Abstract

There has been a striking generational increase in life-threatening food allergies in Westernized societies1,2. One hypothesis to explain this rising prevalence is that twenty-first century lifestyle practices, including misuse of antibiotics, dietary changes, and higher rates of Caesarean birth and formula feeding have altered intestinal bacterial communities; early-life alterations may be particularly detrimental3,4. To better understand how commensal bacteria regulate food allergy in humans, we colonized germ-free mice with feces from healthy or cow’s milk allergic (CMA) infants5. We found that germ-free mice colonized with bacteria from healthy, but not CMA, infants were protected against anaphylactic responses to a cow’s milk allergen. Differences in bacterial composition separated the healthy and CMA populations in both the human donors and the colonized mice. Healthy and CMA colonized mice also exhibited unique transcriptome signatures in the ileal epithelium. Correlation of ileal bacteria with genes upregulated in the ileum of healthy or CMA colonized mice identified a clostridial species, Anaerostipes caccae, that protected against an allergic response to food. Our findings demonstrate that intestinal bacteria are critical for regulating allergic responses to dietary antigens and suggest that interventions that modulate bacterial communities may be therapeutically relevant for food allergy.

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Code availability

The open-source analysis software used in this study is publicly available and referenced as appropriate. Custom codes are available from the corresponding author upon request.

Data availability

The data that support the findings of this study are available from the corresponding author upon request. The 16S rRNA and RNA-seq raw FastQ sequencing files were deposited into the National Center for Biotechnology Information Sequence Read Archive and are available under the accession numbers SRP130620 and SRP130644, respectively. Additional processed data reported in this study are available upon request.

Additional information

Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Change history

  • 25 January 2019

    In the version of the article originally published, there was a hyperlinking error in the callout to Extended Data Fig. 7 at the end of the ‘16s RNA-targeted sequencing section' of the Methods. The hyperlink led to Extended Data Fig. 6 instead of Extended Data Fig. 7. The error has been corrected online.

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Acknowledgements

We thank the children and families for their participation in this study. We are grateful to D. Wesemann, E. Forbes-Blom, G. Nunez, M. Rothenberg, M. Alegre and J. Colson for discussion. We thank S. Wang, M. Bauer and A. Kemter for assistance with some experiments, M. Jarsulic for technical assistance with computing infrastructure, K. Hernandez for discussion of the statistical results and C. Weber for histopathological evaluation of all intestinal sections. Statistical consultation was also provided by M. Giurcanu of the University of Chicago Biostatistics Laboratory. We are grateful to B. Theriault and her staff at the University of Chicago Gnotobiotic Research Animal Facility for superb animal care and experimental support. This work was supported by the Sunshine Charitable Foundation (C.R.N.), a pilot award from the University of Chicago Institute for Translational Medicine (CTSA ULI TR000430, C.R.N.), National Institutes of Health (NIH) grants AI134923 (C.R.N.), DK42086 (D.A.A.) and an Italian Ministry of Health grant PE-2011-02348447 (R.B.C.). The Center for Research Informatics is funded by the Biological Sciences Division at the University of Chicago with additional support provided by the Institute for Translational Medicine/Clinical and Translational Award (NIH 5UL1TR002389-02), and the University of Chicago Comprehensive Cancer Center Support Grant (NIH P30CA014599). Bioinformatics analysis was performed on Gardner High-Performance Computing clusters at the Center for Research Informatics at the University of Chicago. A provisional US patent application (62/755,945) was filed on 5 November 2018.

Author information

Author notes

  1. These authors contributed equally: Taylor Feehley, Catherine H. Plunkett, Riyue Bao.

Affiliations

  1. Department of Pathology and Committee on Immunology, The University of Chicago, Chicago, IL, USA

    • Taylor Feehley
    • , Catherine H. Plunkett
    • , Sung Min Choi Hong
    • , Elliot Culleen
    • , Pedro Belda-Ferre
    • , Evelyn Campbell
    •  & Cathryn R. Nagler
  2. Center for Research Informatics, The University of Chicago, Chicago, IL, USA

    • Riyue Bao
    •  & Jorge Andrade
  3. Department of Pediatrics, The University of Chicago, Chicago, IL, USA

    • Riyue Bao
    •  & Jorge Andrade
  4. Department of Translational Medical Science, Section of Pediatrics, University of Naples Federico II, Naples, Italy

    • Rosita Aitoro
    • , Rita Nocerino
    • , Lorella Paparo
    •  & Roberto Berni Canani
  5. Department of Medicine, The University of Chicago, Chicago, IL, USA

    • Dionysios A. Antonopoulos
  6. Biosciences Division, Argonne National Laboratory, Argonne, IL, USA

    • Dionysios A. Antonopoulos
  7. European Laboratory for the Investigation of Food-Induced Diseases, University of Naples Federico II, Naples, Italy

