Toxoplasma Gondii Immunity Against Trefoil Factor 2 Negatively

Trefoil Factor 2 Negatively Regulates Type 1 Immunity against Toxoplasma gondii Cortez McBerry, Charlotte E. Egan, Reena Rani, Yanfen Yang, David Wu, Nicholas Boespflug, Louis Boon, Barbara This information is current as Butcher, Julie Mirpuri, Simon P. Hogan, Eric Y. Denkers, of September 23, 2021. Julio Aliberti and De'Broski R. Herbert J Immunol 2012; 189:3078-3084; Prepublished online 15 August 2012;

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Supplementary http://www.jimmunol.org/content/suppl/2012/08/15/jimmunol.110337 Material 4.DC1 http://www.jimmunol.org/ References This article cites 43 articles, 15 of which you can access for free at: http://www.jimmunol.org/content/189/6/3078.full#ref-list-1

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2012 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Trefoil Factor 2 Negatively Regulates Type 1 Immunity against 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 De’Broski R. Herbert‡

IL-12–mediated type 1 inflammation confers host protection against the parasitic protozoan Toxoplasma gondii. However, production 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 Downloaded from 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 production 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 http://www.jimmunol.org/ Immunology, 2012, 189: 3078–3084.

oxoplasma gondii is an obligate intracellular protozoan that, movement of epithelia to cover exposed areas of basement mem- upon peroral infection, rapidly crosses the gastrointestinal brane tissue following mucosal insult (9, 10). The predominant T epithelium and disseminates through lymphatic, hepatic, sources of TFF2 are stromal cells (epithelia, endothelia, and fibro- and nervous tissues (1, 2). Host-mediated control of parasite repli- blasts), but TFF2 mRNA transcripts are also expressed by tissue cation is dependent on the production of IL-12 and IFN-g from macrophages (10, 11). Although TFF2 and TFF3 can both down- myeloid and lymphoid lineages, respectively (3–6). Oral inocu- regulate gastric and colonic inflammation (11–13), the nonredundant by guest on September 23, 2021 lation of C57BL/6 mice with T. gondii tissue cysts causes severe mechanisms of regulation of intestinal homeostasis or pathogen- inflammatory bowel disease (IBD), characterized by weight loss, specific immunity by TFF2 are currently unclear. massive granulocytic inflammation, excessive production of Th1- This report demonstrates that TFF2 functions as a regulator of associated cytokines, epithelial invasion of enteric microbes, and intestinal homeostasis that suppresses T. gondii-driven type 1 in- mortality within 9–15 d (1, 7). TLR activation and excessive inflammation. TFF2 suppresses p38 MAPK activation and IL-12p70 flammatory cytokine release are considered to drive the epithelial release from CD8+ dendritic cells (DC) and limits IL-12/23p40 cell injury that results from T. gondii infection in C57BL/6 mice production from macrophages. Oral inoculation of TFF22/2 mice (8), but the pathogenesis of oral toxoplasmosis remains poorly with T. gondii results in the rapid clearance of parasites, preventing understood. the development of infection-induced immunopathology. These Trefoil factor (TFF)2 is one of three “trefoil-motif” containing data extend the importance of TFF2 from mucosal barrier function proteins (TFF1–3) that promotes restitution, the rapid and directed to a previously unrecognized role in the suppression of the IL-12/ IFN-g axis that drives host immunity against parasitic protozoa.

