Nature | Vol 577 | 16 January 2020 | 413Data Fig. 5b, d–h), or Treg cells and TH17 cells in the spleen, mesenteric
lymph node and small intestine (Extended Data Fig. 5i–l).
Host BAs are metabolically modified by microorganisms and used
as signalling molecules, serving as ligands that activate BA receptors
(BARs)^4. Thus, we explored whether BARs modulate gut RORγ+ Treg cell
homeostasis. By comparing mouse colonic tissue expression of various
BARs, we found that nuclear receptors, especially vitamin D receptor
(VDR), were more abundant (Fig. 3a and Extended Data Fig. 6a). Next,
we compared the numbers of colonic RORγ+ Treg cells in mice genetically
deficient in each of several BARs. G-protein-coupled receptor defi-
ciency did not affect the number of colonic RORγ+ Treg cells (Fig. 3b). The
nuclear receptors NR1I2/3 (also known as PXR/CAR) and NR1H3 (also
known as LXRα) were also non-contributory (Fig. 3c and Extended Data
Fig. 6b). However, deficiency in either of the two BA-sensing nuclear
receptors—VDR and NR1H4 (also known as FXR)^19 ,^20 —compromised the
number of colonic RORγ+ Treg cells (Fig. 3c and Extended Data Fig. 6b).
The colonic RORγ+ Treg cell phenotype of mice with loss of both of these
genes resembled that of mice with loss of only Vdr or in minimal-diet
littermate controls (Fig. 3c and Extended Data Fig. 6b). This result indi-
cated a prominent role for VDR signalling in modulating this colonic
Treg cell population. That all BAR-deficient mice had normal colonic
counts of total FOXP3+ Treg cells and TH17 cells (Extended Data Fig. 6c–g)
as well as RORγ+ Treg cells in the spleen, mesenteric lymph node and
small intestine (Extended Data Fig. 6h–n) indicated that BAR signalling
control of RORγ+ Treg cells is tissue dependent.
To determine whether intestinal BAs modulate colonic RORγ+ Treg
cells via VDR or NR1H4, we treated nuclear receptor-deficient mice
with a mixture of three murine primary BAs (cholic/chenodeoxycholic/
ursodeoxycholic acids) or a mixture of eight murine secondary BAs
(deoxycholic acid/lithocholic acid/oxidized BAs). Both mixtures ofCo RORγ– Treg0204060Gpbar1+/+ or +/Gpbar1–/–Chrm2 +/+ or +/Chrm2 –/–Chrm3 +/+ or +/–Chrm3 –/–S1pr2+/+ or +/S1pr2–/–abefgcor Vdr+/–Nr 1h4+/–+ minimal diet0204060Nr 1i 2+/–Nr 1i3+/–Nr 1i2–/–Nr 1i3–/–Nr 1h 3+/+ or +/Nr 1h 3–/–Vdr+/+Nr 1h4+/+Vdr+/+Nr 1h 4+/+
Vdr–/–
Nr 1h 4–/–Vdr–/–Nr 1h4–/–********
***Gpbar1 Nr 1i 2Nr 1i 3Nr 1h 3Nr 1h 4VdrColonic mRNA expression(relative toGpbar1)Chrm2
Chrm3
S1pr2G-protein-
coupled receptorsNuclear receptorsSp Treg
Co Treg
Tconv Treg DCEpithdVdr+/+
Nr1h4+/++/+
+/++/++/++/++/+
Minimal diet+ +
- –
+/+
+/+
+- +––––––
–––+ +
++ +
++ +
Primary BAs +
Secondary BAs–/– –/–
+/+++–/–
–/– –/–+/+++–/– –/– –/––/– –/–++–/––/–01020304050
