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J. Exp. Med. Vol. 206 No. 13 3047-3059
Within 8 d after peroral infection with Toxo-
plasma gondii, susceptible C57BL/6 mice develop
massive necrosis in the ileum, leading to death
(Liesenfeld et al., 1996). T. gondii–induced ile-
itis is characterized by a CD4 T cell–dependent
overproduction of proinflammatory mediators,
including IFN-, TNF, and NO (Khan et al.,
1997; Mennechet et al., 2002). Activation of
CD4+ T cells by IL-12 and IL-18 is critical for
the development of small intestinal pathology
(Vossenkämper et al., 2004). Recently, we
showed that LPS derived from gut flora via Toll-
like receptor (TLR)–4 mediates T. gondii–in-
duced immunopathology (Heimesaat et al., 2006).
Thus, the immunopathogenesis of T. gondii–
induced small intestinal pathology resembles
key features of the inflammatory responses in
inflammatory bowel disease (IBD) in humans
and in models of experimental colitis in rodents
(Liesenfeld, 2002). However, most animal models
Abbreviations used: HPRT,
transferase; IBD, inflammatory
bowel disease; MLN, mesen-
teric LN; MMP,
M. Muñoz and M.M. Heimesaat contributed equally to this
Interleukin (IL)-23 mediates Toxoplasma
gondii–induced immunopathology in the gut
via matrixmetalloproteinase-2 and IL-22
but independent of IL-17
Melba Muñoz,1 Markus M. Heimesaat,1 Kerstin Danker,3 Daniela Struck,1
Uwe Lohmann,1 Rita Plickert,1 Stefan Bereswill,1 André Fischer,1
Ildikò Rita Dunay,1,13 Kerstin Wolk,4 Christoph Loddenkemper,2
Hans-Willi Krell,5 Claude Libert,6 Leif R. Lund,7 Oliver Frey,8
Christoph Hölscher,9 Yoichiro Iwakura,10 Nico Ghilardi,11 Wenjun Ouyang,12
Thomas Kamradt,8 Robert Sabat,4 and Oliver Liesenfeld1
1Institute of Microbiology and Hygiene and 2Department of Pathology/Research Center ImmunoSciences, Campus Benjamin
Franklin, Charité Medical School, 12203 Berlin, Germany
3Institute of Biochemistry and 4Interdisciplinary Group of Molecular Immunopathology, Dermatology/Medical Immunology,
Campus Mitte, Charité Medical School, 10117 Berlin, Germany
5Roche Diagnostics GmbH, 82377 Penzberg, Germany
6Molecular Mouse Genetics, Department for Molecular Biomedical Research, Flanders Institute for Biotechnology, Ghent
University, 9052 Ghent, Belgium
7Department of Cellular and Molecular Medicine, Faculty of Health Science, University of Copenhagen, 2200 Copenhagen, Denmark
8Institute of Immunology, School of Medicine, Friedrich Schiller University Jena, 07743 Jena, Germany
9Infection Immunology, Research Center Borstel, 23845 Borstel, Germany
10Center for Experimental Medicine, Institute of Medical Science, University of Tokyo, Tokyo 108-8639, Japan
11Molecular Biology Department and 12Immunology Department, Genentech, Inc., South San Francisco, CA 94080
13Department of Neuropathology, University of Freiburg, 79106 Freiburg, Germany
Peroral infection with Toxoplasma gondii leads to the development of small intestinal
inflammation dependent on Th1 cytokines. The role of Th17 cells in ileitis is unknown.
We report interleukin (IL)-23–mediated gelatinase A (matrixmetalloproteinase [MMP]-2)
up-regulation in the ileum of infected mice. MMP-2 deficiency as well as therapeutic or
prophylactic selective gelatinase blockage protected mice from the development of
T. gondii–induced immunopathology. Moreover, IL-23–dependent up-regulation of IL-22
was essential for the development of ileitis, whereas IL-17 was down-regulated and dis-
pensable. CD4+ T cells were the main source of IL-22 in the small intestinal lamina propria.
Thus, IL-23 regulates small intestinal inflammation via IL-22 but independent of IL-17.
Gelatinases may be useful targets for treatment of intestinal inflammation.
© 2009 Muñoz et al. This article is distributed under the terms of an Attribu-
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The Journal of Experimental Medicine
IL-23–mediated T. gondii–induced immunopatholgy | Muñoz et al.
IL-22 was mostly produced by CD4+ T cells in the small in-
testinal lamina propria.
IL-23 mediates T. gondii–induced small intestinal
We have previously demonstrated that peroral infection of
susceptible C57BL/6 mice with 100 cysts of T. gondii ME49
leads to Th1-type small intestinal necrosis (panileitis; Liesenfeld
et al., 1996). To investigate the role of IL-23 in the develop-
ment of ileal immunopathology, IL-23p19/ mice were in-
fected orally. All WT mice died within 10 d after infection,
whereas 80% of IL-23p19/ mice survived the acute phase of
infection (Fig. 1 A). Severe necrosis of the villi and mucosa
was observed in the ilea of WT mice at 8 d after infection,
whereas IL-23p19/ mice showed mild to moderate signs
of small intestinal inflammation but no necrosis (Fig. 1 B).
Histopathological scores in the ilea of IL-23p19/ mice were
significantly lower compared with WT mice (Fig. 1 C). Nei-
ther the amount of T. gondii DNA nor the number of para-
sitophorous vacuoles containing T.gondii tachyzoites in the
ileum differed significantly between both groups at the same
time point (Fig. 1 D). Furthermore, no differences were ob-
served in the number of inflammatory foci in the parenchyma
and perivascular areas of the liver in WT and IL-23p19/
–infected mice (unpublished data). There was a significant in-
crease in IL-23p19 mRNA expression in the ileum at day 3
after infection, but levels decreased to those observed in naive
mice by day 5 after infection (Fig. 1 E). IL-23 concentrations
were below the limit of detection in the ileum at all time
points (unpublished data). In mesenteric LNs (MLNs) of naive
mice and at day 3 after infection, IL-23 concentrations were
below the limit of detection; however, IL-23 concentrations
increased markedly on day 5 after infection (Fig. 1 E). Inter-
estingly, CD11b+ but not CD11c+ cells were the main source
of IL-23p19 in the small intestinal lamina propria (Fig. S1).
