Mechanism of interferon-gamma mediated down-regulation of interleukin-10 gene expression.

Expression of the anti-inflammatory cytokine IL-10 is suppressed by the pro-inflammatory interferon gamma but the mechanism of this action is unknown. We analysed activity of IL-10 promoter luciferase reporter constructs in transfected RPMI 8226.1 B cells that were treated at -2 h with IFNgamma (1000 U/ml) followed by stimulation with LPS (100 ng/ml) at 0 h. IFNgamma treatment suppressed LPS-induced IL-10 promoter activity in a construct carrying the -1044 promoter and also one containing the -195 promoter. The suppression was independent of the IRF-motif at -182 but involved the Stat-motif at -120. In gelshift analysis this Stat motif did bind LPS-induced Stat3 and with IFNgamma treatment it did, in addition, bind Stat1. ChIP analysis for detection of transcription factor binding to chromatin in intact cells demonstrated Stat3 binding to the proximal IL-10 promoter when cells are stimulated with LPS only. Treatment with IFNgamma only led to Stat1 binding in ChIP analysis and treatment with IFNgamma plus LPS led to reduced Stat3 binding while Stat1 binding remained high. Finally, LPS-induced activity of the trimeric Stat-motif in front of the luciferase reporter was suppressed by IFNgamma. These data demonstrate that IFNgamma down-regulates expression of the IL-10 gene by a novel mechanism that involves displacement of transactiving Stat3 by IFNgamma-induced Stat1.