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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 acts as a TLR2 agonist. The reporter system is based on NIH3T3 cells stably transfected with the NF-κB–inducible reporter gene SEAP together with the corresponding combinations of human TLRs. The amount of SEAP upon TLR induction can be compared in cell supernatants by addition of its substrate pNPP and measurement of the product OD at 405 nm. (A) OD at 405 nm 30 min after substrate incubation with supernatants from treated cells. Cells were treated for 18 h with indicated concentrations of IpaD in comparison to negative, positive, and spiked controls. The TLR2-1 and 2–6 reporter cell lines are shown in comparison to the control SEAP cell line without TLRs. (B) Induction of the TLR2-1 reporter cell line. The OD at 405 nm is presented over time for selected concentrations of IpaD in comparison to positive (Pam3CSK4) and negative (NIH3T3 control cell line) controls. (C) Induction of the TLR2-6 reporter cell line. The OD at 405nm is presented over time for selected concentrations of IpaD in comparison to positive (FSL-1) and negative (NIH3T3 control cell line) controls. Two independent experiments were performed in duplicate and all data are presented as mean ± SEM. Statistically significant differences to the control SEAP cell line were determined by two-way ANOVA with Bonferroni post-test. *, P < 0.05; **, P < 0.01; ***, P < 0.001.
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fig8: IpaD acts as a TLR2 agonist. The reporter system is based on NIH3T3 cells stably transfected with the NF-κB–inducible reporter gene SEAP together with the corresponding combinations of human TLRs. The amount of SEAP upon TLR induction can be compared in cell supernatants by addition of its substrate pNPP and measurement of the product OD at 405 nm. (A) OD at 405 nm 30 min after substrate incubation with supernatants from treated cells. Cells were treated for 18 h with indicated concentrations of IpaD in comparison to negative, positive, and spiked controls. The TLR2-1 and 2–6 reporter cell lines are shown in comparison to the control SEAP cell line without TLRs. (B) Induction of the TLR2-1 reporter cell line. The OD at 405 nm is presented over time for selected concentrations of IpaD in comparison to positive (Pam3CSK4) and negative (NIH3T3 control cell line) controls. (C) Induction of the TLR2-6 reporter cell line. The OD at 405nm is presented over time for selected concentrations of IpaD in comparison to positive (FSL-1) and negative (NIH3T3 control cell line) controls. Two independent experiments were performed in duplicate and all data are presented as mean ± SEM. Statistically significant differences to the control SEAP cell line were determined by two-way ANOVA with Bonferroni post-test. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Mentions: To formally demonstrate that IpaD signals via TLR2, we used a cell-based reporter system previously described (Burger-Kentischer et al., 2010). The reporter system is based on NIH3T3 cells stably transfected with the NF-κB–inducible reporter gene SEAP (secreted alkaline phosphatase) together with the corresponding combinations of human TLRs. The amount of SEAP upon TLR induction can be compared in cell supernatants by addition of its substrate pNPP and measurement of the product OD at 405 nm. As shown in Fig. 8 A, a concentration-dependent induction of the TLR2-1 and TLR2-6 reporter cell lines occurred when adding IpaD. At a concentration of 5 µg/ml IpaD, the activation levels of TLR2-1 and TLR2-6 were comparable to 1 µg/ml of the positive controls Pam3CSK4 and FSL-1, respectively (Fig. 8, B and C). These results demonstrate that IpaD binds to and induces activation of the human TLR2 signaling pathway.


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 acts as a TLR2 agonist. The reporter system is based on NIH3T3 cells stably transfected with the NF-κB–inducible reporter gene SEAP together with the corresponding combinations of human TLRs. The amount of SEAP upon TLR induction can be compared in cell supernatants by addition of its substrate pNPP and measurement of the product OD at 405 nm. (A) OD at 405 nm 30 min after substrate incubation with supernatants from treated cells. Cells were treated for 18 h with indicated concentrations of IpaD in comparison to negative, positive, and spiked controls. The TLR2-1 and 2–6 reporter cell lines are shown in comparison to the control SEAP cell line without TLRs. (B) Induction of the TLR2-1 reporter cell line. The OD at 405 nm is presented over time for selected concentrations of IpaD in comparison to positive (Pam3CSK4) and negative (NIH3T3 control cell line) controls. (C) Induction of the TLR2-6 reporter cell line. The OD at 405nm is presented over time for selected concentrations of IpaD in comparison to positive (FSL-1) and negative (NIH3T3 control cell line) controls. Two independent experiments were performed in duplicate and all data are presented as mean ± SEM. Statistically significant differences to the control SEAP cell line were determined by two-way ANOVA with Bonferroni post-test. *, P < 0.05; **, P < 0.01; ***, P < 0.001.
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fig8: IpaD acts as a TLR2 agonist. The reporter system is based on NIH3T3 cells stably transfected with the NF-κB–inducible reporter gene SEAP together with the corresponding combinations of human TLRs. The amount of SEAP upon TLR induction can be compared in cell supernatants by addition of its substrate pNPP and measurement of the product OD at 405 nm. (A) OD at 405 nm 30 min after substrate incubation with supernatants from treated cells. Cells were treated for 18 h with indicated concentrations of IpaD in comparison to negative, positive, and spiked controls. The TLR2-1 and 2–6 reporter cell lines are shown in comparison to the control SEAP cell line without TLRs. (B) Induction of the TLR2-1 reporter cell line. The OD at 405 nm is presented over time for selected concentrations of IpaD in comparison to positive (Pam3CSK4) and negative (NIH3T3 control cell line) controls. (C) Induction of the TLR2-6 reporter cell line. The OD at 405nm is presented over time for selected concentrations of IpaD in comparison to positive (FSL-1) and negative (NIH3T3 control cell line) controls. Two independent experiments were performed in duplicate and all data are presented as mean ± SEM. Statistically significant differences to the control SEAP cell line were determined by two-way ANOVA with Bonferroni post-test. *, P < 0.05; **, P < 0.01; ***, P < 0.001.
Mentions: To formally demonstrate that IpaD signals via TLR2, we used a cell-based reporter system previously described (Burger-Kentischer et al., 2010). The reporter system is based on NIH3T3 cells stably transfected with the NF-κB–inducible reporter gene SEAP (secreted alkaline phosphatase) together with the corresponding combinations of human TLRs. The amount of SEAP upon TLR induction can be compared in cell supernatants by addition of its substrate pNPP and measurement of the product OD at 405 nm. As shown in Fig. 8 A, a concentration-dependent induction of the TLR2-1 and TLR2-6 reporter cell lines occurred when adding IpaD. At a concentration of 5 µg/ml IpaD, the activation levels of TLR2-1 and TLR2-6 were comparable to 1 µg/ml of the positive controls Pam3CSK4 and FSL-1, respectively (Fig. 8, B and C). These results demonstrate that IpaD binds to and induces activation of the human TLR2 signaling pathway.

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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Related in: MedlinePlus