Interleukin 23 production by intestinal CD103(+)CD11b(+) dendritic cells in response to bacterial flagellin enhances mucosal innate immune defense.
ABSTRACT Microbial penetration of the intestinal epithelial barrier triggers inflammatory responses that include induction of the bactericidal C-type lectin RegIIIγ. Systemic administration of flagellin, a bacterial protein that stimulates Toll-like receptor 5 (TLR5), induces epithelial expression of RegIIIγ and protects mice from intestinal colonization with antibiotic-resistant bacteria. Flagellin-induced RegIIIγ expression is IL-22 dependent, but how TLR signaling leads to IL-22 expression is incompletely defined. By using conditional depletion of lamina propria dendritic cell (LPDC) subsets, we demonstrated that CD103(+)CD11b(+) LPDCs, but not monocyte-derived CD103(-)CD11b(+) LPDCs, expressed high amounts of IL-23 after bacterial flagellin administration and drove IL-22-dependent RegIIIγ production. Maximal expression of IL-23 subunits IL-23p19 and IL-12p40 occurred within 60 min of exposure to flagellin. IL-23 subsequently induced a burst of IL-22 followed by sustained RegIIIγ expression. Thus, CD103(+)CD11b(+) LPDCs, in addition to promoting long-term tolerance to ingested antigens, also rapidly produce IL-23 in response to detection of flagellin in the lamina propria.
- SourceAvailable from: Toshimitsu Uede[Show abstract] [Hide abstract]
ABSTRACT: Gut microbiota are implicated in many liver diseases. Concanavalin A (ConA)-induced hepatitis is a well-characterized murine model of fulminant immunological hepatic injury. Oral administration of pathogenic bacteria or gentamycin to the mice before ConA injection, liver injury and lymphocyte distribution in liver and intestine were assessed. Our data show that administration of pathogenic bacteria exacerbated the liver damage. There was more downregulation of activation-induced natural killer T (NKT) cells in the liver of pathogenic bacteria-treated ConA groups. Also, there was a negative correlation between the numbers of hepatic NKT cells and liver injury in our experiments. Moreover, intestinal dendritic cells (DCs) were increased in pathogenic bacteria-treated ConA groups. The activation of DCs in Peyer's patches and the liver was similar to the intestine. However, depletion of gut gram-negative bacteria alleviated ConA-induced liver injury, through suppressed hepatic NKT cells activation and DCs homing in liver and intestine. In vitro experiments revealed that DCs promoted NKT cell cytotoxicity against hepatocyte following stimulation with pathogenic bacteria. Our study suggests that increased intestinal pathogenic bacteria facilitate immune-mediated liver injury, which may be due to the activation of NKT cells that mediated by intestinal bacterial antigens activated DCs.Scientific Reports 12/2014; 4:7259. · 5.08 Impact Factor
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ABSTRACT: Interleukin-22 (IL-22) plays a critical role in mucosal defense, although the molecular mechanisms that ensure IL-22 tissue distribution remain poorly understood. We show that the CXCL16-CXCR6 chemokine-chemokine receptor axis regulated group 3 innate lymphoid cell (ILC3) diversity and function. CXCL16 was constitutively expressed by CX3CR1(+) intestinal dendritic cells (DCs) and coexpressed with IL-23 after Citrobacter rodentium infection. Intestinal ILC3s expressed CXCR6 and its ablation generated a selective loss of the NKp46(+) ILC3 subset, a depletion of intestinal IL-22, and the inability to control C. rodentium infection. CD4(+) ILC3s were unaffected by CXCR6 deficiency and remained clustered within lymphoid follicles. In contrast, the lamina propria of Cxcr6(-/-) mice was devoid of ILC3s. The loss of ILC3-dependent IL-22 epithelial stimulation reduced antimicrobial peptide expression that explained the sensitivity of Cxcr6(-/-) mice to C. rodentium. Our results delineate a critical CXCL16-CXCR6 crosstalk that coordinates the intestinal topography of IL-22 secretion required for mucosal defense. Copyright © 2014 Elsevier Inc. All rights reserved.Immunity 11/2014; 41(5):776-88. · 19.75 Impact Factor
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ABSTRACT: Activators of innate immunity may have the potential to combat a broad range of infectious agents. We report that treatment with bacterial flagellin prevented rotavirus (RV) infection in mice and cured chronically RV-infected mice. Protection was independent of adaptive immunity and interferon (IFN, type I and II) and required flagellin receptors Toll-like receptor 5 (TLR5) and NOD-like receptor C4 (NLRC4). Flagellin-induced activation of TLR5 on dendritic cells elicited production of the cytokine interleukin-22 (IL-22), which induced a protective gene expression program in intestinal epithelial cells. Flagellin also induced NLRC4-dependent production of IL-18 and immediate elimination of RV-infected cells. Administration of IL-22 and IL-18 to mice fully recapitulated the capacity of flagellin to prevent or eliminate RV infection and thus holds promise as a broad-spectrum antiviral agent.Science 11/2014; 346(6211):861-5. · 31.48 Impact Factor
Interleukin 23 Production by Intestinal CD103+CD11b+
Dendritic Cells in Response to Bacterial Flagellin
Enhances Mucosal Innate Immune Defense
Melissa A. Kinnebrew,1Charlie G. Buffie,1Gretchen E. Diehl,2Lauren A. Zenewicz,3Ingrid Leiner,1Tobias M. Hohl,4
Richard A. Flavell,3,5Dan R. Littman,2,5and Eric G. Pamer1,*
1Infectious Diseases Service, Department of Medicine, Immunology Program, Memorial Sloan-Kettering Cancer Center, New York,
NY 10065, USA
2Molecular Pathogenesis Program, The Kimmel Center for Biology and Medicine of the Skirball Institute, New York University School of
Medicine, New York, NY 10016, USA
3Department of Immunobiology, Yale School of Medicine, New Haven, CT 06510, USA
Seattle, WA 98109, USA
5Howard Hughes Medical Institute, Chevy Chase, MD 20815, USA
Microbial penetration of the intestinal epithelial
barrier triggers inflammatory responses that include
induction of the bactericidal C-type lectin RegIIIg.
Systemic administration of flagellin, a bacterial pro-
tein that stimulates Toll-like receptor 5 (TLR5), in-
duces epithelial expression of RegIIIg and protects
mice from intestinal colonization with antibiotic-
resistant bacteria. Flagellin-induced RegIIIg expres-
sion is IL-22 dependent, but how TLR signaling leads
to IL-22 expression is incompletely defined. By using
conditional depletion of lamina propria dendritic
cell (LPDC) subsets, we demonstrated that CD103+
CD11b+LPDCs, but not monocyte-derived CD103?
CD11b+LPDCs, expressed high amounts of IL-23
after bacterial flagellin administration and drove IL-
22-dependent RegIIIg production. Maximal expres-
sion of IL-23 subunits IL-23p19 and IL-12p40
occurred within 60 min of exposure to flagellin.
