of January 26, 2012
This information is current as
; Prepublished online 20 January 2012;
Renauld, Susanne Hartmann and Richard Lucius
Zajic, Anja A. Kühl, Catherine Uyttenhove, Jean-Christophe
Jörg Stange, Matthew R. Hepworth, Sebastian Rausch, Lara
the Cost of Th17-Driven Immunopathology
Intracellular Parasite in the Absence of IFN-
IL-22 Mediates Host Defense against an Intestinal
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on January 26, 2012
The Journal of Immunology
IL-22 Mediates Host Defense against an Intestinal
Intracellular Parasite in the Absence of IFN-g at the Cost of
Jo ¨rg Stange,*,1Matthew R. Hepworth,*,1Sebastian Rausch,* Lara Zajic,*
Anja A. Ku ¨hl,†Catherine Uyttenhove,‡Jean-Christophe Renauld,‡Susanne Hartmann,*
and Richard Lucius*
The roles of Th1 and Th17 responses as mediators of host protection and pathology in the intestine are the subjects of intense
research. In this study, we investigated a model of intestinal inflammation driven by the intracellular apicomplexan parasite
Eimeria falciformis. Although IFN-g was the predominant cytokine during E. falciformis infection in wild-type mice, it was found
to be dispensable for host defense and the development of intestinal inflammation. E. falciformis-infected IFN-gR2/2and IFN-
g2/2mice developed dramatically exacerbated body weight loss and intestinal pathology, but they surprisingly harbored fewer
parasites. This was associated with a striking increase in parasite-specific IL-17A and IL-22 production in the mesenteric lymph
nodes and intestine. CD4+T cells were found to be the source of IL-17A and IL-22, which drove the recruitment of neutrophils and
increased tissue expression of anti-microbial peptides (RegIIIb, RegIIIg) and matrix metalloproteinase 9. Concurrent neutrali-
zation of IL-17A and IL-22 in E. falciformis-infected IFN-gR2/2mice resulted in a reduction in infection-induced body weight loss
and inflammation and significantly increased parasite shedding. In contrast, neutralization of IL-22 alone was sufficient to
increase parasite burden, but it had no effect on body weight loss. Treatment of an E. falciformis-infected intestinal epithelial
cell line with IFN-g, IL-17A, or IL-22 significantly reduced parasite development in vitro. Taken together, to our knowledge these
data demonstrate for the first time an antiparasite effect of IL-22 during an intestinal infection, and they suggest that IL-17A and
IL-22 have redundant roles in driving intestinal pathology in the absence of IFN-g signaling.
spp., Toxoplasma gondii, and Plasmodium spp. (1–4). IFN-g is
a potent proinflammatory cytokine derived from multiple cells of
both the innate and adaptive immune system, including CD4+Th1
cells, CD8+T cells, and NK cells. It also plays a key role in
driving immunopathology in response to microbial stimuli or
pathogen-induced tissue damage, resulting in the development of
inflammatory disorders of the intestine (5). Recently, many studies
have attributed similar roles to the cytokines IL-17A and IL-22.
Cells of the Th17 lineage are the predominant source of IL-17A
The Journal of Immunology, 2012,
nterferon-g is classically considered the key protective cy-
tokine in response to infections with numerous intracellular
bacteria, viruses, and protozoan parasites, including Eimeria
and IL-22, although innate immune cells such as lymphoid tissue
inducer cells and NK cells have also been shown to be potent
producers of these cytokines (6). In particular, both IL-17A and
IL-22 have been implicated in the development of colitis in
humans and multiple murine models (7, 8) and have host-
protective effects during bacterial infections, including Klebsi-
ella pneumoniae, Citrobacter rodentium, and Staphylococcus au-
reus (9–11). Currently little is known about the roles of IL-17A
and IL-22 in driving immunopathology or mediating host defense
during intestinal protozoan parasite infections.
The development of Th1- and Th17-driven inflammation is
closely linked by the common requirement for IL-12p40, which
constitutes one subunit of both the biologically active IL-12 and
IL-23 heterodimers. Th17 differentiation from naive CD4+T cells
is characterized by the expression of the master transcription
factor retinoic acid receptor-related orphan receptor gt (RORgt)
(12), which is driven by IL-6 and TGF-b and stabilized by IL-23
(13, 14). Interestingly, the priming of IFN-g–producing Th1 and
IL-17A–/IL-22–producing Th17 lineages can be cross-regulated.
For example, IFN-g can directly inhibit the development of Th17
cells via the suppression of T cell IL-23R expression, or amelio-
rate IL-17A–driven pathology via inhibition of IL-23 (15, 16).
Furthermore, the development of Th1 and Th17 lineages is cross-
regulated via their master transcription factors. T-box transcription
factor (T-bet), expressed during Th1 commitment, concurrently
suppresses Th17 development via interaction with Runx-1 and
subsequently prevents transcription of the Rorc gene that codes for
Eimeria falciformis is a natural intracellular pathogen of the
murine cecum and proximal colon, where it develops a self-limiting
*Molekulare Parasitologie, Humboldt Universita ¨t zu Berlin, D-10115 Berlin, Ger-
many;†Institute of Pathology/Research Center ImmunoSciences, Charite ´–Campus
Benjamin Franklin, 12203 Berlin, Germany; and‡Ludwig Institute for Cancer Re-
search, B-1200 Brussels, Belgium
1J.S. and M.R.H. contributed equally to this work.
Received for publication July 19, 2011. Accepted for publication December 20, 2011.
This work was partially supported by Deutsche Forschungsgemeinschaft Grant
LU325/12-1 (to R.L.) and a Novartis Animal Health fellowship (to J.S.).
Address correspondence and reprint requests to Jo ¨rg Stange and Matthew R. Hep-
worth, Molekulare Parasitologie, Humboldt Universita ¨t zu Berlin, Philippstrasse 13,
Haus 14, D-10115 Berlin, Germany. E-mail addresses: firstname.lastname@example.org
(J.S.) and email@example.com (M.R.H.)
The online version of this article contains supplemental material.
Abbreviations used in this article: HPF, high power field; Lin, lineage; LPL, lamina
propria lymphocyte; MLN, mesenteric lymph node; MMP, matrix metalloproteinase;
PAS, periodic acid–Schiff; p.i., postinfection; RORgt, retinoic acid receptor-related
orphan receptor gt; Sca-1, stem cell Ag-1; T-bet, T-box transcription factor; Wt, wild
Published January 20, 2012, doi:10.4049/jimmunol.1102062
on January 26, 2012
infection in epithelial cells of the crypts (18). Moreover, intestinal
E. falciformis infection induces strong inflammation in the cecum
and colon and has many parallels with colitic disease, thus making
it an excellent model for studying mucosal inflammation in the
large intestine. This coccidian parasite belongs to the phylum of
Apicomplexa that also contains major human pathogens such as T.
gondii, Plasmodium spp., and Cryptosporidium parvum. Infections
with Eimeria spp. are of economic relevance, particularly in the
poultry industry, with the costs attributed to lost revenue and drug
treatment estimated to be in excess of £2 billion per year worldwide
(19). Administration of infective oocysts via the natural oral route
typically leads to the rapid development of an IFN-g–dominated
response, which is associated with body weight loss and the de-
velopment of transient inflammation and immunopathology in the
large intestine (2). Although adoptive transfer of IFN-g–producing
CD8+T cells was previously shown to partially control E. falci-
formis infection (2), it is currently not known whether other cyto-
kines can compensate in the absence of IFN-g. Moreover, there is
currently very little information available concerning the roles of
IL-17A and IL-22 in the control of intracellular parasite infections
at mucosal barriers or their ability to induce inflammation and im-
munopathology in response to parasite infections.
