Nod/Ripk2 signaling in dendritic cells activates

IL-17Asecreting innate lymphoid cells and drives

colitis in T-bet
/
.Rag2
/
(TRUC) mice
Joerg Ermann
a,b
, Tracy Staton
c
, Jonathan N. Glickman
d,e
, Rene de Waal Malefyt
f
, and Laurie H. Glimcher
g,1
a
Division of Rheumatology, Immunology, and Allergy, Brigham and Womens Hospital, Boston, MA 02115;
b
Harvard Medical School, Boston, MA 02115;
c
Department of Immunology and Infectious Diseases, Harvard School of Public Health, Boston, MA 02115;
d
Department of Pathology, Harvard Medical School,
Boston, MA 02115;
e
Miraca Life Sciences, Newton, MA 02464;
f
Biologics Discovery, Merck Research Laboratories, Palo Alto, CA 94304; and
g
Weill Cornell
Medical College, New York, NY 10065
Contributed by Laurie H. Glimcher, May 15, 2014 (sent for review April 12, 2014)
T-bet
/
.Rag2
/
(TRUC) mice spontaneously develop microbiota-
driven, TNF-mediated large bowel inflammation that resembles
human ulcerative colitis. We show here that IL-23 and IL-1depen-
dent secretion of IL-17A by innate lymphoid cells (ILCs; defined
as CD45
+
lin

Thy1
hi
NKp46

) is a second critical pathway in this

model. Using an in vitro coculture system of bone marrow-derived
dendritic cells (DCs) and freshly isolated FACS-purified ILCs, we
demonstrate that IL-23 and IL-1 secreted by DCs in response to
microbial stimulation work together to induce IL-17A production
by ILCs. TNF is not required for IL-17A secretion by ILCs in vitro but
synergizes with IL-17A to induce the expression of neutrophil-
attracting chemokines. Upstream, activation of the IL-23/IL-17A
axis is regulated by nucleotide-binding oligomerization domain
containing (Nod)/receptor-interacting serine-threonine kinase 2
(Ripk2) signals in DCs. Genetic ablation of the Nod/Ripk2 signaling
pathway protects TRUC mice from developing colitis without af-
fecting the colitogenicity of the intestinal microbiota. Our data
provide insight into the complex network of interactions between
IL-17Asecreting ILCs and other components of the innate immune
system in the development of colitis.
T
he spontaneous colitis in T-bet
/
.Rag2
/
(TRUC) mice
resembles in many respects human ulcerative colitis. In-
flammation is predominantly distal, limited to the mucosa/
submucosa and associated with a high incidence of epithelial
dysplasia and colorectal carcinoma. TRUC colitis responds to
treatment with anti-TNF (1). The disease is driven by intestinal
microbiota and can be prevented by oral treatment with antibiotics
targeting Gram-negative bacteria (2).
The transcription factor T-bet controls the differentiation and
function of CD4
+
effector T cells, cytotoxic T cells, and several
types of innate immune cells (3). T-betdeficient dendritic cells
(DCs) are an important cellular element in the pathogenesis of
TRUC colitis. CD11c
+
lamina propria DCs are the main source of
TNF in the inflamed TRUC colon, and depletion of CD11c
+
cells
abrogates colitis (4). T-bet negatively regulates the expression
of TNF in DCs by binding to the TNF promoter and inhibiting
nuclear factor kappa B (NFB)-dependent TNF transcription (1).
T-bet is also expressed in subsets of RAR-related orphan receptor
(ROR)t
+
innate lymphoid cells (ILCs) shown to mediate disease
in innate immune-driven murine colitis models including TRUC
(5, 6). ILCs have been identified in intestinal biopsies from
patients with inflammatory bowel disease (IBD) and likely par-
ticipate in the pathogenesis of human IBD (7, 8). The signals
controlling ILC function and the cellular interactions of ILCs
with other immune cells are incompletely understood.
Intestinal epithelial and immune cells sense intestinal bacte-
ria through a variety of pattern recognition receptors (PRRs),
including cytosolic nucleotide-binding oligomerization domain
containing (Nod)-like receptors. NOD2 mutations are strongly
associated with Crohns disease (9, 10). A weaker and less con-
sistent association between a NOD1 insertion/deletion variant
and IBD has been described (11). Nod2 recognizes the ubiqui-
tous bacterial cell wall component muramyl dipeptide (MDP) (12),
whereas Nod1 detects -D-glutamyl-meso-diaminopimelic acid
(iE-DAP) present in Gram-negative bacteria (13). Ligand bind-
ing activates a signaling cascade that involves the kinase re-
ceptor-interacting serine-threonine kinase 2 (Ripk2) resulting in
NFB target gene induction (14). Nod/Ripk2 activation has also
been linked to the induction of autophagy (15, 16), and Nod1
signaling is required for the formation of isolated lymphoid
follicles in the small intestine (17). Mice with genetic disruption
of the Nod/Ripk2 signaling pathway have a dysbiotic intestinal
flora resulting in altered susceptibility to intestinal inflammation
(1719). Whether ILCs can sense bacteria directly through the
Nod/Ripk2 pathway has not been studied.
We report here that CD45
+
lin

