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Toxoplasma gondiiMicrobial Antigen of The Induction of Acute Ileitis by a Single

Boothroyd and Lloyd H. KasperMinns, Michael E. Grigg, Stanislas Tomavo, John C.Franck J. D. Mennechet, Souphalone Luangsay, Laurie A. Nicolas Rachinel, Dominique Buzoni-Gatel, Chaitali Dutta,

http://www.jimmunol.org/content/173/4/2725doi: 10.4049/jimmunol.173.4.2725

2004; 173:2725-2735; ;J Immunol 

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Print ISSN: 0022-1767 Online ISSN: 1550-6606. Immunologists All rights reserved.Copyright © 2004 by The American Association of1451 Rockville Pike, Suite 650, Rockville, MD 20852The American Association of Immunologists, Inc.,

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The Induction of Acute Ileitis by a Single Microbial Antigen ofToxoplasma gondii1

Nicolas Rachinel,*† Dominique Buzoni-Gatel,2*† Chaitali Dutta,* Franck J. D. Mennechet,*Souphalone Luangsay,* Laurie A. Minns,* Michael E. Grigg,‡ Stanislas Tomavo,§

John C. Boothroyd,‡ and Lloyd H. Kasper2*

The role of specific microbial Ags in the induction of experimental inflammatory bowel disease is poorly understood. Oral infectionof susceptible C57BL/6 mice withToxoplasma gondii results in a lethal ileitis within 7–9 days postinfection. An immunodominantAg of T. gondii (surface Ag 1 (SAG1)) that induces a robust B and T cell-specific response has been identified and a SAG1-deficientparasite (�sag1) engineered. We investigated the ability of�sag1 parasite to induce a lethal intestinal inflammatory response insusceptible mice. C57BL/6 mice orally infected with�sag1 parasites failed to develop ileitis. In vitro, the mutant parasites replicatein both enterocytes and dendritic cells. In vivo, infection with the mutant parasites was associated with a decrease in the chemokineand cytokine production within several compartments of the gut-associated cell population. RAG-deficient (RAG1�/�) mice areresistant to the development of the ileitis afterT. gondii infection. Adoptive transfer of Ag-specific CD4� effector T lymphocytesisolated from C57BL/6-infected mice into RAG�/� mice conferred susceptibility to the development of the intestinal disease. Incontrast, CD4� effector T lymphocytes from mice infected with the mutant �sag1 strain failed to transfer the pathology. Inaddition, resistant mice (BALB/c) that fail to develop ileitis following oral infection with T. gondii were rendered susceptiblefollowing intranasal presensitization with the SAG1 protein. This process was associated with a shift toward a Th1 response. Thesefindings demonstrate that a single Ag (SAG1) ofT. gondii can elicit a lethal inflammatory process in this experimental model ofpathogen-driven ileitis. The Journal of Immunology, 2004, 173: 2725–2735.

T he intestinal epithelium provides both a physiologic andimmunologic barrier to a range of microorganisms andforeign substances. When an imbalance does occur in the

regulation of this response, lethal inflammatory disease may de-velop (1). Complex interactions between invading pathogen, com-mensal bacteria, and the host immune response are involved in thedevelopment of inflammatory bowel disease (IBD)3 in humans (2).Individuals with Crohn’s disease mount T cell proliferative re-sponses to autologous microbial Ags, suggesting that chronic in-flammation of the gastrointestinal mucosa may be sustained by anAg. Germfree conditions in murine models generally do not favorthe development of IBD (3, 4). Similarly, Crohn’s disease andulcerative colitis in humans respond to antibiotics (5, 6). A numberof bacteria including Helicobacter hepaticus (7, 8) Citrobacter ro-

dentium (9), mycobacteria, Campylobacter, and Clostridium (10)can elicit intestinal inflammation. In addition, viruses such as her-pes simplex (11) and Epstein-Barr, the primary measles virus (12),can cause IBD-like disease under specific circumstances (13). Therole of microbial Ags in influencing the local inflammatory re-sponse and the contribution of these Ags to the development oflesions have yet to be well understood.

Oral infection with Toxoplasma gondii in susceptible C57BL/6,but not in the resistant BALB/c mice, leads to a Th1-type acute andlethal ileitis (14, 15). In the absence of genetic or chemical ma-nipulation, this pathogen-driven experimental model develops re-markable similarities to human ileitis (14). Following oral infec-tion, C57BL/6 mice exhibit discontinuous areas of transmuralintestinal inflammation, throughout the distal ileum. Histologicalexamination demonstrates mononuclear and polymorphonuclearcell (PMN) infiltrates in the lamina propria (LP), submucosa, andmuscle layers. Inflamed small intestinal mucosa exhibits partialvillous blunting and hemorrhages. The intestinal pathology in-duced by T. gondii requires the induction of proinflammatory cy-tokines such as IFN-�, TNF-�, and inducible NO synthase (iNOS)as well as the regulatory effects of TGF-� (16). Neutralization ofeither IFN-� or CD4� T cells during oral infection prevents ne-crosis of the ilea and acute mortality (14, 17).

