Microbes and Infection 10 (2008) 1346e1354www.elsevier.com/locate/micinf

Original article

Brucella lipoproteins mimic dendritic cell maturation induced byBrucella abortus

Astrid Zwerdling a,b, M. Victoria Delpino a, Paula Barrionuevo a,b, Juliana Cassataro a,b,Karina A. Pasquevich a,b, Clara Garcıa 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 Bioquımica,

Universidad de Buenos Aires (U.B.A.), Buenos Aires, Argentinab Laboratorio de Inmunogenetica, Hospital de Clınicas ‘‘Jose de San Martın’’, 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 bywhich this bacterium triggers this response is unknown. Dendritic cells (DC) are crucial mediators at the host-pathogen interface and are potentTh1-inducing antigen-presenting cells. Thus, we examined the mechanism whereby B. abortus stimulate human DC maturation. B. abortus-infected DC increased the expression of CD86, CD80, CCR7, CD83, MHCII, MHCI and CD40 and induced the production of TNF-a, IL-6, IL-10 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 membraneprotein 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 activityand displayed enhanced T cell stimulatory activity in a MLR. Pre-incubation of DC with anti-TLR2 mAb blocked L-Omp19-mediated cytokineproduction. These results demonstrate that B. abortus lipoproteins can stimulate DC maturation providing a mechanism by which these bacteriagenerate a Th1-type immune response.� 2008 Elsevier Masson SAS. All rights reserved.

Keywords: Brucella; Brucellosis; Dendritic cells; Lipoproteins

1. Introduction

Brucella abortus has been shown to potently activate theinnate as well as the adaptive immune system, leading toa potent pro-inflammatory response that favors a T helper 1(Th1) profile [1]. This recognized ability to induce a Th1response, indicates a long lasting recruitment of a pro-inflammatory mechanism by Brucella-derived products.

* Corresponding author. Instituto de Estudios de la Inmunidad Humoral

(IDEHU), Facultad de Farmacia y Bioquımica, Universidad de Buenos Aires,

Junın 956 4� Piso, 1113 Buenos Aires, Argentina. Tel.: þ54 11 5950 8755;

fax: þ54 11 5950 8758.

E-mail address: ggiambart@ffyb.uba.ar (G.H. Giambartolomei).

1286-4579/$ - see front matter � 2008 Elsevier Masson SAS. All rights reserved.

doi:10.1016/j.micinf.2008.07.035

Nevertheless, the means whereby B. abortus induces thisresponse have not been completely elucidated.

Dendritic cells (DC) play an integral role in host defense in thatthey are the only antigen (Ag)-presenting cells capable of acti-vating naive lymphocytes, resulting in the initiation of protectiveimmune responses. The efficiency of this process relies ona maturation process in which DC modulate the expression of cellsurface molecules and produce immune stimulatory cytokines thatwill dictate the fate of the immune response [2].

Microbial-induced DC maturation provides a crucialelement required for the selective induction of a Th1 response[2] and B. abortus has the ability to infect and multiply insidethese cells [3]. Although it is known that microbial products,particularly enterobacterial lipopolysaccharide (LPS), induce

1347A. Zwerdling et al. / Microbes and Infection 10 (2008) 1346e1354

the maturation of DC [4], LPS from B. abortus is an unlikelycandidate to induce DC maturation since we and others haveshown that this molecule is a weak cellular activator [5] anda poor inducer of pro-inflammatory cytokines [6,7]. Similarly,DNA from B. abortus has also been shown to be relativelyinefficient in eliciting cytokine production in murine spleencells [6]. On the other hand, bacterial lipoproteins are knownto have multiple effects on the immune system in mice andhumans [8,9]. In that respect, we have recently shown that B.abortus lipoproteins, and not its LPS, are the moleculesresponsible for the activation of monocytes/macrophages andthe induction of the pro-inflammatory response elicited byheat-killed B. abortus (HKBA) [5].

