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B lymphocytes undergo TLR2-dependent apoptosis upon Shigella infection.

Nothelfer K, Arena ET, Pinaud L, Neunlist M, Mozeleski B, Belotserkovsky I, Parsot C, Dinadayala P, Burger-Kentischer A, Raqib R, Sansonetti PJ, Phalipon A - J. Exp. Med. (2014)

Bottom Line: The induction of a type three secretion apparatus (T3SA)-dependent B cell death is observed in the human CL-01 B cell line in vitro, as well as in mouse B lymphocytes in vivo.The presence of bacterial co-signals is required to sensitize B cells to apoptosis and to up-regulate tlr2, thus enhancing IpaD binding.Apoptotic B lymphocytes in contact with Shigella-IpaD are detected in rectal biopsies of infected individuals.

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Affiliation: Institut Pasteur, INSERM U786, Unité de Pathogénie Microbienne Moléculaire, 75015 Paris, FranceInstitut Pasteur, INSERM U786, Unité de Pathogénie Microbienne Moléculaire, 75015 Paris, France.

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IpaD binds to human CL-01 B lymphocytes and is internalized. (A) Confocal images of CL-01 B cells incubated with T3SA− bacteria and IpaD or MxiH coupled to Alexa Fluor 488. An overview image (bar, 50 µm) is presented alongside single confocal slices of B cells (bars, 5 µm) after 2 h of incubation at 37°C. Cells were co-stained for GM1 (red, membrane) and DAPI (blue, nuclei). (B and C) Quantification of the AF488 mean fluorescence intensity (MFI) in a 15-µm radius around the center of the nuclei. (B) MFI of IpaD after co-incubation with T3SA− for 2 h at 37°C. Values are compared with the MxiH control protein and to incubation at 4°C. (C) Time dependence of the MFI measured for IpaD. Cells were incubated with IpaD for different times at 37°C in the presence of T3SA− bacteria. A minimum of 100 cells were analyzed for each condition for B and C. Data are presented as median ± interquartile range for one representative experiment and statistically significant differences were determined by Kruskal-Wallis test with Dunn’s post-test. ***, P < 0.0001. (D) IpaD internalization. Cells were incubated with fluorescent AF488-IpaD for 1 h at 4°C (time 0), and unbound AF488-IpaD was washed before incubation at 37°C for 30 min or 1 h. At each time point, IpaD bound to the cell surface was detected by flow cytometry using an anti-IpaD polyclonal serum, and a secondary antibody coupled to Alexa Fluor 647. Data are presented as mean ± SEM for three independent experiments and statistically significant differences to time 0 were determined by one-way ANOVA with Bonferroni post-test. **, P < 0.01; ***, P < 0.001.
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fig6: IpaD binds to human CL-01 B lymphocytes and is internalized. (A) Confocal images of CL-01 B cells incubated with T3SA− bacteria and IpaD or MxiH coupled to Alexa Fluor 488. An overview image (bar, 50 µm) is presented alongside single confocal slices of B cells (bars, 5 µm) after 2 h of incubation at 37°C. Cells were co-stained for GM1 (red, membrane) and DAPI (blue, nuclei). (B and C) Quantification of the AF488 mean fluorescence intensity (MFI) in a 15-µm radius around the center of the nuclei. (B) MFI of IpaD after co-incubation with T3SA− for 2 h at 37°C. Values are compared with the MxiH control protein and to incubation at 4°C. (C) Time dependence of the MFI measured for IpaD. Cells were incubated with IpaD for different times at 37°C in the presence of T3SA− bacteria. A minimum of 100 cells were analyzed for each condition for B and C. Data are presented as median ± interquartile range for one representative experiment and statistically significant differences were determined by Kruskal-Wallis test with Dunn’s post-test. ***, P < 0.0001. (D) IpaD internalization. Cells were incubated with fluorescent AF488-IpaD for 1 h at 4°C (time 0), and unbound AF488-IpaD was washed before incubation at 37°C for 30 min or 1 h. At each time point, IpaD bound to the cell surface was detected by flow cytometry using an anti-IpaD polyclonal serum, and a secondary antibody coupled to Alexa Fluor 647. Data are presented as mean ± SEM for three independent experiments and statistically significant differences to time 0 were determined by one-way ANOVA with Bonferroni post-test. **, P < 0.01; ***, P < 0.001.