T. gondii is an obligate intracellular protozoan that infects awide variety of vertebrate hosts, including humans. Following oralinfection of tissue cyst bradyzoites, the parasite transforms throughan as of yet unknown mechanism into a rapidly replicatingtachyzoite that can then actively invade target cells, beginning thelytic cycle of the parasite. The surface of T. gondii comprises afamily of developmentally regulated GPI-linked proteins (surfaceAg (SAG)-related sequences), of which SAG1 is the prototypicmember (18). SAG1 protein is exclusively expressed on thetachyzoite (19, 20). The biological role for this superfamily of

*Departments of Medicine and Microbiology/Immunology, Dartmouth MedicalSchool, Lebanon, NH 03756; †Department of Parasitology, Institut Pasteur, Paris,France; ‡Department of Microbiology and Immunology, School of Medicine, Stan-ford University, Stanford, CA 94305; and §Equipe de Parasitologie Moleculaire,Laboratoire de Glycobiologie Structurale et Fonctionnelle, Centre National de la Re-cherche Scientifique Unite Mixte de Recherche 8576, Universite des Sciences etTechnologies de Lille, Villeneuve d’Ascq, France

Received for publication January 12, 2004. Accepted for publication May 21, 2004.

The costs of publication of this article were defrayed in part by the payment of pagecharges. This article must therefore be hereby marked advertisement in accordancewith 18 U.S.C. Section 1734 solely to indicate this fact.1 This work was supported by National Institutes of Health Grants AI19613, AI30000,and TW011003.2 Address correspondence and reprint requests to Dr. Lloyd H. Kasper, 710 RubinResearch Building, Department of Medicine and Microbiology, Dartmouth MedicalSchool, Lebanon, NH 03756; or Dr. Dominique Buzoni-Gatel, Department of Parasitol-ogy, Institut Pasteur, 75015, Paris, France. E-mail addresses: Lloyd.H.Kasper@Dartmouth.edu or buzoni@pasteur.fr3 Abbreviations used in this paper: IBD, inflammatory bowel disease; CT, choleratoxin; DC, dendritic cell; IEL, intraepithelial lymphocyte; iNOS, inducible NO syn-thase; KO, knockout; LP, lamina propria; LPMC, LP mononuclear cell; PMN, poly-morphonuclear cell; RPA, RNase protection assay; SAG, surface Ag.

The Journal of Immunology

Copyright © 2004 by The American Association of Immunologists, Inc. 0022-1767/04/$02.00

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surface proteins remains mostly enigmatic, although there is evi-dence for a role in parasite attachment. SAG1 induces the domi-nant Ab response during infection (21, 22) and a strong, systemicTh1-like T cell response characterized by high titer IFN-� produc-tion by CD4 and CD8 T lymphocytes (23).

A SAG1 null mutant was engineered by homologous recombi-nation and used to infect C57BL/6 mice. This mutant was shownin vitro to adhere and to replicate in fibroblasts at the same or evenat a better rate than the control parental strain (M. Grigg and J.Boothroyd, manuscript in preparation). In vivo, we demonstratethat this Ag-deficient parasite is not able to induce ileitis followingintralumenal infection. Although this mutant can replicate in boththe host and in vitro cell culture, infection is associated with adecrease in both innate and adaptive inflammatory immune re-sponses. Host infection with the mutant strain affects the capacityof enterocytes to secrete chemokines, of dendritic cells (DCs) toproduce IL-12, and of LP CD4� T cells to elicit a proinflammatoryTh1-like cytokine response. Adoptive transfer of T lymphocytessensitized with the wild-type parasite, but not with the mutant,confers susceptibility to the infection of RAG�/� mice, normallyresistant to the development of the ileitis. SAG1 sensitization ofresistant BALB/c mice, followed by Ag re-exposure, results in thedevelopment of acute ileitis.

Materials and MethodsParasites and mouse infection

Tachyzoites from the wild-type (RH�) (24), the �sag1 (lacking the sag1gene), the sag1-complemented M34 (generously provided by J. Boothroydand M. Grigg, Stanford, CA), and the SAG3 knockout (KO) strains (gen-erously provided by S. Tomavo, Lille, France) were used for intraintestinalinfection. The 76K strain cysts were maintained in the brain of chronicallyinfected mice and were used for the BALB/c mouse infection. All of theseToxoplasma strains are isogenic.

Five- to 7-wk-old female C57BL/6, BALB/c, and RAG-deficient mice(RAG�/�) on a C57BL/6 background (The Jackson Laboratory, Bar Har-bor, ME), housed under approved conditions of the animal research facilityat Dartmouth Medical School, were used in this study. Infection ofC57BL/6 was established by surgery injection of 10,000 tachyzoites di-rectly into the intestines of anesthetized mice. For presensitization proce-dures, BALB/c mice received intranasally two equivalent doses 28 daysapart of 10 �g of purified SAG1, 10 �g of SAG1 plus 0.5 �g of choleratoxin (CT; List Biological Laboratories, Campbell, CA), or 0.5 �g of CTalone. The sensitization efficiency was verified, as described (25). Oneweek after the last boost, BALB/c mice were challenged by gavage with100 cysts from the 76K strain. RAG�/� mice were challenged with 35cysts from the 76K strain.

Cell culture

mICcl2 cells (26), derived from C57BL/6 mice, were grown in completeDMEM/Ham’s F12 (1/1, v/v; Invitrogen Life Technologies, Grand Island,NY) and were plated on collagen I-coated plate (2 � 106 cells/well forRNA study and 1 � 105 cells/well for [3H]uracil incorporation).

For cell culture, DCs were purified from the spleen of Fms-like tyrosinekinase 3 ligand-treated mice (27) by a negative selection with anti-CD3-coated magnetic beads, followed by a positive selection with anti-CD11c-coated magnetic beads using MidiMACS columns (Miltenyi Biotec, Au-burn, CA). Purity of magnetically sorted CD11c cell population was�95%, as attested by FACScan analysis (BD Biosciences, San Jose, CA).DCs were plated at 1 � 105 cells/well for [3H]uracil incorporation in com-plete medium.

