Bile acid metabolites control TH17 and Treg cell differentiation

Abstract

Bile acids are abundant in the mammalian gut, where they undergo bacteria-mediated transformation to generate a large pool of bioactive molecules. Although bile acids are known to affect host metabolism, cancer progression and innate immunity, it is unknown whether they affect adaptive immune cells such as T helper cells that express IL-17a (TH17 cells) or regulatory T cells (Treg cells). Here we screen a library of bile acid metabolites and identify two distinct derivatives of lithocholic acid (LCA), 3-oxoLCA and isoalloLCA, as T cell regulators in mice. 3-OxoLCA inhibited the differentiation of TH17 cells by directly binding to the key transcription factor retinoid-related orphan receptor-γt (RORγt) and isoalloLCA increased the differentiation of Treg cells through the production of mitochondrial reactive oxygen species (mitoROS), which led to increased expression of FOXP3. The isoalloLCA-mediated enhancement of Treg cell differentiation required an intronic Foxp3 enhancer, the conserved noncoding sequence (CNS) 3; this represents a mode of action distinct from that of previously identified metabolites that increase Treg cell differentiation, which require CNS1. The administration of 3-oxoLCA and isoalloLCA to mice reduced TH17 cell differentiation and increased Treg cell differentiation, respectively, in the intestinal lamina propria. Our data suggest mechanisms through which bile acid metabolites control host immune responses, by directly modulating the balance of TH17 and Treg cells.

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Fig. 1: 3-OxoLCA inhibits TH17 cell differentiation and isoalloLCA enhances Treg cell differentiation.
Fig. 2: 3-OxoLCA binds to RORγt and inhibits its transcriptional activity.
Fig. 3: MitoROS is necessary and sufficient for the isoalloLCA-dependent enhanced expression of FOXP3.
Fig. 4: 3-OxoLCA inhibits TH17 development and isoalloLCA enhances Treg cells in vivo.

Data availability

The 16S rDNA datasets are available through NCBI under accession number PRJNA528994. Source Data for Figs. 14 and Extended Data Figs. 29 are provided with the paper. Any other relevant data are available from the corresponding authors upon reasonable request.

Change history

  • 25 February 2020

    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 N. Lee and K. Hattori for technical assistance; M. Trombly for critical reading of the manuscript; A. Rudensky and S. Smale for sharing FOXP3–CNS- and REL-knockout mice; R. Bronson and the Rodent Histopathology Core at Harvard Medical School for performing H & E analysis and disease score; the BPF Next-Gen Sequencing Core at Harvard Medical School for their expertise and instrument availability with microbiota sequencing. We acknowledge NIH grant P30DK034854 and the use of the Harvard Digestive Disease Center’s (HDDC’s) core services, resources, technology and expertise. This study was supported by a Charles A. King Trust Fellowship to S. Hang, Harvard Medical School Dean’s Innovation Grant in the Basic and Social Sciences to A.S.D. and J.R.H., the Howard Hughes Medical Institute to D.R.L. and National Institutes of Health grants R01AI080885 to D.R.L. and R01 DK110559 to J.R.H.

Author information

M.A.F., J.R.H., and D.R.L. conceptualized the study. S. Hang, D.P., A.S.D., M.R.K., M.A.F., D.R.L., and J.R.H. conceived and designed the experiments; S. Hang and D.P. performed most of the experiments; L.Y., E.K., T.J., A.S.D., J.L., S. Ha, B.N.N., S.P.K., and L.W. provided help with experiments; J.L. and F.R. designed and performed the RORγt binding assay; B.N.N., S.P.K., and M.R.K. synthesized some of the bile acid derivatives; L.Y. and A.S.D. performed in vivo bile acid analyses; R.S.L. and Y.Z. provided critical materials; and S. Hang, D.P., and J.R.H. wrote the manuscript, with contributions from all authors.

Correspondence to Michael A. Fischbach or Dan R. Littman or Jun R. Huh.

Ethics declarations

Competing interests

A.S.D. is an ad hoc consultant for Kintai Therapeutics. D.R.L. is a scientific co-founder of Vedanta Biosciences.

Additional information

Peer review information Nature thanks Navdeep S. Chandel, Richard Steven Blumberg 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 Chemical structures of bile acid derivatives.

