©20

14 N

atu

re A

mer

ica,

Inc.

All

rig

hts

res

erve

d.

l e t t e r s

nature medicine  advance online publication �

Regulatory B cells (Breg cells) differentiate in response to inflammation and subsequently restrain excessive immune responses via the release of interleukin-10 (IL-10)1. However, the precise inflammatory signals governing their differentiation remain to be elucidated. Here we show that the gut microbiota promotes the differentiation of Breg cells in the spleen as well as in the mesenteric lymph nodes. Perturbation of the gut microbiome imposed either by antibiotic treatment or by changes in the sterility of housing conditions reduces the number and function of Breg cells. Following the induction of arthritis, IL-1b and IL-6 are produced only in conventionally housed mice and both cytokines directly promote Breg cell differentiation and IL-10 production. Mice lacking IL-6 receptor (IL-6R) or IL-1 receptor 1 (IL-1R1) specifically on B cells have a reduced number of IL-10–producing B cells and develop exacerbated arthritis compared to control animals. Thus, in response to inflammatory signals induced by both the gut flora and arthritis, Breg cells increase in number and restrain excessive inflammation.

To examine the link between the gut microbiota, inflammation and Breg cell differentiation, we treated conventionally housed (CNV) C57BL/6 wild-type (WT) mice with a combination of different anti-biotics (vancomycin, neomycin and metronidazole) to eliminate the majority of intestinal bacteria2 and left a control group untreated. Antibiotic-treated mice (ABX mice) developed milder antigen-induced arthritis compared to control mice (Fig. 1a), confirming the dependency of arthritis on commensal gut microbiota3. Amelioration of disease in ABX mice was associated with reduced percentages and absolute numbers (numbers) of CD4+IFNγ+ T helper type 1 (TH1) and CD4+IL-17+ T helper type 17 (TH17) cells, and decreased concentra-tion of IL-17 and interferon-γ (IFN-γ), in draining lymph nodes (LNs) (Supplementary Fig. 1a–d). We also detected decreased production of IL-1β, IL-6, tumor necrosis factor-α (TNF-α), IFN-γ and IL-17 by stimulated splenocytes from ABX mice compared to control mice (Fig. 1b,c). In agreement with previously published data showing that ABX mice have fewer Treg cells4, we found that the percentage and

number of Treg cells were reduced in the draining LNs of ABX mice compared to control mice (Supplementary Fig. 1e). Analysis of the B cell compartment revealed that both the percentage and number of CD19+CD21hiCD23hiCD24hiCD1dhi transitional two-marginal-zone precursor B cells (T2-MZPs), which were previously shown to have immunosuppressive Breg cell function, were reduced in ABX mice compared to control mice (Fig. 1d). There were no significant differ-ences in total, transitional 1 (T1), marginal zone or follicular B cells in ABX mice versus control mice (Supplementary Fig. 1f–i).

To determine whether the microbiota instructs T2-MZPs to become functionally suppressive, we isolated T2-MZPs from ABX mice and control mice and transferred them into syngeneic hosts. Unlike control T2-MZPs, T2-MZPs from ABX mice failed to suppress antigen-induced arthritis (Fig. 1e).

IL-10 is produced by Breg cells and is required for Breg cell–mediated suppression1. Thus, we assessed IL-10 expression by donor T2-MZPs ex vivo using reporter mice5 that express eGFP under control of the IL-10 promoter (IL-10–eGFP). The number and frequency of IL-10–eGFP+ T2-MZPs and IL-10–eGFP+ B cells were reduced in ABX mice as compared to control mice (Fig. 1f,g). Moreover, ABX mice showed reduced IL-10 production from B cells stimulated with an agonistic monoclonal antibody against CD40 (anti-CD40) or lipopolysaccharide (LPS) compared to control mice (Fig. 1h). Additionally, numbers of total and IL-10–eGFP+T2-MZPs, and other Breg subsets including CD5+ B cells6, TIM-1+ B cells7 and CD5+CD1dhi B cells (B10)8, were reduced in ABX mice compared to control mice on days 1–3 and 7 following the induction of arthritis (Supplementary Fig. 2a–h). The numbers of total and IL-10–eGFP+ T2-MZPs in ABX mice were equivalent to those in untreated naive mice (data not shown). Previously, it was reported that ABX treatment induces a population of CD5+ Breg cells9. The conflict-ing results here could be due to differences in animal husbandry, diet and source of mice. The treatment of mice with ampicillin, vancomycin, neomycin and metronidazole in the other study compared to vanco-mycin, neomycin and metronidazole in our study may also further contribute to the differences observed between the two studies. Such factors would alter the composition of the microbiota and the baseline immunological phenotype of the mice before ABX treatment.

Regulatory B cells are induced by gut microbiota–driven interleukin-1β and interleukin-6 productionElizabeth C Rosser1, Kristine Oleinika1, Silvia Tonon2, Ronan Doyle3, Anneleen Bosma1, Natalie A Carter1,6, Kathryn A Harris4, Simon A Jones5, Nigel Klein3 & Claudia Mauri1

1Centre for Rheumatology, Division of Medicine, University College London, London, UK. 2Universita’ degli Studi di Udine, Dipartimento di Medicina e Scienze Biologiche, Udine, Italia. 3Infectious Diseases and Microbiology Unit, Institute of Child Health, University College London, London, UK. 4Department of Microbiology, Virology and Infection Control, Great Ormond Street Hospital National Health Service Foundation Trust, London, UK. 5Cardiff Institute for Infection & Immunity, The School of Medicine, Cardiff University, Cardiff, Wales, UK. 6Present address: Arthritis Research UK, London, UK.Correspondence should be addressed to C.M. (c.mauri@ucl.ac.uk).

Received 5 April; accepted 6 August; published online 19 October 2014; doi:10.1038/nm.3680

©20

14 N

atu

re A

mer

ica,

Inc.

All

rig

hts

res

erve

d.

l e t t e r s

�  advance online publication nature medicine

To confirm the importance of the gut microbiota in arthritis devel-opment and Breg cell expansion, we recolonized ABX mice with fecal materials obtained from control mice. Recolonized ABX mice devel-oped an antigen-induced arthritis with similar knee swelling to that observed in control mice. In addition, Breg cells isolated from ABX mice recolonized with fecal materials obtained from control mice sup-pressed the development of antigen-induced arthritis upon adoptive transfer into recipient mice (Supplementary Fig. 3a–d).

To establish whether subtler changes in the microbiota due to vari-ations in housing conditions and sterility also affect Breg cell differ-entiation, we compared the suppressive activity of Breg cells isolated from mice housed in specific pathogen–free (SPF) versus conven-tional (CNV) facilities. SPF mice developed a milder disease, asso-ciated with reduced production of IL-1β, IL-6, TNF-α, IFN-γ and IL-17 by splenocytes, compared to CNV mice (Fig. 2a–c). Analysis of draining LNs from SPF mice revealed a reduction in percentages and numbers of TH1 cells, TH17 cells and Foxp3+ Treg cells and in IL-17 and IFN-γ production by lymphocytes compared to drain-ing LNs from CNV mice (Supplementary Fig. 4a–e). Furthermore, unlike T2-MZPs from CNV mice, T2-MZPs from SPF mice failed to suppress arthritis upon adoptive transfer (Fig. 2d). The number and frequency of IL-10–eGFP+ T2-MZPs and IL-10–eGFP+ B cells were reduced in SPF compared to CNV mice at day 7 after disease induc-tion (Fig. 2e and Supplementary Fig. 4f).

