Microbiota therapy acts via a regulatory T cell MyD88/RORγt pathway to suppress food allergy

An Author Correction to this article was published on 16 August 2019

This article has been updated

Abstract

The role of dysbiosis in food allergy (FA) remains unclear. We found that dysbiotic fecal microbiota in FA infants evolved compositionally over time and failed to protect against FA in mice. Infants and mice with FA had decreased IgA and increased IgE binding to fecal bacteria, indicative of a broader breakdown of oral tolerance than hitherto appreciated. Therapy with Clostridiales species impacted by dysbiosis, either as a consortium or as monotherapy with Subdoligranulum variabile, suppressed FA in mice as did a separate immunomodulatory Bacteroidales consortium. Bacteriotherapy induced expression by regulatory T (Treg) cells of the transcription factor ROR-γt in a MyD88-dependent manner, which was deficient in FA infants and mice and ineffectively induced by their microbiota. Deletion of Myd88 or Rorc in Treg cells abrogated protection by bacteriotherapy. Thus, commensals activate a MyD88/ROR-γt pathway in nascent Treg cells to protect against FA, while dysbiosis impairs this regulatory response to promote disease.

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Fig. 1: Infants with FA exhibit an evolving gut dysbiosis.
Fig. 2: Altered mucosal antibody responses to the gut microbiota in FA.
Fig. 3: A consortium of Clostridiales species prevents FA.
Fig. 4: Clostridiales and Bacteroidales consortia suppress established FA.
Fig. 5: ROR-γt+ Treg cell deficiency promotes FA.
Fig. 6: Protection against FA by commensals requires ROR-γt+ Treg cells.

Data availability

Data presented in the manuscript, including de-identified patient results, will be made available to investigators following request. All requests for raw and analyzed data and materials will be promptly reviewed by the Boston Children’s Hospital Technology & Innovation Development Office to verify whether the request is subject to any intellectual property or confidentiality obligations. Any data and materials that can be shared will be released via a Material Transfer Agreement. The 16S bacterial rRNA datasets generated in the course of this project have been deposited at the National Center for Biotechnology Information Sequence Read Archive under BioProject ID: PRJNA525231. Detailed age and disease attributes of the deposited BioProject sample data will be made available on request.

Change history

  • 16 August 2019

    An amendment to this paper has been published and can be accessed via a link at the top of the paper.

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Acknowledgements

We thank H. Oettgen for the provision of Igh7–/–Il4raF709 mice, L.-M. Charbonnier for critical review of the manuscript and M. Delaney for support with microbiology. This work was supported by NIH NIAID grants Nos. 1R56AI117983 and 1R01AI126915 (to T.A.C.), NIDDK grant No. P30DK056338 (to L.B.), the Clinical and Translational Science Center/Harvard Catalyst, the Bunning Food Allergy Fund, the Jasmine and Paul Mashikian Fund, the Massachusetts Life Sciences Center and a Partners Healthcare Innovations Development grant.

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Contributions

T.A.C., R.R. and D.T.U. conceived the human microbiota studies, and T.A.C. conceived the mechanistic studies and directed the overall project. L.B. conceived the bacterial consortia and oversaw their development for use as a therapeutic. G.K.G., N.L. and X.D. carried out the bioinformatic analyses of human fecal microbiota composition. N.D. designed multiplex probes for the consortia and carried out the persistence studies. A.A.-G., E.S.-V., M.N.R., S.W., H.H. and L.W. carried out the experiments and evaluated the data. R.R. oversaw the design and execution of the human studies. S.S., W.S., E.C. and H.B. were involved in human subject recruitment and/or the collection of fecal samples. T.A.C. and A.A.-G. wrote the manuscript.

Corresponding authors

Correspondence to Rima Rachid or Talal A. Chatila.

