Inflammatory bowel disease (IBD), which is characterized by a dysregulated intestinal immune response, is postulated to be controlled 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 therapeutic 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+CD25− 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 RORγt 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-β1 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+CD25− T cell transfer in an Ag-specific manner by inducing the differentiation of regulatory T cells.
Human inflammatory bowel diseases (IBDs), including Crohn’s disease and ulcerative colitis, are characterized by inflammation in the large and/or small intestine. The exact cause and subsequent development of IBD is not established, but uncontrolled innate and adaptive immunity against normal intestinal constituents, including intestinal epithelial cells, commensal bacteria, various microbial products, and/or foodstuffs, are known to contribute to dysregulation of the immune system and subsequent development of disease (1–3). None of the current treatments have proved curative, emphasizing the need for research in the development of new and better therapeutics.
Microenvironmental immunoregulation is constantly fine-tuned to maintain local homeostasis. This tuning can be specific to the site involved (such as the gut environment) or induced by chronic exposure to microbes. Dendritic cells (DCs) play a critical role in orchestrating this tuning (4). DCs are the most potent and efficient dedicated APCs, and they are critical for inducing primary immune 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 central lymphoid organs and in the periphery (4). Although the tolerogenic mechanisms are not entirely understood, there is also new evidence that DCs mediate the induction of peripheral T cell tolerance by stimulating the differentiation of CD4+CD25− T cells to CD4+CD25+Foxp3+ regulatory T cells (5).
The selective enhancement of the tolerogenicity of DCs has been achieved using immature DCs, created by pharmacological inhibition of DC maturation, or using genetically engineered DCs expressing immunosuppressive molecules (6). In addition, studies using murine models have demonstrated that several types of tolerogenic or regulatory DCs (Reg-DCs) can induce an Ag-specific amelioration of pathology in a wide range of contexts, including autoimmunity, allergy, and graft rejection (7–9). Some studies found that transfer of CD4+ T cells or Th17 cells reactive to fecal extracts called cecal bacterial Ag (CBA) or enteric bacterial Ag induced severe colitis in SCID mice (10, 11). In contrast, normal lamina propria CD4+ T cells cocultured with APCs in the presence of CBA led to the generation of regulatory T cells (Tregs) that could ameliorate experimental colitis (12). These findings indicate that fecal extracts may harbor a colitogenic Ag in IBD.
In the current study, we investigated the mechanisms mediating the therapeutic effect of CBA-pulsed Reg-DCs in experimental murine colitis. We analyzed CBA by two-dimensional difference gel electrophoresis (2D-DIGE) and MALDI-TOF mass spectrometry and found that carbonic anhydrase I (CA I) was a main protein component of CBA. We then assessed whether regulatory dendritic cells pulsed with carbonic anhydrase I (Reg-DCsCA1) induced Ag-specific protection from colitis in a murine model of the disease.
Materials and Methods
CB-17 SCID and BALB/c (H-2d) female mice bred under specific pathogen-free conditions were purchased from CLEA Japan (Tokyo, Japan). All mice were between 8 and 12 wk of age and were maintained at the animal center of Ehime University Graduate School of Medicine (Ehime, Japan). All animals received adequate care according to good laboratory practice guidelines. The Committee of Animal Experimentation, Ehime University Graduate School of Medicine, approved the study.
Preparation of CBA
CBA was prepared as previously described with some modifications (10). Briefly, BALB/c mice were euthanized, and their ceca were removed. Ceca (n = 5) were opened and placed in 10 ml PBS with 1.0-mm silica spheres (Lysing Matrix C; MP Biomedicals, Solon, OH). After vortexing this mixture for 5 min, the silica spheres and residual cecal tissue were removed by centrifugation at 5000 × g for 5 min at 4°C. Subsequently, the supernatant was centrifuged at 18,000 × g for 30 min at 4°C. The lysates were sterilized by passage through an 0.2-μm pore-size syringe filter. The protein concentrations in lysates were measured using the DC protein assay kit (Bio-Rad, Hercules, CA).