To identify the constituents of the microbiota involved in arthritis development and Breg cell differentiation, we collected feces from CNV, ABX and SPF mice and used a 16S rDNA–based amplicon sequencing approach to compare microbiota diversity and to identify whether

specific bacterial signatures differed between groups. Analysis of feces from ABX mice confirmed an ablation of most bacterial species, with the exception of Lactobacillus spp., Sutterella spp. and Klebsiella spp. (Fig. 2f and data not shown). A comparison of clonal diversity between CNV and SPF mice revealed subtler compositional changes (Fig. 2g–i). The population diversities as determined by the Chao1 diversity index in CNV and SPF mice were similar owing to the domi-nance of Bacteroidales and Clostridiales orders (Fig. 2g and data not shown). Unweighted UniFrac principal component analysis revealed that the largest differences in bacterial species diversity were observed between ABX and CNV mice and ABX and SPF mice. Comparison of samples from CNV and SPF mice revealed they were less clustered than samples from ABX mice with an average variation of 8.9% in principal component 2 (Fig. 2h). This clustering between SPF and CNV mice was associated with variation in nine taxa (Fig. 2i). As recently reported, there was a large proportion of Lactobacillus spp. in CNV mice that was associated with a higher percentage and number of Treg cells compared to SPF mice10,11 (Supplementary Fig. 4e). These findings indicate that small variations in the microbiota influence arthritis development and Breg cell differentiation.

Gut bacteria induce IL-1β and IL-6 production by macrophages and dendritic cells (DCs) in gut-associated lymphoid tissues (GALTs) and in the periphery12–15. The impaired production of IL-1β and IL-6 by splenocytes from arthritic ABX mice compared to con-trol arthritic mice suggested a similar effect in our model (Fig. 1b and Supplementary Fig. 5a,b). By comparing the frequency and number of DC subsets and macrophages, we noted a decrease in CD11b+CD11c− macrophages but an increase in both CD11b+CD11c+

Figure 1 Impaired Breg cell function in mice treated with antibiotics. (a) Mean clinical score of untreated control (Ctrl) and ABX-treated mice following induction of arthritis; y axis shows percentage swelling in antigen-injected knee compared to control knee (n = 21 per group, one of five experiments is shown). (b) Production of IL-1β, IL-6 and TNF-α by splenocytes from control and ABX mice at 24 h after disease onset, as measured by ELISA (n = 9 per group per cytokine, one of two experiments is shown; ND, not detectable). (c) Production of IFN-γ and IL-17 by splenocytes from control and ABX mice at day 7 after disease onset, as measured by ELISA (n = 9 per group per cytokine, one of two experiments is shown). (d) Representative FACS plots (left) and bar charts (right) showing the percentage and number of T2-MZPs in control and ABX mice at day 7 after disease onset (n = 21 per group, one of five experiments is shown). (e) Mean clinical score of C57BL/6 WT mice following adoptive transfer of T2-MZPs purified from control and ABX mice administered upon disease onset. Control (no transfer) group received PBS. (n = 7 per group, one of two experiments is shown). (f,g) Representative FACS plots (left) and bar charts (right) showing percentage and number of splenic IL-10–eGFP+ T2-MZPs (f) or IL-10–eGFP+CD19+ B cells (g) from control and ABX mice at day 7 after disease onset (n = 18 per group, one of four experiments is shown). (h) IL-10 production by B cells, isolated from ABX and control mice at day 7 after disease onset, after stimulation with anti-CD40 (aCD40) or LPS, as measured by ELISA (n = 9 per group, one of three experiments is shown). Data represent mean ± s.e.m.; *P < 0.05, **P < 0.01, ***P < 0.005 (b–d,f–h, unpaired t test; a, e two-way analysis of variance (ANOVA)).

b c150

IL-1

β (p

g/m

l)

100

ND

50

0

TN

F-α

(ng

/ml)

4

3

2

1

0

4

IL-6

(ng

/ml)

*3

2

1

0

*

% T

2-M

ZP

IL-1

0–eG

FP

+

15

10

5

0

*

% C

D19

+IL

-10–

eGF

P+

5

4

3

2

1

0

*

CD

19+

IL-1

0–eG

FP

+ (×

105 ) 20

15

10

5

0

h

aCD40

***

IL-1

0 (p

g/m

l)

300

200

100

0

a60

% s

wel

ling

(mB

SA

-PB

S k

nee)

50403020

0

100

1 2 3

CtrlABX

Time (d)

******

******

***

4 5 6 7

% s

wel

ling

(mB

SA

-PB

S k

nee)

eNo transfer

**

*

0 1 2 3Time (d)

4 5 6 7

605040302010

0

T2-MZPfrom CtrlT2-MZPfrom ABX

IFN

-γ (

ng/m

l)

40

30

20

10

0

CtrlABX

IL-1

7 (n

g/m

l)

10

8

6

4

2

0

CtrlABX

*

% T

2-M

ZP

IL-1

0–eG

FP

+ (×

105 ) 5

4

3

2

1

0

CtrlABX

LPS

*

IL-1

0 (n

g/m

l)

10.0

7.5

5.0

2.5

0

10

8

6

4

2

0

% T

2-M

ZP Ctrl

ABX

**50

T2-

MZ

Ps

(×10

5 )

40

30

20

10

0

dCtrl

CD24

CD

21

ABX

CD19+ gated

16.261.6 60.7

4.914.11

14.1105

105

104

104

103

103

102

102

0

105

104

103

102

0

0 1051041031020CD23

CD

21

Ctrl ABX

CD19+CD21hiCD24hi gated

37.2 61

T2-MZP T2-MZP

42.5 55.9105

105

104

104

103

103

102

102

0

105

104

103

102

0

0 1051041031020

f

CD19

FM

O

IL-1

0/eG

FP

Ctrl ABX

0.81 13.9 4.95

T2-MZP+ gated

105

104

103

102

0

1051041031020

105

104

103

102

0

1051041031020

105

104

103

102

0

1051041031020

gCtrl

CD19

IL-1

0/eG

FP

FM

O

ABX

CD19+ gated

0.73 5.88 2.98105

104

103

102

0

1051041031020

105

104

103

102

0

1051041031020

105

104

103

102

0

1051041031020

©20

14 N

atu

re A

mer

ica,

Inc.

All

rig

hts

res

erve

d.

l e t t e r s

nature medicine  advance online publication �

and CD11b−CD11c+ DC frequencies and numbers in arthritic ABX mice compared to arthritic control mice (Supplementary Fig. 5c). Control mice displayed higher numbers of IL-1β+CD11b+CD11c− and IL-6+CD11b+CD11c− macrophages than of IL-1β+ or IL-6+ CD11b+CD11c+ or CD11b−CD11c+ DCs. The numbers of IL-1β+ and IL-6+CD11b+CD11c− macrophages were reduced in ABX mice compared to control mice (Supplementary Fig. 5d,e).

As IL-1β and IL-6 are present in the inflammatory phase of arthritis and their production and expression is reduced in both ABX and SPF mice (Fig. 1b and Fig. 2b), we addressed whether these cytokines are perceived as ‘hazardous’ signals by splenic Breg cells. We used naive B cells as they had not yet been exposed to arthritogenic signals in vivo. In vitro stimulation with anti-CD40 alone or in combination with either IL-1β or IL-6 resulted in three-, five and fourfold increases, respectively, in the frequency of IL-10–eGFP+ B cells compared to unstimulated B cells. Stimulation with IL-1β, IL-6 and anti-CD40 in combination induced the greatest increase in the frequency of IL-10–eGFP+ B cells and secretion of IL-10 compared to that in unstimulated B cells (Fig. 3a–c). All Breg subsets including IL-10–eGFP+ T2-MZPs, IL-10–eGFP+CD5+ cells, IL-10–eGFP+ Tim-1+ cells and IL-10–eGFP+ B10 cells expanded in response to IL-1β, IL-6 and anti-CD40 (Supplementary Fig. 6a–d). Treatment with IL-1β, IL-6

and anti-CD40 increased IL-10 production by B cells from arthritic mice raised under CNV conditions, to a greater extent than by B cells from naive mice, suggesting that inflammatory stimuli in vivo further contribute to Breg cell expansion (Supplementary Fig. 7a–c). Notably, stimulation of B cells with TNF-α or IL-17 in vitro, or IL-12 in vivo16, had no effect on IL-10 production, either alone or with anti-CD40 (data not shown).