Thy1
hi
NKp46

ILCs secreting
IL-17A in response to combined stimulation with IL-23 and IL-1
are critical for colitis development in TRUC mice. Inhibition of
IL-23 or IL-1 signals, antibody-mediated ILC depletion, or
blockade of IL-17A all abrogate disease. We demonstrate that
IL-17A and TNF synergistically induce the expression of neu-
trophil-attracting chemokines, and that neutrophil depletion,
like antibody-mediated inhibition of TNF or IL-17A, abrogates
colitis. Furthermore, we link ILC activation to proximal Nod/
Ripk2 signals. TRUC mice with genetic ablation of the Nod/
Ripk2 signaling pathway do not develop colitis despite having
a colitogenic intestinal flora. Using an in vitro coculture system,
we show that IL-17A induction in ILCs is controlled indirectly
by activation of the Nod/Ripk2 bacterial sensor in DCs.
Significance
Disease mechanisms in inflammatory bowel disease (IBD) are
incompletely understood. In this study, we analyzed the role of
IL-17Asecreting innate lymphoid cells (ILCs) in a mouse model
of microbiota-driven innate immune-mediated colitis. We re-
port that the pathogenic IL-17A response in ILCs is controlled
indirectly by microbial stimulation of dendritic cells (DCs) via
the nucleotide-binding oligomerization domain containing (Nod)/
receptor-interacting serine-threonine kinase 2 (Ripk2) signaling
pathway and requires the cytokines IL-23 and IL-1. Insight into
the complex interactions between various immune cells as dem-
onstrated here for DCs and ILCs is a prerequisite for the de-
velopment of more efficacious therapies in IBD.
Author contributions: J.E., T.S., and L.H.G. designed research; J.E., T.S., and J.N.G. per-
formed research; R.D.W.M. contributed new reagents/analytic tools; J.E., T.S., and L.H.G.
analyzed data; and J.E., T.S., and L.H.G. wrote the paper.
Conflict of interest statement: L.H.G. holds equity in and sits on the Corporate Board of
Directors of the Bristol-Myers Squibb Pharmaceutical Company.
1
To whom correspondence should be addressed. E-mail: lglimche@med.cornell.edu.
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.
1073/pnas.1408540111/-/DCSupplemental.
www.pnas.org/cgi/doi/10.1073/pnas.1408540111 PNAS | Published online June 9, 2014 | E2559E2566
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Results
Inhibition of IL-23R Signaling Abrogates TRUC Disease. Multiple
single nucleotide polymorphisms in the IL-23 signaling pathway
are associated with the development of IBD (2022). A role for
IL-23 in the pathogenesis of IBD is further supported by findings
in a variety of IBD mouse models. To study the role of IL-23 and
IL-23 receptor expressing cells in the TRUC colitis model, we
crossed an IL-23R
GFP
allele (23) into the C57BL/6 TRUC line.
This IL-23R
GFP
allele functions as a reporter in heterozygous
mice and as a knockout (KO) in homozygous animals. We found
that TRUC.IL-23R
GFP/GFP
mice were protected from developing
colitis compared with TRUC.IL-23
GFP/+
control mice at 8 wk of
age (Fig. 1A; mean colitis score 0.5 vs. 1.7, P = 0.0183). We
confirmed this finding in the BALB/c TRUC strain that develops
more severe colitis (24) by antibody-mediated blockade of IL-23
signaling. Compared with recipients of an isotype control, ani-
mals treated from weaning with antibodies against either the
IL-23p19 subunit or IL-23R were protected from disease de-
velopment (Fig. 1B; mean colitis score 0.4 and 0.1 vs. 3.6 in
controls, P < 0.0001). Using the TRUC.IL-23R
GFP/+
mice as
reporters (Fig. 1C), we determined that the majority of cells in
the CD45
+
GFP
+
gate were CD11b

and CD11c

, representing
lineage-negative cells in Rag2-deficient TRUC mice. Most of
these cells were Thy1
hi
Sca1
hi
, which corresponds to the surface
phenotype of the RORt-positive ILC population that mediates
colitis in the Helicobacter hepaticus model and secretes IL-17A in
response to IL-23 stimulation in vitro (5).
IL-17A Secreting Thy1-Positive Cells Mediate TRUC Colitis. To explore
the role of these ILCs and of IL-17A in TRUC disease, we de-
pleted Thy1-positive cells in BALB/c TRUC mice. Administra-
tion of a depleting anti-Thy1 antibody from weaning abrogated
colitis in 8-wk-old animals (Fig. 1D; mean colitis score 0.6 vs. 3.9,
P < 0.0001). Treatment with a blocking antiIL-17A antibody
was equally protective (Fig. 1E; mean colitis score 0.9 vs. 3.4, P <
0.0001). Together, these results suggest that an IL-17Asecreting
Thy1-positive cell population is pathogenic in TRUC mice. We
then stimulated bulk colon lamina propria (LP) cells with
recombinant IL-23 overnight and stained for intracellular IL-17A.
Within the CD45
+
gate, we identified a sizable population of
Thy1
hi
IL-17A
+
cells that included most of the IL-17Aexpress-
ing cells (Fig. S1). Additional stains characterized these cells as
CD11b

CD11c

(lin

) as well as Sca1
hi
and NKp46

, which is
the surface phenotype of ILC3 cells according to a recent con-
sensus paper (25). These data demonstrate that IL-23 and an IL-
23Rpositive ILC subset secreting IL-17A play central roles in
the pathogenesis of TRUC colitis.
DC-Derived IL-23 and IL-1 Trigger IL-17A Secretion by ILCs. To further
characterize the signals leading to IL-17A secretion by ILCs, we
set up an in vitro coculture system modeled after a described
T-cell assay (26). DCs were generated in vitro by culturing bone
marrow (BM) cells with GM-CSF for 6 d followed by positive
selection of CD11c
+
cells by using magnetic beads. ILCs (defined
as CD45
+
lin

Thy1
hi
NKp46

) were FACS sorted from mesen-

teric lymph nodes (mLN) and colon LP. DCs and ILCs were
stimulated alone or in coculture for 24 h, and cytokine levels in
the supernatant (SN) were quantified by ELISA.
Peptidoglycan (PGN) stimulation of cocultured ILCs and DCs,
but not ILCs or DCs cultured alone, resulted in secretion of
IL-17A, whereas TNF production required the presence of DCs
and was unaffected by ILCs (Fig. 2A). We confirmed ILCs as the
source of IL-17A by intracellular staining after coculture of DCs
and ILCs (Fig. S2A). A series of additional control experiments
was performed: First, we demonstrated that the IL-17A responses
of ILCs isolated from mLN and colon LP were equivalent (Fig.
S2B), allowing us to pool cells from these two locations for in
vitro experiments. Second, we sorted CD45
+
lin

cells into the

three subsets Thy1
hi
NKp46

(ILCs), Thy1
hi
NKp46
+
, and Thy1
lo
.
C A
isotype a-p19 a-IL-23R
0
3
6
9
h
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s
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p<0.0001
p<0.0001
B
isotype anti-Thy1
0
3
6
9
h
i
s
t
o
p
a
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o
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s
c
o
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p<0.0001
isotype anti-IL-17A
0
3
6
9
h
i
s
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o
p
a
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o
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s
c
o
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p<0.0001 E
D
IL-23R+/ IL-23R/
0
3
6
9
h
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s
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p=0.0183
GFP
S
S
C
Thy1
S
c
a
-
1
CD11c
C
D
1
1
b
7.7
81
89.7
GFP
neg
GFP
pos
Fig. 1. TRUC disease is mediated by IL-23Rpositive IL-17Asecreting ILCs. (A) TRUC.IL-23R
GFP/+
(IL-23R+/) and TRUC.IL-23R
GFP/GFP
(IL-23R/) mice (n = 30)
were killed at 8 wk of age. Standard H&E sections of the colon were scored in a blinded fashion. Histopathology scores for individual mice are shown with
horizontal lines representing the group mean. (B) BALB/c TRUC mice were treated from weaning with weekly i.p. injections of isotype control or antibodies
blocking IL-23p19 (a-p19) or the IL-23 receptor (a-IL-23R). Histopathology was analyzed at 8 wk of age. Results were pooled from two experiments with n =
910 mice per group. (C) IL-23R expression on colon LP cells was analyzed by FACS in an 8-wk-old TRUC.IL-23R
GFP/+
reporter mouse. The majority of GFP
+
cells
were Thy1
hi
Sca1
hi
and lineage-negative (CD11b

