Indoleamine 2,3-dioxygenase-expressing dendritic cells are involved in the generation of CD4+CD25+ regulatory T cells in Peyer's patches in an orally tolerized, collagen-induced arthritis mouse model

Abstract

Introduction: The present study was devised to understand the role of systemic indoleamine 2,3-dioxygenase (IDO) in the tolerance induction for orally tolerized mice in collagen-induced arthritis (CIA). We examined whether IDO-expressing dendritic cells (DCs) are involved in the generation of CD4+CD25+ regulatory T cells during the induction of oral tolerance in a murine CIA model.

Methods: Type II collagen was fed six times to DBA/1 mice beginning 2 weeks before immunization, and the effect on arthritis was assessed. To examine the IDO expression, the DCs of messenger RNA and protein were analyzed by RT-PCR and Flow cytometry. In addition, a proliferative response assay was also carried out to determine the suppressive effects of DCs through IDO. The ability of DCs expressing IDO to induce CD4+CD25+ T regulatory cells was examined.

Results: CD11c+ DCs in Peyer's patches from orally tolerized mice expressed a higher level of IDO than DCs from nontolerized CIA mice. IDO-expressing CD11c+ DCs were involved in the suppression of type II collagen-specific T-cell proliferation and in the downregulation of proinflammatory T helper 1 cytokine production. The suppressive effect of IDO-expressing CD11c+ DCs was mediated by Foxp3+CD4+CD25+ regulatory T cells.

Conclusion: Our data suggest that tolerogenic CD11c+ DCs are closely linked with the induction of oral tolerance through an IDO-dependent mechanism and that this pathway may provide a new therapeutic modality to treat autoimmune arthritis.

Figure 1

Inhibition of arthritis development in tolerized mice. Mice in the tolerance group were fed 100 μg type II collagen (CII) six times for 2 weeks before immunization. For collagen-induced arthritis (CIA) induction, CII emulsified with complete Freund's adjuvant was injected into the tail of mice in the tolerance group and in the CIA group as a primary immunization. Two weeks later, CII emulsified with incomplete Freund's adjuvant was injected into a hind leg as a booster injection. The mean arthritis index was significantly lower in the tolerance group than in the CIA group throughout the examination period. Values are presented as the mean ± standard deviation of three independent experiments involving 20 tolerized mice and 20 CIA mice per group. *P < 0.05, **P < 0.005.

Figure 2

Oral tolerance induction in indoleamine 2,3-dioxygenase-expressing CD11c⁺ dendritic cells of tolerized mice. The induction of oral tolerance increases the proportion of indoleamine 2,3-dioxygenase (IDO)-expressing CD11c⁺ dendritic cells (DCs) in Peyer's patches of tolerized mice. (a) Flow cytometric analysis of IDO in CD11c⁺ DCs isolated from Peyer's patches. Mononuclear cells obtained from Peyer's patches of tolerized mice and of CIA mice were probed with Fluorescein isothiocyanate-labeled anti-CD11c mAb and were fixed with CytoPerm/CytoFix for 20 minutes. Cells were probed for intracellular IDO using anti-mouse IDO antibody and were analyzed by flow cytometry. The histograms were gated on CD11c⁺ DCs. Dotted histogram lines represent cells stained with isotype-matched control monoclonal antibodies. Results are the mean ± standard deviation of replicate samples from seven independent experiments. SSC. (b) Analysis of IDO transcription in tolerized mice and CIA mice. CD11c⁺ DCs were isolated from Peyer's patch mononuclear cells using the magnetic-activated cell sorting system. The expression of IDO mRNA was analyzed using RT-PCR. β₂-Actin was used as an internal control. Each value is the mean ± standard deviation of replicate determinations in one of four experiments. *P < 0.05. (c) Immunofluorescent confocal microscopic examination of IDO expression by CD11c⁺ DCs. Mononuclear cells obtained from Peyer's patches of tolerized and CIA mice were stained with Fluorescein isothiocyanate-labeled anti-CD11c (green) and anti-IDO (red), fixed, and were examined using confocal microscopy. Isotype-matched control antibody staining was negative (data not shown). Data are representative of three independent experiments.