To verify the importance of IL-1β and IL-6 in Breg cell generation, we stimulated B cells from naive or arthritic Il1r1−/− or Il6r−/− mice raised under CNV conditions with IL-1β, IL-6 and anti-CD40. IL-10 production by B cells was reduced in both naive and arthritic Il1r1−/− and Il6r−/− mice, compared to production from B cells from WT mice, upon in vitro stimulation with anti-CD40 alone or in combination with IL-1β and/or IL-6 (Fig. 3d and Supplementary Fig. 7d). IL-10 production was also reduced following stimulation of naive B cells with LPS and CpG compared to the levels measured in WT mice (Supplementary Fig. 7e).

To understand how IL-1R1 and IL-6R signaling induces B cell–derived IL-10 production, we assessed phosphorylation of nuclear factor-κB (NF-κB) p65 and signal transducer and activator of tran-scription 3 (STAT3), which are phosphorylated downstream of IL-1R1 and IL-6R and are known to bind the IL-10 promoter17. Activation of

70605040

% s

wel

ling

(mB

SA

-PB

S k

nee)

3020100

CNVSPF* *

*

Time (d)0 1 2 3 4 5 6 7

a

IFN

-γ (

ng/m

l)

40

30

20

10

0

c

IL-1

7 (n

g/m

l)

10

8

6

4

2

0

TN

F-α

(ng

/ml)

2.5

2.0

1.5

1.0

0.5

0

*150

100

50

0

CNVSPF

b

IL-1

β (p

g/m

l)

IL-6

(ng

/ml)

*4

3

2

1

0

PC2 (8.9%)

SPF-1

SPF-2

SPF-3

CNV-1 CNV-2

CNV-3ABX-1

ABX-2

ABX-3PC3 (7.5%)

PC1 (72%)

h

CNVSPF

Relative increase in number of reads

RikenellaceaeRuminococcaceae

Bacteroides acidifaciensLactobacillusClostridiales

LachnospirceaeOscillospira

Akkermansia muciniphiliaPrevotella

0 0.005 0.010 0.015 0.020 0.025

i

Cha

o1 d

iver

sity

1,000

800

600

400

200

0

CNVABX

0 20,000 40,000 60,000 80,000Number of sequences sampled

f

Cha

o1 d

iver

sity

1,000

800

600

400

200

00 20,000 40,000 60,000 80,000Number of sequences sampled

g CNVSPF

% s

wel

ling

(mB

SA

-PB

S k

nee)

**

*

50

40

30

20

10

00 1 2 3 4 5 6

Time (d)7

No transferT2-MZP from CNVT2-MZP from SPF

d

CD19

T2-MZP+ gated

e

SPF105

104

103

102

0

1051041031020

3.58

FM

O

105

105

104

104

103

103

102

102

0

0

0.86CNV

IL-1

0e/G

FP

105

104

103

102

0

1051041031020

12.9

% T

2-M

ZP

IL-1

0–eG

FP

+

15

10

5

0

**

T2-

MZ

PIL

-10e

–GF

P+ (

×105 ) *

CNVSPF

6

4

2

0

Figure 2 Breg cells from mice housed in an SPF facility have impaired function. (a) Mean clinical score of SPF mice and CNV mice following induction of arthritis; y axis shows percentage swelling in antigen-injected knee compared to control knee (n = 19 per group, one of three experiments is shown). (b) Production of IL-1β, IL-6, and TNF-α by splenocytes at 24 h after disease onset, as measured by ELISA (n = 9 per group per cytokine, one of two experiments is shown). (c) Production of IFN-γ and IL-17 by splenocytes at day 7 after disease onset, as measured by ELISA, (n = 9 per group per cytokine, one of two experiments is shown). (d) Mean clinical score of C57BL/6 mice following adoptive transfer of T2-MZPs purified from CNV and SPF mice administered on the day of disease onset. Control (no transfer) group received PBS (n = 7 per group, one of two experiments is shown). (e) Representative FACS plots (left) and bar charts (right) showing the percentage and number of splenic IL-10–eGFP+ T2-MZPs from CNV and SPF mice at day 7 after disease onset (n = 11 per group, one of three experiments is shown). (f,g) Rarefaction curve of the Chao1 diversity index comparing feces from CNV and ABX groups (f) and from SPF and CNV groups (g). Each curve was rarified at 100 simulations from 10 up to 80,000 reads with Chao1 diversity calculated ten times at each simulation. (h) Principal component analysis plot of unweighted UniFrac distances sequenced at an even sampling depth across samples. (i) Nine rare taxa contributing to the differences between CNV and SPF mice relative to total number of reads recovered from each group (n = 3 per group). Taxonomy classification level is based on a bootstrap confidence of 80% after hierarchical structuring using the Ribosomal Database Project (RDP) classifier. Data represent mean ± s.e.m.: *P < 0.05, **P < 0.01 (b,c,e, unpaired t test; a,d, two-way ANOVA).

©20

14 N

atu

re A

mer

ica,

Inc.

All

rig

hts

res

erve

d.

l e t t e r s

�  advance online publication nature medicine

B cells with IL-1β, IL-6 and anti-CD40 increased the abundance of phosphorylated NF-κB p65 and STAT3 compared to stimulation with anti-CD40 alone or with IL-1β or IL-6 individually (Fig. 3e). Analysis by western blotting revealed that stimulation with IL-1β, IL-6 and anti-CD40 resulted in increased and prolonged phosphorylation of NF-κB p65 and STAT3 than did stimulation with anti-CD40 alone or in combination with IL-1β or IL-6 (Fig. 3f).

To assess whether IL-1β and IL-6 are essential for differentiation of IL-10–producing B cells in vivo, we generated mixed bone marrow chimeric mice lacking IL-1R1 or IL-6R specifically on B cells (B-Il1r1−/− or B-Il6r−/−, respectively) and a group of control chimeras (B-WT) as previously shown18 . Chimeric mice were kept under CNV conditions. Both B-Il1r1−/− and B-Il6r−/− chimeric mice developed an exacerbated disease course compared to B-WT chimeric mice (Fig. 3g). Although the frequency of T2-MZPs was unaffected (Supplementary Fig. 8a), we noted a decrease in IL-10 secretion by B cells and a reduced frequency of IL-10+ T2-MZPs in B-Il1r1−/− and B-Il6r−/− compared to B-WT chimeric mice (Fig. 3h and Supplementary Fig. 8b). Notably, ABX treatment reduced the severity of arthritis in B-Il1r1−/−, B-Il6r−/− and B-WT mice, confirming that arthritis is modulated by the microbiota (Supplementary Fig. 8c).

How does the microbiota directly influence splenic Breg cell differ-entiation in the context of arthritis? We found that 70% of T2-MZPs residing in the spleen of arthritic mice raised under CNV conditions

expressed integrin α4β7 (Fig. 4a), a marker that allows lymphocytes to home to the GALT19. Furthermore, up to 80% of IL-10–eGFP+ T2-MZPs expressed integrin α4β7 (Fig. 4b). α4β7

+ T2-MZPs expressed more IL-10–eGFP and were more efficient at inducing CD4+Foxp3+ T cells upon co-culture with autologous CD4+CD25− T cells and at suppressing TNF-α+CD4+ T cells in vitro than α4β7

− T2-MZPs (Supplementary Fig. 9a–e). This suggests that the majority of splenic Breg cells traffic to, or from, the gut.

