Exploiting ELIOT for Scaffold-Repurposing Opportunities: TRIM33 a Possible Novel E3 Ligase to Expand the Toolbox for PROTAC Design.

The field of targeted protein degradation, through the control of the ubiquitin-proteasome system (UPS), is progressing considerably; to exploit this new therapeutic modality, the proteolysis targeting chimera (PROTAC) technology was born. The opportunity to use PROTACs engaging of new E3 ligases that can hijack and control the UPS system could greatly extend the applicability of degrading molecules. To this end, here we show a potential application of the ELIOT (E3 LIgase pocketOme navigaTor) platform, previously published by this group, for a scaffold-repurposing strategy to identify new ligands for a novel E3 ligase, such as TRIM33. Starting from ELIOT, a case study of the cross-relationship using GRID Molecular Interaction Field (MIF) similarities between TRIM24 and TRIM33 binding sites was selected. Based on the assumption that similar pockets could bind similar ligands and considering that TRIM24 has 12 known co-crystalised ligands, we applied a scaffold-repurposing strategy for the identification of TRIM33 ligands exploiting the scaffold of TRIM24 ligands. We performed a deeper computational analysis to identify pocket similarities and differences, followed by docking and water analysis; selected ligands were synthesised and subsequently tested against TRIM33 via HTRF binding assay, and we obtained the first-ever X-ray crystallographic complexes of TRIM33α with three of the selected compounds.

Melatonin modulates Nrf2 activity to protect porcine pre-pubertal Sertoli cells from the abnormal H2 O2 generation and reductive stress effects of cadmium.

Melatonin (MLT) is a cytoprotective agent holding potential to prevent cadmium (Cd) toxicity and its impact in testicular function and fertility. In this study, we explored such potential in porcine pre-pubertal Sertoli cells (SCs). Cd toxicity resulted in impaired SC viability and function, abnormal cellular H2 O2 generation and efflux, and induction of reductive stress by the upregulation of Nrf2 expression and activity, cystine uptake and glutathione biosynthesis, glutathione-S-transferase P (GSTP) expression, and protein glutathionylation inhibition. Cd toxicity also stimulated the activity of cellular kinases (MAPK-ERK1/2 and Akt) and NFkB transcription factor, and cJun expression was increased. MLT produced a potent cytoprotective effect when co-administered with Cd to SCs; its efficacy and the molecular mechanism behind its cytoprotective function varied according to Cd concentrations. However, a significant restoration of cell viability and function, and of H2 O2 levels, was observed both at 5 and 10 µM Cd. Mechanistically, these effects of MLT were associated with a significant reduction of the Cd-induced activation of Nrf2 and GSTP expression at all Cd concentrations. CAT and MAPK-ERK1/2 activity upregulation was associated with these effects at 5 µM Cd, whereas glutathione biosynthesis and efflux were involved at 10 µM Cd together with an increased expression of the cystine transporter xCT, of cJun and Akt and NFkB activity. MLT protects SCs from Cd toxicity reducing its H2 O2 generation and reductive stress effects. A reduced activity of Nrf2 and the modulation of other molecular players of MLT signaling, provide a mechanistic rational for the cytoprotective effect of this molecule in SCs.

Endoplasmic reticulum stress and NF-kB activation in SARS-CoV-2 infected cells and their response to antiviral therapy.

Unfolded protein response (UPR) and endoplasmic reticulum (ER) stress are aspects of SARS-CoV-2-host cell interaction with proposed role in the cytopathic and inflammatory pathogenesis of this viral infection. The role of the NF-kB pathway in these cellular processes remains poorly characterized. When investigated in VERO-E6 cells, SARS-CoV-2 infection was found to markedly stimulate NF-kB protein expression and activity. NF-kB activation occurs early in the infection process (6 hpi) and it is associated with increased MAPK signaling and expression of the UPR inducer IRE-1α. These signal transduction processes characterize the cellular stress response to the virus promoting a pro-inflammatory environment and caspase activation in the host cell. Inhibition of viral replication by the viral protease inhibitor Nelfinavir reverts all these molecular changes also stimulating c-Jun expression, a key component of the JNK/AP-1 pathway with important role in the IRE-1α-mediated transcriptional regulation of stress response genes with anti-inflammatory and cytoprotection function. The present study demonstrates that UPR signaling and its interaction with cellular MAPKs and the NF-kB activity are important aspects of SARS-CoV-2-host cell interaction that deserve further investigation to identify more efficient therapies for this viral infection.