The preparation of DCs has been described previously (9, 13). Mature DCs were prepared by culturing bone marrow cells (2 × 106) obtained from BALB/c mice with murine GM-CSF (20 ng/ml; Wako Pure Chemical, Osaka, Japan) for 8 d; subsequently, these cells were stimulated with ultrapure LPS (1 μg/ml; InvivoGen, San Diego, CA) for 24 h. Reg-DCs were generated from bone marrow cells (2 × 106) obtained from BALB/c mice and cultured with murine GM-CSF (20 ng/ml), murine IL-10 (20 ng/ml; Wako Pure Chemical), and human TGF-β1 (20 ng/ml; Wako Pure Chemical) for 8 d, followed by stimulation with ultrapure LPS (1 μg/ml) for 24 h. Subsequently, the Reg-DCs were stained with FITC-conjugated anti-CD40 (3/23), anti-CD80 (16-10AI), and anti-CD86 (GL1) mAbs (BD Biosciences, San Diego, CA) for 30 min at 4°C. After the cells were magnetically labeled with anti-FITC MicroBeads (Miltenyi Biotec, Bergisch Gladbach, Germany), Reg-DCs were purified by depletion of CD40+CD80+CD86+ cells using the AutoMACS (Miltenyi Biotec).
In some experiments, mature DCs or Reg-DCs were pulsed with CA I (6 μg/ml), CBA (50 μg/ml), or keyhole limpet hemocyanin (KLH; 50 μg/ml; Thermo Scientific, Rockford, IL) for 24 h.
Measurements of cytokine levels in vitro
DCs (2 × 105) were cultured in complete RPMI 1640 medium (10% FBS, 20 mM HEPES, 2-mercaptoethanol, penicillin, and streptomycin; Nipro, Osaka, Japan) with polyinosinic-polycytidylic acid (poly-IC; 1 μg/ml; Sigma Chemical, St. Louis, MO), ultrapure LPS (1 μg/ml), R848 (1 μM; InvivoGen), or phosphorothiolated CpG oligonucleotide (CpG-ODN; 1 μg/ml; InvivoGen) for 24 h, and the culture supernatants were assayed for IL-10 and IL-6 using cytometric bead array kits (BD Biosciences) and TGF-β using ELISA kits (R&D Systems, Minneapolis, MN). Mesenteric lymph node (MLN) cells (1 × 106) were cultured for 72 h. For the last 6 h of the culture period, 25 ng/ml PMA (Sigma Chemical) and 1 μg/ml ionomycin (Sigma Chemical) were added; at the end of the culture period, the culture supernatants were assayed for cytokines using cytometric bead array kits (BD Biosciences) and IL-17 using ELISA kits (R&D Systems).
Measurement of nuclear NF-κB p65
DCs (1 × 106) were treated with 1 μg/ml CpG-ODN for 60 min. The nuclear extracts were obtained by using a Nuclear Extract Kit (Active Motif, Tokyo, Japan) and nuclear NF-κB p65 was measured using TransAM NF-κB p65 Kit (Active Motif).
Methods of scanning electron microscopy
DCs were conductive stained with 1% buffered osmium tetroxide and 1% tannic acid. They were dehydrated and coated with a thin layer (3 nm) of osmium coater. The suture was observed with a field-emission scanning electron microscope (Hitachi S-4800, Tokyo, Japan) at 2 kV acceleration voltage.
Induction of Foxp3+CD4+CD25+ T cells differentiation from CD4+CD25− T cells in vitro
Mature DCs, Reg-DCs, or Reg-DCsCA1 (5 × 105) were cultured in 35-mm culture dishes (Corning, Horseheads, NY) for 7 d with CD4+CD25− T cells (5 × 106), which were isolated from the spleens of BALB/c mice using the CD4+CD25+ Regulatory T Cell Isolation Kit and the AutoMACS (Miltenyi Biotec).
Flow cytometric analysis and intracellular cytokine synthesis analysis
Unless otherwise noted, materials were purchased from BD Biosciences. After blocking the FcR with purified rat anti-mouse CD16/CD32 (2.4G2), the DCs were stained with FITC-conjugated anti-H-2Kd (AMS-32.1), anti-CD40, anti-CD80, anti-CD86, anti-F4/80 (BM8), PE-conjugated anti–I-A/I-E (2G9), CD11c (HL3), CD11b (M1/70), CD3 (17A2) mAbs and allophycocyanin-conjugated anti-B220 (RA3-6B2) mAbs. Isotype-matched Abs were used as controls. The frequencies of Foxp3+CD4+CD25+ Tregs were determined by the PE Anti-Mouse/Rat Foxp3 Staining Set (eBioscience, San Diego, CA), PE-conjugated anti-Foxp3 mAb (FJK-16s; eBioscience), allophycocyanin-conjugated anti-CD25 mAb (PC61), and PerCP-conjugated anti-CD4 mAb (RM4-5) after MLN cells were cultured for 72 h. To determine the frequencies of IL-10–producing CD4+CD25+ T cells, the MLN cells were cultured for 24 h. These cells were stimulated with PMA/ionomycin for the last 5 h, and GolgiStop was added for the last 3 h of the incubation period. The cells were stained with PerCP-conjugated anti-CD4 mAb and allophycocyanin-conjugated anti-CD25 mAb, followed by fixation and permeabilization with a FIX & PERM Kit (Caltag Laboratories, Burlingame, CA). The cells were stained with PE-conjugated rat anti-mouse IL-10 mAb (JES5-16E3) for 20 min at room temperature. Isotype-matched Abs were used as controls. Fluorescence staining was analyzed with FACS using FlowJo software version 7.5 (Tree Star, Ashland, OR).