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Competing interests

L.B., G.K.G., T.A.C., R.R. and A.A.-G. are inventors on published US patent application No. 15/801,811, submitted by The Brigham and Women’s Hospital, Inc. and Children’s Medical Center Corporation, that covers methods and compositions for the prevention and treatment of food allergy using microbial treatments. T.A.C., R.R., A.A.-G. and E.S.-V. have pending patent applications related to the use of probiotics in enforcing oral tolerance in food allergy (Nos. 62/758, 161, and 62/823,866). L.B., G.K.G. and T.A.C. are founders of, and have equity in, Consortia Tx. R.R. has equity in Consortia Tx. A.A.-G. is currently an employee of, and owns shares in, Seed Health Inc. The rest of the authors declare no competing interests.

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Extended data

Extended Data Fig. 1 FMT from WT mice protects against FA in GF Il4raF709 mice.

a, Temperature changes in GF Il4raF709 mice that were either left uncolonized or reconstituted with FMT from WT or Il4raF709 mice, then sensitized with OVA/SEB and challenged with OVA (n = 15 WT and 14 Il4raF709 mice). b,c, Total and OVA-specific serum IgE (n = 15 WT and 14 Il4raF709 mice). d, MMCP-1 concentrations post-OVA challenge (n = 6 per group). e,f, Analysis of ROR-γt and GATA3 expression in MLN HeliosNRP1 and Helios+NRP1+ Treg cells (n = 6 per group). Each dot represents one mouse. Data represent mean ± s.e.m. from two or three independent experiments. P values were derived by repeat measures two-way ANOVA (a) or by Student’s unpaired two-tailed t-test with Welch correction (bf). Source data

Extended Data Fig. 2 Analysis of IgA- and IgE-bound bacteria in fecal samples.

a,c, Representative FACS plots showing the gating strategy for human (a) and mouse (c) fecal bacteria. Bacteria present in the feces were identified by gating on SYTO-BC+ events (right-side panels). b,d, Frequencies of IgA- and IgE-bound bacteria as assessed by gating on bacteria bound with the respective PE-labeled anti-IgA and anti-IgE antibodies, as shown in a and c. e,f, Analysis of sIgA+ (e) and IgE+ (f) fecal bacteria from Il4raF709 mice sensitized with OVA/SEB without or with bacterial therapy. Fecal pellets from Rag2–/– and Igh7–/–Il4raF709 mice were used as negative controls. Each symbol in the scatter plots represents one mouse (no treatment: n = 11 per group; Clostridiales: n = 8 per group; Proteobacteria: n = 9 and 7). Data represent mean ± s.e.m. from two independent experiments. Flow panels in c,d are representative of two two independent experiments. P values were derived by one-way ANOVA with Dunnett’s post hoc analysis. Source data

Extended Data Fig. 3 Antibiotic therapy potentiates the therapeutic efficacy of the Clostridiales consortium in Il4raF709 mice.

a, Temperature changes in the respective OVA/SEB-sensitized and OVA-challenged SPF Il4raF709 mouse groups treated as follows: no antibiotics (n = 6), Clostridiales (n = 5) and antibiotics without or with Clostridiales (n = 5 per group). P values were derived by two-way ANOVA. b,c, Total and OVA-specific IgE: no antibiotics (n = 4 per group), Clostridiales (n = 5 per group) and antibiotics without (n = 5 per group) or with Clostridiales (n = 4 per group). d, MMCP-1 concentrations: no antibiotics (n = 5), Clostridiales (n = 5) and antibiotics without (n = 4) or with Clostridiales (n = 4). e, Frequencies of total CD4+Foxp3+, HeliosNRP1Foxp3+, ROR-γt+CD4+Foxp3+ and IL-4+ CD4+Foxp3+ Treg cells in the MLN of the respective mouse group: no antibiotics (n = 6), Clostridiales (n = 5) and antibiotics without (n = 5) or with Clostridiales (n = 5). Each dot represents one mouse. Throughout, data represent mean ± s.e.m. from two independent experiments. Unless otherwise indicated, P values were derived by one-way ANOVA with Dunnett’s post hoc analysis. Source data

Extended Data Fig. 4 Bacteriotherapy with S. variabile protects against FA.

