The Journal of Immunology
Central Nervous System Demyelinating Disease Protection by
the Human Commensal Bacteroides fragilis Depends on
Polysaccharide A Expression
Javier Ochoa-Repa ´raz,* Daniel W. Mielcarz,†Lauren E. Ditrio,†Ashley R. Burroughs,‡
Sakhina Begum-Haque,* Suryasarathi Dasgupta,‡Dennis L. Kasper,‡and
Lloyd H. Kasper*
The importance of gut commensal bacteria in maintaining immune homeostasis is increasingly understood. We recently described
that alteration of the gut microflora can affect a population of Foxp3+Tregcells that regulate demyelination in experimental
autoimmune encephalomyelitis (EAE), the experimental model of human multiple sclerosis. We now extend our previous obser-
vations on the role of commensal bacteria in CNS demyelination, and we demonstrate that Bacteroides fragilis producing a
bacterial capsular polysaccharide Ag can protect against EAE. Recolonization with wild type B. fragilis maintained resistance
to EAE, whereas reconstitution with polysaccharide A-deficient B. fragilis restored EAE susceptibility. Enhanced numbers of
Foxp3+Tregcells in the cervical lymph nodes were observed after intestinal recolonization with either strain of B. fragilis. Ex vivo,
CD4+T cells obtained from mice reconstituted with wild type B. fragilis had significantly enhanced rates of conversion into IL-10–
producing Foxp3+Tregcells and offered greater protection against disease. Our results suggest an important role for commensal
bacterial Ags, in particular B. fragilis expressing polysaccharide A, in protecting against CNS demyelination in EAE and perhaps
human multiple sclerosis.The Journal of Immunology, 2010, 185: 4101–4108.
autoreactive cells respond to self-Ags by inducing inflammation
and attack the CNS, causing the axonal damage. Experimental
autoimmune encephalomyelitis (EAE), the most widely used an-
imal model for human MS, has provided significant information
suggesting the role of T cells in the induction of this autoimmune
disorder (2). It has been hypothesized that encephalitogenic
T cells are activated in the periphery and then cross the blood-
brain barrier into the CNS, where they encounter the self-Ag, and
become reactivated. This reactivation could induce the release of
proinflammatory cytokines, provoking the activation of resident
macrophage–microglia cells to release NO that is directly in-
volved in the demyelination of the neuronal myelin sheath (3).
There is substantial evidence collected in EAE suggesting that Th
ultiple sclerosis (MS) is a human disease of the CNS
that is characterized by an inflammatory process fol-
lowed by demyelination and axonal loss (1). In MS,
immune responses might be polarized toward Th1 and Th17 (1).
Recent findings have further supported a primary role for IL-17 in
the pathogenesis of human MS (4, 5).
Gut commensal microorganisms can modulate immune ho-
meostasis (6–13). Studies in germ-free animals, born and raised in
sterile conditions, showed that monocolonization with Bacteroides
fragilis was sufficient to stimulate early development of the
GALT, to induce normal organogenesis in the spleen and thymus,
and balanced immune development (14). Bacteroides species are
gram-negative bacteria that compose ∼25% of the microbiota in
both humans and other mammals. B. fragilis is symbiotic with the
host, but if it reaches sterile then extraluminal sites can be re-
sponsible for tissue infection, bacteremia, and abscess formation
in the peritoneal cavity, brain, liver, pelvis, or lungs (15, 16).
The administration of a mixture of gut commensal bacteria can
protect against EAE (17). The protection observed was found to be
IL-10 dependent. We (18) and others (19) recently demonstrated
that modification of the bacterial populations of the gut alters the
clinical outcome of EAE in mice. Oral treatment of mice with
antibiotics reduced EAE severity by diminishing proinflammatory
responses and the enhancement of Foxp3+Tregcells that signifi-
cantly accumulated in mesenteric and cervical lymph nodes (LNs).
Adoptive transfer of these IL-10–producing T regulatory (Treg)
cellsconferred protectionagainstEAE. Inthisstudy,we investigate
the effect of oral antibiotic treatment followed by gut reconstitution
with a human isolate of B. fragilis that produces the zwitterionic
capsular polysaccharide A (PSA), or with an isogenic mutant of B.
fragilis deficient in the production of PSA in the development and
protection against EAE. PSA has been found to be determinant in
the regulatory effect of B. fragilis, restoring the default Th2-
immune bias of germ-free animals (14, 16, 20). Moreover, IL-
10–producing CD4+CD45rblowT cells induced in response to PSA
administration were protective in a Helicobacter hepaticus model
of experimental colitis (21), and IL-10–producing Tregcells pro-
*Section of Neurology, Department of Medicine and†Department of Microbiol-
ogy and Immunology, Dartmouth Medical School, Lebanon, NH 03756; and
‡Department of Medicine, Channing Laboratory, Brigham and Women’s Hospital,
Harvard Medical School, Boston, MA 02115
Received for publication May 3, 2010. Accepted for publication July 28, 2010.
This work was supported by the Dartmouth-Hitchcock Foundation (Tiffany Blake
Fellowship No. 205-702B to J.O.-R.), the National Center for Research Resources–
Centers of Biomedical Research Excellence (Pilot Project No. 4, YR06), and training
grants from TEVA Neuroscience (50-2033, TEVA Neuroscience Murray B. Bornstein
Fund to L.H.K.)and the National Multiple Sclerosis Society (CA1027A1/3 to L.H.K.).
Address correspondence and reprint requests to Dr. Javier Ochoa-Repa ´raz, Dart-
mouth Medical School, 1 Medical Center Drive, Lebanon, NH 03576. Email address:
The online version of this article contains supplemental material.
