Giada Mondanelli

A Relay Pathway between Arginine and Tryptophan Metabolism Confers Immunosuppressive Properties on Dendritic Cells

SUMMARY Arginase 1 (Arg1) and indoleamine 2,3‐dioxygenase 1 (IDO1) are immunoregulatory enzymes catalyzing the degradation of l‐arginine and l‐tryptophan, respectively, resulting in local amino acid deprivation. In addition, unlike Arg1, IDO1 is also endowed with non‐enzymatic signaling activity in dendritic cells (DCs). Despite considerable knowledge of their individual biology, no integrated functions of Arg1 and IDO1 have been reported yet. We found that IDO1 phosphorylation and consequent activation of IDO1 signaling in DCs was strictly dependent on prior expression of Arg1 and Arg1‐dependent production of polyamines. Polyamines, either produced by DCs or released by bystander Arg1+ myeloid‐derived suppressor cells, conditioned DCs toward an IDO1‐dependent, immunosuppressive phenotype via activation of the Src kinase, which has IDO1‐phosphorylating activity. Thus our data indicate that Arg1 and IDO1 are linked by an entwined pathway in immunometabolism and that their joint modulation could represent an important target for effective immunotherapy in several disease settings. HighlightsDendritic cells (DCs) can co‐express Arg1 and IDO1 immunosuppressive enzymesArg1 activity is required for IDO1 induction by TGF‐&bgr; in DCsSpermidine, a downstream Arg1 product, but not arginine starvation, induces IDO1 in DCsArg1+ myeloid derived suppressor cells (MDSCs) can render DCs immunosuppressive via IDO1 &NA; Arginase 1 (Arg1) and indoleamine 2,3‐dioxygenase 1 (IDO1) are immunosuppressive enzymes known to operate in distinct immune cells. Mondanelli and colleagues demonstrate that Arg1 and IDO1 cooperate in conferring long‐term, immunosuppressive effects to dendritic cells.

Forced IDO1 expression in dendritic cells restores immunoregulatory signalling in autoimmune diabetes.

Indoleamine 2,3‐dioxygenase (IDO1), a tryptophan catabolizing enzyme, is recognized as an authentic regulator of immunity in several physiopathologic conditions. We have recently demonstrated that IDO1 does not merely degrade tryptophan and produce immunoregulatory kynurenines, but it also acts as a signal‐transducing molecule, independently of its enzymic function. IDO1 signalling activity is triggered in plasmacytoid dendritic cells (pDCs) by transforming growth factor‐β (TGF‐β), an event that requires the non‐canonical NF‐κB pathway and induces long‐lasting IDO1 expression and autocrine TGF‐β production in a positive feedback loop, thus sustaining a stably regulatory phenotype in pDCs. IDO1 expression and catalytic function are defective in pDCs from non‐obese diabetic (NOD) mice, a prototypic model of autoimmune diabetes. In the present study, we found that TGF‐β failed to activate IDO1 signalling function as well as up‐regulate IDO1 expression in NOD pDCs. Moreover, TGF‐β‐treated pDCs failed to exert immunosuppressive properties in vivo. Nevertheless, transfection of NOD pDCs with Ido1 prior to TGF‐β treatment resulted in activation of the Ido1 promoter and induction of non‐canonical NF‐κB and TGF‐β, as well as decreased production of the pro‐inflammatory cytokines, interleukin 6 (IL‐6) and tumour necrosis factor‐α (TNF‐α). Overexpression of IDO1 in TGF‐β‐treated NOD pDCs also resulted in pDC ability to suppress the in vivo presentation of a pancreatic β‐cell auto‐antigen. Thus, our data suggest that a correction of IDO1 expression may restore its dual function and thus represent a proper therapeutic manoeuvre in this autoimmune setting.

The immune regulation in cancer by the amino acid metabolizing enzymes ARG and IDO

Some enzymes degrading amino acids have evolved in mammals to dampen immune responses and maintain peripheral tolerance. The enzymes metabolizing l-arginine and l-tryptophan are particularly powerful, contributing to restrain immunity towards fetal tissues and establish neonatal tolerance. Solid tumors can hijack these formidable pathways to construct a microenvironment highly unfavorable to anti-tumor T lymphocytes able to recognize them, one of mechanisms for their immune evasion. In this review, we analyze emerging concepts in the cross-talk between cells expressing these enzymes, their immune regulatory functions and pharmacological approaches that can target them to enhance cancer immunotherapy.