T. gondii intracellular multiplication

Ten-day differentiated mICcl2 cells (1 � 105 cells/well) and fresh splenicCD11c� cells (1 � 105 cells/well) were infected with either wild-type (RH�)or mutant �sag1 (at a ratio 1:2 parasite-cell). Two hours later, unbound par-asites were removed, and new medium with 0.5 �Ci of [3H]uracil (Amersham,Piscataway, NJ) was added. Twenty-four hours later, the radioactivity wascounted with a scintillation counter (Beckman Coulter, Fullerton, CA).

Quantitation of parasite DNA by real-time PCR

DNA from tissues was extracted using the Qiamp tissue kit (Qiagen, Chats-worth, CA); amplification of parasite DNA was performed using specificprimers for the Toxoplasma B1 gene (5�-GGAACTGCATCCGTTCATGAG-3� and 5�-TCTTTAAAGCTTCGTGGTC-3�). The mouse �-actinhousekeeping gene (5�-AGAGGGAAATCGTGCGTGAC and 5�-CAATAGTGATGACCTGGCCGT) was also amplified at the same time to nor-malize the quality and quantity of DNA between samples. A plasmid con-taining the same primer template sequences was used as a standard tocalculate the number of parasites per �g of DNA. Real-time PCR wasconducted with the SYBR Green PCR Core Kit (Applied Biosystems, War-rington, U.K.) on an iCycler iQ instrument (Bio-Rad, Hercules, CA). Am-plification conditions were 95°C for 8 min, followed by 40 cycles of 94°Cfor 15 s, 63°C for 45 s, and 72°C for 15 s.

Purification of LP CD4� T lymphocytes and intestinal CD11c�

cells

LP CD4� T cells and CD11c� were purified from at least 12 mice, asdescribed (17). Peyer’s patches were removed, and sliced intestines wereplaced in RPMI 1640 with 25 mM EDTA for 45 min. Intestinal fragmentswere then incubated for 2 h at 37°C in RPMI 1640 containing 125 UI/mlcollagenase VIII (Sigma-Aldrich, St. Louis, MO). After centrifugation ona Ficoll layer, the CD4� LP T cells were purified by positive selectionusing anti-CD4 beads and CD11c� cells using anti-CD11c (L3T4 mi-croBeads; Miltenyi Biotec). The purity of CD4� LP cell population was�90%, as attested by FACScan analysis (clone L3T4; BD Biosciences).CD11c� cells were also isolated from the mesenteric lymph nodes follow-ing the same purification procedures as for the LP.

CD4�CD45RBhighCD25� T cell transfer

Mononuclear cells isolated either from the LP or the mesenteric lymph nodeswere stained with anti-CD25 PE, anti-CD45RB FITC, and anti-CD4 Cy-Chrome (BD Pharmingen, San Diego, CA) Abs. CD4�CD45RBhighCD25� Tcells (named T effector cells) were then sorted by FACSorter (BD Bio-sciences). The purified cells (1 � 105) were adoptively transferred by i.v. routeinto immunocompromised RAG1�/� mice. The purity of the sorted cells was�97%. One day after the transfer, recipient mice were infected orally with theparasites.

SAG1 Ag purification

The SAG1 protein was purified by immunoaffinity (28), using the mono-clonal IgG anti-SAG1 (mAb 1E5) (29). A sonicated Ag was prepared fromRH tachyzoites, as described elsewhere, and referred to as crude extract(30). SAG1 was purified using an anti-SAG1 mAb affinity column, aspreviously described (28). The purity was determined by electrophoresis,followed by a transfer to a nitrocellulose paper probed with the mAb 1E5.The quantification of SAG1 was performed with a bicinchoninic acid pro-tein assay reagent kit (Pierce, Rockford, IL).

Cytokine assays

To detect total TGF-�, 100 mg of infected tissues was homogenized andassayed by ELISA (BioSource International, Camarillo, CA). IL-12p70production by the CD11c� cells isolated from the mesenteric lymph nodesand from the LP (4 � 105 cells/well; DC-parasites ratio 1:1) was assayedby ELISA.

RNase protection assay (RPA)

Total mRNA either from freshly isolated LP mononuclear cells (LPMC)(infected or uninfected) or from the mICcl2 cells (infected or uninfected)was extracted using the TRIzol reagent (Invitrogen Life Technologies).Cytokine and chemokine expression was detected using the RiboQuantmultiProbe RPA system kit (BD Pharmingen). For quantification, banddensities were analyzed by NIH Image 1.61/ppc software using a Macin-tosh computer. Results were expressed as the percentage of the band in-tensity relative to the intensity of the housekeeping (GADPH andL32) RNA.

Histological assessment of intestinal inflammation

Intestines were embedded in paraffin and stained with H&E for histologicexamination. Only specimens exhibiting longitudinally oriented sectionsthrough the crypts were measured. Histological inflammatory score rangingfrom 0 to 4, as previously described (31), was applied in a blinded fashionto estimate intestinal inflammation: 0, no inflammation; 1, slight infiltratingcells in LP with focal acute infiltration; 2, mild infiltrating cells in the LP

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with increased blood flow and edema; 3, diffuse and massive infiltratingcells leading to disturbed mucosal architecture; 4, crypt abscess and ne-crosis of the intestinal villi.

Statistical analysis

For ELISA experiments, results were expressed as the mean of cytokineconcentration � SD. Statistical differences between groups were analyzedusing Student’s t test. Value of p � 0.05 was considered as significant.

