Research ArticleMICROBIOME

Cell surface polysaccharides of Bifidobacterium bifidum induce the generation of Foxp3+ regulatory T cells

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Science Immunology  19 Oct 2018:
Vol. 3, Issue 28, eaat6975
DOI: 10.1126/sciimmunol.aat6975
  • Fig. 1 Bb monocolonization enhances Treg population in the cLP.

    (A) Localization analysis of Bb in the intestinal niches of GF mice by HISTO-FISH staining with DNA-Cy5 probes (EUB338, red) 3 weeks after colonization. (B) Representative flow cytometry plots and percentage analyses of CD4+Foxp3+ cells in cLP of GF mice colonized with indicated bacterial strains. (C to F) Representative flow cytometry plots and frequencies of CD103+ and CD62LloCD44hi, CTLA4+, and IL10+ in Treg cells in GF mice or those monocolonized with Lpa or Bb. Numbers in the quadrants represent cell percentage, and circles in the graph plots represent individual mouse corresponding to each parameter. Data are representative of three to five independent experiments with similar results (n ≥ 3 mice). All graph plots show means ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 (Student’s t test). MFI, mean fluorescence intensity; ns, not significant.

  • Fig. 2 Bb monocolonization facilitates de novo generation of pTreg cells.

    cLP cells were isolated from the GF mice or mice monocolonized with Lpa or Bb 3 weeks after colonization. (A) Absolute numbers of Nrp1+ and Nrp1 Treg cells. (B to F) Representative flow cytometry plots and frequencies for Nrp1Foxp3+ (B), HeliosFoxp3+ (C), HeliosNrp1Foxp3+ (D), RORγt+Helios Foxp3+ (E), and RORγt+Foxp3+, RORγtFoxp3+ (F) Treg cells in the cLP of GF or Bb- or Lpa-monocolonized mice. (G) Naïve CD4+Foxp3 T cells sorted from CD45.1+Foxp3GFP reporter mice were transferred into GF mice. Animals were either left GF or monocolonized with Bb for 3 weeks. Foxp3+ Treg population was analyzed by GFP expression in the cLP. (H) Naïve CD4+Foxp3 T cells sorted from CD45.1+Foxp3GFP reporter mice were transferred into SPF mice, and then mice were fed with mock (PBS) or Bb (5 × 108 CFU) every other day for 3 weeks and analyzed for Foxp3+ Treg cells. Data are representative of at least five independent experiments with similar results (n ≥ 3 mice). All graph plots show means ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 (Student’s t test).

  • Fig. 3 Bb colonization induces dietary Ag– and microbiota-reactive Treg cells.

    (A and B) CTV-labeled naïve allelically marked CD4+Thy1.1+Foxp3 T cells from OT-II.Thy1.1+Foxp3GFP mice were adoptively transferred into GF mice or mice premonocolonized with Bb or Lpa for 14 days. The mice were fed with OVA (20 mg) every other day for 7 days. The cLP CD4+Foxp3+ population of the donor (Thy1+OT-II) cells was analyzed. Representative fluorescence-activated cell sorting (FACS) plots (A) and frequencies of Treg cells generated in vivo (B) are shown. (C to E) Naïve CD4+CBir+CD45.1+Foxp3 T cells sorted from CBirTgCD45.1+Foxp3GFP mice were adoptively transferred into the SPF Rag1−/− recipients. The recipient mice were fed with either Bb or PBS every other day until the end of the experiment. Changes in the body weight (C), colon length (D), and histopathology and histological score (E) in colonic tissue were measured and analyzed. (F and G) Representative FACS plots and frequencies of CBir+CD45.1+Foxp3+ Treg cells (F) and CD4+IFNγ+ cells (G) within cLP of mock- or Bb-administered mice analyzed at the end of experiment. Numbers indicate cell percentages in the quadrants, and circles in the graph plots represent individual mouse corresponding to each parameter, respectively. Data are representative of at least three independent experiments with similar results (n ≥ 3 mice). Graphs with error bars show means ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 (Student’s t test).

  • Fig. 4 Bb monocolonization induces Bb-reactive colonic Treg cells with distinctive TCR repertoires.