Abbreviations used in this paper: DC, dendritic cell; EAE, experimental autoimmune
encephalomyelitis; LN, lymph node; MS, multiple sclerosis; PSA, polysaccharide A;
DPSA, polysaccharide A-deficient; RA, retinoic acid; T-bet, T-box transcription fac-
tor TBX21; Treg, T regulatory; WT, wild type.
tective against EAE (22). In this study, we demonstrate that the
absence of PSA production by a human isolate of B. fragilis used to
reconstitute disease-resistant mice restores clinical disease sus-
ceptibility, whereas the reconstitution with PSA-producing B. fra-
gilis maintains resistance by the induction of highly potent IL-10–
producing Tregcells. Our results suggest a potentregulatoryrolefor
this specific bacterial Ag in the control of CNS demyelination in
this experimental model of human MS.
Materials and Methods
Mice and treatments
Female 6-wk old SJL/J mice were obtained from The Jackson Laboratories
(Bar Harbor, ME). All animal care and procedures were in accordance with
at Dartmouth College Animal Resources Center institutional policies for
animal health and well-being. The animal supplier confirmed that all mice
used in the studies were free of exposure to Helicobacter. Dartmouth
College Animal Resources Center routinely screens for a wide range of
infectious agents, including Helicobacter. Because germ-free housing was
not available, mice were maintained in a restricted, access-controlled en-
vironment. All water, feed, and cages used were previously sterilized and
changed daily. Mice were treated with the following antibiotics dissolved
in drinking water for 1wk: ampicillin (1 g/ml), vancomycin (0.5 g/ml),
neomycin sulfate (1 g/ml), and metronidazole (1 g/ml) (23). Wild type
(WT) B. fragilis (National Collection of Type Culture 9343) and the iso-
genic mutant of PSA-deficient (DPSA) B. fragilis were provided by D.L.
Kasper (Harvard Medical School). One week after treatment with anti-
biotics, mice were infected with 1010WTor DPSA B. fragilis resuspended
in 200 ml sterile PBS by a single-time oral gavage.
Serial dilutions of intestinal and fecal samples were collected 1 wk after the
end of treatment with antibiotics and/or bacterial reconstitution and cultured
in general bacteriologic agar plates (CDC blood agar; BD Diagnostic Sys-
tems, Sparks, MD) and Bacteroides selective media plates (Bacteroides Bile
Esculin Agar; BD Diagnostic Systems) for 48 h at 37˚C. Plates were cultured
in aerobic and anaerobic conditions. Total bacteria per gram of sample was
calculated based on the colony forming units counted in each serial dilution.
SJL mice were challenged s.c. with 200 mg PLP139–151 (Peptides In-
ternational, Louisville, KY) in 200 ml CFA (Sigma-Aldrich, St. Louis,
MO). On days 0 and 2 after challenge, mice received 200 ng Bordetella
pertussis toxin i.p. (List Biological Laboratories, Campbell, CA) (24).
Mice were monitored and scored daily for disease progression (24).
Cytokine detection by Luminex and cytokine ELISA
Luminex and specific cytokine ELISAwere used to quantify triplicate sets
of supernatants. Cells were culturedin 24-welltissueplates at 23 106cells/
ml in the presence of anti-CD3 mAb-coated wells (10 mg/ml; BD Bio-
sciences, San Diego, CA), plus the soluble anti-CD28 mAb (5.0 mg/ml;
BD Biosciences) for 3 d, or media (24).
PCR detection of cytokine mRNA
was reverse transcribed using Multiscribe RT (Amersham Biosciences AB,
Uppsala, Sweden). cDNA (200 ng) was amplified using the 32 SYBR
green mix (Applied Biosystems, Foster City, CA) on a Bio-Rad (Hercules,
CA) iCycler. Relative expressions were normalized (b-actin sample–b-
actin of naive samples) and expressed using the cycle threshold method,
where relative mRNA expression = 2^-(DCt)p1000.
Single cervical LN lymphocyte preparations were stained using con-
ventional methods. T cell subsets were analyzed using fluorochrome-
conjugated mAbs (BD Biosciences) for CD4 and CD25. Intracellular
staining for Foxp3 was performed using fluorochrome-labeled anti-Foxp3
mAb (clone FJK-16s; eBioscience, San Diego, CA). Fluorochrome-labeled
anti-rat IgG2a (eBioscience) isotype control for Foxp3 expression was
used. Dendritic cells (DCs) were analyzed using CD11c, CD11b, and
CD103 (BD Biosciences). Bound fluorescence was analyzed with an FACS
Canto (BD Biosciences).
CD11c+cells were enriched with magnetic beads (StemCell Technologies,
Vancouver, British Columbia, Canada) and then sorted (FACSVantage with
Turbo-Sort; BD Biosciences) after staining with FITC-anti–CD103 into
CD11chighCD103+/2cells. CD4+T cells were obtained with magnetic
beads (Dynal Biotech ASA, Oslo, Norway). The enriched CD4+T cells
were cell-sorted for CD4+CD252T cells using FITC-anti–CD4 and PE-
anti–CD25 mAbs (BD Biosciences).
