, 1317 (2012);
et al.Patrice Nancy
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drug (fig. S3). Fluconazole treatment during co-
litis led to reduced weight loss (Fig. 4A), and
milder histological disease characteristics (Fig.
4B), specifically in Clec7a−/−mice. We similarly
observed decreased TH1 and TH17 responses
(Fig. 4, C and D, and fig. S21, A and B) and
decreased production of inflammatory cytokines
(fig. S21, C and D). Taken together, these results
further support the conclusion that an inability to
control fungi in the gut leads to more severe co-
litis in Dectin-1 knockout mice.
Having established a role for Dectin-1 in
fungal control during colitis in mice, we next
explored whether there is an association between
inflammatory bowel disease (IBD) and genetic
Because the mouse model suggested that Dectin-1
is involved in the severity of colonic disease, we
focused our human studies on ulcerative colitis
(UC), a disease of the colon, and in particular
on severe UC. Up to 30% of patients with UC
require colectomy, usually for severe disease that
will not respond to medical therapy, including
systemic corticosteroids, cyclosporine, and bi-
ological therapies [that is, medically refractory
UC (MRUC)]. We compared CLEC7A alleles
in an MRUC group with those from a group of
patients with UC who had not required colectomy
(non-MRUC) (25).We identified an association
of CLEC7A single-nucleotide polymorphism
rs2078178 in patients with MRUC (logistic re-
gression, P = 0.007). Notably, a two-marker
haplotype, rs2078178 to rs16910631, was more
strongly associated with MRUC (AG haplotype;
logistic regression, P = 0.00013; and Fisher’s
test, P = 0.0005) (Fig. 4E and fig. S22 and table
severe UC (Fig. 4F). Compared with healthy
controls, the haplotype is strongly associated
with MRUC and not with non-MRUC, further
consistent with the idea that the haplotype is
has not been identified in any genome-wide as-
sociation study yet as an IBD susceptibility gene.
Unlike susceptibility genes that predispose to
disease, severity gene variants aggravate disease
that is initially established through other mech-
fits the latter situation and agrees with our obser-
vation that Clec7a−/−mice do not develop spon-
studies onthe roleofthe CLEC7Agene andpath-
way on the natural historyof UC and willrequire
further validation in independent cohorts.
A deeper understanding of the mechanisms
by which fungi stimulate inflammatory immune
responses in the gut may lead to better therapies
for IBD and may be especially beneficial to
patients with particularly severe forms of UC
carrying the risk haplotype of the gene for
Dectin-1. Overall, the idea that fungi are present
in the gut and that they interact strongly with the
immune system will fundamentally alter how we
think about the gut microflora and inflammatory
References and Notes
1. C. Lupp et al., Cell Host Microbe 2, 204 (2007).
2. B. P. Willing et al., Gastroenterology 139, 1844, e1 (2010).
3. E. Elinav et al., Cell 145, 745 (2011).
4. M. Arumugam et al., Nature 473, 174 (2011).
5. J. Henao-Mejia et al., Nature 482, 179 (2012).
6. M. Vijay-Kumar et al., Science 328, 228 (2010).
7. J. Qin et al., Nature 464, 59 (2010).
8. A. J. Scupham et al., Appl. Environ. Microbiol. 72, 793
9. S. J. Ott et al., Scand. J. Gastroenterol. 43, 831 (2008).
10. S. C. Cheng et al., J. Leukoc. Biol. 90, 357 (2011).
11. S. I. Gringhuis et al., Nat. Immunol. 13, 246 (2012).
13. H. R. Conti et al., J. Exp. Med. 206, 299 (2009).
14. B. Ferwerda et al., N. Engl. J. Med. 361, 1760 (2009).
15. E. O. Glocker et al., N. Engl. J. Med. 361, 1727 (2009).
16. P. R. Taylor et al., Nat. Immunol. 8, 31 (2007).
17. A. Franke et al., Nat. Genet. 42, 292 (2010).
18. D. P. McGovern et al., Nat. Genet. 42, 332 (2010).
19. C. H. Seow et al., Am. J. Gastroenterol. 104, 1426 (2009).
20. S. Joossens et al., Gastroenterology 122, 1242 (2002).
21. B. N. Gantner, R. M. Simmons, D. M. Underhill, EMBO J.
24, 1277 (2005).
