Search

Engineered skin substitutes (ESSs) comprising both keratinocytes and fibroblasts can afford many advantages over the use of autologous keratinocyte grafts for the treatment of full-thickness and partial-thickness burns. In this study, we investigated the efficacy of a novel ESS containing both genetically altered fibroblasts that express the immunosuppressive factor indoleamine 2,3-dioxygenase (IDO) and primary keratinocytes from a nonautologous source to confer immune protection of xenogeneic cells cultured in a bilayer ESS. The results show that engraftment of IDO expressing skin substitutes on the back of rats significantly improves healing progression over 7 days compared with both nontreated and non-IDO-expressing skin substitutes (p<0.001). Immuno-staining of CD3 and CD31 suggests that IDO-expressing skin substitutes significantly suppress T cell infiltration (p<0.001) and improve neovascularization by four-fold (12.6±1.2 vs. 3.0±1.0 vessel-like structure/high power field), respectively. In conclusion, we found that IDO expression can improve the efficacy of nonautologous ESS for the purpose of wound healing by mitigating T-cell infiltration as well as promoting vascularization of the graft., (© 2010 by the Wound Healing Society.)

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., ((c) 2010 Wiley-Liss, Inc.)

Objective: The requirement of systemic immunosuppression after islet transplantation is of significant concern and a major drawback to clinical islet transplantation. Here, we introduce a novel composite three-dimensional islet graft equipped with a local immunosuppressive system that prevents islet allograft rejection without systemic antirejection agents. In this composite graft, expression of indoleamine 2,3 dioxygenase (IDO), a tryptophan-degrading enzyme, in syngeneic fibroblasts provides a low-tryptophan microenvironment within which T-cells cannot proliferate and infiltrate islets., Research Design and Methods: Composite three-dimensional islet grafts were engineered by embedding allogeneic mouse islets and adenoviral-transduced IDO-expressing syngeneic fibroblasts within collagen gel matrix. These grafts were then transplanted into renal subcapsular space of streptozotocin diabetic immunocompetent mice. The viability, function, and criteria for graft take were then determined in the graft recipient mice., Results: IDO-expressing grafts survived significantly longer than controls (41.2 +/- 1.64 vs. 12.9 +/- 0.73 days; P < 0.001) without administration of systemic immunesuppressive agents. Local expression of IDO suppressed effector T-cells at the graft site, induced a Th2 immune response shift, generated an anti-inflammatory cytokine profile, delayed alloantibody production, and increased number of regulatory T-cells in draining lymph nodes, which resulted in antigen-specific impairment of T-cell priming., Conclusions: Local IDO expression prevents cellular and humoral alloimmune responses against islets and significantly prolongs islet allograft survival without systemic antirejection treatments. This promising finding proves the potent local immunosuppressive activity of IDO in islet allografts and sets the stage for development of a long-lasting nonrejectable islet allograft using stable IDO induction in bystander fibroblasts.

Islet transplantation is a promising treatment for diabetes. However, it faces several challenges including requirement of systemic immunosuppression. Indoleamine 2,3-dioxygenase (IDO), a tryptophan degrading enzyme, is a potent immunomodulatory factor. Local expression of IDO in bystander fibroblasts suppresses islet allogeneic immune response in vitro. The aim of the present study was to investigate the impact of IDO on viability and function of mouse islets embedded within IDO-expressing fibroblast-populated collagen scaffold. Mouse islets were embedded within collagen matrix populated with IDO adenovector-transduced or control fibroblasts. Proliferation, insulin content, glucose responsiveness, and activation of general control nonderepressible-2 kinase stress-responsive pathway were then measured in IDO-exposed islets. In vivo viabilities of composite islet grafts were also tested in a syngeneic diabetic animal model. No reduction in islet cells proliferation was detected in both IDO-expressing and control composites compared to the baseline rates. Islet functional studies showed normal insulin content and secretion in both preparations. In contrast to lymphocytes, general control nonderepressible-2 kinase pathway was not activated in islets cocultured with IDO-expressing fibroblasts. When transplanted to diabetic mice, syngeneic IDO-expressing composite islet grafts were functional up to 100 days tested. These findings collectively confirm normal viability and functionality of islets cocultured with IDO-expressing cells and indicate the feasibility of development of a functional nonrejectable islet graft.