IL-23 subsequently induced a burst of IL-22 followed
by sustained RegIIIg expression. Thus, CD103+
CD11b+LPDCs, in addition to promoting long-term
tolerance to ingested antigens, also rapidly produce
IL-23 in response to detection of flagellin in the
The mammalian gastrointestinal microbiota influences mucosal
innate and adaptive immune defenses. Commensal microbes
provide protection against infection byactivatingmucosal innate
immune defenses (Brandl et al., 2008; Cash et al., 2006) but also
induce regulatory cells that prevent detrimental inflammatory
responses (Honda and Takeda, 2009; Round and Mazmanian,
2010). The commensal microbe-host relationship has existed
throughout vertebrate evolution and, over the course of nearly
bial populations that colonize the mammalian gut (Ley et al.,
2008). The introduction of potent antibiotics to medical and agri-
cultural practice has inadvertently disrupted intestinal microbial
populations with untoward effects that are becoming increas-
ingly apparent. One of the most important adverse effects
of the broad use of antibiotics is the emergence of antibiotic-
resistant pathogens, such as vancomycin-resistant Entero-
coccus (VRE), carbapenem-resistant Klebsiella pneumoniae,
and Clostridium difficile. The antibiotic-conditioned gastrointes-
tinal tract has become a major reservoir for multidrug-resistant
organisms (Donskey, 2004; Ubeda et al., 2010). Strategies to
enhance mucosal innate immune defenses to inhibit pathogen
colonization of the intestinal lumen and/or prevent pathogen
translocation across mucosal barriers may provide opportuni-
ties to complement the shrinking repertoire of antibiotics that
are effective against increasingly antibiotic-resistant bacterial
A common and clinically important scenario, particularly in
antibiotic-treated patients, involves mucosal invasion by other-
wise innocuous but highly antibiotic-resistant bacteria subse-
quent to their dense colonization of the intestine. VRE has this
potential and thus is a leading cause of bacteremia in hospital-
ized patients (CDC, 1993; Hidron et al., 2008). Broad-spectrum
antibiotic administration kills commensal bacteria in the gut
and decreases Toll-like receptor (TLR)-dependent mucosal ex-
pression of antimicrobial factors, such as the bactericidal
C-type lectin RegIIIg, leading to a predisposition to VRE coloni-
zation (Brandl et al., 2008; Vaishnava et al., 2008). We have
shown that exogenous administration of TLR ligands in the
setting of antibiotic treatment restores the inflammatory tone of
the intestinal mucosal barrier. Lipopolysaccharide (LPS) and
RegIIIg expression and restrict colonization of the gut by VRE
(Brandl et al., 2008; Kinnebrew et al., 2010). Expression of
RegIIIg by intestinal epithelial cells is inducible by both direct
TLR signaling in epithelial cells (Brandl et al., 2008; Vaishnava
et al., 2008), and TLR signaling in cells of hematopoietic origin
276 Immunity 36, 276–287, February 24, 2012 ª2012 Elsevier Inc.
(Kinnebrew et al., 2010; Van Maele et al., 2010). Induction of
RegIIIg expression by exogenously administered flagellin is
TLR5 and interleukin-22 (IL-22) dependent and mediated by
hematopoietic cells (Kinnebrew et al., 2010; Van Maele et al.,
2010). The identities of the TLR5-expressing cell subset and
the in vivo signals involved in flagellin-mediated IL-22 and
RegIIIg induction remain incompletely defined.
IL-22 is critical for early defense against intestinal infections
with Citrobacter rodentiumand Candida albicans and pulmonary
infection with Klebsiella pneumoniae (Aujla et al., 2008; Sonnen-
berg et al., 2011; Zheng et al., 2008). IL-6, IL-1b, and IL-23
produced by antigen-presenting cells regulate IL-22 expression
in innate cells. It has also been suggested that direct stimulation
of TLRs on innate immune cells can induce IL-22 expression
(Crellin et al., 2010; Martin et al., 2009; Sutton et al., 2009).
Although Th17 cells and gd T cells that express the transcription
factors RORgt and the aryl hydrocarbon receptor (AHR) can
produce IL-22, the primary source for flagellin-mediated IL-22
expression in the intestine is CD3 3?CD127+innate lymphoid
cells (ILC) (Van Maele et al., 2010). How TLR5 signaling mediates
IL-22 secretion by ILCs is not clear.
Tissue-resident lamina propria dendritic cells (LPDCs) are
heterogeneous and arise from two principal lineages. LPDCs
express classIIMHCandCD11cbut canbedivided intosubsets
on thebasis of CD103and CX3CR1 expression. CD103+CD11b+
LPDCs are derived from the common dendritic cell progenitor
(CDP) through Flt3L-dependent pathways (Bogunovic et al.,
2009; Varol et al., 2009). In contrast, CX3CR1+CD103?CD11b+
LPDCs are derived from Ly6chimonocytes through a M-CSF-
dependent pathway (Bogunovic et al., 2009; Varol et al., 2009).
The relative contributions of these DC subsets to immune toler-
ance or antimicrobial defense remain controversial. CD103+
CD11b+LPDCs can upregulate CCR7 and traffic to mesenteric
lymph nodes, where they have been implicated in T regulatory
cell and oral tolerance development, whereas CX3CR1+
CD103?CD11b+LPDCs are nonmigratory and proinflammatory
in the setting of colitis (Coombes et al., 2007). CD103+CD11b+
LPDCs regulate T cell tolerance, but it is unclear whether they
contribute to innate immune surveillance for mucosal invasion
by microbial pathogens. Although CD103+CD11b+LPDCs are
the primary TLR5-expressing cell in the small intestine (Uematsu
et al., 2008), it is not known whether these cells contribute to
flagellin-induced IL-22 expression.
Although systemic flagellin administration protects mice
nization (Kinnebrew et al., 2010), and radiation-induced mortality
(Burdelya et al., 2008), the underlying mechanisms of flagellin-
mediated protection remain incompletely defined. Herein we
show that IL-22 was rapidly induced after flagellin administration
and required IL-23 production by TLR5-expressing CD103+
CD11b+LPDCs. Although CD103+CD11b+DCs and CX3CR1+
CD103?CD11b+DCs express TLR5, CD103+CD11b+LPDCs
were the major source of IL-23 induced by systemic TLR5 stim-
ulation. The rapidity of IL-23 production by CD103+CD11b+
LPDCssuggeststhatthisresponseisprincipally involved inearly
innate antimicrobial defense. CD103+CD11b+LPDCs, in con-
junction with IL-22-producing ILCs, provide a cellular surveil-
lance and effector mechanism to induce antimicrobial defenses
in intestinal epithelial cells in the event microbes or microbial
molecules enter the lamina propria. Pharmacologic activation
of the LPDC and ILC populations involved in this process may
provide a novel therapeutic approach to enhance mucosal resis-
tance to bacterial pathogens.
Flagellin-Mediated IL-22 Induction Involves
the Expression of TLR5 and IL-22 in Distinct
To characterize the response to systemic administration of
flagellin, we first analyzed the kinetics of RegIIIg expression
in the small intestine after flagellin administration. RegIIIg
messenger RNA (mRNA) transcript expression was increased
in the small intestine of wild-type (WT) mice within 3 hr of flagellin
administration and remained elevated for 24 hr postinjection
(Figure 1A). Flagellin administration did not induce RegIIIg
expression in IL-22-deficient mice as shown in previous studies
(Figure 1B; Kinnebrew et al., 2010). Induction of IL-22 in the
intestinal lamina propria (LP) preceded RegIIIg induction, with
peak IL-22 mRNA transcript and detectable IL-22 protein
amounts occurring 2 hr and 3 hr, respectively, after flagellin
administration (Figure 1C). We also detected elevated IL-22
mRNA transcripts in the liver, lung, and spleen; however, IL-22
mRNA transcripts were atthe highest amounts in the small intes-
tine lamina propria (Figure S1A available online).