Although T2-MZPs are reported to be sessile and reside exclu-sively in the spleen of naive mice20, we found that T2-MZPs were also detectable in mesenteric LNs (MLNs), although not in other GALT, of arthritic mice (Fig. 4c). MLN T2-MZPs were mostly located in the B cell follicle (Supplementary Fig. 10a–e) and were phenotypically (based on the high expression levels of IgM and IgD) and molecularly (based on the expression levels of HES-1 (ref. 21)) indistinguishable from splenic T2-MZPs (Supplementary Fig. 11a–c and data not shown). Although the distribution of T2-MZPs in the GALT was unaffected by ABX treatment, the percentage of MLN IL-10–eGFP+ T2-MZPs was reduced in ABX mice compared to control mice, mirroring the reduction observed in the spleen (Fig. 4c,d). IL-10 pro-duction by MLN B cells from CNV mice was upregulated in response to stimulation with IL-1β, IL-6 and anti-CD40, albeit less efficiently than in splenic B cells (Supplementary Fig. 12a).

b10

8

6

% C

D19

+IL

-10–

eGF

P+

4

2

0– – –+ +IL-1β

– + ++ +aCD40– – +– +IL-6

***

*

*

***

***

*****

c400

300

IL-1

0 (p

g/m

l)

200

100

0– – –+ +IL-1β

– + ++ +aCD40– – +– +IL-6

*

**

*

***

***

****

d Naive B cells

***

****

250

200

IL-1

0 (p

g/m

l)

150

100

50

0– – –+ +IL-1β

– + ++ +aCD40– – +– +IL-6

WTIlr1–/–

Il6r–/–

60

h******

40

IL-1

0 (p

g/m

l)

20

0mBSA+aCD40

1.5

***

1.0

IL-1

0 (n

g/m

l)

0.5

0LPS

B-WT B-Ilr1–/– B-Il6r–/–

1.5*

1.0

IL-1

0 (n

g/m

l)

0.5

0CpG

45 ******

* **

*

403530

% s

wel

ling

(mB

SA

-PB

S k

nee)

252015

0 1 2 3Time (d)

4 5 6 7

1050

g B-WTB-Ilr1–/–

B-Il6r–/–

a

fe

0 102 103 104 105 0 102 103 104 105

pSTAT3

aCD40 +IL-1β + IL-6

aCD40 + IL-6

aCD40

Medium

aCD40 + IL-1β

MF

I

p65

CD19

IL-1

0–eG

FP

0 102 103 104 105

0102

103

104

105 1.17Medium

0 102 103 104 105

0102

103

104

105 4.21IL-6

0 102 103 104 105

0102

103

104

105 8.59IL-1β + IL-6Medium

3.9

0 102 103 104 105

0102

103

105

104

IL-1β4.68

0 102 103 104 105

0102

103

105

104

Naive B cells + aCD40 (10 µg/ml)

Time (min)10 30 60

pSTAT3

β-actin

pNF-κB p65

10 30 6010 30

Naive B cells + aCD40 (10 µg/ml)

6010

Medium Il-1β Il-1β + IL-6IL-6

0

NS

30 60

Figure 3 IL-1β and IL-6 induce the differentiation of IL-10–producing B cells. (a,b) Representative FACS plots (a) and bar chart (b) showing the percentage of IL-10–eGFP+CD19+ cells (n = 15 per group, one of five experiments is shown). (c) IL-10 production, as measured by ELISA (n = 15 per group, one of five experiments is shown). In these experiments, B cells were isolated from naive IL-10–eGFP reporter mice and stimulated with anti-CD40 (aCD40) either alone or in combination with IL-1β and/or IL-6. (d) IL-10 production by B cells isolated from naive Il1r1−/− or Il6r−/− and WT mice following stimulation with aCD40 either alone or in combination with IL-1β and/or IL-6, as measured by ELISA (n = 6 per group, one of two experiments is shown). (e,f) Representative histograms showing the expression of pSTAT3 and pNF-κB p65, as determined by flow cytometry (e), and western blot analysis of total pSTAT3, pNF-κB p65 and β-actin (f). In these experiments, B cells were isolated from naive IL-10–eGFP reporter mice and stimulated with aCD40 either alone or in combination with IL-1β and/or IL-6 (n = 6 per group, one of two experiments is shown). MFI, mean fluorescence intensity. (g) Mean clinical score of B-Il1r1−/−, B-Il6r−/− or B-WT chimeric mice following induction of arthritis; y axis shows percentage swelling in antigen-injected knee compared to control knee (n = 19 per group, one of four experiments is shown). (h) IL-10 production by B cells isolated at day 7 after disease onset from B-Il1r1−/−, B-Il6r−/−, or B-WT chimeric mice following stimulation with mBSA and aCD40, LPS or CpG, as measured by ELISA. Data represent mean ± s.e.m.; *P < 0.05, **P < 0.01, ***P < 0.005 (a–f,h, one-way ANOVA; g, two-way ANOVA).

©20

14 N

atu

re A

mer

ica,

Inc.

All

rig

hts

res

erve

d.

l e t t e r s

nature medicine  advance online publication �

To test whether MLN T2-MZPs could also regulate inflammatory T cell differentiation, we isolated splenic and MLN T2-MZPs from arthritic CNV mice and compared their suppression of pro-arthritogenic CD4+ T cells from arthritic CNV mice in vitro. Both splenic and MLN T2-MZPs induced Foxp3+CD4+ T cells and suppressed TNF-α+CD4+ T cells. However, MLN T2-MZPs were only partially suppressive compared to splenic T2-MZPs (Fig. 4e,f). Owing to the low number of MLN T2-MZPs, we could not assess suppressive function in vivo.

One possible reason for the reduced suppressive function of MLN T2-MZPs as compared to splenic T2-MZPs might be differences in the MLN cytokine microenvironment. Unlike in the spleen, we could detect very few IL-1β+cells in the MLN of CNV mice with antigen-induced arthritis before and after ABX treatment (Fig. 4g). In con-trast, IL-6+cells were detectable in the MLN of control mice but were reduced in number in ABX mice (Fig. 4h). The findings showing that CD11b+CD11c− macrophages were absent from the MLN of arthritic

i j

105

104

103

102

102 103 104 105

0

0

105

104

103

102

102 103 104 105

0

0

105

104

103

102

102 103 104 105

0

0

1.42 6.87 1.91

CD45.2 CD19

CD

45.1

Isot

ype

IL-1

0

Ctrl +MLN T2 transfer

ABX +MLN T2 transferIsotype

Spleen: CD19+ gated Spleen: CD45.2+ gated

105

104

103

102

102 103 104 105

0

0

0.003

No transfer

105

104

103

102

102 103 104 105

0

0

0.19

Ctrl +MLN T2 transfer

105

104

103

102

102 103 104 105

0

0

0.16

ABX +MLN T2 transfer

*10

8

6

4% IL

-6+

2

0

25

20

15

10

5

0

IL-1

β+ c

ells

(×1

03 )0.3

0.2

0.1

0

% IL

-1β+

*6

CtrlABX4

2

0

IL-6

+ c

ells

(×1

05 )