Induction of colitis and treatment of mice with DCs
Colitis was induced according to methods described in a previous report with some modifications (14). Briefly, CD4+CD25− T cells (3 × 105 /mouse) obtained from BALB/c mice were suspended in 0.2 ml PBS and i.p. injected into SCID mice. SCID controls received 0.2 ml PBS alone. The day of this transfer was designated as day 0. Subsequently on day 0, DCs (1 × 106 cells/mouse) generated from BALB/c mice were injected i.p. The body weight of each mouse was measured weekly.
Histological assessment of colitis
Colons were removed from euthanized mice 4 wk after cell transfer. The transverse colons were removed and fixed with 10% neutral buffered formalin and then embedded in paraffin. Thin tissue sections were stained with H&E stain or periodic acid–Schiff stain. The grade of inflammation in tissue sections was evaluated as described previously (14). Histology was scored as follows: 1) severity of inflammation: 0, none; 1, mild lymphoid infiltration; 2, marked lymphoid infiltration or focal degeneration of crypts; 3, severe inflammation or multifocal crypt degeneration and/or erosions; 2) extent of inflammation: 0, none; 1, mucosal; 2, submucosal; 3, transmural; 3) amount of mucus: 0, normal; 1, slight decrease of mucus; 2, moderate decrease or focal absence of mucus; 3, severe depletion of mucus; 4, total absence of mucus; and 4) degree of cell proliferation: 0, none; 1, mild increase in cell numbers and crypt length; 2, moderate increase or focally marked increase; 3, marked increase in entire section. The cumulative histological score was calculated as the sum of the four individual parameters.
The frozen sections of colons (5 μm thick) were fixed with acetone. Endogenous peroxidase activity was inactivated by methanol containing 1% hydrogen peroxidase for 20 min. The sections, inactivated with Endogenous Avidin/Biotin Blocking Kit (Nichirei, Tokyo, Japan), were pretreated with undiluted rabbit serum (Nichirei) and then incubated with 1:50 diluted biotinylated goat anti-human CA I (Rockland, Philadelphia, PA) at 4°C overnight. The tissue sections were treated with HRP-conjugated streptavidin (Nichirei) and incubated with Simple Stain DAB Solution (Nichirei). Finally, the sections were counterstained with hematoxylin, dehydrated, and mounted.
Colon culture ex vivo
Sections of 1 cm of the middle parts of the colon were excised, the feces were removed, and the sections were then washed three times with sterile PBS and halved longitudinally. The colon sections were then placed into culture in complete RPMI 1640 medium and cultured for 3 d before supernatants were harvested. For viability assay, the lamina propria cells were isolated from colonic specimens using a modification of a described technique (15). The viability of the lamina propria cells was ∼90% of total cells and was assessed with the trypan blue exclusion method (Sigma Chemical).
RT-PCR and quantitative real-time PCR
A tissue sample from the middle part of the colon and the MLN were homogenized with the TissueLyser (Qiagen, Tokyo, Japan). Total RNA was isolated with the RNAeasy Plus Mini Kit (Qiagen). cDNA was generated with a high-capacity cDNA reverse transcription kit (Applied Biosystems, Foster City, CA). PCR analysis was performed using a KOD-Plus-Ver.2 polymerase kit (Toyobo, Osaka, Japan) with a pair of primers specific for CD200 receptor 3 (CD200R3) and GAPDH. Quantitative RT-PCR reactions of IL-10, IL-6, IL-17A, TGF-β1, Foxp3, and RORγt proceeded using a LightCycler real-time PCR system (Roche, Basel, Switzerland) with LightCycler Fast Start DNA Master SYBRF Green I (Roche). The sequences of the primers used in these analyses are shown in Supplemental Table I. Quantitative RT-PCR reactions of aldehyde dehydrogenase family 1a2 (ALDH1a2) were performed using TaqMan Gene Expression Assays (Applied Biosystems). ALDH1a2 primers were purchased from Applied Biosystems (Assay ID: Mm00501312-m1).
