The Journal of Immunology

Regulatory Dendritic Cells Pulsed with Carbonic Anhydrase I Protect Mice from Colitis Induced by CD4+CD252 T Cells

Hirofumi Yamanishi,* Hidehiro Murakami,† Yoshiou Ikeda,* Masanori Abe,* Teru Kumagi,* Yoichi Hiasa,* Bunzo Matsuura,* and Morikazu Onji*

Inflammatory bowel disease (IBD), which is characterized by a dysregulated intestinal immune response, is postulated to be con- trolled by intestinal self-antigens and bacterial Ags. Fecal extracts called cecal bacterial Ag (CBA) have been implicated in the pathogenesis of IBD. In this study, we identified a major protein of CBA related to the pathogenesis of IBD and established a ther- apeutic approach using Ag-pulsed regulatory dendritic cells (Reg-DCs). Using two-dimensional gel electrophoresis and MALDI- TOF mass spectrometry, carbonic anhydrase I (CA I) was identified as a major protein of CBA. Next, we induced colitis by transfer of CD4+CD252 T cells obtained from BALB/c mice into SCID mice. Mice were treated with CBA- or CA I-pulsed Reg-DCs (Reg-

DCsCBA or Reg-DCsCA1), which expressed CD200 receptor 3 and produced high levels of IL-10. Treatment with Reg-DCsCBA and Reg-DCsCA1 ameliorated colitis. This effect was shown to be Ag-specific based on no clinical response of irrelevant Ag (keyhole limpet hemocyanin)-pulsed Reg-DCs. Foxp3 mRNA expression was higher but RORgt mRNA expression was lower in the mesenteric lymph nodes (MLNs) of the Reg-DCsCA1–treated mice compared with those in the MLNs of control mice. In the MLNs, Reg-DCsCA1–treated mice had higher mRNA expression of IL-10 and TGF-b1 and lower IL-17 mRNA expression and + + + protein production compared with those of control mice. In addition, Reg-DCsCBA–treated mice had higher Foxp3 CD4 CD25 and IL-10–producing regulatory T cell frequencies in MLNs. In conclusion, Reg-DCsCA1 protected progression of colitis induced by CD4+CD252 T cell transfer in an Ag-specific manner by inducing the differentiation of regulatory T cells. The Journal of Immunology, 2012, 188: 2164–2172.

uman inflammatory bowel diseases (IBDs), including current treatments have proved curative, emphasizing the need for Crohn’s disease and ulcerative colitis, are characterized research in the development of new and better therapeutics. H by inflammation in the large and/or small intestine. The Microenvironmental immunoregulation is constantly fine-tuned exact cause and subsequent development of IBD is not estab- to maintain local homeostasis. This tuning can be specific to the site lished, but uncontrolled innate and adaptive immunity against involved (such as the gut environment) or induced by chronic normal intestinal constituents, including intestinal epithelial cells, exposure to microbes. Dendritic cells (DCs) play a critical role in commensal bacteria, various microbial products, and/or food- orchestrating this tuning (4). DCs are the most potent and efficient stuffs, are known to contribute to dysregulation of the immune dedicated APCs, and they are critical for inducing primary im- system and subsequent development of disease (1–3). None of the mune responses. Conversely, DCs also play a crucial role in the induction of immune tolerance. There is increasing evidence that DCs induce Ag-specific unresponsiveness or tolerance in situ in *Department of Gastroenterology and Metabology, Ehime University Graduate central lymphoid organs and in the periphery (4). Although the School of Medicine, Shitsukawa, To¯on, Ehime 791-0295, Japan; and †Endoscopic tolerogenic mechanisms are not entirely understood, there is also Center, Ehime University Hospital, Shitsukawa, To¯on, Ehime 791-0295, Japan new evidence that DCs mediate the induction of peripheral T cell Received for publication February 25, 2011. Accepted for publication December 13, tolerance by stimulating the differentiation of CD4+CD252 T cells 2011. to CD4+CD25+Foxp3+ regulatory T cells (5). This work was supported by Grants-in-Aid (18790464 and 21590814) from the Japanese Ministry of Education, Culture, Sports, Science, and Technology and by The selective enhancement of the tolerogenicity of DCs has the Department of Biological Resources, Integrated Center for Science, Ehime Uni- been achieved using immature DCs, created by pharmacological versity. inhibition of DC maturation, or using genetically engineered Address correspondence and reprint requests to Prof. Morikazu Onji, Department of DCs expressing immunosuppressive molecules (6). In addition, Gastroenterology and Metabology, Ehime University Graduate School of Medicine, Shitsukawa, To¯on, Ehime 791-0295, Japan. E-mail address: [email protected] studies using murine models have demonstrated that several types of tolerogenic or regulatory DCs (Reg-DCs) can induce an Ag- The online version of this article contains supplemental material. specific amelioration of pathology in a wide range of contexts, Abbreviations used in this article: ALDH1a2, aldehyde dehydrogenase family 1a2; APCCA1, APC pulsed with carbonic anhydrase I; APCKLH, APC pulsed with keyhole including autoimmunity, allergy, and graft rejection (7–9). Some limpet hemocyanin; CA I, carbonic anhydrase I; CBA, cecal bacterial Ag; CD200R3, studies found that transfer of CD4+ T cells or Th17 cells reactive CD200 receptor 3; CpG-ODN, CpG oligonucleotide; DC, dendritic cell; 2D-DIGE, to fecal extracts called cecal bacterial Ag (CBA) or enteric bac- two-dimensional difference gel electrophoresis; IBD, inflammatory bowel disease; KLH, keyhole limpet hemocyanin; MLN, mesenteric lymph node; poly-IC, terial Ag induced severe colitis in SCID mice (10, 11). In contrast, + polyinosinic-polycytidylic acid; Reg-DC, regulatory dendritic cell; Reg-DCsCA1, reg- normal lamina propria CD4 T cells cocultured with APCs in the ulatory dendritic cells pulsed with carbonic anhydrase I; Reg-DCsCBA, regulatory presence of CBA led to the generation of regulatory T cells (Tregs) dendritic cells pulsed with cecal bacterial Ag; Reg-DCsCBA-CA1, regulatory dendritic cells pulsed with cecal bacterial Ag depleted of carbonic anhydrase I; Reg-DCsKLH, that could ameliorate experimental colitis (12). These findings regulatory dendritic cells pulsed with keyhole limpet hemocyanin; Tr1 cell, IL-10– indicate that fecal extracts may harbor a colitogenic Ag in IBD. producing type 1 regulatory T cell; Treg, regulatory T cell. In the current study, we investigated the mechanisms mediating Copyright Ó 2012 by The American Association of Immunologists, Inc. 0022-1767/12/$16.00 the therapeutic effect of CBA-pulsed Reg-DCs in experimental www.jimmunol.org/cgi/doi/10.4049/jimmunol.1100559 The Journal of Immunology 2165 murine colitis. We analyzed CBA by two-dimensional difference coater. The suture was observed with a field-emission scanning electron gel electrophoresis (2D-DIGE) and MALDI-TOF mass spec- microscope (Hitachi S-4800, Tokyo, Japan) at 2 kV acceleration voltage. trometry and found that carbonic anhydrase I (CA I) was a main Induction of Foxp3+CD4+CD25+ T cells differentiation from protein component of CBA. We then assessed whether regulatory CD4+CD252 T cells in vitro dendritic cells pulsed with carbonic anhydrase I (Reg-DCsCA1) 5 Mature DCs, Reg-DCs, or Reg-DCsCA1 (5 3 10 ) were cultured in 35-mm induced Ag-specific protection from colitis in a murine model of culture dishes (Corning, Horseheads, NY) for 7 d with CD4+CD252 the disease. T cells (5 3 106), which were isolated from the spleens of BALB/c mice using the CD4+CD25+ Regulatory T Cell Isolation Kit and the AutoMACS Materials and Methods (Miltenyi Biotec). Mice Flow cytometric analysis and intracellular cytokine synthesis CB-17 SCID and BALB/c (H-2d) female mice bred under specific path- analysis ogen-free conditions were purchased from CLEA Japan (Tokyo, Japan). Unless otherwise noted, materials were purchased from BD Biosciences. All mice were between 8 and 12 wk of age and were maintained at the After blocking the FcR with purified rat anti-mouse CD16/CD32 (2.4G2), the animal center of Ehime University Graduate School of Medicine (Ehime, DCs were stained with FITC-conjugated anti-H-2Kd (AMS-32.1), anti- Japan). All animals received adequate care according to good laboratory CD40, anti-CD80, anti-CD86, anti-F4/80 (BM8), PE-conjugated anti–I-A/ practice guidelines. The Committee of Animal Experimentation, Ehime I-E (2G9), CD11c (HL3), CD11b (M1/70), CD3 (17A2) mAbs and University Graduate School of Medicine, approved the study. allophycocyanin-conjugated anti-B220 (RA3-6B2) mAbs. Isotype-matched + + + Preparation of CBA Abs were used as controls. The frequencies of Foxp3 CD4 CD25 Tregs were determined by the PE Anti-Mouse/Rat Foxp3 Staining Set (eBio- CBA was prepared as previously described with some modifications (10). science, San Diego, CA), PE-conjugated anti-Foxp3 mAb (FJK-16s; eBio- Briefly, BALB/c mice were euthanized, and their ceca were removed. Ceca science), allophycocyanin-conjugated anti-CD25 mAb (PC61), and PerCP- (n = 5) were opened and placed in 10 ml PBS with 1.0-mm silica spheres conjugated anti-CD4 mAb (RM4-5) after MLN cells were cultured for 72 h. (Lysing Matrix C; MP Biomedicals, Solon, OH). After vortexing this To determine the frequencies of IL-10–producing CD4+CD25+ T cells, the mixture for 5 min, the silica spheres and residual cecal tissue were re- MLN cells were cultured for 24 h. These cells were stimulated with PMA/ moved by centrifugation at 5000 3 g for 5 min at 4˚C. Subsequently, the ionomycin for the last 5 h, and GolgiStop was added for the last 3 h of the supernatant was centrifuged at 18,000 3 g for 30 min at 4˚C. The lysates incubation period. The cells were stained with PerCP-conjugated anti-CD4 were sterilized by passage through an 0.2-mm pore-size syringe filter. The mAb and allophycocyanin-conjugated anti-CD25 mAb, followed by fixation protein concentrations in lysates were measured using the DC protein and permeabilization with a FIX & PERM Kit (Caltag Laboratories, Bur- assay kit (Bio-Rad, Hercules, CA). lingame, CA). The cells were stained with PE-conjugated rat anti-mouse IL-10 mAb (JES5-16E3) for 20 min at room temperature. Isotype-matched Cell preparation Abs were used as controls. Fluorescence staining was analyzed with FACS The preparation of DCs has been described previously (9, 13). Mature DCs using FlowJo software version 7.5 (Tree Star, Ashland, OR). 