on TLR2 and TLR4 We have previously demonstrated that TLR2 mediates responses to HKBA and B. abortus lipoproteins in cells of the monocytic lineage [5]. Consequently, we further analyzed the role of TLR2 in the HKBA- and L-Omp19-induced cytokine secretion from DC. Immature DC were pre-incubated with anti-TLR2, anti-TLR4 or the corresponding isotype control and then cultured with HKBA or L-Omp19. The production of

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Fig. 4. Omp19 induces DC maturation. Immature DC were stimulated with complete medium (RPMI), E. coli LPS (EcLPS) (10 ng/ml), Pam3Cys (50 ng/ml) or various concentrations of U-Omp19 or L-Omp19 (10, 100 and 1000 ng/ml) and were analyzed after 24 h for expression of the indicated cell surface markers by flow cytometry (A), or TNF-a, IL-6, IL-10 and IL-12 were quantified by ELISA in the culture supernatants (B). Bars show MFI  SEM of five experiments. ELISA results are expressed as the mean (pg/ml)  SEM. These experiments were performed five times in duplicate. ***P < 0.001, **P < 0.01, *P < 0.05 vs. RPMI.

cytokines was evaluated in culture supernatants by ELISA. E. coli LPS and L-OspA were used as controls. As expected, preincubation of DC with anti-TLR4 significantly blocked (P < 0.001) the E. coli LPS-mediated production of TNF-a, IL-6, IL-10 and IL-12, whereas anti-TLR2 inhibited significantly (P < 0.05) the cytokine production induced by L-OspA

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(Fig. 6). Pre-incubation of DC with anti-TLR2 significantly blocked (P < 0.05) L-Omp19-mediated production of all cytokines investigated. Anti-TLR2 also inhibited significantly (P < 0.05) the HKBA-mediated production of TNF-a, IL-6, IL-10 and IL-12 (Fig. 6). Surprisingly, anti-TLR4 also blocked significantly (P < 0.05) the production of cytokines in

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Ratio DC: PBMCs Fig. 5. Omp19-matured DC have decreased endocytic activity and enhanced T cell stimulatory activity in a MLR. (A) Immature DC were cultured for 24 h with the indicated stimuli and were then analyzed for endocytic activity by uptake of OVA-FITC. Cells were incubated with OVA-FITC (100 mg/ml) for 1 h at 37  C to measure specific binding or at 4  C to measure non-specific binding, and analyzed by flow cytometry. Data shown represent one of five experiments performed with DC from different donors. (B) Immature DC were stimulated with L-Omp19 (1000 ng/ml), U-Omp19 (1000 ng/ml), HKBA (1  109 bacteria/ml) or E. coli LPS (EcLPS) (10 ng/ml) and then were further used as stimulating cells in a MLR. Proliferation was assessed as [3H] thymidine uptake (cpm). Results are the mean  SEM of five independent experiments performed in triplicate. ***P < 0.001, **P < 0.01 vs. RPMI.

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Fig. 6. HKBA-induced production of cytokines in DC is TLR2 and TLR4 dependent. Immature DC were left untreated (no Ab), pre-incubated with anti-TLR4, anti-TLR2 or IgG2a isotype control for 30 min at 4  C before the addition of HKBA (108 bacteria/ml), L-Omp19 (500 ng/ml), E. coli LPS (EcLPS) (10 ng/ml) or L-OspA (500 ng/ml). After 24 h, TNF-a, IL-6, IL-10 and IL-12 were quantified by ELISA in the culture supernatants. Results are expressed as the mean (pg/ ml)  SEM. These experiments were performed five times in duplicate. ***P < 0.001, **P < 0.01 vs. No Ab.

response to HKBA. The isotype-control antibodies had no effect on any of the responses investigated. These results indicate that, in DC, the secretion of cytokines induced by B. abortus depends on TLR2 and TLR4. Our results strongly suggest that the TLR2 ligands on HKBA are B. abortus lipoproteins. Since B. abortus LPS, which signals through TLR4; is not involved in DC maturation, our results also suggest that there might be another TLR4 ligand involved.

