supplementary information - media.nature.com · | 5 supplementary information research 0.01 legend:...

W W W. N A T U R E . C O M / N A T U R E | 1

SUPPLEMENTARY INFORMATIONdoi:10.1038/nature12721

0

1

2

3

4

Tota

l num

ber o

f Neu

ropi

lin1-‐

Tota

l num

ber o

f CD

103+

cel

ls

GF HFD LFD CRB

GF HFD LFD CRB

0

1

2

3

4

5

Supplementary Figure 1 | The number of total regulatory T cells in colonic lamina propria. Colonic lamina propria cells were prepared from GF and CRB mice fed with HFD and LFD. CD3ε+CD4+ T cells were further analyzed for expression of Foxp3, Neuropilin1 and CD103. Data are representative of three independent experiments. The Error bars indicate s.d. (n = 10). P values were determined by the one-way ANOVA test followed by Tukey’s test.

0

1

2

3

4

GF HFD LFD CRB

Tota

l num

ber o

f Neu

ropi

lin1+

0

1

2

3

4

5

GF HFD LFD CRB

Tota

l num

ber o

f CD

103-‐

cel

ls

(x104) (x104)

(x104) (x104)

P < 0.05 P < 0.01

P < 0.05 P < 0.01

SUPPLEMENTARY INFORMATION

2 | W W W. N A T U R E . C O M / N A T U R E

RESEARCH

Tota

l num

ber o

f He

lios-‐

cel

ls

GF HFD LFD

CRB

0

2

4

6

0

5

10

15

20

25

[%] o

f He

lios-‐

Fox

p3+

GF HFD LFD

CRB

0

2

4

6

8

10

GF HFD LFD

CRB

[%] o

f He

lios+

Fox

p3+

GF LFD HFD

Helios

Foxp

3

Supplementary Figure 2 | Helios expression in Foxp3+ cells in the colonic lamina propria. Colonic lamina propria cells were prepared from GF and CRB mice fed with HFD and LFD. CD3ε+CD4+ T cells were further analyzed for expression of Foxp3 and Helios. Data are representative of three independent experiments. The Error bars indicate s.d. (n = 10). P values were determined by the one-way ANOVA test followed by Tukey’s test.

0.0

0.5

1.0

1.5

2.0

2.5

GF HFD LFD

CRB

Tota

l num

ber o

f He

lios+

cel

ls

(x104) (x104)

CRB

P < 0.01

P < 0.01

P < 0.01

P < 0.01

P < 0.01

P < 0.01

Gated on CD3ε+ CD4+


SUPPLEMENTARY INFORMATION RESEARCH

Supplementary Figure 3 | Flow cytometric analysis of lymphocytes in spleen and mesenteric lymph node (MLN). MLN (a) and spleen (b) lymphocytes were prepared from GF and CRB mice fed with HFD and LFD. CD3ε+CD4+ T cells were further analyzed for expression of the indicated markers. The Error bars indicate s.d. (n = 5). Data are representative of three independent experiments.

CD103

Helios

Foxp

3

GF CRB-‐LFD CRB-‐HFD

a

b

GF CRB-‐LFD CRB-‐HFD

Foxp

3

CD103

Helios

Foxp

3 Fo

xp3





RESEARCH

Supplementary Figure 4 | 16S rRNA sequence-based analysis of the cecal microbiota of CRB-associated mice fed with LFD or HFD. a-d, 16S rRNA gene was PCR amplified and sequenced on the 454 platform (n = 2). Phylum (a), class (b), order (c) and family; genus in Clostridiales (d) are shown.

a

b

c

d

0% 50% 100%

HFD

LFD

0% 50% 100%

HFD

LFD

0% 50% 100%

HFD

LFD

0% 50% 100%

HFD

LFD

Peptostreptococcaceae; Unclassified

Ruminococcaceae; Unclassified

Lachnospiraceae; Unclassified

Clostridia

Mollicutes

Bacilli

Others

Clostridiales

Unclassified Clostridia

Anaeroplasmatales

Turicibacterales

Others

Ruminococcaceae; Oscillospira

Ruminococcaceae; Ruminococcus

Unclassified; Unclassified

Others

Firmicutes

Tenericutes

Others



0.01Legend:

