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Immune evasion by Yersinia enterocolitica: differential targeting of dendritic cell subpopulations in vivo.

Autenrieth SE, Linzer TR, Hiller C, Keller B, Warnke P, Köberle M, Bohn E, Biedermann T, Bühring HJ, Hämmerling GJ, Rammensee HG, Autenrieth IB - PLoS Pathog. (2010)

Bottom Line: The decreased number of DC subsets, which was dependent on TLR4 and TRIF signaling, was the result of a faster proliferation and suppressed de novo DC generation.The data suggest that these effects might be caused directly by injection of Yops into DCs and indirectly by affecting the homeostasis of CD4(+) and CD8α(+) DCs.These events may contribute to reduced T-cell proliferation and immune evasion of Ye.

View Article: PubMed Central - PubMed

Affiliation: Interfakultäres Institut für Zellbiologie, Universität Tübingen, Tübingen, Germany. Stella.Autenrieth@medizin.uni-tuebingen.de

ABSTRACT
CD4(+) T cells are essential for the control of Yersinia enterocolitica (Ye) infection in mice. Ye can inhibit dendritic cell (DC) antigen uptake and degradation, maturation and subsequently T-cell activation in vitro. Here we investigated the effects of Ye infection on splenic DCs and T-cell proliferation in an experimental mouse infection model. We found that OVA-specific CD4(+) T cells had a reduced potential to proliferate when stimulated with OVA after infection with Ye compared to control mice. Additionally, proliferation of OVA-specific CD4(+) T cells was markedly reduced when cultured with splenic CD8α(+) DCs from Ye infected mice in the presence of OVA. In contrast, T-cell proliferation was not impaired in cultures with CD4(+) or CD4(-)CD8α(-) DCs isolated from Ye infected mice. However, OVA uptake and degradation as well as cytokine production were impaired in CD8α(+) DCs, but not in CD4(+) and CD4(-)CD8α(-) DCs after Ye infection. Pathogenicity factors (Yops) from Ye were most frequently injected into CD8α(+) DCs, resulting in less MHC class II and CD86 expression than on non-injected CD8α(+) DCs. Three days post infection with Ye the number of splenic CD8α(+) and CD4(+) DCs was reduced by 50% and 90%, respectively. The decreased number of DC subsets, which was dependent on TLR4 and TRIF signaling, was the result of a faster proliferation and suppressed de novo DC generation. Together, we show that Ye infection negatively regulates the stimulatory capacity of some but not all splenic DC subpopulations in vivo. This leads to differential antigen uptake and degradation, cytokine production, cell loss, and cell death rates in various DC subpopulations. The data suggest that these effects might be caused directly by injection of Yops into DCs and indirectly by affecting the homeostasis of CD4(+) and CD8α(+) DCs. These events may contribute to reduced T-cell proliferation and immune evasion of Ye.

