Commensal bacteria protect against food
allergen sensitization
Andrew T. Stefka
a,1
, Taylor Feehley
a,1
, Prabhanshu Tripathi
a
, Ju Qiu
b
, Kathy McCoy
c
, Sarkis K. Mazmanian
d
,
Melissa Y. Tjota
e
, Goo-Young Seo
a
, Severine Cao
a
, Betty R. Theriault
f
, Dionysios A. Antonopoulos
e,g
, Liang Zhou
b
,
Eugene B. Chang
e
, Yang-Xin Fu
a
, and Cathryn R. Nagler
a,e,2
Departments of
a
Pathology,
e
Medicine, and
f
Surgery, The University of Chicago, Chicago, IL 60637;
b
Departments of Pathology and Microbiology-
Immunology, Feinberg School of Medicine, Northwestern University, Chicago, IL 60611;
c
Department of Clinical Research, University of Bern, 3010 Bern,
Switzerland;
d
Department of Biology, California Institute of Technology, Pasadena, CA 91125; and
g
Argonne National Laboratory, Argonne, IL 60439
Edited* by Dan R. Littman, New York University Medical Center, New York, NY, and approved August 5, 2014 (received for review June 25, 2014)
Environmentally induced alterations in the commensal microbiota
have been implicated in the increasing prevalence of food allergy.
We show here that sensitization to a food allergen is increased in
mice that have been treated with antibiotics or are devoid of a
commensal microbiota. By selectively colonizing gnotobiotic mice,
we demonstrate that the allergy-protective capacity is conferred by
a Clostridia-containing microbiota. Microarray analysis of intestinal
epithelial cells from gnotobiotic mice revealed a previously un-
identified mechanism by which Clostridia regulate innate lymphoid
cell function and intestinal epithelial permeability to protect against
allergen sensitization. Our findings will inform the development of
novel approaches to prevent or treat food allergy based on modu-
lating the composition of the intestinal microbiota.
microbiome
|
barrier
|
IL-22
L
ife-threatening anaphylactic responses to food are an in-
creasingly important public health problem (1). Rising dis-
ease prevalence over a short period cannot be explained by
genetic variation alone, renewing interest in the role of the en-
vironment in shaping allergic sensitization to food (2, 3). First
proposed more than 20 years ago, the hygiene hypothesis sug-
gested that societal efforts to reduce exposure to infectious
microbes early in life have deprived the immune system of im-
munoregulatory stimulation necessary for protection against al-
lergic disease (4). As our understanding of the profound influence
of commensal microbes on the maturation of the immune system
has grown, more recent iterations of this hypothesis have sup-
ported the idea that alterations in the composition of the in-
testinal microbiota induced by environmental factors (e.g.,
antibiotics, diet, vaccination, sanitation) play a central role in the
regulation of allergic sensitization (5
–
7). In particular, antibiotic
use during infancy potently perturbs intestinal bacterial pop-
ulations and has often been cited as a contributing factor to the
rising prevalence of allergic disease (8). However, the mechanisms
by which changes in the composition of the intestinal microbiota
regulate allergic responses to food remain poorly understood.
The gastrointestinal tract must maintain nonresponsiveness to
both an enormous variety of food antigens and the trillions of
bacteria that comprise the commensal microbiota (9). Mucosal
IgA and regulatory T-cell (Treg) responses induced by com-
mensal bacteria are critical for sustaining the homeostatic host
–
microbe relationship and preventing intestinal inflammation
(10). In addition, recent work has revealed that a heterogeneous
population of innate immune cells, known collectively as innate
lymphoid cells (ILCs), plays a critical role in integrating signals
from the commensal microbiota to maintain homeostasis at ep-
ithelial barriers and guide adaptive immunity (11). In this report
we show that sensitization to a food allergen is enhanced in mice
that have been treated with antibiotics (Abx) or are devoid of
commensal microbes (germ free, GF). Selective colonization of
gnotobiotic mice demonstrated that the allergy-protective capac-
ity is contained within the Clostridia, a class of anaerobic spore-
forming Firmicutes that reside in close proximity to the intestinal
epithelium. Reintroduction of a Clostridia-containing microbiota
to Abx-treated mice blocks sensitization to a food allergen. Using
microarray analysis of intestinal epithelial cells from gnotobiotic
mice, we identify an innate mechanism by which Clostridia pro-
tect against sensitization to dietary antigens. Defects in intestinal
permeability have been implicated in aberrant allergic responses
to food, but the mechanisms governing uptake of dietary antigen
have not been clear. We show here that Clostridia colonization
induces IL-22 production by both RAR-related orphan receptor
gamma (ROR
γ
t)
+
ILCs and T cells in the intestinal lamina
propria (LP) and that this cytokine acts to reduce uptake of orally
administered dietary antigen into the systemic circulation, con-
tributing, in part, to protection against sensitization.
Results
Neonatal Abx Exposure Alters the Commensal Microbiota and
Enhances Food Allergen Sensitization.
We evaluated the suscepti-
bility of mice to food allergen sensitization by intragastric co-
administration of peanut (PN) allergens and the mucosal adjuvant
cholera toxin (CT), which induces PN-specific IgE, IgG1, and
symptoms typical of systemic allergic hyperreactivity (12). Mice
treated with Abx showed marked elevation in PN-specific IgE and
Significance
The prevalence of food allergy is rising at an alarming rate; the
US Centers for Disease Control and Prevention documented an
18% increase among children in the United States between
1997 and 2007. Twenty-first ce
ntury environ
mental inter-
ventions are implicated by this dramatic generational increase.