*Division of Molecular Immunology, Cincinnati Children’s Research Foundation, Materials and Methods Cincinnati, OH 45229; †Department of Microbiology and Immunology, Cornell Uni- Mice and T. gondii infection model versity College of Veterinary Medicine, Ithaca, NY 14853; ‡Division of Immunobi- 2 2 ology, Cincinnati Children’s Research Foundation, Cincinnati, OH 45229; xDivision Six- to 10-wk-old, sex-matched, wild-type (WT), or TFF2 / C57BL/6 mice of Allergy and Immunology, Cincinnati Children’s Research Foundation, Cincinnati, T. gondii { ‖ bred in-house were used for all studies. For oral (ME49 strain) OH 45229; Bioceros B.V., 3584 CM Utrecht, The Netherlands; and University of infections, brain cyst homogenates were obtained from chronically infected Texas Southwestern Medical Center, Dallas, TX 75390 mice, and cyst suspensions were prepared at the concentrations indicated. Received for publication November 23, 2011. Accepted for publication July 13, Mice were infected by oral gavage with 15–50 cysts using a 21-gauge ball- 2012. tipped feeding needle. Weight was monitored daily. Moribund mice (.20% Address correspondence and reprint requests to Dr. De’Broski R. Herbert at the current weight loss) were sacrificed according to the Institutional Animal Care and address: Division of Experimental Medicine, University of California, San Francisco, Use Committee at the Cincinnati Children’s Hospital Medical Center. San Francisco, CA 94122. E-mail address: [email protected] Histological staining and immunohistochemistry The online version of this article contains supplemental material. Toxoplasma Ag-specific immunohistochemistry on paraffin-embedded tis- Abbreviations used in this article: BMDM, bone marrow-derived macrophage; DC, dendritic cell; IBD, inflammatory bowel disease; mLN, mesenteric lymph node; sue was performed with anti-T. gondii primary Ab (US Biologicals) as STAg, soluble tachyzoite Ag; TER, transepithelial cell resistance; TFF, trefoil described previously (14). For immunofluoresence, paraffin-embedded tissue factor; TFF22/2, trefoil factor 2-deficient; WT, wild-type. sections were immersed in 4% donkey serum (Millipore) for 2 h at room temperature to prevent nonspecific binding of primary Abs. Rabbit anti-CD3 Copyright Ó 2012 by The American Association of Immunologists, Inc. 0022-1767/12/$16.00 (1:100; DakoCytomation) and 5 mg/ml rat anti-mouse F4/80 or anti-CD11b www.jimmunol.org/cgi/doi/10.4049/jimmunol.1103374 The Journal of Immunology 3079

(eBioscience) were applied to tissue sections overnight, washed, and in- infection-induced mucosal inflammation (9, 12). However, it was cubated with 1% BSA incubated with Donkey anti-Rat 594 and Donkey anti- unclear whether TFF2 regulated mucosal barrier function under Rabbit 488 (Invitrogen) for 2 h for detection of primary Ab. DAPI–Fluo- homeostatic conditions. To address this issue, jejunal tissue seg- rmount (Southern Biotechnology Associates) was used for nuclear staining. ments from naive WT and TFF22/2 mice were mounted on Ussing Quantitative RT-PCR chambers to evaluate both TER (V cm2) (Fig. 1A) and paracellular Total RNA was purified from bone marrow-derived macrophages (BMDM) permeability (basolateral to apical flux of FITC–dextran) (Fig. 2/2 or DC cultures using TRIzol reagent, according to the manufacturer’s 1B). Strikingly, jejunum tissue from TFF2 mice had signifi- instructions (Invitrogen). cDNA was prepared using the TaqMan cDNA cantly less TER and significantly more permeability to FITC– synthesis kit (Roche). Gene expression was measured using the Light- dextran than WT tissues (Fig. 1A, 1B). This indicated that TFF2 b cycler 480, and data were normalized to -actin. Biopsies of small intes- served a nonredundant role in the mucosal barrier function within tine (duodenum, jejunum, and ileum) were pooled and weighed, and DNA was extracted using the DNeasy blood