IFN-g receptor (IFN-gR2/2) or the cytokine itself (IFN-g2/2) are
impaired in their ability to control E. falciformis infection, or in
the development of infection-driven immunopathology. We iden-
tified a novel compensatory role for IL-22 in the control of par-
asite infection in the large intestine at the expense of severe
immunopathology and mortality driven by both IL-22 and IL-17A.
Furthermore, our data highlight the interplay between the devel-
opment of IFN-g– and IL-17A–/IL-22–producing lineages during
Materials and Methods
Mice and parasites
Female-specific pathogen-free C57BL/6 wild-type (Wt) mice (Charles
River), C57BL/6 IFN-g2/2mice (T. Schu ¨ler, Charite ´ University Medicine,
Berlin) and C57BL/6 IFN-gR2/2mice (U. Klemm, Max Planck Institute
for Infection Biology, Berlin) were used for parasite infections. All
experiments were performed in accordance with the National Animal
Protection Guidelines, approved by the Animal Ethics Committee. E.
falciformis was maintained by serial passage in NMRI mice, bred at the
Department of Molecular Parasitology (Humboldt University of Berlin),
and oocysts were purified by flotation in sodium hypochloride as described
Infections and quantification of parasite burden
For experimental infections, mice aged 10–14 wk were inoculated with 50
E. falciformis oocysts in 100 ml water via oral gavage, weighed daily, and
sacrificed at the end of patency, or at day 10 postinfection (p.i.) where
indicated. Mice were euthanized upon 20% body weight loss. For analysis
of challenge infections mice received a dose of 10 oocysts followed by a
challenge infection with 50 oocysts 4 wk after the primary dose. During
patency, feces were collected every 24 h (62 h), soaked in water, ho-
mogenized, and floated in saturated sodium chloride solution. The number
of oocysts was quantified using a McMaster chamber.
Neutralization of cytokines
Two hundred micrograms of anti–IL-17A (MM17AF3; C. Uyttenhove,
Brussels) and/or anti–IL-22 (AM22.1; Jean-Christophe Renauld, Brussels)
mAb in PBS was injected i.p. into mice on days 21, 2, 5, and 8 p.i. Control
groups received 200 mg mouse IgG control Ab (Dianova) according to the
same treatment regimen.
For histopathological analysis, colon and cecum samples were fixed with
deparaffinized, and stained with H&E. To quantify intestinal inflammation,
infiltration (0, none; 1, mild, minimal mucosal; 2, moderate, mucosal and
submucosal; 3, marked mucosal and submucosal, sometimes transmural; 4,
marked infiltrates, often transmural; 5, marked transmural inflammation) and
crypt architecture (0, normal; 1, mild hyperplasia, normal architecture and
goblet cell number; 2, moderate hyperplasia, goblet cell loss; 3, architecture
disrupted, severe goblet cell loss; 4, severe loss of crypt architecture; 5,
complete loss of crypt architecture and ulceration) were scored separately
and values were combined to yield a score from 0 to 10. Periodic acid–Schiff
(PAS) histochemistry was conducted in a Dako Autostainer Plus according
to the manufacturer’s instructions. E. falciformis was quantified in PAS-
stained cecal thin sections from day 5 p.i. by counting the number of mature
schizonts per high power field (HPF; 3400 magnification, 20 replicates).
In vitro assays with CMT-93 cells
CMT-93 cells (a gift from T. Schu ¨ler, Berlin) were cultured in DMEM
supplemented with 10% FCS, 20 mM L-glutamine, 100 U/ml penicillin,
and 100 mg/ml streptomycin. Cells were harvested using 13 trypsin/EDTA
solution (PAA Laboratories) and incubated with 2 mg/ml mitomycin C
(AppliChem) for 30 min at 37˚C to inhibit proliferation. After washing,
cells were resuspended in media containing 50 ng/ml respective cytokine
(IFN-g, IL-17A, IL-22, and IL-10; PeproTech) and 5 3 105cells were
seeded in 24-well plates on coverslips (diameter 12 mm). E. falciformis
sporozoites were purified as described before (20), and 8 h after seeding
cells were infected with 1 3 105E. falciformis sporozoites suspended in
DMEM without FCS. Extracellular parasites were washed off 4 h p.i., and
DMEM with 10% FCS was added for further culture. For histological
examination, monolayers were fixed for 10 min using 4% paraformalde-
hyde and stained with PAS. Invasion was determined 4 h p.i. by counting
intracellular sporozoites per HPF (3400 magnification, 10 HPFs per
sample). Development was assessed at 39 h p.i. by scanning the whole
coverslip for mature schizonts. Results are displayed relative to the mean
of the control group.
Lymphocyte isolation and culture
Mesenteric lymph nodes (MLN) and spleens were isolated from euthanised
mice and single cell suspensions were obtained by passing through a 70mm
cell strainer (BD Biosciences). Cells were resuspended in RPMI 1640
containing 10% FCS, 20 mM L-glutamine, 100 U/ml penicillin, and 100
mg/ml streptomycin (complete RPMI 1640; PAA Laboratories) and
counted using a Casy model TT cell counter (Innovatis). Lamina
propria lymphocytes (LPL) were isolated from the cecum and proximal
colon. Briefly, mesenteric fat and the cecal patch were removed and the
intestine was opened longitudinally, washed thoroughly in HBSS con-
taining 2% FCS, and cleaned further by vigorous shaking in the same
buffer twice. Epithelial cells were removed by a 20-min incubation in
HBSS 2% FCS containing 2 mM EDTA. Tissue was then minced and
incubated for 1 h at 37˚C in RPMI 1640 medium containing 10% FCS, 200
U/ml collagenase VIII, and 0.1 U/ml collagenase D (Sigma-Aldrich). The
digested tissue was filtered, centrifuged, and the cell pellet was resus-
pended and layered onto a 40/70% Percoll gradient (GE Healthcare) and
centrifuged for 20 min at 2000 rpm. LPL were isolated from the interface
and analyzed by flow cytometry.
MLN cells (5 3 105) were cultured in a total volume of 200 ml in 96-well
round-bottom plates in complete RPMI 1640. Cultures were restimulated
with total sporozoite Ag equivalent to 1.2 3 105sporozoites per well as
described before (2) or 1 mg/ml anti-CD3 Ab (BD Biosciences) at 37˚C
and 5% CO2for 48 h. Supernatants were harvested for cytokine detection,
and in some experiments medium was replaced and 1 mCi methyl-[3H]thy-
midine (Amersham Pharmacia Biotech) was added. Cultures were placed
at 37˚C and 5% CO2for an additional 20 h to measure radiolabeled thy-
midine incorporation, which was detected using a 1450 MicroBeta TriLux
microplate scintillation and luminescence counter (PerkinElmer).
