Microbial bile acid metabolites modulate gut RORγ+ regulatory T cell homeostasis (original) (raw)

Data availability

The microarray, RNA-seq and 16S rRNA profiling data are available in the NCBI database under accession numbers GSE68009, GSE137405 and PRJNA573477, respectively. The MS data are available in the MetaboLights database with the identifier MTBLS1276.

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Acknowledgements

We thank D. Gardner, Y. Li and T. Hla for providing mouse strains, and C. Fu for help with microscope. We also thank T. Sherpa and J. Ramos for help with GF mice and J. McCoy for manuscript editing. This work was supported in part by a Sponsored Research Agreement from UCB Pharma and Evelo Biosciences. S.F.O. was supported by NIH K01 DK102771.

Author information

Author notes

  1. Naama Geva-Zatorsky
    Present address: Department of Cell Biology and Cancer Science, Rappaport Faculty of Medicine, Technion Integrated Cancer Center, Technion–Israel Institute of Technology, Haifa, Israel
  2. These authors contributed equally: Xinyang Song, Ximei Sun

Authors and Affiliations

  1. Department of Immunology, Blavatnik Institute, Harvard Medical School, Boston, MA, USA
    Xinyang Song, Ximei Sun, Sungwhan F. Oh, Meng Wu, Yanbo Zhang, Wen Zheng, Naama Geva-Zatorsky, Diane Mathis, Christophe Benoist & Dennis L. Kasper
  2. Center for Experimental Therapeutics and Reperfusion Injury, Department of Anesthesiology, Perioperative and Pain Medicine, Brigham and Women’s Hospital, Harvard Medical School, Boston, MA, USA
    Sungwhan F. Oh
  3. UCB Pharma, Slough, UK
    Ray Jupp

Authors

  1. Xinyang Song
  2. Ximei Sun
  3. Sungwhan F. Oh
  4. Meng Wu
  5. Yanbo Zhang
  6. Wen Zheng
  7. Naama Geva-Zatorsky
  8. Ray Jupp
  9. Diane Mathis
  10. Christophe Benoist
  11. Dennis L. Kasper

Contributions

D.L.K. and X. Song designed the experiments and wrote the manuscript. X. Song, X. Sun, S.F.O., M.W., Y.Z., W.Z. and N.G.-Z. conducted or helped with the experiments. X. Song, X. Sun and Y.Z. analysed the data. R.J., D.M. and C.B. were involved in data discussions and edited the manuscript. D.L.K. supervised the study.

Corresponding author

Correspondence toDennis L. Kasper.

Ethics declarations

Competing interests

The authors declare no competing interests.

Additional information

Peer review information Nature thanks Pieter Dorrestein, Hiroshi Ohno and the, other, anonymous reviewer(s) for their contribution to the peer review of this work.

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

Extended data figures and tables

Extended Data Fig. 1 Both dietary and microbial factors control the number of colonic RORγ+ Treg cells.

a, Beginning at 3 weeks of age, three groups of mice were fed special diets for 4 weeks. SPF mice were fed either a nutrient-rich or a minimal diet, and GF mice were fed the nutrient-rich diet. Colonic Treg cells were analysed, and absolute numbers of RORγ+Helios− in the FOXP3+CD4+TCRβ+ Treg cell population are shown. b, c, Three-week-old SPF mice were fed as in a, and Treg cells in different tissues were analysed after 4 weeks. Representative plots (b) and frequencies of RORγ+Helios− in the FOXP3+CD4+TCRβ+ Treg cell population (c) are shown. iLN, inguinal lymph node. d–f, SPF mice were fed a nutrient-rich or a minimal diet at birth and were either maintained on that diet or switched to the opposite diet at 3 weeks of age. Colonic Treg cells were analysed after 4 weeks. Representative plots of RORγ+Helios− in the FOXP3+CD4+TCRβ+ Treg cell population (d), and the frequencies of FOXP3+ in the CD4+TCRβ+ cell population (e) and RORγ+Helios− in the colonic FOXP3+CD4+TCRβ+ Treg cell population (f) are shown. g–i, LC–MS quantification of faecal acetate (g), propionate (h) and butyrate (i) from SPF mice fed a nutrient-rich or a minimal diet, and from GF mice fed a nutrient-rich diet. j, Three-week-old SPF mice were fed a nutrient-rich diet, a minimal diet, or a minimal diet supplemented with individual or mixed SCFAs in drinking water. Colonic Treg cells were analysed after 4 weeks. Frequencies of RORγ+Helios− in the FOXP3+CD4+TCRβ+ Treg cell population are shown. Data are representative of two independent experiments. n represents biologically independent animals. Data are mean ± s.e.m. (a, c and e–j). *P < 0.05, **P < 0.01, ***P < 0.001, one-way ANOVA followed by the Bonferroni post hoc test (a, e–i) or two-tailed Student’s _t_-test (c).

