• An Erratum to this article was published on 12 February 2014

Abstract

Gut commensal microbes shape the mucosal immune system by regulating the differentiation and expansion of several types of T cell1,2,3,4,5. Clostridia, a dominant class of commensal microbe, can induce colonic regulatory T (Treg) cells, which have a central role in the suppression of inflammatory and allergic responses3. However, the molecular mechanisms by which commensal microbes induce colonic Treg cells have been unclear. Here we show that a large bowel microbial fermentation product, butyrate, induces the differentiation of colonic Treg cells in mice. A comparative NMR-based metabolome analysis suggests that the luminal concentrations of short-chain fatty acids positively correlates with the number of Treg cells in the colon. Among short-chain fatty acids, butyrate induced the differentiation of Treg cells in vitro and in vivo, and ameliorated the development of colitis induced by adoptive transfer of CD4+ CD45RBhi T cells in Rag1−/− mice. Treatment of naive T cells under the Treg-cell-polarizing conditions with butyrate enhanced histone H3 acetylation in the promoter and conserved non-coding sequence regions of the Foxp3 locus, suggesting a possible mechanism for how microbial-derived butyrate regulates the differentiation of Treg cells. Our findings provide new insight into the mechanisms by which host–microbe interactions establish immunological homeostasis in the gut.

Access optionsAccess options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Accessions

DDBJ/GenBank/EMBL

Gene Expression Omnibus

Data deposits

The microarray and ChIP-seq analysis data have been deposited at the Gene Expression Omnibus (GEO) under accession number GSE49655. The microbiome analysis data have been deposited at the DDBJ database (http://getentry.ddbj.nig.ac.jp/) under accession number DRA001105.

References

  1. 1.

    et al. Gut immune maturation depends on colonization with a host-specific microbiota. Cell 149, 1578–1593 (2012)

  2. 2.

    et al. Induction of intestinal Th17 cells by segmented filamentous bacteria. Cell 139, 485–498 (2009)

  3. 3.

    et al. Induction of colonic regulatory T cells by indigenous clostridium species. Science 331, 337–341 (2011)

  4. 4.

    et al. Intestinal bacterial colonization induces mutualistic regulatory T cell responses. Immunity 34, 794–806 (2011)

  5. 5.

    & Inducible Foxp3+ regulatory T-cell development by a commensal bacterium of the intestinal microbiota. Proc. Natl Acad. Sci. USA 107, 12204–12209 (2010)

  6. 6.

    & Characterization of clostridia isolated from faeces of limited flora mice and their effect on caecal size when associated with germ-free mice. Lab. Anim. 19, 111–118 (1985)

  7. 7.

    et al. Expression of Helios, an Ikaros transcription factor family member, differentiates thymic-derived from peripherally induced Foxp3+ T regulatory cells. J. Immunol. 184, 3433–3441 (2010)

  8. 8.

    et al. Neuropilin-1 distinguishes natural and inducible regulatory T cells among regulatory T cell subsets in vivo. J. Exp. Med. 209, 1713–1722 (2012)

  9. 9.

    et al. Neuropilin 1 is expressed on thymus-derived natural regulatory T cells, but not mucosa-generated induced Foxp3+ T reg cells. J. Exp. Med. 209, 1723–1742 (2012)

  10. 10.

    , & Acetylated, propionylated or butyrylated starches raise large bowel short-chain fatty acids preferentially when fed to rats. J. Nutr. 133, 3523–3528 (2003)

  11. 11.

    et al. Regulatory T cell-derived interleukin-10 limits inflammation at environmental interfaces. Immunity 28, 546–558 (2008)

  12. 12.

    , , , & Recognition of commensal microflora by Toll-like receptors is required for intestinal homeostasis. Cell 118, 229–241 (2004)

  13. 13.

    , & A microbial symbiosis factor prevents intestinal inflammatory disease. Nature 453, 620–625 (2008)

  14. 14.

    , & Sodium butyrate inhibits histone deacetylation in cultured cells. Cell 14, 105–113 (1978)

  15. 15.

    Inhibition of histone deacetylase activity by butyrate. J. Nutr. 133, 2485S–2493S (2003)

  16. 16.

