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. 2014 Sep 9;111(36):13145-50.
doi: 10.1073/pnas.1412008111. Epub 2014 Aug 25.

Commensal bacteria protect against food allergen sensitization

Andrew T Stefka  1 , Taylor Feehley  1 , Prabhanshu Tripathi  1 , Ju Qiu  2 , Kathy McCoy  3 , Sarkis K Mazmanian  4 , Melissa Y Tjota  5 , Goo-Young Seo  1 , Severine Cao  1 , Betty R Theriault  6 , Dionysios A Antonopoulos  7 , Liang Zhou  2 , Eugene B Chang  5 , Yang-Xin Fu  1 , Cathryn R Nagler  8
Affiliations

Commensal bacteria protect against food allergen sensitization

Andrew T Stefka et al. Proc Natl Acad Sci U S A. .

Abstract

Environmentally induced alterations in the commensal microbiota have been implicated in the increasing prevalence of food allergy. We show here that sensitization to a food allergen is increased in mice that have been treated with antibiotics or are devoid of a commensal microbiota. By selectively colonizing gnotobiotic mice, we demonstrate that the allergy-protective capacity is conferred by a Clostridia-containing microbiota. Microarray analysis of intestinal epithelial cells from gnotobiotic mice revealed a previously unidentified mechanism by which Clostridia regulate innate lymphoid cell function and intestinal epithelial permeability to protect against allergen sensitization. Our findings will inform the development of novel approaches to prevent or treat food allergy based on modulating the composition of the intestinal microbiota.

Keywords: IL-22; barrier; microbiome.

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Conflict of interest statement

Conflict of interest statement: A provisional US patent application (61/937952) was filed on February 10, 2014.