4. Discussion The induction of a Th1 response by Brucella spp. [1] manifest the ability of these organisms to recruit and activate key cellular components of the inflammatory and immune response. In this regard, DC have a key role in the initiation of long lasting immune mechanisms that will dictate the fate of the adaptive immune response [12]. To fulfill their role as initiators and modulators of the immune response, DC must complete a maturation program which includes up-regulation of co-stimulatory molecules and production of cytokines. Activation of DC by components present in pathogens has been studying from a long time [12,13] providing a mechanism by which Brucella organisms could modulate the immune response towards a Th1 profile.

In this work we present evidence indicating that B. abortusinfected DC increased the expression of the cell surface markers CD86, CD80, CCR7, CD83, MHCII, MHCI and CD40. Together with the up-regulation of these surface molecules, B. abortus also induced the production of cytokines necessary for the initiation and modulation of the adaptive immune response. The above-mentioned effects were observed at different multiplicities of infection and up to 48 h post-infection (data not shown), and were not due to a reversion of cell phenotype, as after infection cells maintain DC phenotype (CD14þ, CD1aþ DC-SIGNþ) (data not shown). These results are in agreement with the work of Macedo et al., where it is shown that B. abortus exposure induces murine DC maturation [14]; while conflicting with those of Billard et al. [15,16] and Salcedo et al. [17]. Similar discrepancies were presented for Mycobacterium tuberculosis, for which several studies observed human and murine infected DC maturation [18,19] whereas other study reported inhibition of maturation in M. tuberculosis-infected DC [20]. Although we can only speculate the reasons for these discrepancies, several considerations can be made: first, different cell isolation methods; in the work of Billard et al. monocytes were isolated by magnetic positive selection of CD14þ cells whereas we used Percollgradient to purified monocytes from PBMC. Second, we incubated infected DC in 48-well plates at a concentration of

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106 DC/ml compared to Billard et al. who used only 2  105 DC/well, which may have resulted in reduced cellecell interaction and therefore in decreased autocrine stimulation. Third, our infection experiments were conducted with wild type B. abortus while Billard et al. infected DC with a mutant GFP-expressing bacterium. Despite these differences, it is noteworthy that while expression of maturation markers are diminished when compared to the positive control of infection, the expression of these markers in B. abortus-infected DC are at levels comparable to our results when compared to uninfected cells [15,17]. Indeed, B. abortus-infected DC did induce significant levels of IL-12 [17]. Disregarding the differences of the various in vitro systems used, activation of DC with B. abortus is likely to be relevant at the onset of immune response when a Th1 response is triggered. At later time points Brucella might be able to circumvent this Th1 response to establish a chronic infection by means of different evasion mechanism such as down-modulation of MHCII molecules [21] in macrophages or even the prevention of DC maturation postulated by Billard et al. [15]. Both, up-regulation of co-stimulatory molecules and production of cytokines were not dependent on bacterial viability, since they were also induced by exposure of DC to HKBA, suggesting that they were elicited by a structural bacterial component. In contrast to B. abortus LPS, which was unable to induce DC maturation, following exposure to L-Omp19, DC increased the expression of surface markers. In addition, LOmp19 also induced the production of cytokines in a dosedependent fashion. Accordingly, changes induced by LOmp19 were followed by other functional changes, such as the down-modulation of Ag-capture activity and the enhanced T cell stimulatory capacity. Similar to its action in macrophages [5], Omp19 effects on DC were related to the lipid moiety of the protein; as none of the above mentioned modifications in DC were observed by stimulation with the unlipidated version of the protein, U-Omp19. The use of Omp19 (or any other) Brucella lipoprotein as a model is justified in so far as their immunological effects are elicited by the lipid, not the protein moiety. The lipid moiety is likely shared by all bacterial lipoproteins. Since the B. abortus genome contains no less than 80 genes encoding putative lipoproteins [22], it follows that lipoproteins present in Brucella would suffice to induce the maturation of DC. Altogether, our results are in agreement with previous observations showing the ability of other microbial lipoproteins to induce DC maturation [9,10]. We have previously demonstrated that, in cells of the monocytic lineage, HKBA-induced cytokine production is dependent upon TLR2 stimulation [5]. Along the same line, cytokine release by DC, including IL-12, was dependent on TLR2 stimulation. Conversely, other authors reported that in murine DC HKBA-induced IL-12 production was TLR9 dependent [14,23]. The apparent discrepancies between these authors’ results and ours may be explained by the several differences that display human and murine DC [24e26]. While murine DC have considerable plasticity regarding cytokine production [27], human DC subsets are constrain by