Results�sag1 parasites fail to induce lethal necrosis of the ilea

C57BL/6 mice are susceptible to infection with T. gondii, andacute mortality occurs within 7 days (15) as a result of IFN-�-mediated necrosis and hemorrhage of the ileum (14). To evaluatethe ability of the SAG1 Ag to induce intestinal inflammation,C57BL/6 were infected with a lethal dose of either parental RH�or �sag1 parasites. Disease progression was assessed daily. Boththe time to death and survival were significantly reduced after

infection with the wild-type compared with the �sag1 mutant (Fig.1A). Morphologic and histologic examination of intestines at days5, 7, and 9 postinoculation demonstrated an absence of inflamma-tion in those mice infected with �sag1 parasites. By day 7, severenecrosis of the ilea, predominantly within the villi, a total absenceof columnar epithelial cells, and massive accumulation of poly-morphonuclear inflammatory cells were detected in the intestinalepithelium of mice infected with RH� (Fig. 1B). In striking con-trast at both days 7 (Fig. 1D) and 9 (data not shown) postchallenge,the intestinal epithelium of mice infected with �sag1 parasites wasconsistent with the saline controls and without evidence of signif-icant inflammation.

Genetically manipulated strains that express a functional sag1gene and the corresponding protein on their surface were added asadditional controls and tested for their ability to induce the intes-tinal pathology. These controls included the M34 strain that hasbeen engineered by reinserting the sag1 gene in the �sag1 strain

FIGURE 1. A, Cumulative percentage of sur-vival of C57BL/6 mice infected with T. gondii.C57BL/6 mice were infected intraintestinallywith either 10,000 parental RH� or mutant�sag1 tachyzoites. Data are representative offive independent experiments (n 9 mice/group). The delay to death was significantly in-creased (p � 0.05) after infection with the �sag1mutant compared with parental strain. B–F, His-tological examination of small intestine fromC57BL/6 mice. Histological sections of the in-testine stained with H&E were performed at day7 postinoculation with either RH (B), or �sag1parasites (D), or M34 (E), or SAG3KO (F)tachyzoites, or on naive mice (C). M34 is a mu-tant complemented for the sag1 gene, andSAG3KO is a strain genetically invalidated forthe expression of SAG3 protein. Data are repre-sentative of five independent experiments (n 9mice/group).

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and a deficient parasite for SAG3, SAG3KO strain (M. E. Grigg,manuscript in preparation) (32). Intestinal infection with 10,000parasites from either the M34 or the SAG3KO strains induced anintestinal inflammation 7 days later (Fig. 1, E and F).

Taken together, these results emphasized the important role ofSAG1 in intestinal inflammation following T. gondii infection inC57BL/6.

Increased parasite burden associated with �sag1 parasiteinfection

One possible explanation for �sag1 weak intestinal pathogenicitymight be that the �sag1 mutant is less capable of colonizing andreplicating in its host. For the wild-type RH�, parasite burdenpeaked 5–7 days postinfection before declining to undetectablelevels in the liver, intestine, and spleen of surviving mice. In con-trast, parasite burden in �sag1-infected mice was comparable toRH� infections over the first 5 days, but then rose exponentiallyover the next 4 days, peaking by day 9 postinfection. Between days7 and 9, parasite load in the intestine and spleen was typically2-fold greater in mice infected with the Ag-deficient parasite thanRH�-infected mice (Fig. 2A).

Both enterocytes and DCs are important as first lines of defenseagainst the invading parasite. An vitro assay was developed toevaluate whether the replication of the �sag1 mutant parasite wasconsistent with that of the wild-type strain in these different cellcompartments.

The mICcl2 cell line that is derived from the enterocytes ofC57BL/6 mice as well as splenic DCs was infected either with thewild-type (RH�) or mutant �sag1 parasites, and 24 h later, para-site replication was measured via the [3H]uracil incorporation test.The rate of parasite replication in the enterocyte line was consis-tent among the different parasite strains tested (Fig. 2B). Similarrates of replication were observed in the splenic derived DCs (Fig.2C). These data suggest that the reduced pathogenicity of the�sag1 parasites is not related to a decrease in replication in thesetwo important cell populations or in the whole host.

Reduced production of inflammatory innate immune responseafter infection with �sag1 parasites

Oral infection with the wild-type strain enhances the production ofchemokines in the small intestine (17). Chemokines are essentialfor the attraction of inflammatory cells, including macrophages,DCs, PMNs, and T and B cells. All of these cells have been shownto play a role in the clearance of parasites from the infected host.Compared with mice infected with the wild-type parasites, miceinfected with the Ag-deficient parasites display decreased levels ofchemokine mRNA in their intestinal tissue, in particular for MIP-2and MCP-1 (Fig. 3, A and B). Enterocytes that are the first barrierof defense against pathogen invasion are important for the produc-tion of these molecules (17). Enterocytes from the mICcl2 cell linewere examined for their ability to produce a number of chemo-kines in response to parasite infection. mRNA was isolated at 6 hafter infection with either the mutant �sag1 or the wild-type(RH�) parasites. The mRNA expression for MIP-2 (Fig. 3C) andMCP-1 (CCL-2) (Fig. 3D) was significantly higher in the mICcl2

cells infected with the wild-type parasites in comparison with�sag1 parasites. The DCs might also be a crucial cell populationfor intestinal chemokine production. Ex vivo CD11c� cells iso-lated from the mesenteric lymph nodes display a lower ability toproduce chemokine MIP-1� (CCL3) and RANTES (CCL5) (Fig.3, E and F). Similar results were obtained with CD11c� isolatedfrom the LP (data not shown). These data indicate that SAG-1protein can induce the expression of several chemokines that maybe important in the initiation of the inflammatory response in the