Flow cytometric analysis
The following mouse mAbs were used for flow cytometry: CD8-PE, GR-1-
Cy5 (Deutsches Rheuma-Forschungszentrum Berlin), CD4-FITC, IL-17A-
PE (BD Biosciences), RORgt-allophycocyanin, T-bet-PE, CD49b-PE-Cy7,
IL-17A-FITC, IL-22-PE, IFN-g-eFluor 450, CD4-PercP-eFluor 710, stem
cell Ag-1 (Sca-1)-PE-Cy7, CD117-allophycocyanin (c-Kit), lineage-eFluor
450 (eBioscience), and Ly-6C-Pacific Blue (BioLegend) in pretitrated
concentrations. For intracellular detection of IFN-g, IL-17A, and IL-22, 5 3
106cells were restimulated with 50 ng/ml PMA and 1 mg/ml ionomycin for
3 h at 37˚C and 5% CO2. After 30 min initial incubation, 5 mg/ml brefeldin
A was added. Subsequently, intracellular cytokines were detected using the
Cytofix/Cytoperm kit (BD Biosciences). Fixation/permeabilization buffers
(eBioscience) were used for intracellular detection of cytokines in combi-
2 Th17 RESPONSES DURING EIMERIA FALCIFORMIS INFECTION
on January 26, 2012
nation with T-bet or RORgt. Stained cells were acquired using a BD For-
tessa cytometer and data were analyzed using FlowJo software (Tree Star).
Cytokines were quantified in cell culture supernatants in duplicates using
an IFN-g ELISA (eBioscience) and IL-22 and IL-17A DuoSet ELISAs
(R&D Systems) according to the manufacturers’ instructions. ELISA
plates were read with a Synergy HT reader together with Gen5 data
analysis software (BioTek).
Quantitative real-time PCR
Cecum samples were snap frozen in liquid nitrogen and stored at 280˚C.
The tissue was homogenized using a tissue homogenizer (MP Bio-
medicals), samples were centrifuged (10 min, 20,000 3 g, 4˚C), and total
RNA was isolated using an innuPREP RNA kit (Analytik Jena) according
to the manufacturer’s instructions. Following reverse transcription of 2 mg
RNA using the High Capacity RNA-to-cDNA kit (Applied Biosystems),
quantitative real-time PCR was performed in duplicates with 10 ng cDNA
using the FastStart Universial SYBR Green Master Mix (Roche) in an ABI
7300 real-time PCR system (Applied Biosystems). The following primers
(MWG Biotech) were used for detection: Actb forward, 59-TCTTGGGT-
ATGGAATCCTGTGGCA-39, reverse, 59-TCTCCTTCTGCATCCTGTC-
AGCAA-39; Cxcl9 forward, 59-TCCTTTTGGGCATCATCTTCC-39,
reverse, 59-TTTGTAGTGGATCGTGCCTCG-39; Cxcl10 forward, 59-CC-
AAGTGCTGCCGTCATTTTC-39, reverse, 59-GGCTCGCAGGGATGAT-
TTCAA-39; Nos2 forward, 59-CCCTTCCGAAGTTTCTGGCAGCAGC-
39, reverse, 59-GGCTGTCAGAGCCTCGTGGCTTTGG-39; Il23r forward,
59-TTCAGATGGGCATGAATGTTTCT-39, reverse, 59-CCAAATCCGA-
GCTGTTGTTCTAT-39; Il23a forward, 59-ATGCTGGATTGCAGAGCA-
GTA-39, reverse, 59-ACGGGGCACATTATTTTTAGTCT-39; Tgfb1 for-
ward, 59-TACGTCAGACATTCGGGAAGCAGT-39, reverse, 59-AAAGA-
CAGCCACTCAGGCGTATCA-39; Il6 forward, 59-TAGTCCTTCCTACC-
CCAATTTCC-39, reverse, 59-TTGGTCCTTAGCCACTCCTTC-39; Reg3b
forward, 59-CTCTCCTGCCTGATGCTCTT-39, reverse, 59-GTAGGAGC-
CATAAGCCTGGG-39; Reg3g forward, 59-TCAGGTGCAAGGTGAAGT-
TG-39, reverse, 59-GGCCACTGTTACCACTGCTT-39; Mmp9 forward, 59-
GCTTCAGAAGCAGCTCTCC-39, reverse, 59-GTTTTGGATCCAGTATG-
TG-39; Hprt forward, 59-TGGACAGGACTGAAAGACTTGCTC-39, re-
verse, 59-AGTCTGGCCTGTATCCAACACTTC-39; Ifngr1 forward, 59-
CTGGCAGGATGATTCTGCTGG-39, reverse, 59-TGCAGGAATCAGTC-
CAGGAAC-39; Il17ra forward, 59-AGTGTTTCCTCTACCCAGCAC-39,
reverse, 59-GTGGTTTGGGTCCCCATCA-39; Il22ra1 forward, 59-ATGA-
AGACACTACTGACCATCCT-39, reverse, 59-CAGCGAGCTGAAACGAT-
CA-39; Il10rb forward, 59-ACCTGCTTTCCCCAAAACGAA-39, reverse,
59-TGCATCTCAGGAGGTCCAATG-39. Following normalization to b-
actin (housekeeping gene), the results were plotted as relative expression
compared with naive controls using the 22DDCtmethod.
All experiments were performed with four to eight mice per group, and
representative data are shown as means 6 SEM for three independent
experiments unless stated otherwise. The nonparametric Mann–Whitney U
test was used for statistical analysis. Values of p # 0.05 were considered to
be statistical significant.
IFN-g– and IFN-gR–deficient mice show increased weight loss
and intestinal pathology following E. falciformis infection
To determine whether IFN-g is required for host protection during
E. falciformis infections, we assessed infection-induced immu-
nopathology by monitoring body weight loss in mice deficient in
IFN-g (IFN-g2/2) and its receptor (IFN-gR2/2) in comparison
with C57BL/6 wild-type (Wt) mice. In contrast to Wt mice, which
only transiently lost weight during primary infection (days 8–10 p.i.;
Fig. 1A), IFN-gR2/2mice showed a dramatically exacerbated
body weight loss and an increased histopathology score at day 10
p.i. (IFN-gR2/2, 18.7 6 1.4% versus Wt, 2.2 6 1.7%, p # 0.01;
Fig. 1A–C). Morbidity was also clearly aggravated in IFN-gR2/2
mice, which was apparent from bloody feces, crouched posture,
and ruffled fur (data not shown). Infection of mice lacking the
cytokine (IFN-g2/2) resulted in a comparable body weight loss
and signs of morbidity at day 10 p.i. (IFN-g2/2, 19.7 6 1.0%
versus Wt, 2.4 6 1.0%, p # 0.001; Supplemental Fig. 1A). The
increased infection-induced pathology was even more pronounced
during secondary infections of IFN-gR2/2mice; a primary dose
of 50 oocysts followed by a challenge infection with the same
dose led to severe intestinal pathology and mortality in 100% of
IFN-gR2/2mice (Fig. 1A–C). In contrast, Wt mice showed no
significant weight loss during challenge infection and developed
only mild pathology (Fig. 1A–C).
MLN and spleen cells from IFN-gR2/2mice isolated at day 10
after primary infection showed a 14.9-fold (MLN) and 6.9-fold
(spleen) increase in proliferation to parasite Ag in comparison
with cells isolated from Wt mice (p # 0.01; Fig. 1D), implying
a dysregulation of the immune response to E. falciformis in the
absence of IFN-g signaling.
IFN-gR2/2mice infected with E. falciformis. (A) Body weight loss of Wt
and IFN-gR2/2mice after primary (1st) and challenge (2nd) infections
with 50 (E. falciformis) oocysts. Mice were euthanized upon loss of 20%
body weight (dotted line). (B) H&E-stained cecal thin sections of Wt and
IFN-gR2/2mice and (C) pathological scoring. (D) Parasite Ag-specific
proliferation of MLN and spleen cells of Wt (filled bars) and IFN-gR2/2
(open bars) mice isolated 10 d p.i. with E. falciformis. (E) Quantification of
shed oocysts from Wt and IFN-gR2/2mice following primary or chal-
lenge E. falciformis infection. (F) Quantification of the asexual develop-
ment (schizogony, day 5 p.i.) of E. falciformis in cecal crypts of Wt and
IFN-gR2/2mice during primary infection. Scale bars, 20 mm. Data shown
are means 6 SEM of six mice per group and representative of four in-
dependent experiments. *p # 0.05, **p # 0.01, ****p # 0.0001.