IL-22 production by RORgt-dependent innate lymphoid cells
(ILC) is critical for defense against the intestinal bacterial path-
ogen Citrobacter rodentium (Sonnenberg et al., 2011; Zheng
et al., 2008). Inoculation of Rag1-deficient mice with flagellin
resulted in IL-22 induction, indicating that T cells and B cells
istration (Figures S1B and S1C). In contrast, IL-22 induction was
abrogated in mice lacking both Rag2 and the IL-2R g chain, sug-
gesting that an innate lymphocyte population was the source of
IL-22 (Figures S1B and S1C). Flagellin administration also failed
to stimulate IL-22 expression in mice deficient in either aryl
hydrocarbon receptor (AHR) or RORg (Figures S1D–S1F), which
is consistent with the previously described role of these tran-
scription factors in development of CD3 3?CD127+ILCs and
expression of IL-22. We characterized the phenotype of IL-22+
lamina propria cells and found that they were similar to previ-
ously described immature innate lymphoid cells that lack
expression of NK cell receptors and CD4 but were Thy1+,
c-kit+, and CD127+(Figure S1G; Sanos et al., 2011; Spits and
Di Santo, 2010). These results are consistent with the recent
ulation of ILCs (Van Maele et al., 2010); however, it remains
unclear how IL-22 production by ILCs is regulated in response
to microbial infection or flagellin administration.
To determine whether direct TLR5 stimulation of cells capable
of producing IL-22 is required for the flagellin response, we
generated mixed bone marrow (BM) chimeric mice by trans-
ferring a 1:1 mixture of congenically marked TLR5-deficient
bone marrow and IL-22-deficient bone marrow into lethally irra-
diated WT recipient mice. If TLR5 signaling and IL-22 expression
occurs in the same cell, flagellin administration should fail to
induce IL-22 in these chimeric mice. We, however, found that
IL-22 expression after flagellin administration was normal in
Intestinal DC Subset Triggers IL-23-IL-22 Pathway
Immunity 36, 276–287, February 24, 2012 ª2012 Elsevier Inc. 277
Tlr5?/?Il22?/?mixed BM chimeric mice (Figures 1E and 1F).
RegIIIg induction in the small intestine in response to flagellin
was also not substantially different among the groups of mixed
BM chimeric mice (Figure 1D). Although previously suspected,
these experiments demonstrate that TLR5 signaling and IL-22
expression occur in distinct cell populations, with a TLR5-
expressing cell population responding to flagellin administration
and relaying a second, distinct signal to cells that produce IL-22.
TLR5-Expressing DCs Are Required for Flagellin-
Mediated Induction of IL-22
To determine whether dendritic cells (DCs) are required for
TLR5-mediated induction of IL-22, we generated mixed BM
chimeric mice in which TLR5-expressing DCs could be selec-
tively depleted. Bone marrow cells from TLR5-deficient mice
and CD11c.DTR mice were mixed at a 1:1 ratio and used to
reconstitute irradiated WT recipient mice. CD11c.DTR mice are
transgenic mice that express the diphtheria toxin receptor
(DTR) under control of the CD11c promoter. We confirmed that
diphtheria toxin (DTX) treatment depleted intestinal MHCII+
CD11c+lamina propria DCs (LPDCs) derived from CD11c.DTR
BM cells (Figure S2A). Other cell populations that may contribute
to IL-22 production, including ILCs and NKp46+cells, were not
depleted by DTX treatment (Figure S2B). Specific depletion
of DCs capable of expressing TLR5 completely abrogated
flagellin-induced IL-22 expression in the lamina propria (Fig-
ure 2A). Serum titers of IL-22 were also undetectable after
flagellin treatment in these mice (Figure 2B). The lack of IL-22
induction could not be explained by a 50% loss of LPDCs
WT:CD11c.DTR mixed BM chimeric mice (Figures 2A and 2B).
We also analyzed flagellin-induced RegIIIg expression in mice
depleted of CD11c+dendritic cells to verify the role that DCs
play in this pathway. RegIIIg upregulation was completely
abrogated in mice that lacked DCs (Figure 2C). These results
demonstrate that dendritic cells that express TLR5 rapidly in-
duce IL-22 expression in the lamina propria in response to stim-
ulation with flagellin.
IL-23 Is Rapidly Induced after Flagellin Administration
We reasoned that TLR5 signaling in LPDCs stimulates cytokine
production that drives IL-22 expression by intestinal ILCs.
Soluble factors that mediate IL-22 expression have been identi-
fied and may play a role in flagellin-mediated IL-22 expression
(Ouyang et al., 2011). Given that IL-22 was induced in the lamina
propria within 2 hr of flagellin administration, it seemed likely that
TLR5-stimulated DCs express an IL-22-inducing factor at an
earlier time point. Therefore, we measured cytokine mRNA tran-
script expression in the lamina propria 1 hr after flagellin admin-
istration. Whereas IL-6 (Figure 3E) and IL-1b (Figure 3F) were
Figure 1. Flagellin-Mediated Upregulation of RegIIIg Requires TLR5 Signaling and IL-22 Expression in Distinct Cell Populations
(A) Messenger RNA (mRNA) transcript expression for RegIIIg in the small intestine (SI) was measured by quantitative PCR (qPCR) at the indicated time periods
after intravenous (i.v.) injection of flagellin (Flg) or PBS (Ctrl). (n = 3–4 mice per time point).
(B) Messenger RNA was extracted from the SI lamina propria of wild-type (WT) or IL-22-deficient (Il22?/?) mice 24 hr after i.v. injection of flagellin (n = 3 mice per
group) and quantified by qPCR.
(C) IL-22 messenger RNA expression was measured in the SI lamina propria at the indicated time points after i.v. flagellin administration. IL-22 protein
concentrations were detected in the serum by ELISA (n = 3 mice per time point).
(D and E) Mixed bone marrow (BM) chimeric mice reconstituted with equal ratios of Tlr5?/?,Il22?/?, or WT BM, as indicated on the horizontal axis, were assessed
for responsiveness to flagellin by qPCR analysis of RegIIIg mRNA transcripts in the SI (D) and IL-22 mRNA in the SI lamina propria (E).
(F) IL-22 protein concentrations in the serum were measured by ELISA (n = 3–4 mice per group). RegIIIg expression was measured 24 hr after flagellin
administration whereas serum IL-22 protein and lamina propria IL-22 expression were measured 3 hr after flagellin administration.
Error bars show ±SEM. Data are representative of at least two independent experiments.
