B lymphocytes undergo TLR2-dependent apoptosis upon Shigella infection.
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.
Affiliation: Institut Pasteur, INSERM U786, Unité de Pathogénie Microbienne Moléculaire, 75015 Paris, FranceInstitut Pasteur, INSERM U786, Unité de Pathogénie Microbienne Moléculaire, 75015 Paris, France.
- Dysentery, Bacillary/immunology*/metabolism/microbiology
- Shigella flexneri/genetics/immunology*/physiology
- Toll-Like Receptor 2/genetics/immunology*/metabolism
- Antigens, Bacterial/genetics/immunology/metabolism
- Apoptosis Regulatory Proteins/immunology/metabolism
- Bacterial Proteins/genetics/immunology/metabolism
- Cell Line
- Cells, Cultured
- Flow Cytometry
- Host-Pathogen Interactions/immunology
- Mice, Inbred C57BL
- Microscopy, Confocal
- NIH 3T3 Cells
- Protein Binding/immunology
- RNA Interference
- Reverse Transcriptase Polymerase Chain Reaction
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fig7: 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 shares structural and functional similarities with the Yersinia effector LcrV that has been shown to signal in a TLR2-dependent manner (Sing et al., 2002; Espina et al., 2007). Induction of apoptosis by a TLR2-mediated signaling pathway has been reported in different cell types (Aliprantis et al., 1999); however, to our knowledge, no information is available regarding B lymphocytes. Based on the similarity between IpaD and LcrV and the need of bacterial co-signals for the induction of B cell death, we aimed at investigating the involvement of TLRs in the signaling pathways resulting in human B cell death. Therefore, we first assessed basal levels of mRNA expression of different tlr genes by quantitative RT-PCR in the CL-01 B cell line. tlr2 was expressed at low levels as compared with tlr1, 7, and 10 (highly expressed) and tlr3, 6, and 9 (expressed at intermediate levels). Expression of tlr4, 5, and 8 was not detected (Fig. 7 A). Then, we assessed tlr expression upon incubation of cells with T3SA−, T3SA− + IpaD, or WT bacteria for 24 h. Interestingly, only tlr2 showed significant changes in its expression levels upon infection with its mRNA levels significantly up-regulated upon incubation with the T3SA− strain (eightfold change, Fig. 7 B). Whereas IpaD alone had no effect, a combination of IpaD and T3SA− bacteria or infection with WT bacteria led to a significant decrease in tlr2 as compared with T3SA− bacteria alone (Fig. 7 B). Bacterial co-signals thus lead to up-regulation of tlr2.