22. M. A. Ghannoum et al., PLoS Pathog. 6, e1000713 (2010).
23. W. S. Garrett et al., Cell Host Microbe 8, 292 (2010).
24. S. Jawhara et al., J. Infect. Dis. 197, 972 (2008).
25. T. Haritunians et al., Inflamm. Bowel Dis. 16, 1830 (2010).
Acknowledgments: This study was supported in part by the
National Institute of Allergy and Infectious Diseases, NIH
(AI071116 to D.M.U.) and the Janis and William Wetsman
Family Chair in Inflammatory Bowel Disease Research
(D.M.U.). I.D.I. held a Research Fellowship Award (3064)
from the Crohn’s and Colitis Foundation of America. Further
support came from National Institute of Diabetes and
Digestive and Kidney Diseases, NIH, grant P01-DK046763;
UCLA Clinical and Translational Science Institute grant
UL1RR033176; Cedars-Sinai Medical Center Inflammatory
Bowel and Immunobiology Research Institute Funds; The
Feintech Family Chair in IBD (S. R. Targan); The Cedars-Sinai
Board of Governors’ Chair in Medical Genetics (J.I.R.);
The Abe and Claire Levine Chair in Pediatric IBD (M.D.); and
The Joshua L. and Lisa Z. Greer Chair in IBD Genetics (D.M.).
G.D.B. is supported by the Wellcome Trust. Clec7a−/−mice
are available through a Material Transfer Agreement with the
University of Aberdeen. The data presented in this paper are
tabulated in the main paper and in the supplementary
materials. All sequences generated in this study have been
deposited in the National Center for Biotechnology
Information, NIH, Short Read Archive (www.ncbi.nlm.nih.
gov/Traces/sra, accession no. SRA051853.1). G.D.B. is an
advisory board member of MiniVax and Fiberbiotics. M.D. is a
consultant for Prometheus Labs.
Materials and Methods
Figs. S1 to S22
Tables S1 and S2
12 March 2012; accepted 27 April 2012
Chemokine Gene Silencing in Decidual
Stromal Cells Limits T Cell Access to
the Maternal-Fetal Interface
Patrice Nancy,1Elisa Tagliani,1Chin-Siean Tay,1Patrik Asp,2*
David E. Levy,1,2Adrian Erlebacher1,2†
The chemokine-mediated recruitment of effector T cells to sites of inflammation is a central feature
of the immune response. The extent to which chemokine expression levels are limited by the
intrinsic developmental characteristics of a tissue has remained unexplored. We show in mice
that effector T cells cannot accumulate within the decidua, the specialized stromal tissue
encapsulating the fetus and placenta. Impaired accumulation was in part attributable to the
epigenetic silencing of key T cell–attracting inflammatory chemokine genes in decidual stromal
cells, as evidenced by promoter accrual of repressive histone marks. These findings give insight
into mechanisms of fetomaternal immune tolerance, as well as reveal the epigenetic
modification of tissue stromal cells as a modality for limiting effector T cell trafficking.
esides being essential for reproductive
success, the ability of the allogeneic fetus
and placenta to avoid rejection by the
maternal immune system during pregnancy (i.e.,
fetomaternal tolerance) has served as a paradigm
for the study of organ-specific immune tolerance
(1). Recent work on this problem has made use
of a mouse mating system in which wild-type
females are crossed with males hemizygous for
expressing a transmembrane form of the model
antigen chicken egg ovalbumin (OVA) from the
ubiquitously active b-actin promoter (3, 4). As
early as embryonic day 7.5 (E7.5), OVA is ex-
pressed at high levels by trophoblasts directly
contacting the uterus (i.e., at the maternal/fetal
interface) (3), thus exposing maternal tissue to a
surrogate fetal/placental antigen that should in
principle allow for T cell priming and render the
cytotoxic T lymphocytes (CTLs).
1Department of Pathology, New York University School of
Medicine, New York, NY 10016, USA.2New York University
Cancer Institute, New York University School of Medicine, New
York, NY 10016, USA.