Intestinal DC Subset Triggers IL-23-IL-22 Pathway
278 Immunity 36, 276–287, February 24, 2012 ª2012 Elsevier Inc.
upregulated approximately 7-fold in comparison to controls,
mRNA transcript expression of IL-12p40 (Figure 3A) and IL-
23p19 (Figure 3B) were increased 150-fold and 45-fold, respec-
tively, in flagellin-treated mice. As a heterodimeric protein, IL-23
is composed of two covalently linked subunits, IL-12p40 and
IL-23p19. IL-12p40 can also join with IL-12p35 to form IL-12.
We measured IL-12p35 mRNA transcript expression after
flagellin administration, but no increase was detected (Fig-
ure 3C). To further characterize the flagellin-induced response,
we measured expression of IL-12p40, IL-23p19, and IL-22 in
the lamina propria at several early time points after flagellin
administration. Messenger RNA transcripts for IL-23p19 and
IL12p40 were transiently elevated, peaking at 30 min and 1 hr,
respectively, whereas IL-22 mRNA transcripts reached the
maximum amount at 2 hr postinjection (Figure S3A). In order to
ensure that the resolution of IL-23 lamina propria expression to
baseline after a peak induction at 30–60 min is due to the down-
regulation of the IL-23 and not the migration of IL-23-expressing
cells out of the lamina propria or the recruitment of cells to the
lamina propria, we analyzed the lamina propria cell composition
2.5 hr after flagellin administration, during which IL-23 mRNA
transcript expression returned to baseline. We found that at
this early time point there were no significant shifts in DC popu-
lations that indicated migration of these cells out of the lamina
propria (Figure S3C). We observed recruitment of monocytes
and neutrophils to the lamina propria, but these cells composed
no more than 10% of total CD45+cells in the lamina propria (Fig-
ure S3D). No changes in T cell populations were detected.
To determine whether IL-1b, IL-6, or IL-23 contribute to
flagellin-mediated IL-22 expression, we stimulated purified
lamina propria cells with flagellin in vitro in the presence of
soluble IL-1Ra, anti-IL-6R, or anti-IL-23p19 to block cytokine
signaling. Only the IL-23p19 antibody blocked flagellin-induced
IL-22 production, whereas blockade of IL-1b and IL-6 had no
effect (Figures 3G and S3B). Addition of IL-23 to lamina propria
cells in the absence of flagellin or TLR5 induced high amounts
Figure 2. TLR5-Expressing Dendritic Cells Are Required for Flagellin-Mediated IL-22 Expression
(A and B)Mixed bone marrow (BM) chimeric mice in which the hematopoietic compartment is composed of equal ratios of Tlr5?/?, CD11c.DTR,or wild-type (WT)
BM cells, in combinations indicated on the horizontal axes, were depleted of WT dendritic cells by intraperitoneal injection of diphtheria toxin. Messenger RNA
(mRNA) expression in the small intestine (SI) lamina propria (A) and serum protein amounts (B) of IL-22 in response to flagellin (Flg) administration were assessed
3 hr after injection (n = 2–4 mice per group). Control (Ctrl) groups received PBS.
(C)Bonemarrowchimeric mice,generatedbytransferringbonemarrow fromCD11c.DTRmiceintoirradiatedWT mice,weredepleted ofDCswithdiptheriatoxin
as described in the Experimental Procedures. RegIIIg mRNA expression in the small intestine was assessed 24 hr after intravenous injection of flagellin. (n = 3
mice per group).
Error bars denote ±SEM. Data are pooled from two independent experiments.
Inhibits Flagellin-Mediated Expression of
(A–F) Wild-type (WT) mice received flagellin (Flg)
or PBS (Ctrl) intravenously. One hour after injec-
intestine lamina propria and cytokine expression
was assessed by quantitative PCR (n = 3 mice per
group; *p < 0.05).
(G) Lamina propria cells were isolated from the
small intestine of WT and Tlr5?/?mice and stim-
ulated in vitro with or without flagellin in the
presence of IL-23p19 blocking antibody (a-IL-
23p19) under the indicated conditions for 24 hr.
Recombinant mouse IL-23 (rmIL-23) was used as
a positive control. IL-22 protein was quantified in
culture supernatants by ELISA.
Data are representative of three independent ex-
periments. Error bars denote ±SEM.
Intestinal DC Subset Triggers IL-23-IL-22 Pathway
Immunity 36, 276–287, February 24, 2012 ª2012 Elsevier Inc. 279
of IL-22 expression in vitro (Figure 3G), demonstrating that IL-23
alone can induce lamina propria cells to secrete IL-22. These
results suggest that rapid and transient expression of IL-23 in
response to TLR5 stimulation leads to the upregulation of IL-22
and subsequent production of RegIIIg.
Flagellin-Mediated Induction of IL-22 Requires IL-23
Expression by Dendritic Cells
IL-23 has been demonstrated to induce IL-22 expression. IL-23-
deficient mice have impaired IL-22 expression during T cell-
mediated colitis, Citrobacter rodentium infection, and experi-
mental autoimmune encephalitis (Ahern et al., 2010; Kreymborg
et al., 2007; Zheng et al., 2008). However, a role for IL-23 in TLR-
mediated IL-22 expression has been implicated only in experi-
ments performed in vitro and by correlative upregulation after
in vivo administration of TLR ligands (Takatori et al., 2009; Van
Maele et al., 2010). To determine the in vivo role of IL-23 in
flagellin-mediated IL-22 expression, we generated mixed BM
chimeric mice in which the hematopoietic compartment is
composed of both IL-23p19-deficient and TLR5-deficient cells
in equal proportions (Figures S4A–S4D). In these mice, flagellin
did not induce IL-22 mRNA expression (Figure 4A) or elevate
IL-22 protein in the serum (Figure 4B), demonstrating that
TLR5-expressing cells must produce IL-23 in order to induce
IL-22 expression. This finding was not a consequence of the
fact that only half of the hematopoietic compartment was
capable of expressing IL-23 because the flagellin response
was normal in control mice reconstituted with equal parts WT
bone marrow cells and IL-23p19-deficient bone marrow cells
(Figures 4A and 4B).
To determine whether expression of IL-23 by DCs is required,
we again generated mixed BM chimeric mice by transferring
a 1:1 mixture of BM cells from IL-23p19-deficient and
IL-23p19-deficient DCs efficiently populated the lamina propria
in similar numbers compared to TLR5-deficient or WT DCs (Fig-
ure S4E). Flagellin failed to induce IL-22 expression in vivo when
DTX-sensitive, IL-23p19-sufficient DCs were depleted but DTX-
resistant, IL-23p19-deficient DCs persisted (Figures 4C and 4D).
Mice in which the DC compartment was composed of both
DTX-sensitive wild-type DCs and DTX-resistant wild-type DCs
responded normally to flagellin administration despite depletion
of half of the DC population. Depletion was specific to DCs as
shown by the fact that we did not observe loss of ILCs or
NKp46+cells (Figure S4F). Collectively, these data demonstrate
that IL-23 production by TLR5-expressing lamina propria
dendritic cells drives the rapid upregulation of IL-22 in response
to stimulation with flagellin.