Pathology and parasite development in C57BL/6 Wt and
The Journal of Immunology3
on January 26, 2012
Signaling via the IFN-g receptor is not required for the control
of parasite replication or for the development of immunity
Because IFN-g is a key cytokine for the control of numerous in-
tracellular pathogens (1–5), we analyzed the development of E.
falciformis in IFN-g2/2and IFN-gR2/2mice. Surprisingly, sig-
nificantly fewer parasites developed in IFN-gR2/2mice compared
with Wt mice, as indicated by a reduced number of shed oocysts
(278%, p # 0.01; Fig. 1E). As described above (Fig. 1A–C), IFN-
gR2/2mice developed severe pathology, which was associated
with hemorrhagic diarrhea. To confirm the reliability of the
quantification of shed oocysts as an adequate parameter, we
assessed parasite development directly at the site of infection at an
early time point. In line with the oocyst data, analysis of cecal thin
sections on day 5 p.i. revealed 86% (p # 0.01) fewer parasites in
IFN-gR2/2mice compared with Wt mice (Fig. 1F). Because
a primary infection with a dose of 50 E. falciformis oocysts fol-
lowed by a secondary challenge infection with the same dose led
to 100% mortality in IFN-gR2/2mice (Fig. 1A), we instead
infected mice with a primary dose of 10 oocysts and challenged
them with 50 oocysts to assess whether signaling via the IFN-gR
is required for the development of immunity. Quantification of
shed parasites after challenge infection revealed a comparable
reduction of oocyst shedding in both IFN-gR2/2and Wt mice in
comparison with primary infected control mice (55 versus 58%,
p # 0.01; Fig. 1E). Thus, IFN-gR signaling is dispensable for the
control of E. falciformis replication.
Deficiency in IFN-g signaling leads to an expansion of
IL-17A– and IL-22–producing Th17 cells
Signaling via the IFN-gR has the ability to limit effector T cell
responses and to reciprocally regulate other arms of the adaptive
immune system, thereby preventing immunopathology caused by
an overactive immune response (21). Because enhanced prolifer-
ation of draining lymph node cells was seen following E. falci-
formis infection in IFN-gR2/2mice (Fig. 1D), we sought to assess
the cytokine profile and phenotype of the exaggerated immune
response 10 d after E. falciformis infection.
Strikingly, both E. falciformis-infected IFN-g2/2and IFN-gR2/2
mice had higher frequencies and numbers of CD4+T cells pro-
ducing IL-17A and IL-22 in the MLN and LPL and produced
higher amounts of IL-17A (IFN-gR2/2, 8.9 6 1.0 ng/ml versus Wt,
ND) and IL-22 (IFN-gR2/2, 0.43 6 0.08 versus Wt, 0.05 6 0.02
ng/ml) in comparison with E. falciformis-infected Wt controls, as
measured in Ag-specific restimulated MLN cultures via ELISA
(Fig. 2A, 2B, Supplemental Figs. 1B–D, 2A, 2B). No increase in
Th2-associated cytokine production was detected in IFN-gR2/2
mice (data not shown).
Surprisingly, IFN-g levels were also strongly increased in IFN-
gR2/2mice (IFN-gR2/2, 59.4 6 7.8 ng/ml versus Wt, 7.2 6 3.9
ng/ml, p # 0.01; Fig. 2A, 2B, Supplemental Fig. 2A, 2B). In line
with previous studies in our group (2), the major cellular source of
IFN-g during E. falciformis infection of Wt mice was found to be
CD8+T cells, with smaller numbers of IFN-g+CD49b+NK cells
and relatively few CD4+T cells (Supplemental Fig. 2B). In con-
trast, the enhanced IFN-g production observed in culture super-
natants of cells from IFN-gR2/2mice was found to be almost
entirely derived from an increase in CD4+IFN-g–producing
T cells (MLN: IFN-gR2/2, 0.36 6 0.05 3 106versus Wt, 0.08 6
0.01 3 106, p # 0.01; Supplemental Fig. 2B). CD4+IFN-g+
T cells also expressed T-bet (Fig. 2C), confirming an increase in
committed Th1 cells following loss of IFN-gR signaling.
Similarly, E. falciformis-elicited IL-17A+T cells expressed the
master transcription factor RORgt, indicating committed Th17 cells
(Fig.2C).Interestingly, we noted a subset ofT cells thatcoexpressed
T cells in the MLN and (B) total cell numbers of IL-17A–, IL-22–, and IFN-g–producing CD4+cells in the MLN and isolated LPL of Wt and IFN-gR2/2
mice 10 d after primary infection with E. falciformis. (C) Coexpression of IL-17A/RORgt and IFN-g/T-bet in the MLN cells of E. falciformis-infected IFN-
gR2/2mice. (D) Frequencies and total cell numbers of LPL Ly-6C+(Gr-12) monocytes, Gr-1+neutrophils, and CD4+T cells. Data shown are means 6
SEM of four to six mice per group and representative of three independent experiments. *p # 0.05, **p # 0.01.
Expansion of Th17 cells in IFN-gR2/2mice infected with E. falciformis. (A) Frequencies of IL-17A–, IL-22–, and IFN-g–producing CD4+
4Th17 RESPONSES DURING EIMERIA FALCIFORMIS INFECTION
on January 26, 2012
both IL-17A and IFN-g in the MLN and LPL (Fig. 2A). These cells
were found to coexpress both Th1 (T-bet)- and Th17 (RORgt)-
associated transcription factors (Supplemental Fig. 2C), indicating
the presence of a mixed Th1/Th17 cell phenotype and suggesting
a degree of T cell plasticity during E. falciformis infection.
Although CD4+T cells were the major source of IL-17A and
IL-22 in both the MLN and LPL compartments (Supplemental
Figs. 1D, 2B), we also noted production of both cytokines by non-
CD4+T cells. In the MLN, ∼14% of IL-17A– and IL-22–pro-
ducing cells lacked CD4 expression, but were found to be
lineage-positive (CD42Lin+) (Supplemental Fig. 3A). In contrast,
a significant proportion of IL-17A– and IL-22–producing cells in
the intestinal LPL compartment were found to be CD42lineage
marker negative (Lin2) (Supplemental Fig. 3B). The number of
Lin2IL-17A–/IL-22–producing cells was significantly increased
in the LPL compartment following E. falciformis infection
(Supplemental Fig. 3E). The Lin2populations expressing IL-17A
and IL-22 were apparently heterogeneous, as only a small pro-
portion of cells coproduced both cytokines (Supplemental Fig.
3C). Despite this, lamina propria Lin2IL-17A+and Lin2IL-22+
cells were phenotypically similar and were found to express
Sca-1, but not c-Kit (Supplemental Fig. 3D).
Next we assessed recruitment of Th1/Th17-associated innate
inflammatory cells to the intestinal tissue. In comparison with E.
falciformis-infected Wt mice, the number of Gr1+cells was found
to increase by 3.8-fold in the LPL compartment in IFN-gR2/2
mice (0.15 6 0.03 3 106versus 0.04 6 0.01 3 106, p # 0.01; Fig.