6 were prepared by culturing bone marrow cells (2 3 10 ) obtained from Induction of colitis and treatment of mice with DCs BALB/c mice with murine GM-CSF (20 ng/ml; Wako Pure Chemical, Osaka, Japan) for 8 d; subsequently, these cells were stimulated with ul- Colitis was induced according to methods described in a previous report 2 trapure LPS (1 mg/ml; InvivoGen, San Diego, CA) for 24 h. Reg-DCs were with some modifications (14). Briefly, CD4+CD25 T cells (3 3 105 / generated from bone marrow cells (2 3 106) obtained from BALB/c mice mouse) obtained from BALB/c mice were suspended in 0.2 ml PBS and and cultured with murine GM-CSF (20 ng/ml), murine IL-10 (20 ng/ml; i.p. injected into SCID mice. SCID controls received 0.2 ml PBS alone. Wako Pure Chemical), and human TGF-b1 (20 ng/ml; Wako Pure The day of this transfer was designated as day 0. Subsequently on day 0, Chemical) for 8 d, followed by stimulation with ultrapure LPS (1 mg/ml) DCs (1 3 106 cells/mouse) generated from BALB/c mice were injected i.p. for 24 h. Subsequently, the Reg-DCs were stained with FITC-conjugated The body weight of each mouse was measured weekly. anti-CD40 (3/23), anti-CD80 (16-10AI), and anti-CD86 (GL1) mAbs (BD Histological assessment of colitis Biosciences, San Diego, CA) for 30 min at 4˚C. After the cells were magnetically labeled with anti-FITC MicroBeads (Miltenyi Biotec, Ber- Colons were removed from euthanized mice 4 wk after cell transfer. The + gisch Gladbach, Germany), Reg-DCs were purified by depletion of CD40 transverse colons were removed and fixed with 10% neutral buffered formalin + + CD80 CD86 cells using the AutoMACS (Miltenyi Biotec). and then embedded in paraffin. Thin tissue sections were stained with H&E In some experiments, mature DCs or Reg-DCs were pulsed with CA I (6 stain or periodic acid–Schiff stain. The grade of inflammation in tissue mg/ml), CBA (50 mg/ml), or keyhole limpet hemocyanin (KLH; 50 mg/ml; sections was evaluated as described previously (14). Histology was scored Thermo Scientific, Rockford, IL) for 24 h. as follows: 1) severity of inflammation: 0, none; 1, mild lymphoid infiltra- Measurements of cytokine levels in vitro tion; 2, marked lymphoid infiltration or focal degeneration of crypts; 3, severe inflammation or multifocal crypt degeneration and/or erosions; 2) DCs (2 3 105) were cultured in complete RPMI 1640 medium (10% FBS, extent of inflammation: 0, none; 1, mucosal; 2, submucosal; 3, transmural; 20 mM HEPES, 2-mercaptoethanol, penicillin, and streptomycin; Nipro, 3) amount of mucus: 0, normal; 1, slight decrease of mucus; 2, moderate Osaka, Japan) with polyinosinic-polycytidylic acid (poly-IC; 1 mg/ml; Sig- decrease or focal absence of mucus; 3, severe depletion of mucus; 4, total ma Chemical, St. Louis, MO), ultrapure LPS (1 mg/ml), R848 (1 mM; absence of mucus; and 4) degree of cell proliferation: 0, none; 1, mild in- InvivoGen), or phosphorothiolated CpG oligonucleotide (CpG-ODN; 1 mg/ crease in cell numbers and crypt length; 2, moderate increase or focally ml; InvivoGen) for 24 h, and the culture supernatants were assayed for IL- marked increase; 3, marked increase in entire section. The cumulative his- 10 and IL-6 using cytometric bead array kits (BD Biosciences) and TGF-b tological score was calculated as the sum of the four individual parameters. using ELISA kits (R&D Systems, Minneapolis, MN). Mesenteric lymph node (MLN) cells (1 3 106) were cultured for 72 h. For the last 6 h of the Immunohistochemistry culture period, 25 ng/ml PMA (Sigma Chemical) and 1 mg/ml ionomycin The frozen sections of colons (5 mm thick) were fixed with acetone. En- (Sigma Chemical) were added; at the end of the culture period, the culture dogenous peroxidase activity was inactivated by methanol containing 1% supernatants were assayed for cytokines using cytometric bead array kits hydrogen peroxidase for 20 min. The sections, inactivated with Endoge- (BD Biosciences) and IL-17 using ELISA kits (R&D Systems). nous Avidin/Biotin Blocking Kit (Nichirei, Tokyo, Japan), were pretreated Measurement of nuclear NF-kB p65 with undiluted rabbit serum (Nichirei) and then incubated with 1:50 di- luted biotinylated goat anti-human CA I (Rockland, Philadelphia, PA) at DCs (1 3 106) were treated with 1 mg/ml CpG-ODN for 60 min. The 4˚C overnight. The tissue sections were treated with HRP-conjugated nuclear extracts were obtained by using a Nuclear Extract Kit (Active streptavidin (Nichirei) and incubated with Simple Stain DAB Solution Motif, Tokyo, Japan) and nuclear NF-kB p65 was measured using (Nichirei). Finally, the sections were counterstained with hematoxylin, TransAM NF-kB p65 Kit (Active Motif). dehydrated, and mounted. Methods of scanning electron microscopy Colon culture ex vivo DCs were conductive stainedwith 1% bufferedosmium tetroxide and 1% tannic Sections of 1 cm of the middle parts of the colon were excised, the feces acid. They were dehydrated and coated with a thin layer (3 nm) of osmium were removed, and the sections were then washed three times with sterile 2166 CA I-PULSED REGULATORY DCs PREVENT IBD