SARS-CoV2 infection impairs the metabolism and redox function of cellular glutathione.

Viral infections sustain their replication cycle promoting a pro-oxidant environment in the host cell. In this context, specific alterations of the levels and homeostatic function of the tripeptide glutathione have been reported to play a causal role in the pro-oxidant and cytopathic effects (CPE) of the virus. In this study, these aspects were investigated for the first time in SARS-CoV2-infected Vero E6 cells, a reliable and well-characterized in vitro model of this infection. SARS-CoV2 markedly decreased the levels of cellular thiols, essentially lowering the reduced form of glutathione (GSH). Such an important defect occurred early in the CPE process (in the first 24 hpi). Thiol analysis in N-acetyl-Cys (NAC)-treated cells and membrane transporter expression data demonstrated that both a lowered uptake of the GSH biosynthesis precursor Cys and an increased efflux of cellular thiols, could play a role in this context. Increased levels of oxidized glutathione (GSSG) and protein glutathionylation were also observed along with upregulation of the ER stress marker PERK. The antiviral drugs Remdesivir (Rem) and Nelfinavir (Nel) influenced these changes at different levels, essentially confirming the importance or blocking viral replication to prevent GSH depletion in the host cell. Accordingly, Nel, the most potent antiviral in our in vitro study, produced a timely activation of Nrf2 transcription factor and a GSH enhancing response that synergized with NAC to restore GSH levels in the infected cells. Despite poor in vitro antiviral potency and GSH enhancing function, Rem treatment was found to prevent the SARS-CoV2-induced glutathionylation of cellular proteins. In conclusion, SARS-CoV2 infection impairs the metabolism of cellular glutathione. NAC and the antiviral Nel can prevent such defect in vitro.

Novel mutations in the WFS1 gene are associated with Wolfram syndrome and systemic inflammation.

Mutations in the WFS1 gene, encoding wolframin (WFS1), cause endoplasmic reticulum (ER) stress and are associated with a rare autosomal-recessive disorder known as Wolfram syndrome (WS). WS is clinically characterized by childhood-onset diabetes mellitus, optic atrophy, deafness, diabetes insipidus and neurological signs. We identified two novel WFS1 mutations in a patient with WS, namely, c.316-1G > A (in intron 3) and c.757A > T (in exon 7). Both mutations, located in the N-terminal region of the protein, were predicted to generate a truncated and inactive form of WFS1. We found that although the WFS1 protein was not expressed in peripheral blood mononuclear cells (PBMCs) of the proband, no constitutive ER stress activation could be detected in those cells. In contrast, WS proband's PBMCs produced very high levels of proinflammatory cytokines (i.e. TNF-α, IL-1ß, and IL-6) in the absence of any stimulus. WFS1 silencing in PBMCs from control subjects by means of small RNA interference also induced a pronounced proinflammatory cytokine profile. The same cytokines were also significantly higher in sera from the WS patient as compared to matched healthy controls. Moreover, the chronic inflammatory state was associated with a dominance of proinflammatory T helper 17 (Th17)-type cells over regulatory T (Treg) lymphocytes in the WS PBMCs. The identification of a state of systemic chronic inflammation associated with WFS1 deficiency may pave the way to innovative and personalized therapeutic interventions in WS.

Aspergillus fumigatus tryptophan metabolic route differently affects host immunity.

