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BAFF inhibition attenuates fibrosis in scleroderma by modulating the regulatory and effector B cell balance

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Science Advances  11 Jul 2018:
Vol. 4, no. 7, eaas9944
DOI: 10.1126/sciadv.aas9944
  • Fig. 1 CD40 and LPS synergistically induce IL-6 production from B cells.

    (A) B cells were isolated from spleens of naïve mice by magnetic sorting based on CD19 expression. Sorted B cells were cultured for 72 hours with media alone or media containing anti-CD40 mAb, along with the indicated TLR agonists. After in vitro stimulation for 72 hours, IL-6 (left) and IL-10 (right) levels in supernatants were quantified by enzyme-linked immunosorbent assay (ELISA). Bars represent the means ± SD from three independent experiments (n = 3 mice). Significant differences between means of media alone and individual stimuli are indicated: *P < 0.001, **P < 0.0001, analysis of variance (ANOVA) followed by Tukey’s multiple comparison test. Significant differences between cultures with or without anti-CD40 mAb are indicated: #P < 0.05, ##P < 0.01, ###P < 0.001, ####P < 0.0001, Student’s t test. (B) IL-6–producing B cells were determined after in vitro stimulation by LPS, anti-CD40 mAb, and LPS + anti-CD40 mAb, with PIB [phorbol 12-myristate 13-acetate (PMA), ionomycin, and brefeldin A] added during the final 5 hours of cultures (5 to 48 hours). Isotype control Ab was used as negative controls for IL-6 staining. Percentages indicate the frequencies of cytoplasmic IL-6+ B cells within the indicated gates among total CD19+ B cells. Bars represent the means ± SD from three independent experiments (n = 3 mice). *P < 0.0001, ANOVA followed by Tukey’s multiple comparison test. (C) Representative cell surface phenotype of spleen IL-6–producing B cells after stimulation with LPS + anti-CD40 mAb for 24 hours with PIB added during the final 5 hours of culture. Cultured cells were stained for viability and cell surface molecule expression (using LEGENDScreen Mouse PE Kit from BioLegend), permeabilized, stained with anti–IL-6 mAb, and analyzed by flow cytometry. Representative cell surface molecule expression by IL-6+ (red line) and IL-6 (black line) CD19+ B cells from three individuals is shown. Shaded histograms represent isotype-matched control mAb staining.

  • Fig. 2 The MZ B cell subset is a major source of IL-6–producing B cells.

    (A) Splenic B cells from wild-type mice were isolated by Miltenyi MACS enrichment; stained for CD1d, CD5, and CD19 expression; and sorted into follicular B cell (CD1dintCD5), MZ B cell (CD1dhiCD5), and B1 B cell (CD1dintCD5+) populations before stimulation. (B) Sorted B cells were cultured with LPS + anti-CD40 mAb for 24 hours with PIB added during the final 5 hours of culture (for IL-6) or LPS + PIB for 5 hours (for IL-10). IL-6+ or IL-10+ B cells derived from each purified population were then analyzed by flow cytometry. All data are representative of two independent experiments. (C) Bars represent the means ± SD from four mice in each group. Significant differences between pan-B cell versus other B cell subsets are indicated: *P < 0.05, **P < 0.01, ***P < 0.001, #P < 0.05, ##P < 0.01, ###P < 0.001, ANOVA followed by Tukey’s multiple comparison test.

  • Fig. 3 IL-6 is increased in bleomycin-induced scleroderma.

