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 cytokine interleukin IL-35 is known to exert strong immunosuppressive functions. Indoleamine 2,3-dioxygenase 1 (IDO1) and Arginase 1 (Arg1) are metabolic enzymes that, expressed by dendritic cells (DCs), contribute to immunoregulation. Here, we explored any possible link between IL-35 and the activity of those enzymes. We transfected a single chain IL-35Ig gene construct in murine splenic DCs (DC35 ) and assessed any IDO1 and Arg1 activities as resulting from ectopic IL-35Ig expression, both in vitro and in vivo. Unlike Ido1, Arg1 expression was induced in vitro in DC35 , and it conferred an immunosuppressive phenotype on those cells, as revealed by a delayed-type hypersensitivity assay. Moreover, the in vivo onset of a tolerogenic phenotype in DC35 was associated with the detection of CD25+ CD39+ , rather than Foxp3+ , regulatory T cells. Therefore, Arg1, but not Ido1, expression in DC35 appears to be an early event in IL-35Ig-mediated immunosuppression.

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.

Prediabetes and diabetes in nonobese diabetic (NOD) mice have been targeted by a variety of immunotherapies, including the use of a soluble form of cytotoxic T lymphocyte antigen 4 (CTLA-4) and interferon (IFN)-gamma. The cytokine, however, fails to activate tolerogenic properties in dendritic cells (DCs) from highly susceptible female mice early in prediabetes. The defect is characterized by impaired induction of immunosuppressive tryptophan catabolism, is related to transient blockade of the signal transducer and activator of transcription (STAT)1 pathway of intracellular signaling by IFN-gamma, and is caused by peroxynitrite production. Here, we show that soluble CTLA-4 imparts suppressive properties to DCs from early prediabetic NOD female mice through mechanisms that rely on autocrine signaling by IFN-gamma. Although phosphorylation of STAT1 in response to IFN-gamma is compromised in those mice, CTLA-4 obviates the defect. IFN-gamma-driven expression of tryptophan catabolism by CTLA-4-immunoglobulin is made possible through the concomitant activation of the Forkhead Box class O (FOXO) transcription factor FOXO3a, induction of the superoxide dismutase gene, and prevention of peroxynitrite formation.

Fibroblast growth factor-2 (FGF2) plays a major role in angiogenesis. The pattern recognition receptor long-pentraxin 3 (PTX3) inhibits the angiogenic activity of FGF2. To identify novel FGF2-antagonistic peptide(s), four acetylated (Ac) synthetic peptides overlapping the FGF2-binding region PTX3-(97-110) were assessed for their FGF2-binding capacity. Among them, the shortest pentapeptide Ac-ARPCA-NH(2) (PTX3-[100-104]) inhibits the interaction of FGF2 with PTX3 immobilized to a BIAcore sensorchip and suppresses FGF2-dependent proliferation in endothelial cells, without affecting the activity of unrelated mitogens. Also, Ac-ARPCA-NH(2) inhibits angiogenesis triggered by FGF2 or by tumorigenic FGF2-overexpressing murine endothelial cells in chick and zebrafish embryos, respectively. Accordingly, the peptide hampers the binding of FGF2 to Chinese Hamster ovary cells overexpressing the tyrosine-kinase FGF receptor-1 (FGFR1) and to recombinant FGFR1 immobilized to a BIAcore sensorchip without affecting heparin interaction. In all the assays the mutated Ac-ARPSA-NH(2) peptide was ineffective. In keeping with the observation that hydrophobic interactions dominate the interface between FGF2 and the FGF-binding domain of the Ig-like loop D2 of FGFR1, amino acid substitutions in Ac-ARPCA-NH(2) and saturation transfer difference-nuclear magnetic resonance analysis of its mode of interaction with FGF2 implicate the hydrophobic methyl groups of the pentapeptide in FGF2 binding. These results will provide the basis for the design of novel PTX3-derived anti-angiogenic FGF2 antagonists.

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.

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. IDO1's 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 IDO1's 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 IDO1's 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.

Vascular endothelial growth factor-C (VEGF-C)-induced lymphangiogenesis and increased tissue drainage have been reported to inhibit acute and chronic inflammation, and an activated lymphatic endothelium might mediate peripheral tolerance. Using transgenic mice overexpressing VEGF-C in the skin, we found that under inflammatory conditions, VEGF-C-mediated expansion of the cutaneous lymphatic network establishes an immune-inhibitory microenvironment characterised by increased regulatory T (Treg) cells, immature CD11c+CD11b+ dendritic cells (DCs) and CD8+ cells exhibiting decreased effector function. Strikingly, lymphatic endothelial cell (LEC)-conditioned media (CM) potently suppress DC maturation with reduced expression of MHCII, CD40, and IL-6, and increased IL-10 and CCL2 expression. We identify an imbalance in prostaglandin synthase expression after LEC activation, favoring anti-inflammatory prostacyclin synthesis. Importantly, blockade of LEC prostaglandin synthesis partially restores DC maturity. LECs also produce TGF-ß1, contributing to the immune-inhibitory microenvironment. This study identifies novel mechanisms by which the lymphatic endothelium modulates cellular immune responses to limit inflammation.