Center, Albert Einstein College of Medicine, Bronx, NY 10467,
†To whom correspondence should be addressed. E-mail:
VOL 3368 JUNE 2012
on June 15, 2012
This mating system has been used to show
that fetal rejection is in part prevented by mech-
anisms that minimize the activation of naïve T
cells with fetal/placental specificity (3, 5). How-
when systemic antifetal/placental CTL activity is
a fail-safe mechanism also exists to protect the
conceptus from activated CTLs. To visualize this
phenomenon more directly, we asked whether
pregnant mice, immunized with soluble OVA be-
foremating,would showAct-mOVA–specific fe-
tal loss on E10.5 after being rechallenged with
OVA plus the adjuvant combination of agonistic
CD40 antibodies plus polyinosinic:polycytidylic
acid [poly(I:C)] on E5.5 (6). We studied this pe-
riod of early gestation because the behavior of
OVA-specific T cells would not be influenced
by the systemic release of fetal/placental OVA,
which starts on ~E10.5 (3). Strikingly, pregnant
mice bore the expected Mendelian proportion of
Act-mOVA+concepti (17 of 26 total embryos
from n = 3 pregnant mice), even though 14 to
the time the mice were killed (fig. S1). Thus, fetal
rejection does not occur even when memory T
cells with known fetal/placental specificity are re-
activated in early pregnancy.
To find possible explanations for this observa-
tion, we evaluated the distribution of reactivated
memory T cells in the uteri of C57BL/6-mated
mice. Consistent with the ability of effector T
cells to infiltrate peripheral tissues even in the
absence of a localized antigen source (7, 8), E8.5
mice rechallenged with OVA plus adjuvant on
E5.5 showed large numbers of CD3+T cells
distributed throughout the segments of myome-
trium (and associated submyometrial stroma)
overlying each implantation site (Fig. 1, A and
B), as well as throughout the myometrium and
endometrium of the undecidualized uterine seg-
ments between implantation sites (i.e., interim-
plantation sites) (Fig. 1, C and D). In contrast,
CD3+cells in the decidua appeared sparse (Fig.
1B), with tissue densities remaining at levels
similar to those seen throughout the uteri of mice
that were not OVA-rechallenged. CD3+cells in
the decidua were most prominent within blood
vessels; however, most were extravascular in the
implantation site–associated myometrium and
interimplantation sites (Fig. 1, E and F). Togeth-
er, these results suggested that the decidua had a
reduced capacity for T cell accumulation, possi-
bly due to an intrinsic inability to recruit T cells
from the blood. Accordingly, reactivated mem-
ory Tcells were also unable to infiltrate decidual
tissue encapsulating OVA-expressing concepti
(fig. S2) or in hormonally pseudopregnant fe-
males with oil-induced artificial deciduomas.
Because antibodies to CD40 plus poly(I:C)
induce a type 1–polarized Tcell response (9), we
investigated whether impaired decidual T cell
infiltration was due to low expression of T help-
er 1 (Th1)/T cytotoxic 1 (Tc1) chemoattractants.
antibodies to CD40, poly(I:C), and endotoxin-
blood levels of the proinflammatory cytokines
tumor necrosis factor-a (TNFa) and interferon-g
attracting chemokine CXCL9 (a CXCR3 ligand)
overlying each E8.5 implantation site (Fig. 2, A
and B). In contrast, much lower CXCL9 expres-
sion was apparent in the decidua. High CXCL9
expression was also induced in both the endo-
metrium and myometrium of the undecidualized
uteri of pseudopregnant females (fig. S3), with
the vast majority of the expressing cells being
CD45-stromal cells (Fig. 2C). Because endome-
trial stromal cells (ESCs) are the precursors of
of the decidua, these results suggested that the
the cells’ capacity to produce T cell chemo-
attractants under inflammatory conditions.