In this report we examine how alterations in the trillions of
commensal bacteria that normally populate the gastrointesti-
nal tract influence allergic responses to food. We identify a
bacterial community that protects against sensitization and
describe the mechanism by which these bacteria regulate epi-
thelial permeability to food allergens. Our data support the
development of novel adjunctive probiotic therapies to po-
tentiate the induction of tolerance to dietary allergens.
Author contributions: A.T.S., T.F., P.T., B.R.T., D.A.A., Y.-X.F., and C.R.N. designed research;
A.T.S., T.F., P.T., J.Q., M.Y.T., G.-Y.S., S.C., B.R.T., and D.A.A. performed research; K.M., S.K.M.,
D.A.A., L.Z., E.B.C., and Y.-X.F. contributed new reagents/analytic tools; A.T.S., T.F., P.T., J.Q.,
M.Y.T., S.C., D.A.A., and C.R.N. analyzed data; and A.T.S., T.F., and C.R.N. wrote the paper.
Conflict of interest statement: A provisional US patent application (61/937952) was filed
on February 10, 2014.
*This Direct Submission article had a prearranged editor.
Data deposition: The DNA sequences reported in this paper have been deposited in the
MG-RAST database (project no.
7173
). The Microarray data has been deposited in the Gene
Expression Omnibus (GEO) database,
www.ncbi.nlm.nih.gov/geo
(series no.
GSE60039
).
1
A.T.S. and T.F. contributed equally to this work.
2
To whom correspondence should be addressed. Email: cnagler@bsd.uchicago.edu.
This article contains supporting information online at
www.pnas.org/lookup/suppl/doi:10.
1073/pnas.1412008111/-/DCSupplemental
.
www.pnas.org/cgi/doi/10.1073/pnas.1412008111
PNAS
|
September 9, 2014
|
vol. 111
|
no. 36
|
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–
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IMMUNOLOGY AND
INFLAMMATION
IgG1 after sensitization and allergen challenge (Fig. 1
A
and
B
).
Analysis of 16S rRNA genes revealed that 6 wk of Abx treatment
resulted in a significant reduction
inbacterialloadinboththefeces
and ileal contents (Fig. 1
C
) and altered the diversity (Fig. 1
D
)and
composition (Fig. 1
E
) of the fecal and ileal microbiota. Those
members of the Bacteroidetes and Firmicutes phyla most prevalent
under normal conditions (Fig. 1
E
, no treatment, NT) were absent in
fecal samples obtained from Abx-treated mice and were replaced
instead by
Lactobacillaceae
(Fig. 1
E
), consistent with another recent
report (13).
A Clostridia-Containing Microbiota Protects Against Sensitization to
Food Allergens.
To gain insight into the populations of bacteria
responsible for protection against sensitization to food allergens,
we created a gnotobiotic model of food allergy. Upon sensitization
with PN/CT, GF mice exhibit significantly increased levels of
PN-specific IgE and IgG1 relative to mice maintained in typical
specific pathogen-free (SPF) housing conditions and the reduced
core body temperature at challenge characteristic of an anaphy-
lactic response (Fig. 2
A
,
B
,and
D
). In the absence of a colonizing
microbiota, GF mice displayed grossly enlarged cecal size (Fig. 2
E
)
and spontaneously higher levels of circulating IgE with increasing
age (
Fig. S1
A
). Total IgE levels in GF mice were elevated by
treatment with CT or PN/CT (Fig. 2
C
). GF mice colonized with an
SPF microbiota (conventionalized) did not show elevated levels of
PN-specific IgE (Fig. 2
A
) or IgG1 (Fig. 2
B
) or a reduced core body
temperature (Fig. 2
D
) in response to sensitization with PN/CT.
The concentration of total IgE detectable in the serum of con-
ventionalized mice was also reduced to levels similar to those seen
in SPF mice (Fig. 2
C
). In addition, conventionalized mice displayed
the normal cecal size (Fig. 2
E
) and bacterial load characteristic of
SPF mice (Fig. 2
F
).
We next examined the ability of selected members of the SPF
microbiota to influence susceptibility to allergic sensitization to
food. We focused on
Bacteroides
,
Clostridium
cluster XIVa, and
Clostridium
cluster IV, which constitute the numerically pre-
dominant taxa in the murine colon (14). Anaerobic cultures of
fecal material from our SPF colony yielded
Bacteroides uniformis
as a representative
Bacteroides
species. Monocolonization of GF
mice with
B. uniformis
resulted in a bacterial load similar to that
seen in SPF and conventionalized mice (Fig. 2
F
) but did not
reduce cecal size (Fig. 2
E
), rescue the drop in core body tem-
perature in all mice (Fig. 2
D
), or significantly reduce the
PN-specific IgE or IgG1 response seen in GF mice (Fig. 2
A
and
B
). To colonize GF mice with Clostridia, we used chloroform-
extracted spores isolated from a mixed cecal/fecal sample from
a healthy SPF mouse. Sequence analysis showed that this extract
was consistently and predominantly composed of members of
Clostridium
clusters XIVa, XIVb, and IV (
Fig. S1
B
–
D
). Colo-
nization with this Clostridia consortium protected against sen-
sitization to PN/CT, because levels of PN-specific and total IgE
were reduced compared with GF controls (Fig. 2
A
and
C
), and
no temperature drop was seen at challenge (Fig. 2
D
). Cecal size
in Clostridia-colonized mice was comparable to that seen in SPF
mice (Fig. 2
E
), although the bacterial load measured in feces was
significantly lower (Fig. 2
F
). Collectively, these data suggest that
Clostridia play a role in protection against sensitization to a food
allergen. We then examined whether the changes in food allergen
sensitization induced by neonatal Abx administration (Fig. 1)
could be reversed by selectively restoring the intestinal microbial
community. In addition, we examined the response to sensitization
after recovery from 1 wk of preweaning antibiotic treatment (Abx
Recov.) (
Fig. S2
). PN-specific IgE and IgG1 and total IgE levels
were reduced in serum collected at challenge from Abx-treated,
PN/CT-sensitized mice that
had been conventionalized (Abx
conv.), Clostridia-colonized (Abx Clost.), or allowed to recover
(Abx Recov.) (
Fig. S2
A
–
C
), suggesting that restoring a Clostridia-
containing microbiota by either fecal gavage or removal of Abx-
mediated selection is sufficient t
o protect against food allergen
sensitization. In support of these findings, at termination, the
abundance of Clostridia in fecal samples was restored to untreated
levels in mice that received fecal gavage (Abx conv. or Abx Clost.)
