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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.

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.

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S. flexneri induces B cell death dependent on the T3SA in vitro. The human IgA+ CL-01 B cell line was infected for 30 min with WT or T3SA− bacteria before addition of gentamicin. (A) Count of in vitro–infected human CL-01 B cells over time. Asterisks indicate statistical difference to the uninfected control. (B) Percentages of in vitro–infected PI+ human CL-01 B cells over time. Asterisks indicate statistical difference to the uninfected control. (C) Fold changes of live cell number and percentage of PI+ cells are presented for WT infection over infection with the T3SA− mutant as normalized values. Asterisks indicate statistical difference to the T3SA− strain. (D) Flow cytometry analysis of cells infected with GFP-expressing bacteria. GFPhigh PI− B cells were detected 5 h p.i. with WT, but not T3SA− bacteria (P < 0.001), representing 8.47 ± 1.1% (mean ± SEM) invaded cells. At 24 h p.i., GFPhigh cells are PI+. (E) Invasion assay for CL-01 B cells. The number of CFUs per 3 × 105 infected cells is presented for WT and T3SA− bacteria at 2, 4, 6, and 24 h p.i. Three independent experiments were performed in triplicate for A–E and data are presented as mean ± SEM. Statistically significant differences were determined by two-way ANOVA with Bonferroni post-test. *, P < 0.05; **, P < 0.01; ***, P < 0.001.
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fig2: S. flexneri induces B cell death dependent on the T3SA in vitro. The human IgA+ CL-01 B cell line was infected for 30 min with WT or T3SA− bacteria before addition of gentamicin. (A) Count of in vitro–infected human CL-01 B cells over time. Asterisks indicate statistical difference to the uninfected control. (B) Percentages of in vitro–infected PI+ human CL-01 B cells over time. Asterisks indicate statistical difference to the uninfected control. (C) Fold changes of live cell number and percentage of PI+ cells are presented for WT infection over infection with the T3SA− mutant as normalized values. Asterisks indicate statistical difference to the T3SA− strain. (D) Flow cytometry analysis of cells infected with GFP-expressing bacteria. GFPhigh PI− B cells were detected 5 h p.i. with WT, but not T3SA− bacteria (P < 0.001), representing 8.47 ± 1.1% (mean ± SEM) invaded cells. At 24 h p.i., GFPhigh cells are PI+. (E) Invasion assay for CL-01 B cells. The number of CFUs per 3 × 105 infected cells is presented for WT and T3SA− bacteria at 2, 4, 6, and 24 h p.i. Three independent experiments were performed in triplicate for A–E and data are presented as mean ± SEM. Statistically significant differences were determined by two-way ANOVA with Bonferroni post-test. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Mentions: To characterize Shigella–B lymphocyte interactions further, we analyzed the outcomes of infection using the CL-01 B cell line, a germinal center B cell line (Cerutti et al., 1998) which we found to express IgA (unpublished data). Cells were infected in vitro at a multiplicity of infection (MOI) of 50 for 30 min before addition of gentamicin to kill all extracellular bacteria. As shown in Fig. 2 A, after infection with WT and T3SA− bacteria, a significant reduction in cell number was observed at 24 h post infection (p.i.) with WT bacteria only and at 48 h p.i. with both strains, although greater with WT bacteria. As proliferation was similarly reduced upon infection with both strains (unpublished data), the T3SA-specific drop in cell number was not due to an impairment of cell proliferation. Instead, we observed a T3SA-specific induction of cell death, as the percentages of PI+ (propidium iodide–positive) cells were significantly increased upon infection with the WT strain as compared with the T3SA− strain at 24 and 48 h p.i. (Fig. 2 B). The induction of this T3SA-dependent cell death by WT bacteria is summarized in Fig. 2 C by presenting the reduction of cell number (WT = 0.6× T3SA−) and the induction of PI+ cells (WT = 2× T3SA−) as values normalized to the T3SA− mutant. To assess if cell death was related to the ability of WT bacteria to invade B cells, CL-01 cells were infected as described above with GFP-expressing T3SA− and WT bacteria. Invasion was monitored by flow cytometry, focusing on GFPhigh cells, which have previously been reported to represent cells containing multiple intracellular bacteria (Castro-Eguiluz et al., 2009; Konradt et al., 2011). No GFPhigh CL-01 B cells were observed upon incubation with T3SA− bacteria, whereas ∼8.5% of the B cell population was found to be GFPhigh upon infection with WT bacteria at 5 h p.i. (Fig. 2 D), confirming that invasion was dependent on T3SA activity. At 24 h p.i, the GFPhigh cells were found to be PI+ (Fig. 2 D), indicating that Shigella invasion led to B cell death. Consistently, no intracellular bacteria were detected at 24 h p.i. in the gentamicin invasion assay, whereas the number of intracellular WT bacteria increased at earlier time points, revealing bacterial proliferation inside B cells (Fig. 2 E). Despite only ∼8.5% of the CL-01 cell population being invaded (Fig. 2 D), cell death was induced in ∼15 and 20% of the B cells at 24 and 48 h p.i., respectively (Fig. 2 B). This was neither due to the presence of live, extracellular bacteria, which were killed upon gentamicin treatment, nor to cell-to-cell dissemination given the similar rate of cell death detected upon infection with a nonspreading icsA mutant (unpublished data). Altogether, these results indicate that S. flexneri triggers T3SA-dependent B lymphocyte death in vitro in both invaded and noninvaded human B cells.