Two-dimensional gel electrophoresis and imaging
Unless otherwise noted, materials were purchased from GE Healthcare Bio-Sciences (Piscataway, NJ). The CBA sample treated with a commercial 2-D 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 Mix. The sample was rehydrated with Immobiline DryStrip reswelling solution, pH 3–10, 24 cm at 20°C for 10 h. Isoelectric focusing was performed using the IPGphor II isoelectric focusing system for a total of 45 kVh at 20°C. The IPG gel was incubated in equilibration buffer (50 mM 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% iodoacetamide in fresh equilibration buffer for an additional 15 min. The strip was immediately applied to a 12.5% SDS-PAGE on an Ettan DALTsix electrophoresis system at 2 W for 16 h at 30°C.
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 Platinum Software Informer system. The spots were quantified on the basis of relative volume (the spot volume divided by the total volume over the whole set of gel spots).
MALDI-TOF mass spectrometry
Protein spots were excised from the gel, washed, and digested in-gel with porcine modified trypsin protease (Promega, Madison, WI). Tryptic peptides were extracted by sonication. HPLC, MALDI sample preparation, and spotting on a μFOCUS MALDI plate (Shimadzu, Kyoto, Japan) were carried out automatically in a nano LC-AccuSpot apparatus (Shimadzu). Tandem time-of-flight mass spectrometry was performed on an Axima-TOF2 mass spectrometer (Shimadzu). Proteins were identified using the MASCOT MS/MS ion search engine (Matrix Science, London, U.K.) and the National Center for Biotechnology Information protein database.
Preparation of mouse CA I
Mouse CA I was prepared using a cell-free protein synthesis system and wheat germ rRNA for which there is no risk of LPS contamination (16). The expression plasmid (pEU-mCA1-His) was constructed and amplified in the mouse CA I gene with the primers using the KOD-Plus-Ver.2 polymerase kit (Toyobo).
The CA I protein was automatically synthesized by the Robotic Protein Synthesizer Protemist DT (CellFree Sciences, Matsuyama, Japan) according to the manufacturer’s instruction manual as previously reported (17).
Depletion of CA I from CBAs
CA I was depleted using Immunoprecipitation Kit-Dynabeads Protein G and Carbonic Anhydrase I Ab (Gene Tex, Irvine, CA). Depletion of CA I from CBAs was confirmed by Western blotting. Protein (5 μg) was applied to lanes in 4–12% Bis–Tris Gels (Invitrogen). Resolved products were then blotted onto Immunobilon-P membranes (Millipore, Bedford, MA) and probed with CA I Ab. Proteins were detected using the ECL Plus Kit (GE Healthcare Bio-Sciences).
T cell proliferation assay
CD4+CD25− T cells or CD4+ T cells at 1 × 105 cells/well were incubated in the presence of 1 × 105 Ag-pulsed irradiated spleen cells used as APC. After 4 d, [3H]thymidine (1.0 μCi/ml; Amersham Biosciences, Buckinghamshire, U.K.) was added to the cultures for the last 18 h and harvested automatically by a multiple cell harvester (Labo Mash; Futaba Medical, Osaka, Japan) onto filter paper (Labo Mash 101–10; Futaba Medical). The levels of incorporation of [3H]thymidine were determined in a liquid scintillation counter (Beckman LS 6500; Beckman Instruments, Fullerton, CA).
Data are expressed as mean values ± SD of all individual experiments. Statistical differences between two groups were determined by Student t test. One-way ANOVA was used to compare multiple groups. If the ANOVA was significant, the Tukey–Kramer honestly significant difference test was applied for multiple comparisons. A value of p < 0.05 was considered statistically significant. Statistical calculations were performed with JMP software (SAS International, Cary, NC).
Reg-DCs induce the differentiation of CD4+CD25+Foxp3+ T cells from CD4+CD25−Foxp3− T cells in vitro
Mature bone marrow-derived DCs expressed high levels of MHC molecules (H-2kd and I-A/I-E), CD11c, CD11b, and costimulatory molecules (CD40, CD80, and CD86) (Fig. 1A). By contrast, Reg-DCs expressed moderate levels of MHC molecules and CD11b and did not express CD11c or costimulatory molecules (Fig. 1A). Neither mature DCs nor Reg-DCs expressed macrophage marker (F4/80), T cell marker (CD3), or B cell marker (B220) (Fig. 1A). CD200R3 expression has been reported to be crucial for Reg-DCs to suppress Ag-specific CD4+ T cell responses (5). RT-PCR demonstrated that Reg-DCs, but not mature DCs, expressed high levels of CD200R3—splice variants A (860 bp) and D (642 bp) (18) (Fig. 1B). Field-emission scanning electron microscopy confirmed that Reg-DCs had dendrites like mature DCs (Fig. 1C). Reg-DCs stimulated with various TLR ligands produced significantly lower levels of IL-6 and higher levels of IL-10 than did mature DCs (Fig. 1D). We found that Reg-DCs, but not mature DCs, induced the differentiation of CD4+CD25− T cells to Foxp3+CD4+CD25+ Tregs (Fig. 1E, 1F).