In vitro conversion assays and adoptive transfer experiments
Cervical LN CD4+CD252T cells were and cultured for 4 d in the presence
of anti-CD3/CD28 and IL-2 at different concentrations of TGF-b (0, 0.1,
0.5, and 5 ng/ml). To compare the role of CD11chighCD103+/2cells in the
conversion of CD4+CD252T cells into Foxp3+Tregcells, 1 3 104sorted
CD252T cells in the presence of anti-CD3 Ab (plates were precoated with
10 mg/mlanti-CD3Ab[BDBiosciences]). Cultureswere setin the presence
of PBS, purified PSA (100 mg/ml), or 4 nM retinoic acid (RA) and 5 ng/ml
TGF-b. Foxp3 acquisition by CD4+T cells was measured by flow cytom-
etry. For adoptive transfer experiments, 1 3 105in vitro Foxp3+Tregcon-
verted cells were injected i.v. into naive recipients. EAE was induced
1 d after adoptive transfers.
In vivo inactivation of CD25+cells
To inactivate CD25+CD4+T cells, mice were given 0.3 mg anti-CD25 mAb
(ATCC No. TIB-222, clone PC 61.5.3) on days 4 and 2 before EAE
challenge (24). As a control group, treated and naive mice received 0.3 mg
purified rat IgG Ab. A separate control group was immunized with PBS
7 d prior to EAE challenge.
Kruskal-Wallis followed by Dunn’s comparison of multiple groups was
applied to show differences in EAE clinical scores, cumulative clinical
scores, luminex and ELISA detection of cytokines as well as in the flow
numbers of gut bacteria. Reconstitution with both B. fragilis strains re-
stored the numbers. Oral treatment with antibiotics reduced the total
numbers of bacteria present in the gut, compared with naive mice. Re-
constitution with WT or DPSA B. fragilis restored those numbers (A).
Depicted are the means 6 SD from three separate experiments (n = 9 per
group). Bacteroides-selective media plates were used to confirm the col-
onization of the guts with B. fragilis (B).
Oral treatment with antibiotics significantly altered the
4102PSA-DEFICIENT B. FRAGILIS RESTORES EAE SUSCEPTIBILITY
cytometry of Tregcell and DC experiments. The p values ,0.05 and ,0.01
Reconstitution with PSA deficient B. fragilis restores the
susceptibility to EAE
We have reported that oral antibiotic treatment can protect against
CNS demyelination in a murine model of EAE (18). Four experi-
mental groups were used throughout the studies: 1) mice treated
orally with antibiotics for 1 wk, 2) mice treated with antibiotics
and subsequently reconstituted with WT B. fragilis, 3) mice
treated with antibiotics and subsequently reconstituted with DPSA
B. fragilis, and 4) mice sham-treated with PBS. EAE was induced
1 wk after bacterial reconstitution or PBS sham-treated control.
Oral antibiotic treatment significantly reduced the total bacterial
numbers recovered (Fig. 1A). WT or DPSA B. fragilis persist and
replicate similarly in vivo (14). The reconstitution of mice with
either WT or DPSA B. fragilis restored the number of detectable
bacteria (Fig. 1A). Bacteriologic culture of fecal samples in
Bacteroides selective media showed that the treatment with anti-
biotics reduced the Bacteroides spp. counts when compared with
naive mice whereas oral administration of WTor DPSA B. fragilis
resulted in effective reconstitution of the gut (Fig. 1B).
We examined whether reconstitution of mice with WTor DPSA
B. fragilis confers protection against EAE (18, 19). Oral treatment
with antibiotics reduces EAE severity and cumulative scores, and
it delays the clinical onset (18) (Fig. 2A, Table I). Reconstitution
of antibiotic-treated mice with DPSA B. fragilis reversed pro-
tection by restoring susceptibility to disease. Mice recolonized
with the intact strain of B. fragilis were protected against disease
and demonstrated reduced clinical severity when compared with
the PBS control group or DPSA B. fragilis-reconstituted mice
(Table I). There was no significant difference in the protection
between treatment with antibiotics and antibiotic treatment fol-
lowed by reconstitution with WT B. fragilis (Table I). In contrast,
oral antibiotic treatment followed by reconstitution with DPSA B.
fragilis resulted in clinically significant disease severity consistent
with that observed in the PBS control group.
Cytokine analysis of CNS tissue derived from WT B. fragilis-
reconstituted mice versus either control PBS-treated or DPSA
B. fragilis-reconstituted mice was compared (Fig. 2B). T-box
transcription factor TBX21 (T-bet) and IFN-g, both indicators of
Th1 polarization, were reduced in mice treated with antibiotics
compared with PBS treated mice, whereas GATA-3 and IL-13
expression were enhanced. There was a reduction in RORgt and
IL-17–relative expression in mice treated with antibiotics, and in
restores EAE susceptibility in mice protected after
treatment with antibiotics. A, Reconstitution with
PSA-deficient, but not WT, B. fragilis restored
EAE clinical scores. Depicted are the means for
a representative experiment from three separate ex-
from the experimental groups[n=12] are represented
in Table I). pp , 0.01 for PBS versus WT B. fragilis
reconstituted;†p , 0.01 for PBS- versus antibiotics-
treated. B, Brains of mice were harvested in the peak
of the disease (day 13) and relative expression of
IFN-g, IL-17, IL-10 cytokines as well as RORgt,
T-bet, GATA-3, and SMAD-3 transcription factors
were measured by PCR (relative expression is shown
after normalized with naive levels). Depicted are the
means 6 SD from all data combined of three
PSA deficiencyin B. fragilis
Table I.Reconstitution with DPSA B. fragilis restores EAE susceptibility in mice previously treated with antibiotics
Day of Clinical
WT B. fragilis reconstituted
DPSA B. fragilis reconstituted
8.1 6 0.9
9.9 6 0.9*
9.7 6 1.1*
8.2 6 0.8***
aSJL mice were treated with PBS, antibiotics, antibiotics and reconstitution with WT B. fragilis, and antibiotics and reconstitution with DPSA B.