and tissue extraction kit (Qiagen). the proximal small intestine of mice. Primers used for amplification of the T. gondii B1 gene: forward, 59- Given this finding, we postulated that the TFF2 deficiency might CTGGCAAATACAGGTGAAATG-39, and reverse, 59-GTGTACTGCGA- have led to a dysregulation of immune cell composition or a baseline AAATGAATCC-39, as described previously (15). PCRs were setup in a increase in intestinal inflammation. Evaluation of jejunal biopsies via final volume of 20 ml, using 5 ml total tissue DNA, 1 ml20mM forward 2/2 and reverse primer, and 23 SYBR Green I Master Mix (Roche). RT-PCR H&E staining revealed that TFF2 mice had a moderate accu- analysis was performed on a Light Cycler 480 System (Roche). Relative mulation of leukocytes within the lamina propria but did not possess quantification was performed using standard curve analysis of purified signs of overt immunopathology (Fig. 1C). Immunofluorescence parasite DNA from a defined number of parasites and expressed as the staining for F4/80+ and CD3+ cell populations was used to indicate number of parasites per milligram of tissue. the relative abundance of myeloid phagocytes and T lymphocytes, Downloaded from 2 2 Flow cytometry respectively. Results show that TFF2 / mice had a greater accumulation of both cell populations in the lamina propria, as Mesenteric lymph node (mLN) cells were washed in FACS buffer (HBSS, 1% FCS, and 0.2% sodium azide) and incubated with anti-FcgRII/RIII mAb compared with WT mice (Fig. 1D). (2.4G2). Lamina propria cells were isolated as described previously (16). To determine whether cytokine dysregulation accompanied these Single-cell suspensions were stimulated with PMA/ionomycin/GolgiStop cell compositional changes, 3-cm segments of jejunum were digested (BD Pharmingen) and stained with allophycocyanin-F4/80 (clone BM8), with Liberase and evaluated for intracellular IL-12/23p40 levels http://www.jimmunol.org/ FITC–anti-CD11b (clone M1/70) and intracellularly stained with PE–anti- within the lamina propria macrophage population (CD11b+/F4/80+). IL-12/23p40 (R&D Systems). For T cell staining, MLN were stimulated + + 2/2 with anti-CD3 (1 mg/ml) 16 h with GolgiPlug added during the last 4 h, Strikingly, the CD11b /F4/80 cell population from TFF2 followed by anti–TCR-b, anti-CD4, anti-CD8, and anti–IFN-g mAbs (eBio- mice expressed 4-fold greater levels of IL-12/23p40 than WT (Fig. science). Intracellular staining with mAb specific for p38 or p42/44 MAPK 1E). IL-12p40 mRNA levels within the jejunum were also higher (Cell Signaling Technology) was performed according to the manu- in TFF22/2 tissues than WT, but there were no differences in the facturer’s protocol. Acquisition was performed with a BD FACSCalibur and analyzed with FlowJo 7.5.5 software. baseline expression for IFN-g or IL-17A as determined by quantitative RT-PCR (data not shown). Taken together, these results BMDM and splenic DC indicated that TFF2 served an essential role in the baseline reg- BMDM were grown from the mononuclear fraction of bone marrow cultures ulation of small intestinal barrier function and inflammatory cell by guest on September 23, 2021 for 6 d in M-CSF generated by the CMG cell line as described previously composition/function within the small intestine at baseline. (16). Spleen-derived CD11c cells were obtained after collagenase D di- gestion, followed by CD11c-MACS beads magnetic separation (cell purity TFF22/2 mice control early parasite dissemination without . 