Figure 7.
Figure 7. IpaD induces B cell apoptosis via interaction with TLR2. (A) Relative TLR mRNA expression levels in the CL-01 B cell line as assessed by quantitative RT-PCR. (B) TLR2 mRNA expression levels after 24 h of incubation with IpaD alone, T3SA−, WT, or T3SA− + IpaD. mRNA levels are presented as fold change over the uninfected control. Asterisks indicate a statistical difference between T3SA− and T3SA− + IpaD/WT determined by one-way ANOVA with Bonferroni post-test. **, P < 0.01. (C) Apoptotic CL-01 B cells after transfection with TLR-targeting siRNA pools. Transfection was performed 24 h before incubation with T3SA− + IpaD and percentages of AnnV+PI− cells are presented 24 h p.i. Asterisks indicate statistical differences to the nontargeting control siRNA pool determined by one-way ANOVA with Bonferroni post-test. *, P < 0.05; ***, P < 0.001. (D) IpaD binding to B cells in the presence of TLR1 and 2 blocking antibodies. IpaD coupled to Alexa Fluor 488 was added for 2 h to cells preincubated with the T3SA− mutant for 22 h. The blocking antibodies were added 1 h before IpaD addition. Asterisks indicate statistical differences to the control without antibody determined by Kruskal-Wallis test with Dunn’s post-test. ***, P < 0.0001. (E) B cell death in the presence of TLR1 and 2 blocking antibodies or an antibody against IpaD. IpaD was added for 6 h to cells preincubated with the T3SA− mutant for 22 h. The TLR blocking antibodies were added to the cells, and the anti-IpaD antibody to the IpaD solution, 1 h before IpaD addition to the cells. Live cell numbers and percentages of AnnV+PI− and PI+ cells are presented as fold changes over the uninfected control. Asterisks indicate statistical differences to the control without antibody determined by two-way ANOVA with Bonferroni post-test. ***, P < 0.001. (F) TLR signaling at 24 h p.i. as assessed by Western blot analysis. Protein amounts of IκBα were assessed as an indicator for NF-κB activation and FADD as an indicator of the TLR2 death pathway. Pictures of the blots and the correspondent fold change over the uninfected control after normalization to actin are shown for both proteins. (G) Induction of B cell death by a TLR2 agonist. The TLR2-6 agonist FSL-1 and the TLR2-1 agonist Pam3CSK4 were added for 6 h to cells preincubated with the T3SA− mutant for 22 h. Live cell numbers and percentages of AnnV+PI− and PI+ cells are presented as fold changes over the uninfected control. Asterisks indicate statistical differences to T3SA− bacteria alone determined by two-way ANOVA with Bonferroni post-test. ***, P < 0.001. Data are presented as mean ± SEM (A–C, E, and G) and as median ± interquartile range in D. Three independent experiments were performed in triplicate for A–C, E, and G, and one representative out of two independent experiments is shown in D and F.

IpaD induces B cell apoptosis via interaction with TLR2. (A) Relative TLR mRNA expression levels in the CL-01 B cell line as assessed by quantitative RT-PCR. (B) TLR2 mRNA expression levels after 24 h of incubation with IpaD alone, T3SA−, WT, or T3SA− + IpaD. mRNA levels are presented as fold change over the uninfected control. Asterisks indicate a statistical difference between T3SA− and T3SA− + IpaD/WT determined by one-way ANOVA with Bonferroni post-test. **, P < 0.01. (C) Apoptotic CL-01 B cells after transfection with TLR-targeting siRNA pools. Transfection was performed 24 h before incubation with T3SA− + IpaD and percentages of AnnV+PI− cells are presented 24 h p.i. Asterisks indicate statistical differences to the nontargeting control siRNA pool determined by one-way ANOVA with Bonferroni post-test. *, P < 0.05; ***, P < 0.001. (D) IpaD binding to B cells in the presence of TLR1 and 2 blocking antibodies. IpaD coupled to Alexa Fluor 488 was added for 2 h to cells preincubated with the T3SA− mutant for 22 h. The blocking antibodies were added 1 h before IpaD addition. Asterisks indicate statistical differences to the control without antibody determined by Kruskal-Wallis test with Dunn’s post-test. ***, P < 0.0001. (E) B cell death in the presence of TLR1 and 2 blocking antibodies or an antibody against IpaD. IpaD was added for 6 h to cells preincubated with the T3SA− mutant for 22 h. The TLR blocking antibodies were added to the cells, and the anti-IpaD antibody to the IpaD solution, 1 h before IpaD addition to the cells. Live cell numbers and percentages of AnnV+PI− and PI+ cells are presented as fold changes over the uninfected control. Asterisks indicate statistical differences to the control without antibody determined by two-way ANOVA with Bonferroni post-test. ***, P < 0.001. (F) TLR signaling at 24 h p.i. as assessed by Western blot analysis. Protein amounts of IκBα were assessed as an indicator for NF-κB activation and FADD as an indicator of the TLR2 death pathway. Pictures of the blots and the correspondent fold change over the uninfected control after normalization to actin are shown for both proteins. (G) Induction of B cell death by a TLR2 agonist. The TLR2-6 agonist FSL-1 and the TLR2-1 agonist Pam3CSK4 were added for 6 h to cells preincubated with the T3SA− mutant for 22 h. Live cell numbers and percentages of AnnV+PI− and PI+ cells are presented as fold changes over the uninfected control. Asterisks indicate statistical differences to T3SA− bacteria alone determined by two-way ANOVA with Bonferroni post-test. ***, P < 0.001. Data are presented as mean ± SEM (A–C, E, and G) and as median ± interquartile range in D. Three independent experiments were performed in triplicate for A–C, E, and G, and one representative out of two independent experiments is shown in D and F.

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