    , , , & Inhibition of HDAC9 increases T regulatory cell function and prevents colitis in mice. Gastroenterology 138, 583–594 (2010)

  17. 17.

    et al. Deacetylase inhibition promotes the generation and function of regulatory T cells. Nature Med. 13, 1299–1307 (2007)

  18. 18.

    , & Regulatory T cells: mechanisms of differentiation and function. Annu. Rev. Immunol. 30, 531–564 (2012)

  19. 19.

    et al. Role of conserved non-coding DNA elements in the Foxp3 gene in regulatory T-cell fate. Nature 463, 808–812 (2010)

  20. 20.

    et al. Development of Foxp3+ regulatory T cells is driven by the c-Rel enhanceosome. Immunity 31, 932–940 (2009)

  21. 21.

    , , , & Phenotypically distinct subsets of CD4+ T cells induce or protect from chronic intestinal inflammation in C. B-17 scid mice. Int. Immunol. 5, 1461–1471 (1993)

  22. 22.

    et al. Regulation of inflammatory responses by gut microbiota and chemoattractant receptor GPR43. Nature 461, 1282–1286 (2009)

  23. 23.

    et al. The orphan G protein-coupled receptors GPR41 and GPR43 are activated by propionate and other short chain carboxylic acids. J. Biol. Chem. 278, 11312–11319 (2003)

  24. 24.

    et al. The luminal short-chain fatty acid butyrate modulates NF-κB activity in a human colonic epithelial cell line. Gastroenterology 118, 724–734 (2000)

  25. 25.

    et al. Down-regulation of the monocarboxylate transporter 1 is involved in butyrate deficiency during intestinal inflammation. Gastroenterology 133, 1916–1927 (2007)

  26. 26.

    et al. Molecular-phylogenetic characterization of microbial community imbalances in human inflammatory bowel diseases. Proc. Natl Acad. Sci. USA 104, 13780–13785 (2007)

  27. 27.

    et al. Effect of butyrate enemas on the colonic mucosa in distal ulcerative colitis. Gastroenterology 103, 51–56 (1992)

  28. 28.

    , , & Treatment of diversion colitis with short-chain-fatty acid irrigation. N. Engl. J. Med. 320, 23–28 (1989)

  29. 29.

    et al. Plasticity of Foxp3+ T cells reflects promiscuous Foxp3 expression in conventional T cells but not reprogramming of regulatory T cells. Immunity 36, 262–275 (2012)

  30. 30.

    et al. Control of immune responses by antigen-specific regulatory T cells expressing the folate receptor. Immunity 27, 145–159 (2007)

  31. 31.

    et al. Isolation and subsequent analysis of murine lamina propria mononuclear cells from colonic tissue. Nature Protocols 2, 2307–2311 (2007)

  32. 32.

    et al. New monitoring approach for metabolic dynamics in microbial ecosystems using stable-isotope-labeling technologies. J. Biosci. Bioeng. 110, 87–93 (2010)

  33. 33.

    et al. Lymphoid tissue genesis induced by commensals through NOD1 regulates intestinal homeostasis. Nature 456, 507–510 (2008)

  34. 34.

    et al. Robustness of gut microbiota of healthy adults in response to probiotic intervention revealed by high-throughput pyrosequencing. DNA Res. 20, 241–253 (2013)

  35. 35.

    et al. Viruses in the faecal microbiota of monozygotic twins and their mothers. Nature 466, 334–338 (2010)

  36. 36.

    et al. Clustal W and Clustal X version 2.0. Bioinformatics 23, 2947–2948 (2007)

  37. 37.

    & Interactive Tree Of Life (iTOL): an online tool for phylogenetic tree display and annotation. Bioinformatics 23, 127–128 (2006)

  38. 38.

    et al. Bifidobacteria can protect from enteropathogenic infection through production of acetate. Nature 469, 543–547 (2011)

  39. 39.

    et al. Evaluation and characterization of bacterial metabolic dynamics with a novel profiling technique, real-time metabolotyping. PLoS ONE 4, e4893 (2009)

  40. 40.

    , & 1H NMR metabolite fingerprinting and metabolomic analysis of perchloric acid extracts from plant tissues. Nature Protocols 3, 1001–1012 (2008)

  41. 41.

    et al. Visualization of GC/TOF-MS-based metabolomics data for identification of biochemically interesting compounds using OPLS class models. Anal. Chem. 80, 115–122 (2008)

  42. 42.