Dataset read

L_total

H_total

spore

Color ranges:

IV

I

II

XI

III

XIVa

Escherichia coli {NR 102804}

OTU 1359

OTU 1015

OTU 343

OTU 141OTU 1052

OTU 1418

Hydrogenoanaerobacterium saccharovorans {NR 044425}

OTU 1395Clostridium

cellulosi {FJ465164}

Acetanaerobacterium elongatum

{NR 042930}

OTU 878

OTU 119

OTU

1234O

TU 1555

OTU

856

OTU

1580

OTU 149

OTU 1498

Clostridium m

ethylpentosum {Y18181}

OTU 1604

OTU 1087

Ruminococcus bromii {DQ882649}

Clostridium leptum {AJ305238}

Butyricicoccus pullicaecorum {EU410376}

Eubacterium desmolans {L34618}

OTU

105O

TU 1393 O

TU 1

422

Oscillospira guillierm

ondii {AB040495}

OTU

112O

TU 1465

Oscillibacter valericigenes {N

R 074793} O

TU 1

81O

TU 2

33

OTU

608

OTU

135

8O

TU 1

423

OTU

118

1O

TU 6

52

OTU

239

OTU

422

OTU

46

OTU

540

OTU

295

OTU

43

OTU 52

2OTU

529

Clostrid

ium or

biscin

dens

{Y18

187}

Flavonifra

ctor p

lautii {NR 043142}

Pseud

oflav

onifra

ctor c

apillo

sus {

NR 0256

70}

OTU 21

7

OTU 1596

OTU 513OTU 744OTU 1500

OTU 1048OTU 1097

OTU 1

068

OTU 10

83

OTU 1

406

OTU

211

OTU

1641C

lost

ridiu

m v

iride

{NR

026

204}

Faecalibacterium prausnitzii {AY305307}

Faecalibacterium prausnitzii {NR 028961}

Faecalibacterium prausnitzii {AJ270469}


OTU 455

Clostridium sporogenes {AJ579907}

Clostridium tetani {NR 074498}Clostridium tyrobutyricum {M59113}Clostridium peptidovorans {AF156796}