or were allowed to recover (Abx Recov.,
Fig. S2
D
and
E
), although
their community structures remained distinct (
Fig. S2
F
).
Clostridia Colonization Activates Innate Immune Genes in Intestinal
Epithelial Cells.
Several reports suggest that mucosa-associated
Clostridia populations have a unique role in the induction of
Foxp3
+
Tregs and IgA, the two major arms of adaptive mucosal
immunity (15
–
17). The ability of selected indigenous commensal
bacteria to activate innate immune signaling in intestinal epi-
thelial cells (IECs) is less well understood. We first confirmed
that both conventionalized and Clostridia-colonized mice have
0
100
200
300
600
700
NT
Abx
0
50
100
150
200
250
PN+CT
CT only
*
NT
Abx
AB
C
D
E
0.0
0.2
0.4
0.6
0.8
1.0
NT
Abx
S24-7
Prevotellaceae
Rickinellaceae
Bacteroidaceae
Others
Ruminococcaceae
Lachnospiraceae
Others
Lactobacillaceae
Others
Others
All Members
Others
Desulfovibrionales
All Members
All Members
Bacteroidetes
Firmicutes
Proteobacteria
Bacilli
Clostridia
Other Phyla/Unclassified Bacteria
Actinobacteria
Erysipelotrichi
NT
Abx
Feces
Ileal Contents
Abundance
9
10
11
12
13
NT Abx
***
NT
Abx
Feces
Ileal Contents
*
Log Copies
16S rRNA gene / g content
0
2,000 4,000 6,000 8,000
0
200
400
600
800
sequences per sample
OTUs observed
Fig. 1.
Neonatal Abx exposure alters the commensal microbiota and
enhances food allergen sensitization. Abx treatment was initiated before
weaning as described in
Methods
.(
A
and
B
) 3-wk-old mice were sensitized by
intragastric administration of PN plus CT (PN/CT, open symbols) or CT only
(closed symbols) and challenged on day 35; feces and serum were collected on
day 36. Serum concentration of (
A
) PN-specific IgE and (
B
) PN-specific IgG1
was measured by ELISA (
n
=
4
–
9 mice per group from three independent
experiments; each circle represents an individual mouse; bars depict mean and
SEM). (
C
) Bacterial load in the feces or ileal contents of mice treated with Abx
compared with no treatment (NT) controls. (
n
=
4
–
5micepergroup).(
D
)
Bacterial diversity, as shown by operational taxonomic unit (97% identity)
rarefaction curves in Abx-treated mice compared with NT controls: black lines,
NT feces; gray lines, Abx feces; red lines, NT ileal contents; blue lines, Abx ileal
contents. (
E
) Taxonomic classifications for the mice in
C
represented as pro-
portion of total reads (
Methods
). *
P
<
0.05, **
P
<
0.01, ***
P
<
0.001 de-
termined by Student
t
test (
B
) or one-way ANOVA with Tukey posttest (
C
).
13146
|
www.pnas.org/cgi/doi/10.1073/pnas.1412008111
Stefka et al.
significantly increased proportions of Foxp3
+
Tregs in the co-
lonic LP and elevated concentrations of fecal IgA compared with
the baseline levels detected in GF mice (Fig. 3
A
and
B
). Mon-
ocolonization of GF mice with
B. uniformis
partially restored levels
of fecal IgA (Fig. 3
B
) but did not affect the LP Treg compartment
(Fig. 3
A
).
B. uniformis
and Clostridia therefore differed in their
ability both to protect against food allergen sensitization (Fig. 2)
and to induce colonic Tregs and fecal IgA (Fig. 3), suggesting that
they also differentially activate innate immunity. To gain insight
into the role of microbial interactions with IECs in the regulation
of sensitization to food allergens, we examined gene expression in
IECs from GF mice and from mice colonized with
B. uniformis
or
Clostridia. Microarray analysis showed that 38 genes in IEC from
Clostridia-colonized mice and 16 from
B. uniformis
-colonized mice
exhibited
≥
1.5-fold increase in mean expression compared with
GF controls (Fig. 3
C
). We were particularly interested in the
differential up-regulation of re
generating islet-derived 3 beta
(
Reg3b
) in Clostridia-colonized mice (Fig. 3
D
and
E
) because it
encodes an antimicrobial peptide, REG3
β
, which regulates the
composition of the mucosa-associated microbiota (18).
Clostridia Colonization Induces IL-22.
We validated our microarray
results by demonstrating that
Reg3b
and
Reg3g
expression were
increased in whole-tissue extr
acts from Clostridia, but not
B. uniformis
, colonized mice (Fig. 4
A
). IECs produce antimicrobial
peptides in response to IL-22
–
mediated signaling (18). We found
that only Clostridia colonization induced significant up-regulation
of IL-22 transcripts in lamina
propria lymphocyte (LPL) (Fig. 4
B
).