These derivatives were used for the T cell differentiation assay.

Extended Data Fig. 2 3-OxoLCA and isoalloLCA affect TH17 and Treg cell differentiation.

a, b, Gating strategy for the flow cytometric analyses of in vitro cultured T cells (a) and in vivo derived cells from the lamina propria (b). c, Schematic of the screening procedure. d, e, Naive CD4+ T cells isolated from B6 Jax mice (n = 2 biologically independent samples) were cultured under TH17 (IL-6 = 10 ng ml−1; TGFβ = 0.5 ng ml−1) (d) and Treg (IL-2 = 100 U ml−1; TGFβ = 0.1 ng ml−1) (e) cell polarization conditions for 3 days. DMSO or various bile acids at 20 μM concentration were added to the cell cultures on day 1. Data are mean. Source Data

Extended Data Fig. 3 IsoalloLCA-induced Treg cell expansion requires TGFβ.

ac, Flow cytometry and histogram of CD4+ T cells, cultured for 3 days with different amounts of TGFβ (1, 0.1, 0.01 or 0 ng ml−1) and IL-2 (100 U ml−1) in the presence of DMSO or isoalloLCA (20 μM) and intracellularly stained for FOXP3 (n = 3 biologically independent samples per group). d, e, Flow cytometry of CD4+ T cells, cultured for 3 days in the presence of DMSO, isoalloLCA (20 μM) or TGFβ (0.05 ng ml−1). In addition, anti-TGFβ antibody (10 μg ml−1, 1D11) or isotype control were added to the culture (n = 3 biologically independent samples per group). fh, 3-OxoLCA and isoalloLCA do not affect key transcription factor expression. T cells were cultured under TH0, TH1, TH2 or TH17 conditions, in the presence of DMSO, 3-oxoLCA (20 μM) or isoalloLCA (20 μM). T-cell-lineage-determining transcription factors such as T-bet, GATA3 or RORγt were intracellularly stained (n = 3 biologically independent samples per group). MFI, mean fluorescence intensity. Data are mean ± s.d., by unpaired t-test with two-tailed P value. Source Data

Extended Data Fig. 4 Effects of isoalloLCA on FOXP3 expression require strong TCR stimulation.

a, 3-OxoLCA and isoalloLCA demonstrate dose-dependent effects on TH17 cell and Treg cell differentiation, respectively (n = 2 biologically independent samples). A low concentration of TGFβ (0.01 ng ml−1) was used for Treg cell culture. bd, 3-OxoLCA and isoalloLCA do not significantly affect cell proliferation, cell viability or T cell activation. b, Naive CD4+ T cells were labelled with a cell proliferation dye CFSE and cultured for 3 days in the presence of DMSO, 3-oxoLCA or isoalloLCA under TH17 or Treg cell polarization conditions. c, Live-cell percentages at the end of the 3-day culture were determined based on both annexin V and fixable live/dead staining (n = 3 biologically independent samples per group). d, Both DMSO and isoalloLCA treatment lead to comparable levels of expression of CD25, CD69, NUR77 and CD44. Naive CD4+ T cells were used as a negative control. e, f, T cells were cultured with different concentrations of anti-CD3 antibody, in the presence of DMSO or isoalloLCA (20 μM). Representative FACS plots of CD4+ T cells cultured for 3 days and stained intracellularly for FOXP3 (e). Quantification of FOXP3+ and viable T cells after 3-day culture (f) (n = 2 biologically independent samples per group). Data are representative of two independent experiments (b, d). Data in c are mean ± s.d. Source Data

Extended Data Fig. 5 REL, VDR and FXR are dispensable for isoalloLCA-dependent induction of FOXP3.

