Research ArticleHost-Pathogen Interactions

Carbohydrate-dependent B cell activation by fucose-binding bacterial lectins

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Science Signaling  05 Mar 2019:
Vol. 12, Issue 571, eaao7194
DOI: 10.1126/scisignal.aao7194
  • Fig. 1 BCR and Syk are essential for BambL-induced Ca2+ release in primary B cells.

    (A) Flow cytometric analysis of Ca2+ release in splenic (SP) CD93 MFo B cells from wild-type (WT) mice upon stimulation with BambL (10 μg/ml), LecB (10 μg/ml), or anti-kappa F(ab′)2 [10 μg/ml; hereafter, a-kappa F(ab′)2] antibody (Ab) as a control. BambL preincubated with l-fucose served as a control for BambL fucose-binding specificity. Data are representative of four independent experiments. (B) Dose-dependent stimulation of MFo B cells with BambL was monitored by flow cytometric analysis of Ca2+ release (left) and binding of BambL (right) to these cells. For the binding analysis, the relative fluorescence of BambL-Cy5 over time is shown. Data are representative of three independent experiments. (C) Flow cytometric analysis of Ca2+ release in WT (black) and Syk−/− (red, top) or Lyn−/− (red, bottom) MFo B cells, stimulated with BambL (10 μg/ml; left) or anti-IgM F(ab′)2 [10 μg/ml; hereafter, a-IgM F(ab′)2] Ab (right). Data are representative of three independent experiments. (D) Flow cytometric analysis of Ca2+ release in BCR-negative B cells (HC−/−, red) as well as in BCR-sufficient B cells from the same mouse (HC+/+, black). Cells were stimulated with BambL (10 μg/ml; left) or anti-IgM F(ab′)2 (10 μg/ml; right). Data are representative of four independent experiments. The gating strategy for HC−/− cells and the HC+/+ control is depicted in fig. S1C.

  • Fig. 2 IgD-expressing B cell subsets release Ca2+ upon treatment with BambL.

    (A) Flow cytometric analysis of Ca2+ release in WT B cells at different developmental stages: bone marrow (BM)–derived pre–B cells (gray), immature B cells (red), and mature B cells (black). Cells were stimulated with BambL (5 μg/ml; left) or anti-IgM F(ab′)2 (10 μg/ml; right). The BM gating strategy is shown in fig. S2A. The splenic (SP) subsets are displayed in the middle and bottom panels. Middle: CD93+; transitional 1 (T1, red) and T2/3 (black). Bottom, CD93; MFo (red) B cells and MZ (black) B cells. The SP gating strategy is depicted in fig. S2C. The relative cell surface IgD expression of the respective B cell subtypes is shown in fig. S2D. Data are representative of five independent experiments. (B) Flow cytometric analysis of Ca2+ release in IgM−/− BM-derived B cells at different developmental stages. Cells were stimulated with BambL (5 μg/ml; left) or anti-kappa F(ab′)2 (10 μg/ml; right). BM subsets are displayed as pre–B cells (gray), immature B cells (red), and mature B cells (black). Data are representative of three independent experiments. (C) Flow cytometric analysis of Ca2+ release in WT (black) and IgM−/− (red) plasmablasts after 4 days of incubation with LPS. Top: Representative analysis of the cell surface expression of CD19, IgD, and IgM. Cells were selected on the basis of Blimp-GFP and Syndecan-1 (CD138) expression. The cells were stimulated with BambL (5 μg/ml; bottom left) or anti-kappa F(ab′)2 (10 μg/ml; bottom right). Data are representative of three independent experiments.

  • Fig. 3 IgD and CD19 play a major role in BambL-induced signaling.

