B lymphocytes undergo TLR2-dependent apoptosis upon Shigella infection.
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.
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
License 1 - License 2
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using the CL-01 B cell line, a germinal center B cell line (Cerutti et al., 1998) which we found to express IgA (unpublished data).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 As proliferation was similarly reduced upon infection with both strains (unpublished data), the T3SA-specific drop in cell number was not due to an impairment of cell proliferation.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.
Affiliation: Institut Pasteur, INSERM U786, Unité de Pathogénie Microbienne Moléculaire, 75015 Paris, FranceInstitut Pasteur, INSERM U786, Unité de Pathogénie Microbienne Moléculaire, 75015 Paris, France.