2D), indicating a strong neutrophil influx, an effect commonly
associated with Th17 responses. Conversely, although a 17-fold
increase in the total number of Ly6C+(Gr-12) inflammatory
monocytes in the LPL was seen following infection of Wt mice
(infected, 0.51 6 0.01 3 106versus naı ¨ve, 0.03 6 0.02 3 106),
this was dramatically reduced in IFN-gR2/2mice (0.25 6 0.04 3
106, p # 0.01), suggesting that the monocyte influx was IFN-g
dependent (Fig. 2D).
The expression profile in the infected cecum of IFN-gR2/2
mice reflects the dominant Th17 response
Analysis of the expression profile of downstream effector genes in
the cecum tissue of Wt mice infected with E. falciformis revealed
a strong increase in Th1-associated genes. The mRNA encoding
for CXCL9 and CXCL10, two chemokines that are important for
migration and function of Th1 cells, and inducible NO synthetase,
a major downstream effector of IFN-g, were strongly induced in
Wt mice compared with naive mice (Cxcl9, 90-fold; Cxcl10, 17-
fold; Nos2, 107-fold). This effect was almost completely abro-
gated in IFN-gR2/2mice with only low expression of Cxcl9,
Cxcl10, and Nos2 in comparison with naive mice (Cxcl9, 0.9-fold;
Cxcl10, 0.6-fold; Nos2, 18-fold; Fig. 3A).
Although the levels of several transcripts important for com-
mitment of cells to the Th17 lineage, including Il6, Tgfb1, and
Il23a, did not differ in the cecal tissue of Wt and IFN-gR2/2mice,
the mRNA coding for the IL-23R was significantly upregulated
(6-fold, p # 0.05; Fig. 3B). Furthermore, the dominant Th17 re-
sponse observed in IFN-gR2/2mice was reflected by a dramatic
upregulation of the transcripts encoding two IL-22-dependent
antimicrobial peptides, RegIIIb and RegIIIg (Reg3b, 2976-fold;
Reg3g, 663-fold; p # 0.01; Fig. 3C). The expression of matrix
metalloproteinase (MMP) 9 was also found to be significantly
increased in IFN-gR2/2mice (13-fold, p # 0.05; Fig. 3C).
IL-17A and IL-22 inhibit parasite development in vitro
The correlation between increased levels of IL-17A and IL-22
and decreased parasite development observed during infections
of IFN-gR2/2mice indicated a possible anti-parasite role of IL-
17A and/or IL-22 against E. falciformis. To test this hypothesis,
the mouse intestinal epithelial cell line CMT-93 was treated with
IL-17A, IL-22, and IFN-g (or IL-10 as a control) and the devel-
opment of the parasite was assessed in vitro. The cell line was
found to be immunocompetent, as demonstrated by the expression
of the relevant cytokine receptors (Fig. 4A). E. falciformis spo-
rozoites invaded CMT-93 cells efficiently and developed into the
first asexual generation of their life cycle (schizonts, Fig. 4B).
Invasion of CMT-93 cells by E. falciformis was unaffected by
treatment with any of the cytokines (data not shown), whereas
IL-17A, IL-22, and IFN-g all reduced the development of E. fal-
ciformis to the first asexual parasite generation (Fig. 4C). IL-22
and IL-17A significantly reduced the number of mature schizonts
by 52% (p # 0.001) and 47% (p # 0.01), respectively, whereas the
Th1 cytokine IFN-g inhibited the development to a lesser extent
(35%, p , 0.01). In contrast, treatment with IL-10 had no sig-
nificant effect on development of E. falciformis in this intestinal
epithelial cell line.
IL-17A and IL-22 contribute to immunopathology, and IL-22
has antiparasite effects during E. falciformis infection
To further dissect the roles of IL-17A and IL-22 in our model
in vivo, we concurrently depleted IL-17A and IL-22 during
infections of Wt and IFN-gR2/2mice with E. falciformis. Si-
multaneous neutralization of IL-17A and IL-22 significantly re-
duced E. falciformis-induced body weight loss in IFN-gR2/2
mice, whereas Ab neutralization had no significant effects in Wt
mice in comparison with control/IgG-treated mice (Fig. 5A).
Furthermore, combined neutralization of IL-17A and IL-22 led to
a significant reduction in E. falciformis-induced histopathology
and an almost complete restoration of normal intestinal architec-
ture (Fig. 5B, 5C). IL-17A/IL-22 neutralization correlated with
a significant decrease in inflammation as indicated by a reduction
in the total number of infiltrating CD4+T cells and Gr-1+neu-
cecum of E. falciformis-infected Wt and IFN-gR2/2mice. (A) Th1-reg-
ulated genes, (B) Th17 commitment-associated genes, and (C) Th17 cy-
tokine-regulated genes were assessed by quantitative real-time PCR in the
cecum of E. falciformis-infected Wt (filled bars) and IFN-gR2/2mice
(open bars) 10 d p.i. Data shown are means 6 SEM of five to six mice
per group and representative of two independent experiments. *p # 0.05,
**p # 0.01.
Expression profile of Th1/Th17-associated genes in the
The Journal of Immunology5
on January 26, 2012
trophils in the lamina propria of E. falciformis-infected IFN-gR2/2
mice following Ab treatment (p # 0.05, Fig. 5D). Moreover,
concurrent neutralization of IL-17A and IL-22 led to significantly
higher numbers of shed oocysts in comparison with IgG-treated
IFN-gR2/2controls (anti–IL-17/IL-22, 0.23 6 0.02 3 106versus
IgG, 0.13 6 0.01 3 106, p # 0.01; Fig. 5E). The increased oocyst
shedding was further found to correlate with significantly reduced
expression of the IL-22–dependent antimicrobial peptides RegIIIb
and RegIIIg in the intestinal tissue of infected mice following
cytokine neutralization (p # 0.01, Fig. 5F).
In contrast to double neutralization of IL-17A and IL-22, single
neutralization of either IL-17A or IL-22 failed to reverse E. fal-
ciformis-induced body weight loss in IFN-gR2/2mice (Fig. 5G),
although a slight but significant reduction in histopathology score
was seen in IFN-gR2/2mice following neutralization of IL-22
(Fig. 5H, 5I). This correlated with a marked trend toward fewer
infiltrating CD4+T cells in IL-22–depleted IFN-gR2/2mice,
whereas the number of Gr-1+neutrophils was significantly re-
duced in in IFN-gR2/2mice following either IL-17A or IL-22
neutralization (Fig. 5J). Importantly, total numbers of both cell
populations were still significantly higher than in infected Wt
mice, and the decrease was less pronounced than in combined IL-
17A/IL-22–neutralized mice (Fig. 5D, 5J). Whereas neutralization
of both IL-17A and IL-22 resulted in enhanced E. falciformis
oocyst output in IFN-gR2/2mice, neutralization of IL-17A alone
had no effect (Fig 5K). In contrast, single neutralization of IL-22
led to a significant increase in oocyst shedding in IFN-gR2/2mice
in comparison with IgG-treated controls (Fig. 5K). The increase in
oocyst shedding following IL-22 single neutralization was com-
parable to the effect seen in IL-17A/IL-22 double-neutralized
mice, suggesting that IL-22 was the dominant anti-parasite cyto-
kine in E. falciformis-infected IFN-gR2/2mice.
In this study we used a potent model of intestinal inflammation
driven by the large intestine-dwelling intracellular parasite E. fal-
ciformis to dissect the relative contributions and interplay of Th1
and Th17 immune responses in the generation of mucosal inflam-
mation and host immunity. We demonstrate that in the absence of
IFN-g signaling, E. falciformis-infected mice developed an increase
in immunopathology and morbidity driven by the Th17 cytokines
IL-17A and IL-22. Moreover, we identify a novel anti-parasite role
for IL-22, which reduced parasite development in the absence of
IFN-g during infection in vivo and in an epithelial cell line (sum-
marized in Supplemental Fig. 4). Importantly, these findings rep-
resent the first report, to our knowledge, that demonstrates a role for
IL-22 in host defense against intracellular intestinal parasites.