MMP-2 and MMP-9 are up-regulated in the small intestine
of T. gondii–infected mice
Given that MMPs have been proposed to play an important
role in the pathology of IBD (Gao et al., 2005), we deter-
mined the enzymatic activity of gelatinases A (MMP-2) and
B (MMP-9) in the small intestine of T. gondii–infected WT
mice using gelatin zymography. The activity of both MMP-2
and MMP-9 markedly increased after infection and peaked at
day 5 after infection (the time point when intestinal pathol-
ogy starts to develop; Fig. 2 A). An additional band charac-
teristic of active MMP-2 was also observed at the same time
point. Because IL-23 was shown to induce the expression of
MMP-9, we investigated the enzymatic activity of MMP-9
and MMP-2 in the ileum of WT and IL-23p19/ mice be-
fore and after T. gondii infection. The enzymatic activity of
MMP-9 and MMP-2 was lower in IL-23p19/ compared
with WT mice (Fig. 2 B); furthermore, the band of active
MMP-2 observed in WT mice after infection was absent in
IL-23p19/ animals at day 8 after infection.
of IBD assessed inflammatory responses in the large intestine,
and models of small intestinal pathology are scarce (Kosiewicz
et al., 2001; Strober et al., 2002; Olson et al., 2004; Heimesaat
et al., 2006).
IL-12 shares the p40 subunit, IL-12R1, and components
of the signaling transduction pathways with IL-23 (Parham
et al., 2002). There is strong evidence that IL-23, rather than
IL-12, is important in the development of colitis (Yen et al.,
2006). The association of IL-23R encoding variant Arg381Gln
with IBD (Duerr et al., 2006) and the up-regulation of
IL-23p19 in colon biopsies from patients with Crohn’s dis-
ease (Schmidt et al., 2005) underline the importance of IL-23
in intestinal inflammation. Effector mechanisms of IL-23 in-
clude the up-regulation of matrixmetalloproteinases (MMPs;
Langowski et al., 2006), a large family of endopeptidases that
mediate homeostasis of the extracellular matrix. MMPs were
significantly up-regulated in experimental models of colitis
(Tarlton et al., 2000; Medina et al., 2003) and in colonic tis-
sues of IBD patients (von Lampe et al., 2000).
Studies in mouse models of autoimmune diseases have
associated the pathogenic role of IL-23 with the accumu-
lation of CD4+ T cells secreting IL-17, termed Th17 cells
(Aggarwal et al., 2003; Cua et al., 2003). Moreover, increased
IL-17 expression was reported in the intestinal mucosa of
patients with IBD (Fujino et al., 2003; Nielsen et al., 2003;
Kleinschek et al., 2009).
In addition to IL-17, Th17 cells also produce IL-22, a
member of the IL-10 family (Dumoutier et al., 2000). IL-22,
although secreted by certain immune cell populations, does
not have any effects on immune cells in vitro or in vivo but
regulates functions of some tissue cells (Wolk et al., 2009).
Interestingly, IL-22 has been proposed to possess both pro-
tective as well as pathogenic roles. In fact, IL-22 mediated
psoriasis-like skin alterations (Zheng et al., 2007; Ma et al.,
2008; Wolk et al., 2009). In contrast, IL-22 played a protec-
tive role in experimental models of colitis (Satoh-Takayama
et al., 2008; Sugimoto et al., 2008; Zenewicz et al., 2008;
Zheng et al., 2008), in a model of Klebsiella pneumoniae infec-
tion in the lung (Aujla et al., 2007), and against liver damage
caused by concanavalin A administration (Radaeva et al.,
2004; Zenewicz et al., 2007). IL-22 has been reported to be
produced by CD4+ T cells (Wolk et al., 2002; Zheng et al.,
2007), cells (Zheng et al., 2007), CD11c+ cells (Zheng
et al., 2008), and natural killer cells (Cella et al., 2008; Luci
et al., 2008; Sanos et al., 2009; Satoh-Takayama et al., 2008;
Zheng et al., 2008). The role of IL-22 in small intestinal in-
flammation remains to be determined.
In the present study, we investigated the role of the
IL-23–IL-17 axis in T. gondii–induced small intestinal immuno-
pathology. We show that IL-23 is essential in the develop-
ment of small intestinal immunopathology by inducing local
MMP-2 up-regulation that could be inhibited by prophy-
lactic or therapeutic chemical blockage. Interestingly, IL-23–
dependent IL-22 production was markedly up-regulated and
essential for the development of ileal inflammation, whereas
IL-17 production was down-regulated after T. gondii infection.
JEM VOL. 206, December 21, 2009
respectively, whereas MMP-2/ mice displayed significantly
less weight loss (9.4 ± 4.3%; Fig. 3 A). Furthermore, infection
resulted in significantly less shortening of the small intestinal
length in MMP-2/ compared with MMP-9/ and WT
mice on day 8 after infection (9.3 ± 7.4% vs. 26.4 ± 8.7 and
27.7 ± 10.1%, respectively (Fig. 3 B). Although WT and
MMP-9/ mice displayed severe necrosis at 8 d after infec-
tion, only mild inflammatory changes but no necrosis were
observed in the small intestine of MMP-2/ mice (Fig. 3 C).