a, b, In vitro suppression assay. CD4+ effector T cells (Tconv) were labelled with CFSE and mixed with DMSO- or isoalloLCA-treated Treg cells (tester) at different ratios (n = 2 biologically independent samples per group). c, Expression of GFP in DMSO- or isoalloLCA-treated T cells cultured with anti-CD3/28, IL-2 and TGFβ (0.01 ng ml−1). Naive CD4+ T cells were isolated from FOXP3–IRES–GFP mice. d, Flow cytometry of CD4+ T cells stained intracellularly for FOXP3. Naive CD4+ T cells isolated from wild-type, CNS1-, CNS2- or CNS3-knockout mice (n = 3 biologically independent samples per group) were cultured with anti-CD3/28 and IL-2, LCA (20 μM), TGFβ (0.05 ng ml−1) and additional retinoic acid (1 ng ml−1). e, f, Flow cytometry (e) and its quantification (f) of CD4+ T cells stained intracellularly for FOXP3. Naive CD4+ T cells were isolated from wild-type control mice or REL-knockout mice (n = 4 biologically independent samples per group) and cultured with anti-CD3/28 and IL-2 in the presence of DMSO, isoalloLCA (20 μM) or LCA (20 μM). g, h, Naive CD4+ T cells isolated from wild-type control, VDR-knockout or FXR-knockout (n = 2 biologically independent samples per group) were cultured with anti-CD3/28 and IL-2 (g) or anti-CD3/28, IL-6 and TGFβ (h) for 3 days in the presence of DMSO, isoalloLCA (20 μM), or 3-oxoLCA (20 μM). Representative FACS plots of T cells intracellularly stained for FOXP3 or IL-17a. i, Chemical structures of glycine-conjugated 3-oxoLCA (glyco-3-oxoLCA) and isoalloLCA (glyco-isoalloLCA). j and k, Quantifications of TH17 (j) and Treg (k) cell differentiation in vitro. T cells were cultured with anti-CD3/28, IL-6 and TGFβ (j) or anti-CD3/28 and IL-2 (k) in the presence of DMSO, 3-oxoLCA (20 μM), glyco-3-oxoLCA (20 μM), isoalloLCA (5 or 20 μM) or glyco-isoalloLCA (5, 10 or 20 μM). Glyco-isoalloLCA exhibited enhanced cytotoxicity at 10 or 20 μM compared to isoalloLCA (n = 3 biologically independent samples per group). Data are representative of two independent experiments (c, d). Data are mean ± s.d., by unpaired t-test with two-tailed P value. Source Data

Extended Data Fig. 6 IsoalloLCA-dependent FOXP3 transcription requires mitoROS and H3K27ac.