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their receptor expression pattern [28]. In humans, myeloid DC express TLR2 and TLR4; and plasmocytoid DC only express TLR7 and TLR9 [24,28]. Therefore, unlike mouse DC, human myeloid DC e the main producers of cytokines and triggers of the adaptive immune response due to its ability to activate T cells e do not express TLR9. These differences between mouse and human DC preclude from making simple extrapolation of mouse DC to the human system. Though tempting, the picture proposed by Huang and colleagues is unlikely to occur in humans on the grounds of the differences between mice and human DC. Nonetheless, it seems reasonable to envision a further contribution of TLR9 at later time points during the course of infection following plasmocytoid DC activation. Thus, it is conceivable to speculate about a collaboration between TLR2 and TLR9 in the development of the immune response in Brucella infection as it was reported by Bafica et al. for M. tuberculosis [29]. Strikingly for us, TLR4 also participated in the HKBAmediated production of all cytokines studied. This denotes that HKBA-induces the production of TNF-a, IL-12, IL-10 and IL6 by using a TLR2 ligand, B. abortus lipoproteins, and a TLR4 ligand distinct from B. abortus LPS. A possible alternative player for this TLR4 stimulation could be the enzyme lumazine synthase from Brucella spp., which has been very recently postulated to induce murine DC maturation through TLR4 [30]. In summary, this study shows the ability of B. abortus to induce human DC maturation; not only at the level of cell surface markers expression, but also at the production of cytokines. Additionally, using Omp19 as a model stimulant, we revealed the capacity of B. abortus lipoproteins to induce the phenotypic and functional maturation of DC. The fact that Brucella lipoproteins are, not only the molecules responsible for the B. abortus-mediated activation of monocyte/macrophages [5], but also one of the bacterial components that can activate DC inducing the maturation process crucial for the development of a proper adaptive immune response; places Brucella lipoproteins as key components of the B. abortuselicited immune response. Acknowledgements We thank Dr. Ignacio Moriyo´n (University of Navarra, Pamplona, Spain) for B. abortus and E. coli LPS, Dr. John Dunn (Brookhaven National Laboratory) for purified recombinant OspA and Dr. Monica Vermeulen (Institute of Haematological Research, National Academy of Medicine, Buenos Aires, Argentina) for OVA-FITC. This work was supported by grants PICT 05-14304 and 05-14305 from the Agencia Nacional de Promocio´n Cientı´fica y Tecnolo´gica (ANPCYT-Argentina), PIP 5213 from CONICET (Argentina), 4248-72 from Fundacio´n Antorchas (Argentina), B819 from the Universidad de Buenos Aires (Argentina) and 17-2004 from Centro Argentino Brasile~no de Biotecnologı´a (CABBIO). A. Z. and K. A. P. and C. G. S. are recipients of a fellowship from CONICET (Argentina). P. B., J. C., C. A. F. and G. H. G. are members of the Research Career of

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CONICET. C. A. F. is also member of the Facultad de Ciencias Exactas, Universidad Nacional de La Plata.