    (A) Serum samples were collected from bleomycin-induced scleroderma mice. Serum IL-6 or IL-10 levels were determined by ELISA. Bars represent the means ± SD from four mice in each group. Significant differences between means of naïve mice and bleomycin (Bleo)–treated mice are indicated: *P < 0.05, **P < 0.001, ANOVA followed by Tukey’s multiple comparison test. (B) Splenocytes were isolated from bleomycin-induced scleroderma mice on day 21 after treatment. Splenocytes were cultured with LPS + anti-CD40 mAb for 24 hours with PIB added during the final 5 hours of culture (for IL-6) or LPS + PIB for 5 hours (for IL-10). Left: Percentages indicate the frequencies of cytoplasmic IL-6+ or IL-10+ B cells within the indicated gates among total CD19+ B cells. Right: Bars represent the means ± SD from four mice in each group. *P < 0.001, Student’s t test. (C) Skin-infiltrating cells were isolated from bleomycin-induced scleroderma mice on day 21 after treatment. Lymphocytes were cultured with LPS + anti-CD40 mAb for 24 hours with PIB added during the final 5 hours of culture (for IL-6) or LPS + PIB for 5 hours (for IL-10). Left: Percentages indicate the frequencies of cytoplasmic IL-6+ or IL-10+ B cells within the indicated gates among total CD19+ B cells. Right: Bars represent the means ± SD from four mice in each group. *P < 0.001, Student’s t test. (D and E) Inflamed skin sample was collected from mice treated with bleomycin. Immunofluorescence histology of frozen skin sections detecting IL-6 production by B220+ skin B cells. DAPI (4′,6-diamidino-2-phenylindole) was used to visualize nuclei. Scale bars, 25 μm (D) and 5 μm (E). All data are representative of two independent experiments.

  • Fig. 4 Skin fibrosis is attenuated in mice with B cell–specific IL-6 deficiency.

    (A to D) Bleomycin-induced scleroderma was induced in mice with B cell–specific IL-6 deficiency (B-IL-6−/−; wild-type mice lethally irradiated and reconstituted with 80% μMT plus 20% Il6−/− bone marrow) or B cell–specific IL-10 deficiency (B-IL-10−/−; wild-type mice lethally irradiated and reconstituted with 80% μMT plus 20% Il10−/− bone marrow) and control chimera groups [wild-type mice lethally irradiated and reconstituted with 80% wild-type plus 20% Il6−/− or Il10−/−bone marrow (IL-620% or IL-1020%, respectively)]. (A and C) Skin samples were harvested 4 weeks after PBS or bleomycin treatment. Masson’s trichrome stain. Representative images. Arrows indicate dermis. Scale bars, 100 μm. Right: Dermal thickness (distance from the dermal-epidermal junction to the adipose layer), shown as the means ± SD of triplicate determinations per hpf from 10 mice per group. (B and D) Expression of col1a2 mRNA in the skin was measured by real-time polymerase chain reaction (PCR), shown as the means ± SD of triplicate determinations per hpf (high power field) from 10 mice per group. Open circles, PBS; closed circles, bleomycin. *P < 0.05, ***P < 0.001, ****P < 0.0001, one-way ANOVA followed by Tukey’s multiple comparison test. (E and F) B cells and fibroblasts were cocultured. Collagen release by fibroblasts was determined by the Sirius red assay in 72-hour culture supernatants of fibroblast cultured alone, cocultured with B cells, or recombinant TGF-β1 (5 ng/ml). IL-6 was determined by ELISA in 72-hour culture supernatants. B cells from wild-type, Il6−/−, or Il10−/− mice were isolated by Miltenyi MACS enrichment. B cells were either in cell-cell contact with fibroblast (contact) or seeded in the upper chamber of a Transwell culture insert (Transwell). Bars represent the means ± SD from two independent experiments (n = 4 mice). Significant differences between fibroblast only versus other groups are indicated: *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, #P < 0.05, ##P < 0.01, ###P < 0.001, ####P < 0.0001, ANOVA followed by Tukey’s multiple comparison test.

  • Fig. 5 BAFF enhances IL-6 production from B cells, while BAFF attenuates IL-10 production from B cells.