The number of parasitophorous vacuoles containing T. gondii
tachyzoites in the ileum did not differ significantly between
the groups at the same time point (Fig. 3 D). Ileal immuno-
pathology in MMP-9/ and WT mice was accompanied by
significantly elevated NO and IFN- levels in culture superna-
tants of ileal biopsies compared with MMP-2/ mice (Fig. 3, E
and F, respectively). All MMP-9/ as well as WT mice had
died by day 9 after infection, whereas 82% of MMP-2/
The increased enzymatic activity of gelatinases in the
ileum of infected WT but not IL-23p19/ mice was paral-
leled by increased mRNA and protein levels of either gelati-
nase at day 8 after infection compared with naive WT mice
(Fig. 2, C and D). These results indicate that IL-23 is re-
quired for the up-regulation of both MMP-2 and MMP-9 in
T. gondii–induced ileitis.
Small intestinal immunopathology is mediated
by MMP-2 but not MMP-9
To investigate whether IL-23–dependent up-regulation of
MMP-2 and MMP-9 is critical for the development of
T. gondii–induced ileitis, we determined the mortality, clini-
cal conditions, and histopathological changes in the ileum of
WT, MMP-2/, and MMP-9/ mice after T. gondii infec-
tion. On day 8 after infection, MMP-9/ and WT mice had
lost 19.3 ± 4.3% and 20.3 ± 3.1% of their body weight,
Figure 1. IL-23 mediates T. gondii–induced immunopathology in the gut. (A) Survival of WT and IL-23p19/ mice after oral infection with 100
cysts of T. gondii. (B) Histopathology of hematoxylin and eosin–stained ileal sections of naive and infected (Inf.) WT and IL-23p19/ mice 8 d after infec-
tion. (C) Histopathology scores of the ileum of naive and infected WT and IL-23p19/ mice. The horizontal line indicates the border between mild inflam-
mation (<3) and necrosis (>3). (D) T. gondii DNA concentration in ileal biopsies of WT and IL-23p19/ mice 8 d after infection (top), and the number of
T. gondii parasitophorous vacuoles that contained tachyzoites stained with a rabbit anti–T. gondii IgG antibody and counted in the terminal ileum in WT
and IL-23p19/ mice 8 d after infection (bottom). (E) RT-PCR of IL-23p19 mRNA of ileal biopsies of WT mice at different time points after infection (top).
Results are expressed as fold changes relative to HPRT mRNA expression. IL-23p19 concentration in supernatants of MLN of WT mice detected by ELISA
(bottom). Data (one representative out of three independent experiments is shown) from five mice per group are given as means ± SD, and p-values were
determined by the Mann-Whitney U test or by Kaplan-Meier analysis and the log-rank test (A). Bar, 100 µm.
IL-23–mediated T. gondii–induced immunopatholgy | Muñoz et al.
not differ between neutrophil-depleted (anti-Gr1 mAb)
and control WT mice (Fig. S3). Collectively, MMP-2 but not
MMP-9 appears to be an essential downstream mediator of
immunopathology in T. gondii–induced ileitis.
A selective gelatinase inhibitor protects mice
against T. gondii–induced immunopathology
Because MMP-2 mediates the development of necrosis in
the small intestine after T. gondii infection, we investigated the
efficacy of gelatinase-blocking drugs to prevent or treat the
mice survived the acute phase of infection and 40% survived
for >3 wk (Fig. S2). Interestingly, MMP-2/ mice that sur-
vived the acute phase of infection did not show signs of intes-
tinal inflammation around the time of death (unpublished
data), indicating that the deficiency in MMP-2 does not delay
but prevents the development of intestinal pathology. Immuno-
histochemical staining for MMP-2 in the ileum of naive and
infected WT and MMP-2/ mice indicates that cells in the
lamina propria produce MMP-2 (Fig. S2). Granulocytes did
not seem to secrete MMP-2, because ileal MMP-2 levels did
Figure 2. MMP-2 and MMP-9 are up-regulated in the small intestine of T. gondii–infected mice. (A) MMP-9 and MMP-2 enzymatic activity
was measured by gelatin zymography in the ileum of WT-infected mice at different time points after infection. (B) MMP-9 and MMP-2 enzymatic activ-
ity measured in the ileum of WT and IL-23p19/ mice 8 d after infection by gelatin zymography. (C) mRNA of MMP-9 and MMP-2 in ileal biopsies of
naive and infected (Inf.) WT and IL-23p19/ mice 8 d after infection measured by quantitative real-time PCR. Results are expressed as fold changes
relative to HPRT mRNA expression. (D) MMP-9 and MMP-2 concentrations in supernatants of ileal biopsies from naive and infected WT and IL-23p19/
mice detected by ELISA 8 d after infection. Data (one representative out of two independent experiments is shown) of three mice per group are given as
means ± SD, and p-values were determined by the Mann-Whitney U test.
