Research ArticleIMMUNOLOGY

Food antigens drive spontaneous IgE elevation in the absence of commensal microbiota

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Science Advances  22 May 2019:
Vol. 5, no. 5, eaaw1507
DOI: 10.1126/sciadv.aaw1507
  • Fig. 1 Ingested food Ags are responsible for spontaneous IgE elevation in GF mice.

    (A) Serum IgE levels of SPF, GF, and AF mice with ages were measured by enzyme-linked immunosorbent assay (ELISA) (n = 4). Statistically significant difference between GF and AF mice at indicated age was shown. (B) AF and GF pups were weaned onto NCD (AF weaned on NCD) and AF diet (GF weaned on AFD), respectively. After 7 weeks of feeding, serum IgE levels were measured by ELISA. Age-matched AF and GF mice were used as control mice (n = 6). (C) GF mice were weaned onto NCD or AAD. After 6 weeks, serum IgE levels were measured by ELISA (n = 7). (D) Levels of IgE specific to water-soluble fraction of chow diet (diet extract) in sera from 12-week-old AF and GF mice were measured by direct ELISA. OVA at an equivalent amount was used as an irrelevant control (n = 4 for AF sera and n = 18 for GF sera). (E) GF mice were weaned onto AAD mixed with indicated proteins (W.Glu: wheat gluten and EW). After 6 weeks of feeding, serum IgE levels were measured by ELISA (n = 4). Each symbol represents an individual mouse. (F) Levels of serum IgE specifically bound to wheat gluten (n = 4 for AF sera and n = 9 for GF sera). Data in (A) and (E) are representative data of two or three independent experiments. Data are pooled from two or three independent experiments (B, C, D, and F). Statistical differences were determined by one-way analysis of variance (ANOVA) with Tukey’s multiple comparisons test (A, B, and D to F) or by unpaired two-tailed Student’s t test (C). *P < 0.05, **P < 0.01, ***P < 0.001. Error bars represent SEM.

  • Fig. 2 Food Ag–driven TFH cells generated in GALT mediate IgE elevation in GF conditions.

    (A to C) Single-cell suspension was prepared from MLN and PP from 10-week-old SPF, GF, and AF mice. (A) Representative fluorescence-activated cell sorting (FACS) plot of CXCR5 and PD-1 (left) and frequency of PD-1hi CXCR5+ TFH cells (right) gated on CD4+ TCRβ+ Foxp3 CD44hi cells (n = 5). (B) Number of PD-1hi CXCR5+ TFH cells at indicated tissues. (C) Representative FACS plot showing PD-1hi CXCR5+ TFH cells among IL-4+ CD4+ T cells (left) and frequency of PD-1hi CXCR5+ TFH cells gated on IL-4–producing CD4+ T cells in indicated tissues from 10-week-old GF mice (n = 4) (right). (D) Number of IL-4–producing TFH cells in indicated tissues from SPF, GF, and AF mice. (E and F) GF mice (4.5 weeks old) were treated with isotype (Control) or anti-ICOSL antibody (TFH-depleted) every 3 days for 3 weeks to deplete TFH cells. Representative FACS plot showing PD-1hi CXCR5+ TFH cells in MLN and PP (E) and serum IgE levels (n = 4) (F). (G) FACS-sorted TFH cells and non-TFH cells in MLN and TFH cells in SPL from 9- to 12-week-old GF mice were cotransferred into GF Rag1−/− mice with naïve B cells isolated from SPF mice. Serum IgE levels at 1 and 2 weeks after transfer were shown (n = 3). Each symbol represents an individual mouse. Data are pooled from two independent experiments (A and B). Data in (C), (D), (F), and (G) are representative of two or three independent experiments. Statistical differences were determined by one-way (A to D) or two-way (G) ANOVA with Tukey’s multiple comparisons test or by unpaired two-tailed Student’s t test (F). *P < 0.05, **P < 0.01, ***P < 0.001. Error bars represent SEM.

  • Fig. 3 Food Ag–induced CD40 elevation on DCs promotes ICOS up-regulation on activated T cells.

