Figure 4.

AhR-dependent increase in IDO2 is associated with CCR6 expression in DC subsets. (a–c) Effect of TCDD on IDO1, IDO2 and CCR6 mRNA expression in BMDC subsets from B6 wt (AhR^+/+) and AhR^−/− mice as determined using real-time qRT-PCR 7 days after treatment with 0.1% DMSO (Ctrl) or 1 nM TCDD. Results are expressed as Relative Expression, which indicates the expression of each gene relative to its expression in control of double-negative cells (DN). (d) Detection of IDO1 and IDO2 protein expressions. Cell lysates were prepared from BMDC subsets IDC and MDC using RIPA buffer. Twenty microgram of the total protein lysate was separated on 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). After transfer to polyvinylidene difluoride (PVDF) membranes, protein loading was monitored using Actin. IDO1 and IDO2 proteins were detected using a rabbit polyclonal antibody (IDO1, sc-25809, Santa Cruz Biotechnology, Santa Cruz, CA, USA) and IDO2 preparation (kindly provided by Dr Richard Metz from NewLink Genetics, Wynnewood, PA, USA) and anti-rabbit IgG-horseradish peroxidase (HRP; Santa Cruz Biotechnology), and visualized by chemiluminescence (ECL; Amersham, Piscataway, NJ, USA). One representative experiment out of three independently performed experiments is shown. (e) Densitometric evaluation of band intensities of the IDO1 and IDO2 protein bands. Results of three independent experiments are shown as mean values±s.d. (f) Determination of IDO enzymatic activity. After 7 days of exposure to TCDD, BMDC subsets were harvested. Cells were washed twice with phosphate-buffered saline (PBS) and frozen at −70 °C. Cell pellets were lyophilized overnight. The powdered cell residues were resuspended in PBS and, after centrifugation, the supernatants were assayed for IDO activity as described.¹⁶ Optic density was measured at 490 nm using a Mithras LB 940 (Berthold Technologies, Germany) microplate reader. Shown are the mean±s.d. of four independent experiments (*, significantly higher than control P<0.05).