Flow cytometric analysis revealed that both ROR
γ
t
+
ILCs and
CD4
+
TCR
β
+
T cells produced elevated levels of IL-22 in response
to Clostridia colonization (Fig. 4
C
and
Fig. S3
A
). The proportion
of ROR
γ
t
+
ILCs within the LTi0, LTi4, and NK22 ILC3 subsets in
the colonic LP was unchanged in Clostridia-colonized mice (
Fig.
S3
B
). IL-22 also protects the intestinal epithelial barrier by
promoting mucus secretion by g
oblet cells (19); the numbers of
mucus-producing goblet cells were significantly increased in mice
colonized with Clostridia but not in those colonized with
B
.
uniformis
(Fig. 4
D
). Having identified its cellular sources and
confirmed a known barrier protective functional activity in our
model, we asked whether IL-22 also plays a role in regulating
epithelial permeability to protein antigens. Because antigen uptake
from the intestinal lumen is the first step in sensitization to a food
allergen, we reasoned that Clostridia-induced IL-22 production
reinforces the epithelial barri
er to reduce intestinal permeabil-
ity to dietary proteins. To explore this hypothesis, we developed
an assay to measure the transient presence of allergen in the
blood after intragastric gavage. Several Ara h proteins have been
identified as the immunodominant allergens of PN (
Arachis hypo-
gaea)
(20). We used sensitive capture ELISAs to measure the
concentration of two of these prote
ins in the systemic circulation.
BothArah6andArah2werereadilydetectableintheserum
of GF mice (Fig. 4
E
). Colonization with Clostridia, but not
A
B
C
D
E
F
0
100
200
300
400
500
1,000
2,000
3,000
4,000
GF
B.
uniformis
SPF
Clost.
Conv.
**
**
CT only
PN+CT
0
100
200
300
400
*
GF
B.
uniformis
SPF
Clost.
Conv.
-6
-4
-2
0
2
***
GF
B.
uniformis
SPF
Clost.
Conv.
0
5,000
10,000
20,000
30,000
GF
B.
uniformis
SPF
Clost.
Conv.
Total IgE (ng/mL)
9
10
11
12
13
SPF
Conv.
B.
uniformis
Clost.
***
Log Copies
16S rRNA gene / g feces
SPF
GF
Conv.
B. uni for mi s
Cl ostr i di a
Fig. 2.
A Clostridia-containing microbiota protects against sensitization to
food allergens. (
A
–
D
) Groups of SPF (white), GF (red), or gnotobiotic mice
colonized with fecal/cecal material from SPF mice (Conventionalized, blue),
B. uniformis
(gray), or with a consortium of Clostridia (green) were sensitized
with either CT only or PN/CT at weaning and challenged on day 35. (
A
)
Concentration of PN-specific IgE, (
B
) IgG1, and (
C
) total IgE in serum of
sensitized mice collected 24 h after challenge. (
D
) Change in core body
temperature in sensitized mice (
n
=
4
–
10 mice per group from two in-
dependent experiments; closed circles, CT only; open circles, PN/CT). In
A
–
D
,
each circle represents an individual mouse; bars depict median. (
E
) Cecal size
at 13 d after colonization. (
F
) Bacterial load in feces collected from 5- to 10-
wk-old SPF and gnotobiotic mice 14 d after colonization.
n
=
3
–
5 mice per
group.
F
depicts mean and SEM. *
P
<
0.05, **
P
<
0.01, ***
P
<
0.001 de-
termined by two-way ANOVA with the Kruskal-Wallis test (
A
–
D
) or one-way
ANOVA with Tukey posttest (
F
).
GF
B. uniformis
Clostridia
Row Mean
+3 SD
-3 SD
Cyp2c55
Tmod4
Arg2
Reg3b
ProS1
Wfs1
Vegfc
Fbn1
Col16a1
Gadd45a
Pdk4
0
2
4
6
8
Germ free
B. uniformis
Clostridia
Reg3b
Reg3g
**
Relative Expression
0
10
20
30
40
SPF
Germ free
Conventionalized
Clostridia
Spleen
MLN
Colonic
LP
***
***
***
*
B. uniformis
% Foxp3
+
of CD4
+
0
200
400
600
Germ
free
B.
uniformis
SPF
Clost.
Conv.
***
***
***
***
***
IgA (ng/100 mg feces)
B. uniformis
-induced
Clostridia-induced
Common
AB
C
D
E
Fig. 3.
Clostridia colonization activates innate immune genes in IECs. (
A
)
Proportion of Foxp3
+
Tregs among CD4
+
T cells in the spleen, MLN, and
colonic LP of age-matched SPF (white) and GF (red) mice and 14 d after
colonization of GF mice with an SPF microbiota (Conventionalized, blue),
B. uniformis
(gray), or Clostridia (green) (
n
=
4
–
8 per group). (
B
) Concentration
of IgA in feces collected from sensitized mice in Fig. 2. (
C
) Number of genes
up-regulated in IECs relative to GF by
B. uniformis
(gray), Clostridia (green), or
both (black) at 6 d after colonization. Genes shown exhibited significant ex-
pression above background in all samples (detection
P
value
<
0.05) and
≥
1.5-fold increase in mean expression in comparison with values obtained for
GF mice. (
D
) Heatmap depicting differential gene expression for 11 genes of
interest. Samples with the highest and lowest transcript levels are red and
blue, respectively. (
E
) Quantitative PCR verification of microarray data for
selected genes. *
P
<
0.05, **
P
<
0.01, ***
P
<
0.001 by two-way ANOVA with
Bonferroni posttest (
A
and
E
) or one-way ANOVA with Tukey posttest (
B
).