    (A) Representative histograms (left) and mean fluorescence intensity (MFI) of ICOS expression (right) gated on CD44hi or CD44lo CD4+ T cells at indicated tissues from 9- to 10-week-old GF and AF mice (n = 3). (B) MFI of CD40 expression gated on DCs (Lin CD11c+ MHCII+) or B cells at indicated tissues from 9- to 10-week-old GF and AF mice (n = 4 to 5). (C and D) GF mice (4.5 weeks old) were treated with isotype or anti-CD40L antibody every 3 days for 2.5 weeks (n = 3). (C) Representative histograms (left) and MFI of ICOS expression (right) gated on CD44hi or CD44lo CD4+ T cells at indicated tissues. (D) Representative FACS plot of CXCR5 and PD-1 (left) and frequency of PD-1hi CXCR5+ TFH cells (right) gated on CD4+ TCRβ+ Foxp3 CD44hi cells. (E) Number of TFH cells in indicated tissues from GF mice treated with isotype and anti-CD40L antibody. (F) Total serum IgE levels in GF mice treated with isotype and anti-CD40L antibody. Each symbol represents an individual mouse. Data in (A) and (C) to (F) are representative of three independent experiments. Data were pooled from two independent experiments (B). Statistical differences were determined by two-way ANOVA with Tukey’s multiple comparisons test. *P < 0.05, **P < 0.01, ***P < 0.001. Error bars represent SEM.

  • Fig. 4 TFH cell generation and IgE elevation in response to food Ags are developmentally regulated.

    (A to D) GF mice (6.5 weeks old) were treated with anti-ICOSL antibody for 2 weeks to deplete TFH cells. Mice were analyzed to examine the level of TFH cells at days 2 and 28 after cessation of anti-ICOSL antibody treatment (n = 3). (A) Schematic view of experimental design and time plan. (B) Representative FACS plots of PD-1 and CXCR5 gated on CD4+ TCRβ+ Foxp3 CD44hi cells. (C) Number of PD-1hi CXCR5+ TFH cells. (D) Serum IgE levels of control mice and mice with TFH depletion at days 0 and 28 were measured by ELISA. (E to G) Young and adult AF mice (4 and 8 weeks of age, respectively) were switched to NCD for 4 weeks (n = 4). Representative FACS plots showing PD-1hi CXCR5+ TFH cells gated on CD4+ TCRβ+ Foxp3 CD44hi cells (E) and number of PD-1hi CXCR5+ TFH cells (F) from MLN and PP of indicated mice. (G) Serum IgE levels in indicated mice by ELISA. Each symbol represents an individual mouse. All data are representative of two independent experiments. Statistical differences were determined by one-way (C, F, and G) or two-way (D) ANOVA with Tukey’s multiple comparisons test. **P < 0.01, ***P < 0.001. Error bars represent SEM.

  • Fig. 5 Long-lived IgE-producing plasma cells contribute to sustained serum IgE levels regardless of the maintenance of TFH cells in adult mice.

    (A and D) GF mice (10 to 12 weeks old) were fed with NCD and AFD for 8 weeks (NCD versus AFD, n = 4 to 5). (A) Representative FACS plots showing PD-1hi CXCR5+ TFH cells gated on CD4+ TCRβ+ Foxp3 CD44hi cells in MLN and PP (left) and ratios of TFH cell frequency (right). (B and E) GF mice (12 weeks old) were untreated or treated with anti-ICOSL antibody every 3 days for 3 weeks (control versus anti-ICOSL, n = 4). (B) Representative FACS plots showing TFH cells in MLN and PP (left) and ratios of TFH cell frequency (right). (C) B cells in 10-week-old GF mice were gradually depleted by treating once with 0, 25, 100, and 250 μg of anti-CD20–depleting antibody (n = 2 per each dose). The correlation between the frequency of TFH cells and B220+ cells in MLN (left) and PP (right) was examined at 2 weeks after anti-CD20 antibody treatment. (D) Serum IgE levels in GF control (NCD, n = 5) and GF on AFD (AFD, n = 4) for indicated periods. (E) Serum IgE levels in GF untreated control (n = 3) and GF mice treated with anti-ICOSL antibody for indicated periods (n = 4). (F) GF mice (14 weeks old) were treated with isotype and anti-CD20 antibody (250 μg) every 3 days for 2 weeks. Serum IgE levels were shown. Each symbol represents an individual mouse. Data in (A) to (D) are pooled from two or three independent experiments. Data in (E) and (F) are representative of two independent experiments. Statistical differences were determined by two-way (A, B, D, and E) ANOVA with Tukey’s multiple comparisons test and by unpaired two-tailed Student’s t test (F). *P < 0.05, **P < 0.01, ***P < 0.001. NS, not significant. Error bars represent SEM.

  • Fig. 6 Commensal microbiota suppresses IgE elevation by restraining food Ag–driven TFH cell generation.

