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Brucella control of dendritic cell maturation is dependent on the TIR-containing protein Btp1.

Salcedo SP, Marchesini MI, Lelouard H, Fugier E, Jolly G, Balor S, Muller A, Lapaque N, Demaria O, Alexopoulou L, Comerci DJ, Ugalde RA, Pierre P, Gorvel JP - PLoS Pathog. (2008)

Bottom Line: In vitro, we found that Brucella replicates within dendritic cells and hinders their functional activation.In addition, we identified a new Brucella protein Btp1, which down-modulates maturation of infected dendritic cells by interfering with the TLR2 signaling pathway.These results show that intracellular Brucella is able to control dendritic cell function, which may have important consequences in the development of chronic brucellosis.

View Article: PubMed Central - PubMed

Affiliation: Centre d'Immunologie de Marseille-Luminy, Aix Marseille Université, Faculté de Sciences de Luminy, Marseille, France.

ABSTRACT
Brucella is an intracellular pathogen able to persist for long periods of time within the host and establish a chronic disease. We show that soon after Brucella inoculation in intestinal loops, dendritic cells from ileal Peyer's patches become infected and constitute a cell target for this pathogen. In vitro, we found that Brucella replicates within dendritic cells and hinders their functional activation. In addition, we identified a new Brucella protein Btp1, which down-modulates maturation of infected dendritic cells by interfering with the TLR2 signaling pathway. These results show that intracellular Brucella is able to control dendritic cell function, which may have important consequences in the development of chronic brucellosis.

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B. abortus Protein Btp1 Modulates TLR Signaling(A) Representative images obtained by immunofluorescence microscopy of DCs from wild-type mice or TLR2 knockout mice labelled for MHC II (blue), DALIS (red), and in the case of lower panel also labelled for Brucella (green). Cell were either stimulated with PAM (500 ng/μl, upper panel) or infected with the Brucella wild-type and btp1− mutant strains (lower panel).(B) DCs obtained from wild-type mice or different knockout mice were infected for 24 h with either B. abortus wild-type or btp1− mutant strain. The percentage of DALIS in infected cells was then quantified and the data represent means ± standard errors of four independent experiments. The average value for the wild-type Brucella is indicated with a red line. For btp1− we observed a statistical difference (*) between the wt and TLR2−/− DCs (p = 0.031).(C) HEK293 cells were transiently transfected for 24 h with three vectors: a luciferase reporter vector and either empty vector, myc-Btp1 or myc-PipB2 along with either TLR2 (upper panel) or TLR9 (lower panel). The amounts of Btp plasmids are indicated in the graph (ng). Data represent the means ± standard errors of relative luciferase activity obtained from triplicates of a representative experiment. In the case of TLR2, cells were stimulated with PAM (500 ng/μl) and for TLR9 with CpG (1 μM) for 6 h before measuring the luciferase activity.
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ppat-0040021-g007: B. abortus Protein Btp1 Modulates TLR Signaling(A) Representative images obtained by immunofluorescence microscopy of DCs from wild-type mice or TLR2 knockout mice labelled for MHC II (blue), DALIS (red), and in the case of lower panel also labelled for Brucella (green). Cell were either stimulated with PAM (500 ng/μl, upper panel) or infected with the Brucella wild-type and btp1− mutant strains (lower panel).(B) DCs obtained from wild-type mice or different knockout mice were infected for 24 h with either B. abortus wild-type or btp1− mutant strain. The percentage of DALIS in infected cells was then quantified and the data represent means ± standard errors of four independent experiments. The average value for the wild-type Brucella is indicated with a red line. For btp1− we observed a statistical difference (*) between the wt and TLR2−/− DCs (p = 0.031).(C) HEK293 cells were transiently transfected for 24 h with three vectors: a luciferase reporter vector and either empty vector, myc-Btp1 or myc-PipB2 along with either TLR2 (upper panel) or TLR9 (lower panel). The amounts of Btp plasmids are indicated in the graph (ng). Data represent the means ± standard errors of relative luciferase activity obtained from triplicates of a representative experiment. In the case of TLR2, cells were stimulated with PAM (500 ng/μl) and for TLR9 with CpG (1 μM) for 6 h before measuring the luciferase activity.