85% as determined by flow cytometry). Soluble tachyzoite Ag (STAg) developing intestinal immunopathology following oral was generated from RH–strain tachyzoites grown from HS27 fibroblasts. CpG was purchased from a commercial vendor (oligodoxyucleotide 1826; infection with T. gondii tissue cysts Coley Pharmaceutical). Because there were marked perturbations in TFF22/2 mice at Ab neutralization baseline, we sought to determine whether these abnormalities would alter the course of disease caused by an oral infection with T. gondii. Rat–anti-mouse IL-12/IL-23p40 (C17.8), rat–anti-mouse IFN-g (XMG1.2), 2/2 or rat Ig control Ab (GL113) were purified from culture supernatant using WT and TFF2 mice were orally inoculated with Me49 tissue thiophilic agarose chromatography, dialyzed against PBS and sterile cysts at varying inoculum doses and monitored for changes in filtered through a 0.2-mm filter. weight as an indicator of disease progression. Oral inoculation with Evaluation of intestinal permeability 50 cysts (high dose) caused rapid weight loss in both WT and TFF22/2 strains (Fig. 2A), accompanied by marked infection- One-centimeter segments of mucosa were mounted in U2500 Dual Channel Ussing chambers that exposed 0.30 cm2 tissue to 10 ml Krebs buffer. Agar– induced immunopathology within the liver and intestine (Supple- salt bridges and electrodes were used to measure the potential difference. mental Fig. 1). However, infection with 25 cysts (medium dose) or Following a 15-min equilibrium period, Basal short-circuit current, trans- 15 cysts (data not shown) did not cause cachexia or mortality in epithelial cell resistance (TER), and permeability to FITC–dextran (2.2 mg/ml; TFF22/2 mice, whereas WT animals lost .20% of their original molecular mass, 4.4 kDa; Sigma-Aldrich) were determined as described weight and experienced 30–40% mortality by 8–9 d postinoculation previously (17). (Fig.2B).TFF22/2 mice inoculated with 25 tissue cysts did not Statistical analysis develop splenomegaly (Fig. 2C) or intestinal inflammation (Fig. Statistical significance was assessed by either two-tailed Student t test (two 2D), whereas infected WT mice developed severe transmural il- groups) or ANOVA for multiple groups with a post hoc test to determine eitis characterized by extensive granulocytic infiltration (Fig. 2C, significance using Prism GraphPad 4.0 software. 2D). Congruent with reduced intestinal inflammation in TFF22/2 mice, the expression levels of IL-10, a key immunosuppressive Results cytokine, were significantly higher in the ileal tissue of TFF22/2 TFF 2 is necessary to maintain mucosal barrier function within mice compared with WT at day 7 postinoculation (Fig. 2E). the small intestine and limits baseline production of IL-12/23 Furthermore, the marked differences between strains in the p40 from myeloid phagocytes susceptibility to infection-induced ileitis prompted experiments to Treatment of rodents with recombinant hTFF2 ameliorates mucosal test whether reduced immunopathology in TFF22/2 mice corre- injury and TFF2 deficiency in mice exacerbates Helicobacter lated with parasite burden. Tachyzoite-specific PCR revealed that 3080 TFF2 THWARTS IMMUNITY AGAINST Toxoplasma gondii Downloaded from http://www.jimmunol.org/ FIGURE 2. Low-dose oral infection with T. gondii in TFF22/2 mice does not lead to cachexia, parasite dissemination, or intestinal immunopathology. WT and TFF22/2 mice were orally inoculated with 50 Me49 brain cysts (A) or 25 cysts (B) and monitored for weight change. Data show mean 6 SE from four to six mice per group. Experiment was repeated four times. Death indicated by cross (**p , 0.01). (C) Representative photo- graphs of spleens from orally infected WT (top panel) and TFF22/2 mice (bottom panel) at 8 d postinoculation. Original magnification 34. (D) H&E-stained small intestine from WT (top panel) and TFF22/2 (bottom panel) mice at 8 d postinoculation following oral inoculation from ex- by guest on September 23, 2021 periment described in (B). Original magnification 3200. (E) IL-10 mRNA transcript levels within the jejunum from naive or T. gondii-infected mice at 7 d postinfection with 25 Me49 cysts. Data show mean 6 SE from four to six mice per group. Experiment performed twice with similar results.

