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-dioxygenase (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.

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.

In the original publication of the article, last author's name was misspelt. The correct name is given here.

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.

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 phagocytose 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.

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.

Preconditioning of primary human renal proximal tubular epithelial cells without tryptophan increases survival under hypoxia by inducing autophagy.

PURPOSE: Hypoxia plays a significant role in the pathogenesis of acute kidney injury (AKI). Autophagy protects from AKI. Amino acid deprivation induces autophagy. The effect of L-tryptophan depletion on survival and autophagy in cultures of renal proximal tubular epithelial cells (RPTECs) under hypoxia was evaluated. METHODS: RPTECs were preconditioned in a medium containing or not tryptophan, following culture under hypoxia and treatment with or without the autophagy inhibitor chloroquine. Cell survival was assessed by cell imaging, the level of certain proteins by western blotting and cellular ATP fluorometrically. RESULTS: Preconditioning of RPTECs in a medium without tryptophan activated general control nonderepressible 2 kinase and induced changes that favored autophagy and cell survival under hypoxic conditions. Additionally, it increased cellular ATP, while it inhibited apoptosis. Inhibition of autophagy nullified the induced increase in cellular ATP and cell survival by the absence of tryptophan. The absence of tryptophan increased p53, although its effect on p53's transcriptional targets was heterogeneous. In accordance with the decreased apoptosis, expression of p21 increased, while expression of Bax decreased. The expression of BNIP3L, which may be pro-apoptotic or pro-autophagic, increased. Considering the decreased apoptosis, it is likely that tryptophan depletion enhances autophagy through a p53-mediated increase of BNIP3L. CONCLUSION: Preconditioning of primary human RPTECs in a medium without tryptophan increases their survival under hypoxia by inducing autophagy. Identifying new molecular mechanisms that protect renal tissue from hypoxia could be proved clinically important in the prevention of AKI.

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.

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.

Indoleamine 2,3-dioxygenase, by degrading L-tryptophan, enhances carnitine palmitoyltransferase I activity and fatty acid oxidation, and exerts fatty acid-dependent effects in human alloreactive CD4+ T-cells.

Indoleamine 2,3-dioxygenase (IDO) is expressed in antigen-presenting cells and by degrading L-tryptophan along the kynurenine pathway suppresses CD4+ T-cell proliferation, induces apoptosis and promotes differentiation towards a regulatory as opposed to an effector phenotype. Recent findings revealed that the above effects may be mediated through alterations in T-cell metabolism. In this study, the effect of IDO on fatty acid ß-oxidation in CD4+ T-cells was evaluated in human mixed lymphocyte reactions (MLRs) using the IDO inhibitor, 1-DL-methyl-tryptophan. Protein analysis of CD4+ T-cells isolated from the MLR showed that L-tryptophan degradation acts by activating the general control non­derepressible 2 kinase and aryl-hydrocarbon receptor in T-cells. In the absence of IDO inhibition, fatty acid oxidation increased along with increased activity of carnitine palmitoyltransferase I (CPT1), the latter due to the increased expression of CPT1 isoenzymes and alterations in acetyl-CoA carboxylase 2, the enzyme that controls CPT1 activity. Increased fatty acid oxidation due to the action of IDO was accompanied by an increased expression of forkhead box P3 (FoxP3) and a decreased expression of related orphan receptor Î³t (RORγt), the signature transcription factors of regulatory T-cells and T helper 17 cells, respectively. However, in MLR and in the presence of fatty acid in the culture medium, IDO did not inhibit proliferation. Additionally, fatty acid protected the CD4+ T-cells against apoptosis. Thus, IDO, by degrading L-tryptophan, enhances CPT1 activity and fatty acid oxidation, and exerts fatty acid-dependent effects in human alloreactive CD4+ T-cells.

Differential effects of the two amino acid sensing systems, the GCN2 kinase and the mTOR complex 1, on primary human alloreactive CD4⁺ T-cells.