JEM VOL. 206, December 21, 2009
Differential regulation of IL-17 and IL-22 after infection
with T. gondii
Because IL-23 was required for small intestinal pathology but
MMP-2/ mice still show significant mortality but rather
mild pathological changes in the small intestine, we investi-
gated the expression of IL-23–dependent cytokines involved
in mucosal inflammatory responses. In naive mice, IL-17
mRNA was similarly expressed in both WT and IL-23p19/
mice but was absent in IL-17/ mice (Fig. 4 A). Interest-
ingly, IL-17 was markedly down-regulated in WT and IL-
23p19/ mice 8 d after infection, although no difference was
detected between both groups (Fig. 4 A). In contrast, we did
not detect IL-22 mRNA in naive and infected IL-23p19/
mice, but IL-22 mRNA was up-regulated in infected WT
mice and detectable at similar levels in naive and infected
IL-17/ mice (Fig. 4 A). Moreover, IL-6 mRNA was not
detected in any group of naive mice, whereas infected WT
mice displayed higher IL-6 mRNA expression compared with
IL-23p19/– and IL-17/–infected mice (Fig. 4 A). Com-
parable levels of IL-1 mRNA were found in all naive groups;
upon infection, IL-1 transcripts increased significantly in
infected WT mice compared with IL-23p19/– and IL-17/–
infected mice (Fig. 4 A). The expression of IL-12p35 mRNA
was comparable in naive WT and IL-23p19/ mice but
undetectable in naive IL-17/ mice. However, IL-12p35
mRNA was significantly up-regulated in infected WT mice
as compared with both infected IL-23p19/ and IL-17/
mice (Fig. 4 A). Furthermore, levels of IFN- and IL-10
development of immunopathology. A nonselective MMP
inhibitor (doxycycline) as well as a more “selective” gelatinase
inhibitor, RO28-2653, were evaluated. Infected mice treated
prophylactically with RO28-2653 survived longer than
PBS- or doxycycline-treated animals (Fig. S4 A). Mice treated
prophylactically with either doxycycline or RO28-2653 dis-
played significantly less weight loss as well as less shortening
of the small intestine as compared with the PBS control group
on day 8 after infection (Fig. S4, B and C). The lengths of
small intestines did not differ between individual treatment
groups (Fig. S4 C).
Although PBS-treated mice displayed severe ileal necrosis,
mice treated prophylactically or therapeutically with doxycy-
cline showed mild signs of inflammation but no necrosis on
day 8 after infection (Fig. S4 D). Moreover, therapeutic treat-
ment (initiated 5 d after ileitis induction) with RO28-2653
led to an even more pronounced amelioration of inflamma-
tion than doxycycline treatment, and mice only showed
minor signs of inflammation (Fig. S4 D). Neither doxycycline
nor RO28-2653 affected the numbers of parasites in the small
intestinal lamina propria after infection (unpublished data). In
addition, significantly lower NO as well as IFN- levels were
found in the ilea of mice prophylactically treated with either
doxycycline or RO28-2653 compared with the PBS-treated
group on day 8 after infection (Fig. S4, E and F). Collectively,
these results indicate that prophylactic and therapeutic treat-
ment with selective gelatinase inhibitors prevents the devel-
opment of T. gondii–induced small intestinal necrosis.
Figure 3. Small intestinal immunopathology is mediated by MMP-2 but not MMP-9. (A) Relative body weight loss of WT (n = 43), MMP-9/ (n =
28), and MMP-2/ (n = 11) mice 8 d after infection. (B) Relative shortening of small intestine of WT (n = 13), MMP-9/ (n = 6), and MMP-2/ (n = 11)
mice 8 d after infection. (C) Histopathology score of WT (n = 42), MMP-9/ (n = 17), and MMP-2/ (n = 11) mice 8 d after infection. Horizontal bars
indicate means for each group. (D) T. gondii parasitophorous vacuoles that contained tachyzoites were stained with a rabbit anti–T. gondii IgG antibody
and counted in the terminal ileum in WT, MMP-2/, and MMP-9/ mice 8 d after infection. (E) NO levels and (F) IFN- concentration in supernatants of
ileal biopsies from WT (n = 15), MMP-9/ (n = 6), and MMP-2/ (n = 10) mice 8 d after infection. Data are pooled from at least three independent ex-
periments. Mean values, SDs, and significance levels were determined by the Student’s t test or by Kaplan-Meier analysis and the log-rank test (A).
IL-23–mediated T. gondii–induced immunopatholgy | Muñoz et al.
Figure 4. Differential regulation of IL-17 and IL-22 after infection with T. gondii. (A) RT-PCR of IL-17, IL-22, IL-6, IL-1, IL-12p35, IFN-, and
IL-10 mRNA in the ileum of naive and infected (Inf.) WT, IL-23p19/, and IL-17/ mice 8 d after infection. Results are expressed as fold changes relative
to HPRT mRNA expression. (B) IL-17, IL-22, IFN-, and IL-6 concentrations (ELISA) in supernatants of ileal biopsies from naive and infected (Inf.) WT, IL-
23p19/, and IL-17/ mice 8 d after infection. (C) IL-17 and IL-22 concentrations in supernatants of ileal biopsies from WT mice at different time points
after T. gondii infection (ELISA). Data are pooled from at least three independent experiments of five mice per group and are given as means ± SD, and
p-values were determined by the Mann-Whitney U test. ND, not detected.
mRNA increased similarly in all groups upon infection (Fig. 4 A).
In accordance with the mRNA expression data described,
protein concentration of IL-17 was markedly down-regulated
in infected WT mice and undetectable in infected IL-23p19/
or IL-17/ mice. In contrast, upon infection, IL-22 secre-
tion was up-regulated three- to fourfold in WT and IL-17/
JEM VOL. 206, December 21, 2009
inflammation but no necrosis by day 8 after infection (Fig. 5 A).
Interestingly, IL-17/ mice also developed severe necrosis
of the mucosa (Fig. 5 A). WT mice displayed a significantly
higher ileal histopathological score than IL-22/ but not
IL-17/ mice (Fig. 5 B). Furthermore, IL-22/ mice sur-
vived significantly longer than WT mice (Fig. 5 C). Interest-
ingly, IL-22/ mice that survived the acute phase of infection
did not show ileal necrosis around the time of death but more
frequently displayed bacterial translocation to the spleen and
liver than WT mice (unpublished data). The amount of
T. gondii DNA in the ileum did not differ between groups of
mice 8 d after infection (Fig. 5 D).
Lastly, we examined IL-22 levels in the ilea of infected
gnotobiotic mice, because we have previously shown that these
mice do not develop ileitis after T. gondii infection (Heimesaat
et al., 2006). Interestingly, IL-22 was almost undetectable in
infected gnotobiotic mice, whereas mice with a normal specific
pathogen-free gut flora had significantly higher IL-22 levels
in their ilea 8 d after infection (Fig. 5 E). Thus, IL-22 but not
IL-17 is a crucial mediator in the development of small intesti-
nal pathology after oral infection with T. gondii.