IL-17A production is highly correlated with disease severity
in patients suffering intestinal inflammatory disorders such as
ulcerative colitis and Crohn’s disease, and RORgt-expressing
Th17 cells are known to be essential for the development of co-
litis in murine models (7). In contrast, IL-22 has seemingly
pleiotropic functions during colitis. It is abundantly expressed in
the mucosa of patients suffering from inflammatory bowel disease
and drives production of proinflammatory cytokines and MMPs
(22). Conversely, IL-22 also has protective roles in murine ul-
cerative colitis models by promoting mucosal wound healing
responses (23). Following E. falciformis infection of IFN-gR2/2
or IFN-g2/2mice, CD4+RORgt+Th17 cells were found to be the
predominant cellular source of IL-17A and IL-22, although we
also noted a significant increase in innate cells producing IL-17A
and IL-22 in the lamina propria of E. falciformis-infected IFN-
gR2/2mice. These cells were found to be Lin2c-Kit2Sca-1+,
a phenotype resembling a population of IL-17A–/IL-22–produc-
ing innate intestinal lymphoid cells recently identified in a T cell-
independent model of colitis (24).
Our data suggest that IL-17A and IL-22 have redundant
proinflammatory roles during E. falciformis infection, as dramatic
reductions in cellular infiltrate and, in particular, attenuation of
body weight loss were only seen following concurrent neutrali-
zation of both cytokines. However, we cannot rule out the possi-
bility that other Th17 cytokines such as IL-17F and GM-CSF
contribute to E. falciformis-induced inflammation (7, 25). The
severe Th17-driven inflammation and pathology seen during the
self-limiting E. falciformis infection in the absence of IFN-g have
many parallels to murine models of colitis, and thus may prove to
be a powerful model for dissecting the immunological mecha-
nisms behind Th1/Th17 disease. For example, MMPs are potent
mediators of intestinal pathology, and mice deficient for MMP9
develop less severe inflammation and disease during a mouse
model of colitis (26). In line with these findings an increase in
MMP9 was observed in IFN-gR2/2mice following E. falciformis
infection. Furthermore, IL-22 but not IL-17A was shown to be
necessary to drive small intestinal inflammation and tissue damage
following oral infection of mice with T. gondii via induction of
proinflammatory cytokines and MMP2 (27).
Th17 responses are implicated in the etiology of many diseases
of intestinal inflammation. For example, polymorphisms in IL-23R
are highly associated with altered susceptibility to colitis (28, 29).
During E. falciformis infection of IFN-gR2/2mice we observed
an increased expression of IL-23R in the large intestine. Because
IFN-g can directly prevent Th17 expansion via suppression of IL-
23R (15), it is possible that IFN-g regulates parasite-induced Th17
responses in this manner during E. falciformis infection. We were
in untreated CMT-93 cells detected by RT-PCR. (B) Representative images of intracellular sporozoites (4 h p.i., left panel) and a mature schizont (39 h p.i.,
right panel) in E. falciformis-infected CMT-93 cells (parasites indicated by arrowheads), PAS stain. Original magnification 31000. (C) Development
(schizogony) of E. falciformis parasites assessed at 39 h p.i. of CMT-93 cells that were pretreated for 8 h with either media alone or supplemented with
50 ng/ml IL-17A, IL-22, IFN-g, and IL-10 prior to infection. Data are from three independent experiments and are shown as means 6 SEM of 7–11
samples per group. **p # 0.01, ***p # 0.001.
In vitro development of E. falciformis in cytokine-pretreated CMT-93 cells. (A) Expression of Th1/Th17-associated cytokine receptor chains
6Th17 RESPONSES DURING EIMERIA FALCIFORMIS INFECTION
on January 26, 2012
unable to detect any significant changes in the expression of es-
sential Th17-inducing cytokines, including TGF-b, IL-6, and IL-
23, in the intestines of IFN-gR2/2mice, and thus it is possible that
IFN-g may limit Th17 differentiation and expansion via direct
effects on Th17 cells, such as induction of apoptosis (30). Fur-
thermore, IFN-g was shown to suppress Th17 expansion via
induction of IDO expression in the lung during tuberculosis in-
fection (31). Interestingly, IDO expression was completely abro-
gated in the intestine during E. falciformis infection of IFN-g2/2
and IFN-gR2/2mice (data not shown). The regulation of Th17
cytokines by IFN-g may be of relevance in a wide range of dis-
eases, and deficiences in IFN-g or its receptor result in aggravated
disease associated with enhanced IL-17A and/or IL-22 and neu-
trophilia in murine models of arthritis, cancer, and tuberculosis
(31–33). Interestingly, in human populations, polymorphisms in
IFN-g signaling-associated genes correlate with decreased Th17
immune responses. In particular, it was recently shown that
patients with gain of function mutations in STAT-1, an essential
IFN-g signaling factor, have impaired IL-17A and IL-22 pro-
duction (34). Intriguingly, IFN-g and IL-22 are found on a highly
conserved locus in a wide range of species, suggesting a close
evolutionary relationship between these two important cytokines
(35). Thus, understanding the interplay between IFN-g signaling
and development of Th17 lineages may be of clinical relevance,
and IFN-g signals may directly regulate Th17 cytokine production
during E. falciformis infection.
Surprisingly, the loss of IFN-g or IFN-gR and the subsequent
increase in Th17 responses were not associated with attenuated
mice infected with 50 E. falciformis oocysts and treated with 200 mg anti–IL-17A and anti–IL-22 mAb (top panel), 200 mg anti–IL-22 or anti–IL-17A mAb
alone (bottom panel), or control mouse IgG Ab. Mice were euthanized upon 20% body weight loss (dotted line). (B and H) Representative image of H&E-
stained cecal thin sections. Scale bars, 100 mm. (C and I) Histopathology score. (D and J) Total cell numbers of LPL CD4+T cells and Gr-1+cells. (E and K)
Quantification of shed oocysts from day 8 to 10 p.i. of Wt (circles) and IFN-gR2/2(triangles) mice infected with 50 E. falciformis oocysts following
concurrent anti–IL-17A/anti–IL-22 treatment or single neutralization of anti–IL-17A or anti–IL-22. (F) Relative expression of the antimicrobial peptides
RegIIIb and RegIIIg in cecum samples of Wt and IFN-gR2/2mice infected with 50 E. falciformis oocysts and treated with anti–IL-17A and anti–IL-22 or
control mouse IgG Ab. Data shown are means 6 SEM of four to eight mice per group representative of three independent experiments. *p # 0.05, **p #
Neutralization of IL-17A and IL-22 during E. falciformis infection. (A and G) Body weight loss of Wt (circles) and IFN-gR2/2(triangles)
The Journal of Immunology7
on January 26, 2012
host defense, but rather with a decreased parasite shedding. Pre-
vious reports utilizing the large intestine-dwelling species Eimeria
pragensis also noted that host defense was not impaired in the
absence of IFN-g, but clinical signs were exacerbated (36). Al-
though CD8+cell-derived IFN-g may partially protect against
E. falciformis (2), we found that the switch to Th17-dominated
responses in the absence of IFN-g resulted in enhanced control of
asexual replication, decreased oocyst shedding, and comparable
protection from reinfection. IL-22 mediated parasite control, both
in vitro and in vivo, whereas IL-17A reduced parasite development
in our in vitro system only. The mechanisms by which these
cytokines inhibit E. falciformis development in vitro and in vivo are
currently unclear; however, both cytokines have critical roles in host
protection to other pathogens. IL-17A and IL-22 are important for
host defense during oral candidiasis (37, 38). Similarly, IL-22 is
essential for maintaining mucosal barrier function and for the en-
hanced epithelial cell proliferation and cytokine production required
to control K. pneumoniae infection (9). High levels of IL-17A and
IL-22 also strongly correlate with protective immunity in human
populations infected with Leishmania donovani (39), and IL-17A2/2
mice fail to control Trypanosoma cruzi infection, which leads to
multiple organ failure and mortality (40). However, to date no
protective role for IL-22 has been shown in parasite infections.