α4β7+

α4β7–

% e

xpre

ssio

n on

T2-

MZ

P-I

L-10

/eG

FP

+ 100

80***

60

40

20

0

Spleena

105

104

103

102

102 103 104 105

0

0 102 103 104 1050

105

104

103

102

0

0.067 70.6

FM

O

CD19α4

β7

T2-MZP+

b Spleen

102 103 104 1050

105

104

103

102

0

102 103 104 1050

105

104

103

102

0

102 103 104 1050

105

104

103

102

0FM

O

CD19

α 4β7

IL-1

0–eG

FP

1.35 5.86 72

22.2

T2-MZP+T2-MZP+

IL-10–eGFP+

dABX

Ctrl

4

3

2

1

0MLN

*

% o

f T2-

MZ

P IL

-10–

eGF

P+

c105

104

103

102

102 103 104 105

0

0

105

104

103

102

102 103 104 105

0

0

105

104

103

102

102 103 104 105

0

0

105

104

103

102

102 103 104 105

0

0

105

104

103

102

102103 104 105

0

0

105

104

103

102

102 103 104 105

0

0

105

104

103

102

102 103 104 105

0

0

105

104

103

102

102 103 104 105

0

0

105

104

103

102

102 103 104 105

0

0

105

104

103

102

102 103 104 105

0

0

14.8

12.5

2.74

72.5 83.3 87.6 86.5

0.16 0.2

0.72

0

1.5

76.5

9.53

1.241.21

1.95

69.9 80.1

0.2

1.95 1.35

0.51

2.21

0.67

0.74

10.7

7781.2 86.7

CD24

CD

21

Ctrl: CD19+ gated

ABX: CD19+ gated

Spleen IELs LPLs PPs MLNs

Spleen IELs LPLs PPs MLNs

e

f

105

104

103

102

102 103 104 105

0

0

105

104

103

102

102 103 104 105

0

0

105

104

103

102

102 103 104 105

0

0

105

104

103

102

102 103 104 105

0

0

105

104

103

102

102 103 104 105

0

0

105

104

103

102

102 103 104 105

0

0

1.5 4.62 2.89

60.9 25.4 48.7

CD24

CD4

TN

F-α

150

60

40

20

0

% in

duct

ion

of F

oxp3

% s

uppr

essi

on o

f TN

F-α

100

50

0

Fox

p3

aCD3 (1 µg/ml)

aCD3 (1 µg/ml)

CD4+ T cells +spleen T2-MZPs

CD4+ T cells +MLN T2-MZPs

CD4+ T cells +spleen T2-MZPs

CD4+ T cells

CD4+ T cells +MLN T2-MZPs *CD4+ T cells

Splenic T2-MZPsMLN T2-MZPs

g105

104

103

102

0

105

104

103

102

0

050

K10

0K15

0K20

0K25

0K 050

K10

0K15

0K20

0K25

0K

0.3 0.23

FSC-A

IL-1

β

Ctrl ABX

MLN: live gated h

050

K10

0K15

0K20

0K25

0K 050

K10

0K15

0K20

0K25

0K

105

104

103

102

0

105

104

103

102

0

8.2 5.59

FSC-A

IL-6

Ctrl ABX

MLN: live gated

Figure 4 Breg cells are present in the mesenteric lymph nodes of inflamed mice and exhibit suppressive function. (a) Representative FACS plots showing expression of integrin α4β7 on splenic T2-MZPs from IL-10–eGFP reporter mice at day 7 after disease onset. (b) Representative FACS plots (left) and bar chart (right) showing IL-10–eGFP expression in T2-MZPs and α4β7 expression on splenic IL-10/eGFP+ T2-MZPs at day 7 after disease onset. (n = 21 in total; one of three experiments is shown). (c) Representative FACS plots showing percentage of T2-MZPs in the spleen, intraepithelial lymphocytes (IELs), lamina propria lymphocytes (LPLs), Peyer’s patches (PPs) and mesenteric lymph nodes (MLNs) in control and ABX mice at day 7 after disease onset; gates containing T2-MZPs are highlighted (n = 24 per organ, one of four experiments is shown). (d) Bar chart showing the percentage of IL-10–eGFP+ T2-MZPs in MLN of control and ABX mice (n = 5 per group, one of three experiments is shown). (e) Left, representative FACS plots showing the expression of Foxp3 in CD4+ T cells after coculture with isolated T2-MZPs from the spleen and MLN after stimulation with anti-CD3 (aCD3). Right, bar chart showing percentage induction of Foxp3+CD4+ T cells by splenic-T2-MZPs or MLN-T2-MZPs following stimulation with aCD3 (n = 7, cumulative data are shown). (f) Left, representative FACS plots showing the expression of TNF-α in CD4+ T cells following co-culture with isolated T2-MZPs from the spleen and MLN after stimulation with aCD3. Right, bar chart showing percentage suppression of TNF-α+CD4+ T cells by splenic T2-MZPs or MLN T2-MZPs following stimulation with aCD3 (n = 7, cumulative data are shown). (g,h) Representative FACS plots (left) and bar charts (right) showing the percentage and number of IL-1β+ (g) and IL-6+ (h) cells in the MLN of control and ABX mice at day 7 after disease onset (n = 6 per group, one of two experiments is shown). (i,j) MLN T2-MZPs from CD45.2+ C57BL/6 WT mice were isolated from mice in remission from arthritis and transferred into either control or ABX congenic CD45.1+ C57BL/6 mice on the same day as disease induction. (i) Representative FACS plots showing the expression of CD45.1 and CD45.2 in CD19+ gated cells, at 72 h after transfer (n = 3 per group; one of three experiments is shown). (j) Representative FACS plots showing the expression of IL-10 in CD19+CD45.2+ gated cells at 72 h after transfer (n = 3 per group, one of three experiments is shown). Data represent mean ± s.e.m.; *P < 0.05, ***P < 0.005 (unpaired t-test).

©20

14 N

atu

re A

mer

ica,

Inc.

All

rig

hts

res

erve

d.

l e t t e r s

�  advance online publication nature medicine

ABX and control mice may explain our inability to detect IL-1β+cells in the MLN, as we found this population to be the primary source of IL-1β in the spleen (Supplementary Fig. 13a). We did not observe differences in IL-6+CD11b+CD11c+ or IL-6+CD11b−CD11c+ DCs. However, there was a decrease in the number of IL-6+CD11c+CD103− DCs in ABX mice compared to control mice; this DC subset was previously described as a specialized MLN pro-inflammatory sub-set22. The percentage and number of IL-6+ non-myeloid cells was also decreased in the MLN of ABX mice compared to control mice (Supplementary Fig. 13d,e).

To test whether MLN T2-MZPs can recirculate to the spleen, we transferred MLN T2-MZPs from arthritic CD45.2+ mice into recipient congenic CD45.1+ ABX or CD45.1+ control mice concurrently with disease induction. A similar frequency of CD45.2+ MLN T2-MZPs homed to the spleens of recipient control mice and ABX mice (Fig. 4i). IL-10 expression by transferred cells was reduced in ABX mice compared to control mice (Fig. 4j). Administration of a mono-clonal antibody specific to integrin α4β7 reduced the frequency of T2-MZPs and IL-10–eGFP+ T2-MZPs in the spleen of arthritic mice (Supplementary Fig. 14a,b). Finally, we found that MLN T2-MZPs preconditioned with IL-1β, IL-6 and anti-CD40 overnight before co-culture with splenic proarthritogenic CD4+ T cells induced Foxp3+CD4+ T cells and suppressed TNF-α+CD4+ T cells equiva-lently to splenic T2-MZPs (Supplementary Fig. 15a,b).

The gut microbiota induces DCs and macrophages to produce IL-1β and IL-6, which promotes both TH17 differentiation and arthritis3,12,13. In this study, we show that the same signals induce the differentiation of IL-10–producing Breg cells. We suggest that the commensal microbiota is important for establishing both proinflam-matory and regulatory responses and for maintaining equilibrium, such that the immune system can promptly clear infections but is able to minimize any inflammatory collateral damage.

MeTHodsMethods and any associated references are available in the online version of the paper.

Note: Any Supplementary Information and Source Data files are available in the online version of the paper.

ACKNOwlEDgMENTSThis work is funded by an Arthritis Research United Kingdom programme grant (MP/17707) awarded to C.M. E.C.R. is funded by an Arthritis Research United Kingdom PhD studentship (MP/19314) also awarded to C.M. R.D. is funded by a Food and Nutritional Technical Assistance III project (FANTA III). S.T. is funded by an Erasmus Placement programme (Consorzio KTEU EP6). We would like to thank the University College London Biological Service Unit staff for all their help controlling animal husbandry, J. Evans for cell sorting, K. Nistala for comments and P. Blair for critically reviewing the manuscript.

AUTHOR CONTRIBUTIONSC.M. and E.C.R. designed and conceived the experiments. E.C.R. performed the experiments. C.M., S.T., A.B. and N.A.C. helped with large in vivo experiments.