    (A) Total colonic CD4+ T cells containing Foxp3+ Treg cells from the GF mice or Bb-monocolonized mice were sorted and labeled with CTV and then cocultured with T cell–depleted splenic APC in the presence of indicated fecal Ags from GF or Bb- or Lpa-monocolonized mice. Three days later, relative proliferation of Treg cells was analyzed by FACS. (B) Comparison of Shannon diversity from TCR α and β chains of Treg cell repertoires obtained from different lymphoid organs of GF or Bb-monocolonized mice. (C and D) Frequency distribution two-way bar plot representation of common TCR-CDR3 α regions (85 from colon, 1469 from mLN, and 1381 from spleen) (C) and β regions (118 from colon, 1724 from mLN, and 1381 from spleen) (D). (E) Comparative enrichment frequency distribution of unique α and β chains of TCR-CDR3 regions of Treg cells isolated from colon, mLN, and spleen of Bb-monocolonized mice compared with GF mice.

  • Fig. 5 Cell surface CSGG of Bb mediates Treg cell induction.

    (A to C) Quantitative polymerase chain reaction analysis of mRNA for cytokines and inhibitory and costimulatory molecules plotted as fold change in the cLP-DCs (MHCII+CD11c+CD11b+ CD103+F4/80) of GF or Bb-monocolonized mice. Data are representative of three independent experiments with similar results. (D) Graph shows absolute number of cLP-DCs from GF mice or monocolonized mice with Lpa or Bb for CD103, CD11b, CX3CR1, and pDCs (Bst2+CD11clo). Numbers represent the frequency of each population from the total CD11c+MHCII+ cells. Data are representative of two independent experiments with similar results (n = 4 mice). (E) CD11c+DCs pretreated with Bb or Lpa were cocultured with naïve CD4+ T cells in suboptimal Treg-inducing conditions for 3 days, after which Foxp3+ Treg cells were analyzed within live cells. Representative flow cytometric analysis and mean frequencies of CD4+Foxp3+ Treg cells are shown. From culture supernatant, IL-10 secretion was determined by enzyme-linked immunosorbent assay. Data are representative of five independent experiments with similar results. (F) Structures of neutral (CSGG) or negatively charged polysaccharides (PGβG) isolated from the tCSPS of Bb. Glcp, glucopyranos; Galp, galactopyranose; Galf, glucofuranose; Gro, glycerol. n = repeating unit. Relative abundances of each polysaccharide are shown as % (mol/mol). (G) Comparison of CD4+Foxp3+ Treg-inducing activity between the neutral (CSGG, 50 μg/ml) and negatively charged polysaccharides (PGβG, 100 μg/ml) compared with tCSPS (100 μg/ml). (H) Effect of β-1,6-glucanase treatment on CSGG-induced iTreg cells. (I) Populations and phenotypes of Treg cells in GF mice or mice monocolonized with Bb or Lpa or intraperitoneally injected with CSGG (100 μg per dose) every other day for 3 weeks. (J) Dose-dependent induction of human CD4+CD25+Foxp3+ Treg cells by coculturing of DCs pretreated with CSGG with naïve CD4+ T cells from PBMCs of healthy donors. Data are representative of three independent experiments with similar results. Graphs with error bars show means ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 (Student’s t test).

  • Fig. 6 CSGG facilitates iTreg induction through TLR2-dependent generation of regulatory DCs.

    (A) RNA-seq analysis of CD11c+ splenic DCs treated with mock or CSGG for 4 hours. (B) Cytokine levels in the culture supernatants after coculturing naïve CD4+ T cells with CD11c+ DCs pretreated with mock or CSGG. Data are representative of four independent experiments with similar results. nd, not detected. (C) Effect of anti–TGF-β Ab treatment on in vitro CD4+Foxp3+ Treg induction after Bb or CSGG treatment. (D) CD11c+ DCs from the WT or IL-10−/− mice pretreated with CSGG were cocultured with WT naïve CD4+ T cells, and iTreg induction was determined after 3 days. Data are representative of three independent experiments with similar results (C and D). (E) RNA-seq data were analyzed to determine the expression of genes encoding the TLR subtypes upon CSGG treatment. (F) Naïve CD4+ T cells and CD11c+ DCs derived from indicated mice, pretreated with mock or CSGG, were cocultured in suboptimal iTreg generation condition. CD4+Foxp3+ iTreg population was determined by flow cytometry. Data are representative of five independent experiments with similar results. (G and H) IL-10 and TGF-β1 cytokine levels in the culture supernatants after naïve CD4+ T cells were cocultured with WT or TLR2-deficient CD11c+ DCs pretreated with mock or CSGG. Data are representative of at least three independent experiments with similar results. All bar graphs show means ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001 (Student’s t test).