Distinct roles of immunoreceptor tyrosine‐based motifs in immunosuppressive indoleamine 2,3‐dioxygenase 1

The enzyme indoleamine 2,3‐dioxygenase 1 (IDO1) catalyses the initial, rate‐limiting step in tryptophan (Trp) degradation, resulting in tryptophan starvation and the production of immunoregulatory kynurenines. IDO1s catalytic function has long been considered as the one mechanism responsible for IDO1‐dependent immune suppression by dendritic cells (DCs), which are master regulators of the balance between immunity and tolerance. However, IDO1 also harbours immunoreceptor tyrosine‐based inhibitory motifs, (ITIM1 and ITIM2), that, once phosphorylated, bind protein tyrosine phosphatases, (SHP‐1 and SHP‐2), and thus trigger an immunoregulatory signalling in DCs. This mechanism leads to sustained IDO1 expression, in a feedforward loop, which is particularly important in restraining autoimmunity and chronic inflammation. Yet, under specific conditions requiring that early and protective inflammation be unrelieved, tyrosine‐phosphorylated ITIMs will instead bind the suppressor of cytokine signalling 3 (SOCS3), which drives IDO1 proteasomal degradation and shortens the enzyme half‐life. To dissect any differential roles of the two IDO1s ITIMs, we generated protein mutants by replacing one or both ITIM‐associated tyrosines with phospho‐mimicking glutamic acid residues. Although all mutants lost their enzymic activity, the ITIM1 – but not ITIM2 mutant – did bind SHPs and conferred immunosuppressive effects on DCs, making cells capable of restraining an antigen‐specific response in vivo. Conversely, the ITIM2 mutant would preferentially bind SOCS3, and IDO1s degradation was accelerated. Thus, it is the selective phosphorylation of either ITIM that controls the duration of IDO1 expression and function, in that it dictates whether enhanced tolerogenic signalling or shutdown of IDO1‐dependent events will occur in a local microenvironment.

Allosteric modulation of metabotropic glutamate receptor 4 activates IDO1-dependent, immunoregulatory signaling in dendritic cells.

Metabotropic glutamate receptor 4 (mGluR4) possesses immune modulatory properties in vivo, such that a positive allosteric modulator (PAM) of the receptor confers protection on mice with relapsing-remitting experimental autoimmune encephalomyelitis (RR-EAE). ADX88178 is a newly-developed, one such mGluR4 modulator with high selectivity, potency, and optimized pharmacokinetics. Here we found that application of ADX88178 in the RR-EAE model system converted disease into a form of mild—yet chronic—neuroinflammation that remained stable for over two months after discontinuing drug treatment. In vitro, ADX88178 modulated the cytokine secretion profile of dendritic cells (DCs), increasing production of tolerogenic IL-10 and TGF-β. The in vitro effects required activation of a Gi-independent, alternative signaling pathway that involved phosphatidylinositol-3-kinase (PI3K), Src kinase, and the signaling activity of indoleamine 2,3-dioxygenase 1 (IDO1). A PI3K inhibitor as well as small interfering RNA targeting Ido1—but not pertussis toxin, which affects Gi protein-dependent responses—abrogated the tolerogenic effects of ADX88178-conditioned DCs in vivo. Thus our data indicate that, in DCs, highly selective and potent mGluR4 PAMs such as ADX88178 may activate a Gi-independent, long-lived regulatory pathway that could be therapeutically exploited in chronic autoimmune diseases such as multiple sclerosis.

Amino-acid sensing and degrading pathways in immune regulation

Indoleamine 2,3-dioxygenases (IDOs) - belonging in the heme dioxygenase family and degrading tryptophan - are responsible for the de novo synthesis of nicotinamide adenine dinucleotide (NAD+). As such, they are expressed by a variety of invertebrate and vertebrate species. In mammals, IDO1 has remarkably evolved to expand its functions, so to become a prominent homeostatic regulator, capable of modulating infection and immunity in multiple ways, including local tryptophan deprivation, production of biologically active tryptophan catabolites, and non-enzymatic cell-signaling activity. Much like IDO1, arginase 1 (Arg1) is an immunoregulatory enzyme that catalyzes the degradation of arginine. Here, we discuss the possible role of amino-acid degradation as related to the evolution of the immune systems and how the functions of those enzymes are linked by an entwined pathway selected by phylogenesis to meet the newly arising needs imposed by an evolving environment.