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)

S. flexneri induces B cell death dependent on the T3SA in vitro. The human IgA+ CL-01 B cell line was infected for 30 min with WT or T3SA− bacteria before addition of gentamicin. (A) Count of in vitro–infected human CL-01 B cells over time. Asterisks indicate statistical difference to the uninfected control. (B) Percentages of in vitro–infected PI+ human CL-01 B cells over time. Asterisks indicate statistical difference to the uninfected control. (C) Fold changes of live cell number and percentage of PI+ cells are presented for WT infection over infection with the T3SA− mutant as normalized values. Asterisks indicate statistical difference to the T3SA− strain. (D) Flow cytometry analysis of cells infected with GFP-expressing bacteria. GFPhigh PI− B cells were detected 5 h p.i. with WT, but not T3SA− bacteria (P < 0.001), representing 8.47 ± 1.1% (mean ± SEM) invaded cells. At 24 h p.i., GFPhigh cells are PI+. (E) Invasion assay for CL-01 B cells. The number of CFUs per 3 × 105 infected cells is presented for WT and T3SA− bacteria at 2, 4, 6, and 24 h p.i. Three independent experiments were performed in triplicate for A–E and data are presented as mean ± SEM. Statistically significant differences were determined by two-way ANOVA with Bonferroni post-test. *, P < 0.05; **, P < 0.01; ***, P < 0.001.
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fig2: S. flexneri induces B cell death dependent on the T3SA in vitro. The human IgA+ CL-01 B cell line was infected for 30 min with WT or T3SA− bacteria before addition of gentamicin. (A) Count of in vitro–infected human CL-01 B cells over time. Asterisks indicate statistical difference to the uninfected control. (B) Percentages of in vitro–infected PI+ human CL-01 B cells over time. Asterisks indicate statistical difference to the uninfected control. (C) Fold changes of live cell number and percentage of PI+ cells are presented for WT infection over infection with the T3SA− mutant as normalized values. Asterisks indicate statistical difference to the T3SA− strain. (D) Flow cytometry analysis of cells infected with GFP-expressing bacteria. GFPhigh PI− B cells were detected 5 h p.i. with WT, but not T3SA− bacteria (P < 0.001), representing 8.47 ± 1.1% (mean ± SEM) invaded cells. At 24 h p.i., GFPhigh cells are PI+. (E) Invasion assay for CL-01 B cells. The number of CFUs per 3 × 105 infected cells is presented for WT and T3SA− bacteria at 2, 4, 6, and 24 h p.i. Three independent experiments were performed in triplicate for A–E and data are presented as mean ± SEM. Statistically significant differences were determined by two-way ANOVA with Bonferroni post-test. *, P < 0.05; **, P < 0.01; ***, P < 0.001.
Mentions: To characterize Shigella–B lymphocyte interactions further, we analyzed the outcomes of infection using the CL-01 B cell line, a germinal center B cell line (Cerutti et al., 1998) which we found to express IgA (unpublished data). Cells were infected in vitro at a multiplicity of infection (MOI) of 50 for 30 min before addition of gentamicin to kill all extracellular bacteria. As shown in Fig. 2 A, after infection with WT and T3SA− bacteria, a significant reduction in cell number was observed at 24 h post infection (p.i.) with WT bacteria only and at 48 h p.i. with both strains, although greater with WT bacteria. As proliferation was similarly reduced upon infection with both strains (unpublished data), the T3SA-specific drop in cell number was not due to an impairment of cell proliferation. Instead, we observed a T3SA-specific induction of cell death, as the percentages of PI+ (propidium iodide–positive) cells were significantly increased upon infection with the WT strain as compared with the T3SA− strain at 24 and 48 h p.i. (Fig. 2 B). The induction of this T3SA-dependent cell death by WT bacteria is summarized in Fig. 2 C by presenting the reduction of cell number (WT = 0.6× T3SA−) and the induction of PI+ cells (WT = 2× T3SA−) as values normalized to the T3SA− mutant. To assess if cell death was related to the ability of WT bacteria to invade B cells, CL-01 cells were infected as described above with GFP-expressing T3SA− and WT bacteria. Invasion was monitored by flow cytometry, focusing on GFPhigh cells, which have previously been reported to represent cells containing multiple intracellular bacteria (Castro-Eguiluz et al., 2009; Konradt et al., 2011). No GFPhigh CL-01 B cells were observed upon incubation with T3SA− bacteria, whereas ∼8.5% of the B cell population was found to be GFPhigh upon infection with WT bacteria at 5 h p.i. (Fig. 2 D), confirming that invasion was dependent on T3SA activity. At 24 h p.i, the GFPhigh cells were found to be PI+ (Fig. 2 D), indicating that Shigella invasion led to B cell death. Consistently, no intracellular bacteria were detected at 24 h p.i. in the gentamicin invasion assay, whereas the number of intracellular WT bacteria increased at earlier time points, revealing bacterial proliferation inside B cells (Fig. 2 E). Despite only ∼8.5% of the CL-01 cell population being invaded (Fig. 2 D), cell death was induced in ∼15 and 20% of the B cells at 24 and 48 h p.i., respectively (Fig. 2 B). This was neither due to the presence of live, extracellular bacteria, which were killed upon gentamicin treatment, nor to cell-to-cell dissemination given the similar rate of cell death detected upon infection with a nonspreading icsA mutant (unpublished data). Altogether, these results indicate that S. flexneri triggers T3SA-dependent B lymphocyte death in vitro in both invaded and noninvaded human B cells.

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