Source of IL-22 in the lamina propria of the small intestine
Given the evidence that both T cells and non–T cells can pro-
duce IL-22 (Wolk and Sabat, 2006), we investigated the poten-
tial source of IL-22 in the ileum of mice after ileitis induction.
mice but was undetectable in naive and infected IL-23p19/
mice (Fig. 4 B). IFN- was not detectable in naive mice but
levels increased after infection. IFN- and IL-6 concentra-
tions did not differ significantly in infected WT, IL-23p19/,
or IL-17/ mice (Fig. 4 B). Collectively, IFN- and IL-22
are up-regulated upon infection with T. gondii, whereas IL-17
is markedly down-regulated.
Because IL-17 and IL-22 were differentially expressed in
WT-infected mice, we assessed their expression in the ileum
at different time points after ileitis induction. IL-17 concentra-
tions decreased gradually during the course of infection with
maximum levels in naive mice and minimum levels at day 8
after infection (Fig. 4 C). In contrast, IL-22 was undetectable
in WT naive mice, but IL-22 concentrations increased during
the course of the ileitis development and peaked at day 8 after
infection (Fig. 4 C). These results indicate that IL-22 and
IL-17 are inversely regulated during T. gondii–induced ileitis,
and IL-22 but not IL-17 is induced by IL-23.
IL-22–deficient mice are resistant to T. gondii–induced
Given that the production of IL-22 but not IL-17 was induced
by IL-23 and up-regulated in mice developing intestinal
immunopathology, we assessed the development of ileal in-
flammation in WT, IL-22/, and IL-17/ mice after T. gondii
infection. Severe necrosis of the mucosa was observed in WT
mice, whereas IL-22/ mice showed only mild signs of
Figure 5. IL-22–deficient mice are resistant to T. gondii–induced immunopathology. (A) Histopathology of hematoxylin and eosin–stained ileal
sections of WT, IL-22/, and IL-17/ mice 8 d after infection. (B) Histopathology scores of the ileum of WT, IL-22/, and IL-17/ mice 8 d after infection.
The horizontal line indicates the border between mild inflammation (<3) and necrosis (>3). (C) Survival of WT and IL-22/ mice after oral infection with 100
cysts of T. gondii. (D) T. gondii DNA concentration in ileum of WT, IL-22/, and IL-17/ mice 8 d after infection. (E) IL-22 concentration in culture superna-
tants of ileal biopsies from gnotobiotic (GB) and specific pathogen-free (SPF) mice 8 d after infection (ELISA). Data (one representative out of three indepen-
dent experiments) from three to five mice per group are given as means ± SD, and p-values were determined by the Mann-Whitney U test. Bar, 100 µm.
IL-23–mediated T. gondii–induced immunopatholgy | Muñoz et al.
cells in the small intestinal lamina propria, but IL-22 produc-
tion does not appear to be restricted to T cells.
A growing body of evidence from human studies and mouse
models of IBD has shown that IL-23 promotes acute as well
as chronic intestinal inflammation through the induction of
a plethora of proinflammatory mediators. Because the vast
majority of these studies have used models of large intestinal
inflammation, in the present study, we investigated the role
of IL-23 using a well-established mouse model of small intes-
tinal inflammation. In this model, peroral infection with
T. gondii induces a hyperinflammatory Th1-type immune
response characterized by overproduction of IL-12, IL-18,
IFN-, TNF, and NO leading to small intestinal immuno-
pathology with massive necrosis (panileitis). Although IL-23
has been shown to induce and/or maintain mucosal inflam-
matory responses via the induction of Th17 cells, we show in
this paper that IL-23 caused small intestinal immunopathology
via the up-regulation of MMP-2 and, surprisingly, via the
induction of IL-22 but in an IL-17–independent way.
The expression and enzymatic activity of MMP-2 and
MMP-9 were enhanced in the small intestine when pathol-
ogy started to develop. However, only MMP-2–deficient
We performed flow cytometry to identify the source of
IL-22 in isolated cells from MLNs and in the small intestinal
lamina propria of infected WT mice. Although IL-22–
producing cells in MLNs of infected mice were almost unde-
tectable (Fig. 6 A), 2.8% of CD3+ cells produced IL-22 in
the small intestinal lamina propria and 45.2% of all IL-22–
producing cells were CD3+. IL-22 production was also
observed in 7.4% of all CD4+ cells. Interestingly, we were
unable to detect IL-22 secretion among NK22 cells because
cells that expressed NK1.1 or NKp46 did not secrete IL-22.
To confirm the presence of non–T cells producing IL-22
in the ilea of infected mice, we compared the production of
IL-22 in the ileum of infected RAG1/ (which lack T cells)
and WT mice. After ileitis induction, RAG1/ mice dis-
played lower IL-22 concentrations in the ileum compared
with WT mice, but this difference did not reach statistical
significance (Fig. 6 B). IL-17 was not detected in RAG1/
-infected mice (Fig. 6 B). We obtained similar results by mea-
suring IL-22 levels in culture supernatants of sorted CD4+
and CD4 cells from the MLN and the small intestinal lam-
ina propria cells (Fig. 6 C). Furthermore, supernatants ob-
tained from CD11c+ cells sorted from both MLN and the
lamina propria produced very low levels of IL-22 (Fig. 6 C).