Considering the relevance of DC in linking innate andadaptive immune responses, and that Brucella-induced DCmaturation may provide the crucial element required for theselective induction of a Th1 response, we focused our presentwork on the interactions between B. abortus and human DC. Wefirst investigated the ability of B. abortus to induce the pheno-typic and functional changes associated with DC maturation. Inaddition, given the notion that other bacterial lipoproteins areable to activate DC [10], together with the finding made in ourlaboratory indicating that B. abortus lipoproteins can activatecells of the innate immune system [5], we investigated the role ofBrucella lipoproteins on Brucella-induced DC maturation. Forthat purpose, we used purified recombinant outer membraneprotein 19 (Omp19) as a B. abortus lipoprotein model.

2. Materials and methods

2.1. Bacteria

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B. abortus S2308 and Escherichia coli strain 11105 (ATCC)were cultured in tryptose-soy agar supplemented with yeastextract (Merck). Bacterial numbers were determined asdescribed [5]. To obtain Heat-killed B. abortus, bacteria werewashed five times for 10 min each in PBS and heat-killed byboiling for 20 min. Absence of B. abortus viability subsequentto heat-killing was verified by the absence of bacterial growth.

2.2. Lipoproteins and LPS

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Lipidated Omp19 (L-Omp19) and unlipidated Omp19 (U-Omp19) were obtained as described [5]. Both recombinantproteins contained less than 0.25 endotoxin U/mg of protein asassessed by Limulus amebocyte assay (Associates of CapeCod). B. abortus 2308 LPS and Escherichia coli O111k58H2LPS were provided by I. Moriyon. The synthetic lip-ohexapeptide (tripalmitoyl-S-glyceryl-Cys-Ser-Lys4-OH[Pam3Cys]) was purchased from Boehringer Mannheim.Lipidated B. burgdorferi outer surface protein (L-OspA) wasobtained from Dr. John Dunn.

Fig. 1. Intracellular growth of B. abortus in DC. DC were infected with B.

2.3. Generation of monocyte-derived DC 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 post-

infection, as described in Section 2. Data are means � SD of the means of

three experiments performed in duplicate.

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

gradient (GE Bio-Sciences). Monocytes were then purifiedfrom the PBMC by Percoll gradient (GE Bio-Sciences). Purityof the isolated CD14þ monocytes was more than 90% asdetermined by flow cytometry. Monocytes were cultured at2 � 106 cell/ml under a humidified atmosphere of 5% CO2 at37 �C in complete medium (RPMI 1640, 10% FBS, 1 mMglutamine, 100 U/ml penicillin, 100 mg/ml streptomycin;Invitrogen) supplemented with 50 ng/ml recombinant gran-ulocyte-monocyte colony stimulating factor (GM-CSF)(Gautier laboratories) and 10 ng/ml recombinant interleukin(IL)-4 (Prepotech). At day 6, cells exhibited morphologytypical 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 (asindicated below) or re-cultured in fresh medium containing differentstimulants for an additional 24 h. The concentrations of the stimuliused 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 DCDC (1 � 106/ml) were infected with B. abortus or E. coli at

a MOI of 5 for 1 h in medium containing no antibiotics. Cellswere extensively washed to remove uninternalized bacteriaand infection was maintained for an additional 24 h in thepresence of antibiotics (100 mg/ml gentamicin and 50 mg/ml ofstreptomycin) in order to kill remaining extracellular bacteria.To monitor Brucella intracellular replication, infected cells(5 � 105/well) were washed and lysed at several intervalspost-infection with 0.1% (vol/vol) Triton X-100. The numberof intracellular viable bacteria (in CFU/well) was determinedby plating serial dilutions onto TSB agar plates (Fig. 1).

2.4. Flow cytometry

FITC-conjugated mAbs to CD14 and CD1a and, PE-conjugated mAbs to CD86, CD80, CCR7, CD83, MHCII,

1348 A. Zwerdling et al. / Microbes and Infection 10 (2008) 1346e1354

MHCI, CD40 and DC-SIGN were obtained from BD Biosci-ences. After staining, cells were washed and fixed in 1%paraformaldehyde before analysis on a FacsCa1ibur FlowCytometer�. Gating was on large granular cells, and 10,000gated events were collected from each sample. Data wereprocessed 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 inculture supernatants were quantified by ELISA (BDBioscience).