The lymphatic vascular system plays an active role in immune cell trafficking, inflammation and cancer spread. In order to provide an in vivo tool to improve our understanding of lymphatic vessel function in physiological and pathological conditions, we generated and characterized a tdTomato reporter mouse and crossed it with a mouse line expressing Cre recombinase under the control of the lymphatic specific promoter Prox1 in an inducible fashion. We found that the tdTomato fluorescent signal recapitulates the expression pattern of Prox1 in lymphatic vessels and other known Prox1-expressing organs. Importantly, tdTomato co-localized with the lymphatic markers Prox1, LYVE-1 and podoplanin as assessed by whole-mount immunofluorescence and FACS analysis. The tdTomato reporter was brighter than a previously established red fluorescent reporter line. We confirmed the applicability of this animal model to intravital microscopy of dendritic cell migration into and within lymphatic vessels, and to fluorescence-activated single cell analysis of lymphatic endothelial cells. Additionally, we were able to describe the early morphological changes of the lymphatic vasculature upon induction of skin inflammation. The Prox1-Cre-tdTomato reporter mouse thus shows great potential for lymphatic research.

Extracellular S100B is known to affect astrocytic, neuronal and microglial activities, with different effects depending on its concentration. Whereas at relatively low concentrations S100B exerts trophic effects on neurons and astrocytes, at relatively high concentrations the protein causes neuronal apoptosis and activates astrocytes and microglia, thus potentially representing an endogenous factor implicated in neuroinflammation. We have reported that RAGE ligation by S100B in BV-2 microglia results in the upregulation of expression of the pro-inflammatory cyclo-oxygenase 2 (COX-2) via parallel Ras-Cdc42-Rac1-dependent activation of c-Jun NH(2) terminal protein kinase (JNK) and Ras-Rac1-dependent stimulation of NF-kappaB transcriptional activity. We show here that: (1) S100B also stimulates AP-1 transcriptional activity in microglia via RAGE-dependent activation of JNK; (2) S100B upregulates IL-1beta and TNF-alpha expression in microglia via RAGE engagement; and (3) S100B/RAGE-induced upregulation of COX-2, IL-1beta and TNF-alpha expression requires the concurrent activation of NF-kappaB and AP-1. We also show that S100B synergizes with IL-1beta and TNF-alpha to upregulate on COX-2 expression in microglia. Given the crucial roles of COX-2, IL-1beta and TNF-alpha in the inflammatory response, we propose that, by engaging RAGE, S100B might play an important role in microglia activation in the course of brain damage.

Indoleamine 2,3-dioxygenase 1 (IDO1) catalyzes the first step in the kynurenine pathway of tryptophan (Trp) degradation that produces several biologically active Trp metabolites. L-kynurenine (Kyn), the first byproduct by IDO1, promotes immunoregulatory effects via activation of the Aryl hydrocarbon Receptor (AhR) in dendritic cells (DCs) and T lymphocytes. We here identified the nuclear coactivator 7 (NCOA7) as a molecular target of 3-hydroxyanthranilic acid (3-HAA), a Trp metabolite produced downstream of Kyn along the kynurenine pathway. In cells overexpressing NCOA7 and AhR, the presence of 3-HAA increased the association of the two molecules and enhanced Kyn-driven, AhR-dependent gene transcription. Physiologically, conventional (cDCs) but not plasmacytoid DCs or other immune cells expressed high levels of NCOA7. In cocultures of CD4+ T cells with cDCs, the co-addition of Kyn and 3-HAA significantly increased the induction of Foxp3+ regulatory T cells and the production of immunosuppressive transforming growth factor β in an NCOA7-dependent fashion. Thus, the co-presence of NCOA7 and the Trp metabolite 3-HAA can selectively enhance the activation of ubiquitary AhR in cDCs and consequent immunoregulatory effects. Because NCOA7 is often overexpressed and/or mutated in tumor microenvironments, our current data may provide evidence for a new immune check-point mechanism based on Trp metabolism and AhR.

Target-dependent TCB activity can result in the strong and systemic release of cytokines that may develop into cytokine release syndrome (CRS), highlighting the need to understand and prevent this complex clinical syndrome.

The predisposition of nonobese diabetic (NOD) mice to develop autoimmunity reflects deficiencies in both peripheral and central tolerance. Several defects have been described in these mice, among which aberrant antigen-presenting cell function and peroxynitrite formation. Prediabetes and diabetes in NOD mice have been targeted with different outcomes by a variety of immunotherapies, including interferon (IFN)-gamma. This cytokine may be instrumental in specific forms of tolerance by virtue of its ability to activate immunosuppressive tryptophan catabolism. Here, we provide evidence that IFN-gamma fails to induce tolerizing properties in dendritic cells from highly susceptible female mice early in prediabetes. This effect is associated with impaired tryptophan catabolism, is related to transient blockade of the Stat1 pathway of intracellular signaling by IFN-gamma, and is caused by peroxynitrite production. However, the use of a peroxynitrite inhibitor can rescue tryptophan catabolism and tolerance in those mice. This is the first report of an experimental autoimmune disease in which defective tolerance is causally linked to impaired tryptophan catabolism.

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.