We have previously demonstrated that indoleamine 2, 3-dioxygenase (IDO) expressed by dermal fibroblasts generated a tryptophan deficient environment in which immune cells, but not skin cells, undergo apoptosis. However, the mechanism by which primary skin cells such as fibroblasts and keratinocytes are resistant to this culture environment is not elucidated. Here, we asked the question of whether the activity of the general control nondepressing-2 (GCN2) kinase pathway in primary immune and skin cells is differently regulated in response to IDO-induced tryptophan deficient environment. Before addressing this question, the expression of IDO in IDO-adenoviral infected fibroblasts, as a source of IDO expression, was validated. We then demonstrated a significant immunosuppressive effect of IDO expression in primary human T cells co-cultured with IDO expressing fibroblasts in the presence of allogeneic pieces of either epidermis or full thickness skin. Evaluating the mechanism by which skin cells, but not T cells, are resistant to IDO induced low tryptophan environment, we then co-cultured IDO-expressing fibroblasts with bystander human T cells, the fibroblasts, or keratinocytes for 3 days. The results showed a significant activation of apoptotic pathway as analyzed by caspase-3 induction as well as the expression of CHOP, a downstream effector of GCN2 kinase pathway in T cells, but not in skin cells.

We have previously demonstrated that indoleamine 2,3-dioxygenase (IDO) expression by skin cells generates a tryptophan deficient environment in which THP-1, Jurkat cells as well as human PBMC are unable to survive. However, the subsets of primary human T cells that are sensitive to tryptophan depletion have not been identified. In this study, we asked whether the proliferation and viability of bystander CD4+ and CD8+ T cells are modulated in response to IDO induced tryptophan deficient environment and if so, whether their response is different. To address these questions, we co-cultured IDO-expressing fibroblasts with bystander human CD4+ and CD8+ T cells for 4 days and then the survival and proliferation rates as well as downstream metabolic pathway of tryptophan degradation in these cells were evaluated. The results showed a marked immunosuppressive effect of IDO expression on both subsets of primary human T cells. Interestingly, there was also a significant difference in the suppressive effect of IDO on proliferation of CD8+ compared to that of CD4+ T cells. The results of subsequent experiments showed that this discrepancy is due to differences in GCN2 kinase pathway activation between these two sets of immune cells. In conclusion, the finding of this study revealed that the proliferation of CD8+ and CD4+ T cells are suppressed in response to tryptophan deficient environment caused by IDO expression and it is more so for CD8+ T than CD4+ T cells.

Indolemine 2, 3-dioxygenase (IDO) is a cytosolic monomeric hemoprotein enzyme that catalyses tryptophan, the least available essential amino acid in the human body, to N-formylkynurenine, which in turn rapidly degrades to give kynurenine. IDO is expressed in different tissues, especially and prominently in some subsets of antigen presenting cells (APCs) of lymphoid organs and also in the placenta of human and other mammals. Expression of IDO by certain dendritic cells, monocytes and macrophages has a regulatory effect on T cells probably by providing a tryptophan-deficient microenvironment and/or accumulation of toxic metabolites of tryptophan. This immunomodulatory function of IDO plays an essential role in different physiological and pathological states. IDO was shown to prevent rejection of the fetus during pregnancy, possibly by inhibiting alloreactive T cells. Moreover, IDO expression in APCs was suggested to control autoreactive immune responses. In this review we discuss the molecular and biological characteristics of IDO and its function in immune system as well as the potential application of this enzyme in improving the outcome of allogeneic transplantation as a local immunosuppressive factor.