ac, ChIP analysis of H3K27ac, p300 and H3K4 mono-methylation (H3K4me1) on the Foxp3 gene locus. Chromatin obtained from DMSO- and isoalloLCA-treated wild-type cells were immunoprecipitated with IgG, anti-H3K27ac, anti-p300 or anti-H3K4me1 antibodies, followed by real-time PCR analysis (n = 3 biologically independent samples per group). Primers targeting Foxp3 promoter (Pro), CNS1, CNS2 and CNS3 region and Hsp90ab1 promoter were used for qPCR quantification. Relative enrichment was calculated as fold change relative to the ChIP signal at the Foxp3 promoter of the DMSO-treated control. d, e, Flow cytometry and quantification of CD4+ T cells stained intracellularly for FOXP3. Naive CD4+ T cells isolated from wild-type mice (n = 2 biologically independent samples per group) were cultured with anti-CD3/28, IL-2 and TGFβ (0.05 ng ml−1) in the presence of DMSO or isoalloLCA (20 μM) in the presence or absence of iBET. f, ChIP analysis of H3K27ac on the Foxp3 promoter region. Naive CD4+ T cells isolated from wild-type or CNS3-knockout mice (n = 3 biologically independent samples per group) were treated with DMSO or isoalloLCA (20 μM). g, Seahorse analysis of oxygen consumption rate (OCR) with naive CD4+ T cells isolated from wild-type or CNS3-knockout mice cultured with anti-CD3/28 and IL-2 for 48 h, in the presence of DMSO or isoalloLCA (20 μM). Measurements from six wells from two mice for each genotype. hk, T cells were cultured with DMSO, LCA, isoLCA, alloLCA, isoalloLCA or 3-oxoLCA at 20 μM for 48 h. Their mitochondrial and cytoplasmic ROS were measured by mitoSOX (h) and 2′,7′-dichlorofluorescein diacetate (DCFDA) (i), respectively. Total mitochondria mass was measured by MitoTracker (j) and the mitochondrial membrane potential measured by JC-1 dye (k). Mean fluorescence intensities of different treatments were normalized as fold changes of those of the DMSO control (n = 3 biologically independent samples per group). l, MitoROS production measured by mitoSOX with T cells cultured with DMSO, isoalloLCA (20 μM), retinoic acid (1 nM), or isoalloLCA (20 μM) + mitoQ (0.5 μM) for 48 h. m, ChIP analysis (n = 3 biologically independent samples per group) of H3K27ac on the Foxp3 promoter of T cells, treated with DMSO, isoalloLCA, isoalloLCA + mitoQ or isoalloLCA + anti-TGFβ for 72 h. nq, MitoROS production measured by mitoSOX with T cells cultured with different concentrations of anti-CD3 and treated with DMSO, isoalloLCA (20 μM), TGFβ (0.05 ng ml−1) or isoalloLCA plus TGFβ (n = 2 biologically independent samples per group) (n); or with T cells treated with DMSO or isoalloLCA (20 μM) plus an isotype control or anti-TGFβ antibody (n = 4 biologically independent samples per group) (o); or with T cells cultured under TH1, TH2, TH17 or Treg cell conditions (n = 3 biologically independent samples per group) (p); or with naive CD4+ T cells isolated from wild-type or CNS3-knockout mice and cultured with anti-CD3/28 and IL-2 (n = 3 biologically independent samples per group) (q). r, MitoROS production measured by mitoSOX with T cells cultured with DMSO or mitoPQ (5 μM) for 48 h. s, Dose-dependent effects of mitoPQ on Treg cell differentiation (n = 3 biologically independent samples per group). t, Quantification of Treg cell differentiation in vitro on naive CD4+ T cells cultured in the presence of DMSO or mitoPQ (5 μM) and treated with isotype control or anti-TGFβ antibody (n = 3 biologically independent samples per group). u, A model showing the mechanism of isoalloLCA enhancement of Treg cell differentiation. Data are representative of two independent experiments (l, r) and shown as mean ± s.d., by unpaired t-test with two-tailed P value. Source Data

Extended Data Fig. 7 3-OxoLCA inhibits the differentiation of TH17 cells but not Treg cells, and isoalloLCA alone does not enhance Treg cell differentiation in vivo.

a, UPLC–MS spectra of LCA and its isomers isoalloLCA, alloLCA, and isoLCA, as well as 3-oxoLCA. b, Quantification of unconjugated LCA and its derivatives in the caecal contents of B6 Tac mice fed on a control or bile-acid-containing diet (n = 7, 5 and 4 mice for control (ctrl), 3-oxoLCA and 3-oxoLCA + isoalloLCA, respectively). c, Quantification of unconjugated 3-oxoLCA and isoalloLCA in human stool samples from patients with ulcerative colitis (n = 16 donors). d, Quantification of unconjugated 3-oxoLCA, isoalloLCA and LCA in mouse caecal contents from germ-free (GF) or conventionally housed (CNV) mice (n = 15 mice per group). e, B6 Jax mice gavaged with SFB. SFB colonization measured by qPCR analysis calculated as copy number (n = 5 mice per group). f, Diagram showing experimental design. B6 Tac mice were fed a 3-oxoLCA (0.3%)-containing diet for 7 days. g, SFB colonization measured by qPCR analysis calculated as SFB copy number (n = 5 mice per group). h, i, Flow cytometric analysis and quantification of TH17 (h) and Treg (i) cells of the ileal lamina propria (n = 7 mice per group). jl, Experimental scheme of anti-CD3 experiment with 3-oxoLCA (j). Flow cytometric analysis and quantification of CD4+ cells of the lamina propria following an anti-CD3 injection from B6 Tac mice fed with control or 3-oxoLCA (0.3%) diet (n = 9 mice per group) (k), or 3-oxoLCA (1%) diet (n = 7 mice per group) (l). m, n, Flow cytometric analysis and quantification of CD4+ cells of the ileal lamina propria in steady-state (m) (n = 6 mice per group) or following an anti-CD3 injection (n) (n = 5 mice per group). B6 Tac mice were fed with control or isoalloLCA (0.03%) diet. o, p, Flow cytometry (o) and quantification (p) of CD4+ T cells stained intracellularly for FOXP3, showing that the combination of 3-oxoLCA and isoalloLCA further increases Treg cell differentiation. Naive CD4+ T cells isolated from wild-type B6 mice (n = 3 biologically independent samples) treated with DMSO, isoalloLCA (20 μM), a mixture of 3-oxoLCA (20 μM) and isoalloLCA (20 μM) or a mixture of 3-oxoCA (20 μM) and isoalloLCA (20 μM) and cultured with anti-CD3/28 and IL-2, with or without the addition of IL-6 (62.5 pg ml−1). q, MitoROS production in total CD4+ T cells isolated from the ileal lamina propria. Mice were fed a control diet or diet containing a mixture of 3-oxoLCA (0.3%) + isoalloLCA (0.03%) (n = 9 or 10 mice, respectively) and injected with 10 μg of anti-CD3 to induce inflammation. Data are mean ± s.d., by unpaired t-test with two-tailed P value. Source Data