a, Temperature changes in SPF Il4raF709 mice that were antibiotic-treated then sensitized with OVA/SEB while receiving no treatment (n = 8) or treatment with S. variabile (n = 11), and thereafter challenged with OVA. P values were derived by two-way ANOVA. b,c, Total and OVA-specific IgE (no bacteria: n = 8; S. variabile: n = 11). d, MMCP-1 concentrations (no bacteria: n = 8; S. variabile: n = 11). e,f, Analysis of MLN ROR-γt+ and GATA3+ cells among HeliosNRP1 and Helios+NRP1+ Foxp3+ Treg cells, respectively (no bacteria: n = 8; S. variabile: n = 5). g, Analysis of MLN IL-4+ CD4+Foxp3+ Treg cells and IL-4+ CD4+Foxp3 Teff cells (no bacteria: n = 8; S. variabile: n = 5). Each dot represents one mouse. Throughout, data represent mean ± s.e.m. from two independent experiments. For bg, P values were derived by Student’s unpaired two-tailed t-test with Welch correction. Source data

Extended Data Fig. 5 Clostridiales protects against percutaneous sensitization-induced FA.

a, Temperature changes in SPF WT BALB/c mice that were antibiotic-treated then percutaneously sensitized with OVA/SEB while receiving either no treatment (n = 14) or treatment with Clostridales (n = 11), and thereafter challenged with OVA. P values were derived by two-way ANOVA. b,c, Total and OVA-specific IgE concentrations (n = 7 per group). d, MMCP-1 concentrations (n = 7 per group). e, Analysis of small intestinal LPL ROR-γt+ CD4+Foxp3+ Treg cells (n = 7 per group). Each dot represents one mouse. Data represent mean ± s.e.m. from two independent experiments. For be, P values were derived by Student’s unpaired two-tailed t-test with Welch correction. Source data

Extended Data Fig. 6 A Bacteroidales consortium prevents FA.

a, Left: experimental schema; right: temperature changes in GF Il4raF709 mice that were colonized and sensitized as indicated then challenged with OVA (n = 5 per group). b,c, Total and OVA-specific IgE (b) and MMCP-1 concentrations (c). GF, OVA/SEB (n = 5 per group), Bacteroidales, PBS (n = 6, 7 and 7), Bacteroidales, OVA/SEB (n = 6, 6 and 7). d, Frequencies of MLN CD4+Foxp3+, IL-4+Foxp3+ and GATA3+Foxp3+ T cells. GF, OVA/SEB (n = 5 per group), Bacteroidales, PBS (n = 4, 8 and 5), Bacteroidales, OVA/SEB (n = 6, 5 and 6). e, Frequencies of HeliosNrp1Foxp3+ and ROR-γt+Foxp3+ T cells. GF, OVA/SEB (n = 5 and 7), Bacteroidales, PBS (n = 5 per group), Bacteroidales, OVA/SEB (n = 6 per group). f, Left: experimental schema; right: temperature changes in Il4raF709 mice sensitized and treated as indicated. OVA/SEB (n = 6), OVA/SEB, Bacteroidales, (n = 5). g, Total and OVA-specific IgE and MMCP-1 concentrations: OVA/SEB (n = 7, 9 and 9), OVA/SEB, Bacteroidales, (n = 5, 10 and 5). h,i, Frequencies of MLN CD4+Foxp3+, IL-4+Foxp3+ and GATA3+Foxp3+ (h) and HeliosNrp1Foxp3+ and ROR-γt+Foxp3+ T cells (i). OVA/SEB (n = 5, 8 and 3, 5 and 5), OVA/SEB, Bacteroidales (n = 5, 10, 4, 6 and 9). j, IgE and IgA staining of fecal bacteria. OVA/SEB (n = 11 per group), OVA/SEB, Bacteroidales (n = 8 per group). Each dot represents one mouse. Data represent mean ± s.e.m. from two independent experiments. P values were derived by repeat measures two-way ANOVA (a,f), by one-way ANOVA with Dunnett’s post hoc analysis (be) or by Student’s unpaired two-tailed t-test (hj). Source data

Extended Data Fig. 7 Depletion of Treg cells abrogates protection by the microbiota.

