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Toxoplasma gondiiImmunity against Trefoil Factor 2 Negatively Regulates Type 1

Julio Aliberti and De'Broski R. HerbertButcher, Julie Mirpuri, Simon P. Hogan, Eric Y. Denkers,Yang, David Wu, Nicholas Boespflug, Louis Boon, Barbara Cortez McBerry, Charlotte E. Egan, Reena Rani, Yanfen

http://www.jimmunol.org/content/189/6/3078doi: 10.4049/jimmunol.1103374August 2012;

2012; 189:3078-3084; Prepublished online 15J Immunol

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4.DC1http://www.jimmunol.org/content/suppl/2012/08/15/jimmunol.110337

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The Journal of Immunology

Trefoil Factor 2 Negatively Regulates Type 1 Immunityagainst Toxoplasma gondii

Cortez McBerry,* Charlotte E. Egan, Reena Rani, Yanfen Yang, David Wu,x

Nicholas Boespflug,* Louis Boon,{ Barbara Butcher, Julie Mirpuri, Simon P. Hogan,x

Eric Y. Denkers, Julio Aliberti,* and DeBroski R. Herbert

IL-12mediated type 1 inflammation confers host protection against the parasitic protozoan Toxoplasma gondii. However, pro-

duction of IFN-g, another type 1 inflammatory cytokine, also drives lethality from excessive injury to the intestinal epithelium. As

mechanisms that restore epithelial barrier function following infection remain poorly understood, this study investigated the role

of trefoil factor 2 (TFF2), a well-established regulator of mucosal tissue repair. Paradoxically, TFF2 antagonized IL-12 release

from dendritic cells (DCs) and macrophages, which protected TFF2-deficient (TFF22/2) mice from T. gondii pathogenesis.

Dysregulated intestinal homeostasis in naive TFF22/2 mice correlated with increased IL-12/23p40 levels and enhanced T cell

recruitment at baseline. Infected TFF22/2 mice displayed low rates of parasite replication and reduced gut immunopathology,

whereas wild-type (WT) mice experienced disseminated infection and lethal ileitis. p38 MAPK activation and IL-12p70 produc-

tion was more robust from TFF22/2CD8+ DC compared with WT CD8+ DC and treatment of WT DC with rTFF2 suppressed

TLR-induced IL-12/23p40 production. Neutralization of IFN-g and IL-12 in TFF22/2 animals abrogated resistance shown by

enhanced parasite replication and infection-induced morbidity. Hence, TFF2 regulated intestinal barrier function and type 1

cytokine release from myeloid phagocytes, which dictated the outcome of oral T. gondii infection in mice. The Journal of

Immunology, 2012, 189: 30783084.

Toxoplasma gondii is an obligate intracellular protozoan that,upon peroral infection, rapidly crosses the gastrointestinalepithelium and disseminates through lymphatic, hepatic,

and nervous tissues (1, 2). Host-mediated control of parasite repli-cation is dependent on the production of IL-12 and IFN-g frommyeloid and lymphoid lineages, respectively (36). Oral inocu-lation of C57BL/6 mice with T. gondii tissue cysts causes severeinflammatory bowel disease (IBD), characterized by weight loss,massive granulocytic inflammation, excessive production of Th1-associated cytokines, epithelial invasion of enteric microbes, andmortality within 915 d (1, 7). TLR activation and excessive in-flammatory cytokine release are considered to drive the epithelialcell injury that results from T. gondii infection in C57BL/6 mice(8), but the pathogenesis of oral toxoplasmosis remains poorlyunderstood.Trefoil factor (TFF)2 is one of three trefoil-motif containing

proteins (TFF13) that promotes restitution, the rapid and directed

movement of epithelia to cover exposed areas of basement mem-brane tissue following mucosal insult (9, 10). The predominantsources of TFF2 are stromal cells (epithelia, endothelia, and fibro-blasts), but TFF2 mRNA transcripts are also expressed by tissuemacrophages (10, 11). Although TFF2 and TFF3 can both down-regulate gastric and colonic inflammation (1113), the nonredundantmechanisms of regulation of intestinal homeostasis or pathogen-specific immunity by TFF2 are currently unclear.This report demonstrates that TFF2 functions as a regulator of