2.6. Endocytic activity

Endocytic activity was measured by the uptake of FITC-conjugated ovalbumin (OVA-FITC) (kindly gifted by Dr.Vermeulen) as previously described [11]. Briefly, DC wereincubated in complete medium plus 100 mg/ml OVA-FITC for1 h at 4 �C to measure non-specific binding, or at 37 �C tomeasure specific uptake. Cells were then washed extensivelyand analyzed by flow cytometry.

2.7. MLR

L-Omp19, U-Omp19, HKBA or E. coli LPS-stimulated DCwere treated with 30 mg/ml of mitomycin C (Sigma) at 37 �Cfor 30 min, extensively washed and incubated in fresh mediumin 96-wells round bottom plates (Costar) for 5 days withPBMC (2 � 105/well) from an unrelated donor to obtainedfinal DC:PBMC ratios of 1:20; 1:50; 1:100. For measuringlymphocyte proliferation 1.0 mCi/well of [3H] thymidine (ICNPharmaceuticals Inc.) was added and the culture was incu-bated for another 18 h. The assay was then harvested and theradioactive incorporation was measured in a liquid scintilla-tion counter (Beckman Instruments).

2.8. Blocking of TLRs

DC (1 � 106/ml) were incubated with 20 mg/ml of anti-hTLR2 (clone TL2.1), anti-hTLR4 (clone HTA125) or IgG2aisotype control (eBioscience) for 30 min at 4 �C and thenincubated with E. coli LPS, L-OspA, HKBA or L-Omp19 toreach 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 ofL-Omp19 in a final volume of 0.4 ml. Culture were incubatedfor 24 h and supernatants were assayed for cytokine produc-tion as described.

2.9. Statistical analysis

Results were logarithmically transformed and analyzed byone-way analysis of variance followed by post-hoc Bonferronianalysis (InStat; GraphPad).

3. Results

3.1. B. abortus induces DC maturation

We first evaluated the ability of B. abortus to induce humanDC maturation. DC phenotype was confirmed by the expres-sion of CD1a and DC-SIGN; and the absence of CD14. E. coliinfection was used as positive control for DC maturation. B.abortus infection induced DC maturation as evidenced by theup-regulated expression of CD86, CD80, CCR7, CD83,MHCII, MHCI and CD40 (Fig. 2A,B). Additionally, theculture supernatants were analyzed for cytokine production byELISA. 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 testwhether viable bacteria were necessary to induce DC matu-ration, the ability of HKBA to up-regulate the expression ofco-stimulatory molecules and induce the secretion of cyto-kines was examined. E. coli infection also induced up-regu-lation of cell surface markers and cytokine production (Fig. 2).As observed with live B. abortus, HKBA-induced the up-regulation of CD86, CD80, CCR7, CD83, MHCII, MHCI andCD40. The level of expression of these markers was dependenton the amount of bacteria present in the culture (Fig. 3A,B).HKBA also induced a significant (P < 0.05) production ofTNF-a, IL-6, IL-10 and IL-12 in a dose-dependent fashion(Fig. 3C). E. coli LPS, a known maturation stimulus used asa positive control, also induced up-regulation of cell surfacemarkers and cytokine production (Fig. 3).

These results demonstrate the ability of B. abortus toinduce human DC maturation. The fact that HKBA alsoinduces DC maturation, suggest that this phenomenon ismediated by a structural component of B. abortus.

3.2. Omp19 induces DC maturation

When we analyzed the contribution of B. abortus LPS onHKBA-induced DC maturation, we found that highly purifiedB. abortus LPS was unable to induce DC maturation, atconcentrations comparable to the ones estimated to be presentin the concentration of bacteria used [5] (data not shown). As B.abortus LPS is not involved in the Brucella-induced DCmaturation we examined if B. abortus lipoproteins were able toinduce the phenotypic and functional changes associated withDC maturation using L-Omp19 as a Brucella lipoprotein model.Immature DC cultured with L-Omp19 increased the cell surfaceexpression of CD86, CD80, CCR7, CD83, MHCII, MHCI andCD40 in a dose-dependent fashion (Fig. 4A). DC maturationinduced by Brucella lipoproteins was dependent on the lipidmoiety since unlipidated Omp19 (U-Omp19) induced onlysmall or none increase in the levels of CD80, CCR7, CD83,MHCII, MHCI and CD40 at all the concentrations tested. Therequirement for lipidation was further supported by the fact thatPam3Cys, a lipohexapeptide with an irrelevant peptidesequence, also increased the expression of the moleculesinvestigated (Fig. 4A). Additionally, L-Omp19-stimulated DCsecreted 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).