Flt3L-Dependent Dendritic Cells Are Required
for Flagellin-Mediated IL-22 Expression
The MHCII+CD11c+lamina propria DC (LPDC) population is
composed of two major groups, one derived from classic DC
precursors and dependent on Flt3L and the other derived from
monocyte precursors and dependent on M-CSF (Bogunovic
et al., 2009; Varol et al., 2009). The relative functions of these
distinct populations remain incompletely defined, and it is not
clear whether either, neither, or both subsets produce IL-23.
To determine whether these LPDC subsets respond to flagellin
administration, we selectively depleted CD103?CD11b+LPDCs
by using transgenic mice in which the CCR2 promoter drives
expression of the diphtheria toxin receptor (CCR2.DTR) (Hohl
et al., 2009). The CCR2 chemokine receptor is selectively ex-
pressed by Ly6Chimonocytes, and DTX administration depletes
Figure 4. IL-23p19 Expression by TLR5+
Dendritic Cells Is Required for Flagellin-
Mediated IL-22 Induction
(A and B) Flagellin (Flg) or PBS (Ctrl) was admin-
istered intravenously to mixed bone marrow (BM)
chimeric mice reconstituted with equal parts
Tlr5?/?and Il23a?/?(gene name for IL-23p19)
bone marrow cells, as indicated. Three hours
after flagellin administration, lamina propria IL-22
messenger RNA (mRNA) expression was as-
sessed by quantitative PCR (qPCR) (A) and the
serum concentration of IL-22 was assessed by
ELISA (B) (n = 3–4 mice per group).
(C and D) Irradiated mice were reconstituted with
a 1:1 mixture of IL-23p19-deficient (Il23a?/?) BM
cells and CD11c.DTR BM cells to generate mixed
BM chimeric mice, which were subsequently
depleted of IL-23p19-sufficient dendritic cells
by treatment with diphtheria toxin as described
in Experimental Procedures. Three hours after
mRNA expression was assessed by qPCR (C) and
the serum concentration of IL-22 was assessed
by ELISA (D) (n = 3–4 mice per group).
Error bars denote ±SEM. Data are representative
of two independent experiments.
Intestinal DC Subset Triggers IL-23-IL-22 Pathway
280 Immunity 36, 276–287, February 24, 2012 ª2012 Elsevier Inc.
CCR2.DTR mice when the toxin is administered over the course
of 6 days (Figures S5A–S5E). Ly6Chimonocytes express the
CCR2 receptor and thus are depleted in CCR2 mice; however,
monocyte-derived CD103?CD11b+LPDCs do not actively ex-
press CCR2 (Figure S5F), but because of the short turnover
rate of these cells, elimination of their precursor population
(Ly6Chimonocytes) leads to loss of monocyte-derived LPDCs
(Figure 5A). Although flagellin-mediated IL-12p40 and IL-23p19
induction was completely abrogated in DTX-treated CD11c.DTR
mice, we found that 80% depletion of CD103?CD11b+lamina
propria DCs in CCR2.DTR mice resulted in enhanced induction
of IL-12p40 (Figure 5B) and IL-23p19 (Figure 5C). Depletion of
CD11c+cells in CD11c.DTR mice did not deplete Ly6Chimono-
cytes, indicating that Ly6Chimonocytes did not directly drive
IL-23 induction (not shown). Consistent with the enhanced
induction of IL-23 in mice depleted of CD103?CD11b+lamina
propria DCs, we found that IL-22 expression was also increased
compared to the response observed in WT mice (Figure 5D).
Enhanced induction of IL-22 was not due to proliferation of
CD103+CD11b+LPDCs (Figures S5A–S5D). These results sug-
gest that CD103+CD11b+DCs orchestrate the IL-22 response
and that monocyte-derived CD103?CD11b+lamina propria
DCs play a more regulatory role.
To determine whether CD103+CD11b+LPDCs contribute to
flagellin-mediated induction of IL-23 in the lamina propria, we
evaluated flagellin responses in mice that lack Flt3L, which is
required for the development of CD103+CD11b+DCs but not
monocyte-derived DCs (Figure 5E). Consistent with a role for
CD103+CD11b+LPDCs in the flagellin-induced response, we
found that IL-12p40 (Figure 5F) and IL23p19 (Figure 5G)
induction were significantly reduced in Flt3L-deficient mice. In
Flt3L-deficient mice, we also observed impaired flagellin-
induced IL-22 and RegIIIg expression, indicating that CD103+
DCsplay a critical role in initiating IL-22-dependent antimicrobial
defense (Figures 5H–5J). This result was not a consequence of
impaired development of innate lymphoid cells in the absence
of Flt3L as shown by the fact that ILC populations in the lamina
propria were found intact in Flt3L-deficient mice (Figures S5H
and S5I). Although the degree of IL-23 induction was reduced
in Flt3L-deficient mice, a greater than 90% reduction in CD103+
CD11b+LPDCs reduced IL-12p40 and IL-23p19 by only 60%
and 80%, respectively. This result suggests that Flt3L-indepen-
dent cell populations, particularly in the setting of congenital
Flt3L deficiency, may also contribute to the production of IL-23
after flagellin administration.
IL-10 production from monocyte-derived DCs and macro-
phages have been shown to limit inflammatory responses in
the intestinal lamina propria (Takeda et al., 1999). To determine
whether IL-10 expression by monocytes or monocyte-derived
DCs restricts the responsiveness of CD103+CD11b+DCs to
TLRstimulation, wegenerated mixed BMchimeric mice in which
CCR2.DTR bone marrow cells were combined in equal parts
with either WT bone marrow cells or IL-10-deficient bone
marrow cells and transferred into lethally irradiated WT recipi-
ents. Depletion of WT but not IL-10-deficient monocyte-derived
DCs did not affect the flagellin-mediated IL-22 induction
compared to control mice (Figure S5G). These data suggest
that the hyperresponsiveness of CD103+CD11b+LPDCs that
we observed is not due to the loss of IL-10 produced by mono-
cyte-derived DCs and their precursors.
TLR5-Expressing CD103+CD11b+LPDCs Upregulate
IL-23 in Response to Stimulation with Flagellin
To determine whether TLR5 is differentially expressed by lamina
propria DC subsets, we sorted CD103+CD11b+LPDCs and
CD103?CD11b+LPDCs (Figure 6A) and quantified TLR5 mRNA
transcripts in each population by qPCR (Figure 6B). Both DC
population of lamina propria cells, but we detected more TLR5
mRNA transcripts in CD103+CD11b+DCs compared to CD103?
CD11b+DCs as previously demonstrated (Uematsu et al., 2008).
To further evaluate IL-23 expression by lamina propria DCs,
we individually stimulated sorted populations of CD103+CD11b+
LPDCs and CD103?CD11b+LPDCs with flagellin in vitro.
Flagellin induced the expression of IL-12p40 (Figure 6C) and
IL-23p19 (Figure 6D) mRNA transcripts in CD103+CD11b+DCs
but not CD103?CD11b+DCs.