Treatment with Reg-DCs pulsed with CBA protects mice against experimental colitis
CB-17 SCID mice were divided into four groups, and CD4+CD25− colitogenic effector T cells were transferred into all mice in each group. At the time of T cell transfer, the groups of mice were treated with PBS or with Reg-DCs (1 × 106 /mouse) that had been pulsed with either CBA (Reg-DCsCBA) or KLH (Reg-DCsKLH) or left untreated with Ag. Four weeks after T cell transfer, body weights were significantly higher in mice treated with Reg-DCsCBA than in mice treated with PBS (p < 0.01) (Fig. 2A). Treatment of mature DCs pulsed with CBA was insufficient for protection against colitis (Supplemental Fig. 1A). Macroscopic examinations at week 4 after transfer revealed that the colon length was significantly longer in mice treated with Reg-DCs or Reg-DCsCBA than in PBS-treated mice (p < 0.01) (Fig. 2B, 2C). Upon histological examination, colitis was characterized by severe epithelial hyperproliferation, mucus depletion, massive infiltration of inflammatory cells, crypt degeneration, reduced numbers of goblet cells, and erosions. CB-17 SCID mice injected with CD4+CD25− T cells and treated with PBS had severe colitis. The mice injected with unpulsed Reg-DCs or with Reg-DCsKLH had colonic changes similar to those seen in mice treated with PBS. In contrast, treatment with Reg-DCsCBA improved these histological signs; moreover, there was a significant reduction of inflammatory cell infiltration, and goblet cells and mucus were preserved (Fig. 2D).
The cumulative histological score (14) was significantly lower in mice treated with Reg-DCsCBA than in PBS-treated mice (p < 0.01) (Fig. 2E). Unpulsed Reg-DC–treated mice showed moderate weight loss and mild shortening of the colon; however, they also had moderate to severe colitis upon histological examination.
Treatment with Reg-DCsCBA reduces inflammatory cytokine responses in the colon
The effect of Reg-DCsCBA treatment on the production of inflammatory cytokines in the colon of mice subjected to CD4+CD25− T cell transfer was evaluated. The colonic expression of IL-6 and IL-17A mRNA was reduced and IL-10 mRNA expression was increased in Reg-DCsCBA–treated mice compared with PBS-treated mice (p < 0.05) (Supplemental Fig. 1B). There was no difference in the expression of ALDH1a2 (which converts retinoids to retinoic acid and induces Tregs) between Reg-DCsCBA–treated mice and PBS-treated mice (Supplemental Fig. 1B) (19, 20). Foxp3 and RORγt, transcription factors that define the Treg and Th17 lineages, respectively, were examined (21). There was no significant difference in the expression of Foxp3 in the colons of Reg-DCsCBA–treated mice and PBS-treated mice (Supplemental Fig. 1B). RORγt mRNA expression in the colon was lower in mice treated with Reg-DCsCBA than in PBS-treated mice (p < 0.05) (Supplemental Fig. 1B). Culture supernatants from colonic explants indicated that the production of IL-17 and TNF-α was lower in mice treated with Reg-DCsCBA than in PBS-treated mice (p < 0.05), but significant differences in IFN-γ, IL-6, TGF-β, and IL-10 levels were not detected between the two groups (Supplemental Fig. 1C).
Administration of Reg-DCsCBA induced Foxp3 expression and reduced RORγt expression in the MLN
To elucidate the mechanism underlying downregulation of inflammatory processes mediated by Reg-DCsCBA, the levels of expression of transcription factors and cytokines in the MLN were assessed (Fig. 3). Foxp3 mRNA expression in the MLN was significantly higher in mice treated with Reg-DCsCBA compared with PBS-treated mice (p < 0.05). In contrast, RORγt mRNA expression in the MLN was lower in mice treated with Reg-DCsCBA than in PBS-treated mice (p < 0.05) (Fig. 3A). In addition, expression of IL-10 and TGF-β1 mRNA in the MLN was significantly higher in mice treated with Reg-DCsCBA than in PBS-treated mice (p < 0.05). Significant differences in the expression of IL-6, IL-17A, and ALDH1a2 in the MLN of mice treated with Reg-DCsCBA versus PBS-treated mice were not detected (Fig. 3B). In addition, Reg-DCsCBA administration resulted in a striking reduction of IL-6, IFN-γ, TNF-α, and MCP-1 production from the MLN cells of colitic mice (p < 0.01) (Fig. 3C).