Amino acid deprivation activates general control nonderepressible 2 (GCN2) kinase and inhibits mammalian target of rapamycin (mTOR), affecting the immune response. In this study, the effects of GCN2 kinase activation or mTOR inhibition on human alloreactive CD4+ T-cells were evaluated. The mixed lymphocyte reaction, as a model of alloreactivity, the GCN2 kinase activator, tryptophanol (TRP), and the mTOR complex 1 inhibitor, rapamycin (RAP), were used. Both TRP and RAP suppressed cell proliferation and induced cell apoptosis. These events were p53-independent in the case of RAP, but were accompanied by an increase in p53 levels in the case of TRP. TRP decreased the levels of the Th2 signature transcription factor, GATA-3, as RAP did, yet the latter also decreased the levels of the Th1 and Th17 signature transcription factors, T-bet and RORγt, whereas it increased the levels of the Treg signature transcription factor, FoxP3. Accordingly, TRP decreased the production of interleukin (IL)-4, as RAP did, but RAP also decreased the levels of interferon-γ (IFN-γ) and IL-17. Both TRP and RAP increased the levels of IL-10. As regards hypoxia-inducible factor-1α (HIF-1α), which upregulates the Th17/Treg ratio, its levels were decreased by RAP. TRP increased the HIF-1α levels, which however, remained inactive. In conclusion, our findings indicate that, in primary human alloreactive CD4+ T-cells, the two systems that sense amino acid deprivation affect cell proliferation, apoptosis and differentiation in different ways or through different mechanisms. Both mTOR inhibition and GCN2 kinase activation exert immunosuppressive effects, since they inhibit cell proliferation and induce apoptosis. As regards CD4+ T-cell differentiation, mTOR inhibition exerted a more profound effect, since it suppressed differentiation into the Th1, Th2 and Th17 lineages, while it induced Treg differentiation. On the contrary, the activation of GCN2 kinase suppressed only Th2 differentiation.

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+ T­cells were then isolated from the MLRs and the expression of cleaved caspase­3, various enzymes involved in glycolysis, and the signature transcription factors of CD4+ T­cell 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+ T­cells, DCA induced apoptosis, and decreased the expression of glucose trasporter­1, hexokinase II, lactate dehydrogenase­A 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 T­box transcription factor TBX21, trans­acting T-cell-specific transcription factor GATA­3 and retinoic acid receptor related orphan receptor­Î³t. In conclusion, in alloreactive CD4+ T­cells, DCA inhibits aerobic glycolysis, induces apoptosis and favors differentiation towards the regulatory T­cell subset. These characteristics render it a promising immunosuppressive agent in the field of transplantation.

Indoleamine 2,3­dioxygenase downregulates T­cell receptor complex ζ­chain and c­Myc, and reduces proliferation, lactate dehydrogenase levels and mitochondrial glutaminase in human T­cells.

Indoleamine 2,3­dioxygenase (IDO), through L­tryptophan depletion, activates general control non­derepressible (GCN) 2 kinase and suppresses T­cell 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 IDO­induced p53 upregulation. In two­way mixed lymphocyte reactions (MLRs), IDO reduced cellular proliferation. In MLR­derived T­cells, IDO induced the expression levels of p53 and p21, however concurrently reduced the levels of ζ­chain, c­Myc, lactate dehydrogenase A (LDH­A) and glutaminase (GLS)2. However, p53 had no effect on the expression of the above proteins. These results were recapitulated in T­cells activated with anti­CD2, anti­CD3 and anti­CD28 by direct activation of the GCN2 kinase with tryptophanol. In conclusion, IDO, through GCN2 kinase activation, downregulates the levels of TCR­complex ζ­chain and c­Myc, resulting in the suppression of T­cell proliferation and a reduction in the levels of LDH­A and GLS2, which are key enzymes involved in aerobic glycolysis and glutaminolysis, respectively.

Gluten induces coeliac-like disease in sensitised mice involving IgA, CD71 and transglutaminase 2 interactions that are prevented by probiotics.

Coeliac disease (CD) is a malabsorptive enteropathy resulting from intolerance to gluten. Environmental factors and the microbiota are suggested to have critical roles in the onset of CD. The CD71 IgA receptor on epithelial cells is responsible for abnormal retrotranscytosis of IgA-gluten peptide complexes from the intestinal lumen into the lamina propria, inducing intestinal inflammation. However, understanding the role of gluten in the CD physiopathology has been hindered by the absence of relevant animal models. Here, we generated a mouse model for CD to study the factors controlling its pathogenesis as well as to investigate the influence of oral delivery of probiotics on disease development. Gluten sensitivity was established by feeding three generations of BALB/c mice a gluten-free diet (G-) followed by gluten challenge (G+) for 30 days. The G+ mice developed villous atrophy, crypt hyperplasia and infiltration of T cells and macrophages in the small intestine. Inflammation was associated with an overexpression of CD71 on the apical side of enterocytes and an increase of plasma cells producing IgA, which colocalised with the CD71. Moreover, IgA colocalised with the transglutaminase 2 (TG2), the production of which was increased in the lamina propria of G+ mice. These mice displayed increased production of cyclooxygenase-2 (COX-2), pro-inflammatory cytokines and IL-15, as well as anti-gliadin and anti-TG2 autoantibodies. The commensal flora-isolated presumptive probiotic Saccharomyces boulardii KK1 strain hydrolysed the 28-kDa α-gliadin fraction, and its oral delivery in G+ mice improved enteropathy development in association with decrease of epithelial cell CD71 expression and local cytokine production. In conclusion, the G+ BALB/c mouse represents a new mouse model for human CD based on histopathological features and expression of common biomarkers. The selected probiotic treatment reversing disease development will allow the study of the role of probiotics as a new therapeutic approach of CD.