These results suggest that IL-22 is mostly produced by CD4+
Figure 6. Source of IL-22 in the lamina propria of the small intestine upon induction of ileitis. (A) Flow cytometric analysis of cells isolated
from the MLNs and small intestinal lamina propria of infected WT mice 8 d after infection, stained for CD4, CD3, NK1.1, NKp46, and IL-22 (percentages
are shown). (B) IL-22 and IL-17 concentration in supernatants of ileal biopsies from WT and RAG1/ mice 8 d after infection measured by ELISA. (C) IL-22
concentration in supernatants of CD11c+, CD4+, and CD4 sorted cells isolated from the small intestinal lamina propria and MLNs from infected WT mice
8 d after infection. Data shown are representative of three independent experiments. LPL, lamina propria of the small intestine.
JEM VOL. 206, December 21, 2009
(Kim et al., 2008) and during mycobacterial infection (Cruz
et al., 2006).
Importantly, we found that IL-23–induced up-regulation
of IL-22 was essential for the development of small intestinal
immunopathology. IL-22/ mice did not develop small in-
testinal necrosis although they harbored the same number
of parasites as both WT and IL-17/ mice. These data
support a new pathogenic rather than protective role of IL-22
in the small intestine.
Although IL-22 has been reported to promote psoriasis-
like skin alterations (Wolk et al., 2004; Wolk et al., 2006;
Zheng et al., 2007; Ma et al., 2008; Wolk et al., 2009), an in-
creasing number of studies have reported a rather protective role
of IL-22, especially in hepatitis and colitis experimental mod-
els (Zenewicz et al., 2007; Cella et al., 2008; Satoh-Takayama
et al., 2008; Sugimoto et al., 2008; Zenewicz et al., 2008).
In regard to intestinal inflammation, IL-22 protected mice
from colitis in a CD45RBhi transfer model (Zenewicz et al.,
2008), and IL-22 deficiency rendered mice susceptible to
Citrobacter rodentium– and dextran sulfate sodium–induced
colitis (Satoh-Takayama et al., 2008; Zheng et al., 2008).
Furthermore, IL-22 induces the expression of antimicrobial
peptides in epithelial cells (Liang et al., 2006; Aujla et al., 2007;
Zheng et al., 2008).
These contrasting features of IL-22 raise important ques-
tions about how the same protein could behave in opposite
ways. First, the role of IL-22 in inflammation could be tissue
specific. IL-22 might exert pathogenic functions in keratino-
cytes and epithelial cells of the small intestine while playing
a protective role in the large intestinal and lung epithelium,
as well as in hepatocytes. Second, IL-22 may exert different
functions dependent on its amount and duration in tissues. In
the present study, increasing levels of IL-22 in the ileum were
found during ileitis, and they peaked at day 8 after infection
when intestinal pathology was full blown and mice began
to succumb to infection. Third, the diversity of experimental
models used (pathogen and chemical induced) may contribute
to the contrasting roles of IL-22. At this point, we can only
speculate on the potential pathogenic effector mechanisms
mediated by IL-22 in the small intestine. Neutralization of
IL-22 has recently been reported to block CXCL-8 expression
by intestinal epithelial cells after stimulation with T memory
cells (Kleinschek et al., 2009). Lastly, pathogenic IL-22–
producing cells might constitute a subpopulation of cells, dif-
ferent from Th17 and NK22 cells. IL-22 production is higher
in Th1 cells than in Th17 cells (Volpe et al., 2008), and a
unique IL-22–producing population of NKp46+RORt+
natural killer cells, termed NK22 cells, was identified in the
dermis, lamina propria, and other mucosa-associated lymphoid
tissues (Cella et al., 2008; Luci et al., 2008; Sanos et al., 2009;
Satoh-Takayama et al., 2008; Zenewicz et al., 2008). Further-
more, a human Th cell population that secretes IL-22 but not
IL-17 nor IFN- has been recently reported (Duhen et al.,
2009; Trifari et al., 2009). In the present study, we used a
well-characterized IL-22 mAb (Zheng et al., 2007) to identify
the source of IL-22 after ileitis induction. CD4+ T cells were
mice were protected against the development of intestinal
immunopathology and early death. IL-22 levels in the ileum
of WT and MMP-2/ mice as well as the levels of MMP-2
in the ileum of WT and IL-22/ mice did not differ be-
tween WT and knockout mice, respectively, indicating that
IL-22 and MMP-2 mediate T. gondii–induced ileitis through
In accordance with our data, several studies have demon-
strated that MMP-2 and MMP-9 are up-regulated during ac-
tive episodes of IBD in humans as well as in animal models of
colitis (Baugh et al., 1998; Castaneda et al., 2005; Yen et al.,
2006; Gordon et al., 2008), and that IL-23 is able to induce
their expression (Ivanov et al., 2007). Epithelial barrier dys-
function may be involved in the MMP-mediated effects in
T. gondii–induced ileitis, because MMP-2/ mice showed a
lower rate of bacterial translocation into the spleen compared
with WT mice (unpublished data). Interestingly, granulocytes
have been proposed as an important source of MMPs. We
found an increased number of granulocytes in the lamina pro-
pria after infection (unpublished data). However, neutrophil
depletion in WT mice did not prevent the development of
ileitis, and MMP concentrations did not differ after depletion
of granulocytes. In addition, IL-22 levels in the ileum were
similar in granulocyte-depleted and control mice, suggesting
that neutrophils are not a source of IL-22 (unpublished data).
We observed that nonselective (doxycycline) and selec-
tive (RO28-3653) gelatinase inhibitors ameliorated intestinal
pathology when given either prophylactically or therapeuti-
cally. Dosages of RO28-2653 used in the present study were
similar to those administered in pharmacodynamic studies in
rat and mouse models (Kilian et al., 2006; Abramjuk et al.,
2007). Although nonselective MMP-blocking agents may cause
severe adverse side effects (Bernardo et al., 2002), RO28-2653
did not show major side effects in rat and monkey toxicologi-
cal studies (unpublished data).
Moreover, RO28-2653 also blocked large intestinal in-
flammation in a model of dextran sulfate sodium–induced
colitis (unpublished data). Thus, selective blockage of gelati-
nases may be a safe and effective new strategy in the preven-
tion and treatment of intestinal inflammation.