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Extended Data Fig. 2 Intestinal BAs regulate the number of colonic Treg cells.

a, b, Absolute numbers of RORγ+Helios− in the colonic FOXP3+CD4+TCRβ+ Treg cell population (a) and of FOXP3+ Treg cells in the CD4+TCRβ+ population (b) in SPF mice fed a nutrient-rich diet, a minimal diet, or a minimal diet supplemented with mixtures of primary or secondary BAs in drinking water. The primary BAs were CA, CDCA and UDCA (2 mM of each). The secondary BAs were 3-oxo-CA, 3-oxo-LCA, 7-oxo-CA, 7-oxo-CDCA, 12-oxo-CA, 12-oxo-DCA, DCA and LCA (1 mM of each). c, Three-week-old SPF mice were fed a nutrient-rich diet, a minimal diet, or a minimal diet supplemented with one or more primary or secondary BAs in drinking water. Colonic TH17 cells were analysed after 4 weeks. CA, CDCA, UDCA, DCA, LCA, 3-oxo-CA, 3-oxo-LCA, 7-oxo-CA, 7-oxo-CDCA, 12-oxo-CA, 12-oxo-DCA and the indicated BA combinations were tested. Frequencies of RORγ+FOXP3− in the CD4+TCRβ+ cell population are shown. d, e, Three-week-old SPF mice were fed a nutrient-rich diet, a minimal diet, or a minimal diet supplemented with the indicated primary BAs (CA/CDCA/UDCA, 2 mM of each) or the secondary BAs (oxo-BAs/LCA/DCA, 1 mM of each) in drinking water. Treg cells and TH17 cells in the spleen, mesenteric lymph node and ileum were analysed after 4 weeks. Frequencies of RORγ+Helios− in the FOXP3+CD4+TCRβ+ Treg cell population (d) and of RORγ+FOXP3− in the CD4+TCRβ+ cell population (e) are shown. Data are pooled from two or three independent experiments. n represents biologically independent animals. Data are mean ± s.e.m. **P < 0.01, ***P < 0.001, one-way ANOVA followed by the Bonferroni post hoc test.

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Extended Data Fig. 3 Colonic microbial profiling of rich-diet mice versus minimal-diet mice.

a–d, Three-week-old SPF mice were fed a nutrient-rich or a minimal diet, and the microbial compositions in the colonic lumen were analysed after 4 weeks by 16S rRNA sequencing. Observed operational taxonomic units (OTUs) (a), Shannon index (b), principal coordinates analysis (PCoA) (c) and the relative abundance of bacteria at the phylum and family levels (d) are shown. e, Quantitative PCR analysis of 16S rDNA of Clostridium cluster IV and Clostridium cluster XIVα in colonic luminal specimens from SPF mice fed a nutrient-rich diet, a minimal diet, or a minimal diet supplemented with the indicated primary BAs (CA/CDCA/UDCA, 2 mM of each) or the secondary BAs (oxo-BAs/LCA/DCA, 1 mM of each) in drinking water. f, Four-week-old GF mice or GF mice receiving transferred faecal materials (FMTs) from minimal-diet or rich-diet SPF mice were fed a nutrient-rich diet or a minimal diet, and colonic Treg cells were analysed after 2 weeks. Frequencies of colonic RORγ+Helios− in the FOXP3+CD4+TCRβ+ Treg cell population are shown. Data are pooled from three independent experiments in a–d. Data are representative of two independent experiments in e and f. n represents biologically independent animals. Data are mean ± s.e.m. ***P < 0.001, one-way ANOVA followed by the Bonferroni post hoc test.

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Extended Data Fig. 4 Generation of BA metabolic pathway mutants in Bacteroides.

a, Schematic diagram of pNJR6 suicide vector-mediated BA gene deletion in Bacteroides. b, Genotyping of B. thetaiotaomicron and B. fragilis BA metabolic pathway mutants by PCR. PCR primers were designed to target the flanking regions of an intact gene. PCR of an untouched gene plus its flanking regions generated a PCR product of around 1,150–1,500 bp, while deletion of an interested BA metabolic gene resulted in only an approximately 350–450-bp PCR amplicon of its two flanking regions. c, d, Bacterial load (measured as colony-forming unit (CFU) per gram of faeces) of B. thetaiotaomicron (c) and B. fragilis (d) BA metabolic pathway mutants and their wild-type control strains in monocolonized GF mice. e, f, LC–MS quantification of faecal conjugated primary BAs (e) and deconjugated primary BAs (f) in GF mice monocolonized with B. thetaiotaomicron or B. fragilis BA metabolic pathway mutants and their wild-type control strains. Data are representative of two independent experiments in b, e and f. Data are pooled from three independent experiments in c and d. n represents biologically independent animals. Data are mean ± s.e.m. (c–f).