Mentions: As extracellular exposure to IpaD in the presence of bacterial co-signals triggers B cell apoptosis, we assessed IpaD binding to CL-01 B cells. His-tagged IpaD and MxiH were coupled to Alexa Fluor 488 (AF488) and incubated with B cells in the presence of T3SA− bacteria for 2 h at 37°C. Whereas no fluorescence was detected with AF488-MxiH, labeling of B cells was detected upon incubation with AF488-IpaD (Fig. 6 A). As illustrated in Fig. 6 A, the detection of AF488-IpaD in orthogonal slices suggested a specific interaction of IpaD with B cells. To further assess IpaD–CL-01 association, we compared the interaction of AF488-IpaD with these cells in different conditions by measuring relative mean AF488 fluorescence intensities within 15-µm-diameter spheres centered on B cell nuclei. In agreement with results from Fig. 6 A, the relative mean intensity was significantly higher upon incubation at 37°C with IpaD as compared with MxiH (Fig. 6 B). AF488-IpaD relative mean intensity was significantly increased upon incubation at 37°C as compared with 4°C (Fig. 6 B) and with time (Fig. 6 C), suggesting an active internalization process. Indeed, ∼50 and 75% of IpaD bound to B cells at 4°C was internalized after 30 and 60 min incubation at 37°C, respectively (Fig. 6 D). It is noteworthy that the presence of T3SA− bacteria significantly reduced IpaD association after 30 min of incubation with the cells, while significantly increasing it at 24 h after incubation (unpublished data). These findings show that exposure of IpaD to B cells results in its binding and internalization and suggests the existence of a specific interaction partner for IpaD at the B cell surface.


B lymphocytes undergo TLR2-dependent apoptosis upon Shigella infection.

Nothelfer K, Arena ET, Pinaud L, Neunlist M, Mozeleski B, Belotserkovsky I, Parsot C, Dinadayala P, Burger-Kentischer A, Raqib R, Sansonetti PJ, Phalipon A - J. Exp. Med. (2014)