CD11c

). The Inset in the FACS plot on the right shows cells in the GFP

gate for comparison. The experiment

was performed three times with identical results. BALB/c TRUC mice were treated from weaning with weekly i.p. injections of a depleting anti-Thy1 antibody
or isotype control (D) or a neutralizing antibody against IL-17A or isotype control (E). Histopathology was analyzed at 8 wk of age. Results were pooled from
two experiments with n = 10 mice per group.
E2560 | www.pnas.org/cgi/doi/10.1073/pnas.1408540111 Ermann et al.
Upon stimulation with PGN in the presence of BM-DCs, only the
Thy1
hi
NKp46

but not the other two subsets produced significant

amounts of IL-17A (Fig. S2C). Third, we isolated CD11c
+
cells
from the spleen (spleen DCs) and CD11b
+
/CD11c
+
bead-
enriched cells from the colon LP (LP DCs) of TRUC mice and
cocultured them with ILCs in the presence of PGN. Data in Fig.
S2D demonstrate that primary DCs could drive IL-17A in this
system, suggesting that the in vitro coculture assay of BM-DCs
and ILCs correctly models a cellcell interaction occurring in vivo.
We then asked which factors were needed to induce IL-17A in
ILCs. Direct cellcell contact between DCs and ILCs did not
appear to be important, because supernatant from PGN-stimu-
lated DCs was sufficient to induce IL-17A secretion by ILCs
(Fig. S2E). As expected, the addition of a blocking antiIL-23R
antibody abrogated the IL-17A response in cocultured DCs and
ILCs. An antibody blocking IL-1 receptor (IL-1R1) signals
similarly prevented IL-17A induction, whereas inhibition of TNF
using blocking antibodies against TNF or the TNF receptor
TNFR1 had no effect (Fig. 2B). We also took the reciprocal
approach and stimulated ILCs with recombinant IL-23, IL-1,
and TNF either alone or in combination (Fig. 2C). Only the
combination of IL-23 and IL-1 induced IL-17A, demonstrating
that a combination of IL-23 and IL-1 signals is necessary and
sufficient to induce IL-17A secretion in freshly isolated highly
purified ILCs.
In apparent conflict with these results, IL-17A is readily in-
duced in ILCs when bulk cultures of mLN or colon LP cells are
stimulated with IL-23 alone (5, 27) (Fig. S1) and a costimulatory
effect of TNF on IL-17A secretion has been reported under
these conditions (6). We thus stimulated mLN cells with IL-23,
TNF, or IL-23 + TNF in the absence or presence of a blocking
antiIL-1R1 antibody and measured IL-17A in the SN (Fig. 2D).
Both IL-23induced IL-17A production and the synergistic effect
of TNF on IL-17A production disappeared in the presence of
a blocking antiIL-1R1 antibody demonstrating, that in bulk
mLN culture, non-ILCs provide the IL-1 signal required for
IL-17A secretion by ILCs.
Given the importance of IL-1 signals for IL-17A secretion by
ILCs in vitro, we would expect that blockade of IL-1 signals in
TRUC mice in vivo protects the mice from developing colitis. We
therefore generated TRUC.IL1R1
/
mice by crossing an IL1R1
KO allele (28) into the C57BL/6 TRUC line. Confirming our
hypothesis, colitis severity at 8 wk of age was significantly reduced
in TRUC.IL1R1
/
mice compared with TRUC.IL1R1
+/+
litter-
mates (Fig. 2E, mean colitis score 1.7 vs. 4.3, P = 0.0072). Taken
together, these data demonstrate that colitogenic ILCs require
a combination of IL-23 and IL-1 receptor signals for the in-
duction of IL-17A secretion similar to what has been reported for
T cells (29) and invariant NKT cells (30).
T-Bet Deficiency in ILCs Enhances IL-17A Response. We analyzed the
presence of ILCs in the colon LP of BALB/c TRUC and Rag2
/
mice at 8 wk of age (Fig. S3A; mean colitis score 3.8. vs. 0.3, P <
0.0001) (1). The absolute number of CD45
+
lin

Thy1
hi
NKp46

ILCs was significantly higher in the LP of TRUC mice compared

with Rag2
/
controls (Fig. S3B; 28.7 10
3
vs. 19.3 10
3
cells per
colon, P = 0.0096), reflecting an overall increase in cellularity
in the inflamed TRUC colon, because their relative frequency
among CD45
+
cells was not different between TRUC and Rag2
/
mice. We then cocultured ILCs from 8-wk-old TRUC or Rag2
/
mice with BM-DCs from TRUC or Rag2
/
mice to compare their
function. Upon stimulation with PGN, TRUC ILCs secreted sig-
nificantly more IL-17A regardless of the origin of the DCs (Fig.
S3C). The same enhanced IL-17A response in TRUC versus
Rag2
/
ILCs was seen when ILCs were stimulated with recombi-
nant IL-23 + IL-1 in the absence of accessory cells (Fig. S3D).
In this study, we define the pathogenic ILC population by
their surface marker phenotype as CD45
+
lin

Thy1
hi
NKp46

. This
population is itself heterogenous, and less than 50% of these
cells express RORt (Fig. S3E). Expression of IL-17A in re-
sponse to ILC stimulation with IL-23 + IL-1 in vitro was re-
stricted to RORt
+
cells regardless of T-bet status (Fig. S3E).
However, TRUC mice had both a higher frequency of RORt
+
Thy1
hi
NKp46

cells and a higher frequency of IL-17Apositive

responders among these RORt
+
cells (Fig. S3F). Consistent
with findings in WT mice (31), we found that in Rag2
/
mice,
30% of Thy1
hi
NKp46

cells express T-bet (Fig. S3G). T-bet has

been shown to be required for the differentiation of NKp46
+
ILCs (31, 32). Our data demonstrate that T-bet also controls
A
IL-17A
ILC DC ILC + DC
0
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1500
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D E
Fig. 2. IL-17A secretion by ILCs requires IL-23 and IL-1 signals. (A) CD11c
+
BM-DCs and FACS-sorted ILCs (CD45
+
lin

Thy1
hi
NKp46

) pooled from mLN

and colon LP of Rag2
/
mice were stimulated either alone or in coculture
with 1 g/mL PGN. SN was harvested after 24 h, and the concentration of
IL-17A and TNF was determined by ELISA. (B) BM-DCs and ILCs were stimu-
lated with 1 g/mL PGN for 24 h in the presence of blocking antibodies
against IL-23R, IL-1R1, TNF, TNFR1, or isotype control (all 10 g/mL). (C) ILCs
were incubated alone for 24 h in the presence of recombinant murine IL-23,
IL-1, TNF, IL-23 + IL-1, or IL-23 + TNF (all 10 ng/mL). (D) mLN cells from TRUC
mice were stimulated for 24 h with IL-23, TNF, or IL-23 + TNF (all 10 ng/mL)
together with antiIL-1R1 or isotype control (10 g/mL). In BD, IL-17A was
measured in the SN by ELISA. All experiments were performed at least twice
with similar results. (E) TRUC.IL1R1
/
mice and TRUC.IL1R
+/+
littermate con-
trols were generated by mating TRUC.IL1R1
+/
parents. Colon histopathology
(n = 9 per genotype) was scored at 8 wk of age.
Ermann et al. PNAS | Published online June 9, 2014 | E2561
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the function of Thy1
hi
NKp46