Mentions: TLR signal transduction pathways are essential for the induction of DC maturation and secretion of pro-inflammatory cytokines. The most important TLRs in the context of a bacterial infection are TLR2, TLR4, TLR5 and TLR9 that recognize lipoproteins, LPS, flagellin and CpG DNA, respectively. However, recent work showed that DCs poorly respond to flagellin due to limited TLR5 expression in these cells [25] so we therefore excluded this pathway. To determine if Btp1 was interfering with a specific TLR in the context of an infection we used DALIS formation as a read-out, since the increased number of DCs with DALIS was the clearest phenotype for the btp1 mutant when compared to wild-type Brucella. DCs from different TLR knockout mice were infected with Brucella wild-type or the btp1 mutant and analysed by confocal immunofluorescence microscopy. We observed that DCs from wt mice infected with btp1− contained a very high number of DALIS, contrasting with infected TLR2−/− DCs (Figure 7A), a phenotype reminiscent of DCs treated with the TLR2 ligand PAM. This difference was not noticeable in other TLR knockout mice used. Indeed, quantification of the number of infected DCs containing DALIS confirmed that DCs infected with the btp1 mutant showed increased DALIS formation in wild-type, TRIF−/−, TLR4−/−, TLR7−/− and TLR9−/− DCs but not in TLR2−/− and to a lesser extent in MyD88−/− DCs (Figure 7B). These results suggest that inhibition of DC maturation by Btp1 is at least in part dependent on the TLR2 pathway. Appropriate ligands were used as positive controls for the assay (Figure S6). In addition, we found that DC maturation induced by heat-killed Brucella, as measured by DALIS formation, was also dependent on TLR2 (Figure S7). This result is consistent with previous reports showing that TNF-α secretion induced by heat-killed Brucella in DCs is partially dependent on TLR2 and MyD88 pathways [26]. We then investigated if the ability of Brucella to reduce DC maturation was dependent on the TLR2 pathway. DCs were infected with wild-type B. abortus for 24 h to allow establishment of a replication niche and then incubated with either LPS or PAM for a further 24 h. We found that DCs infected with wild-type Brucella and treated with E. coli LPS were very activated and contained very large DALIS, similar to what has been observed in LPS-treated cells. In contrast, infected cells treated with PAM contained only small DALIS similar to untreated infected cells. However, non-infected cells treated with PAM contained large and numerous DALIS (Figure S8). These results confirm that Brucella is able to control TLR2-dependent DC maturation.


Brucella control of dendritic cell maturation is dependent on the TIR-containing protein Btp1.

Salcedo SP, Marchesini MI, Lelouard H, Fugier E, Jolly G, Balor S, Muller A, Lapaque N, Demaria O, Alexopoulou L, Comerci DJ, Ugalde RA, Pierre P, Gorvel JP - PLoS Pathog. (2008)

B. abortus Protein Btp1 Modulates TLR Signaling(A) Representative images obtained by immunofluorescence microscopy of DCs from wild-type mice or TLR2 knockout mice labelled for MHC II (blue), DALIS (red), and in the case of lower panel also labelled for Brucella (green). Cell were either stimulated with PAM (500 ng/μl, upper panel) or infected with the Brucella wild-type and btp1− mutant strains (lower panel).(B) DCs obtained from wild-type mice or different knockout mice were infected for 24 h with either B. abortus wild-type or btp1− mutant strain. The percentage of DALIS in infected cells was then quantified and the data represent means ± standard errors of four independent experiments. The average value for the wild-type Brucella is indicated with a red line. For btp1− we observed a statistical difference (*) between the wt and TLR2−/− DCs (p = 0.031).(C) HEK293 cells were transiently transfected for 24 h with three vectors: a luciferase reporter vector and either empty vector, myc-Btp1 or myc-PipB2 along with either TLR2 (upper panel) or TLR9 (lower panel). The amounts of Btp plasmids are indicated in the graph (ng). Data represent the means ± standard errors of relative luciferase activity obtained from triplicates of a representative experiment. In the case of TLR2, cells were stimulated with PAM (500 ng/μl) and for TLR9 with CpG (1 μM) for 6 h before measuring the luciferase activity.
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Related In: Results  -  Collection