Neutralization of IL-22 in vivo or treatment of infected cells
in vitro highlighted the anti-parasite role of IL-22 during E. fal-
ciformis infection. This is of particular relevance as IL-22R
expression is restricted to non-hematopoetic cells including in-
testinal epithelial cells (41), which are the host cells for E. falci-
formis infection. Furthermore, IL-22 is a potent inducer of the Reg
family of antimicrobial peptides and innate IL-22 has been shown
to inhibit C. rodentium attachment and to prevent epithelial cell
damage in the gastrointestinal tract via induction of RegIIIb and
RegIIIg (10, 11). In our model, the drastic increase in the ex-
pression of RegIIIb and RegIIIg in IFN-gR2/2mice infected with
E. falciformis was reversed after concurrent neutralization of
IL-17A and IL-22, and it correlated with increased parasite
shedding. Thus, further research is required to determine whether
RegIII family proteins may have antiparasite properties and con-
tribute to host defense during E. falciformis infection.
Taken together, to our knowledge, we report for the first time
a novel compensatory anti-parasite role for IL-22 during intestinal
intracellular parasite infection in the absence of IFN-g, which
comes at the expense of increased intestinal pathology, mediated
partly by both IL-22 and IL-17A. Furthermore, these data provide
new information regarding the interplay between the Th1 and
Th17 arms of the adaptive immune system in mucosal inflam-
mation, with implications for the pathogenesis of inflammatory
diseases of the intestine, including colitis and pathogen-driven
We thank B. Sonnenburg, G. Meusel, M. Schmid, I. Meister, and F. Uhlitz
for excellent technical assistance and S. Spiekermann for histological
The authors have no financial conflicts of interest.
1. Huang, S., W. Hendriks, A. Althage, S. Hemmi, H. Bluethmann, R. Kamijo,
J. Vilcek, R. M. Zinkernagel, and M. Aguet. 1993. Immune response in mice that
lack the interferon-g receptor. Science 259: 1742–1745.
2. Pogonka, T., K. Schelzke, J. Stange, K. Papadakis, S. Steinfelder, O. Liesenfeld,
and R. Lucius. 2010. CD8+cells protect mice against reinfection with the in-
testinal parasite Eimeria falciformis. Microbes Infect. 12: 218–226.
3. Yap, G. S., and A. Sher. 1999. Effector cells of both nonhemopoietic and he-
mopoietic origin are required for interferon (IFN)-g- and tumor necrosis factor
(TNF)-a-dependent host resistance to the intracellular pathogen, Toxoplasma
gondii. J. Exp. Med. 189: 1083–1092.
4. McCall, M. B. B., and R. W. Sauerwein. 2010. Interferon-g: central mediator of
protective immune responses against the pre-erythrocytic and blood stage of
malaria. J. Leukoc. Biol. 88: 1131–1143.
5. Liesenfeld, O., J. Kosek, J. S. Remington, and Y. Suzuki. 1996. Association of
CD4+T cell-dependent, interferon-g-mediated necrosis of the small intestine
with genetic susceptibility of mice to peroral infection with Toxoplasma gondii.
J. Exp. Med. 184: 597–607.
6. Cua, D. J., and C. M. Tato. 2010. Innate IL-17-producing cells: the sentinels of
the immune system. Nat. Rev. Immunol. 10: 479–489.
7. Leppkes, M., C. Becker, I. I. Ivanov, S. Hirth, S. Wirtz, C. Neufert, S. Pouly,
A. J. Murphy, D. M. Valenzuela, G. D. Yancopoulos, et al. 2009. RORg-
expressing Th17 cells induce murine chronic intestinal inflammation via re-
dundant effects of IL-17A and IL-17F. Gastroenterology 136: 257–267.
8. Kamanaka, M., S. Huber, L. A. Zenewicz, N. Gagliani, C. Rathinam,
W. O’Connor, Jr., Y. Y. Wan, S. Nakae, Y. Iwakura, L. Hao, and R. A. Flavell.
2011. Memory/effector (CD45RBlo) CD4 T cells are controlled directly by IL-10
and cause IL-22-dependent intestinal pathology. J. Exp. Med. 208: 1027–1040.
9. Aujla, S. J., Y. R. Chan, M. Zheng, M. Fei, D. J. Askew, D. A. Pociask,
T. A. Reinhart, F. McAllister, J. Edeal, K. Gaus, et al. 2008. IL-22 mediates mucosal
host defense against Gram-negative bacterial pneumonia. Nat. Med. 14: 275–281.
10. Sonnenberg, G. F., L. A. Monticelli, M. M. Elloso, L. A. Fouser, and D. Artis.
2011. CD4+lymphoid tissue-inducer cells promote innate immunity in the gut.
Immunity 34: 122–134.
11. Zheng, Y., P. A. Valdez, D. M. Danilenko, Y. Hu, S. M. Sa, Q. Gong,
A. R. Abbas, Z. Modrusan, N. Ghilardi, F. J. de Sauvage, and W. Ouyang. 2008.
Interleukin-22 mediates early host defense against attaching and effacing bac-
terial pathogens. Nat. Med. 14: 282–289.
12. Ivanov, I. I., B. S. McKenzie, L. Zhou, C. E. Tadokoro, A. Lepelley, J. J. Lafaille,
D. J. Cua, and D. R. Littman. 2006. The orphan nuclear receptor RORgt directs
the differentiation program of proinflammatory IL-17+T helper cells. Cell 126:
13. Veldhoen, M., R. J. Hocking, C. J. Atkins, R. M. Locksley, and B. Stockinger.
2006. TGFb in the context of an inflammatory cytokine milieu supports de novo
differentiation of IL-17-producing T cells. Immunity 24: 179–189.
14. Zhou, L., I. I. Ivanov, R. Spolski, R. Min, K. Shenderov, T. Egawa, D. E. Levy,
W. J. Leonard, and D. R. Littman. 2007. IL-6 programs TH-17 cell differentiation
by promoting sequential engagement of the IL-21 and IL-23 pathways. Nat.
Immunol. 8: 967–974.
15. Harrington, L. E., R. D. Hatton, P. R. Mangan, H. Turner, T. L. Murphy,
K. M. Murphy, and C. T. Weaver. 2005. Interleukin 17-producing CD4+effector
T cells develop via a lineage distinct from the T helper type 1 and 2 lineages.
Nat. Immunol. 6: 1123–1132.
16. Cruz, A., S. A. Khader, E. Torrado, A. Fraga, J. E. Pearl, J. Pedrosa,
A. M. Cooper, and A. G. Castro. 2006. Cutting edge: IFN-g regulates the in-
duction and expansion of IL-17-producing CD4 T cells during mycobacterial
infection. J. Immunol. 177: 1416–1420.