Clostridium histolyticum {M59094}Clostridium limosum {M59096}

Clostridium proteolyticum {X73448}

Clostridium grantii {X75272}

Clostridium acetobutylicum {X68182}

Clostridium butyricum {FJ384369}Clostridium disporicum {Y18176}

Clostridium difficile {AB638443}

Clostridium difficile {X73450}

Clostridium lituseburense {M59107}

OTU 20

Clostridium litorale {X77845}

Clostridium thermolacticum {X72870}Clostridium termitidis {X71854}

Clostridium aldrichii {NR 026099}

Clostridium thermocellum {NR 074629}

OTU 50OTU 1313

OTU 100OTU 1496

OTU 1034

OTU 96OTU 1070

OTU 1250

OTU 1538OTU 998

OTU 1434OTU 979

OTU 634Catonella morbi {NR 026248}

Clostridium scindens {NR 028785}

OTU 1073

OTU 1091

OTU 601OTU 1299

OTU 1426

OTU 600OTU 1215

OTU 1038OTU 1397

OTU 124

OTU 611

OTU

262

OTU

984O

TU 466

OTU

1309O

TU 1142O

TU 7

72

OTU

849

OTU

1099O

TU 89

OTU

614

OTU

1519

OTU

923Butyrivibrio proteoclasticus {U

37378}

Clostridium nexile {NR 029248}Clostridium oroticum {FR749917}

Clostridium coccoides {EF025906}

Roseburia inulinivorans {AJ270473}

Roseburia inulinivorans {NR 042007}

Lachnospira pectinoschiza {AY169414}

Roseburia intestinalis {AJ312385}

Roseburia intestinalis {AY305308}Roseburia intestinalis {NR 027557}

Roseburia hominis {AJ270482}Roseburia faecis {AY305310}

Roseburia cecicola {L14676}

OTU 399

OTU 1386OTU 1653

Clostridium hylemonae {NR 024719}

OTU 268OTU 693

OTU 114OTU 288

Clostridium celerecrescens {NR 026100}

Clostridium xylanolyticum {X71855}Clostridium bolteae {NR 025567}

Clostridium clostridioforme {HM008264}

Clostridium lavalense {EF564278}

OTU 293OTU 571

OTU 179OTU 1656

OTU 139

Rumino

coccu

s obe

um {A

Y1694

11}

OTU 1578

Euba

cteriu

m re

ctale

{AJ2

7047

5}

Euba

cteriu

m re

ctale

{AY3

0531

1}

Butyr

ivibr

io fib

risolv

ens {

AJ25

0365

}

Blau

tia h

ydro

geno

troph

ica {N

R 02

6314

}O

TU 1

449

Anae

rost

ipes

cac

cae

{AJ2

7048

7}

Para

spor

obac

teriu

m p

auciv

oran

s {NR

025

390}

OTU 23

7OTU 1053

OTU 1623

OTU 10

11

Clostrid

ium xy

lanov

oran

s {NR 02

8740

}

OTU 55

6

Clos

tridi

um a

min

ophi

lum

{DQ

2788

62}

Euba

cter

ium

rum

inan

tium

{NR

0246

61}

OTU

141

0

OTU

490

OTU

264

OTU

162

4

OTU

223

OTU

953

OTU

148

4O

TU 6

56

Cluster XIVa

Cluster IV

Cluster III

Cluster I

Cluster II

Cluster XI

Supplementary Figure 5 | Phylogenetic distribution of operational taxonomic units (OTUs) obtained from 16S rRNA sequences in order Clostriales colonized in CRB mice. 12,000 filter-passed reads of 16S rRNA sequences in CRB mice were used for OTUs and a phylogenetic tree was constructed based on the CRB-derived 114 OTUs which were categorized into Clostridiales by RDP-classifier (v2.5) and 69 reference 16S rRNA sequences of Clostridiales obtained from the GenBank database. The 16S rRNA sequence of Escherichia coli was used as the outgroup. The phylogenetic tree was constructed based on the neighbor-joining method, with 100 bootstrap iterations (bootstrap values > 70 are shown on each branch). The scale bar indicates an estimated 0.1 change per nucleotide position. Colors configuring a doughnut shape represent the branches consisting of the distinct clusters of Clostridiales. The reference 16S rRNA sequences from spore forming, non-spore forming, and unknown spore forming capacity bacteria are shown in blue, purple and black, respectively. The outside red and green bars indicate the relative number of sequence reads in each OTU from CRB-HFD and CRB-LFD, respectively. Most of OTUs both in CRB-HFD and CRB-LFD were clustered into Clostridium clusters IV and XIVa.

0.1

0.01Legend:

Dataset read

L_total

H_total

spore

Color ranges:

IV

I

II

XI

III

XIVa

Escherichia coli {NR 102804}

OTU 1359

OTU 1015

OTU 343

OTU 141OTU 1052

OTU 1418

Hydrogenoanaerobacterium saccharovorans {NR 044425}

OTU 1395Clostridium

cellulosi {FJ465164}

Acetanaerobacterium elongatum

{NR 042930}

OTU 878

OTU 119

OTU

1234O

TU 1555

OTU

856

OTU

1580

OTU 149

OTU 1498

Clostridium m

ethylpentosum {Y18181}

OTU 1604

OTU 1087

Ruminococcus bromii {DQ882649}

Clostridium leptum {AJ305238}

Butyricicoccus pullicaecorum {EU410376}

Eubacterium desmolans {L34618}

OTU

105O

TU 1393 O

TU 1

422

Oscillospira guillierm

ondii {AB040495}

OTU

112O

TU 1465

Oscillibacter valericigenes {N

R 074793} O

TU 1

81O

TU 2

33

OTU

608

OTU

135

8O

TU 1

423

OTU

118

1O

TU 6

52

OTU

239

OTU

422

OTU

46

OTU

540

OTU

295

OTU

43

OTU 52

2OTU

529

Clostrid

ium or

biscin

dens

{Y18

187}

Flavonifra

ctor p

lautii {NR 043142}

Pseud

oflav

onifra

ctor c

apillo

sus {

NR 0256

70}

OTU 21

7

OTU 1596

OTU 513OTU 744OTU 1500

OTU 1048OTU 1097

OTU 1

068

OTU 10

83

OTU 1

406

OTU

211

OTU

1641C

lost

ridiu

m v

iride

{NR

026

204}

Faecalibacterium prausnitzii {AY305307}

Faecalibacterium prausnitzii {NR 028961}



OTU 455

Clostridium sporogenes {AJ579907}

Clostridium tetani {NR 074498}Clostridium tyrobutyricum {M59113}Clostridium peptidovorans {AF156796}