We next independently prepared highly en-
riched stromal cells from E7.5 artificial decid-
uomas and overlying myometrium (fig. S4) and
evaluated their inflammatory response in vitro
(Fig. 2D).Aswith othercell types (13,14),myo-
metrial stromal cells (MSCs) treated with a com-
bination of TNFa and IFN-g showed synergistic
mRNA induction of both Cxcl9 and Cxcl10,
which encodesa second CXCR3 ligand.Further-
also been implicated in Th1/Tc1 recruitment to
inflamed tissues (12), was up-regulated ~15-fold
in TNFa-treated MSCs, with expression further
transcript levels in DSCs remained unchanged
after cytokine treatment, whereas TNFa+IFN-g
mildly induced Cxcl10 expression to levels that
barely exceeded those of MSCs at baseline. The
expression pattern of Cxcl11, which encodes the
third known CXCR3 ligand, was similar to that
of Cxcl9 and Cxcl10 (fig. S5). The inability of
tionally confirmed using transwell migration
assays, which furthermore showed that MSCs
ligands and CCL5 (Fig. 2, E and F). Together,
these results suggested that the inability of DSCs
to produce T cell–attracting chemokines under
Fig. 1. The decidua resists infiltration by reactivated memory T cells. (A to D)
B6CBAF1/J (H-2b/k) females were given 3 × 105OVA-specific OT-I CD8+T cells
to maximize potential T cell accumulation and were immunized with OVA
protein 2 to 3 weeks before mating to C57BL/6 males. On E5.5, pregnant mice
either received no additional treatment [(A) and (C)] or were intravenously
injected with 0.5 mg OVA and adjuvant [CD40 antibodies plus poly(I:C)] [(B)
and (D)]. The mice were killed on E8.5, and tissue immunostaining using CD3-
specific antibodies (red) was performed on cross sections of implantation sites
[(A) and (B)] and interimplantation sites [(C) and (D)]. The two poles of the
decidua (mesometrial and antimesometrial) are indicated; 4´,6-diamidino-2-
phenylindole (DAPI) counterstain. (E and F) The decidual/myometrial border (E)
and undecidualized endometrium (F) of a rechallenged mouse. Blood vessels
(arrowheads) are identified by the presence of red blood cells, which appear
green as an artifact of the immunostaining protocol. myo, myometrium; dec,
decidua; asterisk, uterine lumen. Data are representative of three independent
experiments (at least n = 23 implantation sites each per group).
8 JUNE 2012VOL 336
on June 15, 2012
inflammatory conditions in vivo was due to a
cell-intrinsic defect in their inflammatory cyto-
The inability of DSCs to produce CXCR3
ligands and CCL5 was not explained by de-
creased activation of NF-kB or STAT1, the ma-
jor transcription factors mediating TNFa and
IFN-g signaling (fig. S6, A and B). Moreover,
we could find examples of NF-kB and STAT1
target genes that were induced in DSCs in a
relatively robust fashion (fig. S6C). These results
suggested that the DSC chemokine expression
defects were gene-specific and independent of
inflammatory signaling per se. We therefore
using chromatin immunoprecipitation (ChIP)
assays. Elevated basal levels of the repressive
histone H3 trimethyl lysine 27 (H3K27me3)
mark (15) were present on the Cxcl9 and Cxcl10
promoters in DSCs as compared to MSCs (Fig.
3A, top row). Conversely, TNFa+IFN-g treat-
ment increased Cxcl9/10 promoter levels of
acetylated histone H4 (H4Ac), a mark of active
gene transcription, in MSCs but not DSCs (Fig.
3A, bottom row). Both cell populations showed
the inverse patterns of H3K27me3 and H4Ac
occupancies on the Gapdh and Cd8a promoters
expectedfromthese genes’ respectively high and
low constitutive expression levels. Thus, low
cytokine inducibility of Cxcl9/10 in DSCs was
associated with the gene-specific presence of
the repressive H3K27me3 histone mark.
In vivo ChIP assays performed directly on
dissected E7.5 uterine tissue layers also revealed
high levels of the H3K27me3 mark on the
Cxcl9/10 promoters in whole decidua as com-
pared with overlying myometrium, thus demon-
of true, pregnancy-associated decidua in vivo
(Fig. 3B). Recognizing that undecidualized uteri
are comprised equally in volume by endometri-
um and myometrium (5), this result also meant
that in vivo ChIP assays could be used to infer
chromatin configurations in undecidualized
ly purified for ex vivo assays. Accordingly,
Cxcl9/10 H3K27me3 promoter occupancies in
whole nonpregnant uteri and E7.5 interimplanta-
tion sites were similar to those in segments of
implantation site–associated myometrium and
clearly not intermediate between the myome-
trium and decidua (Fig. 3B). This result strongly
suggested thatthe H3K27me3 modification of the
Cxcl9/10 promoters appears upon transformation
of ESCs into DSCs.
The Ccl5 promoter also showed increased
pared with myometrium and undecidualized
minimizing decidual chemokine expression. In-
terestingly, this increase was not readily appar-
ent ex vivo, while H3K27me3 levels on the
Cxcl9/10 promoters also appeared somewhat
reduced upon TNFa+IFN-g treatment (Fig. 3A).
