In human alloreactive CD4+ T-cells, dichloroacetate inhibits aerobic glycolysis, induces apoptosis and favors differentiation towards the regulatory T-cell subset instead of effector T-cell subsets

Although kidney transplantation is the best therapy for end-stage renal disease, rejection remains a concern, and currently available immunosuppressive agents contribute to morbidity and mortality. Thus, novel immunosuppressive drugs are required. Dichloroacetate (DCA) is already used in the treatment of congenital lactic acidosis and characterized by limited toxicity. As DCA inhibits aerobic glycolysis, which is a prerequisite for CD4+ T-cell proliferation and differentiation into effector T-cells, its possible immunosuppressive role in mixed lymphocyte reaction (MLR), a model of alloreactivity, was investigated. Glucose and lactate concentrations were measured in the supernatants, and cell proliferation was assessed immunoenzymatically. CD4+ T-cells were then isolated from the MLRs and the expression of cleaved caspase-3, various enzymes involved in glycolysis, and the signature transcription factors of CD4+ T-cell subsets were evaluated by western blotting. In MLRs, DCA decreased glucose consumption and aerobic glycolysis, while it exerted a negligible effect on cell proliferation. In CD4+ T-cells, DCA induced apoptosis, and decreased the expression of glucose trasporter-1, hexokinase II, lactate dehydrogenase-A and phosphorylated pyruvate dehydrogenase, while it increased total pyruvate dehydrogenase. In addition, DCA increased the expression of transcription factor forkhead box P3, whereas it decreased the expression of T-box transcription factor TBX21, trans-acting T-cell-specific transcription factor GATA-3 and retinoic acid receptor related orphan receptor-γt. In conclusion, in alloreactive CD4+ T-cells, DCA inhibits aerobic glycolysis, induces apoptosis and favors differentiation towards the regulatory T-cell subset. These characteristics render it a promising immunosuppressive agent in the field of transplantation

Indoleamine 2,3-dioxygenase suppresses humoral alloimmunity via pathways that different to those associated with its effects on T cells

Chronic antibody-mediated rejection remains a major cause of late graft loss. Regarding cellular alloimmunity, the immunosuppressive properties of indoleamine 2,3-dioxy-genase (IDO) have been well investigated; however, little is known of its effects on humoral alloimmunity. Therefore, the present study aimed to evaluate the effects of IDO on humoral alloimmunity. We developed a method for the induction of humoral alloimmunity in a one-way mixed lymphocyte reaction (MLR), which was measured with an antibody-mediated complement-dependent cytotoxicity assay using resting cells, which are similar to the stimulator cells of the aforementioned MLR. In parallel, cellular alloimmunity was assessed in two-way MLRs. The IDO inhibitor 1-methyl-DL-tryptophan was used for evaluating the role of IDO. In order to investigate whether the pathways known to serve a role in the effects of IDO on T cells are applied in humoral alloimmunity, the general control nonderepressible-2 (GCN-2) kinase activator tryptophanol and the aryl hydrocarbon receptor (AhR) inhibitor CH223191 were employed. The IDO inhibitor was revealed to increased cellular autoimmunity, but was decreased by the GCN-2 kinase activator. Unexpectedly, the AhR inhibitor decreased cellular alloimmunity. In addition, the IDO inhibitor was observed to suppress humoral alloimmunity, which may occur in manners independent of GCN-2 kinase AhR. The present study proposed that IDO may decrease humoral alloimmunity in primary human peripheral blood mononuclear cells via pathways that differ to those associated with its effect on T cells. © 2019, Spandidos Publications. All rights reserved

Angiogenin is upregulated during the alloreactive immune response and has no effect on the T-cell expansion phase, whereas it affects the contraction phase by inhibiting CD4+ T-cell apoptosis

Under growth conditions, angiogenin is translocated into the nucleus, where it enhances ribosomal RNA transcription, facilitating increased protein synthesis and cellular proliferation. During stress conditions, angiogenin is sequestered in the cytoplasm, where it cleaves transfer RNA (tRNA) to produce tRNA-derived, stress-induced small RNAs (tiRNAs) that inhibit global protein synthesis, but increase the translation of anti-apoptotic factors. In the present study, the role of angiogenin in the human alloreactive immune response was evaluated using mixed lymphocyte reactions (MLRs) and neamine, an inhibitor of angiogenin nuclear translocation. In MLRs, angiogenin production was significantly (P<0.001) increased compared with resting peripheral blood mononuclear cells. The addition of neamine had no effect on cell proliferation, but did significantly (P<0.001) increase expression of Bcl-2-associated X protein and protein levels of activated caspase-3 in CD4+ T-cells isolated from the MLRs, indicating that angiogenin reduces apoptosis. In conclusion, angiogenin is upregulated during the alloreactive immune response, in which it does not affect the T-cell expansion phase, but inhibits the T-cell contraction phase by protecting against CD4+ T-cell apoptosis. © 2016, Spandidos Publications. All rights reserved