K.O. helped with signaling experiments. R.D., K.A.H. and N.K. analyzed fecal samples. C.M. and E.C.R. wrote and prepared the manuscript. S.A.J. provided Il6r−/− mice.

COMPETINg FINANCIAl INTERESTSThe authors declare no competing financial interests.

Reprints and permissions information is available online at http://www.nature.com/reprints/index.html.

1. Mauri, C. & Bosma, A. Immune regulatory function of B cells. Annu. Rev. Immunol. 30, 221–241 (2012).

2. Rakoff-Nahoum, S., Paglino, J., Eslami-Varzaneh, F., Edberg, S. & Medzhitov, R. Recognition of commensal microflora by Toll-like receptors is required for intestinal homeostasis. Cell 118, 229–241 (2004).

3. Wu, H.J. et al. Gut-residing segmented filamentous bacteria drive autoimmune arthritis via T helper 17 cells. Immunity 32, 815–827 (2010).

4. Russell, S.L. et al. Early life antibiotic-driven changes in microbiota enhance susceptibility to allergic asthma. EMBO Rep. 13, 440–447 (2012).

5. Madan, R. et al. Nonredundant roles for B cell–derived IL-10 in immune counter-regulation. J. Immunol. 183, 2312–2320 (2009).

6. O’Garra, A. et al. Ly-1 B (B-1) cells are the main source of B cell–derived interleukin 10. Eur. J. Immunol. 22, 711–717 (1992).

7. Ding, Q. et al. Regulatory B cells are identified by expression of TIM-1 and can be induced through TIM-1 ligation to promote tolerance in mice. J. Clin. Invest. 121, 3645–3656 (2011).

8. Yanaba, K. et al. A regulatory B cell subset with a unique CD1dhiCD5+ phenotype controls T cell–dependent inflammatory responses. Immunity 28, 639–650 (2008).

9. Ochoa-Repáraz, J., Mielcarz, D.W., Haque-Begum, S. & Kasper, L.H. Induction of a regulatory B cell population in experimental allergic encephalomyelitis by alteration of the gut commensal microflora. Gut Microbes 1, 103–108 (2010).

10. Kwon, H.K. et al. Generation of regulatory dendritic cells and CD4+Foxp3+ T cells by probiotics administration suppresses immune disorders. Proc. Natl. Acad. Sci. USA 107, 2159–2164 (2010).

11. Karimi, K., Inman, M.D., Bienenstock, J. & Forsythe, P. Lactobacillus reuteri–induced regulatory T cells protect against an allergic airway response in mice. Am. J. Respir. Crit. Care Med. 179, 186–193 (2009).

12. Shaw, M.H., Kamada, N., Kim, Y.G. & Nunez, G. Microbiota-induced IL-1β, but not IL-6, is critical for the development of steady-state TH17 cells in the intestine. J. Exp. Med. 209, 251–258 (2012).

13. Ivanov, I.I. et al. Induction of intestinal TH17 cells by segmented filamentous bacteria. Cell 139, 485–498 (2009).

14. Hu, B. et al. Microbiota-induced activation of epithelial IL-6 signaling links inflammasome-driven inflammation with transmissible cancer. Proc. Natl. Acad. Sci. USA 110, 9862–9867 (2013).

15. Abt, M.C. et al. Commensal bacteria calibrate the activation threshold of innate antiviral immunity. Immunity 37, 158–170 (2012).

16. Vasconcellos, R., Carter, N.A., Rosser, E.C. & Mauri, C. IL-12p35 subunit contributes to autoimmunity by limiting IL-27–driven regulatory responses. J. Immunol. 187, 3402–3412 (2011).

17. Saraiva, M. & O’Garra, A. The regulation of IL-10 production by immune cells. Nat. Rev. Immunol. 10, 170–181 (2010).

18. Fillatreau, S., Sweenie, C.H., McGeachy, M.J., Gray, D. & Anderton, S.M. B cells regulate autoimmunity by provision of IL-10. Nat. Immunol. 3, 944–950 (2002).

19. Hamann, A., Andrew, D.P., Jablonski-Westrich, D., Holzmann, B. & Butcher, E.C. Role of α4-integrins in lymphocyte homing to mucosal tissues in vivo. J. Immunol. 152, 3282–3293 (1994).

20. Carsetti, R., Rosado, M.M. & Wardmann, H. Peripheral development of B cells in mouse and man. Immunol. Rev. 197, 179–191 (2004).

21. Su, T.T. & Rawlings, D.J. Transitional B lymphocyte subsets operate as distinct checkpoints in murine splenic B cell development. J. Immunol. 168, 2101–2110 (2002).

22. Annacker, O. et al. Essential role for CD103 in the T cell–mediated regulation of experimental colitis. J. Exp. Med. 202, 1051–1061 (2005).

©20

14 N

atu

re A

mer

ica,

Inc.

All

rig

hts

res

erve

d.

nature medicinedoi:10.1038/nm.3680

oNLINe MeTHodsMice. IL-10e–GFP mice were as described5 and provided by K. Cooper, London School of Hygiene & Tropical Medicine. Il6r−/− mice were provided by S. Jones, Cardiff University. B-Il1r1−/− mice were provided by S. Francis, University of Sheffield. CD45.1-expressing mice were provided by D. Gilroy, University College London. C57BL/6 mice were from Harlan, UK. Female mice were used at 6–12 weeks of age and were bred and maintained at the animal facility at University College London. All experiments were approved by the Animal Welfare and Ethical Review Body of University College London and authorized by the United Kingdom Home Office.

Antibiotics treatment and induction of antigen-induced arthritis (AIA). 1 week before induction of arthritis, Vancomycin (500 mg L−1; Sigma-Aldrich), Neomycin (1 g L1; Sigma-Aldrich) and Metronidazole (1 g L−1; Sigma-Aldrich) were added to drinking water as described2. Mice were maintained on antibiotics throughout the duration of the experiment. AIA was induced and assessed as previously described16,23. Briefly, mice were injected subcutane-ously at the tail base with 200 µg of methylated BSA (mBSA; Sigma-Aldrich) emulsified in 100 µl Complete Freund’s Adjuvant (CFA). CFA was made by mixing 3 mg ml−1 of Mycobacterium tuberculosis (Difco) in Incomplete Freund’s Adjuvant (IFA) (Sigma-Aldrich). After 7 d, mice received an intra-articular injection of 10 µl of PBS containing 200 µg mBSA in the right knee and 10 µl PBS alone in the left knee as a control. Joint size was measured using calipers (POCO 2T; Kroeplin Längenmesstechnik), and swelling was calculated as a percentage increase in size between inflamed and control knee.

Gavage. Mice were treated with antibiotics for 7 d and then placed on auto-claved water overnight before first gavage. Fecal suspension was prepared by collecting 0.2 g of fecal pellets and homogenizing pellets in PBS with 3-mm glass beads in a tissue lyser for 5 min at 25 Hz. Following homogenization, solution was passed through a 70-µm cell strainer to a final concentration of 0.05 g ml−1. Mice were gavaged with 150 µl of fecal suspension every 48 h for 1 week before subcutaneous injection, then on days 0, 2 and 4 post-subcutaneous injection.

Treatment of mice with anti-a4b7. Mice treated with anti-α4β7 monoclonal antibody (mAb) (DATK32; BioXcell) received an intraperitoneal injection of 500 µg of anti-α4β7 mAb in HBSS (Sigma-Aldrich) on days 0, 2, 4 and 6 post-subcutaneous injection, as well as on days 0, 2, 4 and 6 post-intra-articular injection. Control group received appropriate isotype control.