These results suggest some level of reversibility
of the H3K27me3 mark at the locations we as-
sessed by ChIP, possibly as a result of just isolat-
ing and culturing the cells. Because H4Ac levels
on the Cxcl9/10 and Ccl5 promoters were none-
theless unchanged in TNFa+IFN-g–treated DSCs
(Fig. 3A), it is likely that the continued repressed
status of these genes after 24 hours culture also
involves either the presence of the H3K27me3
mark in other regions of their respective loci or
the presence of other repressive modifications.
We next determined the effect of ectopic
chemokine expression within the decidua by in-
jecting artificial deciduomas inOVA-rechallenged
mice with control, Cxcl9, or Ccl5-expressing
lentiviruses mixed with enhanced green fluores-
mouse, decidual CD3+Tcell densities were nor-
malized to myometrial CD3+cell densities to ac-
count for systemic differences in the magnitude
of the anti-OVAT cell response. CD3+cell den-
injected mice were elevated as compared to
nonspecific effect of viral infection per se. How-
ever, these densities were not further altered in
mice injected with Cxcl9- or Ccl5-expressing vi-
ruses. In contrast, CD3+cell densities in decidual
areas infected with mixtures of Cxcl9- and Ccl5-
decidual areas was undetectable by tissue im-
munostaining (fig. S7), it was unlikely that Tcell
to β-actin (log10)
Fig. 2. The decidua produces low levels of Th1/Tc1–attracting chemokines in response to inflammation.
(A to C) E8.5 pregnant [(A) and (B)] or pseudopregnant (C) B6CBAF1/J mice were either untreated (A) or
injected with CD40 antibodies, poly(I:C), and OVA [(B) and (C)] 6 hours before they were killed. [(A) and
(B)] CXCL9 immunostaining (red) of implantation site cross sections. DAPI counterstain. (C) Double
CXCL9 (green)/CD45 (red) immunostaining of the endometrium of an inflamed pseudopregnant uterus.
CXCL9 immunoreactivity appears largely confined to the endoplasmic reticulum, giving a punctate
appearance. Data are representative of at least three independent experiments. (D) Quantitative real-
time fluorescence polymerase chain reaction analysis of cultured MSCs and DSCs. Cytokines were added
for the last 6 hours of a 24-hour total culture period. Data show mean T SEM of four independent
experiments. n.s., not significant. (E) Migration of in vitro differentiated Th1 cells to supernatants
collected from MSCs or DSCs treated as indicated over the entirety of a 24-hour culture period. Data
show mean T SEM of three independent experiments. (F) Effect of CXCR3 desensitization (via pre-
incubation of the Th1 cells with CXCL9) or CCL5 neutralization on Th1 cell migration to supernatants
from TNFa+IFN-g–treated MSCs. Nonspecific rat immunoglobulin G (IgG) antibodies served as the
control for CCL5 antibodies. Data show mean T SEM of three independent experiments.
VOL 336 8 JUNE 2012
on June 15, 2012
infiltration into these areas was due to supraphys-
iologic chemokine expression. Together, these
results suggested that inadequate endogenous
expression of CXCR3 ligands and CCL5 was
limiting for decidual Tcell accumulation.
Provocatively, T cells have been reported
to be relatively scarce in the human decidua
(17, 18), implicating the developmental program
of decidual chemokine silencing described here
as a potentially conserved mechanism of fetoma-
ternal tolerance. Consistent with this possibility,
Cxcl10 is expressed only focally in the human
decidua, in association with periglandular leuko-
numbers of activated Tcells at the maternal/fetal
No cytokine, IgG ChIP
TNFα+IFNγ, IgG ChIP
No cytokine, H4ac ChIP
TNFα+IFNγ, H4ac ChIP
No cytokine, IgG ChIP
TNFα+IFNγ, IgG ChIP
No cytokine, H3K27me3 ChIP
TNFα+IFNγ, H3K27me3 ChIP
H3K27me3IgGH3K27me3 IgG H3K27me3IgG H3K27me3IgG
% of input
% of input
Fig. 3. Chromatin configurations in uterine cells and tissue layers. (A) Ex
vivo ChIP assays performed on cultured MSCs and DSCs. Cytokines were
added for the last 6 hours of a 24-hour total culture period. Data show
on dissected uterine tissues and tissue layers. Deciduas were dissected free of
embryos. Data show mean T SD of three independent experiments.