In human cell cultures, everolimus is inferior to tacrolimus in inhibiting cellular alloimmunity, but equally effective as regards humoral alloimmunity

Purpose: Acute cellular rejection is the major cause of immune-mediated graft failure early in the course of kidney transplantation, whereas chronic antibody-mediated rejection is a major contributor to graft loss in the late post-transplant phase. Based mainly on the results of short-term studies, the calcineurin inhibitor tacrolimus prevails over the mammalian target of rapamycin (mTOR) inhibitors. However, the toxicity profile of the two drug categories differs, making the interchange between them appealing. In this study, the effect of tacrolimus and of the mTOR inhibitor everolimus on cellular and humoral alloimmunity was evaluated. Methods: Cellular alloimmunity was assessed by cell proliferation in two-way mixed lymphocyte reaction (MLR) with human peripheral blood mononuclear cells (PBMC). For assessing humoral alloimmunity, we developed a method in which humoral alloimmunity was induced in a one-way MLR. The de novo production of alloantibodies was measured with an antibody-mediated complement-dependent cytotoxicity assay, in which supernatants from the above MLRs were used against resting PBMC similar to the stimulator cells of the forementioned MLRs. Tacrolimus and everolimus were used at concentrations near their upper recommended trough levels. Results: In two-way MLRs, tacrolimus inhibited cell proliferation more than everolimus. In one-way MLRs, tacrolimus and everolimus decreased alloantibody production to the same extent. Conclusions: In human cell cultures, everolimus is inferior to tacrolimus in inhibiting cellular alloimmunity, but equally effective as regards humoral alloimmunity. Thus, everolimus might be a safe alternative in case of tacrolimus toxicity, particularly after the early period of kidney transplantation. © 2017, Springer Science+Business Media Dordrecht

Urate crystals directly activate the T-cell receptor complex and induce T-cell proliferation

Uric acid is a known danger associated molecular pattern molecule able to induce inflammation following internalization of its crystals by cells of the innate immune system. By activating antigen-presenting cells, urate boosts adaptive immunity as well. Furthermore, urate crystals can induce proliferation of isolated T-cells, which are unable to phago-cytose crystal particles. In light of the evidence that urate crystals can also activate dendritic cells and macrophages without prior internalization but through sequestration of lipid rafts (and consequently receptors clustering in a non specific manner), the authors evaluated whether such a mechanism is involved in the direct activation of T-cells by urate crystals. In the present study, isolated human T-cells were cultured with or without urate at a concentration above its crystallization level. The expression and phosphorylation state of the T-cell receptor (TCR) complex zeta chain and the expression of the master regulator of cell proliferation c-Myc were assessed by western blotting. T-cell proliferation was measured by bromodeoxyuridine assay. Collectively, the results indicated that urate increased zeta chain phosphorylation indicating that it induces activation of TCR complex directly, since zeta chain phosphorylation takes place at the cell membrane and is a very proximal event in TCR complex-mediated signal transduction. In parallel, urate increased the expression of the transcription factor c-Myc and induced T-cell proliferation. In conclusion, urate crystals directly activate the TCR complex and induce T-cell proliferation. © 2017, Spandidos Publications. All rights reserved

Uric acid increases cellular and humoral alloimmunity in primary human peripheral blood mononuclear cells