    • Roberto Berni Canani
  8. CEINGE Advanced Biotechnologies, University of Naples Federico II, Naples, Italy

    • Roberto Berni Canani

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Contributions

T.F., C.H.P., R.B., R.B.C., and C.R.N. designed the study. C.H.P. and T.F. performed mouse experiments with help from P.B.-F., R.A., E.Culleen, E.Campbell, and S.M.C.H. R.B., P.B.-F., and J.A. performed bioinformatics analysis. T.F., C.H.P., R.B., P.B.F., and C.R.N. analyzed results. R.B.C., R.N., and L.A. cared for patients and provided donor fecal samples. D.A.A. provided protocols and assisted with the colonization of germ-free mice with human feces and A. caccae. T.F., C.H.P., R.B., R.B.C., and C.R.N. wrote the manuscript. All authors read and commented on the manuscript.

Competing interests

C.R.N. is president and co-founder of ClostraBio, Inc. The other authors declare no competing interests.

Corresponding author

Correspondence to Cathryn R. Nagler.

Extended data

  1. Extended Data Fig. 1 Sensitization of healthy- or CMA-colonized mice with BLG plus cholera toxin does not result in intestinal pathology.

    Representative images of histological samples from BLG plus cholera toxin-sensitized healthy- or CMA-colonized mice 24 h post-challenge for donors 1 (healthy) and 5 (CMA; see Supplementary Table 1). All sections stained with H&E or PAS, as indicated. Scale bars, 100 μm.

  2. Extended Data Fig. 2 Long-term colonization of germ-free mice with feces from healthy or CMA infants does not lead to intestinal pathology.

    Representative images of histological samples from unsensitized healthy- or CMA-colonized mice collected 5 to 6 months post-colonization for donors described in Supplementary Table 1. All sections stained with H&E or PAS, as indicated. Scale bars, 100 μm.

  3. Extended Data Fig. 3 Diversity analysis of fecal samples from healthy- or CMA-colonized mice.

    Shannon diversity index (a) and Pielou’s evenness index (b) in feces from healthy-colonized (orange) and CMA-colonized (blue) mice from Fig. 2a. n = 1–4 mice per colonized mouse group with feces taken at 2 and 3 weeks post-colonization; see Online Methods). Each circle represents one fecal sample; bars represent mean  +s.e.m. The eight human formula-fed fecal donors are described in Supplementary Table 1.

  4. Extended Data Fig. 4 Transfer of a healthy, exclusively breast-fed infant microbiota protects against an anaphylactic response to sensitization with BLG plus  cholera toxin.

    a, Change in core body temperature at indicated time points following first challenge with BLG of mice colonized with feces from breast-fed healthy or CMA infant donors (n = 13 mice per group, collected from at least 2 independent experiments). bd, Serum BLG-specific IgE (b), BLG-specific IgG1 (c) and mMCPT-1 (d) from mice in a. Four of the BLG plus cholera toxin-sensitized CMA-colonized mice died of anaphylaxis following challenge. For a, symbols represent mean, and bars represent s.e.m. For bd, symbols represent individual mice, and bars represent mean + s.e.m. Linear mixed-effect models were used to compare groups in a and two-sided Student’s t-test in b after log transformation. The two human breast-fed fecal donors are described in Supplementary Table 2. *P < 0.05.

  5. Extended Data Fig. 5 Continuous exposure to cow’s milk does not induce tolerance to BLG in germ-free mice fed with water or Enfamil and sensitized with BLG plus cholera toxin.

    a, Change in core body temperature at indicated time points following first challenge with BLG of mice fed with water (n = 12) or Enfamil (n = 10) collected from 3 independent experiments. bd, serum BLG-specific IgE (b), BLG-specific IgG1 (c) and mMCPT-1 (d) from mice in a. For a, circles represent mean, and error bars represent s.e.m. For bd, circles represent individual mice, and bars represent mean + s.e.m. Linear mixed-effect models were used to compare groups in a and two-sided Student’s t-test in bd after log transformation. **P < 0.01. n.s. = not significant (P = 0.36).

  6. Extended Data Fig. 6 Binary representation of protective and non-protective OTUs in CMA and healthy donors and colonized mouse groups.

    a, Binary map of the presence/absence ratio of protective/non-protective OTUs in CMA and healthy donors with the same layout as Fig. 2a. Columns depict each donor (D) or colonized mouse group (m). n = 2–3 technical replicates per donor and n = 1–4 mice per colonized mouse group, with feces taken at 2 and 3 weeks post-colonization; see Online Methods). Rows show 58 OTUs FDR controlled at 0.10 (see Online Methods) in human CMA versus healthy donor comparison, present in at least 4 human fecal samples and at least 2 groups of colonized mice (see Supplementary Table 3). The bar graphs above the grid map represent the total number of potentially protective (more abundant in healthy donors; orange) and potentially non-protective (more abundant in CMA donors; blue) OTUs in each individual donor or mouse group. The grid map represents presence (green) or absence (white) of protective and non-protective OTUs in each sample. b, A protective/non-protective OTU ratio was computed per individual donor or mouse group from a, taking into consideration the presence or absence of 58 OTUs. The donors and their murine transfer recipients are shown in squares and circles, respectively. The vertical dashed line represents a ratio of 2.6.