(relative to myometrium)
Cxcl9 Ccl5 Cxcl9
CD3+ cell density
Fig.4.Effect of ectopic chemokine expression on effector T cell accumulation within the decidua. Mice were immunized
with OVA 2 to 3 weeks before mating, rechallenged with OVA plus adjuvants on E4.5, injected with lentivirus on E5.5,
and killed on E7.5. Viral preparations included samples of EGFP reporter lentiviruses so that T cells within transduced
areas could be identified on anti-CD3 and anti-GFP immunostained serial sections. (A) CD3+cell densities in infected
(GFP+) and uninfected (GFP–) decidual areas relative to myometrial CD3+cell densities. Data show mean T SEM of at
least three independent experiments encompassing n = 4 control (empty vector) virus–infected mice (75 × 107virus
particles each); n = 5 Cxcl9+Ccl5 virus–infected mice (37.5 × 107particles each); and n = 3 each of mice infected with
Cxcl9- or Ccl5-expressing viruses alone (75 × 107particles each). Myometrial CD3+cell densities were not significantly
different between the four groups (average: 0.037 cells per mm2). (B to K) GFP or CD3 immunostaining (as indicated,
red) and DAPI counterstain (blue) of representative serial sections of decidualized uteri infected with Cxcl9+Ccl5 viruses
[(B) and (C) and (H) to (K)] or control viruses [(D) to (G)]. [(B) and (C)] Asterisks denote different areas of the decidual
lumen, which frequently contained GFP+cells, CD3+cells, and granulocytes scattered among necrotic debris; the
dashed line indicates the border between the myometrium and decidua. [(D) to (K)] The dashed lines show the
perimeters of infected areas used to calculate CD3+cell densities. Asterisks show areas excluded from analysis because
of their necrotic appearance. Arrow, decidual lumen. (H) to (K) are close-ups of (B) and (C).
8 JUNE 2012VOL 336
on June 15, 2012
interface might disturb placental development or Download full-text
Conversely, altered chemokine silencing may in-
that genes encoding Th1/Tc1–attracting chemo-
stromal cells and that such regulation can signif-
icantly influence a tissue’s capacity for Tcell ac-
cumulation. This demonstration raises questions
mark is targeted to select chemokine genes and
whether related pathways control Tcell access to
References and Notes
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Acknowledgments: We thank S. K. Dey and K. Johnson
for advice, and A. Frey and J. Ernst for comments on the
manuscript. The Histopathology and Vaccine and Cell
Therapy core facilities of the New York University Cancer
Institute provided histology services and tetramer reagents
and were supported by NIH, National Cancer Institute
(P30CA016087). P.N., E.T., and C.-S. T. performed
experiments; P.A. and D.E.L. provided critical expertise and
reagents; P.N. and A.E. analyzed data; and P.N. and
A.E. designed experiments and wrote the manuscript.
The data reported in the manuscript are tabulated in the
main paper and in the supplementary materials. This work
was supported by grants from NIH (RO1AI062980) and the
American Cancer Society to A.E. (RSG-10-158-01-LIB).
Materials and Methods
Figs. S1 to S7
3 February 2012; accepted 24 April 2012
Innate Lymphoid Cells Promote
Anatomical Containment of
Lymphoid-Resident Commensal Bacteria
Gregory F. Sonnenberg,1Laurel A. Monticelli,1Theresa Alenghat,1Thomas C. Fung,1
Natalie A. Hutnick,2Jun Kunisawa,3,4Naoko Shibata,3,4Stephanie Grunberg,1Rohini Sinha,1
Adam M. Zahm,5Mélanie R. Tardif,6Taheri Sathaliyawala,7Masaru Kubota,7Donna L. Farber,7
Ronald G. Collman,8Abraham Shaked,9Lynette A. Fouser,10David B. Weiner,2
Philippe A. Tessier,6Joshua R. Friedman,5Hiroshi Kiyono,3,4,11Frederic D. Bushman,1
Kyong-Mi Chang,8,12David Artis1,13*
The mammalian intestinal tract is colonized by trillions of beneficial commensal bacteria that
are anatomically restricted to specific niches. However, the mechanisms that regulate anatomical
containment remain unclear. Here, we show that interleukin-22 (IL-22)–producing innate
lymphoid cells (ILCs) are present in intestinal tissues of healthy mammals. Depletion of ILCs
resulted in peripheral dissemination of commensal bacteria and systemic inflammation, which
was prevented by administration of IL-22. Disseminating bacteria were identified as Alcaligenes
species originating from host lymphoid tissues. Alcaligenes was sufficient to promote systemic
inflammation after ILC depletion in mice, and Alcaligenes-specific systemic immune responses
were associated with Crohn’s disease and progressive hepatitis C virus infection in patients.