Indoleamine 2,3-dioxygenases (IDOs) degrade l-tryptophan to kynurenines and drive the de novo synthesis of nicotinamide adenine dinucleotide. Unsurprisingly, various invertebrates, vertebrates, and even fungi produce IDO. In mammals, IDO1 also serves as a homeostatic regulator, modulating immune response to infection via local tryptophan deprivation, active catabolite production, and non-enzymatic cell signaling. Whether fungal Idos have pleiotropic functions that impact on host-fungal physiology is unclear. Here, we show that Aspergillus fumigatus possesses three ido genes that are expressed under conditions of hypoxia or tryptophan abundance. Loss of these genes results in increased fungal pathogenicity and inflammation in a mouse model of aspergillosis, driven by an alternative tryptophan degradation pathway to indole derivatives and the host aryl hydrocarbon receptor. Fungal tryptophan metabolic pathways thus cooperate with the host xenobiotic response to shape host-microbe interactions in local tissue microenvironments.

Class IA PI3Ks regulate subcellular and functional dynamics of IDO1.

Knowledge of a protein's spatial dynamics at the subcellular level is key to understanding its function(s), interactions, and associated intracellular events. Indoleamine 2,3-dioxygenase 1 (IDO1) is a cytosolic enzyme that controls immune responses via tryptophan metabolism, mainly through its enzymic activity. When phosphorylated, however, IDO1 acts as a signaling molecule in plasmacytoid dendritic cells (pDCs), thus activating genomic effects, ultimately leading to long-lasting immunosuppression. Whether the two activities-namely, the catalytic and signaling functions-are spatially segregated has been unclear. We found that, under conditions favoring signaling rather than catabolic events, IDO1 shifts from the cytosol to early endosomes. The event requires interaction with class IA phosphoinositide 3-kinases (PI3Ks), which become activated, resulting in full expression of the immunoregulatory phenotype in vivo in pDCs as resulting from IDO1-dependent signaling events. Thus, IDO1's spatial dynamics meet the needs for short-acting as well as durable mechanisms of immune suppression, both under acute and chronic inflammatory conditions. These data expand the theoretical basis for an IDO1-centered therapy in inflammation and autoimmunity.

Childhood survivors of high-risk neuroblastoma show signs of immune recovery and not immunosenescence.

Neuroblastoma survivors show signs of immunosenescence early after therapy in CD8+ T cell compartment and elevated plasma TNF-α but in later follow-up immune recovery comes into play. Whether the recovery phenotype is long lasting or transient remains to be elucidated, however, late adverse effects often occur in childhood cancer survivors.

Positive allosteric modulation of indoleamine 2,3-dioxygenase 1 restrains neuroinflammation.

l-tryptophan (Trp), an essential amino acid for mammals, is the precursor of a wide array of immunomodulatory metabolites produced by the kynurenine and serotonin pathways. The kynurenine pathway is a paramount source of several immunoregulatory metabolites, including l-kynurenine (Kyn), the main product of indoleamine 2,3-dioxygenase 1 (IDO1) that catalyzes the rate-limiting step of the pathway. In the serotonin pathway, the metabolite N-acetylserotonin (NAS) has been shown to possess antioxidant, antiinflammatory, and neuroprotective properties in experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis (MS). However, little is known about the exact mode of action of the serotonin metabolite and the possible interplay between the 2 Trp metabolic pathways. Prompted by the discovery that NAS neuroprotective effects in EAE are abrogated in mice lacking IDO1 expression, we investigated the NAS mode of action in neuroinflammation. We found that NAS directly binds IDO1 and acts as a positive allosteric modulator (PAM) of the IDO1 enzyme in vitro and in vivo. As a result, increased Kyn will activate the ligand-activated transcription factor aryl hydrocarbon receptor and, consequently, antiinflammatory and immunoregulatory effects. Because NAS also increased IDO1 activity in peripheral blood mononuclear cells of a significant proportion of MS patients, our data may set the basis for the development of IDO1 PAMs as first-in-class drugs in autoimmune/neuroinflammatory diseases.

Effects of probiotic administration on immune responses of children and adolescents with type 1 diabetes to a quadrivalent inactivated influenza vaccine.