fragilis by oral gavage. Seven days after treatment, EAE was induced.
bThe cumulative scores were calculated as the sum of all EAE clinical scores divided by the total number of mice per group (three experiments are
pp , 0.05, Kruskal-Wallis test followed by Dunn’s multiple comparison test, for PBS versus antibiotics and PBS versus WT B. fragilis; ppp , 0.01,
Kruskal-Wallis test followed by Dunn’s multiple comparison test, for PBS versus antibiotics and PBS versus WT B. fragilis; pppp , 0.05, for antibiotics
versus DPSA B. fragilis and WT B. fragilis versus DPSA B. fragilis; ppppp , 0.05, for antibiotics versus DPSA B. fragilis and WT B. fragilis versus
DPSA B. fragilis.
The Journal of Immunology 4103
WT B. fragilis-reconstituted mice when compared with PBS-
treated mice. Reconstitution with WT B. fragilis in EAE mice
enhanced GATA-3, SMAD-3, and IL-10 compared with PBS con-
trol mice. The profile of mice reconstituted with DPSA B. fragilis
showed no alterations of cytokine relative expression compared
with PBS control mice. Enhanced RORgt, IL-17, and T-bet levels
and reduced GATA-3, IL-10, and IL-13 levels of expression were
detected in DPSA B. fragilis-reconstituted mice versus mice treated
with antibiotics. When compared with WT B. fragilis, DPSA B.
fragilis-reconstituted mice showed enhanced RORgt and IL-17 as
well as and reduced SMAD-3 and IL-10.
Previous studies from our laboratory have demonstrated changes
in cytokine production by cells in the cervical LNs of mice after
oral antibiotic treatment (18). IL-13 levels were increased in mice
treated with antibiotics compared with mice reconstituted with
WT or DPSA B. fragilis (Fig. 3). The reconstitution with either
By contrast, colonization with WT B. fragilis enhanced the levels of IL-10 and IL-12 (p40). Both strains increased the levels of IFN-g compared with mice
treated with antibiotics. IL-10, GATA3, and SMAD3 levels were increased in WT B. fragilis compared with DPSA B. fragilis-reconstituted mice, whereas
IL-6 was reduced. IL-13 levels were enhanced in mice treated with antibiotics compared with the rest of experimental groups. Cytokines released to the
culture media were detected by Luminex (A), and relative expressions of GATA3, SMAD3, and IL-12 (p40) were detected by PCR (B). Depicted are the
means 6 SD from all data combined of three experiments (n = 8 per group). pp , 0.05; ppp , 0.01.
PSA-deficient B. fragilis reconstitution enhances production of IL-17 and IL-6 in cervical LN compared with mice treated with antibiotics.
compare the frequencies of Foxp3+CD25+among total CD4+T cells (A, B) gated on cervical LNs in PBS-treated mice, mice treated with antibiotics, and mice
Depicted are results for a representative experiment (A, C) and the means for a representative experiment from two separate experiments (n = 8 per group; B, D).
4104PSA-DEFICIENT B. FRAGILIS RESTORES EAE SUSCEPTIBILITY
WT or DPSA B. fragilis induced the production of IFN-g, com-
pared with mice treated with PBS and with antibiotics. Whereas
treatment with antibiotics significantly reduced the levels of IL-17
and IL-6 produced compared with PBS treatment (18), re-
constitution with DPSA B. fragilis, but not WT B. fragilis, sig-
nificantly augmented those levels. WT B. fragilis reconstitution
induced enhanced levels of IL-10, IFN-g, IL-12 (p40) in cervical
LN cells when compared with mice treated with antibiotics. IL-10
production and GATA3- and SMAD3-relative expressions in cells
obtained from WT B. fragilis-reconstituted mice were also en-
hanced, compared with mice reconstituted with DPSA B. fragilis
and PBS-treated mice. Although reductions in TNF-a and MCP-1
were observed in mice treated with antibiotics and reconstituted
with WT B. fragilis, the differences were found to be not signif-
icant compared with PBS control mice and mice reconstituted
with DPSA B. fragilis (not shown). No significant changes were
observed in IL-4, IL-5, MIP-1a, or MIP-1b levels (not shown).