Reg-DCsCBA induced Foxp3+CD4+CD25+ T cells and IL-10–producing CD4+CD25+ T cells in vivo
To address whether Reg-DCsCBA could induce peripheral differentiation of Foxp3+CD4+CD25+ T cells and IL-10–producing CD4+ T cells from Foxp3−CD4+CD25− T cells in vivo, the numbers of Foxp3+CD4+CD25+ T cells and IL-10–producing CD4+CD25+ T cells were evaluated in the MLN of mice treated with Reg-DCsCBA. Flow cytometric analysis showed that the percentages of Foxp3+CD4+CD25+ Tregs and IL-10–producing 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).
CA I is the major Ag of CBA
Proteomic analysis of CBA using 2D-DIGE imaging (Fig. 5A) showed 14 main spots on the spot map. These spots were excised from the gel for subsequent mass spectrometry. Fourteen spots gave the identifications referred to in the database interrogation shown in Supplemental Table II (the cut spots are indicated with circles in the map in Fig. 5A). Among these proteins, 7.03% (nos. 1, 4, 5, 9, and 10) was CA I, 5.76% (nos. 2, 3, and 7) was serum albumin precursor, and 1.54% (nos. 6 and 14) was selenium binding protein 1. CA I emerged as the principal protein Ag of CBA.
CA I expression in both healthy and inflamed mouse colons was evaluated by immunohistochemical analysis. The inflamed colons of SCID mice transferred with CD4+CD25− T cells showed a significant reduction of CA I expression compared with the colons of healthy SCID control mice or of SCID mice subjected to CD4+CD25− T cells transfer and treatment with Reg-DCsCBA (Fig. 5B).
Reg-DCs pulsed with CA I suppress development of colitis in the SCID transfer model
To assess whether CA I is necessary for protection against colitis, CB-17 SCID mice received CD4+CD25− T cells with Reg-DCsCA1 (Reg-DCsCA1: 1 × 106 cells/mouse) or regulatory dendritic cells pulsed with cecal bacterial Ag depleted of carbonic anhydrase I (Reg-DCsCBA-CA1: 1 × 106 cells /mouse) (Supplemental Fig. 2A). Four weeks after transfer, Reg-DCsCA1–treated mice showed more effective protection against colitis (Fig. 6A–C). Foxp3 mRNA expression in the MLN of Reg-DCsCA1–treated mice was higher than that in the MLN of Reg-DCsCBA-CA1–treated mice (p < 0.05) (Fig. 6D).
Next, we analyzed the underlying mechanism of therapeutic effects by Reg-DCsCA1. The expression of IL-17A mRNA was lower in the colon and MLN of Reg-DCsCA1–treated colitic mice than in those of PBS-treated colitic mice (p < 0.05); however, the expression of IL-10 and TGF-β1 mRNA was higher in the Reg-DCsCA1–treated colitic mice (Fig. 6E). In the MLN, the expression of Foxp3 was significantly higher and that of RORγt was significantly lower in colitic mice treated with Reg-DCsCA1 than in PBS-treated colitic mice (p < 0.05) (Fig. 6E). RORγt mRNA expression in the colon was lower in mice treated with Reg-DCsCA1 than in PBS-treated mice (p < 0.05) (Fig. 6E). Culture supernatants from colonic explants and MLN cells of colitic mice treated with PBS and Reg-DCsCA1 were assayed. IL-10 and TGF-β1 production from the colon did not differ between the two groups. The production of IL-17, TNF-α, and IFN-γ in the colon was significantly lower in colitic mice treated with Reg-DCsCA1 than in PBS-treated colitic mice (p < 0.05) (Fig. 6F). In addition, Reg-DCCA1 administration resulted in a striking reduction in the production of IL-6, IL-17, TNF-α, IFN-γ, and MCP-1 from the MLN cells relative to that from PBS-treated colitic mice (p < 0.01) (Fig. 6F).