parasites within TFF22/2 intestinal tissues at day 5 postinfection (Fig. 3B, 3C). Taken together, these data demonstrate that at low inoculum doses, TFF2 deficiency protects the host from infection- induced immunopathology, which is associated with early control of parasite replication and increased IL-10 expression within the FIGURE 1. TFF2 deficiency impairs mucosal barrier function and increa- intestine. ses the baseline frequency of IL-12+ macrophages in the small intestine. Muscle-free segments of jejunum isolated from naive WT and TFF22/2 Cell-intrinsic expression of TFF2 in macrophages and DC mice were evaluated for TER (A) and permeability (B) to FITC–dextran negatively regulates MAPK activation and IL-12 production (4.4 kDa). Data show mean 6 SEM from six mice per group. (C)Rep- Although epithelial cells are considered as the major source of resentative images of H&E-stained jejunum from naive WT (left panel) 2/2 TFF2, monocyte/macrophage lineage cells also express TFF2 and TFF2 (right panel) mice. Scale bar, 20 mm. Original magnifi- mRNA. Thus, it was possible that enhanced host resistance to T. cation 3200. (D) Immunofluorescence staining for F4/80 (green) and CD3 2 2 gondii infection in TFF2 / mice involved dysregulation of my- (magenta) in paraffin-embedded sections of naive jejunum from WT (left panel) and TFF22/2 (right panel) mice. Original magnification 3400. (E) eloid phagocyte function(s). To test whether TFF2 served a direct Percentage of IL-12/23p40+ events within the FSChiSSClowCD11b+/F4/ role in the regulation of proinflammatory and anti-inflammatory 80+ gate from the lamina propria mononuclear cell fraction of naive WT cytokine release from DC and macrophages, experiments were and TFF22/2 mice. Pooled samples from two to three mice are shown. conducted with TLR-activating microbial Ags. Splenic DC from 2 2 Data are representative of three independent experiments (**p , 0.01, naive WT and TFF2 / mice were isolated by magnetic bead ***p , 0.001). sorting (.85% CD11c+), exposed to STAg, and evaluated for IL-12/23p40 mRNA transcript levels by quantitative RT-PCR. parasite numbers in the intestine of TFF22/2 mice inoculated with Strikingly, TFF22/2 DC upregulated IL-12/23p40 mRNA tran- 25 cysts were significantly reduced compared with WT at days 3 scripts more rapidly than WT DC at 4 h, although there were no and 5 postinfection (Fig. 3A). Tachyzoite-specific immunohisto- differences between strains by 16 h (Fig. 4A). Increased IL-12/23 chemistry verified these findings because there was robust staining p40 message levels were accompanied by higher amounts of IL- for parasite Ag within the lamina propria of WT mice but very few 12p70 produced from TFF22/2 DC cultures as compared with WT The Journal of Immunology 3081 Downloaded from

FIGURE 3. TFF2 deficiency results in early control of T. gondii replication following ingestion. (A) T. gondii B1-gene specific PCR was per- http://www.jimmunol.org/ formed on small intestine from mice orally inoculated with 25 Me49 brain cysts. The experiment was performed three times with similar results (*p , 0.05). T. gondii tachyzoite-specific immunohistochemistry was used to detect parasites (dark brown precipitate) within the small intestine of WT and TFF22/2 mice at 5 d postinfection from the experiment described in (A). (B) Original magnification 3100; (C) original magnification 3400. Arrows indicate parasite-infected cells.

DC following exposure to STAg or CpG (Fig. 4B). Profillin, a by guest on September 23, 2021 selective TLR11 agonist, also induced greater amounts of IL-12p70 in TFF22/2 DC cultures than WT (Fig. 4C). CpG, a TLR9 agonist, 2 2 induced IL-10 production that was significantly higher in TFF2 / FIGURE 4. TFF2 negatively regulates p38 MAPK activation and IL-12 DC cultures as compared with WT (Fig. 4D). Collectively, this production in splenic DC and BMDM. (A) Splenic DC from naive WT and 2/2 demonstrated that TFF22/2 DCs were hyperresponsive to diverse TFF2 mice were stimulated with STAg (10 mg/ml) and monitored for 6 TLR-activating Ags, resulting in proinflammatory and anti- IL-12p40 mRNA transcripts. Data show mean SE from six mice per group. Experiment was performed twice. (B) IL-12p70 levels produced inflammatory cytokine production. 