ILCs, suggesting that enhanced

IL-17A secretion by T-betdeficient ILCs is a key factor in
TRUC pathogenesis.
IL-17A and TNF Synergistically Induce Neutrophil-Attracting Chemokines.
Having established that IL-23responsive IL-17Asecreting ILCs
are as important for the development of TRUC colitis as TNF, we
asked how these two pathways may intersect in the pathogenesis of
the disease. TNF and IL-17A have been shown to synergistically
induce neutrophil-attracting chemokines in several cell types in-
cluding osteoblasts (33), synovial fibroblasts (34), and type II al-
veolar cells (35). Neutrophils are an important component of the
inflammatory infiltrate in TRUC mice (1) and, correspondingly,
expression of the neutrophil-attracting chemokines Cxcl1, Cxcl2,
and Cxcl5 is up-regulated in TRUC colon tissue. To formally test
the role of neutrophils in TRUC pathogenesis, we treated a cohort
of mice from weaning with a neutrophil-depleting antibody (anti-
GR1) or isotype control. Analysis of colitis severity at 8 wk of age
demonstrated that neutrophil depletion abrogated colitis (Fig. 3A;
mean colitis score 0.25 vs. 5.0, P < 0.0001). Next, we analyzed the
induction of neutrophil-attracting chemokines by TNF and IL-17A
in the murine intestinal epithelial cell line MODE-K. Confirming
observations in other cell types, we found that TNF and IL-17A
synergistically up-regulated the expression of Cxcl1, Cxcl2, and
Cxcl5 as measured by real-time quantitative PCR (qPCR) after 6 h
of stimulation (Fig. 3B). Reasoning that neutrophil recruitment is
an early event in disease pathogenesis, we quantified the expression
of Cxcl1, Cxcl2, and Cxcl5 in the colon of 4-wk-old TRUC mice 3
d after administration of a single dose of anti-TNF or antiIL-23R
antibodies (Fig. 3C). Antibody-mediated blockade of either TNF
or the IL-23/IL-17A axis resulted in equivalent down-regulation of
Cxcl1, Cxcl2, and Cxcl5 mRNA in vivo. Taken together these data
suggest that the synergistic effect of TNF and IL-17A on the in-
duction of the neutrophil-attracting chemokines Cxcl1, Cxcl2, and
Cxcl5 demonstrated in vitro is operating in the TRUC colon in
vivo, promoting colitis via neutrophil recruitment.
TRUC.Ripk2
/
Mice Are Protected from Disease. We were interested
in upstream signals driving the development of colitis in TRUC
animals. Colitis in TRUC.Myd88
/
mice lacking the adapter
molecule Myd88 required for signaling through most toll-like
receptors (TLRs) is unchanged compared with TRUC.Myd88
+/+
controls (4). Given the importance of the Nod/Ripk2 pathway in
IBD, we bred a Ripk2 KO allele (36) onto the BALB/c TRUC
background. Ripk2 mediates signals downstream of both Nod1
and Nod2. Hence, disruption of Ripk2 simultaneously abrogates
both Nod1 and Nod2 signaling. Heterozygous TRUC.Ripk2
+/
mice were intercrossed to generate TRUC.Ripk2
+/+
and TRUC.
Ripk2
/
littermates. At 8 wk of age, TRUC.Ripk2
/
animals
had no signs of colitis (Fig. 4A; mean colitis score 0.3 vs. 4.2 in
TRUC.Ripk2
+/+
littermate controls, P = 0.0001).
The influence of Nod signals on shaping the host intestinal
microbiota has been well described (1719). To address whether
the protective effect of Ripk2 deficiency on colitis development
was mediated indirectly via changes in intestinal microbiota,
we performed a reciprocal cross-fostering experiment. TRUC.
Ripk2
+/+
and TRUC.Ripk2
/
breeders were set up in parallel,
and half of the pups were exchanged within 24 h after birth. Fig.
4B demonstrates that with regard to colitis development, the
genotype of the pups was dominant over the genotype of
the foster mother. Compared with TRUC.Ripk2
+/+
littermates,
TRUC.Ripk2
/
mice were always protected, whether they were
raised by a TRUC.Ripk2
+/+
mother with colitis (mean colitis
score 0.1 vs. 4.4, P = 0.0003), or by a TRUC.Ripk2
/
mother
without colitis (mean colitis score 0.2 vs. 3.8, P = 0.0006). Put
another way, TRUC.Ripk2
/
mothers appear to maintain and
pass on a colitogenic flora without developing colitis themselves.
These data are consistent with a model where Ripk2 controls the
inflammatory response in the colon and not the colitogenicity of
the intestinal microbiota in TRUC mice.
Nod1 or Nod2 Indirectly Costimulate IL-17A Secretion by ILCs. We
then investigated how Nod/Ripk2 signals affect the induction
of IL-17A secretion by ILCs in vitro comparing DCs and ILCs
from TRUC.Ripk2
+/+
and TRUC.Ripk2
/
animals. We set up
A
isotype anti-GR1
0
3
6
9
h
i
s
t
o
p
a
t
h
o
l
o
g
y

s
c
o
r
e
p<0.0001
Cxcl1
- TNF IL-17A TNF
0
2
4
6
8
[
r
e
l
a
t
i
v
e

e
x
p
r
e
s
s
i
o
n
]
p=0.0008
IL-17A
Cxcl2
- TNF IL-17A TNF
0
2
4
6
8
10
[
r
e
l
a
t
i
v
e

e
x
p
r
e
s
s
i
o
n
]
p=0.0205
IL-17A
Cxcl5
- TNF IL-17A TNF
0
2
4
6
8
[
r
e
l
a
t
i
v
e

e
x
p
r
e
s
s
i
o
n
]
p=0.0002
IL-17A
Cxcl1
isotype a-TNF a-IL-23R
0.01
0.1
1
10
100
r
e
l
a
t
i
v
e

e
x
p
r
e
s
s
i
o
n
p=0.0223
p=0.0212
Cxcl2
isotype a-TNF a-IL-23R
0.01
0.1
1
10
100
r
e
l
a
t
i
v
e

e
x
p
r
e
s
s
i
o
n
p=0.0429
p=0.0433
Cxcl5
isotype a-TNF a-IL-23R
0.01
0.1
1
10
100
r
e
l
a
t
i
v
e