In order to detect IL-23-expressing DC subsets in vivo, we
evaluated the flagellin response in reporter mice that express
YFP under control of the endogenous IL-12p40 promoter (Rein-
hardt et al., 2006). Because IL-12p35 is not upregulated during
the time frame of the experiment, IL-12p40-driven YFP expres-
sion predominantly reflects induction of IL-23. We detected no
increase in YFP+CD103+CD11b?LPDCs after flagellin adminis-
tration (Figure 6E). Consistent with the previously presented
data of sorted LPDCs stimulated in vitro, the percentage of YFP+
CD103+CD11b+LPDCs dramatically increased from 0.5% to
34% after stimulation of TLR5 in vivo (Figure 6E). In contrast,
the percentage YFP+CD103?CD11b+LPDCs increased by
only three percentage points after flagellin administration; how-
ever, 10% of these cells were YFP+under steady-state condi-
tions, indicating that this population constitutively expresses
IL-12p40 as previously described (Becker et al., 2003). Collec-
tively, these data demonstrate that CD103+CD11b+LPDCs are
the primary source of flagellin-mediated IL-23 expression and,
therefore, play a critical role in inducible IL-22 expression in
the intestinal lamina propria (Figure S6).
The luminal contents of the intestine are separated from the
vasculature, connective tissue, and immune cells of the lamina
propria by a single layer of epithelial cells. Interruption of this
barrier represents a threat to the mammalian host and results
in rapid and robust inflammatory responses that prevent
systemic dissemination of intestinal bacteria. Upon introduction
of bacterial flagellin into the lamina propria, CD103+CD11b+
LPDCs activate antimicrobial inflammatory responses by rapidly
producing IL-23, thereby stimulating ILCs to produce IL-22,
which promotes epithelial cell recovery and induces expression
of RegIIIg. Induction of IL-23 in CD103+CD11b+LPDCs occurs
within 15 to 30 min exposure of the lamina propria to flagellin
and is transient. Thus, it is unlikely that this early and transient
wave of IL-23 induction directly influences subsequent CD4
T cell differentiation into Th17 cells. Instead, TLR5-dependent
production of IL-23 by CD103+CD11b+LPDCs represents a
local, expeditious response that informs ILCs that the epithelial
Intestinal DC Subset Triggers IL-23-IL-22 Pathway
Immunity 36, 276–287, February 24, 2012 ª2012 Elsevier Inc. 281
Figure 5. Flt3L-Dependent CD103+CD11b–DCs, Not Monocyte-Derived DCs, Are Required for Flagellin-Mediated IL-23 Expression
(A) Wild-type (WT), CD11c.DTR, or CCR2.DTR bone marrow chimeric mice received 200 ng of diphtheria toxin by intraperitoneal injection every other day for
6 days. Mice then received 1 mg of flagellin (Flg) or PBS (Ctrl) by intravenous injection and were sacrificed 2 hr later. FACS analysis of purified small intestine (SI)
lamina propria cells, gated on CD45+cells, demonstrates depletion of dendritic cell populations after diphtheria toxin treatment. Numbers on dot plots indicate
the percentage of cells within the gate out of the parent population.
(B–D) Quantitative PCR (qPCR) was performed on SI lamina propria tissue from WT, CCR2.DTR, and CD11c.DTR mice to determine the relative expression of
IL-12p40 (B), IL-23p19 (C), and IL-22 (D) (n = 3 mice per group). Data are representative of two independent experiments.
(E–G) Flagellin or PBS was administered by i.v. injection to Flt3L-deficient (Flt3?/?) mice or WT control mice, and mice were sacrificed 1 hr after injection. Lamina
propria cells were isolated for FACS analysis of dendritic cell populations. Dot plots show cells that were gated on CD45+MHCII+CD11c+cells. The numbers on
the dot plot indicate the percentage of cells out of this population within the gate. SI lamina propria tissue was harvested for analysisof IL-12p40 (F) and IL-23p19
(G) mRNA transcripts by qPCR.
(H) To assess IL-22 mRNA transcript expression in the lamina propria, Flt3l?/?mice were sacrificed 2 hr postinjection with flagellin and processed for analysis
(I) To assess secretion of IL-22, Flt3l?/?mice were sacrificed 3 hr after flagellin administration and blood was collected for analysis of IL-22 protein concentration
(J) Flagellin-induced RegIIIg expression in the small intestine in Flt3l?/?mice was evaluated 24 hr after flagellin injection by qPCR (n = 3 mice per group).
The data shown are representative of two independent experiments. Error bars denote ±SEM.
Intestinal DC Subset Triggers IL-23-IL-22 Pathway
282 Immunity 36, 276–287, February 24, 2012 ª2012 Elsevier Inc.
barrier has been disrupted and that epithelial repair and the
production of antimicrobial factors must be initiated.
therapeutic purposes by administering flagellin systemically to
antibiotic-treated mice in order to restore intestinal epithelial
defenses against colonization with multidrug-resistant bacteria
(Kinnebrew et al., 2010). Stimulation of TLR5 by flagellin induces
intestinal RegIIIg expression and subsequently limits intestinal
colonization with vancomycin-resistant Enterococcus (VRE). In
patients susceptible to infections with antibiotic-resistant organ-
isms, intestinal colonization has been shown to precede blood
infection; therefore, efforts to reduce or prevent colonization
with these pathogens may provide an important prophylactic
therapy (Ubeda et al., 2010). In the current study, we have eluci-
dated a previously unappreciated role for IL-23 and CD103+
CD11b+dendritic cells in the regulation of TLR-induced RegIIIg
expression in the small intestine. A clear understanding of
the innate immune pathways that regulate intestinal mucosal
defenses may lead to the development of therapeutics that
broadly target antibiotic-resistant pathogens that colonize the
Prior studies have shown that homeostatic RegIIIg expres-
pathways in intestinal epithelial cells by microbial products shed
Figure 6. CD103+CD11b+DCs but not CD103–CD11b+DCs Express IL-23p19 and IL-12p40 in Response to TLR5 Stimulation
(A) Small intestinal lamina propria cells were isolated from six mice and pooled for FACS sorting. DAPI?CD45+CD11c+MHCII+lamina propria cells were FACS
sorted into CD103+CD11b+or CD103?CD11b+groups and individually assessed for purity by FACS analysis.
(B) TLR5 mRNA expression by quantitative PCR.
(Cand D) Foldinductionof messenger RNA(mRNA) encoding for IL-23 protein subunits IL-12p40 (C) and IL-23p19(D) after 2hrof invitro incubationwithflagellin.
Data are representative of one out of four independent experiments. Error bars denote ±SEM.
(E) IL-12p40-YFP reporter mice received 1 mg of flagellin intravenously. Three hours after flagellin injection, mice were sacrificed and lamina propria cells were
harvested for FACS analysis. Dot plots show cells gated by CD45+CD11c+MHCII+. DC subsets were analyzed for YFP expression in the following three groups:
CD103+CD11b?, CD103+CD11b+, and CD103?CD11b+LPDCs. Numbers are percentage of gated cells out of the parent population (n = 3 mice per group).