PBS and halved longitudinally. The colon sections were then placed into T cell proliferation assay culture in complete RPMI 1640 medium and cultured for 3 d before + 2 + 3 5 supernatants were harvested. For viability assay, the lamina propria cells CD4 CD25 T cells or CD4 T cells at 1 10 cells/well were incubated in the presence of 1 3 105 Ag-pulsed irradiated spleen cells used as APC. were isolated from colonic specimens using a modification of a described 3 technique (15). The viability of the lamina propria cells was ∼90% of total After 4 d, [ H]thymidine (1.0 mCi/ml; Amersham Biosciences, Buck- cells and was assessed with the trypan blue exclusion method (Sigma inghamshire, U.K.) was added to the cultures for the last 18 h and har- Chemical). vested automatically by a multiple cell harvester (Labo Mash; Futaba Medical, Osaka, Japan) onto filter paper (Labo Mash 101–10; Futaba 3 RT-PCR and quantitative real-time PCR Medical). The levels of incorporation of [ H]thymidine were determined in a liquid scintillation counter (Beckman LS 6500; Beckman Instruments, A tissue sample from the middle part of the colon and the MLN were Fullerton, CA). homogenized with the TissueLyser (Qiagen, Tokyo, Japan). Total RNA was isolated with the RNAeasy Plus Mini Kit (Qiagen). cDNA was generated Statistical analysis with a high-capacity cDNA reverse transcription kit (Applied Biosystems, 6 Foster City, CA). PCR analysis was performed using a KOD-Plus-Ver.2 Data are expressed as mean values SD of all individual experiments. polymerase kit (Toyobo, Osaka, Japan) with a pair of primers specific Statistical differences between two groups were determined by Student for CD200 receptor 3 (CD200R3) and GAPDH. Quantitative RT-PCR t test. One-way ANOVA was used to compare multiple groups. If the reactions of IL-10, IL-6, IL-17A, TGF-b1, Foxp3, and RORgt proceeded ANOVAwas significant, the Tukey–Kramer honestly significant difference , using a LightCycler real-time PCR system (Roche, Basel, Switzerland) test was applied for multiple comparisons. A value of p 0.05 was con- with LightCycler Fast Start DNA Master SYBRF Green I (Roche). The sidered statistically significant. Statistical calculations were performed sequences of the primers used in these analyses are shown in Supplemental with JMP software (SAS International, Cary, NC). Table I. Quantitative RT-PCR reactions of aldehyde dehydrogenase family 1a2 (ALDH1a2) were performed using TaqMan Gene Expression Assays Results (Applied Biosystems). ALDH1a2 primers were purchased from Applied + + + Biosystems (Assay ID: Mm00501312-m1). Reg-DCs induce the differentiation of CD4 CD25 Foxp3 T cells from CD4+CD252Foxp32 T cells in vitro Two-dimensional gel electrophoresis and imaging Mature bone marrow-derived DCs expressed high levels of MHC Unless otherwise noted, materials were purchased from GE Healthcare Bio- molecules (H-2kd and I-A/I-E), CD11c, CD11b, and costimulatory Sciences (Piscataway, NJ). The CBA sample treated with a commercial 2-D molecules (CD40, CD80, and CD86) (Fig. 1A). By contrast, Reg- Clean-Up kit was resuspended in lysis buffer consisting of 30 mM Tris-HCl, pH 8.5, 7 M urea, 2 M thiourea, 4% CHAPS, and PlusOne Protease Inhibitor DCs expressed moderate levels of MHC molecules and CD11b Mix. The sample was rehydrated with Immobiline DryStrip reswelling and did not express CD11c or costimulatory molecules (Fig. 1A). solution, pH 3–10, 24 cm at 20˚C for 10 h. Isoelectric focusing was per- Neither mature DCs nor Reg-DCs expressed macrophage marker formed using the IPGphor II isoelectric focusing system for a total of 45 (F4/80), T cell marker (CD3), or B cell marker (B220) (Fig. 1A). kVh at 20˚C. The IPG gel was incubated in equilibration buffer (50 mM CD200R3 expression has been reported to be crucial for Reg- Tris HCl, pH 8.8, 6 M urea, 30% glycerol, 2% SDS, 0.002% bromophenol + blue) supplemented with 0.5% DTT for 15 min, followed by 4.5% DCs to suppress Ag-specific CD4 T cell responses (5). RT-PCR iodoacetamide in fresh equilibration buffer for an additional 15 min. The demonstrated that Reg-DCs, but not mature DCs, expressed high strip was immediately applied to a 12.5% SDS-PAGE on an Ettan levels of CD200R3—splice variants A (860 bp) and D (642 bp) DALTsix electrophoresis system at 2 W for 16 h at 30˚C. (18) (Fig. 1B). Field-emission scanning electron microscopy con- The gel stained with Deep Purple Total Protein Stain was scanned on an Ettan DIGE Imager, and the image was analyzed with the ImageMaster 2D firmed that Reg-DCs had dendrites like mature DCs (Fig. 1C). Platinum Software Informer system. The spots were quantified on the basis Reg-DCs stimulated with various TLR ligands produced signifi- of relative volume (the spot volume divided by the total volume over the cantly lower levels of IL-6 and higher levels of IL-10 than did whole set of gel spots). mature DCs (Fig. 1D). We found that Reg-DCs, but not mature + 2 + MALDI-TOF mass spectrometry DCs, induced the differentiation of CD4 CD25 T cells to Foxp3 CD4+CD25+ Tregs (Fig. 1E, 1F). Protein spots were excised from the gel, washed, and digested in-gel with porcine modified trypsin protease (Promega, Madison, WI). Tryptic pep- Treatment with Reg-DCs pulsed with CBA protects mice tides were extracted by sonication. HPLC, MALDI sample preparation, and against experimental colitis spotting on a mFOCUS MALDI plate (Shimadzu, Kyoto, Japan) were 2 carried out automatically in a nano LC-AccuSpot apparatus (Shimadzu). CB-17 SCID mice were divided into four groups, and CD4+CD25 Tandem time-of-flight mass spectrometry was performed on an Axima- colitogenic effector T cells were transferred into all mice in each 2 TOF mass spectrometer (Shimadzu). Proteins were identified using the group. At the time of T cell transfer, the groups of mice were MASCOT MS/MS ion search engine (Matrix Science, London, U.K.) and 3 6 the National Center for Biotechnology Information protein database. treated with PBS or with Reg-DCs (1 10 /mouse) that had been pulsed with either CBA (Reg-DCsCBA) or KLH (Reg-DCsKLH)or Preparation of mouse CA I left untreated with Ag. Four weeks after T cell transfer, body Mouse CA I was prepared using a cell-free protein synthesis system and weights were significantly higher in mice treated with Reg- , wheat germ rRNA for which there is no risk of LPS contamination (16). DCsCBA than in mice treated with PBS (p 0.01) (Fig. 2A). The expression plasmid (pEU-mCA1-His) was constructed and amplified Treatment of mature DCs pulsed with CBA was insufficient for in the mouse CA I gene with the primers using the KOD-Plus-Ver.2 protection against colitis (Supplemental Fig. 1A). Macroscopic polymerase kit (Toyobo). examinations at week 4 after transfer revealed that the colon length The CA I protein was automatically synthesized by the Robotic Protein Synthesizer Protemist DT (CellFree Sciences, Matsuyama, Japan) according was significantly longer in mice treated with Reg-DCs or Reg- to the manufacturer’s instruction manual as previously reported (17). DCsCBA than in PBS-treated mice (p , 0.01) (Fig. 2B, 2C). Upon histological examination, colitis was characterized by severe epi- Depletion of CA I from CBAs thelial hyperproliferation, mucus depletion, massive infiltration of CA I was depleted using Immunoprecipitation Kit-Dynabeads Protein G inflammatory cells, crypt degeneration, reduced numbers of goblet and Carbonic Anhydrase I Ab (Gene Tex, Irvine, CA). Depletion of CA I cells, and erosions. CB-17 SCID mice injected with CD4+CD252 from CBAs was confirmed by Western blotting. Protein (5 mg) was applied T cells and treated with PBS had severe colitis. The mice injected to lanes in 4–12% Bis–Tris Gels (Invitrogen). Resolved products were then blotted onto Immunobilon-P membranes (Millipore, Bedford, MA) and with unpulsed Reg-DCs or with Reg-DCsKLH had colonic changes probed with CA I Ab. Proteins were detected using the ECL Plus Kit (GE similar to those seen in mice treated with PBS. In contrast, Healthcare Bio-Sciences). treatment with Reg-DCsCBA improved these histological signs; The Journal of Immunology 2167

FIGURE 1. Phenotypic markers, cytokine production, and differentiation of CD4+CD25+Foxp3+ T cells by Reg-DCs. (A) The expression of cell surface molecules on mature DCs and Reg-DCs was analyzed by FACS, and the data are represented as dot plots. (B) RT-PCR analysis of CD200R3 expression in DCs. GAPDH was used as the internal control. The bands represent the expression of splice variant A (860 bp) and D (642 bp) of CD200R3 and GAPDH. (C) Morphology of mature DCs and Reg-DCs (scale bar, 10 mm) was examined by field-emission scanning electron microscope after stimulation with CpG- ODN (1 mg/ml) for 24 h. Data are representative of four replicate experiments. (D) DCs (1 3 105) were stimulated with poly-IC (1 mg/ml), ultrapure LPS (1 mg/ml), R848 (1 mg/ml), or CpG-ODN (1 mg/ml) for 24 h, and the culture supernatants were analyzed using the cytometric bead array method. (E and F) CD4+CD252 T cells (5 3 106) obtained from BALB/c mice were cultured with mature DCs or Reg-DCs (5 3 105) obtained from BALB/c mice for 7 d. (E) The data are expressed as percentages of cells positive for CD4+CD25+Foxp3+ and CD4+CD25+ Foxp3+/CD4+CD25+.(F) The expression of CD25 and Foxp3 on CD4+ T cells was analyzed by FACS. The data in (A) and (B) are representative of three independent experiments, and the data in (D) and (E) are representative of five independent experiments with similar results. *p , 0.01. moreover, there was a significant reduction of inflammatory cell There was no significant difference in the expression of Foxp3 in infiltration, and goblet cells and mucus were preserved (Fig. 2D). the colons of Reg-DCsCBA–treated mice and PBS-treated mice The cumulative histological score (14) was significantly lower (Supplemental Fig. 1B). RORgt mRNA expression in the colon in mice treated with Reg-DCsCBA than in PBS-treated mice (p , was lower in mice treated with Reg-DCsCBA than in PBS-treated 0.01) (Fig. 