e
x
p
r
e
s
s
i
o
n
p=0.0818
p=0.0774
C B
Fig. 3. TNF and IL-17A synergistically induce the expression of neutrophil-
recruiting chemokines. (A) TRUC mice were treated from weaning with bi-
weekly injections of a depleting anti-GR1 antibody or isotype control (n = 8
per group). Histopathology was analyzed at 8 wk of age. (B) MODE-K cells
were stimulated with recombinant murine TNF, IL-17A, or TNF + IL-17A (all
10 ng/mL) for 6 h. The expression of Cxcl1, Cxcl2, and Cxcl5 was determined
by real-time qPCR using Hprt for normalization. Data represent fold change
relative to unstimulated cells. Mean and SD of biological duplicates are
shown. The experiment was performed twice with similar results. (C) Four-
week-old TRUC mice received a single i.p. injection of a blocking antibody
against TNF (a-TNF) or the IL-23 receptor (a-IL-23R) or isotype control (n =
67 per group). Mice were killed after 3 d, and RNA was isolated from the
distal third of the colon. Cxcl1, Cxcl2, and Cxcl5 mRNA levels were de-
termined by real-time qPCR and normalized to Hprt. Values are expressed
relative to isotype-treated TRUC mice.
E2562 | www.pnas.org/cgi/doi/10.1073/pnas.1408540111 Ermann et al.
reciprocal cocultures and stimulated the cells with lipopolysac-
charide (LPS) with or without the Nod2 ligand L18-MDP. As
expected, the addition of L18-MDP to LPS resulted in increased
TNF production by DCs, which was abrogated by Ripk2 de-
ficiency (Fig. 4C). Activation of the Nod/Ripk2 pathways also
strongly enhanced the secretion of IL-17A in the DC/ILC co-
culture. This effect disappeared when the DCs were derived
from Ripk2-deficient animals, whereas Ripk2-deficiency in ILCs
had no impact (Fig. 4D). Therefore, TLR and Nod2 signals in
DCs synergistically drive IL-17A production in ILCs cocultured in
vitro, but ILCs do not perceive Nod signals themselves. Consistent
with previous reports (26, 37), we found that Nod2 costimulation
of BM-DCs from TRUC.Ripk2
+/+
mice strongly enhanced the
expression of the IL-23 chains IL-23p40 and IL-23p19, an effect
that was absent in BM-DCs from TRUC.Ripk2
/
mice (Fig. 4E).
Both Nod1 and Nod2 signal through Ripk2. To address which
of these sensors affects the development of TRUC disease
in vivo, we crossed Nod1- and Nod2-deficient mice with the
C57BL/6 TRUC strain. TRUC mice heterozygous for the Nod1
or Nod2 KO allele were intercrossed, and histological analysis
was performed in the offspring at 8 wk of age. Single deficiency
of either Nod1 or Nod2 was insufficient to abrogate colitis (Fig. 5
A and B). Although there was a trend toward reduced disease
severity in homozygous TRUC.Nod1
/
and TRUC.Nod2
/
animals, this difference was not statistically significant. In con-
trast, double deficiency of Nod1 and Nod2 reproduced the
phenotype of complete protection seen in TRUC.Ripk2
/
ani-
mals (Fig. 5 C and D; mean colitis score 0.2 in TRUC.Nod1
/
.
Nod2
/
vs. 2.3 in TRUC.Nod1
+/+
.Nod2
/
littermate controls,
P = 0.0070). This result is consistent with the dominant role of
Gram-negative bacteria, which can activate both Nod1 and
Nod2, in TRUC pathogenesis (1, 2, 24). It argues further, that, in
TRUC colitis, there is no alternative upstream signal driving
Ripk2 activation independently of Nod1 or Nod2 (38).
Discussion
TRUC is a model for innate immune-driven chronic intestinal
inflammation and carcinogenesis. Removal of regulatory influ-
ences (T-bet, Treg cells) leads to robust inflammation of the
distal large bowel, allowing for the analysis of disease mecha-
nisms not necessarily related to T-bet deficiency. Previous work
established DCs and TNF as central mediators of disease de-
velopment in TRUC mice (1, 24). We now demonstrate that
a second major pathway involving ILCs and the IL-23/IL-17A
axis is equally important in TRUC pathogenesis. We show that
IL-17A induction in ILCs requires the combined activity of IL-23
and IL-1 but not TNF. Instead, the TNF and IL-23/IL-17A
pathways intersect at the critical step of neutrophil recruitment
to the colon. Both pathways are controlled by Nod/Ripk2 signals
A
Ripk2+/+ Ripk2-/-
0
3
6
9
h
i
s
t
o
p
a
t
h
o
l
o
g
y

s
c
o
r
e
p=0.0001
Ripk2+/+ Ripk2-/- Ripk2+/+ Ripk2-/-
0
3
6
9
h
i
s
t
o
p
a
t
h
o
l
o
g
y

s
c
o
r
e
mother Ripk2+/+ Ripk2+/+ Ripk2-/- Ripk2-/-
pups
p=0.5543
p<0.0001
p=0.9901
p<0.0001
TNF
Ripk2+/+ Ripk2-/-
0
500
1000
1500
2000
T
N
F

[
p
g
/
m
l
]
DC
IL-17A
Ripk2+/+ Ripk2+/+ Ripk2-/- Ripk2-/-
0
500
1000
1500
2000
I
L
-
1
7
A

[
p
g
/
m
l
]
Ripk2+/+ Ripk2-/- Ripk2+/+ Ripk2-/- ILC
DC
IL-12p40
Ripk2+/+ Ripk2-/-
0
50
100
150
200
250
r
e
l
a
t
i
v
e

e
x
p
r
e
s
s
i
o
n
IL-23p19
Ripk2+/+ Ripk2-/-
0
10
20
30
40
r
e
l
a
t
i
v
e

e
x
p
r
e
s
s
i
o
n
medium
LPS
LPS + L18-MDP
C
B
D
E
Fig. 4. TRUC.Ripk2
/
mice are protected from colitis and Nod/Ripk2 stim-
ulated DCs drive IL-17A secretion by ILCs. (A) TRUC.Ripk2
+/
mice backcrossed
to BALB/c for at least seven generations were mated to generate TRUC.
Ripk2
/
and TRUC.Ripk2
+/+
littermates. (B) Crossfostering of TRUC.Ripk2
+/+
and TRUC.Ripk2
/
pups. Breeders were set up in parallel and newborn mice
were exchanged between cages within 24 h after birth. Colon histopathol-
ogy in A and B was analyzed at 8 wk of age. Results are pooled from mul-
tiple litters (n = 813 animals per group). (C and D) BM-DCs and FACS sorted
ILCs were prepared from TRUC.Ripk2
+/+
and TRUC.Ripk2
/
mice and stim-
ulated for 24 h with 1 ng/mL LPS alone or LPS plus 1 g/mL L18-MDP. The
concentration of TNF in the SN of DCs cultured alone (C) and IL-17A in the SN
of cocultures of DCs and ILCs (D) was determined by ELISA. (E) BM-DCs from
TRUC.Ripk
+/+
and TRUC.Ripk2
/
mice were stimulated for 6 h with 1 ng/mL
LPS alone or LPS plus 1 g/mL L18-MDP. Total RNA was isolated and real-time
qPCR analysis for IL-12p40 and IL-23p19 expression was performed. The data
were first normalized to Hprt and then scaled by dividing over the mean of
the unstimulated samples. All in vitro assays described in this figure were
performed twice with similar results.
A
C
B
D
+/+ +/- -/-
0
3
6
9
h
i
s
t
o
p
a
t
h
o
l
o
g
y