intestine. In response to pathogen exposure, enterocytes and DCscan produce additional molecules such as NO and/or IL-12p70 thatwould directly participate in the innate inflammatory process (33).In the intestinal tissue, taken as a whole, the production of iNOS(Fig. 4A) was significantly decreased in mice infected with the�sag1 parasites as compared with the wild-type RH� parasites. Asubstantial difference in iNOS production was observed whenLPMC were compared. LPMC isolated from mice infected withRH� parasites produced a substantially greater quantity of mRNAfor iNOS, compared with those mice infected with the �sag1 mu-tant parasites that failed to express iNOS mRNA (Fig. 4B). Inaddition, we observed that following infection with the �sag1 par-asites, enterocytes from the mICcl2 cell line ( p � 0.05) (Fig. 4C)synthesized less iNOS mRNA. Ex vivo CD11c� cells isolatedfrom the mesenteric lymph nodes have a lower ability to produce

FIGURE 2. A, Parasite burden in the whole intestine. The number ofparasites per 0.5 �g of tissue DNA was determined by real-time PCR atday 7 postinfection from the small intestine of C57BL/6 infected intraint-estinally with RH� or �sag-1 parasites. This is representative of threeindependent experiments (five mice per group). Results are expressed asthe means of triplicate of one experiment � SD. B and C, Parasite prolif-eration in the mICcl2 cell line and DCs. Differentiated enterocytes from themICcl2 cell line (1 � 105 cells/well) (A) or splenic DCs (1 � 105 cells/well)(B) were infected (5 � 104 parasites/well). Two hours later, unpenetratedparasites were washed and fresh medium was added with 0.5 �Ci of[3H]uracil/well. T. gondii proliferation correlates with the cpm values be-cause the only parasite can incorporate [3H]uracil. The number of cpm wasevaluated 24 h after. Results are means of triplicate and represent one ofthree independent experiments.

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Th1 cytokines such as IL-12p40 (Fig. 4D). Similar results wereobtained with CD11c� isolated from the LP (data not shown).These observations suggest that SAG1 protein may be important inboth trafficking and activation of professional and nonprofessionalAPCs. This observation also suggests that the SAG1 protein isimplicated in iNOS activation.

Reduced production of inflammatory cytokines and adaptiveimmune response after infection with �sag1 parasites

IFN-� acts synergistically with TNF-� to activate the inflamma-tory response that limits parasite proliferation (23). If uncontrolled,overexpression of these cytokines leads to the overproduction ofNO. Previous experiments have indicated maximal IFN-� produc-tion at day 7 postinfection in susceptible mice that develop acuteileitis. Cytokine quantitation by RT-PCR shows that intestinalIFN-� and TNF-� mRNA in mice infected with the RH� parasiteswere present at higher levels at day 7 postinfection than those ofmice infected with the �sag1 parasites (Fig. 5, A and B). A similardecrease in proinflammatory cytokine mRNA in response to theAg-deficient mutant was observed in both mesenteric lymph nodesand spleen.

In this model of experimental ileitis, the deleterious effect of thisresponse has been reported to be mediated by TNF-�- and IFN-�-producing CD4� T cells from the LP (CD4� LP T cells) (17).The absence of inflammatory immune response following intraint-estinal inoculation of the �sag1 parasites is due perhaps to the lackof CD4� LP T cell activation secondary to the diminished pro-duction of chemokines, IL-12, and iNOS. CD4� LP T cells weremore abundant in the mice infected with the wild-type parasites(6 � 105� 3 � 104 cells/mouse) compared with the Ag-deficientstrain (3 � 105� 6 � 104 cells/mouse) and uninfected mice (1 �105� 5 � 104 cells/mouse). The failure of CD4� LP T cell acti-vation after infection with the �sag1 parasites was further inves-tigated by mRNA analysis for cytokine production. CD4� LP T

cells from the mice infected with the �sag1 parasites producedsignificantly less ( p � 0.05) IFN-� (Fig. 5C) and TNF-� (Fig. 5D)than the controls isolated from mice infected with the RH� para-sites. This observation suggests that the SAG1 protein is impli-cated in immune cell activation.

The defect in activation of the strong immune response afterinfection with the �sag1 parasites might be related to the over-stimulation of regulatory cytokines. There was no difference inIL-4 mRNA expression in the intestinal tissue regardless of theinfecting strain (Fig. 5E). IL-10 could not be detected in any of thegroups at day 7 after infection (data not shown). The role of TGF-�in the control of the hyperinflammatory response has been previ-ously reported (16). Intraepithelial lymphocytes (IELs) have beenidentified as a major source of TGF-� production in our model.There was no significant difference in the quantity of TGF-� se-creted by IELs purified from mice infected with either RH� or�sag1 parasites (Fig. 5F). These data suggest that although IL-4and TGF-� are increased in response to parasite infection, they donot appear to be responsible for the differences noted in the in-flammatory response to the �sag 1 parasites.