Stefka et al.
PNAS
|
September 9, 2014
|
vol. 111
|
no. 36
|
13147
IMMUNOLOGY AND
INFLAMMATION
B. uniformis
, reduced the circulating concentrations of both
proteins after gavage.
Clostridia-Induced IL-22 Regulates
Allergen Access to the Bloodstream.
To determine whether IL-22 induced by Clostridia gavage is
necessary and sufficient to reduce intestinal barrier permeability
we used the Abx-depletion model.
Il22
expression was signifi-
cantly increased in the colon of Abx-treated Clostridia-colonized
mice (Fig. 5
A
). Significantly higher concentrations of Ara h 6
were detected in the serum of Abx-treated mice compared with
mice that received no treatment (NT; Fig. 5
B
); similar results
were obtained for Ara h 2 (
Fig. S4
A
). Serum Ara h 6 and Ara h 2
were reduced in Abx mice treated with an IL-22-Fc fusion pro-
tein (21) or colonized with Clostridia after 1 wk of Abx gavage
(Fig. 5
B
and
Fig. S4
A
), indicating that either Clostridia gavage or
exogenous IL-22 is sufficient to reduce the concentration of
serum allergen. To demonstrate that Clostridia-induced IL-22
regulates allergen access to the bloodstream, groups of Abx-
treated Clostridia-colonized mice were given i.p. injections of
a neutralizing antibody to IL-22 (22) or an isotype control before
allergen challenge. Serum concentrations of Ara h 6 and Ara h 2
were significantly elevated in Clos
tridia-colonized mice treated with
anti-IL-22 compared with mice treated with an isotype control (Fig.
5
C
and
Fig. S4
B
), directly linking Clostridia-induced IL-22 pro-
duction to the regulation of aller
gen uptake. Anti-IL-22 treatment
did not affect Clostridia-mediated induction of Foxp3
+
Tregs in the
colonic LP (
Fig. S4
C
). Together with the inability of IL-22-Fc to
0
5
10
Small
Intestine
Colon
***
**
Relative expression Il22
A
B
0
2
4
6
Small
Intestine
Colon
**
***
Germ free
B. uniformis
Clostridia
Relative expression Reg3b
0
2
4
6
Small
Intestine
Colon
**
***
Relative expression Reg3g
Pre
15 min
45 min
0
20
40
60
80
100
Germ free
Clostridia
B. uniformis
Ara h 6 in serum (ng/mL)
D
Pre
15 min
45 min
0
10
20
30
Ara h 2 in serum (ng/mL)
C
Germ free
B. uniformis
Clostridia
0
5
10
15
20
***
Germ free
B. uniformis
Clostridia
**
Number of Goblet Cells / Crypt
0
1
2
3
GF
Clostridia
%IL-22
+
cells gated
on CD4
+
TCR
+
cells
0
20
40
60
80
100
GF
Clostridia
**
%IL-22
+
cells gated
on ROR
t
+
ILCs
E
Fig. 4.
Clostridia colonization induces IL-22. (
A
)
Reg3b
and
Reg3g
expres-
sion from whole-tissue extracts isolated 4 d after colonization from the small
intestine or colon of GF (red),
B. uniformis
-colonized (gray), or Clostridia-
colonized (green) mice. Quantitative RT-PCR data are plotted relative to GF
and normalized to
Hprt
(
n
=
8
–
9 mice per group from two independent
experiments). (
B
)
Il22
expression in LPL from mice in
A
.(
C
) IL-22 production
by ROR
γ
t
+
ILCs and T cells 6 d after colonization, determined by flow cyto-
metric analysis of permeabilized cells (
SI Methods
;
n
=
3 mice per group
representative of three independent experiments). (
D
) Representative
images and quantification of goblet cells in distal colon of GF,
B. uniformis
-
colonized, and Clostridia-colonized mice 6 d after colonization.
n
=
3
–
5 mice
per group. (Scale bar, 100
μ
m.) (
E
) Serum Ara h 6 and Ara h 2 levels after PN
gavage in GF,
B. uniformis
-colonized, or Clostridia-colonized mice 6 d after
colonization (
n
=
5
–
12 mice per group from two independent experiments).
*
P
<
0.05, **
P
<
0.01, ***
P
<
0.001 by two-way ANOVA with Bonferroni
posttest (
A
and
B
) or one-way ANOVA with Tukey posttest (
C
).
C
A
D
B
E
0.0
0.5
1.0
1.5
AbxClostridia+isotype
AbxClostridia+
IL-22
Small Intestine
Colon
**
*
Relative expression Reg3b
0
2
4
6
Abx
AbxClostridia
*
Small Intestine
Colon
Relative expression Il22
H
F
G
I
0
100
200
300
400
500
*
CD3
IL-17 (pg/mL)
-0.6
-0.4
-0.2
0.0
0.2
-0.3
-0.2
-0.1
0.0
0.1
0.2
0.3
PC1 (12.7% of var. expl.)
PC2 (5.2% of var. expl.)
AbxClostridia+isotype
AbxClostridia+
IL-22
Day 0 7 14 21 28 36
Pre
1 hr
3 hr
0
20
40
60
80
**
**
**
Abx PN
AbxClostridia PN
Abx+IL-22Fc PN
NT PN
Ara h 6 in serum (ng/mL)
Pre
1 hr
3 hr
0
20
40
60
80
100
*
AbxClostridia+isotype PN
AbxClostridia+
IL-22 PN
Ara h 6 in serum (ng/mL)
0
200
400
600
1200
Abx
Clost.