EAE regulation by PSA exposure depends on Tregcells
We assessed whether PSA expression in B. fragilis could enhance
the percentages of Foxp3+Tregcells. Oral treatment with anti-
biotics increased the frequency of Foxp3+CD25+in CD4+T cells
isolated from the cervical LN (18). No significant differences were
observed in the frequency of Foxp3+CD25+Tregcells detected in
mice reconstituted with either WT or DPSA B. fragilis, compared
with mice treated with antibiotics (Fig. 4A, 4B). The frequency of
Foxp3+was enhanced in the CD25highversus the total CD25+
fraction of CD4+T cells in all groups (Fig. 4C, 4D). Mice sub-
jected to bacterial reconstitution showed similar frequencies of
Foxp3+Tregcell populations in the GALT (Peyer’s patches and
mesenteric LN) and spleens to those observed in antibiotic treated
mice (Supplemental Fig. 1). In vivo CD25+cell depletion dem-
onstrated a significant effect on the protection after reconstitution
with WT B. fragilis and in mice treated with antibiotics, as shown
before (18) (Fig. 5, Table II). Disease susceptibility after re-
constitution with WT B. fragilis or in mice rendered susceptible
after oral antibiotic treatment was enhanced when CD25+T cells
were depleted. Of interest was the increased severity of disease
following reconstitution with WT B. fragilis compared with mice
treated with antibiotics (Table II). As shown in both Fig. 2 and
Table I, no significant differences were observed in the severity of
EAE in mice reconstituted with DPSA B. fragilis and in PBS-
treated mice. Depletion of CD25+cells induced equivalent
increases in the cumulative disease indexes and mortality of both
DPSA B. fragilis reduces T cell conversion into Foxp3+
We further analyzed the role of PSA in the acquisition of regulatory
phenotypes by CD4+T cells. We previously showed that alteration
of the gut flora with antibiotics educated CD103+DCs to enhance
the conversion of CD4+T cells into Foxp3+Tregcells (18). To
compare the effect of B. fragilis recolonization in the induction of
regulatory phenotypes of T cells, CD103+/2CD11chighDCs were
sorted from the cervical LNs of mice treated with antibiotics and
from mice reconstituted with either WT or DPSA B. fragilis. DCs
protection.Invivo depletion of CD25+cells restoresthe susceptibility to EAE
were given 0.3 mg anti-CD25 mAb (clone PC 61.5.3) on days 4 and 2 before
EAE induction. As a control group, treated and naive mice received 0.3 mg
purified rat IgG Ab. When EAE was induced, protection observed in mice
Depicted are the means for a representative experiment from two separate
experiments (n = 4 per group; the combination of all data [n = 8] from the
versus WT B. fragilis reconstituted/aCD25, and DPSA B. fragilis recon-
stituted/IgG versus DPSA B. fragilis reconstituted/aCD25.
Bacteroides reconstitution depends on Tregcells to induce
Table II.In vivo neutralization of CD25+cells exacerbates EAE severity in protected mice
Day of Clinical
WT B. fragilis/IgG
WT B. fragilis/aCD25
DPSA B. fragilis/IgG
DPSA B. fragilis/aCD25
9.1 6 0.6
7.8 6 0.6**
10.6 6 0.5
8.8 6 0.6**
11.1 6 0.4
8.5 6 0.5**
8.6 6 0.5****
8.1 6 0.9
aSJL mice were treated with PBS, antibiotics, antibiotics and reconstitution with WT B. fragilis, and antibiotics and reconstitution with DPSA B.
fragilis by oral gavage. Seven days after treatment, EAE was induced. Four and 2 d before prior disease induction, mice were treated with 0.3 mg anti-
CD25 Ab or rat IgG isotype control.
bThe cumulative scores were calculated as the sum of all EAE clinical scores divided by the total number of mice per group (two experiments are
pp , 0.01, Kruskal-Wallis test followed by Dunn’s multiple comparison test, for PBS/IgG versus PBS/aCD25, PBS/IgG versus antibiotics/IgG, PBS/
IgG versus Antibiotics/aCD25; and PBS/IgG versus WT B. fragilis/IgG; ppp , 0.05, Kruskal-Wallis test followed by Dunn’s multiple comparison test,
for PBS/IgG versus PBS/aCD25, antibiotics/IgG versus antibiotics/aCD25, and WT B. fragilis/IgG versus WT B. fragilis/aCD25; pppp , 0.05, for WT
B. fragilis/IgG versus DPSA B. fragilis/IgG; ppppp , 0.01, for antibiotics/aCD25 versus WT B. fragilis/aCD25 and antibiotics/aCD25 versus DPSA
The Journal of Immunology4105
were cocultured in anti-CD3 Ab precoated plates with splenic
CD4+CD252(∼10% Foxp3+) T cells in the presence of PBS or
purified PSA (100 mg/ml; Fig. 6). When CD4+T cells were cul-
tured with CD1032DCs, the conversion of Foxp32CD4+T cells
into Foxp3+Tregcells was significantly reduced (data not shown).
CD103+DCs from mice reconstituted with DPSA B. fragilis ex-
posed to PSA showed significantly reduced conversion to Foxp3+
Tregcells compared with DCs from WT B. fragilis-reconstituted
mice. The PSA exposure of CD103+DCs from mice treated with
antibiotics did not significantly enhance the conversion rates
compared with exposure to PBS. Enhanced Tregconversion was
observed when DCs and T cells were cocultured with 4 nM RA
and 5 ng/ml TGF-b. However, despite these optimized conditions,
the conversion was significantly reduced in cocultures with
CD103+DCs from DPSA B. fragilis-reconstituted mice, compared
with cocultures with DCs from mice treated with antibiotics and
mice reconstituted with WT B. fragilis.
IL-10 producing Tregcells induced by PSA-producing B.
fragilis reconstitution protect against EAE
We next analyzed whether Foxp32CD4+T cells isolated from the
cervical LN of PSA-producing B. fragilis-reconstituted mice could
be converted into Foxp3+Tregcells (Fig. 7A, 7B, Supplemental Fig.
2). CD4+CD252(∼10% Foxp3+) T cells from WT B. fragilis-
LNs of mice treated with antibiotics, and mice reconstituted with either WTor DPSA B. fragilis and sorted into CD103+CD11c+DCs. Cells were cocultured
with CD4+CD252(∼10% Foxp3+) T cells sorted from spleens of naive mice in the presence of PBS, purified PSA (100 mg/ml), or with RA (4 nM) and
TGF-b (5 ng/ml). Foxp3 acquisition by CD4+T cells was measured by flow cytometry. Depicted are (A) the results from a representative experiment and
(B) the combination of all data (mean 6 SD; n = 6 per group). pp , 0.05; ppp , 0.01.