a, Experimental schema. b, Temperature changes in the respective OVA/SEB-sensitized and OVA-challenged mouse groups: Il4raF709Foxp3EGFP/DTR– (n = 6), Il4raF709Foxp3EGFP/DTR–+Clostridiales+DT (n = 7), Il4raF709Foxp3EGFP/DTR++Clostridiales+DT (n = 8), Il4raF709Foxp3EGFP/DTR–+Bacteroidales+DT (n = 9), Il4raF709Foxp3EGFP/DTR++Bacteroidales+DT (n = 7). c, Total and OVA-specific IgE in the groups listed in b (total IgE: n = 9, 6, 8, 7 and 5; OVA-specific IgE: 6, 5, 5, 5 and 5). d, MMCP-1 concentrations (n = 12 for Il4raF709Foxp3EGFP/DTR–, and n = 8 per group for all other groups). e,f, Frequencies of MLN CD4+Foxp3+ and IL-4+Foxp3+ T cells: Il4raF709Foxp3EGFP/DTR– (n = 6 and 10), Il4raF709Foxp3EGFP/DTR–+Clostridiales+DT (n = 8 and 7), Il4raF709Foxp3EGFP/DTR++Clostridiales+DT (n = 8 and 7), Il4raF709Foxp3EGFP/DTR–+Bacteroidales+DT (n = 8 and 7), Il4raF709Foxp3EGFP/DTR++Bacteroidales+DT (n = 8 and 7). g, Frequencies of ROR-γt+Foxp3+ and GATA3+Foxp3+ T cells: Il4raF709Foxp3EGFP/DTR– (n = 5 per group), Il4raF709Foxp3EGFP/DTR–+Clostridiales+DT (n = 7 and 9), Il4raF709Foxp3EGFP/DTR++Clostridiales+DT (n = 8 per group), Il4raF709Foxp3EGFP/DTR–+Bacteroidales+DT (n = 6 per group), Il4raF709Foxp3EGFP/DTR++Bacteroidales+DT (n = 8 pre group). Each dot represents one mouse. Data represent mean ± s.e.m. from two independent experiments. P values were derived by repeat measures two-way ANOVA (b) or by one-way ANOVA with Dunnett’s post hoc analysis or Student’s unpaired two-tailed t-test (cf). Source data

Extended Data Fig. 8 Oral SCFA supplementation does not protect against FA.

a, SCFA in fecal samples of PBS or OVA/SEB-sensitized WT and Il4raF709 mice. Acetate, propionate and butyrate: WT, PBS or OVA/SEB (n = 5 per group); Il4raF709, PBS (n = 5 per group) or OVA/SEB (n = 10 per group). Isovalerate: WT, PBS or OVA/SEB (n = 5 and 4); Il4raF709, PBS or OVA/SEB (n = 5 and 8). Valerate: WT, PBS or OVA/SEB (n = 5 and 3); Il4raF709, PBS or OVA/SEB (n = 4 and 7). b, Temperature changes in OVA-challenged WT and Il4raF709 mice sensitized with PBS or OVA/SEB without or with SCFA supplementation: WT, PBS+SCFA (n = 10), WT, OVA/SEB (n = 11), WT, OVA/SEB+SCFA (n = 24); Il4raF709, PBS+SCFA (n = 12), Il4raF709, OVA/SEB (n = 7), Il4raF709, OVA/SEB+SCFA (n = 17). c, Total and OVA-specific IgE: WT, PBS+SCFA (n = 5 per group), WT, OVA/SEB (n = 6 per group), WT, OVA/SEB+SCFA (n = 9 per group); Il4raF709, PBS+SCFA (n = 4 per group), Il4raF709, OVA/SEB (n = 8 per group), Il4raF709, OVA/SEB+SCFA (n = 9 per group). d, Frequencies of MLN CD4+Foxp3+ROR-γt+ and CD4+Foxp3ROR-γt+ T cells: WT, PBS+SCFA (n = 5 per group), WT, OVA/SEB (n = 4 per group), WT, OVA/SEB+SCFA (n = 4 per group); Il4raF709, PBS+SCFA (n = 5 per group), Il4raF709, OVA/SEB (n = 5 per group), Il4raF709, OVA/SEB+SCFA (n = 7 per group). Each dot represents one mouse. Data represent mean ± s.e.m. from two independent experiments. P values were derived by the Kolmogorov–Smirnov test (a), by Student’s unpaired two-tailed t-test (c,d) or by two-way ANOVA (b). Source data