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Extended Data Fig. 5 Gut bacteria modulate colonic RORγ+ Treg cells via their BA metabolic pathways.

a, b, Each of four groups of GF mice was colonized with one of the following microorganisms: (1) a wild-type strain of B. thetaiotaomicron; (2) a BSH-mutant strain; (3) a 7α-HSDH-mutant strain; or (4) a triple-mutant (TKO) strain. Colonic Treg cells were analysed after 2 weeks. Absolute numbers of RORγ+Helios− in the colonic FOXP3+CD4+TCRβ+ Treg cell population (a) and of FOXP3+ Treg cells in the CD4+TCRβ+ population (b) are shown. c, d, Each of four groups of GF mice was colonized with one of the following microorganisms: (1) a wild-type strain of B. fragilis; (2) a BSH-KO strain; (3) a 7α-HSDH-KO strain; or (4) a double-mutant (DKO) strain. Absolute numbers of colonic Treg cells are shown as in a and b. e, f, GF mice were colonized with B. thetaiotaomicron BA metabolic pathway mutants or their wild-type control strains. Colonic Treg cells and TH17 cells were analysed after 2 weeks. Frequencies of FOXP3+ in the CD4+TCRβ+ cell population (e) or RORγ+FOXP3− in the CD4+TCRβ+ cell population (f) are shown. g, h, GF mice were colonized with B. fragilis BA metabolic pathway mutants or their wild-type control strains. Frequencies of colonic Treg cells and TH17 cells are shown as in e and f. i, j, GF mice were colonized with B. thetaiotaomicron BA metabolic pathway mutants or their wild-type control strains. Treg cells and TH17 cells in the spleen, mesenteric lymph node and ileum were analysed after 2 weeks. Frequencies of RORγ+Helios− in the FOXP3+CD4+TCRβ+ Treg cell population (i) and RORγ+FOXP3− in the CD4+TCRβ+ cell population (j) are shown. k, l, GF mice were colonized with B. fragilis BA metabolic pathway mutants or their wild-type control strains. Frequencies of Treg cells and TH17 cells in the spleen, mesenteric lymph node and ileum are shown as in i and j. Data are pooled from two or three independent experiments. n represents biologically independent animals. Data are mean ± s.e.m. *P < 0.05, **P < 0.01, ***P < 0.001, one-way ANOVA followed by the Bonferroni post hoc test.

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Extended Data Fig. 6 The effect of BAR deficiency on Treg cells or TH17 cells in gut and peripheral lymphoid organs.

a, Protein expression of VDR, FXR (also known as NR1H4) and GPBAR1 in the colonic tissue of SPF C57BL/6J mice was analysed by western blot. The red asterisks indicate the corresponding molecular weight of VDR (53 kDa), FXR (69 kDa) and GPBAR1 (33 kDa). For gel source data, see Supplementary Fig. 1. b, c, Absolute numbers of RORγ+Helios− in the colonic FOXP3+CD4+TCRβ+ Treg cell population (b) and of FOXP3+ Treg cells in the CD4+TCRβ+ population (c) from mice deficient in nuclear receptors (_Nr1i2_−/−_Nr1i3_−/−, _Nr1h3_−/−, _Vdr_−/−, _Nr1h4_−/− and _Vdr_−/−_Nr1h4_−/−) and their littermate controls. d, e, Frequencies of FOXP3+ in the colonic CD4+TCRβ+ cell population from mice deficient in G-protein-coupled receptors (_Gpbar1_−/−, _Chrm2_−/−, _Chrm3_−/− and _S1pr2_−/−) and their littermate controls (d) and from mice deficient in nuclear receptors (_Nr1i2_−/−_Nr1i3_−/−, _Nr1h3_−/−, _Vdr_−/−, _Nr1h4_−/− and _Vdr_−/−_Nr1h4_−/−) and their littermate controls (e). f, g, Frequencies of RORγ+FOXP3− in the colonic CD4+TCRβ+ cell population from mice described in d and e. h–n, Treg cells in the spleen, mesenteric lymph node and ileum from the indicated mice were analysed. Frequencies of RORγ+Helios− in the FOXP3+CD4+TCRβ+ Treg cell population from _Gpbar1_−/− (h), _Chrm2_−/− (i), _Chrm3_−/− (j), _S1pr2_−/− (k), _Nr1i2_−/−_Nr1i3_−/− (l), _Nr1h3_−/− (m), and _Vdr_−/−, _Nr1h4_−/− and _Vdr_−/−_Nr1h4_−/− (n) mice and their littermate controls are shown. Data are representative of two or three independent experiments in a and d–n, or are pooled from two or three independent experiments in b and c. n represents biologically independent animals. Data are mean ± s.e.m. **P < 0.01, one-way ANOVA followed by the Bonferroni post hoc test.