  • Fig. 7 CSGG-induced iTreg cells are capable of suppressing intestinal inflammation.

    (A to D) Naïve CD4+Foxp3 T cells sorted from Thy1.1+Foxp3GFP reporter mice were adoptively cotransferred into Rag1−/− recipients in indicated combinations of iTreg cells or nTreg cells. Changes in body weight, colon length, histopathology, and histological score of colonic tissue were measured. (E) Analysis of the Foxp3 stability of transferred Treg cells in the cLP at the end of the experiment. At the time of transfer, the purity of the sorted CD4+Foxp3GFP+ cells was more than 98%. (F to H) Naïve CD4+Foxp3 T cells sorted from CD45.1+Foxp3GFP reporter mice were adoptively transferred into Rag1−/− mice, followed by intraperitoneal administration of PBS or CSGG (100 μg/ml). Changes in body weight, colon length, histopathology, and histological score in colonic tissue were measured. (I) Analysis of the IFNγ-producing effector T cells in cLP at the end of the experiment. Data are representative of at least three independent experiments with similar results (n ≥ 3 mice). Graphs with error bars show means ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 (Student’s t test).

Supplementary Materials

  • immunology.sciencemag.org/cgi/content/full/3/28/eaat6975/DC1

    Materials and Methods

    Fig. S1. Identification of Bb as Treg-inducing bacteria.

    Fig. S2. Effect of Bb monocolonization on cytokine levels in Treg and non-Treg cells.

    Fig. S3. Bb monocolonization facilitates de novo generation of pTreg cells.

    Fig. S4. Bb colonization induces dietary Ag– and/or microbiota-reactive Treg cells.

    Fig. S5. Effect of Bb monocolonization on the TCR repertoire of Treg cells.

    Fig. S6. Effect of Bb monocolonization on phenotypes and population of cLP-DC subtypes.

    Fig. S7. Effect of Bb monocolonization on phenotypes and population of DC subtypes in mLN and siLP.

    Fig. S8. CSGG of the Bb enhances Treg cell induction.

    Fig. S9. Role of DC subtypes in inducing Bb/CSGG-mediated iTreg cells.

    Fig. S10. CSGG facilitates iTreg induction through TLR2-mediated generation of regulatory DCs.

    Fig. S11. CSGG-induced iTreg cells are capable of suppressing intestinal inflammation.

    Fig. S12. Confirmation of Bf monocolonization by DNA sequencing.

    Table S1. Peptide sequences of α-chain CDR3 region of Treg cells sorted from colon, mLN, and spleen of Bb-monocolonized mice compared with GF mice.

    Table S2. Peptide sequences of β-chain CDR3 region of Treg cells sorted from colon, mLN, and spleen of Bb-monocolonized mice compared with GF mice.

    Table S3. Raw data (Excel file).

    References (4955)

  • Supplementary Materials

    The PDF file includes:

    • Materials and Methods
    • Fig. S1. Identification of Bb as Treg-inducing bacteria.
    • Fig. S2. Effect of Bb monocolonization on cytokine levels in Treg and non-Treg cells.
    • Fig. S3. Bb monocolonization facilitates de novo generation of pTreg cells.
    • Fig. S4. Bb colonization induces dietary Ag– and/or microbiota-reactive Treg cells.
    • Fig. S5. Effect of Bb monocolonization on the TCR repertoire of Treg cells.
    • Fig. S6. Effect of Bb monocolonization on phenotypes and population of cLP-DC subtypes.
    • Fig. S7. Effect of Bb monocolonization on phenotypes and population of DC subtypes in mLN and siLP.
    • Fig. S8. CSGG of the Bb enhances Treg cell induction.
    • Fig. S9. Role of DC subtypes in inducing Bb/CSGG-mediated iTreg cells.
    • Fig. S10. CSGG facilitates iTreg induction through TLR2-mediated generation of regulatory DCs.
    • Fig. S11. CSGG-induced iTreg cells are capable of suppressing intestinal inflammation.
    • Fig. S12. Confirmation of Bf monocolonization by DNA sequencing.
    • Table S1. Peptide sequences of α-chain CDR3 region of Treg cells sorted from colon, mLN, and spleen of Bb-monocolonized mice compared with GF mice.
    • Table S2. Peptide sequences of β-chain CDR3 region of Treg cells sorted from colon, mLN, and spleen of Bb-monocolonized mice compared with GF mice.
    • References (4955)

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