+isotype
Abx
Clost.
+
IL-22
0
1000
2000
3000
4000
5000
Abx
Clost.
+isotype
Abx
Clost.
+
IL-22
Total IgE (ng/mL)
0
500
1000
1500
2000
2500
3500
4000
4500
5000
Abx
Clost.
+isotype
Abx
Clost.
+
IL-22
0
10
20
30
40
CD3
AbxClostridia+isotype
AbxClostridia+
IL-22
IL-4 (pg/mL)
Fig. 5.
Clostridia-induced IL-22 regulates
allergen access to the bloodstream. (
A
)
Expression of
Il22
in LPL from neonatal Abx-treated mice without Clostridia
colonization, or at 6 d after weaning and colonization. (
B
)SerumArah6at
indicated time points after PN gavage in NT or Abx mice treated with or without
one i.p. injection of IL-22-Fc, or by Clostridia colonization. (
C
)SerumArah6at
indicated time points after PN gavage in Abx-treated Clostridia-colonized mice
injected i.p. with neutralizing antibody to IL-22 or an isotype control. All mice in
B
and
C
received PN at 6 d after weaning, and serum levels of Ara h 6 were
measured by capture ELISA (
n
=
5
–
10 mice per group, pooled from at least two
experiments). (
D
) Expression of
Reg3b
in whole-tissue extracts from Abx-treated
Clostridia-colonized mice treated with neutralizing antibody to IL-22 or an iso-
type control and sensitized with PN/CT (
n
=
11 mice per group, pooled from four
experiments). (
E
) Concentration of IL-4 in culture supernatants from splenocytes
of mice from
D
(
n
=
7 mice per group, representative of two experiments). (
F
)
Concentration of PN-specific and total IgE
in serum collected 24 h after challenge
for mice in
D
(
n
=
11 mice per group, pooled from four experiments). (
G
) Con-
centration of IL-17 in culture supernatants from splenocytes from mice in
D
(
n
=
7
mice per group, representative of two experiments). (
H
) Concentration of
PN-specific IgG in serum collected 24 h after challenge for mice in D (
n
=
11 mice
per group, pooled from four experiments). (
I
) UniFrac analysis of fecal microbiota
throughout the sensitization protocol (
n
=
4 mice per group). *
P
<
0.05, **
P
<
0.01 ***
P
<
0.001 by two-way ANOVA with Bonferroni posttest (
A
,
B
,and
D
)or
Student
t
test (C and G).
13148
|
www.pnas.org/cgi/doi/10.1073/pnas.1412008111
Stefka et al.
induce CD4
+
Foxp3
+
Tregs in the colonic LP of Abx-treated mice
(
Fig. S4
C
), this result suggested that Clostridia-induced IL-22
does not expand the colonic Treg compartment. In addition,
the concentration of Ara h 6 in the serum of Abx-treated mice
3 h after gavage with PN/CT was significantly higher than that
detected in mice that received PN alone (
Fig. S4
D
compared
with Fig. 5
B
;
P
<
0.05), in agreement with the role of adjuvants
such as CT in increasing intestinal permeability to luminal
antigens (23). Serum Ara h 6 and Ara h 2 were reduced in Abx-
treated Clostridia-colonized mice even when PN was adminis-
tered together with CT (
Fig. S4
D
and
E
). To examine whether
Clostridia-induced IL-22 production by ILCs regulates allergen
uptake, we repeated the Abx treatment/Clostridia colonization in
Rag
−
/
−
mice depleted of ILCs with anti-CD90 antibody (as de-
scribed in ref. 24). Elevated concentrations of Ara h 6 and Ara
h 2 were detectable in the serum of Abx-treated Clostridia-colo-
nized ILC-depleted
Rag
−
/
−
mice compared with mice treated with
an isotype control (
Fig. S4
F
and
G
). The efficacy of anti-CD90
treatment in depleting IL-22 transcripts in the intestinal LP was
confirmed by quantitative PCR (
Fig. S4
H
).
Finally, we examined whether Clostridia-induced IL-22 pro-
duction in the intestinal LP regulates sensitization to food aller-
gens. Abx-treated Clostridia-col
onized mice sensitized with PN/CT
as in Fig. 1 and
Fig. S2
were treated with anti-IL-22 or isotype
control throughout the 35-d protocol. Examination at sacrifice
showed that both intestinal
Reg3b
expression (Fig. 5
D
) and goblet
cell numbers (
Fig. S4
I
) were significantly reduced in mice treated
with anti-IL-22 compared with isotype-treated controls, confirm-
ing that IL-22 was effectively neutralized by this treatment pro-
tocol. To assess sensitization to food, splenocytes harvested after
allergen challenge were restimulated in vitro with anti-CD3 or PN
as previously described (12). Oral administration of antigen with
CT as a mucosal adjuvant typically induces a Th2 biased response
to promote allergic sensitization (12). However, treatment of Abx-
depleted Clostridia-colonized mice with anti-IL-22 throughout the
course of the sensitization protocol did not result in elevated levels
of IL-4 (Fig. 5
E
) or an increased PN-specific or total IgE response
(Fig. 5
F
), in agreement with the absence of Th2 skewing (IL-13
and IFN-
γ
were also not significantly changed;
Fig. S4
J
and
K
).
Instead we detected significantly elevated production of IL-17 (Fig.
