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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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Bacterial co-signals sensitize human B lymphocytes to IpaD-mediated mitochondrial apoptosis. (A) Apoptotic CL-01 B cells after incubation for 24 h with live, gentamicin-treated, sonicated, or heat-killed ipaD bacteria or a cocktail of LPS, PGN, and CpG (C). Percentages of AnnV+PI− cells are shown, in the presence (+IpaD) or absence of IpaD. Asterisks indicate statistical differences to the uninfected control (on column) or between conditions (indicated by bars) determined by one-way ANOVA with Bonferroni post-test. *, P < 0.05; **, P < 0.01. (B) Loss of MMP as assessed by flow cytometry with the fluorescent probe JC-1. Fold changes in the percentage of cells that lost MMP are presented for T3SA− and T3SA− + IpaD over the uninfected control. Asterisks indicate statistical differences to the uninfected control determined by two-way ANOVA with Bonferroni post-test. **, P < 0.01; ***, P < 0.001. (C–F) Dynamic regulation of mitochondrial pro- and anti-apoptotic proteins over time. Protein amounts were assessed by Western blot at 1, 3, 5, and 24 h p.i., and are presented as fold change over the uninfected control for IpaD alone, T3SA−, and T3SA− + IpaD after normalization to actin. (C and D) Protein amounts of pro-apoptotic Bad (C) and Bax (D). (E and F) Protein amounts of anti-apoptotic Bcl-2 (E) and Bcl-xL (F). All data are presented as mean ± SEM for three independent experiments.
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fig5: Bacterial co-signals sensitize human B lymphocytes to IpaD-mediated mitochondrial apoptosis. (A) Apoptotic CL-01 B cells after incubation for 24 h with live, gentamicin-treated, sonicated, or heat-killed ipaD bacteria or a cocktail of LPS, PGN, and CpG (C). Percentages of AnnV+PI− cells are shown, in the presence (+IpaD) or absence of IpaD. Asterisks indicate statistical differences to the uninfected control (on column) or between conditions (indicated by bars) determined by one-way ANOVA with Bonferroni post-test. *, P < 0.05; **, P < 0.01. (B) Loss of MMP as assessed by flow cytometry with the fluorescent probe JC-1. Fold changes in the percentage of cells that lost MMP are presented for T3SA− and T3SA− + IpaD over the uninfected control. Asterisks indicate statistical differences to the uninfected control determined by two-way ANOVA with Bonferroni post-test. **, P < 0.01; ***, P < 0.001. (C–F) Dynamic regulation of mitochondrial pro- and anti-apoptotic proteins over time. Protein amounts were assessed by Western blot at 1, 3, 5, and 24 h p.i., and are presented as fold change over the uninfected control for IpaD alone, T3SA−, and T3SA− + IpaD after normalization to actin. (C and D) Protein amounts of pro-apoptotic Bad (C) and Bax (D). (E and F) Protein amounts of anti-apoptotic Bcl-2 (E) and Bcl-xL (F). All data are presented as mean ± SEM for three independent experiments.

Mentions: In an attempt to identify the microbial products acting as co-signals, we generated bacterial lysates of the ipaD strain by killing bacteria by gentamicin treatment, sonication, or heat before their addition to B cells. Additionally, we tested a panel of pathogen-associated molecular patterns (PAMPs). As shown in Fig. 5 A, LPS, PGN, CpG, or combinations of these products did not provide the required co-signals to induce apoptotic AnnV+PI− cells at 24 h after incubation in the presence of IpaD. Surprisingly, whereas live or gentamicin-treated ipaD lysates provided the co-signals, ipaD lysates generated by heat killing or sonication did not. Altogether, these results indicate that S.flexneri induces B lymphocyte apoptosis through the combined action of IpaD and bacterial co-signals and does so in the absence of invasion or translocation of effectors.


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)

Bacterial co-signals sensitize human B lymphocytes to IpaD-mediated mitochondrial apoptosis. (A) Apoptotic CL-01 B cells after incubation for 24 h with live, gentamicin-treated, sonicated, or heat-killed ipaD bacteria or a cocktail of LPS, PGN, and CpG (C). Percentages of AnnV+PI− cells are shown, in the presence (+IpaD) or absence of IpaD. Asterisks indicate statistical differences to the uninfected control (on column) or between conditions (indicated by bars) determined by one-way ANOVA with Bonferroni post-test. *, P < 0.05; **, P < 0.01. (B) Loss of MMP as assessed by flow cytometry with the fluorescent probe JC-1. Fold changes in the percentage of cells that lost MMP are presented for T3SA− and T3SA− + IpaD over the uninfected control. Asterisks indicate statistical differences to the uninfected control determined by two-way ANOVA with Bonferroni post-test. **, P < 0.01; ***, P < 0.001. (C–F) Dynamic regulation of mitochondrial pro- and anti-apoptotic proteins over time. Protein amounts were assessed by Western blot at 1, 3, 5, and 24 h p.i., and are presented as fold change over the uninfected control for IpaD alone, T3SA−, and T3SA− + IpaD after normalization to actin. (C and D) Protein amounts of pro-apoptotic Bad (C) and Bax (D). (E and F) Protein amounts of anti-apoptotic Bcl-2 (E) and Bcl-xL (F). All data are presented as mean ± SEM for three independent experiments.
© Copyright Policy - openaccess
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4042640&req=5

fig5: Bacterial co-signals sensitize human B lymphocytes to IpaD-mediated mitochondrial apoptosis. (A) Apoptotic CL-01 B cells after incubation for 24 h with live, gentamicin-treated, sonicated, or heat-killed ipaD bacteria or a cocktail of LPS, PGN, and CpG (C). Percentages of AnnV+PI− cells are shown, in the presence (+IpaD) or absence of IpaD. Asterisks indicate statistical differences to the uninfected control (on column) or between conditions (indicated by bars) determined by one-way ANOVA with Bonferroni post-test. *, P < 0.05; **, P < 0.01. (B) Loss of MMP as assessed by flow cytometry with the fluorescent probe JC-1. Fold changes in the percentage of cells that lost MMP are presented for T3SA− and T3SA− + IpaD over the uninfected control. Asterisks indicate statistical differences to the uninfected control determined by two-way ANOVA with Bonferroni post-test. **, P < 0.01; ***, P < 0.001. (C–F) Dynamic regulation of mitochondrial pro- and anti-apoptotic proteins over time. Protein amounts were assessed by Western blot at 1, 3, 5, and 24 h p.i., and are presented as fold change over the uninfected control for IpaD alone, T3SA−, and T3SA− + IpaD after normalization to actin. (C and D) Protein amounts of pro-apoptotic Bad (C) and Bax (D). (E and F) Protein amounts of anti-apoptotic Bcl-2 (E) and Bcl-xL (F). All data are presented as mean ± SEM for three independent experiments.
Mentions: In an attempt to identify the microbial products acting as co-signals, we generated bacterial lysates of the ipaD strain by killing bacteria by gentamicin treatment, sonication, or heat before their addition to B cells. Additionally, we tested a panel of pathogen-associated molecular patterns (PAMPs). As shown in Fig. 5 A, LPS, PGN, CpG, or combinations of these products did not provide the required co-signals to induce apoptotic AnnV+PI− cells at 24 h after incubation in the presence of IpaD. Surprisingly, whereas live or gentamicin-treated ipaD lysates provided the co-signals, ipaD lysates generated by heat killing or sonication did not. Altogether, these results indicate that S.flexneri induces B lymphocyte apoptosis through the combined action of IpaD and bacterial co-signals and does so in the absence of invasion or translocation of effectors.

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