2 2 from WT and TFF2 / splenic DC left untreated (med), stimulated with Because of the rapid accumulation of IL-12/23p40 transcripts 6 2/2 STAg (10 mg/ml), or CpG (1 mg/ml) for 48 h. Data show mean SE from and elevated production of IL-12p70 within TFF2 DC, we then six mice per group. Experiment performed three times. Splenic DC from asked whether the proximal signaling molecules responsible for naive WT and TFF22/2 mice were stimulated with rProfilin (C) or CpG IL-12 production were also differentially regulated by TFF2. (D) for 24 h and measured for IL12p70 and IL-10 levels, respectively. Data Given the essential role for MAPK activation in IL-12 release show mean 6 SE from six mice per group. Phospho-p38 levels within from APC (18), the kinetics of p38 MAPK phosphorylation were splenic CD11c+CD8+ (E) and CD11c+CD82 (F) DC populations from 2 2 determined in both CD8+CD11c+ and CD82CD11c+ populations naive WT and TFF2 / mice that were exposed to STAg (10 mg/ml). exposed to STAg. CD8+CD11c+ DC from TFF22/2 mice gener- Mean 6 SE from eight mice per group. Experiment performed twice. G ated more robust phosphorylation of p38, as compared with WT IL-12p40 protein levels produced from WT splenic DC ( )orBMDM H (Fig. 4E, Supplemental Fig. 2). However, p38 activation was no ( ) that were untreated (med), exposed to rTFF2 (40 ng/ml), LPS (100 2 ng/ml), or pre-exposed to rTFF2 (16 h) prior to stimuli for 24 h. different between strains within the CD8 CD11c+ subpopulation Experiments were performed three times (*p , 0.05, **p , 0.01). (Fig. 4F), a minor contributor to IL-12 production (19, 20). No detectable differences were found between strains with regard to p42/44 (ERK) activation in either DC subset (data not shown). and suppressed Escherichia coli LPS-induced IL-12/23p40 ex- In addition, we sought to determine whether TFF2 could function pression and cytokine release from BMDM (Fig. 4H). Combined, as a direct suppressor of microbial Ag-induced IL-12 production. these data indicate that cell-intrinsic TFF2 expression as well as Thus, the reverse experiments were performed to determine whether exogenous rTFF2 pretreatment antagonizes microbial Ag-induced IL-12 production could be inhibited by prior exposure to rTFF2. proinflammatory cytokine production from myeloid APCs. 2/2 Splenic DC cultures isolated from naive WT and TFF2 mice 2/2 were either left untreated or treated with rTFF2 (40 ng/ml), fol- Host immunity in TFF2 mice is dependent on the lowed by stimulation with STAg (10 mg/ml). IL-12/23p40 levels IL-12/IFN-g axis were measured 16 h later. rTFF2 pretreatment markedly reduced IL-12–driven type 1 inflammation has a central role in driving host STAg-induced IL-12/23p40 production from DC cultures (Fig. 4G) protection against T. gondii in mice. Thus, we addressed whether 3082 TFF2 THWARTS IMMUNITY AGAINST Toxoplasma gondii enhanced resistance afforded by TFF2 deficiency resulted in a preferential expansion of IFN-g producing effector lymphocytes (21). Intracellular IFN-g content within TCRb+CD4+ and TCRb+ CD8+ populations was determined 24 h following CD3 stimulation (1 mg/ml) of mLN cells at day 5 (data not shown) and day 7 following oral inoculation with 25 Me49 tissue cysts. Consistent with our prediction, TFF22/2 animals generated a 5-fold greater percentage of IFN-g+ Th1 cells (Fig. 5A) and 3-fold greater percentage of IFN-g+ CD8 effectors (Fig. 5B) than infected WT animals. Importantly, the baseline production of IFN-g from these T cell subsets was no different between strains, which lends fur- ther support to the hypothesis that TFF22/2 APC preferentially induced the expansion of IFN-g–producing lymphocytes following T. gondii infection. To determine whether the lack of TFF2 indeed protected mice against T. gondii infection through accelerated IL-12/IFN-g production during acute infection, we tested whether neutralization of these cytokines prior to infection would abrogate protective im- 2 2 munity. Naive WT and TFF2 / mice were given a single dose of Downloaded from anti–IL-12p40 (1 mg) and anti–IFN-g (1 mg) neutralizing mAbs 1 d prior to oral inoculation with 25 T. gondii tissue cysts. Results show that TFF22/2 mice treated with anti–IL-12p40/anti–IFN-g mAbs lost .20% of their original body weight by 9 d postinoculation, whereas TFF22/2 mice treated with isotype control

mAb did not develop T. gondii-induced cachexia (Fig. 5C). As ex- http://www.jimmunol.org/ pected, WT mice treated with anti–IL-12p40/anti–IFN-g mAbs also developed worse disease than isotype control-treated animals (22). Last, we predicted that the enhanced disease severity in TFF22/2 mice correlated with an inability to control parasite dissemination. The gut-draining mLN were probed with speciﬁc mAb for T. gondii Ags at day 7 postinoculation in both strains. Although TFF22/2 mice given control IgG harbored few parasites, WT mLN con- tained many tachyzoites (Fig. 5D, 5E). In contrast, administration of anti–IL-12p40/anti–IFN-g mAbs to TFF22/2 mice caused a by guest on September 23, 2021 dramatic increase of parasite numbers in the mLN, similar to WT mAb-treated animals (Fig. 5F, 5G). Similar results were observed following immunostaining of splenic tissues (data not shown). These data support our hypothesis that the canonical type 1 cytokines IL-12 and IFN-g protected TFF22/2 mice against susceptibility to T. gondii infection-induced lethality and immunopathology.