5
G
), consistent with other reports showing that depletion of innate
IL-22 promotes an adaptive Th17 response (25). PN-specific IgG
increased in anti-IL-22
–
treated mice compared with isotype con-
trols (
P
=
0.09) (Fig. 5
H
). Interestingly, in keeping with the anti-
microbial activity of REG3
β
, we found that anti-IL-22 treatment
altered the composition of the feca
l microbiota. UniFrac analysis
showed that the microbiota of anti-IL-22
–
treated mice increasingly
diverged from that of their isotype control treated littermates
during the 5 wk of treatment (Fig. 5
I
). Neutralization of IL-22
increased the abundance of Clostridiales throughout most of the
sensitization period, whereas the abundance of Bacteroidales
remained unchanged (
Fig. S4
L
). Taken together, these data
support our hypothesis that mucosa-associated Clostridia play a
critical role in regulating sensitization to food allergens.
Discussion
Dietary antigens are absorbed in the small intestine and carried
to the mesenteric lymph node by CD103
+
dendritic cells, ulti-
mately generating food antigen-specific Tregs that then migrate
to the small intestinal LP and expand to maintain tolerance to
dietary antigen (26). Our data suggest a new paradigm in
which both antigen-specific tol
erance and a bacteria-induced bar-
rier protective response are required to prevent sensitization to
food antigens. We identify an innate mechanism through which
a predominant component of th
e normal mucosa-associated
commensal microbiota regulates sensitization to food. Using
a sensitive capture ELISA to measure the concentration of two
immunodominant PN allergens in serum within hours after gavage,
we show that Clostridia-induced early innate IL-22 production by
ROR
γ
t
+
ILCs and T cells reduces access of allergen to the blood-
stream. Treatment of Abx-depleted
Clostridia-colonized mice with
neutralizing anti-IL-22 throughout the course of the PN/CT sensiti-
zation protocol induces enhanced production of IL-17 upon restim-
ulation in vitro, in agreement with a role for innate IL-22 in regulating
the adaptive Th17 response (25). PN-specific IgG responses increase
in anti-IL-22
–
treated mice but, without Th2 skewing, the IgE re-
sponse is unaltered. The composition of the microbiota was also
transformed by treatment with anti
-IL-22. The antimicrobial activity
of REG3
β
/
γ
is directed against Gram-positive bacteria (18). Clos-
tridia induce both
Il22
and
Reg3b/g
expression and stably colonize
gnotobiotic mic
e. In anti-IL-22
–
treated mice, however, increased
abundance of Clostridiales corre
lates with reduced expression of
Reg3b
, suggesting that this antimicrobial peptide titrates Clostridia
abundance in its colonic niche.
We also confirmed that the presence of a Clostridia-containing
microbiota is associated with the adaptive expansion of the in-
testinal Treg compartment and class switching to IgA (16, 17),
further reinforcing the immunoregulatory environment required to
maintain tolerance to dietary antigen. Indeed, IgA likely contributes
to immune exclusion to reduce allergen uptake; note the acceler-
ated kinetics with which Ara
h 6 and Ara h 2 reach the blood in
Rag
−
/
−
mice in comparison with WT mice
.
Increased bacteria-
induced luminal IgA and decreased sy
stemic allergen-specific Ig in
Clostridia-colonized mice may both be related to reduced systemic
allergen uptake. However, Clostridia
’
s early induction of IL-22 may
not be directly involved in the adaptive Treg and IgA phase of the
Clostridia-induced protective re
sponse, because treatment with an
IL-22Fc fusion protein does not resu
lt in an expansion of Tregs in the
colonic LP. Instead, recent work suggests that microbial metabolites
such as short chain fatty acids can
regulate the proportions and
functional capabilities of Foxp3
+
Tregs in the colonic LP (27
–
29).
Direct evidence for environment-induced dysbiosis in the in-
creasing prevalence of food allergy among children is just be-
ginning to emerge. Studies have tied urinary levels of the
commonly used antibacterial agent triclosan to food and aero-
allergen sensitization (30) and prepartal or neonatal Abx use to
cow
’
s milk allergy in infancy (31). Clostridia are enriched in the
colon of both mice and humans (14). Recent work has shown
that Clostridia strains isolated from healthy human feces po-
tently induce Tregs in the colonic LP upon transfer to GF mice
(17), suggesting our findings may be translatable to human dis-
ease. Oral and s.c. allergen-specific desensitization protocols are
already showing promise for treating food allergy (32). Our data
suggest that tolerance-inducing protocols could be effectively
paired with Clostridia enrichment of gut microbiota to potentiate
antigen-specific tolerance to prevent or treat food allergy.
Methods
Mice.
C57BL/6, C57BL/6Foxp3
gfp
,and
Rag
−
/
−
mice on an inbred C57BL/6
background (33) were maintained in an SPF facility at The University of
Chicago. Breeding pairs of GF C57BL/6 mice were initially provided by
S. Mazmanian. C57BL/6Foxp3
gfp
mice were rederived GF by K. McCoy. All
experiments were performed in accordance with the Institutional Biosafety
and Animal Care and Use Committees.
Neonatal Abx Treatment.
C57BL/6 or C57BL/6Foxp3
gfp
mice were treated with
a mixture of Abx, beginning at 2 wk of age, as previously described (12). For the
first week, mice were given a daily intragastric gavage with 100
μ
L of a mixture
of kanamycin (4 mg/mL), gentamicin (0.35 mg/mL), colistin (8500 U/mL), met-
ronidazole (2.15 mg/mL), and vancomycin (0.45 mg/mL) (Sigma-Aldrich; MP
Biomedicals). After weaning, the Abx were administered in the drinking water
at 50-fold dilution except for vancomycin, which was maintained at 0.5 mg/mL.
