High expression of IMPACT protein promotes resistance to indoleamine 2,3-dioxygenase-induced cell death.

Indoleamine 2,3-dioxygenase (IDO), a tryptophan degrading enzyme, is a potent immunomodulatory factor. IDO expression in fibroblasts selectively induces apoptosis in immune cells but not in primary skin cells. However, the mechanism(s) of this selective effect of IDO-induced low tryptophan environment is not elucidated. The aim of present study was to investigate whether the activity of general control non-derepressible-2(GCN2) kinase stress-responsive pathway and its known inhibitor, protein IMPACT homolog, in immune and skin cells are differentially regulated in response to IDO-induced low tryptophan environment. IDO-expressing human fibroblasts were co-cultured with Jurkat cells, human T cells, fibroblasts, or keratinocytes. Activation of GCN2 pathway was significantly higher in immune cells exposed to IDO-expressing environment relative to that of skin cells. In contrast, IMPACT was highly and constitutively expressed in skin cells while its expression was very low in stimulated T cells and undetectable in Jurkat cells. A significant IDO-induced suppressive as well as apoptotic effect was demonstrated in IMPACT knocked down fibroblasts co-cultured with IDO-expressing fibroblasts. Proliferation of Jurkat cells, stably transduced with IMPACT-expressing vector, was rescued significantly in tryptophan-deficient but not IDO-expressing environment. This may be due to the ability of IMPACT to recover the effects of IDO-mediated tryptophan depletion (GCN2 dependent) but not the effects of IDO-generated cytotoxic metabolites. These findings collectively suggest for the first time that high expression of protein IMPACT homolog in non-immune cells such as skin cells acts as a protective mechanism against IDO-induced GCN2 activation, therefore, makes them resistant to the amino acid-deprived environment caused by IDO.

Habibi D ，Jalili RB ，Forouzandeh F ，Ong CJ ，Ghahary A ... -  《-》

Immune cell proliferation is suppressed by the interferon-gamma-induced indoleamine 2,3-dioxygenase expression of fibroblasts populated in collagen gel (FPCG).

Indoleamine 2,3-dioxygenase (IDO), a tryptophan-catabolizing enzyme, is an intracellular enzyme possessing various immunosuppressive properties. Here, we report the possible use of this enzyme to suppress proliferation of immune cells cocultured with IDO-expressing fibroblasts of an allogenic skin substitute. Fetal skin fibroblasts embedded within bovine collagen were treated with cytokine interferon-gamma (IFN-gamma) to induce expression of IDO mRNA and protein. Expression of IDO mRNA was evaluated by Northern analysis. IDO enzyme activity was evaluated by measurement of kynurenine and tryptophan levels in the IFN-gamma untreated and treated fibroblasts. The results of Northern analysis showed a dose-dependent increase in expression of IDO mRNA in response to various concentrations of IFN-gamma used. The levels of kynurenine and tryptophan measured, as the bioactivity of IDO, were significantly different in the IFN-gamma treated fibroblasts, compared to those of controls (P < 0.001). In a lasting effect experiment, the expression of IDO mRNA was gradually reduced to an undetectable level within 32 h of IFN-gamma removal. The results of Western blot analysis, however, revealed a significantly longer (192 h) lasting effect of IFN-gamma on IDO protein level, relative to that of mRNA expression. To demonstrate immunosuppressive effects of IDO on proliferation of immune cells, IDO-expressing fibroblasts were cocultured with peripheral blood mononuclear cells (PBMC) for a period of 5 days. The results of (3)H-thymidine incorporation showed a significant reduction in proliferation of PBMC when cocultured with IDO-expressing fibroblasts, compared to those cocultured with non-IDO-expressing fibroblasts (P < 0.001). Furthermore, addition of IDO-inhibitor (1-methyl-d-tryptophan) reversed the suppressive effects of IDO on PBMC proliferation in a dose-dependant fashion. To test the viability of immune cells cocultured with IDO-expressing fibroblasts, FACS analysis of the PI stained PBMC was conducted and no significant difference was found between these cells and the controls. In another set of experiments, we showed that migration rate and subsequent proliferation of IDO-expressing fibroblasts are also the same as those of control cells. In conclusion, IDO-expressing allogenic fibroblasts embedded within collagen gel suppress the proliferation of allogenic immune cells, while they still remain viable in this IDO-induced tryptophan-deficient culture environment.

Sarkhosh K ，Tredget EE ，Karami A ，Uludag H ，Iwashina T ，Kilani RT ，Ghahary A ... -  《JOURNAL OF CELLULAR BIOCHEMISTRY》

Skin cells, but not T cells, are resistant to indoleamine 2, 3-dioxygenase (IDO) expressed by allogeneic fibroblasts.

Forouzandeh F ，Jalili RB ，Germain M ，Duronio V ，Ghahary A ... -  《-》

Temperature-sensitive polymer-conjugated IFN-gamma induces the expression of IDO mRNA and activity by fibroblasts populated in collagen gel (FPCG).

Sarkhosh K ，Tredget EE ，Uludag H ，Kilani RT ，Karami A ，Li Y ，Iwashina T ，Ghahary A ... -  《JOURNAL OF CELLULAR PHYSIOLOGY》

GCN2 kinase in T cells mediates proliferative arrest and anergy induction in response to indoleamine 2,3-dioxygenase.

Munn DH ，Sharma MD ，Baban B ，Harding HP ，Zhang Y ，Ron D ，Mellor AL ... -  《IMMUNITY》