2E). Unpulsed Reg-DC–treated mice showed moderate mice (p , 0.05) (Supplemental Fig. 1B). Culture supernatants weight loss and mild shortening of the colon; however, they also from colonic explants indicated that the production of IL-17 and had moderate to severe colitis upon histological examination. TNF-a was lower in mice treated with Reg-DCsCBA than in PBS- treated mice (p , 0.05), but significant differences in IFN-g, IL-6, Treatment with Reg-DCs reduces inflammatory cytokine CBA TGF-b, and IL-10 levels were not detected between the two responses in the colon groups (Supplemental Fig. 1C). The effect of Reg-DCsCBA treatment on the production of in- flammatory cytokines in the colon of mice subjected to CD4+ Administration of Reg-DCsCBA induced Foxp3 expression and CD252 T cell transfer was evaluated. The colonic expression of reduced RORgt expression in the MLN IL-6 and IL-17A mRNA was reduced and IL-10 mRNA expres- To elucidate the mechanism underlying downregulation of in- sion was increased in Reg-DCsCBA–treated mice compared with flammatory processes mediated by Reg-DCsCBA, the levels of PBS-treated mice (p , 0.05) (Supplemental Fig. 1B). There was expression of transcription factors and cytokines in the MLN were no difference in the expression of ALDH1a2 (which converts assessed (Fig. 3). Foxp3 mRNA expression in the MLN was sig- retinoids to retinoic acid and induces Tregs) between Reg- nificantly higher in mice treated with Reg-DCsCBA compared with DCsCBA–treated mice and PBS-treated mice (Supplemental Fig. PBS-treated mice (p , 0.05). In contrast, RORgt mRNA ex- 1B) (19, 20). Foxp3 and RORgt, transcription factors that define pression in the MLN was lower in mice treated with Reg-DCsCBA the Treg and Th17 lineages, respectively, were examined (21). than in PBS-treated mice (p , 0.05) (Fig. 3A). In addition, 2168 CA I-PULSED REGULATORY DCs PREVENT IBD

FIGURE 3. Administration of Reg-DCsCBA–induced Foxp3 expression and reduced RORgt expression in the MLN. (A and B) Transcription factor (A) and cytokines and ALDH1a2 (B) mRNA expression in the MLN was quantified by real-time RT-PCR; mean 6 SD of four mice per group. (C) 6 MLN cells (1 3 10 ) from mice treated with PBS or Reg-DCsCBA were cultured for 72 h, and secreted cytokines in the supernatants were mea- sured; mean 6 SD of seven mice per group. Data shown are representative of two independent experiments. *p , 0.05, **p , 0.01.

CD4+CD25+ T cells in the MLN were significantly higher in mice treated with Reg-DCsCBA than in mice treated with PBS or Reg- DCsKLH on day 7 after transfer (p , 0.05) (Fig. 4).

FIGURE 2. Treatment with Reg-DCsCBA protects mice against experi- CA I is the major Ag of CBA mental colitis. (A) Relative changes in percent body weight over time for Proteomic analysis of CBA using 2D-DIGE imaging (Fig. 5A) no-transfer mice (Δ, n = 7) and for colitis-induced mice (transfer of CD4+ CD252 T cells) subsequently treated with PBS (d, n = 10), Reg-DCs (s, showed 14 main spots on the spot map. These spots were excised from the gel for subsequent mass spectrometry. Fourteen spots gave n = 7), Reg-DCsKLH (N, n = 6), or Reg-DCsCBA (n, n = 8). (B) Macroscopic findings of the colon on day 28 after transfer of CD4+CD252 T cells. Scale the identifications referred to in the database interrogation shown in bar, 10 mm. (C) The colon lengths of colitic mice were measured on day Supplemental Table II (the cut spots are indicated with circles in 28. (D) Histological analysis of the colon was carried out on day 28. PBS- the map in Fig. 5A). Among these proteins, 7.03% (nos. 1, 4, 5, 9, treated mice showed marked increase in mucosal height and inflammatory and 10) was CA I, 5.76% (nos. 2, 3, and 7) was serum albumin cell infiltration and severe loss of mucus from goblet cells. Treatment with precursor, and 1.54% (nos. 6 and 14) was selenium binding protein Reg-DCsCBA improved these histological signs. Representative photos at 1. CA I emerged as the principal protein Ag of CBA. low (top, periodic acid–Schiff staining; scale bar, 200 mm) and high CA I expression in both healthy and inflamed mouse colons was (bottom, H&E staining; scale bar, 100 mm) magnifications are shown. (E) + 2 evaluated by immunohistochemical analysis. The inflamed colons Histological scores of the colons from CD4 CD25 T cell-transferred + 2 mice. There were 6–10 mice per group. Data shown are representative of of SCID mice transferred with CD4 CD25 T cells showed a two independent experiments. Horizontal bars: median. *p , 0.01 (com- significant reduction of CA I expression compared with the colons + pared with PBS-treated mice). of healthy SCID control mice or of SCID mice subjected to CD4 2 CD25 T cells transfer and treatment with Reg-DCsCBA (Fig. 5B). Reg-DCs pulsed with CA I suppress development of colitis in expression of IL-10 and TGF-b1 mRNA in the MLN was sig- the SCID transfer model nificantly higher in mice treated with Reg-DCsCBA than in PBS- treated mice (p , 0.05). Significant differences in the expression To assess whether CA I is necessary for protection against colitis, CB- + 2 of