Research ArticleIMMUNOLOGY

The regulatory B cell–mediated peripheral tolerance maintained by mast cell IL-5 suppresses oxazolone-induced contact hypersensitivity

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Science Advances  17 Jul 2019:
Vol. 5, no. 7, eaav8152
DOI: 10.1126/sciadv.aav8152
  • Fig. 1 Population alterations of MCs, ILC2s, and IL-10+ Breg cells in CHS mice.

    (A) Data for the ear thickness of CHS mice for 4 days after challenge with OXZ are shown. (B) Two days after OXZ challenge, representative histology images of ear tissues [hematoxylin and eosin (H&E); scale bars, 200 μm] are shown. (C) Two days after OXZ challenge, representative MC images in spleen, aLN, iLN (scale bars, 50 μm), and ear tissues [high-power field (HPF); scale bar, 100 μm] with toluidine blue are shown, and (D) the histograms show the number of MCs in spleen, aLN, iLN, and ear tissues. (A to D) The results are expressed as the mean ± SEM (A and D) or representative images (B and C) from three independent experiments (n = 5 per group for each experiment). **P < 0.01; n.s., not significant versus ACE-treated mice by Student’s t test. (E) Representative plot images show IL-13+LinCD45+CD127+Sca-1+ cells (IL-13+ ILC2s) in spleen, aLN, iLN, and ear. (F) The histograms show the frequencies and numbers of IL-13+ ILC2s for (E). (G) Representative flow cytometry images are shown for IL-10+CD19+ B cells or (I) CD1dhiCD5+CD19+ B cells in the spleen, aLN, and iLN or CD5+CD11b+CD19+ B cells in the PeC. (H) Frequencies and numbers are shown for (G) and (J) for (I). (E to J) The results are expressed as representative images (E, G, and I) or the mean ± SEM (F, H, and J) from two independent experiments (n = 5 per group for each experiment). *P < 0.05; **P < 0.01; n.s., not significant versus acetone (ACE)–treated mice by Student’s t test.

  • Fig. 2 Breg cells suppress IL-13+ ILC2s in an IL-10–dependent manner.

    (A) Data for the ear thickness of WT or Cd19Cre mice with CHS for 4 days after challenge with OXZ are shown. (B) The histograms show the numbers of IL-13+ ILC2s isolated from the spleen, aLN, iLN, and ear tissue 2 days after the OXZ challenge. (C) Data for the ear thickness and (D) the number of IL-13+ ILC2s in Cd19Cre mice with CHS are shown after the adoptive transfer of OXZ-sensitized WT or Il10−/− B cell subsets as indicated. (A to D) The results are expressed as the mean ± SEM from two independent experiments (n = 5 per group for each experiment). *P < 0.05; **P < 0.01; n.s., not significant by Student’s t test (A to C) or one-way analysis of variance (ANOVA) with post hoc Tukey’s test (D). (E to G) The histograms for (E) the number of tissue IL-13+ ILC2s, (F) the ear thickness, and (G) the histology images of ear tissues (2 days after OXZ challenge; scale bars, 200 μm) in CHS Rag2−/− mice with or without anti-Thy1.2 mAb treatment are shown. The results are expressed as the mean ± SEM from two independent experiments (n = 4 per group for each experiment). *P < 0.05; **P < 0.01; n.s., not significant versus Rag2−/− + isotype OXZ by Student’s t test. (H) After the adoptive transfer of WT splenic CD19+ B cells into Rag2−/− mice, CHS was induced by OXZ challenge. Representative immunofluorescence images of IL-10 (green) and B220 (red) are shown (n = 5; scale bars, 100 μm). (I) Ear thicknesses and (J) IL-13+ ILC2 numbers in CHS Rag2−/− mice with or without the transfer of WT or Il10−/− CD1dhiCD5+ B cell subsets. (K) Representative flow cytometry images for IL-13+ ILC2s in coculture with WT CD1dhiCD5+, WT non-CD1dhiCD5+, or Il10−/− CD1dhiCD5+ B cell subsets at a 1:1 ratio and (L) frequency data are shown. (I to L) The results are expressed as the mean ± SEM (I, J, and L) or representative images (K) from two independent experiments (n ≥ 3 per group for each experiment). *P < 0.05; **P < 0.01; n.s., not significant by Student’s t test (I) or one-way ANOVA with post hoc Tukey’s test (J and L).

  • Fig. 3 MCs are critical for the maintenance of IL-10+ Breg cells and the inhibition of IL-13+ ILC2s in peripheral tissues.

