Dietary ligands of the aryl hydrocarbon receptor induce anti-inflammatory and immunoregulatory effects on murine dendritic cells

Abstract

Activation of the aryl hydrocarbon receptor (AhR) in immune cells, such as dendritic cells (DCs), can lead to suppressed immune responses. Although AhR activation is most recognized for mediating the effects of its prototypical ligand 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), many compounds existing in dietary sources can also bind the AhR. Because the immunomodulatory effects of indole-3-carbinol (I3C) and indirubin-3'-oxime (IO) have yet to be investigated in DCs, we evaluated the potential immunomodulatory effects of these compounds on murine DCs. We hypothesized that I3C and IO suppress immune and inflammatory responses in DCs. We found that both I3C and IO decreased the expression of CD11c, CD40, and CD54 while they increased expression of MHC2 and CD80. Following lipopolysaccharide (LPS)-activation, I3C and IO suppressed the production of pro-inflammatory mediators including tumor necrosis factor-α, interleukin (IL)-1β, IL-6, IL-12, and nitric oxide but increased IL-10 levels. These effects of I3C and IO were partially mediated by the AhR. Additionally, immunoregulatory genes, such as ALDH1A, IDO and TGFB, were upregulated following treatment with I3C or IO. Both I3C and IO decreased basal levels of nuclear factor-kappa B p65, but only I3C suppressed the LPS-induced activity of RelB. Finally, when cultured with naïve T cells, bone marrow-derived dendritic cells treated with the dietary AhR ligands increased the frequency of Foxp3+ Tregs in an antigen-specific manner. Taken together, these results indicate that I3C and IO exhibit immunosuppressive and anti-inflammatory effects on DCs. Because I3C and IO are significantly less toxic than TCDD, these natural products may ultimately become useful therapeutics for the treatment of autoimmune and inflammatory diseases.

FIG. 1.

Concentration-dependent effects of I3C and IO on BMDC proliferation and viability. BMDCs were grown in the presence of the 0.1% DMSO vehicle control or various concentrations of I3C and IO. After 7 days, nonadherent cells were stained with Trypan blue to determine cell numbers and viability. Results are representative of three separate experiments with n = 3. *Indicates significance of p ≤ 0.05.

FIG. 2.

Alterations in BMDC phenotype following AhR ligand treatment. BMDCs were grown in the presence of the DMSO vehicle control, 50μM I3C, or 1μM IO for 7 days and subsequently evaluated for their relative expression (mean fluorescence intensity) of surface markers via flow cytometry (numerical values are listed in the corresponding table below the histograms). Thin gray lines indicate isotype control, dotted black lines indicate DMSO-treated cells, thick black lines indicate I3C-treated cells, and thick gray lines indicate IO-treated cells. Arrows indicate MHC2/MHC2^hi, CD86, CD80, CD54, and CD40 expression on CD11c+ cells. Results are representative of three separate experiments with n = 3. *Indicates significance of p ≤ 0.05.

FIG. 3.

Dietary AhR ligands alter LPS-induced changes in surface phenotype of BMDCs. BMDCs were grown in the presence of the DMSO vehicle control, 50μM I3C, or 1μM IO for 7 days and purified. The immature BMDCs were subsequently treated with 1 μg/ml LPS for 24 h, and the immunophenotype was evaluated by flow cytometry. Results are representative of three separate experiments with n = 3. #Indicates significance of p ≤ 0.05 compared with unstimulated DMSO control, *Indicates significance of p ≤ 0.05 compared with LPS-stimulated DMSO control.

FIG. 4.

Suppression of LPS-induced pro-inflammatory cytokine production by I3C- and IO-treated BMDCs. BMDCs were grown in the presence of the DMSO vehicle control, 50μM I3C, or 1 μm IO for 7 days and purified. The immature BMDCs were subsequently treated with 1 μg/ml LPS for 24 h, and protein levels of cytokines present in the supernatants were measured by ELISA. Results are representative of three separate experiments with n = 3. #Indicates significance of p ≤ 0.05 compared with unstimulated DMSO control, *Indicates significance of p ≤ 0.05 compared with LPS-stimulated DMSO control.

FIG. 5.

I3C and IO differentially alter NF-κB signaling. Purified I3C- and IO-treated BMDCs were stimulated with 1 μg/ml LPS for 45 min, and nuclear protein extracts were subsequently prepared for evaluation of the binding activity of NF-κB p65 (A) and RelB (B). Results are representative of one experiment with n = 3. #Indicates significance of p ≤ 0.05 compared with unstimulated vehicle control and *indicates significance of p ≤ 0.05 compared with vehicle control of similarly activated samples.

FIG. 6.

DCs treated with dietary AhR ligands increase the frequency of CD4+Foxp3+ Tregs and alter cytokine production. AhR ligand-treated BMDCs and naïve CD4+ OTII T cells were prepared as described in the “Materials and Methods.” (A) The percent of CD4+Foxp3+ T cells is shown in the histogram. (B) Cytokine production of IL-2, IL-10, and IFN-γ was also measured from the culture supernatants as described in the “Materials and Methods.” Results are representative of two separate experiments with n = 3. *Indicates significance of p ≤ 0.05.