Intestinal DC Subset Triggers IL-23-IL-22 Pathway
Immunity 36, 276–287, February 24, 2012 ª2012 Elsevier Inc. 283
by the intestinal microbiota (Brandl et al., 2008; Vaishnava et al.,
2008). However, during active inflammation, IL-22 is required to
stimulate RegIIIg expression in intestinal epithelial cells (Kinne-
brew et al., 2010; Zheng et al., 2008). Thus, it is possible that
IL-22-mediated RegIIIg induction provides an independent
mechanism by which immune cells can regulate antimicrobial
expression under threat of invasion by enteric pathogen. How-
ever, although the intestinal epithelial barrier is intact, microbial
products from the intestinal microbiota provide signals directly
to IECs leading to cell-intrinsic MyD88-mediated RegIIIg expres-
sion. Yet there is some evidence that these pathways are not
completely independent avenues for regulating RegIIIg expres-
sion. Antibody blockade of IL-22 signaling leads to the downre-
gulation of RegIIIg expression in normal mice under steady-state
conditions (Sanos et al., 2009). In addition, RegIIIg expression is
markedly diminished in IL-22-deficient mice under steady-state
conditions and is not further reduced by antibiotic-mediated
depletion of the microbial flora (Kinnebrew et al., 2010). These
observations suggest that MyD88 and IL-22 are necessary
components of the pathway that regulates homeostatic RegIIIg
expression. The precise nature of the interaction between
MyD88 expression by intestinal epithelial cells and IL-22 expres-
sion by immune cells is unclear and requires further research.
A rapid, innate inflammatory role for CD103+CD11b+LPDCs
contrasts with the more widely appreciated tolerance-inducing
role attributed to this DC subset. CD103+CD11b+LPDCs induce
tolerogenic T cell responses by producing TGF-b and retinoic
acid (Coombes et al., 2007). Signals from the intestinal tissue
microenvironment, including epithelial cell-derived thymic stro-
mal lyphopoietin (TSLP) and bile acid retinoids, condition
CD103+CD11b+LPDCs to preferentially generate T regulatory
cells once these DCs have trafficked to the mesenteric lymph
nodes (Manicassamy and Pulendran, 2011). However, in the
setting of chronic colitis, CD103+CD11b+LPDCs are activated
and acquire a proinflammatory phenotype (Laffont et al., 2010).
Our experiments demonstrate that in healthy mice under
steady-state conditions, CD103+CD11b+LPDCs can rapidly
produce IL-23 in response to microbial products, despite
homeostatic conditioning toward a tolerizing state by the tissue
Determining the distinct functions of the different populations
of lamina propria DCs is challenging because LPDCs are difficult
to harvest from the network of connective tissue that forms
the lamina propria. Most experiments characterizing the func-
tions of LPDC subsets have been performed in vitro, requiring
isolation of LPDCs. After lengthy incubation periods and
mechanical and proteolytic disruption of the lamina propria
extracellular matrix and exposure to luminal bacteria, ‘‘freshly’’
isolated LPDCs have almost certainly been altered. Procedural
and microbiota differences, therefore, might explain differences
in the functions assigned to distinct LPDC populations by
different laboratories (Pabst and Bernhardt, 2010; Varol et al.,
2010). We demonstrate here that IL-23 is required for flagellin-
mediated IL-22 expression in vivo. Uematsu et al. (2008) charac-
terized TLR5-expressing CD103+CD11b+LPDCs from the
lamina propria and found that they specifically promote IgA
production and Th17 cell differentiation. The Th17 cell response
DCs to produce IL-6. In contrast, IL-23 was not detected in the
supernatants of flagellin-stimulated CD103+CD11b+DCs. The
differences in results may be due to the DC isolation technique
and whether in vitro or in vivo experiments were performed.
Because IL-23 can drive the development and persistence of
colitis, mechanisms to limit IL-23 expression in the intestine
are critical for intestinal homeostasis (Hue et al., 2006). How
IL-23 responses are controlled is not completely understood.
We show that CD103+CD11b+LPDCs rapidly produce IL-23
upon TLR stimulation. Specific depletion of monocytes and
monocyte-derived DCs leads to enhanced flagellin-mediated
IL-23 and IL-22 expression, suggesting that these cells play
a role in limiting the responsiveness of CD103+CD11b+LPDCs
to TLR5 stimulation. However, the regulatory role of monocytes
IL-10. Whether monocytes or monocyte-derived dendritic cells
directly inhibit IL-23 expression in CD103+DCs via inhibitory
cytokines or indirectly through stimulation of another cell type,
such as T regulatory cells, requires further investigation.
IL-22 has been implicated as an effector cytokine in defense
against mucosal pathogens, but the signals and cells that initiate
IL-22 expression are currently unknown. During Citrobacter
rodentium infection of the gut, IL-23-dependent IL-22 expres-
sion by ILCs is required for clearance of the pathogen (Sonnen-
berg et al., 2011; Zheng et al., 2008). These previous studies,
although demonstrating that IL-22 or IL-23 deficiency result in
enhanced in vivo growth of C. rodentium over the course of 1
to 2 weeks, did not identify the cell populations that produce
IL-23 during infection. Control of Candida albicans infection of
gastrointestinal tract has also been shown to require early acti-
vation of the IL-23-IL-22 axis (De Luca et al., 2010). Further
research is needed to determine the cells responsible for IL-23
expression and whether TLR signaling is required for IL-22-
mediated defense in these infection models.
Flagellin is a potential immunomodulatory agent that activates
innate mucosal defense. TLR5 is restricted to certain tissues,
with the highest expression occurring in the small intestine
(Uematsu et al., 2006). Although TLR5 expression on the baso-
lateral side of intestinal epithelial cells has been reported,
CD103+CD11b+LPDCs express the highest amounts of TLR5
(Uematsu et al., 2008). Subepithelial sensing of flagellin by
TLR5-expressing CD103+CD11b+DCs probably plays animpor-
tant role in keeping commensal intestinal bacteria at bay by
forming an important layer of defense when microperforation
of the intestinal epithelium occurs. Commensal microbes colo-
nizing the intestinal lumen are a major source of TLR ligands,
including flagellin. Crohn’s disease patients, for example, suffer
from frequent damage to the intestinal epithelium, and the domi-
nant antigen against which mucosal adaptive responses are
directed is flagellin derived from commensal bacteria residing
in the cecum (Lodes et al., 2004). The roles of IL-22 in wound
segregation of gut microbes from the intestinal epithelial cells
(Vaishnava et al., 2011) support the idea that the rapid response
of CD103+CD11b+DCs to TLR stimulation is important for the
repair of the intestinal epithelial barrier.
cells stimulates RegIIIg expression in intestinal epithelial cells
through a multicellular model in which TLR5-stimulated dendritic
cells produce IL-23 and ILCs respond by secreting IL-22. This
Intestinal DC Subset Triggers IL-23-IL-22 Pathway
284 Immunity 36, 276–287, February 24, 2012 ª2012 Elsevier Inc.
indirect mechanism of inducing RegIIIg in intestinal epithelial
cells is in contrast to previous work demonstrating that direct,
microbiota-driven activation of TLR-MyD88 signaling pathways
within intestinal epithelial cells drives RegIIIg under steady-state
the microbial molecules, innate immune receptors, and signaling
pathways that drive RegIIIg expression during homeostasis is an
important topic for future research.
