Aryl hydrocarbon receptor signaling mediates expression of indoleamine 2,3-dioxygenase

Abstract

Aryl hydrocarbon receptor (AhR) activation by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) leads to immune suppression associated with the induction of regulatory T cells (T(reg)) expressing the transcription factor Foxp3. The immunological mechanisms of suppression are not well understood however dendritic cells (DC) are considered a key target for AhR-mediated immune suppression. Here we show that activation of AhR by TCDD induces DC indoleamine 2,3-dioxygenase 1 (IDO1) and indoleamine 2,3-dioxygenase-like protein (IDO2). Induction of IDO1 and IDO2 was also found in lung and spleen associated with an increase of the T(reg) marker Foxp3 in spleen of TCDD-treated C57BL/6 mice, which is suppressed by inhibition of IDO. These data indicate that AhR-activation is an important signaling pathway for IDO expression and suggest a critical role of IDO in the mechanism leading to the generation of T(reg) that mediates the immune suppression through activation of AhR.

Figure 1. AhR activation by TCDD increases DC differentiation

(A) Prior to staining U937 cells were treated 3 days with GM-CSF and IL-4 and supplemented with TCDD (heavy lines), or DMSO (thin lines). After treatment cells were labeled with anti-CD86 or anti HLA-DR followed by streptavidin APC. Dead cells were excluded by propidium iodide. (B) Phase contrast micrograph of unlabeled U937 derived DCs. Cells were cultured with GM-CSF and IL-4 for 3 days (1). To activate AhR cells were treated with 10 nM TCDD during DC differentiation (2). X 400.

Figure 2. Induction of IDO1 and IDO2 by TCDD in DC is AhR-dependent

(A) IDO1 and (B) IDO2 expression in monocyte-derived U937 DC after treatment with 100 U/ml INFγ, 100 nM FICZ, or 10 nM TCDD in presence or absence of the AhR antagonist MNF for 3 days. (C) INFγ and TCDD induce IDO enzyme activity in U937-derived DC. Using a colorimetric assay, we measured IDO activity in FICZ-, TCDD- and INFγ- treated DCs. Without INFγ or TCDD, IDO activity was not detectable (n.d.). The same test conditions were performed in presence of the IDO inhibitor 1-MT.

Figure 2. Induction of IDO1 and IDO2 by TCDD in DC is AhR-dependent

(A) IDO1 and (B) IDO2 expression in monocyte-derived U937 DC after treatment with 100 U/ml INFγ, 100 nM FICZ, or 10 nM TCDD in presence or absence of the AhR antagonist MNF for 3 days. (C) INFγ and TCDD induce IDO enzyme activity in U937-derived DC. Using a colorimetric assay, we measured IDO activity in FICZ-, TCDD- and INFγ- treated DCs. Without INFγ or TCDD, IDO activity was not detectable (n.d.). The same test conditions were performed in presence of the IDO inhibitor 1-MT.

Figure 3. Induction of IDO1 and IDO2 in lung and spleen of C57BL/6 mice in response to TCDD but not FICZ

(A) IDO1 expression in female wild type C57BL/6 mice. Mice were injected i.p. with a single dose of 15 µg/kg TCDD or 50 µg/kg FICZ. Control animals received the solvent vehicle. After 10 days total RNA from various tissues was subjected to real time PCR. The values are given as relative units. (B) Induction of IDO enzyme activity by AhR activation in spleen of TCDD-treated BL6 wt mice. BL/6 wt mice were treated with a single dose of 15 µg/kg TCDD or 50 µg/kg FICZ. In addition, TCDD- and FICZ-treated mice received the IDO inhibitor 1-MT. * p ≤ 0.01 vs group control. (C) Induction of IDO1 protein in spleen by TCDD. Wild type C57BL/6 animals were treated with a single dose of 15 µg/kg TCDD or 50 µg/kg FICZ for 10 days. Spleen was removed for Western blot analysis of IDO1 and β-actin.

Figure 3. Induction of IDO1 and IDO2 in lung and spleen of C57BL/6 mice in response to TCDD but not FICZ

(A) IDO1 expression in female wild type C57BL/6 mice. Mice were injected i.p. with a single dose of 15 µg/kg TCDD or 50 µg/kg FICZ. Control animals received the solvent vehicle. After 10 days total RNA from various tissues was subjected to real time PCR. The values are given as relative units. (B) Induction of IDO enzyme activity by AhR activation in spleen of TCDD-treated BL6 wt mice. BL/6 wt mice were treated with a single dose of 15 µg/kg TCDD or 50 µg/kg FICZ. In addition, TCDD- and FICZ-treated mice received the IDO inhibitor 1-MT. * p ≤ 0.01 vs group control. (C) Induction of IDO1 protein in spleen by TCDD. Wild type C57BL/6 animals were treated with a single dose of 15 µg/kg TCDD or 50 µg/kg FICZ for 10 days. Spleen was removed for Western blot analysis of IDO1 and β-actin.

Figure 4. The induction of the T_reg cell marker Foxp3 in spleen by TCDD is suppressed through inhibition of IDO

C57BL/6 wt mice were injected with a single dose of 15 µg/kg TCDD or 50 µg/kg FICZ. In addition, TCDD- and FICZ-treated mice received the IDO inhibitor 1-MT. * p ≤ 0.01 vs group control.