IL-6, IL-17A, and ALDH1a2 in the MLN of mice treated with 17 SCID mice received CD4 CD25 T cells with Reg-DCsCA1 (Reg- 3 6 Reg-DCsCBA versus PBS-treated mice were not detected (Fig. 3B). DCsCA1:1 10 cells/mouse) or regulatory dendritic cells pulsed In addition, Reg-DCsCBA administration resulted in a striking re- with cecal bacterial Ag depleted of carbonic anhydrase I (Reg- 3 6 duction of IL-6, IFN-g, TNF-a, and MCP-1 production from the DCsCBA-CA1:1 10 cells /mouse) (Supplemental Fig. 2A). Four MLN cells of colitic mice (p , 0.01) (Fig. 3C). weeks after transfer, Reg-DCsCA1–treated mice showed more effec- + + + tive protection against colitis (Fig. 6A–C). Foxp3 mRNA expression Reg-DCsCBA induced Foxp3 CD4 CD25 T cells and in the MLN of Reg-DCsCA1–treated mice was higher than that in the IL-10–producing CD4+CD25+ T cells in vivo MLN of Reg-DCsCBA-CA1–treated mice (p , 0.05) (Fig. 6D). To address whether Reg-DCsCBA could induce peripheral differ- Next, we analyzed the underlying mechanism of therapeutic + + + entiation of Foxp3 CD4 CD25 T cells and IL-10–producing effects by Reg-DCsCA1. The expression of IL-17A mRNA was + 2 + 2 CD4 T cells from Foxp3 CD4 CD25 T cells in vivo, the lower in the colon and MLN of Reg-DCsCA1–treated colitic mice numbers of Foxp3+CD4+CD25+ T cells and IL-10–producing than in those of PBS-treated colitic mice (p , 0.05); however, the CD4+CD25+ T cells were evaluated in the MLN of mice treated expression of IL-10 and TGF-b1 mRNA was higher in the Reg- with Reg-DCsCBA. Flow cytometric analysis showed that the DCsCA1–treated colitic mice (Fig. 6E). In the MLN, the expression percentages of Foxp3+CD4+CD25+ Tregs and IL-10–producing of Foxp3 was significantly higher and that of RORgt was signifi- The Journal of Immunology 2169

+ + + FIGURE 4. Reg-DCsCBA induce Foxp3 CD4 CD25 T cells and IL-10–producing CD4+CD25+ T cells in vivo. The generation of Foxp3+ CD4+CD25+ T cells and IL-10–producing CD4+CD25+ T cells from Foxp32CD4+CD252 T cells in the MLN from CD4+CD252 T cell- transferred mice treated with PBS (n = 7), Reg-DCsKLH (n = 5), or Reg- DCs (n = 5). CD4+ T cells were gated and analyzed by FACS. (A and CBA FIGURE 5. CA I is the major Ag of CBA. (A) Deep purple-stained 2D- B) The data are expressed as percentages of cells positive for Foxp3+CD4+ DIGE gel of CBA. The cut spots as green circles are labeled from 1 to 14 CD25+ T cells (A) and IL-10+CD4+CD25+ T cells (B). (C and D) Ex- according to Supplemental Table II. The results were consistent among all pression of Foxp3 or IL-10 and CD25 is represented in dot plots. Data three independent experiments, and a representative image is shown. (B) shown are representative of two independent experiments. *p , 0.05. NS, Immunohistochemical staining of CA I in the mouse colon. Representative Not significant. photos at low (top; scale bar, 200 mm) and high (bottom, scale bar, 100 mm) magnifications are shown. cantly lower in colitic mice treated with Reg-DCsCA1 than in PBS- treated colitic mice (p , 0.05) (Fig. 6E). RORgt mRNA expres- Finally, we checked whether CA I changed the phenotype of sion in the colon was lower in mice treated with Reg-DCsCA1 than in PBS-treated mice (p , 0.05) (Fig. 6E). Culture supernatants Reg-DCs. There was no difference in the cytokine production, from colonic explants and MLN cells of colitic mice treated with NF-kB p65 activation, aldehyde dehydrogenase family 1 expres- sion, and generation of Foxp3+ Treg between Reg-DCs and Reg- PBS and Reg-DCsCA1 were assayed. IL-10 and TGF-b1 produc- tion from the colon did not differ between the two groups. The DCsCA1 (Supplemental Fig. 2D, 2E). production of IL-17, TNF-a, and IFN-g in the colon was signifi- cantly lower in colitic mice treated with Reg-DCsCA1 than in PBS- Discussion treated colitic mice (p , 0.05) (Fig. 6F). In addition, Reg-DCCA1 The four main findings of the current study are 1) Reg-DCsCBA + 2 administration resulted in a striking reduction in the production of inhibited the progression of colitis induced by CD4 CD25 IL-6, IL-17, TNF-a, IFN-g, and MCP-1 from the MLN cells rel- T cells; 2) CA I, a major Ag of CBA, had an important role in ative to that from PBS-treated colitic mice (p , 0.01) (Fig. 6F). suppressing the development of colitis; 3) Reg-DCsCA1 induced Foxp3+ Tregs and reduced IL-17 expression in the MLN; and 4) Reg-DCs pulsed with CBA induce CA I-specific Treg in the + + + Reg-DCsCBA induced Foxp3 CD4 CD25 T cells and IL-10– MLN producing CD4+CD25+ T cells in vivo. To assess whether Reg-DCsCBA induced CA1-specific Treg Many researchers have shown that DCs have Ag-specific pro- 2 in vivo, CD4+CD25 T cells or CD4+ T cells (1 3 105) in the tective effects in murine models of autoimmune diseases (7, 8, 22). MLN from colitic mice treated with Reg-DCsCBA were cultured In addition, Pedersen et al. (23) found that tolerogenic DCs pulsed with spleen cells (used as APC) of BALB/c mice, APC pulsed with fecal extract suppressed the development of colitis. Our re- with carbonic anhydrase I (APCCA1), or APC pulsed with keyhole sults were consistent with these