Figure 3

Indoleamine 2,3-dioxygenase-expressing CD11c⁺ dendritic cells display a phenotype consistent with the immature state. Indoleamine 2,3-dioxygenase (IDO)⁺CD11c⁺ dendritic cells (DCs) were stained for markers of DC maturity and were analyzed using three-color flow cytometry. Mononuclear cells from Peyer's patches were stained for CD11c, IDO, and Major Histocompatibility Complex (MHC) II, or costimulatory molecule markers. All plots were first gated on IDO⁺CD11c⁺ cells. Dotted line shows the isotype-matched controls. Data are the mean ± standard deviation of three experiments. MFI, mean fluorescence index.

Figure 4

Tolerized mice CD11c⁺ dendritic cells induce indoleamine 2,3-dioxygenase-dependent inhibition of type II collagen-specific T-cell proliferation. (a) Type II collagen (CII)-reactive CD4⁺ T cells (1 × 10⁵/well) and irradiated antigen-presenting cells (APCs) (1 × 10⁵/well) isolated from Peyer's patches of collagen-induced arthritis (CIA) mice were cultured with CD11c⁺ dendritic cells (DCs) (1 × 10⁴/well) from tolerized mice or CIA mice in the presence or absence of CII (40 μg/ml) in 96-well, U-bottomed plates for 3 days. Some DCs were pretreated with 1-methyl tryptophan (1-MT) (200 μM) for 2 hours before coculture. Data presented as the mean counts per minute (cpm) of triplicate cultures. Values are the mean ± standard deviation from three independent experiments. *P < 0.05. (b) Cytokine concentrations in the coculture supernatants. Concentrations of IL-17, IL-10, and transforming growth factor beta (TGFβ) in the culture supernatants were measured by ELISA. Values are the mean ± standard deviation from three independent experiments. *P < 0.05, **P < 0.001.

Figure 5

Indoleamine 2,3-dioxygenase-expressing CD11c⁺ dendritic cells essential for antigen-specific CD4⁺CD25⁺ regulatory T-cell induction in tolerized mice. (a) Increased CD4⁺CD25⁺ T-cell proportion through an indoleamine 2,3-dioxygenase (IDO)-dependent mechanism. For regulatory T-cell induction, isolated CD4⁺CD25^- T cells (1 × 10⁵/well) were cultured with CD11c⁺ dendritic cells (DCs) (2 × 10⁴/well) from tolerized mice or collagen-induced arthritis (CIA) mice in the absence or presence of type II collagen (CII) (40 μg/ml) for 3 days. 1-Methyl tryptophan (1-MT) was added to selected cultures. The proportion of CD4⁺CD25⁺ T cells was determined using flow cytometry. Numbers represent the percentage of double-positive cells. (b) Summary of the percentages of CD4⁺CD25⁺ T cells from the coculture experiments in (a). Values are the mean from four independent experiments; individual symbols are the mean in individual animals, and bars show the group means. *P < 0.02. (c) Analysis of Foxp3 expression by converted CD4⁺CD25⁺ T cells. Plots were gated on CD4⁺CD25⁺ DCs. Dotted histogram lines represent cells stained with isotype-matched control monoclonal antibodies. Data represent the mean ± standard deviation and are representative of four independent experiments. (d) Foxp3 mRNA expression in the same conditions as (a). β -Actin was used as an internal control. Results are representative of four independent experiments. (e) Regulatory function of the CII-induced CD4⁺CD25⁺ T cells. CD4⁺CD25⁺ T cells were expanded by exposure to CD11c⁺ DCs from Peyer's patches from tolerized mice in the presence of CII antigen stimulation. Varying numbers of CD4⁺CD25⁺ T cells were cultured for 3 days with CII-reactive CD4⁺ T cells (1 × 10⁵) and irradiated antigen-presenting cells (1 × 10⁵) from mice with CIA in the presence of CII (40 μg/ml). Values are the mean ± standard deviation from three independent experiments. *P < 0.05, **P < 0.001. cpm, counts per minute.