The role of IFN-gamma and TNF-alpha-responsive regulatory elements in the synergistic induction of indoleamine dioxygenase.

Indoleamine 2,3-dioxygenase (IDO), which enzymatically depletes tryptophan, is an important antimicrobial defense mechanism against susceptible pathogens. In human epithelial cells, interferon-gamma (IFN-gamma)-induced IDO expression is transcriptionally enhanced by tumor necrosis factor-alpha(TNF-alpha). The purpose of this study was to identify those regulatory mechanisms responsible for this synergistic transcriptional activation of IDO. Nuclear concentrations of signal transducer and activator of transcription-1 (Stat1) and IFN regulatory factor-1 (IRF-1), transcription factors that bind gamma-activated sequences (GAS) and IFN-stimulated response elements (ISRE), respectively, were found to increase after stimulation with IFN-gamma and TNF-alpha relative to stimulation with individual cytokines. Additionally, CCAAT enhancer binding protein-beta (C/EBP-beta) bound to one of three consensus C/EBP-beta sites in the IDO regulatory region in response to TNF-alpha alone or combined with IFN-gamma. A transcriptional reporter containing green fluorescent protein (GFP) under the control of the IDO regulatory region was used to analyze the contribution of these enhancer elements to synergistic IDO gene expression in response to IFN-gamma and TNF-alpha. Transcriptional activity following mutation of individual enhancers or large deletions within the regulatory region indicates that increased binding of IFN-gamma-transactivated factors to GAS and ISRE sites alone is responsible for synergistic transcriptional activation of the IDO gene.