    (A) Flow cytometric analysis of Ca2+ release in SP-derived MFo B cells (SP CD93) from WT (black) and IgM−/− (red; top, n = 3) and IgD−/− (red; bottom, n = 4) mice. Cells were stimulated with BambL (250 ng/ml; left), anti-IgD F(ab′)2 (10 μg/ml; top right), or anti-IgM F(ab′)2 (10 μg/ml; bottom right). (B) Flow cytometric analysis of BambL-Cy5 binding to the corresponding B cells: WT (left), IgM−/− (middle), and IgD−/− (right). Data are representative of at least three independent experiments. (C) Flow cytometric analysis of Ca2+ release in WT (black), CD19−/− (red; top, n = 3), and CD19−/−IgD−/− (red; bottom, n = 3) B cells. Cells were stimulated with BambL (5 or 10 μg/ml; left) or anti-IgM F(ab′)2 (10 μg/ml; right). (D) Flow cytometric analysis of BambL-Cy5 binding to the corresponding B cells: WT (left), CD19−/− (middle), and CD19−/−IgD−/− (right). Data are representative of at least three independent experiments.

  • Fig. 4 Binding of BambL leads to the internalization of CD19, IgD, and IgM and can be blocked by soluble l-fucose.

    (A) Purified splenic MFo B cells from WT mice were stimulated ex vivo in the presence of BambL (2.5 μg/ml), BambL (2.5 μg/ml) blocked with l-fucose (25 mg/ml), or anti-IgM F(ab′)2 and analyzed by flow cytometry after 16 hours of incubation for the cell surface expression of CD19, IgD, and IgM. Unstimulated cells served as a negative control for receptor expression. The combination of BambL and l-fucose served as a control for the specificity of BambL. Anti-IgM F(ab′)2 served as a positive control for BCR activation. Viable cells were selected using a fixable viability dye. Data are representative of six independent experiments. (B) Statistical analysis of data shown in (A). Mean values of one representative analysis with at least three technical replicates ± SD are shown. For statistical analysis, a Student’s t test was performed. **P < 0.01; ***P < 0.001. MFI, mean fluorescence intensity.

  • Fig. 5 BambL mediates an increase in CD86 expression and TNF-α secretion.

    (A) Purified splenic MFo B cells from WT mice were stimulated ex vivo in the presence of the indicated stimuli (see Materials and Methods for concentrations used). CD86 served as an activation marker, and its cell surface expression was assessed 16 hours after stimulation. BambL (5 μg/ml) was washed out after 15 min of stimulation at 37°C. Unstimulated cells served as a negative control for CD86 expression. The combination of BambL and l-fucose served as a control for the specificity of BambL. Anti-IgM F(ab′)2, LPS, and CpG served as positive controls for BCR and TLR activation. Viable cells were selected using a fixable viability dye. Data are from one experiment and are representative of three independent experiments. (B) Fold changes in the MFI of CD86 from three independent biological replicate experiments. Cells treated with BambL were compared to the untreated control cells. (C) BCR-negative, Igα−/− B cells were treated as described in (A) and examined by flow cytometry to determine the cell surface expression of CD86. The cells were obtained 10 days after tamoxifen treatment. Data are representative of at least three experiments. (D) B cells were stimulated for 16 hours with BambL (2.5 μg/ml), LPS (10 μg/ml), or BambL (2.5 μg/ml) + l-fucose (25 mg/ml). The cell culture media were collected and analyzed by enzyme-linked immunosorbent assay (ELISA) to determine the amount of TNF-α released. Data are means ± SD of three independent experiments and were analyzed by Student’s t test to assess the statistical significance of observed differences. ***P < 0.001; ns, not significant.

  • Fig. 6 BAFF partially reverses B cell death induced by BambL.

    (A) Purified splenic MFo B cells from WT mice were stimulated with the indicated concentrations of BambL. Cell viability was assessed after 24 hours in parallel with that of an unstimulated control by flow cytometric analysis using the viability dye. Data are means ± SD from three independent experiments and were analyzed by Student’s t test to assess statistical significance. ***P < 0.001; ****P < 0.0001. (B) Purified splenic MFo B cells from WT mice were stimulated ex vivo in the presence of BambL (2.5 μg/ml) with or without BAFF costimulation. The cells were analyzed 24 hours later by flow cytometry to generate forward scatter (FSC) and side scatter (SSC) plots and viability dye histograms. Cells negative for the viability dye were considered to be alive, whereas those cells positive for the dye were considered to be dead. Data are representative of four independent experiments. (C) Flow cytometric analysis of the binding of BambL-Cy5 to MFo B cells in the presence and absence of soluble BAFF (100 ng/ml). Data are representative of three independent experiments.

