Elsevier

Cytokine

Volume 35, Issues 1–2, July 2006, Pages 53-61
Cytokine

NF-κB activation contributes to indoleamine dioxygenase transcriptional synergy induced by IFN-γ and tumor necrosis factor-α

https://doi.org/10.1016/j.cyto.2006.07.007Get rights and content

Abstract

Interferon (IFN)-γ-induced expression of indoleamine 2,3-dioxygenase (IDO), an enzyme that inhibits some pathogens by limiting tryptophan availability, is transcriptionally enhanced by tumor necrosis factor (TNF)-α. The expression of interferon responsive factor (IRF)-1, an IFN-γ-induced transcriptional activator critical to IDO regulation, is also enhanced synergistically in response to IFN-γ and TNF-α. The IRF-1 regulatory region contains an IFN-γ-activated sequence (GAS) and a κB site, which bind STAT-1 and NF-κB, respectively. The TNF-α-mediated increase in STAT-1 activation in IFN-γ-treated cells enhances IRF-1 transcription; however, the contribution of TNF-α-mediated increases in nuclear NF-κB is uncertain. To identify whether binding of NF-κB upstream of the IRF-1 gene is rate-limiting in IRF-1 expression in response to IFN-γ and TNF-α, a proteasome inhibitor was utilized to maintain nuclear translocation of NF-κB at constitutive levels; its effect on IRF-1 expression and IDO-specific transcription was evaluated. By limiting NF-κB nuclear translocation, IRF-1 expression in IFN-γ and TNF-α treated cells was maintained at a level comparable to that achieved in response to IFN-γ alone, and the synergistic increase IDO transcription was blocked, suggesting that increases in NF-κB translocation are required for synergistic IDO expression in response to IFN-γ and TNF-α.

Introduction

Indoleamine dioxygenase (IDO) catalyzes the rate-limiting step in catabolism of tryptophan along the kynurenine pathway [1]. Its activity limits infection by intracellular pathogens in vitro by depleting pools of tryptophan available for protein synthesis, thereby restricting growth of tryptophan auxotrophs, including Chlamydia and Toxoplasma [2], [3], [4], [5], [6], [7]. IDO also contributes significantly to immunological tolerance by suppressing T-cell responses [8]. Although the IDO gene is interferon (IFN)-γ-induced, its expression is enhanced synergistically by proinflammatory cytokines such as TNF-α and IL-1 in macrophages, monocytes and epithelial cells [9], [10], [11], [12], [13]. This synergistic expression is regulated at the level of transcription, demonstrated by synergistic increases in IDO mRNA levels and by increased transgene expression when using a transcriptional reporter plasmid [9], [10], [11], [12], [13].

A number of cellular signaling events take place in response to IFN-γ receptor binding of ligand. First, signal transducer and activator of transcription (STAT)-1 becomes phosphorylated by Janus kinases at the cytoplasmic tail of the IFN-γ receptor [14], [15], [16], [17]. It then dimerizes and translocates to the nucleus, where it binds to the regulatory regions of the IDO and interferon regulatory factor (IRF)-1 genes [13], [18], [19], [20]. The addition of TNF-α leads to enhanced STAT-1 binding to a single IFN-γ-activated sequence (GAS) upstream of the IRF-1 gene [21], [22]. In addition, TNF-α enhances recruitment of STAT-1 and IRF-1 to GAS sites and interferon-stimulated response elements (ISRE), respectively, upstream of the IDO gene [13]. While the binding of IRF-1 to ISRE sites contributes more to IDO activation than does STAT-1 binding to GAS sites [22], [23], this increase in IRF-1 and STAT-1 binding is essential for the synergistic increase in promoter activity in response to IFN-γ and TNF-α.

There are multiple mechanisms by which TNF-α may contribute to an increase in IFN-γ-induced IDO. First, it upregulates surface expression of IFN-γ receptors, thereby increasing the sensitivity of the cell to lower IFN-γ concentrations [13], [24]. Second, it increases the amount of STAT-1 phosphorylation in response to IFN-γ [13], [21], [25], [26]. A third possible mechanism would be by transactivating NF-κB. In the absence of appropriate stimuli, a constitutive low level of NF-κB is found in the nucleus, where it participates in the regulation of a variety of genes, including those involved in prevention of apoptosis [27], [28]. Following TNF-α stimulation, IκB is phosphorylated, ubiquitinated, and degraded by a proteasome, resulting in the release of cytoplasmic NF-κB, which then translocates to the nucleus. Although no κB sites are present within the IDO regulatory region, one is present upstream of the IRF-1 coding region. While both the κB site and the GAS site are required for IFN-γ-induced IRF-1 gene expression [21], it is not known if increases in nuclear NF-κB are required for increased IRF-1 production, or if the increase in STAT-1 activation in response to combined IFN-γ and TNF-α is sufficient to promote synergistic IRF-1 production. Since constitutive NF-κB activity is sufficient for IFN-γ-induced, IRF-1-dependent gene expression [29], and since neutralization of NF-κB had no effect on up-regulation of CD40 expression in response to combined IFN-γ and TNF-α treatment [30], this suggests the possibility that the increase in STAT-1 activation in response to combined IFN-γ and TNF-α is sufficient to promote synergistic IRF-1 production and IDO activation.

The focus of this study was to determine if the availability of NF-κB is rate-limiting in synergistic transcriptional activation of IDO, mediated by its effect on expression of IRF-1. Although maintenance of nuclear NF-κB at constitutive levels had no effect on IFN-γ-induced IDO activity, it eliminated the synergistic enhancement of IDO induction by TNF-α. This indicates that increased nuclear translocation of NF-κB is an event essential to synergistic transcriptional activation of IDO.

Section snippets

Cytokines, inhibitors, antibodies, and plasmids

Recombinant IFN-γ (specific activity = 1.6 × 107 U/mg protein) was provided by Biogen (Cambridge, MA). Recombinant human TNF-α (ED50 [50% effective dose] = 0.02–0.05 ng/ml) was purchased from R&D Systems (Minneapolis, MN). Proteasome inhibitor I (PSI) was purchased from Calbiochem (San Diego, CA). Sodium salicylate was purchased from Sigma (St. Louis, MO) and dissolved in distilled water. Polyclonal rabbit anti-human phospho-STAT-1 (Y701) was purchased from Upstate Biotechnology (Lake Placid, NY).

Synergistic IDO transcriptional activation in response to IFN-γ and TNF-α

IDO transcriptional synergy is exhibited in epithelial cells in response to IFN-γ and TNF-α. This activity was demonstrated with use of a transcriptional reporter construct containing the enhanced green fluorescent protein (GFP) coding region under the regulation of the IDO promoter/enhancer region [13], which had been stably maintained in HeLa cells. Cells were treated for 6 or 24 h with TNF-α, IFN-γ, combined IFN-γ and TNF-α, or medium alone; GFP expression was then quantified by flow

Discussion

Synergistic upregulation of IDO expression in response to IFN-γ and TNF-α is dependent upon the activation of three transcription factors, STAT-1, IRF-1, and NF-κB. While the IDO gene has multiple binding sites for both STAT-1 and IRF-1, and both STAT-1 and IRF-1 activation are required for maximum transcriptional activation of the IDO gene [18], [19], [23], mutations in its ISRE sites have a more profound inhibitory effect than do mutation in its GAS sites, indicating that IRF-1 activation

Acknowledgments

We thank Michael Hughes at Miami University for his assistance with statistical analysis. This study was supported by grant No. AI45836 from the National Institute of Allergy and Infectious Disease (JMC).

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