1349A. Zwerdling et al. / Microbes and Infection 10 (2008) 1346e1354

was a function of the amount of lipoprotein present in theculture. Conversely, U-Omp19 induced little or no cytokineproduction (Fig. 4B).

Upon maturation and concomitant with an increase in Agpresenting function, DC have a reduced capacity for Agcapture via endocytic activity. To determine whetherthe mechanisms of Ag capture were also modulated byB. abortus lipoproteins, endocytic activity was measured inL-Omp19-treated DC by measuring the uptake of OVA-FITCby flow cytometry. Similar to E. coli LPS-stimulated cells,L-Omp19-treated DC took up lower levels of OVA-FITC incomparison to immature DC (Fig. 5A). This finding providesfurther evidence that B. abortus lipoproteins can drive DCmaturation.

We observed that L-Omp19-matured DC as well asHKBA-matured DC expressed increased levels of Ag-pre-senting and co-stimulatory molecules. To determine whether,as a result of these phenotypic changes, Omp19 and HKBA-matured DC also had enhanced functional properties, wecompared the ability of immature and Omp19-matured orHKBA-matured DC to stimulate T cells in a MLR. DC weretreated with L-Omp19, U-Omp19, or HKBA for 24 h beforeco-cultured with PBMC from an unrelated donor. HKBA-matured DC were more efficient than untreated cells instimulating a MLR, as observed by an increase in T cellproliferative responses (Fig. 5B). A similar response wasobtained for L-Omp19-matured DC. Again, treatment of DCwith 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.

1350 A. Zwerdling et al. / Microbes and Infection 10 (2008) 1346e1354

proliferation. The greater efficiency of L-Omp19 and HKBA-matured DC than untreated cells in stimulating MLR corre-lates with the capacity of these stimuli to up-regulate theexpression of both MHCII and MHCI.

Taken together, these results indicate that B. abortus andits lipoproteins are able to induce not only the phenotypic butalso the functional changes necessary for DC maturation.They also indicate that L-Omp19-matured DC have enhancedT cell stimulatory activity at the same level of HKBA-matured DC.

3.3. HKBA-induced cytokine production is dependent onTLR2 and TLR4

We have previously demonstrated that TLR2 mediatesresponses to HKBA and B. abortus lipoproteins in cells of themonocytic lineage [5]. Consequently, we further analyzed therole of TLR2 in the HKBA- and L-Omp19-induced cytokinesecretion from DC. Immature DC were pre-incubated withanti-TLR2, anti-TLR4 or the corresponding isotype controland 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.

1351A. Zwerdling et al. / Microbes and Infection 10 (2008) 1346e1354

cytokines was evaluated in culture supernatants by ELISA. E.coli LPS and L-OspA were used as controls. As expected, pre-incubation 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 signifi-cantly (P < 0.05) the cytokine production induced by L-OspA

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indicated stimuli and were then analyzed for endocytic activity by uptake of OVA

measure specific binding or at 4 �C to measure non-specific binding, and analyzed b

DC from different donors. (B) Immature DC were stimulated with L-Omp19 (1000

(EcLPS) (10 ng/ml) and then were further used as stimulating cells in a MLR.

mean � SEM of five independent experiments performed in triplicate. ***P < 0.0

(Fig. 6). Pre-incubation of DC with anti-TLR2 significantlyblocked (P < 0.05) L-Omp19-mediated production of allcytokines 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 blockedsignificantly (P < 0.05) the production of cytokines in

RPMI L-Omp1918000EcLPS U-Omp19HKBA **

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y flow cytometry. Data shown represent one of five experiments performed with

ng/ml), U-Omp19 (1000 ng/ml), HKBA (1 � 109 bacteria/ml) or E. coli LPS

Proliferation was assessed as [3H] thymidine uptake (cpm). Results are the

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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.