This study was planned to evaluate whether a 3-month treatment with Lactobacillus rhamnosus GG (LGG) can modify immune system functions in children and adolescents with type 1 diabetes (T1D), leading to an increased immune response to an injectable quadrivalent inactivated influenza vaccine (QIV). A total of 87 pediatric patients with T1D were screened, although 34 patients in the Probiotic group and 30 in the Control group accepted to be vaccinated with QIV and completed the study. Vaccine immunogenicity and safety and the inflammatory cytokine response were studied. Results showed that QIV was immunogenic and safe in T1D pediatric patients and pre-administration of LGG for three months did not substantially modify the QIV humoral immunity. The combination of QIV and LGG reduced inflammatory responses (i.e., IFN-γ, IL17A, IL-17F, IL-6, and TNF-α) from activated PBMCs of pediatric patients with T1D, without dampening the production of seroprotective antibodies. In conclusion, QIV is associated with an adequate immunogenicity in children and adolescents with T1D in presence of a good safety profile. Although a systematic administration of LGG did not result in an improvement of humoral responses to an influenza vaccine, the probiotic did induce important anti-inflammatory effects.

Targeting the Aryl Hydrocarbon Receptor With Indole-3-Aldehyde Protects From Vulvovaginal Candidiasis via the IL-22-IL-18 Cross-Talk.

Vulvovaginal candidiasis (VVC) is a common mucosal infection caused by Candida spp., most frequently by Candida albicans, which may become recurrent and severely impacting the quality of life of susceptible women. Although it is increasingly being recognized that mucosal damage is mediated by an exaggerated inflammatory response, current therapeutic approaches are only based on antifungals that may relieve the symptomatology, but fail to definitely prevent recurrences. The unrestrained activation of the NLRP3 inflammasome with continuous production of IL-1ß and recruitment of neutrophils is recognized as a pathogenic factor in VVC. We have previously shown that IL-22 is required to dampen pathogenic inflammasome activation in VVC via the NLRC4/IL-1Ra axis. However, IL-22 also regulates IL-18, a product of the inflammasome activity that regulates IL-22 expression. Here we describe a cross-regulatory circuit between IL-18 and IL-22 in murine VVC that is therapeutically druggable. We found that IL-18 production was dependent on IL-22 and NLRC4, and that IL-18, in turn, contributes to IL-22 activity. Like in IL-22 deficiency, IL-18 deficiency was associated with an increased susceptibility to VVC and unbalanced Th17/Treg response, suggesting that IL-18 can regulate both the innate and the adaptive responses to the fungus. Administration of the microbial metabolite indole-3-aldehyde, known to stimulate the production of IL-22 via the aryl hydrocarbon receptor (AhR), promoted IL-18 expression and protection against Candida infection. Should low levels of IL-18 be demonstrated in the vaginal fluids of women with recurrent VVC, targeting the AhR/IL-22/IL-18 pathway could be exploited for future therapeutic approaches in VVC. This study suggests that a deeper understanding of the mechanisms regulating inflammasome activity may lead to the identification of novel targets for intervention in VVC.

Engagement of Nuclear Coactivator 7 by 3-Hydroxyanthranilic Acid Enhances Activation of Aryl Hydrocarbon Receptor in Immunoregulatory Dendritic Cells.

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 ß 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.

Genetic Polymorphisms Affecting IDO1 or IDO2 Activity Differently Associate With Aspergillosis in Humans.