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Injection of Yops into DCs upon infection of mice with Ye.(A & B) C57BL/6 mice were injected i.v. with 5×105 Ye E40-YopE53β-lactamase (YopE-βla), 5×105 Ye E40-YopE53-OVA (YopE-OVA), or PBS. 24 h p.i. the percentage of DC subpopulations in the spleen injected with YopE-βla was analyzed by flow cytometry using CCF4 substrate emitting a blue fluorescence when degraded by β-lactamase in the cytosol. (A) The dot plots show the percentage ± standard deviation of CCF4-blue+CD11chi cells from mice treated with PBS, infected with the negative control Ye YopE-OVA mutant strain, or infected with the Ye YopE-βla mutant strain. R1 indicates the blue cells (Yop injected) and R2 the green cells (not injected). The diagram shows the percentage of blue+ DC subpopulations of mice infected with the Ye YopE-βla mutant strain. Data shown are the summary of 2 independent experiments with 5–6 mice infected with YopE-βla mutant strain in each experiment. (B) The expression of MHC class II and CD86 on DC subpopulations from mice injected with PBS or infected with YopE-βla mutant strain was analyzed as described in (A) (R1: blue cells, R2: green cells). The diagrams show the MHC class II or CD86 expression of the DC subpopulations as mean fluorescence and are representative for 2 independent experiments with 5–6 mice infected with YopE-βla mutant strain in each experiment. * indicate statistically significant differences.
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ppat-1001212-g004: Injection of Yops into DCs upon infection of mice with Ye.(A & B) C57BL/6 mice were injected i.v. with 5×105 Ye E40-YopE53β-lactamase (YopE-βla), 5×105 Ye E40-YopE53-OVA (YopE-OVA), or PBS. 24 h p.i. the percentage of DC subpopulations in the spleen injected with YopE-βla was analyzed by flow cytometry using CCF4 substrate emitting a blue fluorescence when degraded by β-lactamase in the cytosol. (A) The dot plots show the percentage ± standard deviation of CCF4-blue+CD11chi cells from mice treated with PBS, infected with the negative control Ye YopE-OVA mutant strain, or infected with the Ye YopE-βla mutant strain. R1 indicates the blue cells (Yop injected) and R2 the green cells (not injected). The diagram shows the percentage of blue+ DC subpopulations of mice infected with the Ye YopE-βla mutant strain. Data shown are the summary of 2 independent experiments with 5–6 mice infected with YopE-βla mutant strain in each experiment. (B) The expression of MHC class II and CD86 on DC subpopulations from mice injected with PBS or infected with YopE-βla mutant strain was analyzed as described in (A) (R1: blue cells, R2: green cells). The diagrams show the MHC class II or CD86 expression of the DC subpopulations as mean fluorescence and are representative for 2 independent experiments with 5–6 mice infected with YopE-βla mutant strain in each experiment. * indicate statistically significant differences.

Mentions: Yops are the major pathogenicity factors of Ye and are injected into host cells via the TTSS. By this means, Ye inhibits phagocytosis, antigen presentation and both NF-κB and MAPK signaling cascades [44]. Köberle et al. and others revealed that Ye, Y. pseudotuberculosis and Y. pestis preferentially target macrophages, DCs, and Gr-1+ cells [45]–[47]. To analyze whether Ye preferentially targets a splenic DC subpopulation, we adapted a recently described, new reporter system for monitoring injection of bacterial proteins into host cells via the TTSS [45], [48], [49]. We used Ye wild type strain expressing either a YopE-β-lactamase fusion protein or YopE-OVA as control for mouse infection experiments. Spleen cells were stained with the lipophilic CCF4-AM [50], an esterified form of the CCF4 substrate. After entry into living cells (Figure S1), cytoplasmic esterases rapidly convert CCF4-AM into negatively charged CCF4, which is retained in the cytoplasm. Excitation of CCF4 cumarin residue at 409 nm results in a fluorescence resonance energy transfer (FRET) to the fluorescein residue, leading to emission of green fluorescence at 520 nm. Cleavage of CCF4 substrate by β-lactamase interrupts FRET leading to light emission at 447 nm (blue fluorescence). Therefore, injection of YopE-β-lactamase by Ye into DCs can be determined by analyzing the CCF4-green+blue+ DCs by flow cytometry. 24 h p.i. with Ye YopE-β-lactamase mutant strain, 7.1±1.9% CD11chi cells displayed blue fluorescence (Fig. 4A: R1 in dot plots), indicating injection of the YopE-β-lactamase fusion protein into the cells. No β-lactamase+ cells (R1) and only 0.1±0.1% β-lactamase+ cells (R1) could be detected in mice either treated with PBS or infected with the control Ye mutant strain YopE-OVA, respectively. Flow cytometry analysis of the different DC subpopulations for injection of the YopE-β-lactamase revealed that significantly more CD8α+ DCs (11.4±1.2%) were Yop injected compared to CD4+ (6.2±0.4%) and CD4−CD8α− DCs (6.4±0.5%) 24 h p.i. (Fig. 4 A).