Cell preparation. Spleens and mesenteric and inguinal lymph nodes were dis-sected post-mortem and collected in RPMI (Sigma-Aldrich). For splenocyte and lymph node cell preparation, organs were mashed through a 70-µm cell strainer (BD Biosciences), as previously described16,23–25, and erythrocytes from spleens were lysed using Red Cell Lysis Buffer (Sigma-Aldrich). Intestinal intraepithelial lymphocytes (IELs) were isolated by longitudinal opening of the small intestine followed by shaking of 1-cm fragments for 30 min at 37 °C in PBS containing 10% FCS, 10 mM EDTA, 20 mM HEPES, 1 mM Sodium pyruvate and 10 mg ml−1 Polymyxin B (all Sigma-Aldrich); following removal of IEL fraction, remaining tissue was digested with 0.5 mg ml−1 Collagenase D and 0.1 mg ml−1 DNase I (Roche Applied Science) for 60 min at 37 °C to isolate lamina propria lymphocytes (LPLs). Both IEL and LPL isolation were performed as previously described26.

Flow cytometry. Anti-mouse monoclonal antibodies purchased from BD Biosciences: PE-CY7-conjugated CD19 (1D3), FITC- or APC-conjugated CD21 (7G6), PE-conjugated CD23 (B3B4) and PERCP-CY5.5- or APC- conjugated CD24 (M1/69) were diluted 1 in 250 from stock; PE-conjugated α4β7 (LPAM-1; DATK32) was diluted 1 in 100 from stock; PE-conjugated IL-17A (TC11-18H10.1), APC-conjugated IFN-γ (XMG1.2 RUO), APC-conjugated TNF-α (MP6-XT22), PE-conjugated IL-6 (MP5-20F3) and PE-conjugated IL-10 (JES5-16E3) were diluted 1 in 100 from stock. Anti-mouse monoclonal antibodies purchased from eBiosciences: APC-conjugated Foxp3 (FJK-16s) and IL-1β (NJTEN3) were diluted 1 in 100 from stock. Anti-mouse monoclonal antibodies purchased from BioLegend: Brilliant Violet 785- conjugated CD19 (6D5) and Brilliant Violet 711-conjugated CD4 (RM4-5)

were diluted 1 in 250 from stock; PE-CY7-conjugated CD11c (N418), Brilliant Violet 711-conjugated CD11b (M1/70), PE-conjugated TIM-1 (RM71-4), Alexa-647-conjugated CD5 (53-7.3), PERCP-CY5.5-conjugated CD1d (1B1), PE-conjugated CD126 (IL-6R; D7715A7) and APC-conjugated CD120a (IL-1R; JAMA-147) were diluted 1 in 100 from stock. Anti-mouse monoclonal antibodies purchased from Cell Signaling: Alexa-647-conjugated pSTAT3 (Y705; D3A7) and p-NF-κB-p65 (S536; 93H1) were diluted 1 in 50 from stock. Appropriate isotype controls were used. For four- to six-color immunofluo-rescence analysis, cells were stained at 4 °C for 20 min as described16,23–25. Intracellular cytokine analysis was performed as previously described16,23–25. Briefly, for detection of IL-17 and IFN-γ, splenocyte or lymph node cultures were suspended at 5 × 106 cells/ml in complete medium with PMA (50 ng ml−1; Sigma-Aldrich), Ionomycin (250 ng ml−1; Sigma-Aldrich) and GolgiPlug (BD Biosciences) for 5 h. For detection of IL-6 and IL-1β, splenocytes were suspended at 5 × 106 cells/ml in complete medium with PMA (50 ng ml−1; Sigma-Aldrich), Ionomycin (500 ng ml−1; Sigma-Aldrich) and GolgiStop (BD Biosciences) for 5 h. For detection of IL-10, splenocytes were suspended at 5 × 106 cells/ml in complete medium with PMA (100 ng ml−1; Sigma-Aldrich), Ionomycin (1 µg ml−1; Sigma-Aldrich) and GolgiStop (BD Biosciences) for 5 h. Cells were then stained with surface markers followed by permeabilization and incubation with intracellular Abs. IL-10–eGFP expression, unless stated, was analyzed ex vivo without fixation, and DAPI (1 µg ml−1) was added just before acquisition. Splenocytes from WT mice were used as a control to set gates for IL-10–eGFP+ cells. For detection of pSTAT3 and pNF-κB-p65, negatively purified B cells were rested for 3 h in serum-free media at 37 °C, follow by stimulation with anti-CD40 (10 µg ml−1) or anti-CD40 (10 µg ml−1) and IL-1β and/or IL-6 (both 20 ng ml−1) for 7 min. Following stimulation, cells were fixed with 2% PFA and then permeabilized with 90% methanol. pSTAT3 and p-NF-κB-p65 antibodies were used according to manufacturer’s instructions. Unless otherwise stated, lymphocytes were isolated and analyzed 7 d post-disease onset.

Adoptive transfer experiments. B cells were isolated from the spleens of either ABX-treated mice or control mice from mice 7 d post-disease onset and stained with antibodies specific to CD19 (1D3), CD23 (B3B4), CD21 (7G6) and CD24 (M1/69) diluted 1 in 250 from stock (all antibodies were purchased from BD Biosciences). B cell subsets were FACS sorted using gates drawn according to previous reports23–25. Splenic T2-MZPs (2 × 106) were transferred into recipient mice on the day of intra-articular injection. The control group (no transfer) received a PBS injection. For congenic transfer experiments, recipient CD45.1+ mice received between 2 × 105 and 3 × 105 CD45.2+ MLN T2-MZPs.

16S rDNA sequencing. 20–50 mg of fecal material was extracted using the QIAmp DNA mini kit. Extraction was carried out as per the manufacturer’s protocol with an additional cell disruption step where 0.1-mm glass beads were added to each sample and the 2-ml tubes are shaken on the cell disrupter (Vortex Genie 2, Scientific Industries) for 10 min at the highest speed. An extra negative extraction control was included (200 µl buffer AE). Library preparation for the MiSeq included a single amplification step to add indi-vidual 12-bp error-correcting barcodes27 for each sample, Illumina compat-ible adaptor and extra pad and linker sequences. The V5-7 regions of the 16S rDNA were amplified using 785F: 5′-GGATTAGATACCCBRGTAGTC-3′, 1175R: 5′-ACGTCRTCCCCDCCTTCCTC-3′ with each reaction containing 1× Molzym PCR Buffer (Molzym), 200 µM dNTPs (Bioline), 0.4 µM for-ward and reverse primer with barcode attached, 0.025 µM Moltaq (Molzym), 2 µl template DNA and PCR grade water (Bioline) to make a final reaction volume of 25 µl. Cycling parameters for each reaction were 94 °C × 3 min, 36 cycles of 94 °C × 30 s, 60 °C × 40 s, 72 °C × 90 s and one final extension at 72 °C × 10 min. Samples were purified and the correct length amplicon separated using a SizeSelect E-Gel (Life Technologies). After quantification using the Qubit 2.0 (Life Technologies), samples were pooled into an equimolar solution, diluted and loaded into the MiSeq reagent cartridge as per the manufacturer’s instructions. 10% of the total DNA loaded into the cartridge was PhiX control. The MiSeq runs were set to generated 250-bp paired-end reads and a 12-bp index read for each sample. Sequenced reads generated

©20

14 N

atu

re A

mer

ica,

Inc.

All

rig

hts

res

erve

d.

nature medicine doi:10.1038/nm.3680

by the MiSeq were quality filtered and demultiplexed using QIIME. 250-bp paired-end reads were first assembled into 370-bp contigs using fastq-join. Assembled reads were discarded if 90% of read length was not over a Phred quality score of 30. Sequences were also discarded if read or index contained an ambiguous “N” base. Operational Taxonomic Units (OTUs) were picked using uclust28 and clustered at 97% similarity against the Greengenes data-base29. Sequences were aligned to the reference database using NAST30 for comparison of UniFrac distances and chimeric sequences were identified and removed using ChimeraSlayer. Taxonomic information was assigned using the Ribosomal Database Project Classifier at a confidence of 80% (ref. 31).