CD103+DCs of PSA-deficient B. fragilis have reduced ability to induce Foxp3+Tregcell conversion in vitro. DCs were harvested from cervical
(∼10% Foxp3+) T cells from mice treated with PBS, mice treated with antibiotics, and mice reconstituted with either WTor DPSA B. fragilis were cultured in
the presence of anti-CD3/anti-CD28 Abs, IL-2, and increasing concentrations of TGF-b (0, 0.1, 0.5, and 5 ng/ml). Flow cytometry was used to compare the
conversion rates of CD4+CD252(∼10% Foxp3+) T cells into Foxp3+Tregcells. Cells obtained from mice reconstituted with WT B. fragilis and cultured with
RA (4 nM) and TGF-b (5 ng/ml) are used to show the acquisition of CD25+by CD4+T cells, the isotype control for Foxp3 intracellular staining, and the
gating representing the frequency of CD4+cells that acquired a Foxp3+CD25+phenotype (A). Supplemental Fig. 2 shows representative results of Foxp3+
CD25+cells gated on CD4+T cells for all experimental groups. Cervical LN CD4+CD252(∼10% Foxp3+) T cells obtained from mice reconstituted with WT
B. fragilis showed enhanced rates of conversion into CD25+Foxp3+cells versus cells sorted from PBS treated mice, mice treated with antibiotics, and mice
reconstituted with DPSA B. fragilis, when cultured with 0.5 and 5 ng/ml of TGF-b. B, The column graph represents the combined results (n = 8 per group) for
the frequency of Foxp3+CD25+cells. The TGF-b concentrations are represented in the horizontal axes. pp , 0.01. C, IFN-g, IL-17, IL-10, and IL-13
cytokines were measured by specific ELISA in the supernatants of Foxp3+converted cells upon stimulation with 5 ng/ml of TGF-b (n = 8 per group). pp ,
0.01. D, Foxp3+converted cells after stimulation with 5 ng/ml of TGF-b were adoptively transferred (1 3 105cells per mouse) into naive recipient mice, and
EAE was induced one day later. Depicted are the combined results from two separate experiments (n = 8, per group). pp , 0.01.
IL-10 producing Foxp3+Tregcells converted from CD4+T cells of WT B. fragilis confer protection against EAE. Cervical LN CD4+CD252
4106 PSA-DEFICIENT B. FRAGILIS RESTORES EAE SUSCEPTIBILITY
reconstituted mice showed enhanced Tregconversion rates com-
pared with cells obtained from mice reconstituted with DPSA B.
fragilis, mice treated with antibiotics, and PBS-treated mice, when
cultured with 0.5 and 5 ng/ml TGF-b. Conversion rates were
significantly enhanced in all groups when TGF-b and RA ap-
proached the optimal concentration (Supplemental Fig. 2) (25).
We compared the capacity of these converted Foxp3+Tregcells
to protect against EAE (Fig. 7D). Cells cultured with 5 ng/ml
TGF-b were collected after 4 d and adoptively transferred into
naive recipient mice. Cells converted from WT B. fragilis-
reconstituted mice protected against EAE induction, whereas no
protection was observed in cells converted from PBS, mice treated
with antibiotics, or DPSA B. fragilis reconstituted mice. Cells
converted from WT B. fragilis-reconstituted mice produced signifi-
cantly increased levels of IL-10 compared with PBS-treated mice,
mice treated with antibiotics, and DPSA B. fragilis-reconstituted
mice; they produced a modest but significant increase in TGF-b
compared with DPSA B. fragilis-reconstituted mice (Fig. 7C). No
significant differences in the production of IFN-g, IL-17, IL-6, and
IL-13 were observed.
Alterations of the gut commensal bacteria populations by oral
treatment with antibiotics can influence the development of EAE
(18, 19). We now demonstrate that the reconstitution of mice with
B. fragilis deficient in the production of the zwitterionic capsular
PSA restores disease susceptibility in mice that had been rendered
resistant to disease after treatment with oral antibiotics. Recon-
stitution with both B. fragilis strains similarly restored the numbers
of detectable bacteria, which were significantly reduced after oral
treatment with antibiotics. It has been shown that both WT and
DPSA B. fragilis persist and replicate equally in vivo (14), sug-
gesting that it was PSA and not the number nor strain of B. fragilis
that was responsible for disease protection or susceptibility.
Oral treatment with antibiotics enhanced the frequency of
Foxp3+Tregcells within the cervical LN and reduced Th17 re-
sponses (18). The alterations of Th17 responses upon oral treat-
ment of antibiotics have been recently confirmed by others (26,
27). After antibiotic treatment, reconstitution with either WT or
DPSA B. fragilis resulted in a similar number and frequency of
Tregcells. In MS, the in vitro conversion rates of CD4+T cells into
Tregcells are significantly reduced in MS patients compared with
healthy controls (28). Moreover, functional suppression appears to
be impaired (29). CD4+T cells obtained from PSA-producing
B. fragilis-reconstituted mice more efficiently converted into
Foxp3+Tregcells, with increased IL-10 production and enhanced
protective potency after adoptive transfer.
Our results suggest that deficient PSA production could influence
the functional role of DCs and Foxp3+Tregcells induced by B. fra-
gilis. Prior studies have demonstrated that CD4+T cell activation by
PSA is dependent on the presentation of the Ag by CD11c+DCs
(30). Foxp3+Tregcell conversion by CD103+DCs purified from
PSA-deficient B. fragilis-reconstituted mice was significantly re-
duced compared with DCs from PSA-producing B. fragilis. Foxp3+
Tregcell conversion studies showed enhanced conversion rates of
CD4+T cells obtained from WT B. fragilis-reconstituted mice.