    (A) Ear thickness is shown for 4 days after challenge with OXZ in WT or KitW-sh/W-sh mice. (B) Representative images (2 days after OXZ challenge) after staining with H&E (top) or toluidine blue (bottom) of ear tissues (scale bars, 100 μm) and histograms of ear epidermal thickness are shown. (A and B) Data are expressed as the mean ± SEM from three independent experiments (n ≥ 4 per group for each experiment). *P < 0.05; **P < 0.01; n.s., not significant by Student’s t test. (C) Representative immunofluorescence images of IL-10+ Breg cells in the draining LNs from WT and KitW-sh/W-sh mice are shown (IL-10, green; CD19, red) (n = 5; scale bars, 50 μm). (D to F) The results from tissues of WT or KitW-sh/W-sh mice 4 days after challenge with OXZ are shown. (D) The histograms show the frequencies (left) and numbers (right) of IL-10+ Breg cells. (E) The histograms show the numbers of IL-13+ ILC2s. (F) The histograms show the amount of IL-13 by enzyme-linked immunosorbent assay (ELISA). (D to F) Data are expressed as the mean ± SEM from two independent experiments (n ≥ 3 per group for each experiment). *P < 0.05; **P < 0.01; n.s., not significant by one-way ANOVA with post hoc Tukey’s test.

  • Fig. 4 Reconstitution of MCs in KitW-sh/W-sh mice increases IL-10+ Breg cells and suppresses the CHS response.

    (A) The histograms show the frequencies of total CD19+ B cells in peripheral tissues from WT mice or KitW-sh/W-sh mice with or without the adoptive transfer of MCs. (B and C) Representative plot images show IL-10+ CD19+ B cells (B), and histograms show the frequencies of CD5+CD19+ B cells (C) in peripheral tissues from WT mice or KitW-sh/W-sh mice with or without the adoptive transfer of MCs. (D) Ear thickness, (E) the frequency of IL-10+ B cells, and (F) the number of IL-13+ ILC2s are shown during CHS in WT or KitW-sh/W-sh mice with or without the transfer of MCs. Data are expressed as the mean ± SEM (A and C to F) and representative images (B) from two independent experiments (n ≥ 3 per group for each experiment). *P < 0.05; **P < 0.01; n.s., not significant by Student’s t test (D and F) or one-way ANOVA with post hoc Tukey’s test (C and E). BMMC, bone marrow-derived MC; PDMC, PeC-derived MC.

  • Fig. 5 IL-5–mediated signaling is critical for the development of CD1dintCD5+ Breg precursor cells and IL-10+ Breg cells.

    (A) The histograms show the amount of IL-5 in LNs from KitW-sh/W-sh mice with or without the reconstitution of BMMCs. The data are expressed as the mean ± SEM from three independent experiments (n ≥ 3 per group for each experiment). *P < 0.05 versus phosphate-buffered saline (PBS) intravenous group by Student’s t test. (B) Representative flow cytometry images (middle) and histograms for the population of IL-10+ B cells and the amount of IL-10 in the culture medium (right) are shown. Data are expressed as the mean ± SEM from four independent experiments (triplicate for each experiment). *P < 0.05, **P < 0.01 versus medium group by Student’s t test. (C) Splenic CD19+ B cells (2 × 106 cells per well) from WT mice were cultured with or without cytokines [TNF-α, 10 ng/ml; IFN-γ (interferon-γ), 10 ng/ml; IL-6, 10 ng/ml; IL-4, 5 ng/ml; IL-5, 10 ng/ml; and IL-13, 100 ng/ml] for 48 hours. Representative flow cytometry images (left) and histograms (right) show the mean fluorescence intensity (MFI) of CD125 (IL-5Rα) on B cells. The results are expressed as representative images (left) and the mean ± SEM (right) from three independent experiments (duplicate for each experiment). **P < 0.01 versus medium group by Student’s t test. (D) CD19+ B cells were stimulated with IL-4 (5 ng/ml), IL-5 (10 ng/ml), or IL-4 (5 ng/ml) + IL-5 (10 ng/ml) for 43 hours and subsequently stimulated with lipopolysaccharide (LPS) + phorbol 12-myristate 13-acetate (PMA), ionomycin, and monensin (PIM) for an additional 5 hours. The histograms of IL-10+ B cells by flow cytometry (left) and the amount of IL-10 in the culture medium (right) are shown. (E) CD19+ B cells (2 × 106 cells per well) were cultured with or without IL-4 and IL-5 for 43 hours and subsequently stimulated with LPIM for 5 hours. Representative flow cytometry images (left) and histograms (right) show the MFIs of CD5 on B cells. (D and E) The results are expressed as the mean ± SEM from three independent experiments (triplicate for each experiment). *P < 0.05; **P < 0.01; n.s., not significant by one-way ANOVA with post hoc Tukey’s test. (F to H) Representative flow cytometry images are shown for various B cell subsets (F), and histograms show the frequencies of the B cell subsets (G) and IL-10+ B cells (H) in peripheral tissues from WT or Il5ra−/− mice. Data are expressed as the mean ± SEM from three independent experiments (n ≥ 3 per group for each experiment). *P < 0.05; **P < 0.01; n.s., not significant versus WT by Student’s t test.