1352 A. Zwerdling et al. / Microbes and Infection 10 (2008) 1346e1354

response to HKBA. The isotype-control antibodies had noeffect on any of the responses investigated. These resultsindicate that, in DC, the secretion of cytokines induced by B.abortus depends on TLR2 and TLR4. Our results stronglysuggest that the TLR2 ligands on HKBA are B. abortuslipoproteins. Since B. abortus LPS, which signals throughTLR4; is not involved in DC maturation, our results alsosuggest 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 activatekey cellular components of the inflammatory and immuneresponse. In this regard, DC have a key role in the initiation oflong lasting immune mechanisms that will dictate the fate ofthe adaptive immune response [12]. To fulfill their role asinitiators and modulators of the immune response, DC mustcomplete a maturation program which includes up-regulationof co-stimulatory molecules and production of cytokines.Activation of DC by components present in pathogens hasbeen studying from a long time [12,13] providing a mecha-nism by which Brucella organisms could modulate theimmune response towards a Th1 profile.

In this work we present evidence indicating that B. abortus-infected DC increased the expression of the cell surfacemarkers CD86, CD80, CCR7, CD83, MHCII, MHCI andCD40. Together with the up-regulation of these surfacemolecules, B. abortus also induced the production of cytokinesnecessary for the initiation and modulation of the adaptiveimmune response. The above-mentioned effects wereobserved at different multiplicities of infection and up to 48 hpost-infection (data not shown), and were not due to a rever-sion of cell phenotype, as after infection cells maintain DCphenotype (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 DCmaturation [14]; while conflicting with those of Billard et al.[15,16] and Salcedo et al. [17]. Similar discrepancies werepresented for Mycobacterium tuberculosis, for which severalstudies observed human and murine infected DC maturation[18,19] whereas other study reported inhibition of maturationin M. tuberculosis-infected DC [20]. Although we can onlyspeculate the reasons for these discrepancies, several consid-erations can be made: first, different cell isolation methods; inthe work of Billard et al. monocytes were isolated by magneticpositive selection of CD14þ cells whereas we used Percoll-gradient to purified monocytes from PBMC. Second, weincubated infected DC in 48-well plates at a concentration of

1353A. Zwerdling et al. / Microbes and Infection 10 (2008) 1346e1354

106 DC/ml compared to Billard et al. who used only 2 � 105

DC/well, which may have resulted in reduced cellecellinteraction and therefore in decreased autocrine stimulation.Third, our infection experiments were conducted with wildtype B. abortus while Billard et al. infected DC with a mutantGFP-expressing bacterium. Despite these differences, it isnoteworthy that while expression of maturation markers arediminished when compared to the positive control of infection,the expression of these markers in B. abortus-infected DC areat levels comparable to our results when compared to unin-fected cells [15,17]. Indeed, B. abortus-infected DC did inducesignificant levels of IL-12 [17]. Disregarding the differences ofthe various in vitro systems used, activation of DC with B.abortus is likely to be relevant at the onset of immuneresponse when a Th1 response is triggered. At later time pointsBrucella might be able to circumvent this Th1 response toestablish a chronic infection by means of different evasionmechanism such as down-modulation of MHCII molecules[21] in macrophages or even the prevention of DC maturationpostulated by Billard et al. [15].

Both, up-regulation of co-stimulatory molecules andproduction of cytokines were not dependent on bacterialviability, since they were also induced by exposure of DC toHKBA, suggesting that they were elicited by a structuralbacterial component.

In contrast to B. abortus LPS, which was unable to induceDC maturation, following exposure to L-Omp19, DCincreased the expression of surface markers. In addition, L-Omp19 also induced the production of cytokines in a dose-dependent fashion. Accordingly, changes induced by L-Omp19 were followed by other functional changes, such as thedown-modulation of Ag-capture activity and the enhanced Tcell stimulatory capacity. Similar to its action in macrophages[5], Omp19 effects on DC were related to the lipid moiety ofthe protein; as none of the above mentioned modifications inDC were observed by stimulation with the unlipidated versionof the protein, U-Omp19. The use of Omp19 (or any other)Brucella lipoprotein as a model is justified in so far as theirimmunological effects are elicited by the lipid, not the proteinmoiety. The lipid moiety is likely shared by all bacteriallipoproteins. Since the B. abortus genome contains no lessthan 80 genes encoding putative lipoproteins [22], it followsthat lipoproteins present in Brucella would suffice to inducethe maturation of DC. Altogether, our results are in agreementwith previous observations showing the ability of othermicrobial lipoproteins to induce DC maturation [9,10].