intestinal homeostasis that suppresses T. gondii-driven type 1 in-flammation. TFF2 suppresses p38 MAPK activation and IL-12p70release from CD8+ dendritic cells (DC) and limits IL-12/23p40production from macrophages. Oral inoculation of TFF22/2 micewith T. gondii results in the rapid clearance of parasites, preventingthe development of infection-induced immunopathology. Thesedata extend the importance of TFF2 from mucosal barrier functionto a previously unrecognized role in the suppression of the IL-12/IFN-g axis that drives host immunity against parasitic protozoa.

Materials and MethodsMice and T. gondii infection model

Six- to 10-wk-old, sex-matched, wild-type (WT), or TFF22/2C57BL/6micebred in-house were used for all studies. For oral T. gondii (ME49 strain)infections, brain cyst homogenates were obtained from chronically infectedmice, and cyst suspensions were prepared at the concentrations indicated.Mice were infected by oral gavage with 1550 cysts using a 21-gauge ball-tipped feeding needle. Weight was monitored daily. Moribund mice (.20%weight loss) were sacrificed according to the Institutional Animal Care andUse Committee at the Cincinnati Childrens Hospital Medical Center.

Histological staining and immunohistochemistry

Toxoplasma Ag-specific immunohistochemistry on paraffin-embedded tis-sue was performed with anti-T. gondii primary Ab (US Biologicals) asdescribed previously (14). For immunofluoresence, paraffin-embedded tissuesections were immersed in 4% donkey serum (Millipore) for 2 h at roomtemperature to prevent nonspecific binding of primary Abs. Rabbit anti-CD3(1:100; DakoCytomation) and 5 mg/ml rat anti-mouse F4/80 or anti-CD11b

*Division of Molecular Immunology, Cincinnati Childrens Research Foundation,Cincinnati, OH 45229; Department of Microbiology and Immunology, Cornell Uni-versity College of Veterinary Medicine, Ithaca, NY 14853; Division of Immunobi-ology, Cincinnati Childrens Research Foundation, Cincinnati, OH 45229; xDivisionof Allergy and Immunology, Cincinnati Childrens Research Foundation, Cincinnati,OH 45229; {Bioceros B.V., 3584 CM Utrecht, The Netherlands; and University ofTexas Southwestern Medical Center, Dallas, TX 75390

Received for publication November 23, 2011. Accepted for publication July 13,2012.

Address correspondence and reprint requests to Dr. DeBroski R. Herbert at the currentaddress: Division of Experimental Medicine, University of California, San Francisco,San Francisco, CA 94122. E-mail address: debroski.herbert@ucsf.edu

The online version of this article contains supplemental material.

Abbreviations used in this article: BMDM, bone marrow-derived macrophage; DC,dendritic cell; IBD, inflammatory bowel disease; mLN, mesenteric lymph node;STAg, soluble tachyzoite Ag; TER, transepithelial cell resistance; TFF, trefoilfactor; TFF22/2, trefoil factor 2-deficient; WT, wild-type.

Copyright 2012 by TheAmericanAssociation of Immunologists, Inc. 0022-1767/12/$16.00

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(eBioscience) were applied to tissue sections overnight, washed, and in-cubated with 1% BSA incubated with Donkey anti-Rat 594 and Donkey anti-Rabbit 488 (Invitrogen) for 2 h for detection of primary Ab. DAPIFluo-rmount (Southern Biotechnology Associates) was used for nuclear staining.

Quantitative RT-PCR

Total RNAwas purified from bone marrow-derived macrophages (BMDM)or DC cultures using TRIzol reagent, according to the manufacturersinstructions (Invitrogen). cDNAwas prepared using the TaqMan cDNAsynthesis kit (Roche). Gene expression was measured using the Light-cycler 480, and data were normalized to b-actin. Biops