Mice and Reagents
Wild-type C57BL/6 mice were purchased from Jackson Labs. TLR5-deficient,
IL-22-deficient, AHR-deficient, and IL-23p19-deficient mice were obtained as
previously described (Fernandez-Salguero et al., 1995; Ivanov et al., 2009;
Kinnebrew et al., 2010; Zenewicz et al., 2007). Flt3L-deficient mice were
purchased from Taconic Farms. IL-12p40-YFP reporter mice and IL-10-
deficient mice were purchased from Jackson Laboratories. TLR5-deficient
mice were crossed to the Ly5.1+(CD45.1) C57BL/6 background. The genera-
tionof CCR2.DTR mice has been previously described (Hohl et al., 2009).Mice
were maintained in a specific-pathogen-free barrier facility at Memorial Sloan-
Kettering Cancer Institute and experiments were performed according to insti-
tution-approved animal protocols. In experiments where the flagellin response
was assessed in vivo, 1 mg of ‘‘ultrapure’’ flagellin (Invivogen), derived from
Salmonella typhimurium, was injected intravenously through the tail vein.
Control mice received PBS. In vitro stimulation of single-cell suspensions
from the lamina propria was performed with a concentration of 200 ng/ml of
flagellin. Diphtheria toxin (List Biological Laboratories) was administered every
other day at a dose of 10 ng/g starting 6 days prior to injection of flagellin. Mice
received a total of three doses.
Generation of Bone Marrow Chimeric Mice
Six-week-old C57BL/6 mice were lethally irradiated with 950 cGy with a
137Cssource.Within24hrofirradiation,micereceived an intravenousinjection
ofa1:1mixtureof2.53106congenically markedbonemarrow cellsharvested
from the femurs and tibias of wild-type C57BL/6, TLR5-deficient, IL-22-
deficient, IL-23p19-deficient, IL-10-deficient, CD11c.DTR, or CCR2.DTR
mice as indicated in the figures. Mice were allowed to rest for at least 7 weeks
after irradiation to allow for reconstitution of the hematopoietic compartment
before being used for experiments.
Real-Time PCR Analysis
For measurement of RegIIIg expression in the proximal small intestine, a 2 cm
segment located 1 cm distal to the pylorus was excised and preserved in
RNAlater (Ambion) at ?20?C. For cytokine expression in lamina propria tissue,
a 2 cm segment of the proximal small intestine was isolated. Peyer’s patches
were excised from this segment and the remaining tissue was washed with
PBS and incubated for 10 min in a 10 mM buffer of EDTA in HBSS at 37?C
to dissociate the epithelial cells from the lamina propria. The lamina propria
tissue was pulse-vortexed and washed two times in PBS. Tissues were
homogenized in 1 ml of Trizol (Invitrogen) for 2:15 min with the Mini-
Beadbeater-16 (Biospec). The manufacturer’s protocol was followed for
RNA extraction from tissues homogenized in Trizol. RNA was isolated from
single-cell suspensions with the RNAeasy Mini Kit (QIAGEN). Isolated RNA
was reverse-transcribed with the Quantitect Reverse Transcription Kit
(QIAGEN). Gene expression was assessed by quantitative real-time PCR
with Taqman Expression Assay predesigned probes (Applied Biosystems)
and the Step One Plus Real Time PCR system (Applied Biosystems). Signals
were normalized to Hprt1 mRNA expression. Normalized values were used
to calculate relative expression with DDCtanalysis. Taqman Expression Assay
IDs are listed in Table S1.
Detection of Cytokines by ELISA
Mice were anesthetized with isofluorane. Blood was collected retro-orbitally
with a glass Pasteur pipet. Blood was stored at 4?C overnight to allow for
clotting. IL-22 protein was measured in blood serum or lamina propria cell
culture supernatants with an IL-22 ELISA Construction Kit (Antigenix).
Isolation of Lamina Propria Cells
The small intestine was carefully separated from the mesentery. Peyer’s
patches were excised, and the intestine was opened longitudinally and
washed twice in PBS. Epithelial cells were separated from the underlying
lamina propria by incubation in HBSS containing 10 mM EDTA for 10 min at
37?C with gentle rotation in a water bath shaker (125 rpm). Lamina propria
tissuewaspulse-vortexed and washed twotimes inPBS. Theremainingtissue
was finely chopped with a razor blade and digested in a solution of 0.5 mg/ml
Collagenase type IV (Worthington), 40 mg/ml DNase I, and 8% fetal calf serum
in HBSS for 20 min at 37?C with gentle rotation in a shaker (125 rpm). Tissue
digestion was repeated two times. Leukocytes were isolated from the super-
natant with a Percoll (MP Biomedicals) gradient separation method in which
the cells were resuspended in 40% Percoll and underlayered with 80% Percoll
followed by centrifugation at 2,500 rpm for 20 min. The interface was collected
for FACS analysis or FACS sorting. Cells numbers were determined with a Z2
Coulter counter (Beckman Instruments).
Antibodies and Flow Cytometry
The following antibodies were purchased from BD Biosciences: anti-CD45.2
(104, Alexa700 or PerCP Cy5.5); anti-CD45.1 (A20, PE or APC); anti-CD11c
(HL3, APC, or PE-Cy7); anti-CD11b (M1/70, PerCP-Cy5.5); anti-CD45
(30-F11, PE-Cy7); anti-THY1 (53-2.1, APC); anti-CD4 (RM4-5, Pacific Blue).
The following antibodies were purchased from eBioscience: MHC class II
(M5/114.15.2, Alexa700; eBioscience); anti-CD103 (OX62, PE); anti-CD45
(30-F11, Pacific Blue); anti-NKp46 (29A1.4, PE); anti-CD3 3
PE-Cy7); anti-IL-22 (IL22JOP, APC). A BD LSR II flow cytometer was used
for FACS analysis. Lamina propria dendritic cell subsets were sorted with a
FACSAria (BD Biosciences) to 95%–99% purity. DAPI (Sigma) was used to
distinguish live cells from dead cells during cell sorting. Purified, carrier-free
anti-IL-6R (10 mg/ml, BD PharMingen), IL-1Ra (10 mg/ml, R&D), anti-IL-23p19
(10 mg/ml, eBioscience) in the presence or absence of flagellin were used to
block cytokine signaling in overnight cell cultures of cells purified from the
lamina propria. Recombinant mouse IL-23 (rmIL-23) (40 ng/ml, eBioscience)
was used to stimulate IL-22 secretion by lamina propria cells in vitro. Intracel-
lular cytokine staining for IL-22 was performed on freshly isolated lamina prop-
ria cells that were stimulated in vitro with rmIL-23 (40 ng/ml, eBioscience) for
2.5 hr in the presence of Brefeldin A.
Statistical analysis was conducted with GraphPad Prism software. Unless
otherwise noted, the Student’s unpaired t test was used as statistical test
for significance. p values < 0.05 were considered significant. Error bars
denote ± SEM.
Supplemental Information includes six figures and one table and can be found
with this article online at doi:10.1016/j.immuni.2011.12.011.
This work was supported by NIH grants RO1-AI042135, R37-AI039031, and
PO1-CA023766 to E.G.P. M.A.K. and C.G.B. were supported by NIH Medical
Scientist Training Program grant GM07739 to Cornell/RU/MSKCC Tri-Institu-
tional MD-PhD Program. NIH K08 Fellowship AI07998 supported T.M.H.
Received: June 11, 2011
Revised: October 31, 2011
Accepted: December 6, 2011
Published online: February 2, 2012
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