RBCs were eliminated from spleen preps using RBC Lysing Solution (eBioscience). Total RNA was extracted using the reagents: RNeasy Mini Kit and RNaseFree DNase Set (Qiagen, Valencia, CA). A total of 500 ng total RNA in each group was used for RT reaction (iScript cDNA Synthesis Kit, Bio-Rad, Hercules, CA; or High-capacity cDNA Reverse Transcription Kits, Applied Biosystems, Foster City, CA). The relative quantitation PCR for IDO1 (Mm00492586-m1) and GAPDH (4352339-E0806018) were performed in the AppliedBiosystems 7900HT Fast Real-Time PCR System (Applied Biosystems), and TaqMan Universal PCR Master Mix (Applied Biosystems) was used as a reaction reagent. The relative quantitation PCR for Foxp3, CYP1A1, CYP1B1, and GAPDH were processed by the Bio-Rad iCycler (Bio-Rad) and iQ SYBR Green Supermix (Applied Biosystems) was used as the reaction reagent. Naive CD4+ T cells were isolated using the CD4+ CD62L Isolation Kit (Miltenyi Biotec, Auburn, CA) and an autoMACS. This kit includes a depletion mixture, including the addition of a CD25 and an anti-TCRc/d+ Ab. Cells were tested for purity postsorting and consistently showed .90% purity for CD4+CD62+CD252 cells. Viability at the beginning of culture was typically .98% as seen by trypan blue staining. For quantitative PCR (qPCR) analysis, 2? 6 105 cells were cultured in each well of a 96- well round-bottom plate coated with 0.5 mg/ ml anti-CD3 and anti-CD28 overnight and then washed with PBS twice before seeding the cells. The naive T cells were maintained in F10 media supplemented with 10% heat-inactivated FBS, 100 mg/ml streptomycin, 100 U/ml penicillin, 50 mm 2-ME, 25 mM HEPES, and 2 mM L-glutamine. Cells in Treg conditions included the addition of TGF-b (2 ng/ml). Th17 conditions include TGF-b (4 ng/ml) and IL-6 (20 ng/ml) for 3 days.
BNF for 36 hours. Activation of the AHRd was determined by quantifying EROD activity from whole cell lysate. (TIFF)
Figure S2 Duration of action of AHR activation. A. 0.66106 Cells from a mouse hepatoma cell line H1L6.1c3, stably carrying a dioxin-responsive element (DRE)-driven firefly luciferase reporter gene were seeded in each well of a six-well plate overnight and were then treated with SU5416 100 nM or TCDD 1 nM for 4 hours through 96 hours. DRE activity was assayed by a luminometer at the time points shown. Data is presented as a percent of 100 nM TCDD response at those time points. B. Characterization of antagonism of response of SU5416 by CH223191. B. SU5416 100 nM or TCDD 1 nM were tested with DRE-driven luciferase reporter cells with titrating doses of the antagonist for 4 hours as delineated in the figure. Data is presented as % response without inhibitor. C. SU5416 was titrated in culture with and without the antagonist (10 mM) for 4 hours. Results are presented as % maximal TCDD response at 100 nM. (TIFF) Figure S3 Similar to figure 4, splenocytes from wildtype and AHRd mice analyzed by qPCR for CYP1A1. Spleens from these mice were harvested and suspended in culture media, and exposed to titrating doses of A) TCDD B) SU5416. After 4 hours they were analyzed by qPCR for CYP1A1 analysis. The curves represent fold change and show the similar potency of these ligands. Each graph is representative of 3 independent experiments. C. Cells transfected with AHR containing a valine point-mutation show similar ED50 to Cos-1 cells with AHRb isoform. Cos-1 cells were transfected with an AHR containing the same point mutation (valine for alanine) thought to be responsible for the low affinity of the AHRd isoform compared to AHRb, and compared to the wild-type AHR response. These cells also harbor a luciferase gene next to the DRE. C. Cos-1 cells were exposed to TCDD. D. Cos-1 cells were exposed to SU5416. The graphs represent true luciferase values. They are representative of 2 independent experiments. (TIFF) Figure S4Isolation of Naive CD4+ T Cells and T Cell Differentiation
Naive CD4+CD252 T cells were isolated from WT and AHRnull mice and cocultured with BALB/cJ pDCs isolated using the Miltenyi Mouse pDC Isolation Kit (Miltenyi Biotec) at a ratio of 20:1 or 10:1 as previously described (13). SU5416, TCDD (10nM), or FICZ (100 nM) were added at the start of culture. On day 5, cells were harvested and subjected to qPCR analysis.pDC/T Cell CocultureFlow Cytometry
To stain for FoxP3, T cells were first surface stained with antiCD4 and antiCD25 and then fixed and permeabilized with the Fixation/Permeabilization buffer (eBioscience, San Diego, CA) for 30 min at 4uC. Following this, cells were stained with Pacific Blueconjugated anti-FoxP3. For intracellular IL-17 staining, T cells were first stimulated with 50 ng/ml PMA (Signma-Aldrich) and 800 ng/ml ionomycin (Sigma-Aldrich) for 4 h in the presence of GolgiStop (BD Pharmingen, San Diego, CA) for the final 2 h. Cells were then fixed and permeabilized with the Fixation/ Permeabilization buffer (eBioscience) and then stained with PEconjugated anti-IL-17. All abs were from eBioscience. Flow cytometric analysis was performed using an LSR-II (BD Biosciences). CD39 antibodies for flow cytometry were purchased from eBioscience.secretion conditions exposed to of culture, ELISA. (TIFF)SU5416 causes a small amount of IL-17 ?at low doses. Naive T-cells were placed in Th17 in culture (TGF-b 4 ng/ml, IL-6 20 ng/ml) and titrating doses of SU5416 as indicated.
Background: The wearing-OFF phenomenon is a common motor complication of chronic L-3,4-dihydroxyphenylalanine (LDOPA) therapy for Parkinson’s disease. We recently described the discovery of UWA-101, a dual serotonin (SERT) and dopamine (DAT) transporter inhibitor, which increases the duration of “good quality” ON-time provided by L-DOPA in the 1methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-lesioned primate. Here, we further characterise the effects of UWA-101 on this extension of ON-time in terms of L-DOPA-induced side-effects in the MPTP-lesioned common marmoset. Methods: Marmosets were rendered parkinsonian by MPTP injection and “primed” by repeated L-DOPA administration, to exhibit dyskinesia and psychosis-like behaviours. Animals were then administered acute challenges of L-DOPA in combination with UWA-101 (1, 3, 6 and 10 mg/kg) or vehicle. Results: In combination with L-DOPA, UWA-101 (3, 6 and 10 mg/kg) significantly increased duration of ON-time (by 28%, 28%, and 33%, respectively; all P,0.05). UWA-101 (10 mg/kg) significantly extended duration of ON-time without disabling dyskinesia (by 62%, P,0.01). UWA-101 did not exacerbate the severity of dyskinesia (P.0.05). However, at the highest doses (6 and 10 mg/kg), UWA-101 increased the severity of psychosis-like behaviours (P,0.05). Conclusions: Our results demonstrate that dual SERT/ DAT inhibitors can effectively enhance L-DOPA anti-parkinsonian action, without exacerbating dyskinesia and, as such, represent a promising new therapeutic class for wearing-OFF. However, at higher doses, dual SERT/ DAT inhibitors may exacerbate dopaminergic psychosis.