Preparation of 16S rRNA-Based Amplicon Library and Data Analysis.
PCR ampli-
cons of the V4 region of the 16S rRNA gene were sequenced on the Illumina
MiSeq platform and analyzed using QIIME as described in
SI Methods
.
Stefka et al.
PNAS
|
September 9, 2014
|
vol. 111
|
no. 36
|
13149
IMMUNOLOGY AND
INFLAMMATION
Purified PN Extract and Intragastric Sensitization.
Purified PN extract was
prepared from roasted, unsalted PN by a modification of van Wijk et al.,
which omitted high-speed centrifugation at 10,000
×
g
(34). PN/CT sensiti-
zation was performed as in ref. 12 and is described in
SI Methods
.
Ig Detection, Isolation of Lymphocytes, and Flow Cytometry.
Methods were
modified from refs. 12 and 33 and are described in
SI Methods
.
Microbial Isolation and Colonization of GF or Abx-Treated Mice.
B. uniformis
was isolated from SPF feces. Clostridia were isolated from SPF mice by chloro-
form treatment. Some experimental mice were colonized from live gnotobiotic
repository mice; one fecal pellet was homogenized in 1 mL sterile PBS, solids
were allowed to settle, and 100
μ
L of the liquid phase was administered by
gavage. A detailed description of bacterial colonization is given in
SI Methods
.
Quantitative Real-Time PCR.
RNA was prepared from freshly homogenized
intestinal tissue or isolated LP cells from the small intestine and colon of GF,
B. uniformis
, or Clostridia-colonized mice at 4 d after colonization,
Rag
−
/
−
Abx-
treated Clostridia-colonized mice with or without anti-CD90.2 treatment at
6 d after colonization, or sensitized WT Abx-treated Clostridia-colonized
mice with or without anti-IL-22 treatment at 24 h after challenge using the
RNeasy Mini Kit (Qiagen). cDNA was produced using the iScript cDNA syn-
thesis kit (BioRad), and quantitative real-time PCR was performed using the
iQ SYBR Green supermix (Bio-Rad) on the StepOnePlus system (Applied
Biosystems). Primer sequences for
Il22
,
Reg3b
,
Reg3g
, and
Hprt
are described
in ref. 35. Expression of target genes was normalized to
Hprt
.
Microarray Analysis.
IECs were isolated from colons of GF,
B. uniformis
-col-
onized, or Clostridia-colonized mice at 6 d after colonization by shaking
tissue fragments at 100 rpm for 20 min at 37 °C in 5 mM EDTA followed by
vigorous vortexing and Percoll gradient centrifugation. IECs from three mice
were pooled for each RNA sample in two to three independent experiments
per condition. RNA was isolated as above. Samples were run on a single
Illumina MouseRef-8 array at The University of Chicago Functional Genomics
Facility.
SI Methods
provides analysis detail.
In Vivo Antibody Treatment.
SPF mice were treated with Abx by gavage for
1 wk before weaning. At weaning, mice were either placed on Abx-containing
water or were colonized with Clostri
dia, as above. For exogenous IL-22
treatment, 20
μ
g of IL-22 fusion protein (IL-22-Fc, Genentech) was delivered i.p.
at weaning. For depletion of IL-22, 150
μ
g of neutralizing antibody to IL-22
(clone 8E11, Genentech) (22) or an isotype control (GP120 10E7.1D2, Gen-
entech) (36) was administered throughout the sensitization protocol by i.p.
injection three times per week as previously described (37, 38). To deplete ILCs,
250
μ
g of anti-CD90.2 (clone 30H12, BioXCell) or isotype control (LTF-2, Bio-
XCell) was administered i.p. every 3 d beginning 3 d before weaning, modified
from ref. 24. The requirement for Clostridia-induced IL-22 production for the
expansion of colonic Foxp3
+
Tregs was examined by i.p. injection of 500
μ
gof
clone IL-22JOP (eBioscience), as previously described (39).
Assessment of Allergen Uptake.
To assess allergen uptake into serum, mice were
bled before receiving 20 mg PN by gavage (
±
15
μ
gCT).Micewerebledagainat
indicated time points, and PN allergen concentration in serum was measured
with capture ELISAs for Ara h 2 or Ara h 6 (Indoor Biotechnologies).
Statistical Analysis.
Statistical analysis was performed using GraphPad Prism 5.
Normally distributed data were analyzed by one-way ANOVA with Tukey
posttest, two-way ANOVA with Bonferroni correction, or Student
t
test as
appropriate to the number of compa
risons to be made. Data that did not
exhibit a normal distribution were analyzed using the nonparametric
Kruskal-Wallis test with Dunn
’
s posttest.
ACKNOWLEDGMENTS.
We thank the staff of The University of Chicago
Gnotobiotic Research Animal Facility for their expert technical assistance;
T. Karrison (The University of Chicago Biostatistics Core) for advice on statistical
analysis; S. Chervonsky and other colleagues for critical review of the manuscript;
and W. Ouyang (Genentech) for providing neutralizing antibody to IL-22 (8E11),
its isotype control, and an IL-22-Fc fusion protein for this study. This work was
supported by Food Allergy Research and Education; a gift from the Bunning
family; US National Institutes of Health Grants AI106302 (to C.R.N.), DK078938
(to S.K.M.), AI089954 (to L.Z.), AI091962 (to L.Z.), and T32AI007090-33 (to T.F.); and
University of Chicago Digestive Diseases Research Core Center Grant DK42086.
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1124.
13150
|
www.pnas.org/cgi/doi/10.1073/pnas.1412008111
Stefka et al.