  • Fig. 6 MC-derived IL-5 is critical for the development of peripheral IL-10+ B cells to suppress CHS in mice.

    (A and B) Splenic CD19+ B cells (1.5 × 106 cells per well) were cocultured with an equal number of BMMCs with an anti–IL-5Rα mAb or isotype mAb for 43 hours and subsequently cultured with LPIM for 5 hours. Representative flow cytometry images (A) and histograms of the frequency of IL-10+ B cells (B) are shown. PE, phycoerythrin. (C and D) In (A) and (B), WT or Il5ra−/− splenic B cells were cocultured with equal numbers of WT or Il5v/v BMMCs as indicated. Representative flow cytometry images (left) and the frequency of IL-10+ B cells (right) are shown. (A to D) The results are expressed as representative images (A, C, and D) and mean ± SEM (C and D) from three independent experiments (triplicate for each experiment). **P < 0.01 by one-way ANOVA with post hoc Tukey’s test as indicated. (E and F) The histograms show the numbers of CD1dintCD5+ B cells and IL-10+ B cells in LNs (E) or IL-13+ ILC2s in LNs and ear tissues (F) from KitW-sh/W-sh mice with or without intravenous transfer of WT or Il5v/v BMMCs. (E and F) Data are expressed as the mean ± SEM from three independent experiments (n ≥ 3 per group for each experiment). *P < 0.05; **P < 0.01; n.s., not significant by one-way ANOVA with post hoc Tukey’s test. (G) The graphs show the ear thicknesses of KitW-sh/W-sh mice induced by OXZ with or without the reconstitution of WT or Il5v/v BMMCs. (H) The graphs show the ear thicknesses of WT or Il5ra−/− mice induced by OXZ. (I and J) The graphs show the ear thicknesses of Cd19Cre mice (I) or Rag2−/− mice (J) induced by OXZ with or without the transfer of WT or Il5ra−/− CD1dintCD5+ B cells. (G to J) Data are expressed as the mean ± SEM from two independent experiments (n ≥ 3 per group for each experiment). *P < 0.05, **P < 0.01 versus KitW-sh/W-sh mice (G), WT mice (H), Cd19Cre mice (I), or Rag2−/− mice (J) by Student’s t test.

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/5/7/eaav8152/DC1

    Fig. S1. Leukocyte population changes in mice with OXZ-induced CHS.

    Fig. S2. Treg cells are not essential for the development of IL-10+ Breg cells in peripheral tissues.

    Fig. S3. The population changes in MCs, IL-13+ ILC2s, and IL-10+ Breg cells during OXZ-induced chronic atopic dermatitis (AD)–like skin inflammation.

    Fig. S4. Comparison of MCs and Treg cells in WT and Cd19Cre mice with CHS.

    Fig. S5. The treatment of IL-13 mAb suppresses OXZ-induced CHS in mice.

    Fig. S6. Analysis of B cells, Treg cells, and serum antibody isotypes from WT or KitW-sh/W-sh mice.

    Fig. S7. CD40L on MCs is not critical for the suppression of CHS.

    Fig. S8. Amounts of TH1 and TH2 cytokines in peripheral tissues from WT or KitW-sh/W-sh mice.

    Fig. S9. The tissue distribution of MCs in KitW-sh/W-sh mice and Breg cells in Cd19Cre mice after the adoptive transfer of each cell.

    Fig. S10. The sorting strategies for B cell subsets, IL-13+ ILC2s, and IL-10+ B cells.

  • Supplementary Materials

    This PDF file includes:

    • Fig. S1. Leukocyte population changes in mice with OXZ-induced CHS.
    • Fig. S2. Treg cells are not essential for the development of IL-10+ Breg cells in peripheral tissues.
    • Fig. S3. The population changes in MCs, IL-13+ ILC2s, and IL-10+ Breg cells during OXZ-induced chronic atopic dermatitis (AD)–like skin inflammation.
    • Fig. S4. Comparison of MCs and Treg cells in WT and Cd19Cre mice with CHS.
    • Fig. S5. The treatment of IL-13 mAb suppresses OXZ-induced CHS in mice.
    • Fig. S6. Analysis of B cells, Treg cells, and serum antibody isotypes from WT or KitW-sh/W-sh mice.
    • Fig. S7. CD40L on MCs is not critical for the suppression of CHS.
    • Fig. S8. Amounts of TH1 and TH2 cytokines in peripheral tissues from WT or KitW-sh/W-sh mice.
    • Fig. S9. The tissue distribution of MCs in KitW-sh/W-sh mice and Breg cells in Cd19Cre mice after the adoptive transfer of each cell.
    • Fig. S10. The sorting strategies for B cell subsets, IL-13+ ILC2s, and IL-10+ B cells.

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