17. Lazarevic, V., X. Chen, J.-H. Shim, E.-S. Hwang, E. Jang, A. N. Bolm,
M. Oukka, V. K. Kuchroo, and L. H. Glimcher. 2011. T-bet represses TH17
differentiation by preventing Runx1-mediated activation of the gene encoding
RORgt. Nat. Immunol. 12: 96–104.
18. Owen, D. 1975. Eimeria falciformis (Eimer, 1870) in specific pathogen free and
gnotobiotic mice. Parasitology 71: 293–303.
19. Shirley, M. W., A. Ivens, A. Gruber, A. M. Madeira, K. L. Wan, P. H. Dear, and
F. M. Tomley. 2004. The Eimeria genome projects: a sequence of events. Trends
Parasitol. 20: 199–201.
20. Kurth, M., and R. Entzeroth. 2008. Improved excystation protocol for Eimeria
nieschulzi (Apikomplexa, Coccidia). Parasitol. Res. 102: 819–822.
21. Li, X., K. K. McKinstry, S. L. Swain, and D. K. Dalton. 2007. IFN-g acts di-
rectly on activated CD4+T cells during mycobacterial infection to promote
apoptosis by inducing components of the intracellular apoptosis machinery and
by inducing extracellular proapoptotic signals. J. Immunol. 179: 939–949.
22. Andoh, A., Z. Zhang, O. Inatomi, S. Fujino, Y. Deguchi, Y. Araki, T. Tsujikawa,
K. Kitoh, S. Kim-Mitsuyama, A. Takayanagi, et al. 2005. Interleukin-22,
a member of the IL-10 subfamily, induces inflammatory responses in colonic
subepithelial myofibroblasts. Gastroenterology 129: 969–984.
S. Hirth, B. Weigmann, S. Wirtz, et al. 2009. STAT3 links IL-22 signaling in in-
testinal epithelial cells to mucosal wound healing. J. Exp. Med. 206: 1465–1472.
24. Buonocore, S., P. P. Ahern, H. H. Uhlig, I. I. Ivanov, D. R. Littman, K. J. Maloy,
and F. Powrie. 2010. Innate lymphoid cells drive interleukin-23-dependent innate
intestinal pathology. Nature 464: 1371–1375.
25. Codarri, L., G. Gyu ¨lve ´szi, V. Tosevski, L. Hesske, A. Fontana, L. Magnenat,
T. Suter, and B. Becher. 2011. RORgt drives production of the cytokine GM-CSF
in helper T cells, which is essential for the effector phase of autoimmune neu-
roinflammation. Nat. Immunol. 12: 560–567.
26. Castaneda, F. E., B. Walia, M. Vijay-Kumar, N. R. Patel, S. Roser,
V. L. Kolachala, M. Rojas, L. Wang, G. Oprea, P. Garg, et al. 2005. Targeted
deletion of metalloproteinase 9 attenuates experimental colitis in mice: central
role of epithelial-derived MMP. Gastroenterology 129: 1991–2008.
27. Mun ˜oz, M., M. M. Heimesaat, K. Danker, D. Struck, U. Lohmann, R. Plickert,
S. Bereswill, A. Fischer, I. R. Dunay, K. Wolk, et al. 2009. Interleukin (IL)-23
mediates Toxoplasma gondii-induced immunopathology in the gut via
8 Th17 RESPONSES DURING EIMERIA FALCIFORMIS INFECTION
on January 26, 2012
matrixmetalloproteinase-2 and IL-22 but independent of IL-17. J. Exp. Med.
28. Morrison, P. J., S. J. Ballantyne, and M. C. Kullberg. 2011. Interleukin-23 and
T helper 17-type responses in intestinal inflammation: from cytokines to T-cell
plasticity. Immunology 133: 397–408.
29. Duerr, R. H., K. D. Taylor, S. R. Brant, J. D. Rioux, M. S. Silverberg, M. J. Daly,
A. H. Steinhart, C. Abraham, M. Regueiro, A. Griffiths, et al. 2006. A genome-
wide association study identifies IL23R as an inflammatory bowel disease gene.
Science 314: 1461–1463.
30. Wildbaum, G., Y. Zohar, and N. Karin. 2010. Antigen-specific CD252Foxp32
IFN-ghighCD4+T cells restrain the development of experimental allergic en-
cephalomyelitis by suppressing Th17. Am. J. Pathol. 176: 2764–2775.
31. Desvignes, L., and J. D. Ernst. 2009. Interferon-g-responsive nonhematopoietic
cells regulate the immune response to Mycobacterium tuberculosis. Immunity 31:
32. Kelchtermans, H., E. Schurgers, L. Geboes, T. Mitera, J. Van Damme, J. Van
Snick, C. Uyttenhove, and P. Matthys. 2009. Effector mechanisms of interleukin-
17 in collagen-induced arthritis in the absence of interferon-g and counteraction
by interferon-g. Arthritis Res. Ther. 11: R122.
33. He, D., H. Li, N. Yusuf, C. A. Elmets, J. Li, J. D. Mountz, and H. Xu. 2010. IL-
17 promotes tumor development through the induction of tumor promoting
microenvironments at tumor sites and myeloid-derived suppressor cells. J.
Immunol. 184: 2281–2288.
34. Liu, L., S. Okada, X.-F. Kong, A. Y. Kreins, S. Cypowyj, A. Abhyankar,
J. Toubiana, Y. Itan, M. Audry, P. Nitschke, et al. 2011. Gain-of-function human
STAT1 mutations impair IL-17 immunity and underlie chronic mucocutaneous
candidiasis. J. Exp. Med. 208: 1635–1648.
35. Savan, R., S. Ravichandran, J. R. Collins, M. Sakai, and H. A. Young. 2009.
Structural conservation of interferon gamma among vertebrates. Cytokine
Growth Factor Rev. 20: 115–124.
36. Rose, M. E., D. Wakelin, and P. Hesketh. 1991. Interferon-g-mediated effects
upon immunity to coccidial infections in the mouse. Parasite Immunol. 13: 63–74.
37. Conti, H. R., F. Shen, N. Nayyar, E. Stocum, J. N. Sun, M. J. Lindemann,
A. W. Ho, J. H. Hai, J. J. Yu, J. W. Jung, et al. 2009. Th17 cells and IL-17 re-
ceptor signaling are essential for mucosal host defense against oral candidiasis.
J. Exp. Med. 206: 299–311.
38. De Luca, A., T. Zelante, C. D’Angelo, S. Zagarella, F. Fallarino, A. Spreca,
R. G. Iannitti, P. Bonifazi, J.-C. Renauld, F. Bistoni, et al. 2010. IL-22 defines
a novel immune pathway of antifungal resistance. Mucosal Immunol. 3: 361–373.
39. Pitta, M. G. R., A. Romano, S. Cabantous, S. Henri, A. Hammad, B. Kouriba,
L. Argiro, M. el Kheir, B. Bucheton, C. Mary, et al. 2009. IL-17 and IL-22 are
associated with protection against human kala azar caused by Leishmania
donovani. J. Clin. Invest. 119: 2379–2387.
40. Miyazaki, Y., S. Hamano, S. Wang, Y. Shimanoe, Y. Iwakura, and H. Yoshida.
2010. IL-17 is necessary for host protection against acute-phase Trypanosoma
cruzi infection. J. Immunol. 185: 1150–1157.
41. Sonnenberg, G. F., L. A. Fouser, and D. Artis. 2011. Border patrol: regulation of
immunity, inflammation and tissue homeostasis at barrier surfaces by IL-22. Nat.
Immunol. 12: 383–390.
The Journal of Immunology9
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