Discussion The mechanisms that regulate mucosal barrier function, tissue re- FIGURE 5. Lack of TFF2 increases IFN-g effector cell expansion and pair, and inflammation within the gastrointestinal tract are incom- limits tachyzoite dissemination to lymph nodes. Intracellular IFN-g staining within CD4+ (A) and CD8+ (B) T lymphocytes within the mLN of pletely understood. In this study, oral inoculation of mice with the 2 2 WT and TFF2 / mice that were either naive or 7 d postoral inoculation human parasite T. gondii was used to demonstrate a novel role for with 25 Me49 brain cysts. Representative plots from eight individual mice TFF2 (known to promote epithelial restitution) in the negative analyzed per group. (C) WT and TFF22/2 mice were administered regulation of infection-induced IL-12 production and type 1 im- a combination of anti–IL-12p40/anti–IFN-g mAb (1 mg each) or isotype munity. TFF2 deficiency increased baseline intestinal inflammation control mAb (2 mg) on day 21, followed by oral inoculation with 25 and augmented infection-induced type 1 cytokine production from Me49 brain cysts on day 0 and monitored for weight change. Data show CD8+ DC and CD4+ and CD8+ T lymphocytes. This provided a mean 6 SE shown from four to six mice per group. Experiment was selective advantage for the TFF22/2 strain in the control of early performed three times. (D) mLN sections at day 8 postinoculation from 2/2 parasite replication, such that infection-induced immunopathology experiment described in (C) in WT control mAb treated, (E) TFF2 F G was averted. Interestingly, TFF2 functioned through cell-intrinsic control mAb-treated, ( ) WT anti–IL-12/anti–IFN-g mAb-treated, and ( ) 2/2 g and extrinsic mechanisms to suppress TLR-driven IL-12 produc- TFF2 anti–IL-12/anti–IFN- mAb-treated groups stained with tachyzoite- specific IgG anti-sera. Original magnification 3100. Arrowhead indicates the tion from macrophages and DC, suggesting that homeostatic TFF2 area within the inset (original magnification 3400). Representative images are release within the small intestine has multiple functions that shown. **p , 0.01. maintain mucosal barrier integrity and downmodulate myeloid APC activation and cytokine production in response to microbial Ags. The proinflammatory cytokine IL-12 drives host immunity tissue cysts causes rapid and severe weight loss (cachexia), in- against a variety of microbial, fungal, and viral pathogens (5, 21, testinal immunopathology, and host mortality within 7–12 d (25). 23) including rodents infected with T. gondii (24). Unlike human Lethal ileitis caused by oral T. gondii infection bears striking toxoplasmosis, oral inoculation of C57BL/6 strains with T. gondii similarity to murine and human IBD, because both are due to The Journal of Immunology 3083 excessive IL-12– and IL-23–driven inflammatory responses ag- thology when inoculated with 50 T. gondii tissue cysts instead of gravated by gut microbes (26, 27). One hypothesis for the simi- 25 or 15 cysts. Therefore, the selective advantage of TFF2 defi- larities in phenotypes of IBD and murine toxoplasmosis is that ciency is only evident up to a certain threshold of parasite inoc- microbial flora enter the intestinal tissue through areas of damaged ulum. Once this is exceeded, the baseline inflammation within epithelium (28). TFF22/2 mice is no longer sufficient to limit parasite replication Naive TFF22/2 mice had higher baseline levels of IL-12/23 p40 and dissemination throughout the host. This is supported by our in the intestine than WT, which was concurrent with impaired demonstration that neutralization of IL-12/IFN-g prior to infection TER and increased paracellular permeability. These parameters resulted in uncontrolled tachyzoite replication