The Proteasome Inhibitor Bortezomib Controls Indoleamine 2,3-Dioxygenase 1 Breakdown and Restores Immune Regulation in Autoimmune Diabetes

Bortezomib (BTZ) is a first-in-class proteasome inhibitor approved for the therapy of multiple myeloma that also displays unique regulatory activities on immune cells. The enzyme indoleamine 2,3-dioxygenase 1 (IDO1) is a tryptophan metabolizing enzyme exerting potent immunoregulatory effects when expressed in dendritic cells (DCs), the most potent antigen-presenting cells capable of promoting either immunity or tolerance. We previously demonstrated that, in inflammatory conditions, IDO1 is subjected to proteasomal degradation in DCs, turning these cells from immunoregulatory to immunostimulatory. In non-obese diabetic (NOD) mice, an experimental model of autoimmune diabetes, we also identified an IDO1 defect such that the DCs do not develop tolerance toward pancreatic islet autoantigens. We found that BTZ rescues IDO1 protein expression in vitro in a particular subset of DCs, i.e., plasmacytoid DCs (pDCs) from NOD mice. When administered in vivo to prediabetic mice, the drug prevented diabetes onset through IDO1- and pDC-dependent mechanisms. Although the drug showed no therapeutic activity when administered alone to overtly diabetic mice, its combination with otherwise suboptimal dosages of autoimmune-preventive anti-CD3 antibody resulted in disease reversal in 70% diabetic mice, a therapeutic effect similar to that afforded by full-dosage anti-CD3. Thus, our data indicate a potential for BTZ in the immunotherapy of autoimmune diabetes and further underline the importance of IDO1-mediated immune regulation in such disease.

Islet antigen-pulsed dendritic cells expressing ectopic IL-35Ig protect nonobese diabetic mice from autoimmune diabetes

Dendritic cells (DCs) are professional antigen presenting cells capable of orchestrating either stimulatory or regulatory immune responses mediated by T cells. Interleukin 35 (IL-35) is an immunosuppressive, heterodimeric cytokine belonging to the IL-12 family and known to be produced by regulatory T cells but not DCs. In this study, we explored the possible immunosuppressive effect of IL-35 ectopically expressed by splenic DCs from nonobese diabetic (NOD) mice, a prototypical model of autoimmune diabetes. After pulsing with the IGRP peptide (a dominant, diabetogenic autoantigen in NOD mice) and transfer in vivo, IL-35Ig- but not Ig-transfected DCs suppressed antigen specific, T cell-mediated responses in a skin test assay. More importantly, transfer of IL-35Ig-transfected, IGRP-pulsed DCs into prediabetic NOD mice induced a delayed and less severe form of diabetes, an effect accompanied by the increase of CD4(+)CD39(+) suppressive T cells in pancreatic lymph nodes. Our data therefore suggest that DCs overexpressing ectopic IL-35Ig might represent a powerful tool in negative vaccination strategies.

Deficiency of immunoregulatory indoleamine 2,3-dioxygenase 1 in juvenile diabetes

A defect in indoleamine 2,3-dioxygenase 1 (IDO1), which is responsible for immunoregulatory tryptophan catabolism, impairs development of immune tolerance to autoantigens in NOD mice, a model for human autoimmune type 1 diabetes (T1D). Whether IDO1 function is also defective in T1D is still unknown. We investigated IDO1 function in sera and peripheral blood mononuclear cells (PBMCs) from children with T1D and matched controls. These children were further included in a discovery study to identify SNPs in IDO1 that might modify the risk of T1D. T1D in children was characterized by a remarkable defect in IDO1 function. A common haplotype, associated with dysfunctional IDO1, increased the risk of developing T1D in the discovery and also confirmation studies. In T1D patients sharing such a common IDO1 haplotype, incubation of PBMCs in vitro with tocilizumab (TCZ) - an IL-6 receptor blocker - would, however, rescue IDO1 activity. In an experimental setting with diabetic NOD mice, TCZ was found to restore normoglycemia via IDO1-dependent mechanisms. Thus, functional SNPs of IDO1 are associated with defective tryptophan catabolism in human T1D, and maneuvers aimed at restoring IDO1 function would be therapeutically effective in at least a subgroup of T1D pediatric patients.

Loss of IDO1 Expression From Human Pancreatic β-Cells Precedes Their Destruction During the Development of Type 1 Diabetes

Indoleamine 2,3 dioxygenase-1 (IDO1) is a powerful immunoregulatory enzyme that is deficient in patients with type 1 diabetes (T1D). In this study, we present the first systematic evaluation of IDO1 expression and localization in human pancreatic tissue. Although IDO1 was constitutively expressed in β-cells from donors without diabetes, less IDO1 was expressed in insulin-containing islets from double autoantibody-positive donors and patients with recent-onset T1D, although it was virtually absent in insulin-deficient islets from donors with T1D. Scatter plot analysis suggested that IDO1 decay occurred in individuals with multiple autoantibodies, prior to β-cell demise. IDO1 impairment might therefore contribute to β-cell demise and could potentially emerge as a promising therapeutic target.