Extended Data Fig. 8 3-OxoLCA or isoalloLCA does not significantly alter gut microbiota.

a, Box plot showing operational taxonomic unit (OTU) numbers. b, Shannon diversity of faecal microbiota based on 16S rRNA gene amplicon sequencing. For the box plots in a, b, the three horizontal lines of the box represent the third quartile, median and first quartile, respectively, from top to bottom. The whiskers above and below the box show the maximum and minimum. c, Principal coordinates analysis based on weighted UniFrac distances of 16S rRNA amplicon sequencing of faecal microbiota. d, e, Average relative abundance of microbiota at the phylum (d) and the family (e) levels by taxon-based analyses (n = 4, 5 and 5 mice for the control, 3-oxoLCA and isoalloLCA groups, respectively). f, g, Experimental scheme (f) and flow cytometric analysis and quantification (g) of CD4+ cells of the lamina propria of the colon in germ-free B6 mice, infected with C. rodentium. Mice were fed an autoclaved diet with or without 3-oxoLCA (0.3%) (n = 9 mice per group). Data are mean ± s.d., by unpaired t-test with two-tailed P value. Source Data

Extended Data Fig. 9 IsoalloLCA-induced Treg cells suppress transfer colitis.

a, Experimental scheme. Rag1−/− recipient mice were transferred intraperitoneally with 0.5 million CD45RBhigh naive CD4+ T cells (CD45.1) and with or without co-transfer of 0.5 million FOXP3–GFP+ Treg cells (CD45.2). FOXP3–GFP+ cells were cultured under TGFβlow (0.05 ng ml−1), isoalloLCA (20 μM, 0.01 ng ml−1 TGFβ) and TGFβhigh (1 ng ml−1) conditions with GFP naive CD4 T cells, isolated from CD45.2 FOXP3–IRES–GFP mice. b, Flow cytometric analysis of the FOXP3–GFP+ cells, following in vitro culture. The gated cells were sorted and used for co-transfer. cf, Weight change monitored for 8 weeks; week-7 values are used for unpaired t-test with two-tailed P value (c) (n = 5 mice per group). At the end of the experiment, colon length (d) (n = 10 mice per group), H & E staining (e) and the quantification of disease score (f) (n = 5 mice for ‘none’, 4 mice for other groups). gj, Flow cytometric analysis and quantification of the frequency of CD45.1 and CD45.2 (g, i) and the frequency of FOXP3+ cells in the CD45.2 population (h, j) in each condition (n = 5 mice per group). k, Quantification of total CD45.1 cell number in the lamina propria of the colon (n = 5 mice per group). Data are mean ± s.d., by unpaired t-test with two-tailed P value. Source Data

Extended Data Table 1 Lipophilicity of bile acids

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This file contains the Key Reagents Table, details of the Chemical Synthesis of 3-oxoLCA, isoalloLCA, glyco-3-oxoLCA, and glyco-isoalloLCA and Supplementary Figures 1-8.

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Hang, S., Paik, D., Yao, L. et al. Bile acid metabolites control TH17 and Treg cell differentiation. Nature 576, 143–148 (2019). https://doi.org/10.1038/s41586-019-1785-z

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