Aim: Hyperuricaemia is common among kidney transplant recipients and has been associated with worse graft outcome. Since episodes of acute cellular rejection and chronic humoral rejection contribute to decreased graft survival, in this study the effect of uric acid on cellular and humoral alloimmunity was evaluated. Methods: Cellular alloimmunity was assessed by cell proliferation in two-way mixed lymphocyte reaction (MLR) with human peripheral blood mononuclear cells (PBMC). For assessing humoral alloimmunity we developed a method in which humoral alloimmunity was induced in one-way MLR. Then the de novo production of alloantibodies was measured with an antibody-mediated complement-dependent cytotoxicity assay, in which supernatants from the above MRLs were used against resting PBMC similar to the stimulator cells of the above MLRs. Results: Uric acid at a concentration above its crystallization threshold increased cellular proliferation in two-way MLRs. Supernatants from one-way MLRs performed in the presence of uric acid were more cytotoxic against PBMC from individuals that had conferred the stimulator cells for the above MLRs. Conclusions: Uric acid increases both cellular and humoral alloimmunity in human PBMC. These results offer a possible pathogenetic mechanism for the observed relation between hyperuricaemia and worse kidney allograft survival. © 2017 Asian Pacific Society of Nephrolog

Indoleamine 2,3-dioxygenase downregulates T-cell receptor complex ζ-chain and c-Myc, and reduces proliferation, lactate dehydrogenase levels and mitochondrial glutaminase in human T-cells

Indoleamine 2,3-dioxygenase (IDO), through L-tryptophan depletion, activates general control non-derepressible (GCN) 2 kinase and suppresses T-cell proliferation, in addition to suppressing aerobic glycolysis and glutaminolysis, which are required for these rapidly proliferating cells. A number of, however not all of these alterations, are partially mediated through IDO-induced p53 upregulation. In two-way mixed lymphocyte reactions (MLRs), IDO reduced cellular proliferation. In MLR-derived T-cells, IDO induced the expression levels of p53 and p21, however concurrently reduced the levels of ζ-chain, c-Myc, lactate dehydrogenase A (LDH-A) and glutaminase (GLS)2. However, p53 had no effect on the expression of the above proteins. These results were recapitulated in T-cells activated with anti-CD2, anti-CD3 and anti-CD28 by direct activation of the GCN2 kinase with tryptophanol. In conclusion, IDO, through GCN2 kinase activation, downregulates the levels of TCR-complex ζ-chain and c-Myc, resulting in the suppression of T-cell proliferation and a reduction in the levels of LDH-A and GLS2, which are key enzymes involved in aerobic glycolysis and glutaminolysis, respectively

Tryptophan depletion under conditions that imitate insulin resistance enhances fatty acid oxidation and induces endothelial dysfunction through reactive oxygen species-dependent and independent pathways

In atherosclerosis-associated pathologic entities characterized by malnutrition and inflammation, l-tryptophan (TRP) levels are low. Insulin resistance is an independent cardiovascular risk factor and induces endothelial dysfunction by increasing fatty acid oxidation. It is also associated with inflammation and low TRP levels. Low TRP levels have been related to worse cardiovascular outcome. This study evaluated the effect of TRP depletion on endothelial dysfunction under conditions that imitate insulin resistance. Fatty acid oxidation, harmful pathways due to increased fatty acid oxidation, and endothelial dysfunction were assessed in primary human aortic endothelial cells cultured under normal glucose, low insulin conditions in the presence or absence of TRP. TRP depletion activated general control non-derepressible 2 kinase and inhibited aryl hydrocarbon receptor. It increased fatty acid oxidation by increasing expression and activity of carnitine palmitoyltransferase 1. Elevated fatty acid oxidation increased the formation of reactive oxygen species (ROS) triggering the polyol and hexosamine pathways, and enhancing protein kinase C activity and methylglyoxal production. TRP absence inhibited nitric oxide synthase activity in a ROS-dependent way, whereas it increased the expression of ICAM-1 and VCAM-1 in a ROS independent and possibly p53-dependent manner. Thus, TRP depletion, an amino acid whose low levels have been related to worse cardiovascular outcome and to inflammatory atherosclerosis-associated pathologic entities, under conditions that imitate insulin resistance enhances fatty acid oxidation and induces endothelial dysfunction through ROS-dependent and independent pathways. These findings may offer new insights at the molecular mechanisms involved in accelerated atherosclerosis that frequently accompanies malnutrition and inflammation. © 2017, Springer Science+Business Media New York

Correction to: Tryptophan depletion under conditions that imitate insulin resistance enhances fatty acid oxidation and induces endothelial dysfunction through reactive oxygen species-dependent and independent pathways (Molecular and Cellular Biochemistry, (2017), 428, 1-2, (41-56), 10.1007/s11010-016-2915-7)

In the original publication of the article, last author’s name was misspelt. The correct name is given here. © 2018, Springer Science+Business Media, LLC, part of Springer Nature