SMAD3 was significantly increased in WT B. fragilis-reconstituted
mice, and IL-6 was substantially reduced compared with DPSA B.
fragilis-reconstituted mice. Differentiation of Th17 cells requires
TGF-b and IL-6, whereas TGF-b is also required for Tregcell in-
duction in the absence of IL-6 (31). The differences observed in the
conversion rates could be due to the potential capability of WT B.
fragilis CD4+T cells to enhance TGF-b in cultures that could fa-
cilitate their conversion into Foxp3+Tregcells.
PSA may influence a distinct pathway involved in disease pro-
tection, as suggested by our experiments of Tregneutralization.
CD25+cell depletion exacerbated EAE in all groups, but the en-
hancement of disease severity was significantly higher in mice
treated withantibioticscompared withWTB.fragilis-reconstituted
mice. Other subpopulations of regulatory cells such as NKT cells
may participate in the protection against disease-induced treatment
with oral antibiotics (19). Recent observations from our laboratory
suggest that B cells could be important in this protective response
(32). IL-13 production was reduced in mice reconstituted with
WT B. fragilis compared with mice treated with antibiotics, and
only IL-10–producing Treg cells converted from Foxp32CD4+
T cells of WT B.fragilis-reconstituted mice protected against EAE.
Foxp3+Tregcells that were derived from CD4+T cells of PSA-
deficient B. fragilis-recolonized mice failed to protect against the
disease. We recently demonstrated that a highly purified prepara-
tion of PSA is protective against EAE in conventional mice and
that this protection is completely abrogated in IL-10–deficient
mice, suggesting an important role of this cytokine in the PSA-
induced control of the disease (22).
The presence or absence of PSA could determine protective
or pathogenic outcomes in EAE. WT or DPSA B. fragilis re-
constitution induced production of IFN-g, when compared with
mice treated with PBS and with antibiotics; however, IL-10 pro-
duction was enhanced only after reconstitution with WT B. fragilis,
whereas PSA-deficient B. fragilis reconstitution induced enhanced
levels of IL-6. The imbalance created by alterations of PSA ex-
pression within the gut lumen may lead to peripheral systemic
autoimmune disorders, such as EAE or human MS. In the absence
of PSA, the human commensal B. fragilis can no longer regulate
immune homeostasis, leading to autoimmune disease of the in-
CNS. Our previous (18, 22, 32) and present studies suggest that
differing compositionsof gutmicrobiota couldregulate the balance
between protection and disease induction in MS and may offer
a novel therapeutic approach for disease intervention.
We thank Dr. Azizul Haque, Dr. Jacqueline Y. Channon-Smith, Marc
Christy, Yan Wang, John DeLong, Kathleen Smith, and Alan J. Bergeron
for technical support and critical review of the manuscript.
The authors have no financial conflicts of interest.
1. Kasper, L. H., and J. Shoemaker. 2010. Multiple sclerosis immunology: The
healthy immune system vs the MS immune system. Neurology 74(Suppl 1): S2–
2. Wekerle, H. 2008. Lessons from multiple sclerosis: models, concepts, observa-
tions. Ann. Rheum. Dis. 67(Suppl 3): iii56–iii60.
3. Goverman, J. 2009. Autoimmune T cell responses in the central nervous system.
Nat. Rev. Immunol. 9: 393–407.
4. Tzartos, J. S., M. A. Friese, M. J. Craner, J. Palace, J. Newcombe, M. M. Esiri,
and L. Fugger. 2008. Interleukin-17 production in central nervous system-
infiltrating T cells and glial cells is associated with active disease in multiple
sclerosis. Am. J. Pathol. 172: 146–155.
5. Durelli, L., L. Conti, M. Clerico, D. Boselli, G. Contessa, P. Ripellino,
B. Ferrero, P. Eid, and F. Novelli. 2009. T-helper 17 cells expand in multiple
sclerosis and are inhibited by interferon-beta. Ann. Neurol. 65: 499–509.
6. Grainger, J. R., J. A. Hall, N. Bouladoux, G. Oldenhove, and Y. Belkaid. 2010.
Microbe-dendritic cell dialog controls regulatory T-cell fate. Immunol. Rev. 234:
7. Tezuka, H., and T. Ohteki. 2010. Regulation of intestinal homeostasis by
dendritic cells. Immunol. Rev. 234: 247–258.
8. Hand, T., and Y. Belkaid. 2010. Microbial control of regulatory and effector
T cell responses in the gut. Curr. Opin. Immunol. 22: 63–72.
9. Abreu, M. T. 2010. Toll-like receptor signalling in the intestinal epithelium: how
bacterial recognition shapes intestinal function. Nat. Rev. Immunol. 10: 131–144.
The Journal of Immunology4107
10. Troy, E. B., and D. L. Kasper. 2010. Beneficial effects of Bacteroides fragilis
polysaccharides on the immune system. Front. Biosci. 15: 25–34.
11. Boirivant, M., A. Amendola, and A. Butera. 2008. Intestinal microflora and
immunoregulation. Mucosal Immunol. 1(Suppl 1): S47–S49.
12. Izcue, A., J. L. Coombes, and F. Powrie. 2009. Regulatory lymphocytes and
intestinal inflammation. Annu. Rev. Immunol. 27: 313–338.
13. Artis, D. 2008. Epithelial-cell recognition of commensal bacteria and mainte-
nance of immune homeostasis in the gut. Nat. Rev. Immunol. 8: 411–420.