In vitro B cell culture. B cells were negatively purified by magnetic separation (Miltenyi Biotec) (95–98%) and cultured for 72 h with mBSA (10 µg ml−1; Sigma-Aldrich) and/or anti-CD40 (BioXcell; 10 µg ml−1) or LPS (10 µg ml−1; Sigma-Aldrich) or CpG (1 µM; Invivogen). All culture reagents were commer-cially bought. For B cell IL-10 induction experiments, purified splenic B cells (95–98%) from naive IL-10–eGFP reporter mice were cultured with agonistic anti-CD40 (10 µg ml−1) either alone or in combination with recombinant IL-6 (20 ng ml−1) or IL-1β (20 ng ml−1) (all cytokines; R&D Systems) for 48 h before IL-10–eGFP expression was assessed. Supernatants from cell cultures were harvested and analyzed for cytokines using standard sandwich ELISA Kit (IL-17, IFN-γ, and IL-10; R&D Systems, TNF-α, IL-1β and IL-6; eBiosciences) and performed according to manufacturer’s instructions.

Western blot. B cells were negatively purified and stimulated with anti-CD40 (10 µg ml−1) either alone or in combination with IL-1β and/or IL-6 (both 20 ng ml−1) for 10, 30 and 60 min. Stimulated B cells were then lysed and protein from 2 × 106 cells was resolved by SDS-PAGE, transferred to nitrocellulose membranes (Amersham), and blotted using anti-pSTAT3 (Y705; 3E2), anti-pNF-κB-p65 (S536; 93H1) and anti-β-actin (Cell Signaling). Antibodies were diluted 1 in 1,000 from stock. Bound antibodies were revealed with HRP-conjugated species-specific secondary antibodies using ECL substrate (Amersham).

Generation of chimeric mice. Chimeric mice, and appropriate controls, were generated as previously described18,23. Recipient µMT mice received 800 cGy gamma irradiation via a cesium source. 5 h following irradiation, recipients received 10 × 106 donor bone marrow cells. Bone marrow prepara-tions were depleted of T cells by negative selection with a MACS magnetic column (Miltenyi Biotec). To generate mice in which the absence of IL-1R1 or IL-6R was exclusively restricted to B cells, µMT mice were reconstituted with mixture of bone marrow consisting of 80% from µMT (no B cell dif-ferentiation) with 20% from Il1r1−/− or Il6r−/− mice. Control mice received 80% from µMT and 20% bone marrow from WT mice (to give a normal B cell compartment). Chimeras were left to fully reconstitute over at least 8 weeks before use in AIA experiments.

Immunofluorescence. Immunofluorescence analysis was carried out as pre-viously described32. Briefly, frozen tissue sections from the spleen and MLN were taken 7 d post-disease onset and cut into 7-µm-thick sections in optimum cutting temperature compound (OCT). Sections were stained then with Alexa-647 IgD (11-26c.2a) anti-mouse monoclonal antibody (Biolegend), purified IgM goat anti-mouse polyclonal antibody (Abcam; ab9167) plus Alexa-488 donkey anti-goat (Invitrogen; A11055), and purified CD3 rabbit anti-mouse polyclonal antibody (Abcam; ab5690) plus Alexa-555 donkey anti-rabbit (Invitrogen; A31572). Primary antibodies were diluted 1 in 100 from stock; secondary antibodies were diluted 1 in 200 from stock. Images were obtained

using a Leica DM 2500 SPE confocal microscope with LAS AF 2.3.6 software and analyzed using ImageJ (NIH, USA) v1.44o for Apple Mac.

Suppression assays. T2-MZPs were isolated from mice in remission from arthritis and cultured 1:1 with syngeneic CD4+CD25− T cells isolated by mag-netic separation for 72 h in the presence of 1 µg ml−1 plate-bound anti-CD3 (145-2C11; purchased from BD Bioscience). Following stimulation, cells were analyzed for expression of Foxp3 and TNF-α. For priming assay, MLN T2-MZPs were isolated and cultured overnight with anti-CD40 (10 µg ml−1), IL-1β and IL-6 (both 20 ng ml−1). Primed MLN T2-MZPs were then cultured 1:1 with syngeneic CD4+CD25− T cells isolated by magnetic separation for 48 h in the presence of 1 µg ml−1 plate-bound anti-CD3 (145-2C11).

Statistical analyses. All data are expressed as mean ± s.e.m. For in vivo studies, power calculations were performed on data showing mean maximum wild-type arthritic knee swelling of 2 mm with an s.d. of 0.39 mm, and an expected test group (transferred T2-MZPs) arthritic knee swelling of 1.4 mm. Group sizes of three mice or above were sufficient to reach a statistical power of at least 80% (http://www.statisticalsolutions.net/pss_calc.php). Mice were assigned at random to treatment groups for all mouse studies and, where pos-sible, mixed among cages. Clinical scoring was performed in a blinded fashion. Mice that developed adverse reactions to protocols were excluded from data sets. Statistical significance was determined using unpaired t tests (compari-son of two groups), one-way ANOVA (comparison of three or more groups) or two-way ANOVA (clinical data only), as previously published16,23–25,33,34. All data met the assumption of statistical tests and had a normal distribution, and variance was similar between groups that were statistically compared. Replicates used were biological replicates. Results were considered signi-ficant at P ≤ 0.05. Statistical tests were carried out using GraphPad Prism (La Jolla, CA, USA) v. 400 Software for Apple Mac.

23. Carter, N.A. et al. Mice lacking endogenous IL-10–producing regulatory B cells develop exacerbated disease and present with an increased frequency of TH1/TH17 but a decrease in regulatory T cells. J. Immunol. 186, 5569–5579 (2011).

24. Evans, J.G. et al. Novel suppressive function of transitional 2 B cells in experimental arthritis. J. Immunol. 178, 7868–7878 (2007).

25. Blair, P.A. et al. Selective targeting of B cells with agonistic anti-CD40 is an efficacious strategy for the generation of induced regulatory T2-like B cells and for the suppression of lupus in MRL/lpr mice. J. Immunol. 182, 3492–3502 (2009).

26. Horikawa, M., Minard-Colin, V., Matsushita, T. & Tedder, T.F. Regulatory B cell production of IL-10 inhibits lymphoma depletion during CD20 immunotherapy in mice. J. Clin. Invest. 121, 4268–4280 (2011).

27. Caporaso, J.G. et al. Ultra-high-throughput microbial community analysis on the Illumina HiSeq and MiSeq platforms. ISME J. 6, 1621–1624 (2012).

28. Edgar, R.C. Search and clustering orders of magnitude faster than BLAST. Bioinformatics 26, 2460–2461 (2010).

29. DeSantis, T.Z. et al. Greengenes, a chimera-checked 16S rRNA gene database and workbench compatible with ARB. Appl. Environ. Microbiol. 72, 5069–5072 (2006).

30. DeSantis, T.Z. Jr. et al. NAST: a multiple sequence alignment server for comparative analysis of 16S rRNA genes. Nucleic Acids Res. 34, W394–W399 (2006).

31. Wang, Q., Garrity, G.M., Tiedje, J.M. & Cole, J.R. Naive Bayesian classifier for rapid assignment of rRNA sequences into the new bacterial taxonomy. Appl. Environ. Microbiol. 73, 5261–5267 (2007).

32. King, I.L. et al. Invariant natural killer T cells direct B cell responses to cognate lipid antigen in an IL-21–dependent manner. Nat. Immunol. 13, 44–50 (2012).

33. Mauri, C., Williams, R.O., Walmsley, M. & Feldmann, M. Relationship between TH1/TH2 cytokine patterns and the arthritogenic response in collagen-induced arthritis. Eur. J. Immunol. 26, 1511–1518 (1996).

34. Mauri, C., Gray, D., Mushtaq, N. & Londei, M. Prevention of arthritis by interleukin 10–producing B cells. J. Exp. Med. 197, 489–501 (2003).