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.
This is a preview of subscription content, access via your institution
Access options
Access Nature and 54 other Nature Portfolio journals
Get Nature+, our best-value online-access subscription
$29.99 / 30 days
cancel any time
Subscribe to this journal
Receive 51 print issues and online access
$199.00 per year
only $3.90 per issue
Buy this article
- Purchase on Springer Link
- Instant access to full article PDF
Prices may be subject to local taxes which are calculated during checkout
Similar content being viewed by others
Data availability
The 16S rDNA datasets are available through NCBI under accession number PRJNA528994. Source Data for Figs. 1–4 and Extended Data Figs. 2–9 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.
References
Shapiro, H., Kolodziejczyk, A. A., Halstuch, D. & Elinav, E. Bile acids in glucose metabolism in health and disease. J. Exp. Med. 215, 383–396 (2018).
Ridlon, J. M., Kang, D. J. & Hylemon, P. B. Bile salt biotransformations by human intestinal bacteria. J. Lipid Res. 47, 241–259 (2006).
Devlin, A. S. & Fischbach, M. A. A biosynthetic pathway for a prominent class of microbiota-derived bile acids. Nat. Chem. Biol. 11, 685–690 (2015).
Hamilton, J. P. et al. Human cecal bile acids: concentration and spectrum. Am. J. Physiol. Gastrointest. Liver Physiol. 293, G256–G263 (2007).
Bernstein, H., Bernstein, C., Payne, C. M. & Dvorak, K. Bile acids as endogenous etiologic agents in gastrointestinal cancer. World J. Gastroenterol. 15, 3329–3340 (2009).
Barrasa, J. I., Olmo, N., Lizarbe, M. A. & Turnay, J. Bile acids in the colon, from healthy to cytotoxic molecules. Toxicol. In Vitro 27, 964–977 (2013).
Buffie, C. G. et al. Precision microbiome reconstitution restores bile acid mediated resistance to Clostridium difficile. Nature 517, 205–208 (2015).
Duboc, H. et al. Connecting dysbiosis, bile-acid dysmetabolism and gut inflammation in inflammatory bowel diseases. Gut 62, 531–539 (2013).
Martínez-Moya, P. et al. Dose-dependent antiinflammatory effect of ursodeoxycholic acid in experimental colitis. Int. Immunopharmacol. 15, 372–380 (2013).
Schaap, F. G., Trauner, M. & Jansen, P. L. Bile acid receptors as targets for drug development. Nat. Rev. Gastroenterol. Hepatol. 11, 55–67 (2014).
Guo, C. et al. Bile acids control inflammation and metabolic disorder through inhibition of NLRP3 inflammasome. Immunity 45, 944 (2016).
Ma, C. et al. Gut microbiome-mediated bile acid metabolism regulates liver cancer via NKT cells. Science 360, eaan5931 (2018).
Cao, W. et al. The xenobiotic transporter Mdr1 enforces T cell homeostasis in the presence of intestinal bile acids. Immunity 47, 1182–1196 (2017).
Huh, J. R. et al. Digoxin and its derivatives suppress TH17 cell differentiation by antagonizing RORγt activity. Nature 472, 486–490 (2011).
Jin, L. et al. Structural basis for hydroxycholesterols as natural ligands of orphan nuclear receptor RORγ. Mol. Endocrinol. 24, 923–929 (2010).
Santori, F. R. et al. Identification of natural RORγ ligands that regulate the development of lymphoid cells. Cell Metab. 21, 286–298 (2015).
Soroosh, P. et al. Oxysterols are agonist ligands of RORγt and drive Th17 cell differentiation. Proc. Natl Acad. Sci. USA 111, 12163–12168 (2014).
Esplugues, E. et al. Control of TH17 cells occurs in the small intestine. Nature 475, 514–518 (2011).
Huh, J. R. & Littman, D. R. Small molecule inhibitors of RORγt: targeting Th17 cells and other applications. Eur. J. Immunol. 42, 2232–2237 (2012).
Roda, A., Minutello, A., Angellotti, M. A. & Fini, A. Bile acid structure-activity relationship: evaluation of bile acid lipophilicity using 1-octanol/water partition coefficient and reverse phase HPLC. J. Lipid Res. 31, 1433–1443 (1990).