s
c
o
r
e
p=0.3578
Nod1
+/+ +/- -/-
0
3
6
9
h
i
s
t
o
p
a
t
h
o
l
o
g
y

s
c
o
r
e
Nod2
p=0.3945
+/+ +/- -/-
0
3
6
9
h
i
s
t
o
p
a
t
h
o
l
o
g
y

s
c
o
r
e
Ripk2
p=0.0080
+/+ +/- -/-
0
3
6
9
h
i
s
t
o
p
a
t
h
o
l
o
g
y

s
c
o
r
e
-/- -/- -/- Nod2
Nod1
p=0.0070
Fig. 5. Nod1 and Nod2 signals are redundant for Ripk2-dependent in-
duction of TRUC colitis. (A) TRUC.Nod1
+/
mice were mated to generate
TRUC.Nod1
+/+
, TRUC.Nod1
+/
, and TRUC.Nod1
/
littermates. The same ap-
proach was used to generate TRUC.Nod2
/
(B) and TRUC.Ripk2
/
(C) ani-
mals. (D) After fixing the Nod2
/
genotype, TRUC.Nod1
+/
.Nod2
/
mice
were mated to generate TRUC.Nod1
/
.Nod2
/
DKO mice and littermate
controls as indicated in the figure. Histopathology (n = 812 per genotype)
was scored at 8 wk of age.
Ermann et al. PNAS | Published online June 9, 2014 | E2563
I
M
M
U
N
O
L
O
G
Y
P
N
A
S
P
L
U
S
upstream, and genetic ablation of Nod/Ripk2 signaling protects
TRUC mice from colitis development.
ILCs are a heterogeneous group of innate immune cells that
includes NK cells, lymphoid tissue inducer cells, and various subsets
classified based on their cytokine expression pattern as ILC1 (IFN-
), ILC2 (IL-5, IL-13), or ILC3 (IL-17A, IL-22) cells. The CD45
+
lin

Thy1
hi
NKp46

subset driving colitis in the TRUC model rep-

resents NCR

ILC3 cells according to the latest consensus on ILC

nomenclature (25). Our data corroborate a report on the pathogenic
role of IL-17A secreting lin

Thy1
hi
ILCs in TRUC mice (6); similar
cells identified as CD45
+
lin

Thy1
hi
Sca1
hi
had been shown to me-
diate innate immune colitis in the H. hepaticus model (5). We extend
those findings by analyzing the ILCs within the complex network of
cells and mediators in the colon LP. Using an in vitro coculture
system, we were able to dissect the signals required for IL-17A in-
duction in ILCs. As demonstrated for T cells (29) and invariant
NKT cells (30), a combination of IL-23 and IL-1 proved necessary
and sufficient to drive IL-17A secretion by ILCs. Stimulation with
IL-23 alone failed to induce IL-17A in highly purified ILCs. The
induction of IL-17A upon IL-23 stimulation demonstrated in bulk
cultures of mLN or LP cells (5, 6, 27) was abrogated by blocking
antibodies against the IL-1 receptor and can thus be explained
by small amounts of IL-1 secreted by non-ILCs. ILCs express high
levels of IL-1R1 (27), and we found these cells to be exquisitely
sensitive to IL-1 stimulation in vitro. The observation that TRUC.
IL1R1
/
mice are protected from colitis development appears at
odds with the lack of protection in TRUC.MyD88
/
mice (4).
However, Myd88-mediated TLR signals activate both proin-
flammatory and antiinflammatory effector mechanisms in a cell
type and context-dependent manner (39, 40). Presumably, the net
result of MyD88 deficiency in TRUC mice is an imbalanced loss of
TLR-mediated protective signals resulting in the development of
colitis independently of IL-1. This interpretation is supported by
the finding of significant spontaneous early mortality in TRUC.
MyD88
/
mice (4) but not TRUC.IL1R1
/
mice. Although we
used BM-DC in most in vitro assays to drive IL-17A induction in
cocultured ILCs, we could demonstrate the same effect by using
CD11c
+
spleen DCs and a crude preparation of CD11b
+
/CD11c
+
colon LP cells stimulated with PGN. This fraction contains
dendritic cells and also macrophages (41), both cell types
capable of secreting IL-23 and IL-1. The identity of the cells
providing the ILC activating signals in vivo remains to be
determined.
T-bet controls the phenotype of several innate immune cell
types including dendritic cells (1), NK cells (42), and RORt
+
innate lymphoid cells (31, 32). While NKp46
+
IL-22 secreting
ILCs have been demonstrated to require T-bet for development
(31, 32), we show that T-bet also controls IL-17A induction in a
subset of Thy1
hi
NKp46