******
****h–8 1 +2+4+810 –2P value110 –48 –4 4 –2 2 1 + 2 + 4 + 8Colonic Treg
down-signatureup-signatureColonic TregFold change: Vdr+/+ vs Vdr–/–
colonic Treg cells154305
160161
P = 0.623 P = 1.59 × 10–10Il23rIl12rb25,5515,901
Il1r1
Ccl20
Vdr
Rnf240501001502001,0002,0003,000Vdrexpression valuesVdrexpression values0100200300400Co RORγ+ Treg***
***020406080Vdrflox/flox
Vdrflox/flox
Foxp3 YFP-creFoxp3YFP-cre
Vdrflox/flox
Cd11ccre
Vdrflox/flox
Vil1cre0200400600800(n = 4)(n = 3)n = 3n = 3n = 3n = 4n = 4n = 4n = 4n = 5n = 6n = 6n = 5n = 5 n = 7 n = 5 n = 6n = 8 n = 8 n = 5 n = 8 n = 8 n = 8n = 6 n = 3 n = 6 n = 5 n = 5n = 6n = 8n = 7n = 6n = 8n = 5n = 6n = 8n = 8n = 6n = 8n = 7(n = 4)
20.7%Vdr–/–
30.3%Nr1h4–/– Vdr–/–Nr1h4–/–
20.2%Vdr+/–Nr1h4+/–Helios-FITCRORγ-PE44.8%ROR+γ
Helios- T
reg(% of FOXP3+)ROR+γ
Helios- T
reg(% of FOXP3+)ROR+γ
Helios- T
reg(% of FOXP3+)ROR+γ
Helios- T
reg(% of FOXP3+)Fig. 3 | BA metabolites modulate colonic RORγ+ Treg cells via BARs.
a, Quantitative mRNA expression of BARs in the colon of SPF mice.
b, Frequencies of colonic RORγ+Helios− in the FOXP3+CD4+TCRβ+ Treg cell
population from mice deficient in G-protein-coupled receptors (Gpbar1−/−,
Chrm2−/−, Chrm3−/− and S1pr2−/−) and their littermate controls. c, Representative
plots and frequencies of colonic RORγ+Helios− in the FOXP3+CD4+TCRβ+ Treg cell
population from mice deficient in nuclear receptors (Nr1i 2−/−Nr1i 3−/−, Nr1h 3−/−,
Vdr−/−, Nr1h4−/− and Vdr−/−Nr1h4−/−), their littermate controls and minimal-diet
Vdr+/+Nr1h4+/+ mice. d, Three-week-old Vdr+/+Nr1h4+/+, Vdr−/−, Nr1h4−/− and
Vdr−/−Nr1h4−/− mice were fed a minimal diet or a minimal diet supplemented with
primary BAs (CA/CDCA/UDCA, 2 mM of each) or secondary BAs (DCA/LCA/3-
oxo-CA /3-oxo-LCA /7-oxo-CA /7-oxo-CDCA /12-oxo-CA /12-oxo-DCA, 1 mM of
each) in their drinking water for 4 weeks. Frequencies of colonic RORγ+Helios−
in the FOXP3+CD4+TCRβ+ Treg cell population are shown. e, Normalized
expression values of Vdr in colonic Tconv cells, FOXP3+ Treg cells, dendritic cells
(DCs) and epithelial cells (Epith). f, Normalized expression values of Vdr in
splenic Treg cells (Sp Treg), colonic Treg cells (Co Treg), colonic RORγ− Treg cells
(Co RORγ− Treg) and colonic RORγ+ Treg cells (Co RORγ+ Treg). g, Frequencies of
colonic RORγ+Helios− in the FOXP3+CD4+TCRβ+ Treg cell population from
Vdr flox/floxFoxp3YFP-cre, Vdr flox/floxCd11ccre and Vdr flox/floxVil1cre mice and from
Vdr flox/flox and Foxp3YFP-cre mice. h, Volcano plots comparing transcriptomes
of colonic Treg cells from Vdr+/+Foxp3mRFP or Vdr−/−Foxp3mRFP mice (n = 3).
Colonic Treg cell signature genes are highlighted in red (upregulated) or blue
(downregulated). 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 (c, d and g) or χ^2 test (h).