studies. However, little is known + + limpet hemocyanin (APCKLH). When CD4 CD25 cells were re- about the underlying mechanism. Our data showed that treatment moved from the responder CD4+ T cell population, stronger T cell with Reg-DCs pulsed with fecal extract, including CBA, amelio- proliferation was observed when APCCA1 was used as stimulators. rated the clinical and histopathologic severity of the wasting dis- However, this result was not observed when APCKLH was used as ease in a murine model (Fig. 2). As shown in Fig. 2, Reg-DCs stimulators (Supplemental Fig. 2B). In addition, the proliferative alone or Reg-DCsKLH partially (but not significantly) prevented + + response to APCCA1 was not increased when CD4 CD25 cells colitis. This effect might involve the production of IL-10 (Fig. were removed from the MLN of Reg-DCsKLH or PBS-treated mice 1D), consistent with a previous report by Fujita et al. (13). (Supplemental Fig. 2C), indicating that CD4+CD25+ cells of MLN However, Mengs et al. (8) demonstrated that autoantigen-pulsed, from colitic mice treated with Reg-DCsCBA contained CA I-spe- not irrelevant Ag-pulsed, IL-10 produced DCs that prevented cific Tregs. experimental autoimmune encephalomyelitis by inducing Ag- 2170 CA I-PULSED REGULATORY DCs PREVENT IBD

+ FIGURE 6. Reg-DCsCA1 suppress development of colitis. (A) Relative changes in body weight (%) over time for colitis-induced mice (transfer of CD4 2 CD25 T cells) subsequently treated with PBS (d, n = 12), mature DCs pulsed with CA1 (mature DCsCA1; :, n = 8), Reg-DCsCBA-CA1 (N, n = 8), or Reg- DCsCA1 (n, n = 8). (B) The colon lengths of colitic mice were measured on day 28. There were 8–12 mice per group. (C) Histological scores on day 28. There were 8–12 mice per group. Horizontal bars: median. *p , 0.05, **p , 0.01 (compared with Reg-DCsCA1–treated mice). (D) Foxp3 mRNA ex- pression in the MLN were quantified by real-time RT-PCR. There were five mice per group. (E) Cytokine and transcription factor mRNA expression in the colon and MLN were quantified by real-time RT-PCR. There were five mice per group. (F) Secreted cytokine concentrations from the colon and MLN cells (1 3 106) were measured. There were five mice per group. Data shown are representative of two independent experiments. *p , 0.05, **p , 0.01.

specific Tregs. Consistent with this, Reg-DCsCBA and Reg- reduction of CA I expression was also observed in the mucosa of DCsCA1, but not Reg-DCsKLH and Reg-DCsCBA-CA1, prevented patients with mild colitis whose colonic epithelium was not se- colitis by induction of CA I-specific Treg in vivo in this study verely damaged and in areas with apparently normal crypts and (Supplemental Fig. 2B, 2C). These results indicate that Ag pre- surface epithelium (29, 30). In our experiment, CA I reduction sentation is important for the induction of Treg in vivo (24), and was observed in areas of residual goblet cells and enterocytes (Fig. CA-I is a main target Ag in this colitis model. 5B), indicating that the loss of goblet cells and enterocytes is not We used 2D-DIGE and MALDI-TOF mass spectrometry to likely to be responsible for the reduction of CA I expression. reveal that CA I was a major protein in fecal exacts (Fig. 5A). In Accumulating evidence from multiple, distinct model systems our experiment, CD4+CD252 T cells transferred colitis by causing indicates that an important property of tolerogenic or regulatory a significant reduction of CA I expression in the colon, but DCs is the capacity to induce Tregs, such as Foxp3+CD4+CD25+ treatment with Reg-DCsCBA preserved CA I expression (Fig. 5B). Tregs and/or IL-10–producing type 1 Tregs (Tr1 cells) (7, 31–33). Moreover, Reg-DCsCA1 conferred Ag-specific protection from this Although it has been reported that tolerogenic DCs and APCs experimental colitis in mice. These findings indicate that CA I was pulsed with CBA protect against experimental colitis by generating a specific Ag in this colitis model. Cong et al. (25, 26) reported Tr1 cells (12, 34), there is little data on the involvement of DC that proteosome inhibitors or curcumin affected the function of subsets in the differentiation of Foxp3+CD4+CD25+ T cells in APC. However, the generation of Foxp3+ Tregs, cytokine pro- murine experimental colitis. We showed that Reg-DCs efficiently duction, and NF-kB activation of Reg-DCs did not change com- induced CD4+CD252 T cells to differentiate into Foxp3+CD4+ pared with those pulsed with CA I in vitro in this study (Sup- CD25+ T cells in vitro (Fig. 1E, 1F). In addition, both Foxp3+CD4+ plemental Fig. 2D, 2E). CD25+ T cells and IL-10–producing CD4+CD25+ T cells were CA I is expressed on the surface of enterocytes of the colon, and significantly increased in the MLN of Reg-DCCBA–treated mice reduction of CA I expression was reported in colonic mucosa of compared with PBS-treated or Reg-DCsKLH–treated mice (Fig. 4). active ulcerative colitis and experimental colitis (27, 28). However, Although very little is known about the role of Foxp3+ Tregs and The Journal of Immunology 2171

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