Comparison of IFN constructs for induction of IDO. (A) Total mRNA was isolated from macrophages (6 × 106) treated with IFNα21b, IFNα2c, SDM1 or SDM2. SDM1 (S86- > Y) and SDM2 (N90- > Y) induced reduced IDO gene expression in comparison to parental IFN molecules at 1 ng/mL (*P < .02). Representative data, n = 3. (B) Total mRNA analyses of macrophages incubated in the presence or absence of Polymyxin B and exposed to IFNα2c or SDM1 as indicated for 4 hours (*P < .05). (C) Real-time PCR of monocytes treated with indicated concentrations of IFNα2c, SDM1 for 4 hours confirms limited IDO transcription for mutant IFN, *P < .01. Inset: CD118 antibody blocks parental IFN and SDM1 IDO gene induction. Representative data, n = 3, *P < .01 IFNα2c vs SDM1. *P < .01 IFN alone compared with anti-CD118/IFN-treated. (D) Real-time PCR analyses comparing levels of transcriptional activity for IDO in macrophages cultures pretreated with JAK inhibitor (100nM) 60 minutes before 4-hour treatment with 1 ng/mL of IFNα2c or SDM1. *P < .01 for levels of IDO for SDM1 vs IFNα2c and IFN constructs versus inhibitor/IFN-treated. Data represent mean + SEM.
