Nature - USA (2020-01-16)

Nature | Vol 577 | 16 January 2020 | 411

chenodeoxycholic/ursodeoxycholic acids or cholic/chenodeoxycholic/
ursodeoxycholic acids) restored colonic frequencies of both RORγ+ Treg
cells and FOXP3+ Treg cells close to that in rich-diet mice (Fig. 1e and
Extended Data Fig. 2a, b). Similarly, a mixture of eight representative
secondary BAs from bacterial oxidation and dehydroxylation path-
ways significantly increased the number of colonic RORγ+ Treg cells
and FOXP3+ Treg cells in minimal-diet mice (Fig. 1e and Extended Data
Fig. 2a, b). We tested several more combinations of secondary BAs and

found that only a combination of lithocholic/3-oxo-lithocholic acids

restored colonic RORγ+ Treg counts (Fig. 1e). No similar effect of these

BAs was found on colonic type 17 T helper (TH17) cells (Extended Data

Fig. 2c), or on RORγ+ Treg cells or TH17 cells from the spleen, mesenteric

lymph node or small intestine (Extended Data Fig. 2d, e). These results

indicated that certain primary and secondary BA species within a BA

pool preferentially regulate RORγ+ Treg cells and that modulation of this

Treg cell population by BAs is both cell-type and tissue-type specific.

FOXP3

+

(% of TCR

+β

CD4

+)

Helios-FITC

ROR

γ-PE

Helios-FITC

ROR

γ-PE

9.6%

GF-

rich diet

SPF-

minimal diet

21.7%

SPF-

rich diet

46.8%

a b

e

0

10

20

30

40

60

50

ROR

+γ

Helios

• T

reg

(% of FOXP3

+)

***

***

cd

***

***

** ***

***

***

***

**

***

Minimal diet

Rich diet +

• +++++ ++ ++++ +++++++ + +

––––––

+

––

+

–––––

+

––––––

+

–––––

ROR

+γ

Helios

• T

reg

(% of FOXP3

+)

SPF-

minimal diet

CA/CDCA/UDCA

38.0%

SPF-

minimal diet

CA alone

20.1%

SPF-

minimal diet

CDCA alone

19.6%

SPF-

minimal diet

UDCA alone

21.2%

SPF-

minimal diet

Oxo-BAs/

LCA/DCA

35.2%

SPF-

minimal diet

21.0%

2 mM CA 2 mM LCA

2 mM LCA/DCA mix1 mM oxo-BAs mix

2 mM oxo-CAs/DCA mix2 mM 3-oxo-LCA/LCA mix

1 mM oxo-BAs/LCA/DCA mix

2 mM DCA

2 mM 3-oxo-LCA2 mM 3-oxo-CA2 mM 7-oxo-CA

2 mM 7-oxo-CDCA2 mM 12-oxo-DCA2 mM 12-oxo-CA

2 mM CA/CDCA/UDCA mi

x

3 mM CA/CDCA mix3 mM CA/UDCA mix

3 mM CDCA/UDCA mix

2 mM CDCA2 mM UDC

A

6 mM CA

6 mM CDCA6 mM UDCA

Primary BAs Secondary BAs

0

10

20

30

40

50

60

0

10

20

30

40

50

60

SPF-rich diet SPF-minimal diet

GF-rich diet

(n = 7) (n = 7)

(n = 7)

GF-rich diet

SPF-rich diet

(n = 5)

(n = 6)

(n = 5)

SPF-minimal diet

n = 7n = 6n = 4n = 8n = 8n = 6n = 6n = 6n = 5n = 6n = 6n = 10n = 8n = 7n = 6n = 6n = 6n = 7n = 8n = 6n = 8n = 6n = 6n = 6n = 9

GF-rich diet

SPF-rich diet

(n = 5)

(n = 6)

(n = 5)

SPF-minimal diet

GF-rich diet

SPF-rich diet

(n = 5)

(n = 6)

(n = 5)

SPF-minimal diet

CA

CDCAUDCAαMCA βMCA

Deconjugated primary bile acids

(μg per g faeces)

***

** *

**

*

**

(^100) ** ***
20
30
30
90
150
210
210
510
1,110 810
1,410
DC
A
LCA
3-oxo-CA3-oxo-LCA7-oxo-CA
7-oxo-CDCA12-oxo-CA12-oxo-DCA
ωMCA
Secondary bile acids
(μg per g faeces)

---

• 
  


• 
  


• 
  

  0
  25
  50
  75
  100
  350
  850
  1,350
  1,850
  2,350
  TC
  A
  TCDCATUDCATα
  MCA
  Tβ
  MCA
  Conjugated primary bile acids
  (μ
  g per g faeces)

  
  

  ---

  
  

  ---

  
  

  ---

  
  


• 
  

  ---

  
  

  
  
  3
  6
  9
  12
  0
  15
  (^13030)
  230
  330430
  530
  Fig. 1 | Gut BA metabolites are essential for colonic RORγ+ Treg cell
  maintenance. 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. Representative plots and
  frequencies of RORγ+Helios− in the FOXP3+CD4+TCRβ+ Treg cell population are
  shown. b–d, Liquid chromatography–mass spectrometry (LC–MS)
  quantification of faecal conjugated primary BAs (b), deconjugated primary
  BAs (c) and secondary BAs (d) from groups of mice fed as in a. The BAs
  determined were cholic acid (CA), chenodeoxycholic acid (CDCA),
  ursodeoxycholic acid (UDCA), α-muricholic acid (αMCA), βMCA, deoxycholic
  acid (DCA), lithocholic acid (LCA), 3-oxo-CA, 3-oxo-LCA, 7-oxo-CA, 7-oxo-
  CDCA, 12-oxo-CA, 12-oxo-DCA, ωMCA and taurine-conjugated species (TCA,
  TCDCA, TUDCA, TαMCA and TβMCA). e, 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 for 4 weeks. Representative
  plots and frequencies of colonic RORγ+Helios− in the FOXP3+CD4+TCRβ+ Treg cell
  population and of colonic FOXP3+ in the CD4+TCRβ+ cell population are shown.
  The BAs used in the feed were CA, CDCA, UDCA, DCA, LCA, 3-oxo-CA, 3-oxo-
  LCA, 7-oxo-CA, 7-oxo-CDCA, 12-oxo-CA, 12-oxo-DCA and various indicated BA
  combinations. Data are representative of at least two independent
  experiments in a–d. Data are pooled from three independent experiments in e.
  n represents biologically independent animals. Data are mean ± s.e.m.
  P < 0.05, P < 0.01, P < 0.001, one-way analysis of variance (ANOVA) followed
  by the Bonferroni post hoc test.