RORt
+
ILCs. The inhibitory effect of
T-bet on IL-17A secretion by ILCs is not sufficiently explained
by suppression of RORt (43) as RORt expression levels in
RORt-positive ILCs from Rag2
/
and TRUC mice are similar
(Fig. S3E). IL-22 mRNA is strongly up-regulated in the inflamed
TRUC colon, making it unlikely that T-bet deficiency promotes
colitis development in TRUC mice via a lack of IL-22secreting
NKp46
+
ILCs. Both protective and disease-promoting roles have
been described for IL-22 in mouse IBD models (4447), and it is
feasible that IL-22 contributes to the development of colitis and
colorectal carcinoma (48) in the TRUC model.
We have demonstrated that IL-17A production by ILCs is un-
der control of the NOD/Ripk2 pathway. However, this effect
appears to be mediated indirectly by DCs (and possibly other
antigen-presenting cells in vivo). Whether IL-17A production in
ILCs is modulated by PRR signaling in ILCs in other settings
remains to be determined. Our in vitro data suggest that ILCs do
not respond directly to TLR2/4 or Nod/Ripk2 stimulation. Dis-
ruption of the Nod/Ripk2 signaling pathway has been shown
to cause changes in the intestinal flora that result in increased
severity of DSS-induced colitis and inflammation-associated co-
lorectal cancer (19). As the reciprocal cross-fostering experiment
with TRUC and TRUC.Ripk2
/
litters (Fig. 4B) demonstrates,
TRUC.Ripk2
/
females without colitis could raise TRUC pups
that would develop colitis, whereas TRUC.Ripk2
/
pups were
always protected regardless of the genotype or colitis phenotype of
the foster mother. Although fecal reconstitution experiments in
germfree mice would be required for definite proof, our data
suggest that the protective effect of Ripk2 deficiency in the TRUC
model is not an indirect effect mediated by changes in intestinal
microbiota and that development of colitis and maintenance of
a colitogenic flora are uncoupled in TRUC.Ripk2
/
mice.
We propose the following model for the pathogenesis of
TRUC disease (Fig. S4). DCs activated via PRR signaling re-
lease TNF, IL-1, and IL-23. IL-23 in combination with IL-1
drives ILCs to secrete IL-17A. The magnitude of both the TNF
and the IL-23/IL-17A response is controlled by Nod/Ripk2 sig-
nals in the DCs. TNF, which by itself induces colon epithelial
cell apoptosis (1), synergizes with IL-17A to attract neutrophils
whose release of tissue-damaging mediators contributes further
to the barrier dysfunction observed in TRUC mice. T-bet de-
ficiency in DCs and in ILCs contributes to the pathogenesis by
enhancing the production of TNF and IL-17A. The experimental
disruption of this complex network at multiple nodes abrogates
disease. Antibody-mediated blockade of TNF, IL-23, or IL-17A
are equally protective in TRUC mice when started early in the
disease course. It remains to be shown which interventions or
combinations thereof work best in established disease, which is
ultimately the situation in human patients with IBD.
Materials and Methods
Mice. Tbx21
/
.Rag2
/
(T-bet
/
.Rag2
/
, TRUC) mice (1) have been back-
crossed to a BALB/c or C57BL/6 background for at least 10 generations.
IL23R-GFP mice (23) were provided by Mohamed Oukka (University of
Washington and Seattle Childrens Research Institute, Seattle). Nod1
/
mice
(49) were provided by Tak Mak (University of Toronto and Princess Margaret
Hospital, Toronto), Nod2
/
mice (50) and IL1R1
/
mice (28) were purchased
from the Jackson Laboratory. Ripk2
/
mice (36) were provided by Kochi
Kobayashi (Texas A&M Health Science Center, College Station, TX). These
mutant strains on C57BL/6 backgrounds were backcrossed to C57BL/6 TRUC
mice by successive mating with TRUC females to ensure vertical transmission
of the colitogenic TRUC intestinal flora. TRUC mice homozygous for the
mutant allele were generated by mating heterozygous TRUC mice (e.g.,
TRUC.Ripk2
+/
) yielding WT/WT, WT/KO, and KO/KO littermates (e.g., TRUC.
Ripk2
+/+
, TRUC.Ripk2
+/
, TRUC.Ripk2
/
). Although we did not monitor the
composition of the intestinal microbiota with molecular or microbiological
tools and, thus, cannot exclude the possibility of shifts, the development of
colitis in WT/WT littermates proves the presence of colitogenic intestinal
microbiota in the breeding colony. Nod1
/
.Nod2
/
DKO mice were gen-
erated by first fixing the Nod2
/
genotype and then mating TRUC.Nod1
+/
.
Nod2
/
mice. C57BL/6 TRUC.Ripk2
/
mice were backcrossed to BALB/c TRUC
for at least seven generations. Homozygosity for the BALB/c allele at the
major colitis susceptibility locus Cdcs1 (24) was verified at an earlier gener-
ation. Mice were housed in microisolator cages in a specific pathogen-free
animal facility at the Harvard School of Public Health, and studies were
performed according to institutional and National Institutes of Health
guidelines for humane animal use. Sulfatrim (1 g/L Sulfamethoxazole + 0.2 g/L
Trimethoprim; Hi-Tech Pharmacal) was added to the drinking water. Mice
were weaned between 3 and 4 wk of age.
Cross-Fostering. TRUC.Ripk2
+/+
and TRUC.Ripk2
/
pregnant (frommating with
a male of identical genotype) females were monitored daily for delivery. Pups
were exchanged between newborn litters within 24 h after birth.
In Vivo Antibody Blockade. For treatment studies, BALB/c TRUC mice received
weekly i.p. injections of the following antibodies: anti-Thy1 (clone 30H12, rat
IgG2b, 1 mg; BioXcell) or isotype (clone LTF-2, rat IgG2b, 1 mg; BioXcell); anti-
IL-17A (clone 17F3, mouse IgG1, 1 mg; BioXcell) or isotype (clone MOPC-21,
mouse IgG1, 1 mg; BioXcell). Anti-IL-23p19 (clone MB490, rat IgG1, 2 mg),
anti-IL-23R (clone 21A4, rat IgG1, 0.6 mg), or isotype (clone 27F11, rat IgG1,
2 mg) (51) were generously provided by Merck. Anti-GR1 (anti-Ly-6G, clone
E2564 | www.pnas.org/cgi/doi/10.1073/pnas.1408540111 Ermann et al.
RB6-8C5, rat IgG2b, 0.25 mg; BioXcell) or isotype (clone LTF-2, rat IgG2b, 0.25
mg; BioXcells) were injected twice weekly. Injections started at weaning, and
mice were killed at 8 wk of age. For the in vivo chemokine inhibition ex-
periment, 4-wk-old BALB/c TRUC mice received a single i.p. injection of 0.5
mg anti-TNF (clone TN3-19.12, hamster IgG; Leinco), anti-IL-23R (clone 21A4,
rat IgG1; Merck) or isotype (hamster IgG; Leinco, or rat IgG1; Merck) and
mice were killed after 3 d.
Histology. Animals were killed at the age of 8 wk, the colon was isolated, fixed
in 4% (wt/vol) paraformaldehyde (Sigma-Aldrich) and embedded in paraffin.
Standard hematoxylin/eosin (H&E)-stained sections were examined and
scored by an experienced pathologist (J.N.G.) in a blinded fashion. The
parameters mononuclear cell infiltration, polymorphonuclear cell infiltration,
epithelial hyperplasia, and epithelial injury were scored as absent (0), mild (1),
moderate (2), or severe (3), giving a total score of 012 (24).
Cell Preparation from Tissues. Mesenteric lymph nodes (mLN) were minced,
digested with 0.2 mg/mL Liberase TL (Roche), and 0.1 mg/mL DNase (Roche) in
DMEM (Cellgro) for 20 min at 37 C rotating, and then passed through
a 70-m strainer to generate a single cell suspension. For the isolation of LP
cells, the intestine was opened longitudinally, rinsed with PBS, and cut into
1- to 2-cm pieces. These pieces were incubated in HBSS plus 1 mM DTT and
5 mM EDTA for 40 min at 37 C rotating. Mucus and epithelial cells were
removed by straining through a metal kitchen sieve. The retained tissue
pieces were rinsed with DMEM, minced with scissors, and digested with
Liberase/DNase in DMEM for 20 min at 37 C rotating. The cells suspension
obtained after passing the digested tissue through a 100-m strainer, was
stained with anti-CD45 microbeads, and CD45
+
cells were positively selected
by using an autoMACS system (Miltenyi). Viable cell numbers were de-
termined manually by Trypan blue exclusion.
Flow Cytometry. Single cell suspensions were stained in PBS with 0.5%
BSA (Roche) and analyzed on a LSRII (BD Biosciences), or sorted by using
a FACSAria (BD Biosciences). Data were analyzed by using FlowJo software
(Treestar). Intracellular staining was performed as described (43). Anti-NKp46
FITC, anti-CD11b FITC, anti-CD11c PE, anti-IL-17A PE, anti-CD45 PerCP-Cy5.5,
anti-CD11c PerCP-Cy5.5, anti-Sca1 PE-Cy7, anti-NKp46 PE-Cy7, anti-CD45
Pacific Blue, anti-Thy1 Brilliant Violet 510, anti-Thy1 APC, anti-NKp46 APC,
anti-IL-17A APC, anti-CD11b APC-Cy7, anti-CD11c APC-Cy7, and 7-AAD were
purchased from Biolegend. Anti-T-bet PE, anti-RORt APC, and the fixable
viability dye eFluor780 were from eBioscience; Anti-H2k
b
FITC was from
BD Pharmingen.
Preparation of DCs. BM-DCs were prepared as described (24) by incubating
BM single cell suspensions in medium conditioned with supernatant from
GM-CSFproducing J558L cells. On day 6, CD11c
+
cells were purified by using
magnetic microbeads (Miltenyi). For the isolation of spleen DCs, spleen tissue
was digested with Liberase/Dnase, red blood cells were lysed, and CD11c
+
cells were purified with anti-CD11c beads (Miltenyi). For LP DCs, a single cell
suspension was prepared from colon tissue by two-step EDTA and Liberase/
Dnase digestions as described. Mononuclear cells were further purified by
Percoll gradient centrifugation (Sigma-Aldrich). The cells from the 7540%
(vol/vol) interface were washed, incubated with microbeads, and CD11b
+
/CD11c
+
cells were positively selected by using MS magnetic columns (Miltenyi).
ILC Assays. Twenty thousand CD11c
+
BM-DCs and 10,000 FACS sorted ILCs
(CD45
+
CD11b