Reg-DCs pulsed with CBA induce CA I-specific Treg in the MLN
To assess whether Reg-DCsCBA induced CA1-specific Treg in vivo, CD4+CD25− T cells or CD4+ T cells (1 × 105) in the MLN from colitic mice treated with Reg-DCsCBA were cultured with spleen cells (used as APC) of BALB/c mice, APC pulsed with carbonic anhydrase I (APCCA1), or APC pulsed with keyhole limpet hemocyanin (APCKLH). When CD4+CD25+ cells were removed from the responder CD4+ T cell population, stronger T cell proliferation was observed when APCCA1 was used as stimulators. However, this result was not observed when APCKLH was used as stimulators (Supplemental Fig. 2B). In addition, the proliferative response to APCCA1 was not increased when CD4+CD25+ cells were removed from the MLN of Reg-DCsKLH or PBS-treated mice (Supplemental Fig. 2C), indicating that CD4+CD25+ cells of MLN from colitic mice treated with Reg-DCsCBA contained CA I-specific Tregs.
Finally, we checked whether CA I changed the phenotype of Reg-DCs. There was no difference in the cytokine production, NF-κB p65 activation, aldehyde dehydrogenase family 1 expression, and generation of Foxp3+ Treg between Reg-DCs and Reg-DCsCA1 (Supplemental Fig. 2D, 2E).
The four main findings of the current study are 1) Reg-DCsCBA inhibited the progression of colitis induced by CD4+CD25− T cells; 2) CA I, a major Ag of CBA, had an important role in suppressing the development of colitis; 3) Reg-DCsCA1 induced Foxp3+ Tregs and reduced IL-17 expression in the MLN; and 4) Reg-DCsCBA induced Foxp3+CD4+CD25+ T cells and IL-10–producing CD4+CD25+ T cells in vivo.
Many researchers have shown that DCs have Ag-specific protective effects in murine models of autoimmune diseases (7, 8, 22). In addition, Pedersen et al. (23) found that tolerogenic DCs pulsed with fecal extract suppressed the development of colitis. Our results were consistent with these studies. However, little is known about the underlying mechanism. Our data showed that treatment with Reg-DCs pulsed with fecal extract, including CBA, ameliorated the clinical and histopathologic severity of the wasting disease in a murine model (Fig. 2). As shown in Fig. 2, Reg-DCs alone or Reg-DCsKLH partially (but not significantly) prevented colitis. This effect might involve the production of IL-10 (Fig. 1D), consistent with a previous report by Fujita et al. (13). However, Mengs et al. (8) demonstrated that autoantigen-pulsed, not irrelevant Ag-pulsed, IL-10 produced DCs that prevented experimental autoimmune encephalomyelitis by inducing Ag-specific Tregs. Consistent with this, Reg-DCsCBA and Reg-DCsCA1, but not Reg-DCsKLH and Reg-DCsCBA-CA1, prevented colitis by induction of CA I-specific Treg in vivo in this study (Supplemental Fig. 2B, 2C). These results indicate that Ag presentation is important for the induction of Treg in vivo (24), and CA-I is a main target Ag in this colitis model.
We used 2D-DIGE and MALDI-TOF mass spectrometry to reveal that CA I was a major protein in fecal exacts (Fig. 5A). In our experiment, CD4+CD25− T cells transferred colitis by causing a significant reduction of CA I expression in the colon, but treatment with Reg-DCsCBA preserved CA I expression (Fig. 5B). Moreover, Reg-DCsCA1 conferred Ag-specific protection from this experimental colitis in mice. These findings indicate that CA I was a specific Ag in this colitis model. Cong et al. (25, 26) reported that proteosome inhibitors or curcumin affected the function of APC. However, the generation of Foxp3+ Tregs, cytokine production, and NF-κB activation of Reg-DCs did not change compared with those pulsed with CA I in vitro in this study (Supplemental Fig. 2D, 2E).
CA I is expressed on the surface of enterocytes of the colon, and reduction of CA I expression was reported in colonic mucosa of active ulcerative colitis and experimental colitis (27, 28). However, reduction of CA I expression was also observed in the mucosa of patients with mild colitis whose colonic epithelium was not severely damaged and in areas with apparently normal crypts and surface epithelium (29, 30). In our experiment, CA I reduction was observed in areas of residual goblet cells and enterocytes (Fig. 5B), indicating that the loss of goblet cells and enterocytes is not likely to be responsible for the reduction of CA I expression.