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Extended Data Fig. 7 Dietary vitamin D3 does not alter the frequency of colonic RORγ+ Treg cells.

a, b, Beginning at 3 weeks of age, three groups of mice were fed special diets for 4 weeks. SPF mice were fed either a nutrient-rich or a minimal diet, and GF mice were fed the nutrient-rich diet. The levels of 1,25-dihydroxyvitamin D3 in serum (a) and the colon (b) of these mice were determined by ELISA. c, SPF mice were fed a nutrient-rich diet, or a rich diet deficient in vitamin D3 (VitD3) or vitamin A (VitA) at birth. Colonic Treg cells were analysed after 7 weeks. Frequencies of RORγ+Helios− in the colonic FOXP3+CD4+TCRβ+ Treg cell population are shown. d, SPF mice were fed a nutrient-rich diet at birth and were either maintained on that diet or switched to a rich diet deficient in vitamin D3 or vitamin A at 3 weeks of age. Colonic Treg cells were analysed after 4 weeks. Frequencies of RORγ+Helios− in the colonic FOXP3+CD4+TCRβ+ Treg cell population are shown. Data are representative of two independent experiments. n represents biologically independent animals. Data are mean ± s.e.m. ***P < 0.001, one-way ANOVA followed by the Bonferroni post hoc test.

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Extended Data Fig. 8 Comparison of RORγ+ Treg cell signature genes of colonic Treg cells from Vdr+/+ and _Vdr_−/− mice.

Volcano plots comparing transcriptomes of colonic Treg cells from Vdr+/+Foxp3 mRFP and Vdr_−/−_Foxp3 mRFP mice (n = 3). Colonic RORγ+ Treg cell signature genes are highlighted in red (upregulated) or blue (downregulated). The number of genes from each signature preferentially expressed by one or the other population is shown at the bottom. Data are pooled from two independent experiments. n represents biologically independent animals. To determine the enrichment of certain gene signatures in RNA-seq datasets, a _χ_2 test was used. P < 0.05 was considered statistically significant.

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Extended Data Fig. 9 BA supplementation does not cause gut inflammation and cannot ameliorate gut inflammation after the development of colitis.

a–c, Three-week-old SPF mice were fed a nutrient-rich diet, a minimal diet, or a minimal diet supplemented with mixtures of primary BAs (CA/CDCA/UDCA, 2 mM of each) or secondary BAs (oxo-BAs/LCA/DCA, 1 mM of each) in drinking water. Initial body weights were recorded before DSS challenge (a). Clinical scores (b) and haematoxylin and eosin histology (c) for representative colons from mice not challenged with DSS are shown. d, e, Three-week-old SPF mice fed a nutrient-rich or a minimal diet for 4 weeks were then challenged in the DSS-induced colitis model. After the development of colitis at day 5 of the model, the DSS containing drinking water was switched to regular drinking water or to drinking water supplemented with mixtures of primary or secondary BAs. The primary BAs were CA, CDCA and UDCA (2 mM of each). The secondary BAs were 3-oxo-CA, 3-oxo-LCA, 7-oxo-CA, 7-oxo-CDCA, 12-oxo-CA, 12-oxo-DCA, DCA and LCA (1 mM of each). Daily weight loss (d) of mice during the course of DSS-induced colitis and clinical scores (e) on day 10 of colitis are shown. Data are representative of two independent experiments. n represents biologically independent animals. Data are mean ± s.e.m. in a, b, d and e.

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Extended Data Fig. 10 VDR signalling controls gut inflammation.

a–c, Daily weight loss (a) of Vdr+/+ and _Vdr_−/− mice during the course of DSS-induced colitis. Clinical scores (b) and haematoxylin and eosin histology (c) of representative colons on day 10 of colitis are shown. d, Schematic representation of the T cell-adaptive transfer model of colitis. Either Vdr+/+ or _Vdr_−/− naive T cells are transferred to _Rag1_−/− mice. e–g, Weight loss (e) of _Rag1_−/− mice in d during the course of T cell-adaptive transfer-induced colitis. Clinical scores (f) and haematoxylin and eosin histology (g) of representative colons on day 67 of colitis are shown. Data are representative of two independent experiments. n represents biologically independent animals. Data are mean ± s.e.m. in a, b, e and f. *P < 0.05, **P < 0.01, ***P < 0.001, two-way ANOVA followed by the Bonferroni post hoc test (a and e) or two-tailed Student’s _t_-test (b and f).

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Song, X., Sun, X., Oh, S.F. et al. Microbial bile acid metabolites modulate gut RORγ+ regulatory T cell homeostasis.Nature 577, 410–415 (2020). https://doi.org/10.1038/s41586-019-1865-0

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