Aspergillus is the causative agent of human diseases ranging from asthma to invasive infection. Genetic and environmental factors are crucial in regulating the interaction between the host and Aspergillus. The role played by the enzyme indoleamine 2,3-dioxygenase 1 (IDO1), which catalyzes the first and rate-limiting step of tryptophan catabolism along the kynurenine pathway, is increasingly being recognized, but whether and how genetic variation of IDO1 influences the risk of aspergillosis in susceptible patients is incompletely understood. In addition, whether the closely related protein IDO2 plays a similar role remains unexplored. In the present study, we performed genetic association studies in two different cohorts of susceptible patients [cystic fibrosis (CF) patients and recipients of hematopoietic stem cell transplantation (HSCT)], and identified IDO1 polymorphisms that associate with the risk of infection in both cohorts. By using human bronchial epithelial cells and PBMC from CF and HSCT patients, respectively, we could show that the IDO1 polymorphisms appeared to down-modulate IDO1 expression and function in response to IFNγ or Aspergillus conidia, and to associate with an increased inflammatory response. In contrast, IDO2 polymorphisms, including variants known to profoundly affect protein expression and function, were differently associated with the risk of aspergillosis in the two cohorts of patients as no association was found in CF patients as opposed to recipients of HSCT. By resorting to a murine model of bone marrow transplantation, we could show that the absence of IDO2 more severely affected fungal burden and lung pathology upon infection with Aspergillus as compared to IDO1, and this effect appeared to be linked to a deficit in the antifungal effector phagocytic activity. Thus, our study confirms and extends the role of IDO1 in the response to Aspergillus, and shed light on the possible involvement of IDO2 in specific clinical settings.

Deficiency of immunoregulatory indoleamine 2,3-dioxygenase 1in 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.

An intensive lifestyle intervention reduces circulating oxidised low-density lipoprotein and increases human paraoxonase activity in obese subjects.

OBJECTIVE: Obesity has a great impact on cardiovascular morbidity and mortality, the treatment of this pathological state is important given the significant health consequences. Lifestyle and behaviour changes play a significant role in weight management. The purpose of this study was to investigate the impact of an intensive multidisciplinary lifestyle intervention on well-known atherogenic factors in a group of overweight and obese subjects. METHODS: A total of 44 people with overweight/obesity underwent a lifestyle intervention based on nutritional education, psychological support and a 3-month exercise training program with a frequency of twice a week. Several anthropometric and biochemical parameters were measured before and after the lifestyle intervention. RESULTS: Lifestyle intervention led to a significant reduction in metabolic profile including body mass index (BMI), waist circumference, systolic and diastolic blood pressure, plasma glucose, and plasma triglycerides. These reductions were also accompanied by a significant increase in maximal oxygen consumption and muscle strength. Furthermore, paraoxonase and lactonase activities and the concentration of Apoliproteins A1 (APO A1) were significantly increased and the serum levels of oxLDL reduced without any changes in the circulating levels of LDL and HDL. CONCLUSION: In conclusion, our study suggests that an intensive lifestyle intervention in obese subjects promotes a series of beneficial antiatherogenic changes which included increased enzyme activites of paraoxonase and lactonase, concentration of Apoliproteins A1 and decreased circulating levels of oxLDL.

Disease Tolerance Mediated by Phosphorylated Indoleamine-2,3 Dioxygenase Confers Resistance to a Primary Fungal Pathogen.

Resistance to primary fungal pathogens is usually attributed to the proinflammatory mechanisms of immunity conferred by interferon-γ activation of phagocytes that control microbial growth, whereas susceptibility is attributed to anti-inflammatory responses that deactivate immunity. This study challenges this paradigm by demonstrating that resistance to a primary fungal pathogen such as Paracoccidiodes brasiliensis can be mediated by disease tolerance, a mechanism that preserves host fitness instead of pathogen clearance. Among the mechanisms of disease tolerance described, a crucial role has been ascribed to the enzyme indoleamine-2,3 dioxygenase (IDO) that concomitantly controls pathogen growth by limiting tryptophan availability and reduces tissue damage by decreasing the inflammatory process. Here, we demonstrated in a pulmonary model of paracoccidioidomycosis that IDO exerts a dual function depending on the resistant pattern of hosts. IDO activity is predominantly enzymatic and induced by IFN-γ signaling in the pulmonary dendritic cells (DCs) from infected susceptible (B10.A) mice, whereas phosphorylated IDO (pIDO) triggered by TGF-ß activation of DCs functions as a signaling molecule in resistant mice. IFN-γ signaling activates the canonical pathway of NF-κB that promotes a proinflammatory phenotype in B10.A DCs that control fungal growth but ultimately suppress T cell responses. In contrast, in A/J DCs IDO promotes a tolerogenic phenotype that conditions a sustained synthesis of TGF-ß and expansion of regulatory T cells that avoid excessive inflammation and tissue damage contributing to host fitness. Therefore, susceptibility is unexpectedly mediated by mechanisms of proinflammatory immunity that are usually associated with resistance, whereas genetic resistance is based on mechanisms of disease tolerance mediated by pIDO, a phenomenon never described in the protective immunity against primary fungal pathogens.