Immune evasion by Yersinia enterocolitica: differential targeting of dendritic cell subpopulations in vivo.

Autenrieth SE, Linzer TR, Hiller C, Keller B, Warnke P, Köberle M, Bohn E, Biedermann T, Bühring HJ, Hämmerling GJ, Rammensee HG, Autenrieth IB - PLoS Pathog. (2010)

Injection of Yops into DCs upon infection of mice with Ye.(A & B) C57BL/6 mice were injected i.v. with 5×105 Ye E40-YopE53β-lactamase (YopE-βla), 5×105 Ye E40-YopE53-OVA (YopE-OVA), or PBS. 24 h p.i. the percentage of DC subpopulations in the spleen injected with YopE-βla was analyzed by flow cytometry using CCF4 substrate emitting a blue fluorescence when degraded by β-lactamase in the cytosol. (A) The dot plots show the percentage ± standard deviation of CCF4-blue+CD11chi cells from mice treated with PBS, infected with the negative control Ye YopE-OVA mutant strain, or infected with the Ye YopE-βla mutant strain. R1 indicates the blue cells (Yop injected) and R2 the green cells (not injected). The diagram shows the percentage of blue+ DC subpopulations of mice infected with the Ye YopE-βla mutant strain. Data shown are the summary of 2 independent experiments with 5–6 mice infected with YopE-βla mutant strain in each experiment. (B) The expression of MHC class II and CD86 on DC subpopulations from mice injected with PBS or infected with YopE-βla mutant strain was analyzed as described in (A) (R1: blue cells, R2: green cells). The diagrams show the MHC class II or CD86 expression of the DC subpopulations as mean fluorescence and are representative for 2 independent experiments with 5–6 mice infected with YopE-βla mutant strain in each experiment. * indicate statistically significant differences.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2991265&req=5

ppat-1001212-g004: Injection of Yops into DCs upon infection of mice with Ye.(A & B) C57BL/6 mice were injected i.v. with 5×105 Ye E40-YopE53β-lactamase (YopE-βla), 5×105 Ye E40-YopE53-OVA (YopE-OVA), or PBS. 24 h p.i. the percentage of DC subpopulations in the spleen injected with YopE-βla was analyzed by flow cytometry using CCF4 substrate emitting a blue fluorescence when degraded by β-lactamase in the cytosol. (A) The dot plots show the percentage ± standard deviation of CCF4-blue+CD11chi cells from mice treated with PBS, infected with the negative control Ye YopE-OVA mutant strain, or infected with the Ye YopE-βla mutant strain. R1 indicates the blue cells (Yop injected) and R2 the green cells (not injected). The diagram shows the percentage of blue+ DC subpopulations of mice infected with the Ye YopE-βla mutant strain. Data shown are the summary of 2 independent experiments with 5–6 mice infected with YopE-βla mutant strain in each experiment. (B) The expression of MHC class II and CD86 on DC subpopulations from mice injected with PBS or infected with YopE-βla mutant strain was analyzed as described in (A) (R1: blue cells, R2: green cells). The diagrams show the MHC class II or CD86 expression of the DC subpopulations as mean fluorescence and are representative for 2 independent experiments with 5–6 mice infected with YopE-βla mutant strain in each experiment. * indicate statistically significant differences.
Mentions: Yops are the major pathogenicity factors of Ye and are injected into host cells via the TTSS. By this means, Ye inhibits phagocytosis, antigen presentation and both NF-κB and MAPK signaling cascades [44]. Köberle et al. and others revealed that Ye, Y. pseudotuberculosis and Y. pestis preferentially target macrophages, DCs, and Gr-1+ cells [45]–[47]. To analyze whether Ye preferentially targets a splenic DC subpopulation, we adapted a recently described, new reporter system for monitoring injection of bacterial proteins into host cells via the TTSS [45], [48], [49]. We used Ye wild type strain expressing either a YopE-β-lactamase fusion protein or YopE-OVA as control for mouse infection experiments. Spleen cells were stained with the lipophilic CCF4-AM [50], an esterified form of the CCF4 substrate. After entry into living cells (Figure S1), cytoplasmic esterases rapidly convert CCF4-AM into negatively charged CCF4, which is retained in the cytoplasm. Excitation of CCF4 cumarin residue at 409 nm results in a fluorescence resonance energy transfer (FRET) to the fluorescein residue, leading to emission of green fluorescence at 520 nm. Cleavage of CCF4 substrate by β-lactamase interrupts FRET leading to light emission at 447 nm (blue fluorescence). Therefore, injection of YopE-β-lactamase by Ye into DCs can be determined by analyzing the CCF4-green+blue+ DCs by flow cytometry. 24 h p.i. with Ye YopE-β-lactamase mutant strain, 7.1±1.9% CD11chi cells displayed blue fluorescence (Fig. 4A: R1 in dot plots), indicating injection of the YopE-β-lactamase fusion protein into the cells. No β-lactamase+ cells (R1) and only 0.1±0.1% β-lactamase+ cells (R1) could be detected in mice either treated with PBS or infected with the control Ye mutant strain YopE-OVA, respectively. Flow cytometry analysis of the different DC subpopulations for injection of the YopE-β-lactamase revealed that significantly more CD8α+ DCs (11.4±1.2%) were Yop injected compared to CD4+ (6.2±0.4%) and CD4−CD8α− DCs (6.4±0.5%) 24 h p.i. (Fig. 4 A).