Transfer of Ag-specific CD4�CD45RBhigh T cells from the LP ofmice infected with the �sag1 parasites into resistant RAG1�/�

mice failed to confer the intestinal pathology

CD4� T cell population characterized by high expression of theCD45RB Ag contains cells that mediate both protective and patho-genic Th1 responses, and the reciprocal CD45RBlow populationcan suppress both of these responses (34). Functionally specializedregulatory T cells exist as part of the normal immune repertoire,preventing the development of pathogenic responses to both selfand intestinal Ags (35). Based on these observations, the CD4� Tcell population was isolated from the LP of infected mice, and bycell sorting, potential T regulatory cells (CD25�) were discarded,whereas the likely most efficient effector T cells (CD45RBhigh)

FIGURE 3. Chemokine expression in the whole intestine, enterocytes, and intestinal CD11c� cells. At day 7 postinfection, either with RH� or �sag1parasites, intestines were collected. mRNA levels for MIP-2 (A) and MCP-1 (B) were assayed separately using RPA. Uninfected intestines were used ascontrols. This was representative of three independent experiments, and the results are expressed as the means of duplicate of one experiment � SD (�,p � 0.05). Enterocytes from the mICcl2 cell line were infected either with the parental RH� or with the �sag1 parasites at a 1:1 ratio. Six hours afterinfection, mRNA was extracted, and an RPA was performed to evaluate MIP-2 (C) and MCP-1 (D). Each experiment was performed in triplicate, and resultsare the mean � SD of the triplicated values (�, p � 0.05). CD11c� cells were collected from the mesenteric lymph nodes and cultured for 24 without anystimuli. Supernatants were collected and analyzed for different chemokines (E and F). Results are the mean � SD of triplicates, and this experiment wasrepeated twice (�, p � 0.05).

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were sorted. To further investigate the role of immune compo-nents, and more specifically of SAG1-specific T cells, in the de-velopment of the intestinal inflammation after T. gondii oral in-fection, adoptive transfer of CD4�CD45RBhighCD25� T cells(effector T cells) into RAG1�/� mice was performed. RAG1�/�

mice orally infected with T. gondii do not develop the ileitis. How-ever, 13 days after the transfer of T. gondii-specific effector T cellsisolated from mice infected with the wild-type �RH strain,RAG1�/� mice exhibited intestinal inflammation following infec-tion (Fig. 6A). RAG1�/� mice adoptively transferred with specificeffector T cells isolated from �sag1-infected mice (Fig. 6B) failedto develop such a pathology. Control uninfected RAG1�/� micenever developed the acute ileitis at least within the short 13-daywindow observation, regardless of the source of effector cellstransferred (�RH- or �sag1-primed T cells). Similarly, transfer ofunprimed T cells isolated from naive LP was unable to induce theacute ileitis even upon parasite re-exposure in RAG1�/� mice.Together, these observations suggest that T. gondii Ag-specific Teffector cells are able to transfer the inflammatory disease uponrestimulation, and that SAG1-specific T cells are most likely in-volved in this process.

Presensitization with the SAG1 protein increases susceptibilityof resistant BALB/c mice

Intranasal SAG1 immunization triggers a strong Th1-like immuneresponse (25). To assess the ability of SAG1 to induce inflamma-tion in resistant mice, inbred BALB/c mice were sensitized viaintranasal route with SAG1 protein and CT and challenged orallywith tissue cysts. BALB/c mice were chosen for the sensitizationstudies because this strain is resistant to the development of acuteileitis following oral parasite infection. The ability of CT to act as

a mucosal adjuvant has been reported (25). Based on our earlyexperience using the intranasal instillation of the SAG1 protein(25), CT was added to the SAG1 protein (SAG1 � CT), and micewere immunized, as outlined in Materials and Methods. Mice werechallenged with parasites following the completion of the vacci-nation with SAG1 � CT. It was observed at day 7 postinfectionthat mice sensitized with SAG1 � CT developed histologic evi-dence of an acute inflammatory ileitis. Histological score and ex-amination revealed extensive disruption of the villi from SAG1 �CT-presensitized mice. In comparison, neither of the controlgroups, naive mice or mice vaccinated with CT alone, developedintestinal pathology (Fig. 7). Moreover, 3 of 10 SAG1 � CT-vaccinated mice died from lethal ileitis following parasite chal-lenge. These data suggest that resistant inbred mice could be ren-dered susceptible to acute ileitis when sensitized with the singleparasite Ag SAG1 in the presence of an appropriate mucosal ad-juvant such as CT.

Increased Th1 cytokine expression in SAG1 � CT-presensitizedBALB/c mice

The cytokine profile in the intestine was examined after challengefor IFN-� mRNA expression in those mice presensitized withSAG1 � CT (Fig. 8A). Expression was up-regulated for bothIFN-� and TNF-� (data not shown) in mice that had been presen-sitized with SAG1 � CT compared with control mice. NO, essen-tial for the development of experimental ileitis, as well as otherproinflammatory cytokines such as IL-1� were increased in thegroup immunized with SAG1 � CT (Fig. 8B). No difference wasobserved in cytokine production in the control groups either onlychallenged or treated with CT alone. The role of IL-10 and TGF-�in the regulation of the inflammatory response was also evaluated.

FIGURE 4. iNOS and IL-12 expression in the whole intestine, LPMC, enterocytes, and CD11c� cells. At day 7 postinfection either with RH� or �sag1parasites, intestines were collected. mRNA levels for iNOS (A) were assayed using RPA. Uninfected intestines were used as controls. This was repre-sentative of three independent experiments, and the results are expressed as the means of duplicate of one experiment � SD (�, p � 0.05). B, LPMC isolatedfrom the LP of RH�- or �sag1-infected mice at day 7 postinfection were evaluated by RPA for cytokine. This experiment was performed three times, andthe results are representative of one experiment. C, mICcl2 cells were infected either with the parental RH� or with the �sag1 parasites at a 1:1 ratio. Sixhours after infection, mRNA was extracted and an RPA was performed to evaluate iNOS. Each experiment was performed in triplicate, and results are themean � SD of the triplicated values (�, p � 0.05). Results were expressed as the percentage of the band intensity relative to the intensity of thehousekeeping (GADPH and L32) RNA. D, CD11c� cells were collected from the mesenteric lymph nodes and cultured for 24 h without any stimuli.Supernatants were collected and analyzed for IL-12p40 expression. Results are the mean � SD of triplicates, and this experiment was repeated twice (�,p � 0.05).