    , & Stable isotope labeling of Arabidopsis thaliana for an NMR-based metabolomics approach. Plant Cell Physiol. 45, 1099–1104 (2004)

  43. 43.

    et al. Top-down phenomics of Arabidopsis thaliana: metabolic profiling by one- and two-dimensional nuclear magnetic resonance spectroscopy and transcriptome analysis of albino mutants. J. Biol. Chem. 282, 18532–18541 (2007)

  44. 44.

    , & Profiling polar and semipolar plant metabolites throughout extraction processes using a combined solution-state and high-resolution magic angle spinning NMR approach. Anal. Chem. 82, 1643–1652 (2010)

  45. 45.

    et al. PRIMe: a Web site that assembles tools for metabolomics and transcriptomics. In Silico Biol. 8, 339–345 (2008)

  46. 46.

    et al. Statistical indices for simultaneous large-scale metabolite detections for a single NMR spectrum. Anal. Chem. 82, 1653–1658 (2010)

  47. 47.

    , , & The influence of Staphylococcus aureus on gut microbial ecology in an in vitro continuous culture human colonic model system. PLoS ONE 6, e23227 (2011)

  48. 48.

    et al. Resistant proteins alter cecal short-chain fatty acid profiles in rats fed high amylose cornstarch. J. Nutr. 128, 1156–1164 (1998)

  49. 49.

    et al. Epithelial cell-intrinsic Notch signaling plays an essential role in the maintenance of gut immune homeostasis. J. Immunol. 188, 2427–2436 (2012)

  50. 50.

    et al. DNA double-strand breaks induced by cavitational mechanical effects of ultrasound in cancer cell lines. PLoS ONE 7, e29012 (2012)

Download references

Acknowledgements

We would like to thank P. Carninci, Y. Shinkai and M. Yoshida for discussion; Y. Chiba and S. Yamada for technical support; H. Sugahara for technical advice; and P. D. Burrows for critical reading and editing of the manuscript. This work was supported in part by grants from Japanese Ministry of Education, Culture, Sports, Science and Technology (24117524 to S.F.; 21022049 to K.Ha.; 20113003 to H.O.), The Japan Society for the Promotion of Science (24890293 to Y.F.; 252667 to Y.O.; 24380072 and 24658129 to S.F.; 22689017 to K.Ha.; 21390155 to H.O.), The Japan Science and Technology Agency (K.Ha., K.A. and K.Ho.), RIKEN President’s Special Research Grant (H.O.), RIKEN RCAI Young Chief Investigator program (K.Ha.), the Institute for Fermentation, Osaka (S.F.), the Mishima Kaiun Memorial Foundation (S.F.), The Takeda Science Foundation (S.F. and H.O.), The Mitsubishi Foundation (H.O.), and The Uehara Memorial Foundation (S.F. and K.Ha.).

Author information

Author notes

    • Yukihiro Furusawa
    • , Yuuki Obata
    • , Shinji Fukuda
    •  & Koji Hase

    These authors contributed equally to this work.

Affiliations

  1. RIKEN Center for Integrative Medical Sciences (IMS-RCAI), Kanagawa 230-0045, Japan

    • Yukihiro Furusawa
    • , Yuuki Obata
    • , Shinji Fukuda
    • , Takaho A. Endo
    • , Gaku Nakato
    • , Daisuke Takahashi
    • , Chikako Uetake
    • , Keiko Kato
    • , Tamotsu Kato
    • , Masumi Takahashi
    • , Eiji Miyauchi
    • , Koji Atarashi
    • , Satoshi Onawa
    • , Shohei Hori
    • , Osamu Ohara
    • , Haruhiko Koseki
    • , Kenya Honda
    • , Koji Hase
    •  & Hiroshi Ohno
  2. The Institute of Medical Science, The University of Tokyo, Tokyo 108-8639, Japan

    • Yukihiro Furusawa
    • , Yuuki Obata
    • , Yumiko Fujimura
    •  & Koji Hase
  3. Graduate School of Medicine, Chiba University, Chiba 260-8670, Japan