Bottom Line: The decreased number of DC subsets, which was dependent on TLR4 and TRIF signaling, was the result of a faster proliferation and suppressed de novo DC generation.The data suggest that these effects might be caused directly by injection of Yops into DCs and indirectly by affecting the homeostasis of CD4(+) and CD8α(+) DCs.These events may contribute to reduced T-cell proliferation and immune evasion of Ye.

View Article: PubMed Central - PubMed

Affiliation: Interfakultäres Institut für Zellbiologie, Universität Tübingen, Tübingen, Germany. Stella.Autenrieth@medizin.uni-tuebingen.de

ABSTRACT
CD4(+) T cells are essential for the control of Yersinia enterocolitica (Ye) infection in mice. Ye can inhibit dendritic cell (DC) antigen uptake and degradation, maturation and subsequently T-cell activation in vitro. Here we investigated the effects of Ye infection on splenic DCs and T-cell proliferation in an experimental mouse infection model. We found that OVA-specific CD4(+) T cells had a reduced potential to proliferate when stimulated with OVA after infection with Ye compared to control mice. Additionally, proliferation of OVA-specific CD4(+) T cells was markedly reduced when cultured with splenic CD8α(+) DCs from Ye infected mice in the presence of OVA. In contrast, T-cell proliferation was not impaired in cultures with CD4(+) or CD4(-)CD8α(-) DCs isolated from Ye infected mice. However, OVA uptake and degradation as well as cytokine production were impaired in CD8α(+) DCs, but not in CD4(+) and CD4(-)CD8α(-) DCs after Ye infection. Pathogenicity factors (Yops) from Ye were most frequently injected into CD8α(+) DCs, resulting in less MHC class II and CD86 expression than on non-injected CD8α(+) DCs. Three days post infection with Ye the number of splenic CD8α(+) and CD4(+) DCs was reduced by 50% and 90%, respectively. The decreased number of DC subsets, which was dependent on TLR4 and TRIF signaling, was the result of a faster proliferation and suppressed de novo DC generation. Together, we show that Ye infection negatively regulates the stimulatory capacity of some but not all splenic DC subpopulations in vivo. This leads to differential antigen uptake and degradation, cytokine production, cell loss, and cell death rates in various DC subpopulations. The data suggest that these effects might be caused directly by injection of Yops into DCs and indirectly by affecting the homeostasis of CD4(+) and CD8α(+) DCs. These events may contribute to reduced T-cell proliferation and immune evasion of Ye.

Show MeSH
Related in: MedlinePlus