Pellicciari, R. et al. Discovery of 3α,7α,11β-trihydroxy-6α-ethyl-5β-cholan-24-oic acid (TC-100), a novel bile acid as potent and highly selective FXR agonist for enterohepatic disorders. J. Med. Chem. 59, 9201–9214 (2016).
Feng, Y. et al. Control of the inheritance of regulatory T cell identity by a cis element in the Foxp3 locus. Cell 158, 749–763 (2014).
Feng, Y. et al. A mechanism for expansion of regulatory T-cell repertoire and its role in self-tolerance. Nature 528, 132–136 (2015).
Zheng, Y. et al. Role of conserved non-coding DNA elements in the Foxp3 gene in regulatory T-cell fate. Nature 463, 808–812 (2010).
Arpaia, N. et al. Metabolites produced by commensal bacteria promote peripheral regulatory T-cell generation. Nature 504, 451–455 (2013).
Josefowicz, S. Z. et al. Extrathymically generated regulatory T cells control mucosal TH2 inflammation. Nature 482, 395–399 (2012).
Schlenner, S. M., Weigmann, B., Ruan, Q., Chen, Y. & von Boehmer, H. Smad3 binding to the foxp3 enhancer is dispensable for the development of regulatory T cells with the exception of the gut. J. Exp. Med. 209, 1529–1535 (2012).
Makishima, M. et al. Vitamin D receptor as an intestinal bile acid sensor. Science 296, 1313–1316 (2002).
Yu, J. et al. Lithocholic acid decreases expression of bile salt export pump through farnesoid X receptor antagonist activity. J. Biol. Chem. 277, 31441–31447 (2002).
Nanduri, R. et al. The active form of vitamin D transcriptionally represses Smad7 Signaling and activates extracellular signal-regulated kinase (ERK) to inhibit the differentiation of a inflammatory T helper cell subset and suppress experimental autoimmune encephalomyelitis. J. Biol. Chem. 290, 12222–12236 (2015).
Jeffery, L. E. et al. 1,25-Dihydroxyvitamin D3 and IL-2 combine to inhibit T cell production of inflammatory cytokines and promote development of regulatory T cells expressing CTLA-4 and FoxP3. J. Immunol. 183, 5458–5467 (2009).
Gorman, S. et al. Topically applied 1,25-dihydroxyvitamin D3 enhances the suppressive activity of CD4+CD25+ cells in the draining lymph nodes. J. Immunol. 179, 6273–6283 (2007).
Kang, S. W. et al. 1,25-Dihyroxyvitamin D3 promotes FOXP3 expression via binding to vitamin D response elements in its conserved noncoding sequence region. J. Immunol. 188, 5276–5282 (2012).
Etchegaray, J. P. & Mostoslavsky, R. Interplay between metabolism and epigenetics: a nuclear adaptation to environmental changes. Mol. Cell 62, 695–711 (2016).
Gerriets, V. A. & Rathmell, J. C. Metabolic pathways in T cell fate and function. Trends Immunol. 33, 168–173 (2012).
Buck, M. D., O’Sullivan, D. & Pearce, E. L. T cell metabolism drives immunity. J. Exp. Med. 212, 1345–1360 (2015).
Gerriets, V. A. et al. Metabolic programming and PDHK1 control CD4+ T cell subsets and inflammation. J. Clin. Invest. 125, 194–207 (2015).
Xu, T. et al. Metabolic control of TH17 and induced Treg cell balance by an epigenetic mechanism. Nature 548, 228–233 (2017).
Zhang, D. et al. d-mannose induces regulatory T cells and suppresses immunopathology. Nat. Med. 23, 1036–1045 (2017).
Sena, L. A. et al. Mitochondria are required for antigen-specific T cell activation through reactive oxygen species signaling. Immunity 38, 225–236 (2013).
Angelin, A. et al. Foxp3 reprograms T cell metabolism to function in low-glucose, high-lactate environments. Cell Metab. 25, 1282–1293 (2017).