Figures

Fig. 1.
Fig. 1.
Neonatal Abx exposure alters the commensal microbiota and enhances food allergen sensitization. Abx treatment was initiated before weaning as described in Methods. (A and B) 3-wk-old mice were sensitized by intragastric administration of PN plus CT (PN/CT, open symbols) or CT only (closed symbols) and challenged on day 35; feces and serum were collected on day 36. Serum concentration of (A) PN-specific IgE and (B) PN-specific IgG1 was measured by ELISA (n = 4–9 mice per group from three independent experiments; each circle represents an individual mouse; bars depict mean and SEM). (C) Bacterial load in the feces or ileal contents of mice treated with Abx compared with no treatment (NT) controls. (n = 4–5 mice per group). (D) Bacterial diversity, as shown by operational taxonomic unit (97% identity) rarefaction curves in Abx-treated mice compared with NT controls: black lines, NT feces; gray lines, Abx feces; red lines, NT ileal contents; blue lines, Abx ileal contents. (E) Taxonomic classifications for the mice in C represented as proportion of total reads (Methods). *P < 0.05, **P < 0.01, ***P < 0.001 determined by Student t test (B) or one-way ANOVA with Tukey posttest (C).
Fig. 2.
Fig. 2.
A Clostridia-containing microbiota protects against sensitization to food allergens. (A–D) Groups of SPF (white), GF (red), or gnotobiotic mice colonized with fecal/cecal material from SPF mice (Conventionalized, blue), B. uniformis (gray), or with a consortium of Clostridia (green) were sensitized with either CT only or PN/CT at weaning and challenged on day 35. (A) Concentration of PN-specific IgE, (B) IgG1, and (C) total IgE in serum of sensitized mice collected 24 h after challenge. (D) Change in core body temperature in sensitized mice (n = 4–10 mice per group from two independent experiments; closed circles, CT only; open circles, PN/CT). In A–D, each circle represents an individual mouse; bars depict median. (E) Cecal size at 13 d after colonization. (F) Bacterial load in feces collected from 5- to 10-wk-old SPF and gnotobiotic mice 14 d after colonization. n = 3–5 mice per group. F depicts mean and SEM. *P < 0.05, **P < 0.01, ***P < 0.001 determined by two-way ANOVA with the Kruskal-Wallis test (A–D) or one-way ANOVA with Tukey posttest (F).
Fig. 3.
Fig. 3.
Clostridia colonization activates innate immune genes in IECs. (A) Proportion of Foxp3+ Tregs among CD4+ T cells in the spleen, MLN, and colonic LP of age-matched SPF (white) and GF (red) mice and 14 d after colonization of GF mice with an SPF microbiota (Conventionalized, blue), B. uniformis (gray), or Clostridia (green) (n = 4–8 per group). (B) Concentration of IgA in feces collected from sensitized mice in Fig. 2. (C) Number of genes up-regulated in IECs relative to GF by B. uniformis (gray), Clostridia (green), or both (black) at 6 d after colonization. Genes shown exhibited significant expression above background in all samples (detection P value <0.05) and ≥1.5-fold increase in mean expression in comparison with values obtained for GF mice. (D) Heatmap depicting differential gene expression for 11 genes of interest. Samples with the highest and lowest transcript levels are red and blue, respectively. (E) Quantitative PCR verification of microarray data for selected genes. *P < 0.05, **P < 0.01, ***P < 0.001 by two-way ANOVA with Bonferroni posttest (A and E) or one-way ANOVA with Tukey posttest (B).
Fig. 4.
Fig. 4.
Clostridia colonization induces IL-22. (A) Reg3b and Reg3g expression from whole-tissue extracts isolated 4 d after colonization from the small intestine or colon of GF (red), B. uniformis-colonized (gray), or Clostridia-colonized (green) mice. Quantitative RT-PCR data are plotted relative to GF and normalized to Hprt (n = 8–9 mice per group from two independent experiments). (B) Il22 expression in LPL from mice in A. (C) IL-22 production by RORγt+ ILCs and T cells 6 d after colonization, determined by flow cytometric analysis of permeabilized cells (SI Methods; n = 3 mice per group representative of three independent experiments). (D) Representative images and quantification of goblet cells in distal colon of GF, B. uniformis-colonized, and Clostridia-colonized mice 6 d after colonization. n = 3–5 mice per group. (Scale bar, 100 µm.) (E) Serum Ara h 6 and Ara h 2 levels after PN gavage in GF, B. uniformis-colonized, or Clostridia-colonized mice 6 d after colonization (n = 5–12 mice per group from two independent experiments). *P < 0.05, **P < 0.01, ***P < 0.001 by two-way ANOVA with Bonferroni posttest (A and B) or one-way ANOVA with Tukey posttest (C).
Fig. 5.
Fig. 5.
Clostridia-induced IL-22 regulates allergen access to the bloodstream. (A) Expression of Il22 in LPL from neonatal Abx-treated mice without Clostridia colonization, or at 6 d after weaning and colonization. (B) Serum Ara h 6 at indicated time points after PN gavage in NT or Abx mice treated with or without one i.p. injection of IL-22-Fc, or by Clostridia colonization. (C) Serum Ara h 6 at indicated time points after PN gavage in Abx-treated Clostridia-colonized mice injected i.p. with neutralizing antibody to IL-22 or an isotype control. All mice in B and C received PN at 6 d after weaning, and serum levels of Ara h 6 were measured by capture ELISA (n = 5–10 mice per group, pooled from at least two experiments). (D) Expression of Reg3b in whole-tissue extracts from Abx-treated Clostridia-colonized mice treated with neutralizing antibody to IL-22 or an isotype control and sensitized with PN/CT (n = 11 mice per group, pooled from four experiments). (E) Concentration of IL-4 in culture supernatants from splenocytes of mice from D (n = 7 mice per group, representative of two experiments). (F) Concentration of PN-specific and total IgE in serum collected 24 h after challenge for mice in D (n = 11 mice per group, pooled from four experiments). (G) Concentration of IL-17 in culture supernatants from splenocytes from mice in D (n = 7 mice per group, representative of two experiments). (H) Concentration of PN-specific IgG in serum collected 24 h after challenge for mice in D (n = 11 mice per group, pooled from four experiments). (I) UniFrac analysis of fecal microbiota throughout the sensitization protocol (n = 4 mice per group). *P < 0.05, **P < 0.01 ***P < 0.001 by two-way ANOVA with Bonferroni posttest (A, B, and D) or Student t test (C and G).
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