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.