and cachexia in are a central feature of IBD in mice and humans and can be used TFF22/2 mice. to indicate the degree of injury to the mucosal barrier within the In contrast, anti-inflammatory cytokines such as IL 10 have been intestine (29–31). Defective barrier function at baseline in TFF22/2 demonstrated to serve an essential role in limiting excessive IL-12/ mice was unexpected, because TFF family members have func- IFN-g–associated immunopathology following T. gondii infection tionally redundant roles in mucosal epithelial cell repair (32, 33). in rodents. Enhanced IL-10 production from infected TFF22/2 mice Although the explanation(s) for this defective barrier function and TFF22/2 DC could have contributed to suppression of excess remains undefined, one possibility is that TFF22/2 mice have immunopathology. However, rTFF2 treatment of DC or macro- defective expression/function of gap-junction proteins because of phages did not induce IL-10 or TGF-b mRNA expression (data not the role of TFF2 in the regulation of cell adhesion molecule ex- shown). Thus, although the anti-inflammatory effects of TFF2 are pression (34). Alternatively, TFF2 deficiency could result in ex- unlikely to be IL-10 dependent, the enhanced IL-10 in TFF22/2 cess intestinal epithelial cell death because several TFF family mice may have served an important biological role in the context of Downloaded from members block apoptosis (35, 36). Irrespective of the exact T. gondii infection. The mechanism(s) responsible for TFF2’s im- mechanism, our evidence for decreased barrier function in naive munosuppressive effects are currently under investigation. TFF2 mice may partially explain why TFF22/2 mice have in- The role of cross-talk between epithelial cells and leukocytes creased susceptibility to chemical- and infection-induced models in the regulation of mucosal and systemic immunity has become of gastritis and colitis (11). increasingly apparent. Our data show that TFF2, a major com-

This work demonstrates that TFF2 suppressed IL-12 production ponent of mucus, serves an essential role in the negative regulation http://www.jimmunol.org/ in splenic DC and macrophages through extrinsic and intrinsic of IL-12/23p40 and IL-12p70 production from macrophages and mechanisms, which demonstrates a previously unrecognized role DC as well as enforcement of homeostatic mucosal barrier in- for TFF2 in the regulation of type 1 inflammation. CD8+CD11c+ tegrity. Taken together, our work is consistent with the emerging DC, a major source of T. gondii infection-induced IL-12p70, pro- concept that epithelial cell repair proteins have direct immuno- duced more IL-12 when isolated from TFF22/2 mice compared modulatory functions. with WT. This production was suppressed when WT DC were exposed to exogenous rTFF2. IL-12/23p40 production from intes- 2 2 Disclosures tinal lamina propria macrophages was highly elevated in TFF2 / The authors have no financial conflicts of interest. mice as compared with WT. This classically activated macrophage by guest on September 23, 2021 phenotype is consistent with reports showing that TFF22/2 mice 2 2 are hyperresponsive to IL-1b stimulation and that TFF2 / mac- References rophages show enhanced NF-kb activation compared with WT 1. Munoz, M., O. Liesenfeld, and M. M. Heimesaat. 2011. Immunology of Toxo- macrophages (13). In addition, TFF22/2 monocytes release greater plasma gondii. Immunol. Rev. 240: 269–285. 2. Lieberman, L. A., and C. A. Hunter. 2002. The role of cytokines and their amount of reactive nitrogen intermediates and inflammatory cyto- signaling pathways in the regulation of immunity to Toxoplasma gondii. Int. Rev. kines than WT monocytes (11, 13). 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