  • Fig. 7 Intraperitoneal injection of mice with BambL leads to splenomegaly and accumulation of B cells in the spleen with simultaneous lymphopenia in the bone marrow.

    (A) Images of spleens for the indicated times and treatment conditions after mice were injected with BambL. The sizes of the spleens were directly compared after extraction. Scale bar is given in centimeters. (B) Statistical analysis of spleen weights from the indicated groups of treated mice. (C and D) Statistical analysis of the numbers of the indicated B cell populations in the indicated groups of treated mice. (E and F) Absolute counts of the indicated B cell populations in the bone marrow of the indicated groups of treated mice. In (B) to (F), each dot indicates an individual mouse: PBS, n = 10; BambL d3, n = 7; BambL + QVD d3, n = 4; BambL d7, n = 4. The data represent a compilation of values from four to seven independent experiments. All P values were obtained using a two-tailed Student’s t test.

  • Fig. 8 Myeloid cell counts are increased in mice after injection with BambL.

    (A to C) Absolute counts of the indicated myeloid cell populations in the spleens of mice from the indicated treatment groups. (D to F) Absolute counts of the indicated myeloid cell populations in the bone marrow of mice from the indicated treatment groups. In (A) to (F), each dot indicates an individual mouse: PBS, n = 10; BambL d3, n = 7; BambL + QVD d3, n = 4; BambL d7, n = 4. The data represent a compilation of values from four to seven independent experiments. All P values were obtained using a two-tailed Student’s t test. Gating strategy was performed as described previously (67, 68).

Supplementary Materials

  • www.sciencesignaling.org/cgi/content/full/12/571/eaao7194/DC1

    Fig. S1. Lectin binding and gating of HC−/− B cells.

    Fig. S2. Gating schemes and IgD surface expression of specific B cell populations.

    Fig. S3. Characterization of CD19-, IgM-, and IgD-deficient B cells.

    Fig. S4. B220 is not internalized upon BambL binding.

    Fig. S5. Internalization of CD86 and intracellular increase in TNF-α in BambL-treated primary B cells.

    Fig. S6. Stimulation of cells with BambL in the presence of cell death inhibitors.

    Fig. S7. BambL binds to soluble Igs.

    Fig. S8. Analysis of lymphoid lineage cells after in vivo injection of BambL.

    Fig. S9. Analysis of myeloid lineage cells after in vivo injection of BambL.

    Fig. S10. Non–B cells in the spleen show an increase in caspase activity upon BambL injection.

    Fig. S11. BambL injection leads to splenomegaly and B cell accumulation in IgαTMF mice.

    Fig. S12. Mice treated with anti–IL-7R antibody exhibit splenomegaly and B cell proliferation in the spleen.

    Fig. S13. B. ambifaria biofilms do not secrete BambL but retain it in the cytosol.

  • This PDF file includes:

    • Fig. S1. Lectin binding and gating of HC−/− B cells.
    • Fig. S2. Gating schemes and IgD surface expression of specific B cell populations.
    • Fig. S3. Characterization of CD19-, IgM-, and IgD-deficient B cells.
    • Fig. S4. B220 is not internalized upon BambL binding.
    • Fig. S5. Internalization of CD86 and intracellular increase in TNF-α in BambL-treated primary B cells.
    • Fig. S6. Stimulation of cells with BambL in the presence of cell death inhibitors.
    • Fig. S7. BambL binds to soluble Igs.
    • Fig. S8. Analysis of lymphoid lineage cells after in vivo injection of BambL.
    • Fig. S9. Analysis of myeloid lineage cells after in vivo injection of BambL.
    • Fig. S10. Non–B cells in the spleen show an increase in caspase activity upon BambL injection.
    • Fig. S11. BambL injection leads to splenomegaly and B cell accumulation in IgαTMF mice.
    • Fig. S12. Mice treated with anti–IL-7R antibody exhibit splenomegaly and B cell proliferation in the spleen.
    • Fig. S13. B. ambifaria biofilms do not secrete BambL but retain it in the cytosol.

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