Extended Data Fig. 9 Analysis of ROR-γt+ expression in human subjects and mutant mice.

a, Gating strategy for CD4+Foxp3+ (G1) and CD4+Foxp3 T (G2) cells ex vivo. b, Gating strategy for the expression of ROR-γt in Teff cells (G2) from patients with FA, healthy controls (HC) and atopic subjects (atopy), as compared to an isotype control. c, Flow plots and frequencies of peripheral blood CD4+Foxp3+ROR-γt+ T cells in WT and Il4raF709 mice (n = 7 mice per group). d, Flow plots and frequencies of peripheral blood CD4+Foxp3+HeliosNRP1ROR-γt+ T cells in WT and Il4raF709 mice (n = 7 mice per group). e,f, Flow plots and frequencies of MLN CD4+Foxp3+ROR-γt+ T cells from Foxp3YFPCre mice sensitized with OVA/SEB, and Foxp3YFPCreRorcΔ/Δ either sham-sensitized (PBS) or sensitized with OVA/SEB, as indicated (n = 5 mice per group). g, Quantitative PCR with reverse transcription of Rorc gene expression in MLN CD4+Foxp3+ Treg and CD4+Foxp3 Teff cells from Foxp3YFPCre, Foxp3YFPCreRorcΔ/Δ and Il4raF709Foxp3YFPCreRorcΔ/Δ mice. Data were normalized to the endogenous Hprt transcripts (n = 5 mice per group). Each dot represents one mouse. Results represent means ± s.e.m. collated from two independent experiments. P values were derived by Student’s unpaired two-tailed t-test with Welch correction (c,d), or by one-way ANOVA with Dunnett’s post hoc analysis (f,g). Source data

Extended Data Fig. 10 Treg cell-specific deletion of Rorc and Myd88 impairs mucosal tolerance.

ad. Analysis of sIgA+ (a,b) and IgE+ (c,d) fecal bacteria in OVA/SEB-sensitized Foxp3YFPCre, Il4raF709Foxp3YFPCre and Foxp3YFPCreRorcΔ/Δ mice. Fecal pellets from Rag2–/– and Igh7–/–Il4raF709 mice were used as negative controls: Foxp3YFPCre (n = 6 per group), Il4raF709Foxp3YFPCre (n = 11 and 7) and Foxp3YFPCreRorcΔ/Δ mice (n = 10 and 8). ef, Analysis of GATA3+Foxp3+ Treg cells in the following OVA/SEB-sensitized mice that were either untreated or treated with Clostridiales or Bacteroidales consortium: Il4raF709Foxp3YFPCre (n = 9, 5 and 5) and Il4raF709Foxp3YFPCreRorcΔ/Δ (n = 4, 5 and 8). g,h, Analysis of GATA3+Foxp3+ Treg cells in OVA/SEB-sensitized Il4raF709Foxp3YFPCre mice treated with the Bacteroidales consortium (n = 9), and in OVA/SEB-sensitized Il4raF709Foxp3YFPCreMyd88Δ/Δ mice otherwise untreated or treated with the Clostridiales or Bacteroidales consortium (n = 8, 9 and 8). Each symbol represents one mouse. Results represent means ± s.e.m. collated from two independent experiments. P values were derived by one-way ANOVA with Dunnett’s post hoc analysis (b,d,f,h). Source data

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Abdel-Gadir, A., Stephen-Victor, E., Gerber, G.K. et al. Microbiota therapy acts via a regulatory T cell MyD88/RORγt pathway to suppress food allergy. Nat Med 25, 1164–1174 (2019). https://doi.org/10.1038/s41591-019-0461-z

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