Collectively, these data indicate that ILCs regulate selective containment of lymphoid-resident
bacteria to prevent systemic inflammation associated with chronic diseases.
sal bacteria are anatomically restricted to either
the intestinal lumen, the epithelial surface, or
within the underlying gut-associated lymphoid
tissues (GALTs) (1–5). Anatomical containment
is essential to limit inflammation and maintain
normal systemic immune cell homeostasis (1, 2).
Loss of containment and subsequent dissemina-
tion of commensal bacteria to peripheral organs
promotes inflammation and is a hallmark of mul-
tiple chronic human infectious and inflammatory
atitis virus infection, and inflammatory bowel
olonization of the mammalian gastroin-
testinal tract by commensal bacteria is
essential for promoting normal intestinal
ways that promote anatomical containment of
mation may provide targets for treatment and
prevention of chronic human diseases.
Studies in murine models identified a critical
role for the cytokine interleukin-22 (IL-22) in
regulating intestinal immunity, inflammation, and
tissue repair (11, 12). CD4+T cells and innate
however, whether T cell– or ILC-derived IL-22
contributes to the anatomical containment of com-
mensal bacteria and prevention of systemic in-
flammation in the steady state has not been
investigated. To address this issue, we sought to
identify the IL-23–responsive cell populations
of healthy human donors (see supplementary
human donors that lacked expression of lineage
markers CD20, CD56, and CD3 (Fig. 1A) and
RORgt+(Fig. 1B), a phenotype consistent with
ILCs in humans (11, 14). IL-22+cells in the
mesenteric lymphnode (mLN) of healthy human
donors also exhibited an ILC phenotype (fig. S1,
A and B). Examination of tissues from healthy
nonhuman primates revealed an analogous pop-
ulation of IL-22+cells that exhibited an ILC phe-
notype in rectal tissues (Fig. 1, C and D) and
inguinal LNs (fig. S1, C and D). A population of
1Department of Microbiology and Institute for Immunology,
Perelman School of Medicine,University of Pennsylvania,Phil-
adelphia, PA19104,USA.2Department of Pathology and Lab-
oratory Medicine, Perelman School of Medicine, University of
Pennsylvania, Philadelphia, PA 19104, USA.3Division of Mu-
cosal Immunology, Institute of Medical Science, The University
of Tokyo, Tokyo 108-8639, Japan.4Department of Medical Ge-
nome Science, Graduate School of Frontier Science, The
University of Tokyo, Chiba 277-8562, Japan.5Department of
Pediatrics, Division of Gastroenterology, Hepatology, and Nu-
trition, Perelman School of Medicine, University of Pennsylva-
nia, Children’s Hospital of Philadelphia, Philadelphia, PA
19104, USA.6Centre de Recherche en Infectiologie, Centre
Hospitalier de l’Université Laval, Faculty of Medicine, Laval
University, Quebec, Canada.7Department of Surgery and the
versity Medical Center, New York, NY 10032, USA.8Depart-
ment of Medicine, Perelman School of Medicine, University of
Pennsylvania, Philadelphia, PA 19104, USA.9Department of
Surgery, University of Pennsylvania, Philadelphia, PA 19104,
apeutics Research and Development, Pfizer Worldwide R&D,
Cambridge, MA 02140, USA.11Core Research for Evolutional
Science and Technology, Japan Science and Technology Agen-
cy, Tokyo 102-0075, Japan.12Philadelphia VA Medical Center,
Philadelphia, PA 19104, USA.13Department of Pathobiology,
School of Veterinary Medicine, University of Pennsylvania, Phil-
adelphia, PA 19104, USA.
*To whom correspondence should be addressed. E-mail:
VOL 336 8 JUNE 2012
on June 15, 2012