  7. Extended Data Fig. 7 Validation of protective/non-protective OTU ratio using a larger, independent cohort of healthy and CMA infant donors.

    Box plots showing the protective/non-protective OTU ratio (see Fig. 2 and Extended Data Fig. 6) in fecal samples from healthy (n = 19) and CMA (n = 19) infants from ref. 5. The horizontal center line indicates the median, the boxes represent the 25th and 75th percentiles, and the whiskers extend to the farthest data point within a maximum of 1.5 times the interquartile range (IQR). All individual points are shown, with each circle denoting a subject. Out of the 58 OTUs shown in Fig. 2a, 55 OTUs were assigned with known reference IDs and 3 with new reference IDs. The new reference OTU IDs are not comparable across the different analysis cohorts, so we focused on the OTUs with known reference IDs. Among the 55 known OTUs, 52 (29 protective OTUs and 23 non-protective OTUs) were detected in this cohort and were used for the ratio calculation (see Online Methods). The other 3 were not detected. Two-sided Wilcoxon rank sum test was used. *P < 0.05.

  8. Extended Data Fig. 8 The healthy versus CMA OTU abundance ratio is significantly correlated between mouse fecal and ileal samples.

    a, Bubble plots show a similar pattern in fecal (n = 8 mice in healthy group, n = 9 mice in CMA group, with fecal samples collected at 2 and 3 weeks post-colonization, same as in Fig. 2a) and ileal samples (n = 22 mice in healthy group, n = 25 mice in CMA group) from healthy- and CMA-colonized mice; 58 OTUs significantly differentially abundant between CMA and healthy donors are shown in the same order as in Fig. 2a. The size of the circle indicates the magnitude of relative abundance enrichment towards either CMA or healthy. Color intensity indicates the statistical significance computed using the DS-FDR permutation test (see Online Methods). b,c, The healthy versus CMA OTU abundance ratio is significantly correlated between mouse fecal and ileal samples. Each dot represents one individual OTU. For b, for each OTU, its average abundance was calculated at the group level among 8 healthy-colonized and 9 CMA-colonized mice for the fecal samples, and among 22 healthy-colonized and 25 CMA-colonized mice for the ileal samples. The ratios of OTU abundance in the feces are plotted on the x axis with the ratio of OTU abundance in the ileum on the y axis. For c, n = 35 (15 healthy-colonized and 20 CMA-colonized) mice collected from at least 2 independent experiments were used for the calculation of both the fecal and ileal OTU abundance ratio, where fecal and ileal samples were collected from the same individual mice. For further details, see the Online Methods.

  9. Extended Data Fig. 9 Abundance of OTU259772 (Lachnospiraceae) and A. caccae are correlated in fecal samples from healthy- and CMA-colonized mice.

    a,b, Abundance of OTU259772 (Lachnospiraceae) from the 16S dataset (a) and abundance of A. caccae by qPCR (b) in fecal samples from healthy-colonized (n = 7) and CMA-colonized (n = 8) mice from Fig. 2. For each individual mouse, 1–2 fecal samples were collected at 2 and 3 weeks post-colonization. LD indicates samples that were below the limit of detection for the assay. c, Spearman’s correlation between abundance of OTU259772 (Lachnospiraceae; 16S sequencing) and abundance of A. caccae (qPCR) in fecal samples from healthy- and CMA-colonized mice from Fig. 2. Fecal samples that were above LD in both 16S and qPCR experiments are shown (n = 13). Each circle represents one fecal sample. For a and b, bars show mean + s.e.m. For c, shaded bands indicate 95% confidence interval fitted by linear regression. The DS-FDR method was used to compare groups in a and two-sided Student’s t-test in b. ***P < 0.001.

  10. Extended Data Fig. 10 Abundance of A. caccae in ileal samples correlates with gene expression in ileal IECs.

    Spearman’s correlation between abundance of A. caccae by qPCR and RNA-seq gene expression of Ror2, Fbp1, Tgfbr3, Acot12 and Me1 in ileal IECs (see Fig. 3a). Circles show individual mice, and shaded bands indicate 95% confidence interval fitted by linear regression. n = 36 (18 healthy and 18 CMA-colonized) mice collected from at least 2 independent experiments. Samples with values above the limit of detection are shown (A. caccae abundance > 0).

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