Clostridium histolyticum {M59094}Clostridium limosum {M59096}

Clostridium proteolyticum {X73448}

Clostridium grantii {X75272}

Clostridium acetobutylicum {X68182}

Clostridium butyricum {FJ384369}Clostridium disporicum {Y18176}

Clostridium difficile {AB638443}

Clostridium difficile {X73450}

Clostridium lituseburense {M59107}

OTU 20

Clostridium litorale {X77845}

Clostridium thermolacticum {X72870}Clostridium termitidis {X71854}

Clostridium aldrichii {NR 026099}

Clostridium thermocellum {NR 074629}

OTU 50OTU 1313

OTU 100OTU 1496

OTU 1034

OTU 96OTU 1070

OTU 1250

OTU 1538OTU 998

OTU 1434OTU 979

OTU 634Catonella morbi {NR 026248}

Clostridium scindens {NR 028785}

OTU 1073

OTU 1091

OTU 601OTU 1299

OTU 1426

OTU 600OTU 1215

OTU 1038OTU 1397

OTU 124

OTU 611

OTU

262

OTU

984O

TU 466

OTU

1309O

TU 1142O

TU 7

72

OTU

849

OTU

1099O

TU 89

OTU

614

OTU

1519

OTU

923Butyrivibrio proteoclasticus {U

37378}Clostridium nexile {NR 029248}

Clostridium oroticum {FR749917}

Clostridium coccoides {EF025906}

Roseburia inulinivorans {AJ270473}

Roseburia inulinivorans {NR 042007}

Lachnospira pectinoschiza {AY169414}

Roseburia intestinalis {AJ312385}

Roseburia intestinalis {AY305308}Roseburia intestinalis {NR 027557}

Roseburia hominis {AJ270482}Roseburia faecis {AY305310}

Roseburia cecicola {L14676}

OTU 399

OTU 1386OTU 1653

Clostridium hylemonae {NR 024719}

OTU 268OTU 693

OTU 114OTU 288

Clostridium celerecrescens {NR 026100}

Clostridium xylanolyticum {X71855}Clostridium bolteae {NR 025567}

Clostridium clostridioforme {HM008264}

Clostridium lavalense {EF564278}

OTU 293OTU 571

OTU 179OTU 1656

OTU 139

Rumino

coccu

s obe

um {A

Y1694

11}

OTU 1578

Euba

cteriu

m re

ctale

{AJ2

7047

5}

Euba

cteriu

m re

ctale

{AY3

0531

1}

Butyr

ivibr

io fib

risolv

ens {

AJ25

0365

}

Blau

tia h

ydro

geno

troph

ica {N

R 02

6314

}O

TU 1

449

Anae

rost

ipes

cac

cae

{AJ2

7048

7}

Para

spor

obac

teriu

m p

auciv

oran

s {NR

025

390}

OTU 23

7OTU 1053

OTU 1623

OTU 10

11

Clostrid

ium xy

lanov

oran

s {NR 02

8740

}

OTU 55

6

Clos

tridi

um a

min

ophi

lum

{DQ

2788

62}

Euba

cter

ium

rum

inan

tium

{NR

0246

61}

OTU

141

0

OTU

490

OTU

264

OTU

162

4

OTU

223

OTU

953

OTU

148

4O

TU 6

56

Cluster XIVa

Cluster IV

Cluster III

Cluster I

Cluster II

Cluster XI

Supplementary Figure 5 | Phylogenetic distribution of operational taxonomic units (OTUs) obtained from 16S rRNA sequences in order Clostriales colonized in CRB mice. 12,000 filter-passed reads of 16S rRNA sequences in CRB mice were used for OTUs and a phylogenetic tree was constructed based on the CRB-derived 114 OTUs which were categorized into Clostridiales by RDP-classifier (v2.5) and 69 reference 16S rRNA sequences of Clostridiales obtained from the GenBank database. The 16S rRNA sequence of Escherichia coli was used as the outgroup. The phylogenetic tree was constructed based on the neighbor-joining method, with 100 bootstrap iterations (bootstrap values > 70 are shown on each branch). The scale bar indicates an estimated 0.1 change per nucleotide position. Colors configuring a doughnut shape represent the branches consisting of the distinct clusters of Clostridiales. The reference 16S rRNA sequences from spore forming, non-spore forming, and unknown spore forming capacity bacteria are shown in blue, purple and black, respectively. The outside red and green bars indicate the relative number of sequence reads in each OTU from CRB-HFD and CRB-LFD, respectively. Most of OTUs both in CRB-HFD and CRB-LFD were clustered into Clostridium clusters IV and XIVa.

0.1



RESEARCH

b

Supplementary Figure 6 | The number of total commensal microbes and Clostridiales in GF, and CRB mice fed with HFD and LFD. a, b, Total number of commensal microbes (a) and Clostridiales (b) were detected by qPCR with 16S rRNA gene universal primers and Clostridiales-specific primers. The results were calculated as the quantity relative to the copy number detected in the feces of conventionalized mice (CV). Error bars indicate s.e.m. (n = 5). P values were determined using the Kruskal-Wallis test followed by the Scheffé test. n.s., not significantly different. n.d., not detected.

a

(%) C

V

nd

n.s.

GF HFD LFD CRB

100

50

0

(%) C

V

nd

n.s.

GF HFD LFD CRB

100

50

0

b

Supplementary Figure 6 | The number of total commensal microbes and Clostridiales in GF, and CRB mice fed with HFD and LFD. a, b, Total number of commensal microbes (a) and Clostridiales (b) were detected by qPCR with 16S rRNA gene universal primers and Clostridiales-specific primers. The results were calculated as the quantity relative to the copy number detected in the feces of conventionalized mice (CV). Error bars indicate s.e.m. (n = 5). P values were determined using the Kruskal-Wallis test followed by the Scheffé test. n.s., not significantly different. n.d., not detected.

a

(%) C

V

nd

n.s.

GF HFD LFD CRB

100

50

0

(%) C

V

nd

n.s.

GF HFD LFD CRB

100

50

0



Supplementary Figure 7 | Orthogonal partial least squares discriminate analysis (OPLS-DA) on the cecal metabolome data of CRB-LFD and CRB-HFD mice. a, Cross-validated score plots from OPLS-DA of 1H-NMR data of CRB-LFD and CRB-HFD murine cecum (n = 7). The model resulted in one predictive and one orthogonal (1+1) components with the cross-validated predictive ability Q2(Y) = 0.93 and the total explained variance R2(X) = 0.79. The ellipse denotes the 95% significance limit of the model, as defined by Hotelling’s t-test. b, S-plot for predictive component from OPLS-DA of 1H-NMR data of CRB-LFD and CRB-HFD murine cecum. The S-plot visualizes the variable influence in a model and corresponds to combining the contribution or magnitude (covariance) with the effect and reliability (correlation) for the model variables with respect to model component scores. The highlighted signals (|covariance| > 0.16 ) in the S-plot have high contribution and reliability for class separation between CRB-LFD and CRB-HFD mice.