    (A to C) GF mice (10 weeks old) were conventionalized by cohousing them with SPF mice for indicated periods (ConvGF). (A) Serum IgE levels in age-matched SPF, GF, and ConvGF mice at the indicated time point after conventionalization were measured by ELISA (n = 3). (B) Frequency of IL-4+ cells among CD44hi CD4+ T cells in MLN and PP from indicated mice at 8 weeks after conventionalization (n = 4). (C) Representative FACS plots of PD-1 and CXCR5 (left) and frequency of PD-1hi CXCR5+ TFH cells gated on CD4+ TCRβ+ Foxp3 CD44hi cells in MLN and PP from indicated mice (right) (n = 4). (D) Number of TFH cells in MLN and PP from indicated mice. (E) Representative FACS plots showing the frequency of PD-1hi CXCR5+ TFH cells gated on IL-4–producing CD4+ T cells (left) and number of IL-4–producing TFH cells (right). (F and G) SPF mice were weaned onto either NCD or AAD and were untreated or simultaneously treated with broad-spectrum ABX in drinking water for 6 weeks. (F) Serum IgE levels by ELISA (n = 4). (G) Frequency of PD-1hi CXCR5+ TFH cells gated on CD4+ TCRβ+ Foxp3 CD44hi cells (left) and number of TFH cells (right) in MLN and PP from indicated mice (n = 5). Each symbol represents an individual mouse. All data in (A) to (F) are representatives of two independent experiments, and data in (G) are pooled from two independent experiments. Statistical differences were determined by one-way (A to E and G) or two-way (F) ANOVA with Tukey’s multiple comparisons test. *P < 0.05, **P < 0.01, ***P < 0.001. Error bars represent SEM.

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/5/5/eaaw1507/DC1

    Fig. S1. MLN and PP of GF mice are major sites for TH2-skewed immune response against food Ags.

    Fig. S2. TFH cells in MLN and PP of GF mice are generated upon weaning onto NCD, and AF mice displayed impaired generation of GC B cells and IgE-producing cells.

    Fig. S3. Levels of wheat gluten–specific IgE in GF Rag1−/− mice cotransferred with naïve B cells and indicated CD4+ T cell subsets.

    Fig. S4. ICOS up-regulation on activated CD4+ T cells is independent on the presence of TFH cells and B cells.

    Fig. S5. Expression levels of CD80, CD86, and ICOSL on MLN and PP DCs are comparable between GF and AF mice.

    Fig. S6. B cells are required for the generation of food Ag–driven TFH cells in GALT.

    Fig. S7. B cells promote terminal differentiation of food Ag–driven TFH cells by providing ICOS signaling and presenting cognate Ags.

    Fig. S8. Levels of CD4+ T cell activation in MLN and PP are comparable between young and adult AF mice switched to NCD, but the latter shows increased levels of TH2 cells.

    Fig. S9. High serum IgE levels in adult GF mice are sustained by radioresistant long-lived IgE-producing cells in MLN and BM.

    Fig. S10. ICOS expression on activated CD4+ T cells and CD40 expression on DCs in MLN and PP in SPF mice are both comparable with those in GF mice.

  • Supplementary Materials

    This PDF file includes:

    • Fig. S1. MLN and PP of GF mice are major sites for TH2-skewed immune response against food Ags.
    • Fig. S2. TFH cells in MLN and PP of GF mice are generated upon weaning onto NCD, and AF mice displayed impaired generation of GC B cells and IgE-producing cells.
    • Fig. S3. Levels of wheat gluten–specific IgE in GF Rag1−/− mice cotransferred with naïve B cells and indicated CD4+ T cell subsets.
    • Fig. S4. ICOS up-regulation on activated CD4+ T cells is independent on the presence of TFH cells and B cells.
    • Fig. S5. Expression levels of CD80, CD86, and ICOSL on MLN and PP DCs are comparable between GF and AF mice.
    • Fig. S6. B cells are required for the generation of food Ag–driven TFH cells in GALT.
    • Fig. S7. B cells promote terminal differentiation of food Ag–driven TFH cells by providing ICOS signaling and presenting cognate Ags.
    • Fig. S8. Levels of CD4+ T cell activation in MLN and PP are comparable between young and adult AF mice switched to NCD, but the latter shows increased levels of TH2 cells.
    • Fig. S9. High serum IgE levels in adult GF mice are sustained by radioresistant long-lived IgE-producing cells in MLN and BM.
    • Fig. S10. ICOS expression on activated CD4+ T cells and CD40 expression on DCs in MLN and PP in SPF mice are both comparable with those in GF mice.

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