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There was no difference in either IL-10 mRNA expression (Fig.8C) or TGF-� protein in the gut whatever the experimental groups(Fig. 8D). These data demonstrate that sensitization with SAG1 �CT is associated with the induction of an intestinal Th1-like im-mune response and turns the resistant phenotype of BALB/c miceinto susceptible.

DiscussionWe demonstrate that acute inflammatory ileitis in mice can beelicited by exposure to a surface Ag of T. gondii that is recognizedby both acute and convalescent sera from humans infected withthis parasite. Although the �sag1 parasites can infect and replicateboth in vitro and in vivo with equal efficiency to the wild-typeparental strain, these Ag-deficient parasites fail to elicit an inflam-matory response in the infected mouse small intestine. Consistentwith this lack of histologic evidence of inflammation is an insuf-ficient up-regulation of the appropriate chemokines and cytokinesthat initiate and mediate this inflammatory response. The anti-inflammatory response, including TGF-� secretion, IL-10, and Il-4production, does not appear to be implicated. In addition, T. gon-dii-specific effector CD4� T (CD25�CD45RBhigh) lymphocytes,when stimulated with the wild-type parasite, but not with the�sag1 mutant parasite, can transfer the inflammatory disease intonormally resistant RAG1�/� mice following infection. This indi-cates that SAG1-specific T lymphocytes might be involved in thisprocess.

Microbial Ags may provide a local environmental trigger thatinitiates and perpetuates intestinal inflammatory response (36).The most compelling evidence for involvement of microflora in

the pathogenesis of mucosal inflammation models has been exam-ined in several experimental murine models of IBD (37). Previousobservations have shown that oral inoculation of T. gondii caninduce acute ileitis in C57BL/6 mice (14, 33). This T. gondii-driven process of acute ileitis in a genetically unmanipulated strainof mouse provides a reasonable experimental model to evaluate theimportance of specific microbial Ags. Histological analyses of theintestinal tissue from these mice demonstrate many morphologicchanges consistent with human ileitis. We have observed that incontrast to the wild-type parasites, the Ag-deficient parasites in-duce substantially less inflammation in the gut of theparasite-infected host.

The failure to initiate an inflammatory response is not linked toa decreased ability of the mutant parasite to either invade or toreplicate in host cells. In vivo observations demonstrate that themutant replicates at a rate consistent with the wild-type strain. Invitro, no difference was observed between the replicative rate ofthe mutant and the wild-type parasites in host cells. We observedthat mice infected with the mutant parasite eventually died fromparasitemia without evidence of ileitis. The recruitment and acti-vation of leukocytes at sites of intestinal inflammation and injuryare the hallmark of intestinal inflammation. Chemokines are cen-tral to the recruitment and activation of leukocytes and have over-lapping functions during the process of inflammation. Both MIP-2(IL-8) and MCP-1 (CCL2) play an important role in the immuno-pathogenesis of intestinal inflammatory disease (38, 39). In vitro,we show that the SAG1KO parasites were less efficient in stimu-lating the expression of MIP-2 and MCP-1 (CCL-2) in a C57BL/

FIGURE 5. Cytokine expression in the whole intestine and by the CD4� LP T cells. At day 7 postinfection either with RH� or �sag-1 parasites,intestines were collected. mRNA levels for TNF-� (A), IFN-� (B), and IL-4 (E) were assayed separately using RPA. Uninfected intestines were used ascontrols. This was representative of three independent experiments, and the results are expressed as the means of duplicate of one experiment � SD (�,p � 0.05). C, CD4� LP T cells were magnetically sorted from the LP of RH�- or �sag1-infected mice at day 7 postinfection and examined for cytokinesecretion. Results are the mean � SD of triplicates, and this experiment was repeated twice (��, p � 0.01). D, mRNA levels for cytokine production onCD4� LP T cells were assayed by RPA. Results are the mean � SD of the duplicate values (�, p � 0.05). F, IELs isolated from RH�- or �sag1-infectedmice at day 7 postinfection were evaluated by ELISA for TGF-�. This experiment was performed three times, and the results are representative of oneexperiment. Results are the mean � SD of duplicate values (�, p � 0.05).

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FIGURE 7. Presensitization of BALB/cmice with SAG1 protein. Mice were presen-sitized by intranasal route with SAG1 protein(10 �g) together with CT (0.5 �g) or sepa-rately. Twenty-eight days later, mice weresimilarly boosted and orally challenged. His-tological examinations and histologicalscores reveal that presensitized and chal-lenged mice exhibited intestinal lesions con-sistent with acute ileitis. This has been per-formed with 10 mice in each group, and thisis representative of two independentexperiments.

FIGURE 6. Adoptive transfer of Ag-specific CD4�CD45RBhigh T cells intoRAG�/� mice. CD4�CD45RBhigh Tcells were sorted by FACS from the mes-enteric lymph nodes of mice at day 5 af-ter infection with either RH� or �sag1parasites and were adoptively transfer-rred (1 � 105 cells per mouse) intoRAG�/� mice. The recipients were chal-lenged the day after the transfer, and his-tological examination (A) of their intes-tines was performed at day 13 after thetransfer. Histological scores (B) were es-tablished for each recipient. These exper-iments were repeated at least three times.