We have previously demonstrated that, in cells of themonocytic lineage, HKBA-induced cytokine production isdependent upon TLR2 stimulation [5]. Along the same line,cytokine release by DC, including IL-12, was dependent onTLR2 stimulation. Conversely, other authors reported that inmurine DC HKBA-induced IL-12 production was TLR9dependent [14,23]. The apparent discrepancies between theseauthors’ results and ours may be explained by the severaldifferences that display human and murine DC [24e26].While murine DC have considerable plasticity regardingcytokine production [27], human DC subsets are constrain by

their receptor expression pattern [28]. In humans, myeloid DCexpress TLR2 and TLR4; and plasmocytoid DC only expressTLR7 and TLR9 [24,28]. Therefore, unlike mouse DC, humanmyeloid DC e the main producers of cytokines and triggers ofthe adaptive immune response due to its ability to activate Tcells e do not express TLR9. These differences betweenmouse and human DC preclude from making simple extrap-olation of mouse DC to the human system. Though tempting,the picture proposed by Huang and colleagues is unlikely tooccur in humans on the grounds of the differences betweenmice and human DC. Nonetheless, it seems reasonable toenvision a further contribution of TLR9 at later time pointsduring the course of infection following plasmocytoid DCactivation. Thus, it is conceivable to speculate about a collab-oration between TLR2 and TLR9 in the development of theimmune response in Brucella infection as it was reported byBafica et al. for M. tuberculosis [29].

Strikingly for us, TLR4 also participated in the HKBA-mediated production of all cytokines studied. This denotes thatHKBA-induces the production of TNF-a, IL-12, IL-10 and IL-6 by using a TLR2 ligand, B. abortus lipoproteins, and a TLR4ligand distinct from B. abortus LPS. A possible alternativeplayer for this TLR4 stimulation could be the enzyme luma-zine synthase from Brucella spp., which has been very recentlypostulated to induce murine DC maturation through TLR4[30].

In summary, this study shows the ability of B. abortus toinduce human DC maturation; not only at the level of cellsurface markers expression, but also at the production ofcytokines. Additionally, using Omp19 as a model stimulant,we revealed the capacity of B. abortus lipoproteins to inducethe phenotypic and functional maturation of DC. The fact thatBrucella lipoproteins are, not only the molecules responsiblefor the B. abortus-mediated activation of monocyte/macro-phages [5], but also one of the bacterial components that canactivate DC inducing the maturation process crucial for thedevelopment of a proper adaptive immune response; placesBrucella lipoproteins as key components of the B. abortus-elicited immune response.

Acknowledgements

We thank Dr. Ignacio Moriyon (University of Navarra,Pamplona, Spain) for B. abortus and E. coli LPS, Dr. JohnDunn (Brookhaven National Laboratory) for purifiedrecombinant OspA and Dr. Monica Vermeulen (Institute ofHaematological Research, National Academy of Medicine,Buenos Aires, Argentina) for OVA-FITC. This work wassupported by grants PICT 05-14304 and 05-14305 from theAgencia Nacional de Promocion Cientıfica y Tecnologica(ANPCYT-Argentina), PIP 5213 from CONICET (Argentina),4248-72 from Fundacion Antorchas (Argentina), B819 fromthe Universidad de Buenos Aires (Argentina) and 17-2004from Centro Argentino Brasile~no de Biotecnologıa (CAB-BIO). A. Z. and K. A. P. and C. G. S. are recipients ofa fellowship from CONICET (Argentina). P. B., J. C., C. A. F.and G. H. G. are members of the Research Career of

1354 A. Zwerdling et al. / Microbes and Infection 10 (2008) 1346e1354

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

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