References [1] B. Golding, D.E. Scott, O. Scharf, L.Y. Huang, M. Zaitseva, C. Lapham, N. Eller, H. Golding, Immunity and protection against Brucella abortus, Microbes Infect. 3 (1) (2001) 43e48. [2] M. Colonna, B. Pulendran, A. Iwasaki, Dendritic cells at the host-pathogen interface, Nat. Immunol. 7 (2) (2006) 117e120. [3] E. Billard, C. Cazevieille, J. Dornand, A. Gross, High susceptibility of human dendritic cells to invasion by the intracellular pathogens Brucella suis, B. abortus, and B. melitensis, Infect. Immun. 73 (12) (2005) 8418e8424. [4] C. Reis e Sousa, Dendritic cells as sensors of infection, Immunity 14 (5) (2001) 495e498. [5] G.H. Giambartolomei, A. Zwerdling, J. Cassataro, L. Bruno, C.A. Fossati, M.T. Philipp, Lipoproteins, not lipopolysaccharide, are the key mediators of the proinflammatory response elicited by heat-killed Brucella abortus, J. Immunol. 173 (7) (2004) 4635e4642. [6] L. Huang, A.M. Krieg, N. Eller, D.E. Scott, Induction and regulation of Th1-inducing cytokines by bacterial DNA, lipopolysaccharide, and heatinactivated bacteria, Infect. Immun. 67 (12) (1999) 6257e6263. [7] J. Goldstein, T. Hoffman, C. Frasch, E.F. Lizzio, P.R. Beining, D. Hochstein, Y.L. Lee, R.D. Angus, B. Golding, Lipopolysaccharide (LPS) from Brucella abortus is less toxic than that from Escherichia coli, suggesting the possible use of B. abortus or LPS from B. abortus as a carrier in vaccines, Infect. Immun. 60 (4) (1992) 1385e1389. [8] G.H. Giambartolomei, V.A. Dennis, B.L. Lasater, M.T. Philipp, Induction of pro- and anti-inflammatory cytokines by Borrelia burgdorferi lipoproteins in monocytes is mediated by CD14, Infect. Immun. 67 (1) (1999) 140e147. [9] H.D. Brightbill, D.H. Libraty, S.R. Krutzik, R.B. Yang, J.T. Belisle, J.R. Bleharski, M. Maitland, M.V. Norgard, S.E. Plevy, S.T. Smale, P.J. Brennan, B.R. Bloom, P.J. Godowski, R.L. Modlin, Host defense mechanisms triggered by microbial lipoproteins through toll-like receptors, Science 285 (5428) (1999) 732e736. [10] C.J. Hertz, S.M. Kiertscher, P.J. Godowski, D.A. Bouis, M.V. Norgard, M.D. Roth, R.L. Modlin, Microbial lipopeptides stimulate dendritic cell maturation via Toll-like receptor 2, J. Immunol. 166 (4) (2001) 2444e2450. [11] M. Vermeulen, M. Giordano, A.S. Trevani, C. Sedlik, R. Gamberale, P. Fernandez-Calotti, G. Salamone, S. Raiden, J. Sanjurjo, J.R. Geffner, Acidosis improves uptake of antigens and MHC class I-restricted presentation by dendritic cells, J. Immunol. 172 (5) (2004) 3196e3204. [12] J. Banchereau, F. Briere, C. Caux, J. Davoust, S. Lebecque, Y.J. Liu, B. Pulendran, K. Palucka, Immunobiology of dendritic cells, Annu. Rev. Immunol. 18 (2000) 767e811. [13] M. Schnare, G.M. Barton, A.C. Holt, K. Takeda, S. Akira, R. Medzhitov, Toll-like receptors control activation of adaptive immune responses, Nat. Immunol 2 (10) (2001) 947e950. [14] G.C. Macedo, D.M. Magnani, N.B. Carvalho, O. Bruna-Romero, R.T. Gazzinelli, S.C. Oliveira, Central role of MyD88-dependent dendritic cell maturation and proinflammatory cytokine production to control Brucella abortus infection, J. Immunol. 180 (2) (2008) 1080e1087.

