Immune Netw.  2015 Dec;15(6):278-290. 10.4110/in.2015.15.6.278.

3,3'-Diindolylmethane Inhibits Flt3L/GM-CSF-induced-bone Marrow-derived CD103+ Dendritic Cell Differentiation Regulating Phosphorylation of STAT3 and STAT5

• Park JH
• Choi AJ
• Kim SJ
• Jeong SY

• KMID: 2132702
• DOI: http://doi.org/10.4110/in.2015.15.6.278

Abstract

The intestinal immune system maintains oral tolerance to harmless antigens or nutrients. One mechanism of oral tolerance is mediated by regulatory T cell (Treg)s, of which differentiation is regulated by a subset of dendritic cell (DC)s, primarily CD103+ DCs. The aryl hydrocarbon receptor (AhR), a ligand-activated transcription factor, plays an important role in regulating immunity. The intestines are exposed to various AhR ligands, including endogenous metabolites and phytochemicals. It was previously reported that AhR activation induced tolerogenic DCs in mice or in cultures of bone marrow-derived DCs. However, given the variety of tolerogenic DCs, which type of tolerogenic DCs is regulated by AhR remains unknown. In this study, we found that AhR ligand 3,3'-diindolylmethane (DIM) inhibited the development of CD103+ DCs from mouse bone marrow cells stimulated with Flt3L and GM-CSF. DIM interfered with phosphorylation of STAT3 and STAT5 inhibiting the expression of genes, including Id2, E2-2, IDO-1, and Aldh1a2, which are associated with DC differentiation and functions. Finally, DIM suppressed the ability of CD103+ DCs to induce Foxp3+ Tregs.

Keyword

AhR; Oral tolerance; 3,3'-diindolylmethane; CD103+ DC; Foxp3+ Tregs; STAT5

MeSH Terms

Animals
Bone Marrow Cells
Dendritic Cells*
Granulocyte-Macrophage Colony-Stimulating Factor
Immune System
Intestines
Ligands
Mice
Phosphorylation*
Phytochemicals
Receptors, Aryl Hydrocarbon
Transcription Factors
Granulocyte-Macrophage Colony-Stimulating Factor
Ligands
Receptors, Aryl Hydrocarbon
Transcription Factors

Figure

• 

  Figure 1 In vitro differentiation of CD11c+CD103+ DCs from bone marrow cells. BM cells were induced to differentiate into CD11c+CD103+ DCs with Flt3L for the first 7 days and with Flt3L plus GM-CSF for the last 2 days. The cells were harvested, stained with fluorochrome conjugated-anti-CD11c and -anti-CD103 Abs, and analyzed by flow cytometry.

• 

  Figure 2 AhR is functionally expressed in Flt3L/GM-CSF-stimulated bone marrow cells and CD11c+CD103+ DCs. BM cells were induced to differentiate into CD11c+CD103+ DCs with Flt3L for the first 7 days and with Flt3L plus GM-CSF for the last 2 days. Then, the cells (FGBMCs) or CD11c+CD103+ sorted from FGBMCs were assayed for mRNA expression by PCR: AhR (a) and Cyp1A1 (c). The purity of the CD11c+CD103+ population was assessed post sorting (b). DIM (1 µM) was added to the culture 1 h before the addition of Flt3L/GM-CSF.

• 

  Figure 3 DIM and TCDD inhibit the development of CD11c+CD103+ DCs. BM cells were induced to differentiate into CD11c+CD103+ DCs with Flt3L for the first 7 days and with Flt3L plus GM-CSF for the last 2 days. The cells were harvested, stained with fluorochrome conjugated-anti-CD11c and -anti-CD103 Abs, and analyzed by flow cytometry. AhR ligands or DMSO (0.1% v/v) were added to the culture 1 h before the addition of Flt3L/GM-CSF. The results are representative of three separate experiments that showed similar results. Data were analyzed by the paired Student's t-test.

• 

  Figure 4 DIM regulates expression of genes associated with the development and functions of CD11c+CD103+ DCs. BM cells were induced to differentiate into CD11c+CD103+ DCs with Flt3L for the first 7 days and with Flt3L plus GM-CSF for the last 2 days. Next, the cells (FGBMCs) (a, c) or CD11c+CD103+ (b, d) sorted from FGBMCs were assayed for mRNA expression by PCR (a, b) or real-time PCR (c, d). DIM (1 µM) or DMSO (0.1% v/v) was added to the culture 1 h before the addition of Flt3L/GM-CSF. The results are representative of three separate experiments that showed similar results.

• 

  Figure 5 DIM inhibits phosphorylation of STAT3 and STAT5 associated with the development of CD11c+CD103+ DCs. BM cells were induced to differentiate into CD11c+CD103+ DCs with Flt3L for 7 days and then with Flt3L/GM-CSF for 1 h. Next, the cells (FGBMCs) were harvested for protein analysis by western blotting (a). For CD11c+ CD103+ DCs, BM cells were cultured with Flt3L for 7 days, harvested, and sorted for the expression of CD11c/CD103. Then, the CD11c+CD103+ DCs were cultured with Flt3L/GM-CSF for 1 h and harvested for protein analysis (b). DIM (1 µM) or DMSO (0.1% v/v) was added to the culture 1 h before the addition of Flt3L/GM-CSF. The results are representative of three separate experiments that showed similar results.

• 

  Figure 6 DIM inhibits the differentiation of Foxp3+ Tregs induced by tolerogenic DCs. Naïve CD4+CD62+ helper T cells (2×105) were cultured with 4×104 Flt3/GM-CSF-stimulated bone marrow cultures in 1 mL of culture medium (RPMI 1640 with 10 mM HEPES, 2 mM L-glutamine, 10% FBS, and 50 µM β-mercaptoethanol) in the presence of soluble anti-CD3 Ab (1 µg/mL), human TGF-β (3 ng/mL, and murine IL-2 (5 ng/mL) for 5 days. Then cells were harvested, stained with fluorochrome conjugated-anti-CD4 and -anti-Foxp3 Abs, and analyzed by flow cytometry. DIM (1 µM) or DMSO (0.1% v/v) was added to the Flt3/GM-CSF-stimulated bone marrow cultures 1 h before the addition of Flt3L/GM-CSF. The results are representative of three separate experiments that showed similar results. Data were analyzed by the paired Student's t-test.

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