IL-23 has been proposed to induce pathology through
the proliferation and maintenance of IL-17–secreting cells
(Aggarwal et al., 2003; Bettelli et al., 2007). In contrast, our
results demonstrate that the pathogenic role of IL-23 was in-
dependent of IL-17 but dependent on IL-22. In agreement
with our data, several studies have demonstrated that IL-17
and IL-22 possess distinct roles during immune responses
(Cruz et al., 2006; Kreymborg et al., 2007; Schulz et al.,
2008; Sugimoto et al., 2008; Wolk et al., 2009). Moreover,
our study provides strong evidence that IL-22 and IL-17 are
inversely regulated in ileitis.
Although IL-22 was up-regulated, IL-17 production was
turned off in the ileum of infected mice. High concentra-
tions of IFN- in the small intestine of mice might have
contributed to the down-regulation of IL-17 production,
as previously shown in models of adjuvant-induced arthritis
IL-23–mediated T. gondii–induced immunopatholgy | Muñoz et al.
0.2 µM of each oligonucleotide probe (TOX-HP-1, 5-GAGTCGGAGA-
GGGAGAAGATGTT-FAM-3; TOX-HP-2, 5-RED-640-CCGGCTT-
GGCTGCTTTTCCTG-PH-3), and 500 ng of template DNA in a final
volume of 20 µl. The amplification was performed using one cycle at 95°C
for 10 min, 50 cycles at 95°C for 10 s/52°C for 20 s/72°C for 30 s, and one
cycle at 40°C for 30 s with a single fluorescence detection point at the end
of the cycle. A standard curve was performed using 500–5 pg T. gondii
DNA of GFP tachyzoites. Fluorescence was analyzed by LightCycler Data
Analysis software (version 3.5; Roche). T. gondii parasitophorous vacuoles that
contained tachyzoites were stained with a rabbit anti–T. gondii IgG antibody
and counted in the terminal ileum, as previously described (Vossenkämper
et al., 2004).
Gelatin zymography. The activity of MMP-2 and MMP-9 was measured
by zymography under nonreducing conditions. In brief, ileum samples of
infected and noninfected mice were snap frozen in liquid nitrogen and
homogenized in lysis buffer (50 mM Hepes [pH 7.5], 150 mM NaCl, 1 mM
MgCl2, 1 mM CaCl2, and 1% Triton X-100). MMPs were enriched by
binding for 1 h at 4°C to gelatin-agarose beads (Sigma-Aldrich). Bound
MMPs were eluted in 80 µl of 1× nonreducing SDS loading buffer. Samples
were subjected to SDS-PAGE in 8% polyacrylamide gels containing 0.1%
gelatin. After staining with Coomassie brilliant blue and destaining, gels were
scanned and visualized using an image analyzer (LAS-1000; Fujifilm). A mix-
ture of human MMP-2/9 was used as a standard.
Real-time PCR. RNA was isolated from organs using the RNeasy Mini
Kit (QIAGEN). mRNA was reverse transcribed and analyzed in triplicate
assays by TaqMan PCR using a sequence detection system (ABI Prism
7700; Applied Biosystems), as described previously (Wolk et al., 2002). For
detection of mouse IFN-, IL-12, IL-10, IL-6, IL-1, IL-17A, IL-22,
MMP-2, MMP-9, and IL-23p19, assays including double-fluorescent
probes in combination with assays for the mouse housekeeping gene hypo-
xanthine phosphoribosyltransferase (HPRT) were purchased from Applied
Biosystems or developed by us (IL-22). Expression levels were calculated
relative to the HPRT expression.
Cytokine and MMP ELISA. Concentrations of IL-6, IL-10, TNF,
IFN- (BD), IL-22, IL-17, MMP-9, and MMP-2 (R&D Systems) were
determined in supernatants of organ cultures according to the manufac-
Treatment with doxycycline and RO28-2653. C57BL/6 mice were
treated perorally by gavage twice daily with either doxycycline (50 mg/kg
body weight/day; Sigma-Aldrich) or RO28-2653 (75 mg/kg body weight/
day; Roche) in 0.3 ml PBS. One group of mice was treated starting 5 d be-
fore T. gondii infection until 8 d after infection (prophylactic regimen), and
another group from 5 d (the time point when inflammatory changes in the
gut mucosa start to develop) until 8 d after infection (therapeutic regimen).
PBS-treated mice served as negative controls.
Determination of NO concentrations. NO concentrations in superna-
tants were determined by Griess reaction, as previously described (Heimesaat
et al., 2006).
Lamina propria and MLN cell isolation, sorting, and flow cytom-
etry. The small intestine, dissected from its mesentery and Peyer’s patches,
was cut into 2-cm pieces and incubated in 5 mM EDTA, 1 mM dithioth-
reitol (Sigma-Aldrich), and RPMI 1640. Thereafter, pieces were incu-
bated in RPMI 1640 containing 500 µg/ml liberase CI (Roche) and 0.05%
DNase. MLNs were removed and subsequently minced through a 70-µm
filter. For intracellular staining, cells were stimulated for 6 h with 50 ng/ml
12-O-tetradecanoylphorbol-13-acetate (Sigma-Aldrich), 750 ng/ml iono-
mycin (Sigma-Aldrich), and GolgiStop (BD) at 37°C. Stainings and cell
sorting with anti-CD4 (L3T4 clone, allophycocyanin [APC]-Cy7 conju-
gated), anti-CD8 (53-6.7 clone, Pacific blue conjugated), anti-CD11c
the predominant source of IL-22. IL-17 was down-regulated
and dispensable for the development of intestinal necrosis. In-
terestingly, flow cytometry pointed toward non-CD4, non-
CD3 T cells as an additional source of IL-22 in the ilea of
infected mice. This was confirmed by our finding of IL-22
production in the ileum of RAG1/ mice, which do not
have T cells, and in supernatants of CD4 cells sorted from
the lamina propria of infected mice. However, flow cytome-
try did not associate IL-22 production with NK cells, as re-
cently described (Cella et al., 2008; Luci et al., 2008; Sanos et
al., 2009; Satoh-Takayama et al., 2008; Zenewicz et al.,
2008). We cannot formally rule out the presence of NK22
cells, because the mAb used to detect IL-22–producing cells
may have failed to detect IL-22 secretion by non–T cells be-
cause of a short half-life of IL-22 or a low sensitivity of this
mAb. Therefore, we assume that CD4+ T cells are the main
producers of IL-22 but other non–T cells contribute to IL-22
production. In agreement with a previous study, IL-22 pro-
duction was also dependent on the presence of the normal gut
flora, because gnotobiotic mice displayed significantly lower
IL-22 concentrations (Satoh-Takayama et al., 2008).