[15] E. Billard, J. Dornand, A. Gross, Interaction of Brucella suis and Brucella abortus rough strains with human dendritic cells, Infect. Immun. 75 (12) (2007) 5916e5923. [16] E. Billard, J. Dornand, A. Gross, Brucella suis prevents human dendritic cell maturation and antigen presentation through regulation of tumor necrosis factor alpha secretion, Infect. Immun. 75 (10) (2007) 4980e4989. [17] S.P. Salcedo, M.I. Marchesini, H. Lelouard, E. Fugier, G. Jolly, S. Balor, A. Muller, N. Lapaque, O. Demaria, L. Alexopoulou, D.J. Comerci, R.A. Ugalde, P. Pierre, J.P. Gorvel, Brucella control of dendritic cell maturation is dependent on the TIR-containing protein Btp1, PLoS Pathog. 4 (2) (2008) e21. [18] E.J. Cheadle, P.J. Selby, A.M. Jackson, Mycobacterium bovis bacillus CalmetteeGuerin-infected dendritic cells potently activate autologous T cells via a B7 and interleukin-12-dependent mechanism, Immunology 108 (1) (2003) 79e88. [19] R.A. Henderson, S.C. Watkins, J.L. Flynn, Activation of human dendritic cells following infection with Mycobacterium tuberculosis, J. Immunol. 159 (2) (1997) 635e643. [20] W.A. Hanekom, M. Mendillo, C. Manca, P.A. Haslett, M.R. Siddiqui, C. Barry 3rd, G. Kaplan, Mycobacterium tuberculosis inhibits maturation of human monocyte-derived dendritic cells in vitro, J. Infect. Dis. 188 (2) (2003) 257e266. [21] P. Barrionuevo, J. Cassataro, M.V. Delpino, A. Zwerdling, K.A. Pasquevich, C. Garcia Samartino, J.C. Wallach, C.A. Fossati, G.H. Giambartolomei, Brucella abortus inhibits major histocompatibility complex class II expression and antigen processing through interleukin-6 secretion via Toll-like receptor 2, Infect. Immun. 76 (1) (2008) 250e262. [22] P.S. Chain, D.J. Comerci, M.E. Tolmasky, F.W. Larimer, S.A. Malfatti, L.M. Vergez, F. Aguero, M.L. Land, R.A. Ugalde, E. Garcia, Wholegenome analyses of speciation events in pathogenic Brucellae, Infect. Immun. 73 (12) (2005) 8353e8361. [23] L.Y. Huang, K.J. Ishii, S. Akira, J. Aliberti, B. Golding, Th1-like cytokine induction by heat-killed Brucella abortus is dependent on triggering of TLR9, J. Immunol. 175 (6) (2005) 3964e3970. [24] M. Rehli, Of mice and men: species variations of Toll-like receptor expression, Trends Immunol. 23 (8) (2002) 375e378. [25] B. Pulendran, Modulating vaccine responses with dendritic cells and Toll-like receptors, Immunol. Rev. 199 (2004) 227e250. [26] K. Shortman, Y.J. Liu, Mouse and human dendritic cell subtypes, Nat. Rev. Immunol. 2 (3) (2002) 151e161. [27] A.D. Edwards, S.P. Manickasingham, R. Sporri, S.S. Diebold, O. Schulz, A. Sher, T. Kaisho, S. Akira, C. Reis e Sousa, Microbial recognition via Toll-like receptor-dependent and -independent pathways determines the cytokine response of murine dendritic cell subsets to CD40 triggering, J. Immunol. 169 (7) (2002) 3652e3660. [28] N. Kadowaki, S. Ho, S. Antonenko, R.W. Malefyt, R.A. Kastelein, F. Bazan, Y.J. Liu, Subsets of human dendritic cell precursors express different toll-like receptors and respond to different microbial antigens, J. Exp. Med. 194 (6) (2001) 863e869. [29] A. Bafica, C.A. Scanga, C.G. Feng, C. Leifer, A. Cheever, A. Sher, TLR9 regulates Th1 responses and cooperates with TLR2 in mediating optimal resistance to Mycobacterium tuberculosis, J. Exp. Med. 202 (12) (2005) 1715e1724. [30] P.M. Berguer, J. Mundinano, I. Piazzon, F.A. Goldbaum, A polymeric bacterial protein activates dendritic cells via TLR4, J. Immunol. 176 (4) (2006) 2366e2372.