14. Mazmanian, S. K., C. H. Liu, A. O. Tzianabos, and D. L. Kasper. 2005. An
immunomodulatory molecule of symbiotic bacteria directs maturation of the
host immune system. Cell 122: 107–118.
15. Round, J. L., and S. K. Mazmanian. 2009. The gut microbiota shapes intestinal
immune responses during health and disease. Nat. Rev. Immunol. 9: 313–323.
16. Mazmanian, S. K., and D. L. Kasper. 2006. The love-hate relationship between bac-
terial polysaccharides and the host immune system. Nat. Rev. Immunol. 6: 849–858.
17. Lavasani, S., B. Dzhambazov, M. Nouri, F. Fa ˚k, S. Buske, G. Molin,
H. Thorlacius, J. Alenfall, B. Jeppsson, and B. Westro ¨m. 2010. A novel probiotic
mixture exerts a therapeutic effect on experimental autoimmune encephalomy-
elitis mediated by IL-10 producing regulatory T cells. PLoS ONE 5: e9009.
18. Ochoa-Repa ´raz, J., D. W. Mielcarz, L. E. Ditrio, A. R. Burroughs,
D. M. Foureau, S. Haque-Begum, and L. H. Kasper. 2009. Role of gut com-
mensal microflora in the development of experimental autoimmune encephalo-
myelitis. J. Immunol. 183: 6041–6050.
19. Yokote, H., S. Miyake, J. L. Croxford, S. Oki, H. Mizusawa, and T. Yamamura.
2008. NKT cell-dependent amelioration of a mouse model of multiple sclerosis
by altering gut flora. Am. J. Pathol. 173: 1714–1723.
20. Wang, Q., R. M. McLoughlin, B. A. Cobb, M. Charrel-Dennis, K. J. Zaleski,
D. Golenbock, A. O. Tzianabos, and D. L. Kasper. 2006. A bacterial carbohy-
drate links innate and adaptive responses through Toll-like receptor 2. J. Exp.
Med. 203: 2853–2863.
21. Mazmanian, S. K., J. L. Round, and D. L. Kasper. 2008. A microbial symbiosis
factor prevents intestinal inflammatory disease. Nature 453: 620–625.
22. Ochoa-Repa ´raz, J., D. W. Mielcarz, Y. Wang, S. Begum-Haque, S. Dasgupta,
D. L. Kasper, and L. H. Kasper. 2010. A polysaccharide from the human com-
mensal Bacteroides fragilis protects against CNS demyelinating disease. Mu-
cosal Immunol. 3: 487–495.
23. Rakoff-Nahoum,S.,J. Paglino,F.Eslami-Varzaneh,S.Edberg, and R.Medzhitov.
2004. Recognition of commensal microflora by toll-like receptors is required for
intestinal homeostasis. Cell 118: 229–241.
24. Ochoa-Repa ´raz, J., C. Riccardi, A. Rynda, S. Jun, G. Callis, and D. W. Pascual.
2007. Regulatory T cell vaccination without autoantigen protects against ex-
perimental autoimmune encephalomyelitis. J. Immunol. 178: 1791–1799.
25. Benson, M. J., K. Pino-Lagos, M. Rosemblatt, and R. J. Noelle. 2007. All-trans
retinoicacid mediates enhancedTregcell growth, differentiation,andguthoming
in the face of high levels of co-stimulation. J. Exp. Med. 204: 1765–1774.
26. Ivanov, I. I., K. Atarashi, N. Manel, E. L. Brodie, T. Shima, U. Karaoz, D. Wei,
K. C. Goldfarb, C. A. Santee, S. V. Lynch, et al. 2009. Induction of intestinal
Th17 cells by segmented filamentous bacteria. Cell 139: 485–498.
27. Ivanov, I. I., Rde. L. Frutos, N. Manel, K. Yoshinaga, D. B. Rifkin, R. B. Sartor,
B. B. Finlay, and D. R. Littman. 2008. Specific microbiota direct the differen-
tiation of IL-17-producing T-helper cells in the mucosa of the small intestine.
Cell Host Microbe 4: 337–349.
28. Kasper, L., A. Bergeron, R. Noelle, and J. Channon. 2009. Enhanced T regu-
latory cell conversion in relapsing multiple sclerosis: response to treatment with
glatiramer acetate. Mult. Scler. 15(Suppl. 2): S118–S119.
29. Haas, J., A. Hug, A. Vieho ¨ver, B. Fritzsching, C. S. Falk, A. Filser, T. Vetter,
L. Milkova, M. Korporal, B. Fritz, et al. 2005. Reduced suppressive effect of
CD4+CD25high regulatory T cells on the T cell immune response against my-
elin oligodendrocyte glycoprotein in patients with multiple sclerosis. Eur. J.
Immunol. 35: 3343–3352.
30. Duan, J., F. Y. Avci, and D. L. Kasper. 2008. Microbial carbohydrate de-
polymerization by antigen-presenting cells: deamination prior to presentation by
the MHCII pathway. Proc. Natl. Acad. Sci. USA 105: 5183–5188.
31. Korn, T., E. Bettelli, M. Oukka, and V. K. Kuchroo. 2009. IL-17 and Th17 Cells.
Annu. Rev. Immunol. 27: 485–517.
32. Ochoa-Repa ´raz, J., D. W. Mielcarz, S. Haque-Begum, and L. H. Kasper. 2010.
Induction of a regulatory B cell population in experimental allergic encephalo-
myelitis by alteration of the gut commensal microflora. Gut Microbes 1: 103–108.
4108 PSA-DEFICIENT B. FRAGILIS RESTORES EAE SUSCEPTIBILITY