    • Yuuki Obata
    • , Haruhiko Koseki
    •  & Hiroshi Ohno
  4. Institute for Advanced Biosciences, Keio University, Yamagata 997-0052, Japan

    • Shinji Fukuda
    • , Yumiko Nakanishi
    • , Noriko N. Fukuda
    • , Shinnosuke Murakami
    •  & Masaru Tomita
  5. Graduate School of Medical Life Science, Yokohama City University, Kanagawa 230-0045, Japan

    • Keiko Kato
    • , Haruhiko Koseki
    • , Jun Kikuchi
    •  & Hiroshi Ohno
  6. Faculty of Agriculture, Shizuoka University, Shizuoka 422-8529, Japan

    • Shingo Hino
    •  & Tatsuya Morita
  7. PRESTO, Japan Science and Technology Agency, Saitama 332-0012, Japan

    • Koji Atarashi
    •  & Koji Hase
  8. Preventative Health National Research Flagship, CSIRO Food and Nutritional Sciences, South Australia 5000, Australia

    • Trevor Lockett
    • , Julie M. Clarke
    •  & David L. Topping
  9. RIKEN Center for Sustainable Resource Science, Kanagawa 230-0045, Japan

    • Jun Kikuchi
  10. CREST, Japan Science and Technology Agency, Saitama 332-0012, Japan

    • Kenya Honda

Authors

  1. Search for Yukihiro Furusawa in:

  2. Search for Yuuki Obata in:

  3. Search for Shinji Fukuda in:

  4. Search for Takaho A. Endo in:

  5. Search for Gaku Nakato in:

  6. Search for Daisuke Takahashi in:

  7. Search for Yumiko Nakanishi in:

  8. Search for Chikako Uetake in:

  9. Search for Keiko Kato in:

  10. Search for Tamotsu Kato in:

  11. Search for Masumi Takahashi in:

  12. Search for Noriko N. Fukuda in:

  13. Search for Shinnosuke Murakami in:

  14. Search for Eiji Miyauchi in:

  15. Search for Shingo Hino in:

  16. Search for Koji Atarashi in:

  17. Search for Satoshi Onawa in:

  18. Search for Yumiko Fujimura in:

  19. Search for Trevor Lockett in:

  20. Search for Julie M. Clarke in:

  21. Search for David L. Topping in:

  22. Search for Masaru Tomita in:

  23. Search for Shohei Hori in:

  24. Search for Osamu Ohara in:

  25. Search for Tatsuya Morita in:

  26. Search for Haruhiko Koseki in:

  27. Search for Jun Kikuchi in:

  28. Search for Kenya Honda in:

  29. Search for Koji Hase in:

  30. Search for Hiroshi Ohno in:

Contributions

S.F., K.Ha., D.L.T., T.M., K.Ho. and H.O. conceived the study; K.Ha. and S.F. designed the experiments and wrote the manuscript with Y.Fur., Y.O. and H.O.; Y. Fur. and Y.O conducted a large part of experiments together with S.F., G.N., D.T., C.U., K.K., T.K., M.Ta., E.M. and K.Ha; S.F, S.O. and K.Ha. prepared germ-free, CRB-associated and gnotobiotic mice. K.A. and K.Ho. were involved in data discussion. S.F., Y.N., C.U. and J.K. performed metabolome analysis. S.F., T.K., S.M. and M.To. performed microbiome analysis. T.A.E. performed bioinformatic analyses. S.Hi. and T.M. performed HPLC analysis. S.F. and N.N.F. performed GC–MS analysis. Y.Fuj. performed histological analysis. T.L., J.M.C., D.L.T. and S.Ho. provided essential materials and contributed to the design of experiments. Y.Fur. and H.K. contributed to the ChIP assay. H.O. directed the study and took primary responsibility for editing the manuscript.

Competing interests

The authors declare no competing financial interests.

Corresponding authors

Correspondence to Shinji Fukuda or Koji Hase or Hiroshi Ohno.

Supplementary information

PDF files

  1. 1.

    Supplementary Information

    This file contains Supplementary Figures 1-22 and Supplementary Table 1.

About this article

Publication history

Received

Accepted

Published

DOI

https://doi.org/10.1038/nature12721

Further reading Further reading

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.