In conclusion, IL-22 induced by IL-23 but not IL-17 is
a key mediator of immunopathology in the small intestine.
IL-23 also induced the local up-regulation of MMP-2 that
was a crucial downstream effector molecule for the develop-
ment of small intestinal inflammation that could be effec-
tively inhibited by chemical blockage of gelatinases.
MATERIALS AND METHODS
Mice. Female WT, IL-23p19/, IL-17A/, RAG1/, MMP-2/,
MMP-9/ (all on a C57BL/6 background), and NMRI mice were 8–12
wk of age and bred and maintained in the Forschungsinstitut für Experimen-
telle Medizin (Charité Medical School, Berlin, Germany). Clinical condi-
tions and body weights were determined daily, and all experiments were
conducted according to the German animal protection laws. Animal proto-
cols were approved by the Landesamt für Gesundheit und Soziales.
Parasites and infection. Cysts of the T. gondii ME49 strain were ob-
tained from brains of NMRI mice that had been infected i.p. with 10 cysts
for 2–3 mo. For peroral infection, mice were infected with 100 cysts in
a volume of 0.3 ml in PBS (pH 7.4) by gavage, as described previously
(Heimesaat et al., 2006).
Sampling procedures, determination of small intestinal length, his-
tological scores, and parasite load. Mice were sacrificed with Halothan
(Eurim-Pharm) 8 d after infection. The relative shortening of the small in-
testine was calculated by dividing the difference of the mean length of the
small intestine from naive control mice minus the length from infected mice
at day 8 after infection and then multiplied by 100 over the mean length of
naive mice small intestines. Histological scores and parasite loads were deter-
mined in fixed and paraffin-embedded tissue sections taken from the termi-
nal ileum, as previously described (Heimesaat et al., 2006).
Detection of T. gondii. Approximately 1 cm of ileum tissue was homoge-
nized in a rotor–stator in 500 µl of lysis buffer containing 100 mM Tris-HCl
[pH 8], 200 mM NaCl, 5 mM EDTA, 0.2% SDS, and 200 µg/ml proteinase K.
The amplification mixture consisted of 2 µl of 10× reaction mix (Light-
Cycler FastStart Master Hybridization Probes; Roche), 2 mM MgCl2, 1 µM
of each oligonucleotide primer (TOX-9, 5-AGGAGAGATATCAG-
GACTGTAG-3; TOX-10, 5-GCGTCGTCTCGTCTAGATCG-3),
JEM VOL. 206, December 21, 2009
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jugated), anti- (GL3 clone, FITC conjugated; BD), anti-NKp46 (PE
conjugated; R&D Systems), and anti–IL-22 (APC conjugated; Genentech)
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Depletion of neutrophils. Neutrophils were depleted using an anti-Gr1
mAb (RB6-8C5) at days 3 and 5 after infection with T. gondii. Each mouse
was injected i.p. with 0.15 mg/ml anti-Gr1. Control animals were left un-
treated. Neutrophil depletion was confirmed by flow cytometric analyses in
all experimental animals.
Generation of gnotobiotic mice. To remove the commensal gut flora,
C57BL/6 mice were treated by adding 1 g/liter ampicillin (ratiopharm),
500 mg/liter vancomycin (Cell Pharm), 200 mg/liter ciprofloxacin (Bayer
Vital), 250 mg/liter imipenem (MSD), and 1 g/liter metronidazole (Frese-
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Online supplemental material. Fig. S1 shows IL-23p19 mRNA from
sorted lamina propria and MLN CD11b+ and CD11c+ cells. Fig. S2 shows
survival of WT, MMP-2/, and MMP-9/ mice after T. gondii infection.
Fig. S3 A shows immunohistochemistry staining of MMP-2 in ileal biopsies
of WT and MMP-2/ mice, and Fig. S3 B shows MMP-2 concentra-
tion in the ileum of WT mice after neutrophil depletion. Fig. S4 (A–D)
shows survival, body weight loss, small intestinal length shortening, and
histopathology scores, respectively, of mice treated with PBS, doxycycline,
or RO28-2653. Fig. S4 (E and F) show NO and IFN- concentrations,
respectively, in the ileum from mice treated with PBS, doxycycline, or
RO28-2653. Online supplemental material is available at http://www.jem
We would like to thank G. Reifenberger, M. Wattrodt, D. Trautmann, U.B. Göbel,
N. Kassner, S. Rutz, G. Alber, and the staff of the animal facility of the Charité
Medical School for their technical expertise and/or discussion.
This study was supported by grants from the German Research Foundation
to O. Liesenfeld (SFB633, B6), S. Bereswill (SFB633, A7), and C. Loddenkemper
H.-W. Krell is employed by Roche Diagnostics. The authors have no further
conflicting financial interests.
Submitted: 23 April 2009
Accepted: 3 November 2009
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