Accumulating evidence from multiple, distinct model systems indicates that an important property of tolerogenic or regulatory DCs is the capacity to induce Tregs, such as Foxp3+CD4+CD25+ Tregs and/or IL-10–producing type 1 Tregs (Tr1 cells) (7, 31–33). Although it has been reported that tolerogenic DCs and APCs pulsed with CBA protect against experimental colitis by generating Tr1 cells (12, 34), there is little data on the involvement of DC subsets in the differentiation of Foxp3+CD4+CD25+ T cells in murine experimental colitis. We showed that Reg-DCs efficiently induced CD4+CD25− T cells to differentiate into Foxp3+CD4+CD25+ T cells in vitro (Fig. 1E, 1F). In addition, both Foxp3+CD4+CD25+ T cells and IL-10–producing CD4+CD25+ T cells were significantly increased in the MLN of Reg-DCCBA–treated mice compared with PBS-treated or Reg-DCsKLH–treated mice (Fig. 4). Although very little is known about the role of Foxp3+ Tregs and Tr1 cells in human IBD, Foxp3+ T cells and Tr1 cells have been reported to decrease the degree of pathology in the murine colitis model (24). Therefore, we suggest that differentiation of Foxp3+CD4+CD25+ T cells in MLN, induced by Reg-DCsCBA, contributed to the repression of colitis. However, the Foxp3 mRNA expression in the colon was similar between PBS-treated mice and mice treated with Reg-DCsCA1 (Fig. 6E). The reasons for this discrepancy are not clear, but it is possible that other factors, such as inflammatory cytokine production and Foxp3 instability (35), were induced in the inflamed colon tissue of PBS-treated mice. We did not obtain much information about the transferred cells in the colon because a sufficient number of CD4+ T cells from the colons of mice treated with Reg-DCCBA or Reg-DCCA1 could not be harvested. Further studies are necessary in this regard.
In the MLN of mice treated with Reg-DCsCBA or Reg-DCsCA1, the levels of IL-10 and TGF-β mRNA expression were higher than those of PBS-treated mice (Fig. 3B). In addition to these cytokines, retinoic acid plays a critical role in the generation of Tregs and mucosal DCs and their function (19, 20). Feng et al. (19) reported that ALDH1a2, which converts retinoids to retinoic acid, has a role in the induction of Tregs. However, in our study, ALDH1a2 expression in the MLN was not different between PBS-treated mice and mice treated with Reg-DCsCBA (Fig. 3B), indicating that retinoic acid has little effect in the induction of Tregs in our model.
Th17 cells are characterized by the production of a distinct profile of effector cytokines, and serum IL-17 levels are increased in human IBD and several mouse models of IBD (36). Reg-DCCA1 treatment was associated with lower expression of RORγt mRNA, the master transcription factor guiding Th17 differentiation (22), and decreased IL-17 protein production in the MLN and colon relative to that in PBS treatment (Fig. 6F). It is well established that TGF-β1 and IL-6 in cooperation induce the differentiation of naive T cells into Th17 cells in mice (36). Lower production of IL-6 by Reg-DC (Figs. 1D, 3C) might have a role in the inhibition of Th17 cells and the limitation of the development of colitis.
In conclusion, the findings reported in this study highlight the efficacy of DC-based immune therapy for experimental colitis. Reg-DCsCA1 were used to control the balance of Foxp3+CD4+CD25+ T cells and Th17 cells in the MLN and ameliorated colitis arising from the transfer of CD4+CD25− T cells in a murine model of colitis. Generation of such Ag-specific Reg-DCs may lead to the development of treatments for human IBD that minimize the use of nonspecific immunosuppressive drugs. However, further studies are needed to assess whether human Reg-DCs can be generated in vitro and whether they can induce Tregs that have the capacity to inhibit the inflammatory response in human IBD.
We thank K. Tanimoto, T. Fujino, and Dr. T. Mashiba for technical assistance, Dr. H. Iwabuki for technical assistance with MALDI-TOF mass spectrometry, and Dr. Cathryn R. Nagler (Biological Sciences Division/Department of Pathology, Committee on Immunology, The University of Chicago) for critically reading the manuscript.
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 Department of Biological Resources, Integrated Center for Science, Ehime University.
The online version of this article contains supplemental material.
Abbreviations used in this article:
aldehyde dehydrogenase family 1a2
APC pulsed with carbonic anhydrase I
APC pulsed with keyhole limpet hemocyanin
- CA I
carbonic anhydrase I
cecal bacterial Ag
CD200 receptor 3
two-dimensional difference gel electrophoresis
inflammatory bowel disease
keyhole limpet hemocyanin
mesenteric lymph node
regulatory dendritic cell
regulatory dendritic cells pulsed with carbonic anhydrase I
regulatory dendritic cells pulsed with cecal bacterial Ag
regulatory dendritic cells pulsed with cecal bacterial Ag depleted of carbonic anhydrase I
regulatory dendritic cells pulsed with keyhole limpet hemocyanin
- Tr1 cell
IL-10–producing type 1 regulatory T cell
regulatory T cell.
The authors have no financial conflicts of interest.