Robb, E. L. et al. Selective superoxide generation within mitochondria by the targeted redox cycler MitoParaquat. Free Radic. Biol. Med. 89, 883–894 (2015).
Ivanov, I. I. et al. Induction of intestinal Th17 cells by segmented filamentous bacteria. Cell 139, 485–498 (2009).
Gagliani, N. et al. TH17 cells transdifferentiate into regulatory T cells during resolution of inflammation. Nature 523, 221–225 (2015).
Trauner, M., Meier, P. J. & Boyer, J. L. Molecular pathogenesis of cholestasis. N. Engl. J. Med. 339, 1217–1227 (1998).
Vavassori, P., Mencarelli, A., Renga, B., Distrutti, E. & Fiorucci, S. The bile acid receptor FXR is a modulator of intestinal innate immunity. J. Immunol. 183, 6251–6261 (2009).
Pols, T. W. et al. TGR5 activation inhibits atherosclerosis by reducing macrophage inflammation and lipid loading. Cell Metab. 14, 747–757 (2011).
Kakiyama, G. et al. A simple and accurate HPLC method for fecal bile acid profile in healthy and cirrhotic subjects: validation by GC-MS and LC-MS. J. Lipid Res. 55, 978–990 (2014).
Sakai, K., Makino, T., Kawai, Y. & Mutai, M. Intestinal microflora and bile acids. Effect of bile acids on the distribution of microflora and bile acid in the digestive tract of the rat. Microbiol. Immunol. 24, 187–196 (1980).
Robben, J., Caenepeel, P., Van Eldere, J. & Eyssen, H. Effects of intestinal microbial bile salt sulfatase activity on bile salt kinetics in gnotobiotic rats. Gastroenterology 94, 494–502 (1988).
Yao, L. et al. A selective gut bacterial bile salt hydrolase alters host metabolism. eLife 7, e37182 (2018).
van der Windt, G. J., Chang, C. H. & Pearce, E. L. Measuring bioenergetics in T cells using a seahorse extracellular flux analyzer. Curr. Protoc. Immunol. 113, 3.16B.1–13.16B.14 (2016).
Powrie, F. et al. Inhibition of Th1 responses prevents inflammatory bowel disease in scid mice reconstituted with CD45RBhi CD4+ T cells. Immunity 1, 553–562 (1994).
Bokulich, N. A. et al. Optimizing taxonomic classification of marker-gene amplicon sequences with QIIME 2’s q2-feature-classifier plugin. Microbiome 6, 90 (2018).
Bolyen, E. et al. An introduction to applied bioinformatics: a free, open, and interactive text. J Open Source Educ 1, 27 (2018).
Callahan, B. J. et al. DADA2: high-resolution sample inference from Illumina amplicon data. Nat. Methods 13, 581–583 (2016).
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
Authors and Affiliations
Contributions
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.
Corresponding authors
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.
Extended Data Fig. 3 IsoalloLCA-induced Treg cell expansion requires TGFβ.
a–c, 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). f–h, 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.
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. b–d, 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.
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.
Extended Data Fig. 6 IsoalloLCA-dependent FOXP3 transcription requires mitoROS and H3K27ac.
a–c, 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. h–k, 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. n–q, 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.
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). j–l, 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.
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.
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. c–f, 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). g–j, 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.
Supplementary information
Supplementary Information
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.
Source data
Rights and permissions
About this article
Cite this article
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
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1038/s41586-019-1785-z
This article is cited by
-
Cancer immunometabolism: advent, challenges, and perspective
Molecular Cancer (2024)
-
Integrated Metabolomic and transcriptomic analyses reveal deoxycholic acid promotes transmissible gastroenteritis virus infection by inhibiting phosphorylation of NF-κB and STAT3
BMC Genomics (2024)
-
Development of non-alcoholic steatohepatitis is associated with gut microbiota but not with oxysterol enzymes CH25H, EBI2, or CYP7B1 in mice
BMC Microbiology (2024)
-
Metabolic reprogramming in the tumor microenvironment of liver cancer
Journal of Hematology & Oncology (2024)
-
Dopamine receptor D2 confers colonization resistance via microbial metabolites
Nature (2024)
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.