a

b -1.0

-0.5

0.0

0.5

1.0

-0.20-0.15-0.10-0.05-0.000.050.100.150.200.25

p(co

rr)[1

]

p[1]

130509_1HNMR_Treg_IQI&BALB_CRB for Loading and S-plot.M5 (OPLS/O2PLS-DA), L, H: Parp[Comp. 1]/p(corr)[Comp. 1]

R2X[1] = 0.667384 SIMCA-P+ 12.0.1 - 2013-05-20 12:07:30 (UTC+9)

Covariance

Mod

eled

cor

rela

Uon

Acetate Butyrate/Propionate Butyrate L-‐Isoleucine L-‐Leucine GABA Lactate UnidenUfied Others

-

-

- - - - -

-8

-6

-4

-2

0

2

4

6

8

-30-25-20-15-10-5051015202530

to[1

]

t[1]

130509_1HNMR_Treg_IQI&BALB_CRB for Loading and S-plot.M5 (OPLS/O2PLS-DA), L, H: Part[Comp. 1]/to[XSide Comp. 1]Colored according to classes in M5

R2X[1] = 0.667384 R2X[XSide Comp. 1] = 0.124008 Ellipse: Hotelling T2 (0.95)

LH

SIMCA-P+ 12.0.1 - 2013-05-20 12:02:08 (UTC+9)

LFD HFD

PredicUve component

- - - - - -

Ort

hogo

nal c

ompo

nent

1

-

-

-

-



RESEARCH

0

10

20

30

Supplementary Figure 8 | Leucine, Isoleucine and GABA have no effect on the induction of Foxp3+ cells a, b, c, Naïve CD4+ T cells were cultured under Treg-inducing conditions in vitro. The cells were cultured in RPMI 1640 medium, which contains 0.38 mM each of L-leucine and L-isoleucine, that was supplemented with L-leucine (0, 0,12 and 0.62 mM) (a), L-isoleucine (0, 0,12 and 0.62 mM) (b) or GABA (0, 0,001, 0.01 and 0.1 mM) (c). Cells were further analyzed for expression of Foxp3. The Error bars indicate s.d. (n = 3).

0

10

20

30

[%] o

f Fox

p3+

a b c

[%] o

f Fox

p3+

0

10

20

30

[%] o

f Fox

p3+

L-‐leucine L-‐isoeucine GABA



Tota

l num

ber o

f Fo

xp3+ c

ells

CD4

Foxp

3

Supplementary Figure 9 | The number of total Foxp3+ cells in colonic lamina propria of C57BL/6 mice fed with HAMS or HAMSB. Colonic lamina propria cells were prepared from C57BL/6 mice fed with HAMS (Cont) or HAMSB (SB). CD3ε+CD4+ T cells were further analyzed for expression of Foxp3. Data are representative of three independent experiments. The Error bars indicate s.d. (n = 10). P values were determined by the non-parametrical Mann-Whitney U test

0

5

10

15

20 (×104)

P < 0.05

Cont SB

Cont SB



1 0 | W W W. N A T U R E . C O M / N A T U R E

RESEARCH

Supplementary Figure 10 | Butyrate has no effect on proliferation of Treg cells in colonic lamina propria. a, Mice fed with HAMS (Control) or HAMSB (SB) were treated with EdU, a nucleoside analog incorporated into DNA during synthesis phase of cell cycle, for 2 days before the analysis. Colonic lamina propria cells were prepared from those mice and CD3ε+CD4+ T cells were further analyzed for expression of Foxp3 and EdU. The Error bars indicate s.d. (n = 5).

Control

EdU Fo

xp3

SB


W W W. N A T U R E . C O M / N A T U R E | 1 1


Supplementary Figure 11 | Butyrate has no effect on cell survival under Treg-inducing conditions in vitro. The number of total cells under Treg-inducing condition cultures in the absence or presence of butyrate. Cells were cultured as described in Fig.3a. The viability was evaluated by Trypan blue dye exclusion test. The Error bars indicate s.d. (n = 5).