IpaD binds to human CL-01 B lymphocytes and is internalized. (A) Confocal images of CL-01 B cells incubated with T3SA− bacteria and IpaD or MxiH coupled to Alexa Fluor 488. An overview image (bar, 50 µm) is presented alongside single confocal slices of B cells (bars, 5 µm) after 2 h of incubation at 37°C. Cells were co-stained for GM1 (red, membrane) and DAPI (blue, nuclei). (B and C) Quantification of the AF488 mean fluorescence intensity (MFI) in a 15-µm radius around the center of the nuclei. (B) MFI of IpaD after co-incubation with T3SA− for 2 h at 37°C. Values are compared with the MxiH control protein and to incubation at 4°C. (C) Time dependence of the MFI measured for IpaD. Cells were incubated with IpaD for different times at 37°C in the presence of T3SA− bacteria. A minimum of 100 cells were analyzed for each condition for B and C. Data are presented as median ± interquartile range for one representative experiment and statistically significant differences were determined by Kruskal-Wallis test with Dunn’s post-test. ***, P < 0.0001. (D) IpaD internalization. Cells were incubated with fluorescent AF488-IpaD for 1 h at 4°C (time 0), and unbound AF488-IpaD was washed before incubation at 37°C for 30 min or 1 h. At each time point, IpaD bound to the cell surface was detected by flow cytometry using an anti-IpaD polyclonal serum, and a secondary antibody coupled to Alexa Fluor 647. Data are presented as mean ± SEM for three independent experiments and statistically significant differences to time 0 were determined by one-way ANOVA with Bonferroni post-test. **, P < 0.01; ***, P < 0.001.
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fig6: IpaD binds to human CL-01 B lymphocytes and is internalized. (A) Confocal images of CL-01 B cells incubated with T3SA− bacteria and IpaD or MxiH coupled to Alexa Fluor 488. An overview image (bar, 50 µm) is presented alongside single confocal slices of B cells (bars, 5 µm) after 2 h of incubation at 37°C. Cells were co-stained for GM1 (red, membrane) and DAPI (blue, nuclei). (B and C) Quantification of the AF488 mean fluorescence intensity (MFI) in a 15-µm radius around the center of the nuclei. (B) MFI of IpaD after co-incubation with T3SA− for 2 h at 37°C. Values are compared with the MxiH control protein and to incubation at 4°C. (C) Time dependence of the MFI measured for IpaD. Cells were incubated with IpaD for different times at 37°C in the presence of T3SA− bacteria. A minimum of 100 cells were analyzed for each condition for B and C. Data are presented as median ± interquartile range for one representative experiment and statistically significant differences were determined by Kruskal-Wallis test with Dunn’s post-test. ***, P < 0.0001. (D) IpaD internalization. Cells were incubated with fluorescent AF488-IpaD for 1 h at 4°C (time 0), and unbound AF488-IpaD was washed before incubation at 37°C for 30 min or 1 h. At each time point, IpaD bound to the cell surface was detected by flow cytometry using an anti-IpaD polyclonal serum, and a secondary antibody coupled to Alexa Fluor 647. Data are presented as mean ± SEM for three independent experiments and statistically significant differences to time 0 were determined by one-way ANOVA with Bonferroni post-test. **, P < 0.01; ***, P < 0.001.
Mentions: As extracellular exposure to IpaD in the presence of bacterial co-signals triggers B cell apoptosis, we assessed IpaD binding to CL-01 B cells. His-tagged IpaD and MxiH were coupled to Alexa Fluor 488 (AF488) and incubated with B cells in the presence of T3SA− bacteria for 2 h at 37°C. Whereas no fluorescence was detected with AF488-MxiH, labeling of B cells was detected upon incubation with AF488-IpaD (Fig. 6 A). As illustrated in Fig. 6 A, the detection of AF488-IpaD in orthogonal slices suggested a specific interaction of IpaD with B cells. To further assess IpaD–CL-01 association, we compared the interaction of AF488-IpaD with these cells in different conditions by measuring relative mean AF488 fluorescence intensities within 15-µm-diameter spheres centered on B cell nuclei. In agreement with results from Fig. 6 A, the relative mean intensity was significantly higher upon incubation at 37°C with IpaD as compared with MxiH (Fig. 6 B). AF488-IpaD relative mean intensity was significantly increased upon incubation at 37°C as compared with 4°C (Fig. 6 B) and with time (Fig. 6 C), suggesting an active internalization process. Indeed, ∼50 and 75% of IpaD bound to B cells at 4°C was internalized after 30 and 60 min incubation at 37°C, respectively (Fig. 6 D). It is noteworthy that the presence of T3SA− bacteria significantly reduced IpaD association after 30 min of incubation with the cells, while significantly increasing it at 24 h after incubation (unpublished data). These findings show that exposure of IpaD to B cells results in its binding and internalization and suggests the existence of a specific interaction partner for IpaD at the B cell surface.

Bottom Line: The induction of a type three secretion apparatus (T3SA)-dependent B cell death is observed in the human CL-01 B cell line in vitro, as well as in mouse B lymphocytes in vivo.The presence of bacterial co-signals is required to sensitize B cells to apoptosis and to up-regulate tlr2, thus enhancing IpaD binding.Apoptotic B lymphocytes in contact with Shigella-IpaD are detected in rectal biopsies of infected individuals.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institut Pasteur, INSERM U786, Unité de Pathogénie Microbienne Moléculaire, 75015 Paris, FranceInstitut Pasteur, INSERM U786, Unité de Pathogénie Microbienne Moléculaire, 75015 Paris, France.

Show MeSH
Related in: MedlinePlus