Inhibition of indoleamine 2,3-dioxygenase in mixed lymphocyte reaction affects glucose influx and enzymes involved in aerobic glycolysis and glutaminolysis in alloreactive T-cells
Introduction
Indoleamine 2,3-dioxygenase (IDO) catalyzes the initial rate-limiting step of tryptophan degradation along the kynurenine pathway. IDO is inducible by various inflammatory stimuli and it is expressed in antigen presenting cells (APCs), such as monocytes, macrophages and dendritic cells. In the local microenvironment of inflammation, IDO expression in APCs depletes tryptophan and suppresses adaptive immunity [1], [2]. Amino acid depletion is sensed by T-cells through two pathways. Tryptophan depletion by IDO activates the general control nonrepressed 2 (GCN2) kinase, which in turn phosphorylates the eukaryotic translation initiation factor 2α (eIF2α) ultimately altering the translation program of the T-cells [3]. Another pathway able to sense tryptophan depletion is the mammalian target of rapamycin complex 1 (mTORC1) pathway. T-cells fail to proliferate in response to antigen once tryptophan or other essential amino acid becomes sparse, associated with a reduced mTORC1 signaling [4].
Experimental studies showed that IDO mediated immunosuppression ameliorates the clinical course of autoimmune diseases [5], [6], [7] and reduces graft rejection [8], [9], [10]. Inhibition of T-cell function via IDO is also mediated by non-APC cell types. Expression of IDO in tumor cells contributes to escape of tumor by immunosurveillance [11], while its expression in paternally derived placental trophoblast contributes to a successful semi-allogenic pregnancy [12], [13]. Hemodialysis patients are characterized by impaired adaptive immunity and exhibit increased IDO expression, further enhanced in the non-responders to hepatitis B virus vaccination [14]. Interestingly in the above population plasma IDO concentration is inversely related to blood T-cell count [15]. IDO exerts its immunosuppressive action in both CD4+ and CD8+ T-cells [16]. Among others, IDO suppresses adaptive immunity by affecting T-cell differentiation, for instance the differentiation of latter to regulatory T-cells (Treg) [17], [18], [19], [20], [21].
Activated T-cells have increased metabolic requirements in order to support proliferation and effector function. Increased glucose uptake and aerobic glycolysis fuel this demand [22], [23]. Effector CD4+ T-cells require distinct metabolic programs to support their function in opposition to Treg. More precisely, effector CD4+ T-cells express high levels of the glucose transporter-1 (GLUT1) and are reliant on glucose metabolism, whereas Treg express low levels of GLUT1 and are reliant on lipid oxidation. Direct manipulation of cell metabolism determines the fate of CD4+ T-cells [24], [25]. An elegant study showed that in order to fulfill the bioenergetic and biosynthetic demand of proliferation, activated T-cells reprogram their metabolic pathways from pyruvate oxidation and fatty acid β-oxidation via the tricarboxylic acid (TCA) cycle to the glycolytic, pentose-phosphate, and glutaminolytic pathways [26]. Interestingly, in a previous study the IDO inhibitor 1-methyl-dl-tryptophan(1-MT) suppresses mitochondrial function and induces aerobic glycolysis in peripheral blood mononuclear cells (PBMCs) during an immune response against tetanus toxoid [27].
In this study we evaluated the effect of IDO on T-cell metabolism, since IDO affects T-cell activation and differentiation [16], [17], [18], [19], [20], and direct manipulation of glucose metabolism modulates lymphocyte survival, proliferation, differentiation and function [24], [25]. For this purpose the model of alloreactivity, two-way mixed lymphocyte reaction (MLR) [28], and the specific IDO inhibitor 1-MT were used. 1-MT is a competitive, non-toxic IDO inhibitor [29] that has been successfully used to break immune privilege of placenta and tolerance against grafts [8], [12].
Section snippets
Subjects
Blood samples from 10 healthy volunteers (7 men, 37 ± 9 years old) were collected for the evaluation of 1-MT cytotoxicity in resting PMBCs. Blood samples from five non-relative healthy volunteers (all men, 34 ± 8 years old) were also collected in order to perform 10 couples of MLRs. An informed consent was obtained from each individual enrolled into the study and the hospital ethics committee gave its approval to the study protocol.
Peripheral blood mononuclear cell isolation and culture
PBMCs were isolated from whole blood by Ficoll–Hypaque density
1-MT was almost non-toxic in resting peripheral blood mononuclear cells
In resting untreated PBMCs, LDH release assay revealed a cytotoxicity of 11.92 ± 0.73%. In PBMCs treated with 100 μM 1-MT, cytotoxicity was slightly higher 14.12 ± 0.49% (p < 0.001) (Fig. 1A). Despite the statistical significance, the 2% increase in cytotoxicity due to 1-MT treatment compared to spontaneous cell death indicates that 1-MT at the used concentration is almost non-toxic for PMBCs.
1-MT decreased tryptophan consumption, increased proliferation, glucose consumption, aerobic glycolysis and decreased TCA cycle activity in two-way mixed lymphocyte reaction
1-MT decreased l-tryptophan consumption significantly. l-tryptophan concentration in the supernatants of MLRs
Discussion
Indoleamine 2,3-dioxygenase expression in APCs, and more specifically in monocytes in the case of MLR, depletes tryptophan and suppresses adaptive immunity [1], [2]. It is also known that activated T cells in order to fulfill the bioenergetic and biosynthetic demand of proliferation, reprogram their metabolic pathways from pyruvate oxidation and fatty acid β-oxidation via the TCA cycle to the glycolytic, pentose-phosphate, and glutaminolytic pathways [26]. In addition the various T-cell
Declaration of interest
The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.
Contribution of authors
The project and the experimental design were conceptualized by T.E. after significant discussions with I.S. Almost all the experiments were conducted by T.E. and G.P. Flow cytometry analysis was performed by T.E., G.P., E.Y. and D.M. T.E., S.A., G.A. and V.L. analyzed the data. All authors contributed towards writing the manuscript.
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