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ppat-0040021-g007: B. abortus Protein Btp1 Modulates TLR Signaling(A) Representative images obtained by immunofluorescence microscopy of DCs from wild-type mice or TLR2 knockout mice labelled for MHC II (blue), DALIS (red), and in the case of lower panel also labelled for Brucella (green). Cell were either stimulated with PAM (500 ng/μl, upper panel) or infected with the Brucella wild-type and btp1− mutant strains (lower panel).(B) DCs obtained from wild-type mice or different knockout mice were infected for 24 h with either B. abortus wild-type or btp1− mutant strain. The percentage of DALIS in infected cells was then quantified and the data represent means ± standard errors of four independent experiments. The average value for the wild-type Brucella is indicated with a red line. For btp1− we observed a statistical difference (*) between the wt and TLR2−/− DCs (p = 0.031).(C) HEK293 cells were transiently transfected for 24 h with three vectors: a luciferase reporter vector and either empty vector, myc-Btp1 or myc-PipB2 along with either TLR2 (upper panel) or TLR9 (lower panel). The amounts of Btp plasmids are indicated in the graph (ng). Data represent the means ± standard errors of relative luciferase activity obtained from triplicates of a representative experiment. In the case of TLR2, cells were stimulated with PAM (500 ng/μl) and for TLR9 with CpG (1 μM) for 6 h before measuring the luciferase activity.
Mentions: TLR signal transduction pathways are essential for the induction of DC maturation and secretion of pro-inflammatory cytokines. The most important TLRs in the context of a bacterial infection are TLR2, TLR4, TLR5 and TLR9 that recognize lipoproteins, LPS, flagellin and CpG DNA, respectively. However, recent work showed that DCs poorly respond to flagellin due to limited TLR5 expression in these cells [25] so we therefore excluded this pathway. To determine if Btp1 was interfering with a specific TLR in the context of an infection we used DALIS formation as a read-out, since the increased number of DCs with DALIS was the clearest phenotype for the btp1 mutant when compared to wild-type Brucella. DCs from different TLR knockout mice were infected with Brucella wild-type or the btp1 mutant and analysed by confocal immunofluorescence microscopy. We observed that DCs from wt mice infected with btp1− contained a very high number of DALIS, contrasting with infected TLR2−/− DCs (Figure 7A), a phenotype reminiscent of DCs treated with the TLR2 ligand PAM. This difference was not noticeable in other TLR knockout mice used. Indeed, quantification of the number of infected DCs containing DALIS confirmed that DCs infected with the btp1 mutant showed increased DALIS formation in wild-type, TRIF−/−, TLR4−/−, TLR7−/− and TLR9−/− DCs but not in TLR2−/− and to a lesser extent in MyD88−/− DCs (Figure 7B). These results suggest that inhibition of DC maturation by Btp1 is at least in part dependent on the TLR2 pathway. Appropriate ligands were used as positive controls for the assay (Figure S6). In addition, we found that DC maturation induced by heat-killed Brucella, as measured by DALIS formation, was also dependent on TLR2 (Figure S7). This result is consistent with previous reports showing that TNF-α secretion induced by heat-killed Brucella in DCs is partially dependent on TLR2 and MyD88 pathways [26]. We then investigated if the ability of Brucella to reduce DC maturation was dependent on the TLR2 pathway. DCs were infected with wild-type B. abortus for 24 h to allow establishment of a replication niche and then incubated with either LPS or PAM for a further 24 h. We found that DCs infected with wild-type Brucella and treated with E. coli LPS were very activated and contained very large DALIS, similar to what has been observed in LPS-treated cells. In contrast, infected cells treated with PAM contained only small DALIS similar to untreated infected cells. However, non-infected cells treated with PAM contained large and numerous DALIS (Figure S8). These results confirm that Brucella is able to control TLR2-dependent DC maturation.

Bottom Line: In vitro, we found that Brucella replicates within dendritic cells and hinders their functional activation.In addition, we identified a new Brucella protein Btp1, which down-modulates maturation of infected dendritic cells by interfering with the TLR2 signaling pathway.These results show that intracellular Brucella is able to control dendritic cell function, which may have important consequences in the development of chronic brucellosis.

View Article: PubMed Central - PubMed

Affiliation: Centre d'Immunologie de Marseille-Luminy, Aix Marseille Université, Faculté de Sciences de Luminy, Marseille, France.

ABSTRACT
Brucella is an intracellular pathogen able to persist for long periods of time within the host and establish a chronic disease. We show that soon after Brucella inoculation in intestinal loops, dendritic cells from ileal Peyer's patches become infected and constitute a cell target for this pathogen. In vitro, we found that Brucella replicates within dendritic cells and hinders their functional activation. In addition, we identified a new Brucella protein Btp1, which down-modulates maturation of infected dendritic cells by interfering with the TLR2 signaling pathway. These results show that intracellular Brucella is able to control dendritic cell function, which may have important consequences in the development of chronic brucellosis.

Show MeSH
Related in: MedlinePlus