CD11c

Thy1
hi
NKp46

) were incubated in 96-well round bot-

tom plates (Nunc) in 250 L of RPMI 1640 (Cellgro) supplemented with 10%
(vol/vol) FCS (Atlanta Biologicals), 10 mM Hepes, 1 mM sodium pyruvate,
2 mM L-glutamine, 100 U/mL penicillin, 100 g/mL streptomycin, 50 g/mL
gentamicin (all Cellgro), and 50 M -mercaptoethanol (Sigma-Aldrich). For
coculture experiments involving primary DC populations, 100,000 DCs were
incubated with 20,000 ILCs (all isolated from BALB/c TRUC mice). Cells
were stimulated with 1 g/mL peptidoglycan (PGN; Invivogen), 1 ng/mL LPS
(Salmonella enterica typhimurium; Sigma-Aldrich), and 1 g/mL L18-MDP
(Invivogen), and supernatant was harvested after 24 h. The following
blocking antibodies were used at 10 g/mL: antiIL-23R (clone 21A4, rat
IgG1; Merck), or isotype (clone 27F11, rat IgG1; Merck), antiIL-1RI (clone
JAMA147, hamster IgG; R&D), anti-TNF (clone TN3-19.12; hamster IgG,
Biolegend), anti-TNFR1 (clone 55R-170, hamster IgG; Biolegend) or isotype
(clone HTK888, hamster IgG; Biolegend). In other experiments, 10,000 FACS-
sorted ILCs were stimulated in 96-well plates with recombinant murine IL-23
(R&D), IL-1 (Biolegend), TNF (Biolegend), IL-23 + IL-1, or IL-23 + TNF, all at
10 ng/mL BM-DC. SN was generated by pooling the SN from multiple wells
containing 20,000 DCs stimulated with 1 g/mL PGN for 24 h.
ELISA. TNF in cell culture supernatant was measured by using a commercial kit
(Opteia; BD Pharmingen). For the quantification of IL-17A, we used capture
and biotinylated detection antibodies, strepavidin/HPRT, and HPRT substrate
reagent from Biolegend, IL-17A standard was purchased from R&D.
In Vitro Gene Expression Analysis. Four hundred thousand BM-DCs were
stimulated in 24-well plates with 1 ng/mL LPS or LPS + 1 g/mL L18-MDP for
6 h, then harvested into Qiazol (Qiagen). MODE-K cells (ATCC) were grown
to near-confluence in 12-well plates, stimulated with 10 ng/mL recombinant
murine TNF (Biolegend), IL-17A (R&D), or TNF + IL-17A. Cells were harvested
into Qiazol after 6 h.
Real-Time qPCR. Total RNA was prepared by using Qiazol and reverse tran-
scribed with the High Capacity cDNA RT Kit (Applied Biosystems) and ran-
dom primers. Real-time qPCR was performed on a MX3005 cycler (Stratagen)
by using the SYBR Green method. Data were normalized to Hprt. Primer
sequences: Hprt-F GTTAAGCAGTACAGCCCCAAA, Hprt-R AGGGCATATCCA-
ACAACAAAC, Cxcl1-F CTGGGATTCACCTCAAGAACATC, Cxcl1-R CAGGGT-
CAAGGCAAGCCTC, Cxcl2-F GCGCCCAGACAGAAGTCATAG, Cxcl2-R AGCCT-
TGCCTTTGTTCAGTATC, Cxcl5-F GGTCCACAGTGCCCTACG, Cxcl5-R GCGAGT-
GCATTCCGCTTA, IL-12p40-F GAGAAGACATCTACCGAAGTCCAATG, IL-12p40-
R GGAACACATGCCCACTTGCT, IL-23p19-F ACATGCACCAGCGGGACATA, IL-23p19-
R GTGGGTCACAACCATCTTCACA.
Statistical Analysis. Colitis scores and colonic gene expression data were
compared by using the unpaired t test or one-way ANOVA with post hoc
Tukeys test for more than two experimental groups. Statistical analysis and
graphing were done with Prism 6 (Graphpad Software).
ACKNOWLEDGMENTS. We thank Mohamed Oukka, Koichi Kobayashi, and
Tak Mak for providing knockout mice; Jacobo Ramirez for excellent animal
care; Deneen Kozoriz for high quality cell sorting; and Dorothy Zhang for
preparing histology slides. This work was supported by National Institutes of
Health (NIH) Grant CA112663 (to L.H.G.), a grant from the Harvard Institute
of Translational Immunology Helmsley Trust Pilot Grants Program (to L.H.G.
and J.E.), and a Harvard Digestive Diseases Center (NIH Grant 5-P30 DK034854)
pilot award (to J.E.).
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