    (A) B cells were isolated from spleens of naïve mice by magnetic sorting based on CD19 expression. Sorted B cells were cultured for 72 hours with or without BAFF, along with LPS, anti-CD40 mAb, or LPS + anti-CD40 mAb. After in vitro stimulation for 72 hours, IL-6 (left) and IL-10 (right) levels in supernatants were quantified by ELISA. Bars represent the means ± SD from three independent experiments (n = 3 mice). Significant differences between means of media alone and individual stimuli are indicated: *P < 0.001, **P < 0.0001, ANOVA followed by Tukey’s multiple comparison test. Significant differences between cultures with or without BAFF are indicated: #P < 0.01, ##P < 0.0001, Student’s t test. (B) Wild-type mice were treated with BAFFR-Fc or Fc control protein (control). Spleens were collected 1 week after treatment. IL-6– or IL-10–producing B cells were determined after in vitro stimulation. CD1dhiMZ B cells and CD5+ B1 B cells from spleens of mice treated with BAFFR-Fc or Fc control protein were examined by flow cytometry. Percentages indicate the frequencies of various B cell subsets within the indicated gates among total CD19+ B cells. Bars represent the means ± SD from four mice. NS, not significant. *P < 0.001, **P < 0.0001, Student’s t test. All data are representative of two independent experiments.

  • Fig. 6 BAFF inhibition attenuates skin fibrosis in bleomycin-induced scleroderma.

    (A) Serum samples were collected from bleomycin-induced scleroderma mice. Serum BAFF levels were determined by ELISA. Bars represent the means ± SD from five mice in each group. Significant differences between means of naïve mice and bleomycin (Bleo)–treated mice are indicated: *P < 0.001, ANOVA followed by Tukey’s multiple comparison test. (B) Bleomycin-induced scleroderma was induced in mice treated with BAFFR-Fc or Fc control protein (control). Skin samples were harvested 4 weeks after bleomycin treatment. Left: Masson’s trichrome stain. Representative images. Arrows indicate dermis (distance from the dermal-epidermal junction to the adipose layer). Scale bar, 100 μm. (B and C) Analysis of dermal thickness (B, right) and expression of col1a2 mRNA in the skin (C). (D) Lung samples were harvested 4 weeks after bleomycin treatment. Left: Hematoxylin and eosin (H&E). Representative images. Scale bar, 100 μm. (D and E) Analysis of the lungs for determination of lung fibrosis scores (D, right) and collagen content (E). (B to D) Values are means ± SD of five mice per group. Open circles, PBS; closed circles, bleomycin. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, one-way ANOVA followed by Tukey’s multiple comparison test. (F) Spleens from bleomycin-induced scleroderma mice were collected 4 weeks after treatment. IL-6– or IL-10–producing B cells were determined after in vitro stimulation. Percentages indicate the frequencies of IL-6– or IL-10–producing B cells within the indicated gates among total CD19+ B cells. Bars represent the means ± SD from five mice. *P < 0.0001, Student’s t test. All data are representative of two independent experiments. (G) BAFF increased the IL-6–producing Beffs but suppressed the IL-10–producing Bregs. Furthermore, Beffs play a pathogenic role in scleroderma, while Bregs play a protective role.

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/4/7/eaas9944/DC1

    Fig. S1. Phenotypes of IL-6–producing B cells after in vitro culture.

    Fig. S2. CD25 expression on B cell is enhanced after stimulation.

    Fig. S3. IL-6– and/or IL-10–producing B cells.

    Fig. S4. Phenotype of IL-6–producing B cells in the skin.

    Fig. S5. Scheme for the generation of B-IL-6−/− or B-IL-10−/− and corresponding control mice.

    Fig. S6. Lung fibrosis is attenuated in mice with B cell–specific IL-6 deficiency.

  • Supplementary Materials

    This PDF file includes:

    • Fig. S1. Phenotypes of IL-6–producing B cells after in vitro culture.
    • Fig. S2. CD25 expression on B cell is enhanced after stimulation.
    • Fig. S3. IL-6– and/or IL-10–producing B cells.
    • Fig. S4. Phenotype of IL-6–producing B cells in the skin.
    • Fig. S5. Scheme for the generation of B-IL-6−/− or B-IL-10−/− and corresponding control mice.
    • Fig. S6. Lung fibrosis is attenuated in mice with B cell–specific IL-6 deficiency.

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