Day 1 Day 2 Day 3 Live Dead

0

10

20

30 Th

e nu

mbe

r of c

ells

0

10

20

30

0

10

20

30 (x104) (x104) (x104)

(-‐) (+) Butyrate: (-‐) (+) (-‐) (+)



RESEARCH

0

2

4

6

8

10

Tota

l num

ber o

f N

euro

pilin

1-‐ T

reg

cells

Tota

l num

ber o

f H

elio

s-‐ T

reg

cells

0

1

2

3

4

5

Neuropilin1

Foxp

3

Helios

Foxp

3 P < 0.05

P < 0.05

Cecal SCFA Fecal SCFA

Supplementary Figure 12 | Intestinal butyrate induces the accumulation of Neuropilin- and Helios- subsets of Treg cells in mice colonized with Bacteroides thetaiotaomicron. GF mice were colonized with B. thetaiotaomicron. B. thetaiotaomicron-associated mice were fed with HAMS (Cont) or HAMSB (SB) for 4 weeks. a, Colonic lamina propria cells were prepared and CD3ε+CD4+-gated cells were further analyzed for expression of Foxp3, Neuropilin and Helios. P values were determined by the non-parametrical Mann-Whitney U test. b, Cecal and fecal amount of short-chain fatty acids from B. thetaiotaomicron-associated mice fed with HAMS or HAMSB. Ace, acetate; Pro, propionate; But, butyrate. The Error bars indicate s.d. (n = 5).

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Supplementary Figure 13 | Butyrate has no effect on Foxp3+ induction in colonic lamina propria of GF mice fed with HAMS or HAMSB. a, Colonic lamina propria cells were prepared from GF mice fed with HAMS (Cont) or HAMSB (SB). CD3ε+CD4+ T cells were further analyzed for expression of Foxp3. b, Amount of cecal short-chain fatty acids from GF mice fed with HAMS or HAMSB. Ace, acetate; Pro, propionate; But, butyrate. Data are representative of three independent experiments. n.s., not statistically significant. The Error bars indicate s.d. (n = 5).

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Supplementary Figure 14 | Intestinal butyrate has no effect on the accumulation of T-bet+, GATA3+ and RORγt+ T cells. Colonic lamina propria cells were prepared from SPF C57BL/6 mice fed with HAMS (Control or Cont) or HAMSB (SB). CD3ε+CD4+ T cells were further analyzed for expressions of T-bet, GATA3, and RORγt, the lineage-specific transcription factors of TH1, TH2, and TH17, respectively. The Error bars indicate s.d. (n = 10). P-values were calculated by Mann-Whitney U test. Data are representative of three independent experiments.

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Supplementary Figure 15 | Butyrate increases the frequency of Foxp3+ T cells under TH1 and TH17 polarizing conditions. a, b, Naïve CD4+ T cells were stimulated with immobilized anti-CD3 and soluble anti-CD28 antibodies. Cells were cultured in the presence or absence of butyrate and either with rIL-12 (10 ng/mL) plus anti-IL-4 (10 mg/mL) for TH1 polarization, with rIL-4 (10 ng/mL) plus anti-IFNγ (10 mg/mL) for TH2 polarization or with rIL-6 (10 ng/mL), rhTGFβ (0.2 ng/mL), rIL-1β (10 ng/mL), anti-IFNγ plus anti-IL-4 for TH17 polarization. rIL-2 (10 ng/mL) was added to culture medium 2 days later. Cells were further analyzed for expression of Foxp3, T-bet, Gata3 and RORγt on day 4. Representative FACS plots gated on CD3ε+CD4+ are shown in c. The Error bars indicate s.d. (n = 3). P-values were calculated by Mann-Whitney U test. Data are representative of three independent experiments.