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6-derived enterocyte line (mICcl2 cells) when compared with wild-type parasites. We speculate that this reduction in chemokineexpression would lead to decreased recruitment and activation ofinflammatory cells such as PMNs, macrophages, DCs, and B andT lymphocytes with a decrease in the severity of the inflammatoryresponse. Increased iNOS expression has been associated with theinflammatory disorder observed after infection with the parentalstrain (33). In vitro, enterocytes produce less iNOS when infectedwith the �sag1 parasites compared with wild-type parasites. Thisfurther emphasizes the diminished capacity of the mutant strain toinitiate an inflammatory response.

Intestinal inflammation and tissue damage are characterizedby an exaggerated immune response mediated by both TNF-�and IFN-� (40) and heightened sensitivity of intestinal epithe-lial cells to TNF-� (41– 43). IFN-� is the cytokine mediatorassociated with intestinal inflammation following oral infectionwith T. gondii. Recent studies have identified that at least onesource of IFN-� is LP CD4� T lymphocytes (17). Host expo-sure to SAG1 protein affects the LP CD4� T cell response. Weobserved a significant reduction in the expression of bothTNF-� and IFN-� by the CD4� LP T lymphocytes isolatedfrom mice infected with the �sag1 parasites compared withmice infected with RH�. In vivo and in vitro, chemokine ex-pression was significantly reduced following infection with the�sag1 parasites, and this observation is consistent with the lowattraction and activation of the LP CD4� T lymphocytes. Adecrease in the production of the proinflammatory cytokine,IL-12, was seen in those DCs infected with the SAG1-deficientparasite. The diminished IL-12 production may be responsiblefor the reduced capacity of CD4� T lymphocytes to secreteTNF-� and IFN-�. Together, these observations imply that theSAG1 Ag is an important mediator of the increased levels ofIFN-�, TNF-�, and iNOS following oral parasite infection. This

Ag appears to be directly linked to the development of the lethalileitis following oral infection in the C57BL/6 mice. We havegenerated data using another strain of genetically engineeredparasites that is deficient for SAG3, another parasite surface Ag(32), and have found that these KO parasites are capable ofinducing lethal ileitis similar to the parental strains. Althoughthere may be other parasitic Ags that are yet to be identified ascapable of eliciting this lethal inflammatory process, our datasupport a unique immunogenic role for the SAG1 protein. Pre-vious investigations from our laboratory and others have clearlydemonstrated the ability of this particular parasite Ag, SAG1, toinduce a population of IFN-�-producing Ag-specific CD4� Tcells (44). Furthermore, it has been shown that SAG1-specificCD4� T cells are involved in long-term immunity in those in-dividuals with chronic infection (45). To further investigate thespecific role of CD4 T cells in the development of this acuteinflammatory response, we used RAG�/� mice in which theinnate immune response against the parasite is intact, but thatlack all the T and B cell adaptive immune responses. AlthoughDCs from the RAG�/� mice are capable of responding to the T.gondii infection by producing high amounts of IL-12 and IFN-�(data not shown), and despite a normal parasite replication,these mice do not develop the acute inflammatory intestinaldisease after the infection with T. gondii. Transfer of T. gondii-specific effector T cells with the phenotype CD4CD25�CD45RBhigh stimulated with the parental strain into in-fected RAG�/� mice resulted in the development of a lethalinflammatory response day 13 postinfection by T. gondii,whereas the same CD4� cell subpopulation cells stimulatedwith the �sag1-deficient parasites did not. This emphasizes theimportant role of Ag-specific CD4� T cells in the pathogenesisof this infection, and reinforces the idea that this is the unreg-ulated immune response rather than the parasite itself that leads

FIGURE 8. A–C, Cytokine mRNA expres-sion following SAG1 presensitization and infec-tion of BALB/c mice. At day 7 postinfection,intestines from each group were collected. Tenmicrograms of total RNA was used to performRPA. Relative value of cytokine mRNA produc-tion was determined by densitometric analysisand normalized with housekeeping gene. This isrepresentative of three independent experiments.D, For TGF-� expression, supernatants were as-sayed in triplicates by ELISA. The results are themeans of triplicate � SD (�, p � 0.05) and arerepresentative of two independent experiments.

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to the lethal damages observed following infection with T. gon-dii (46, 47).

Susceptible mice can be rendered resistant to ileitis by alterationor deletion of specific regulatory immune products. For example,C57BL/6 mice deficient in IL-12, CD40, or CD40L fail to developgut inflammation in response to parasite infection (48). Con-versely, we have also observed that resistant mice (49) are madesusceptible by either blocking TGF-� (16) or deleting the gene forIL-10 (50), respectively. We demonstrate that intranasal sensitiza-tion of resistant BALB/c mice, followed by Ag re-exposure canrender a resistant animal susceptible to the development of ileitis.This change in phenotype is associated with an increase in theproduction of Th1-type cytokines rather than a reduced anti-inflammatory response from an increased TGF-�, IL-10, or IL-4production. It has been demonstrated that in these conditions ofSAG1 protein stimulation, Ag-specific CD4� T cell-producingIFN-� could be generated (25). We argue that presensitization withSAG1 generates an inflammatory Ag-specific response.

Oral infection with T. gondii in B6 mice provokes a robust in-flammatory response that results in the development of lethal ile-itis. In this study, the identification of a single microbial Ag thatcan elicit this response provides a unique and potentially usefulmodel to study inflammation in the small intestine and itsregulation.

AcknowledgmentsWe gratefully acknowledge Joe Schwartzman for his assistance in histol-ogy slide analysis, and members of the Kasper lab for many helpful dis-cussions. We also thank Dr. F. Velge-Roussel for providing us with puri-fied SAG1 protein.

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