Modulation of natriuretic peptide receptors in human adipose tissue: molecular mechanisms behind the "natriuretic handicap" in morbidly obese patients.

The B-type natriuretic peptide (BNP) hormone plays a crucial role in the regulation of cardiovascular and energy homeostasis. Obesity is associated with low circulating levels of BNP, a condition known as "natriuretic handicap." Recent evidences suggest an altered expression of BNP receptors-both the signaling natriuretic peptide receptors (NPR)-A and the clearance NPR-C receptor-in adipose tissue (AT) as one of the putative causes of natriuretic handicap. The current study aims at clarifying the molecular mechanisms behind the natriuretic handicap, focusing on NPR modulation in the AT of obese and control subjects. The study enrolled 34 obese and 20 control subjects undergoing bariatric or abdominal surgery, respectively. The main clinical and biochemical parameters, including circulating BNP, were assessed. In visceral (VAT) and subcutaneous AT (SAT) samples, collected during surgery, the adipocytes and stromal vascular fraction (SVF) expression of NPR-A and NPR-C and the SVF secretion of interleukin 6 (IL-6) were determined. Both VAT and SAT from obese patients expressed a lower NPR-A/NPR-C ratio in adipocytes and the SVF secreted a higher level of IL-6, compared with the controls. Moreover, NPR-A/NPR-C ratio expressed by VAT and SAT adipocytes negatively correlated with body mass index, insulin, the Homeostasis Model Assessment of Insulin resistance, and IL-6 secreted by SVF, and the expression of the clearance receptor NPR-C, in both the VAT and SAT adipocytes, showed a negative correlation with circulating BNP. Overall, insulin resistance/hyperinsulinemia and AT inflammation (ie, high level of IL-6) are the major determinants of the lower NPR-A/NPR-C ratio in adipocytes, thus contributing to the natriuretic handicap in obese subjects.

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.

CpG Type A Induction of an Early Protective Environment in Experimental Multiple Sclerosis.

Experimental autoimmune encephalomyelitis (EAE) is an inflammatory, demyelinating disease of the CNS that mimics human multiple sclerosis (MS), and it is thought to be driven by Th1 and Th17 myelin-reactive cells. Although adaptive immunity is clearly pivotal in the pathogenesis of EAE, with an essential role of CD4+ T cells, little is known of early, innate responses in this experimental setting. CpG-rich oligodeoxynucleotides (ODNs), typically found in microbial genomes, are potent activators of TLR9 in plasmacytoid dendritic cells (pDCs). In this study, we compared the effects of two types of CpG, namely, type A and type B, on EAE. We found that treatment with CpG type A ODN (CpG-A), known to induce high amounts of IFN-α in pDCs, significantly reduced disease severity in EAE, relative to controls (12.63 ± 1.86 versus 23.49 ± 1.46, resp.; p = 0.001). Treatment also delayed onset of neurological deficits and reduced spinal cord demyelination, while increasing the percentage of splenic regulatory (Foxp3+ CD4+) T cells. CpG-A likewise reduced the levels of IL-17 and IFN-γ in the CNS. Mechanistic insight into those events showed that CpG-A promoted a regulatory phenotype in pDCs. Moreover, adoptive transfer of pDCs isolated from CpG-A-treated mice inhibited CNS inflammation and induced disease remission in acute-phase EAE. Our data thus identify a link between TLR9 activation by specific ligands and the induction of tolerance via innate immunity mechanisms.

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

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.