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Supplementary Figure 16 | Induction of colonic Treg cells by intestinal butyrate in SPF Myd88-/-Ticam1-/- mice. a-b, SPF Myd88-/-Ticam1-/- mice were fed with HAMS (Cont) or HAMSB (SB). Representative FACS plots gated on CD3ε+CD4+ are shown in a. The frequency and total number of Helios-

Foxp3+ cells among colonic CD4+ T cells are shown in b. Data are representative of two independent experiments. The Error bars indicate s.d. (n = 4). P-values were calculated by Mann-Whitney U test.

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Supplementary Figure 16 | Induction of colonic Treg cells by intestinal butyrate in SPF Myd88-/-Ticam1-/- mice. a-b, SPF Myd88-/-Ticam1-/- mice were fed with HAMS (Cont) or HAMSB (SB). Representative FACS plots gated on CD3ε+CD4+ are shown in a. The frequency and total number of Helios-

Foxp3+ cells among colonic CD4+ T cells are shown in b. Data are representative of two independent experiments. The Error bars indicate s.d. (n = 4). P-values were calculated by Mann-Whitney U test.

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Supplementary Fig. 17 | Genome-wide histone H3 acetylation status after butyrate exposure. Naïve CD4+ T cells were cultured under Treg-inducing conditions in the presence or absence (control) of butyrate for 1 day. a, Total and acetylated histone H3 (AcH3) were measured by Western blot analysis of total cell lysates. b, ChIP-Seq analysis was performed using anti-AcH3 antibody. The average values of AcH3 status around the transcription start site (TSS) over total genes are shown.

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Supplementary Figure 18 | Histone acetylation status of Tbx21, Gata3 and Rorc. Naïve CD4+ T cells were cultured under Treg-inducing conditions in the presence or absence (control) of butyrate for 1 day. Histone acetylation in the genes encoding master molecules for effector T cells was analyzed by ChIP-sequencing using anti-acetylated histone H3.

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Supplementary Figure 18 | Histone acetylation status of Tbx21, Gata3 and Rorc. Naïve CD4+ T cells were cultured under Treg-inducing conditions in the presence or absence (control) of butyrate for 1 day. Histone acetylation in the genes encoding master molecules for effector T cells was analyzed by ChIP-sequencing using anti-acetylated histone H3.

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Supplementary Figure 19 | Histone acetylation status of Stat5, Smad3, Nfatc2 and Rel. Naïve CD4+ T cells were cultured under Treg-inducing conditions in the presence or absence (control) of butyrate for 1 day. Histone acetylation in the genes encoding transcriptional regulators controlling Foxp3 expression was analyzed by ChIP-sequencing using anti-acetylated histone H3.

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Supplementary Figure 20 | Expression of STAT5, SMAD3, NFAT1, and c-Rel. Naïve CD4+ T cells were cultured under Treg-inducing conditions in the absence or presence of butyrate. Total cell lysates were subjected to western blotting analysis with antibodies described in Methods. H3 and AcH3 indicate histone H3 and acetylated histone H3, respectively. Data are representative of three independent experiments.

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Supplementary Figure 21 | Intestinal butyrate, but not acetate or propionate, ameliorates T-cell-dependent experimental colitis. a-f, Experimental colitis was induced by adoptive transfer of CD4+CD45RBhi T cells in Rag1-/- mice fed with HAMS (Control), HAMSA (SA), HAMSP (SP) or HAMSB (SB). Fecal amount of SCFAs (a), body weight change (b), and representative colonic specimens stained with hematoxylin-eosin (HE) and Alcian blue (AB) (c) are shown. The Error bars indicate s.d. (n = 5). Scale bars: 200 µm (c). All data are representative of two independent experiments.

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RESEARCH

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Epigenetic modifications

Supplementary Figure 22 | A schematic diagram of epigenetic modifications by commensal microbial fermentation product. Clostridiales abundantly produce butyrate, which promotes histone H3 lysine acetylation in Foxp3 gene locus of CD4+ T cells, and eventually facilitates induction of Treg cells. APC: antigen-presenting cells.

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