Synergistic effect of endurance training and nettle leaf extract on the IDO1-KYN-AHR pathway homeostasis and inhibiting of liver toxicity in rats with STZ-induced diabetes.

Introduction: Diabetes adversely affects a number of hepatic molecular pathways, including the kynurenine (KYN) pathway. KYN is produced by indoleamine 2,3-dioxygenase (IDO) and activates the aryl hydrocarbon receptor (AHR). This study evaluated the effect of endurance training (EndTr) and nettle leaf extract (NLE) on the IDO1-KYN-AHR pathway in the livers of rats with streptozotocin-induced diabetes. Methods: We divided 48 rats into six groups: controls (Ct), treated with EndTr (EndTr), diabetes-induced (D), D treated with NLE (D + NLE), D treated with EndTr (D + EnTr), and D treated with EndTr and NLE (D + EndTr + NLE). EndTr, D + EnTr, and D + EndTr + NLE groups were subjected to training with running on treadmill for 8 weeks, 5 days per week, 25 min in first session to 59 min at last session with intensity of 55% to 65% VO2max. Using real-time PCR gene (Ahr, Cyp1a1, and Ido1) expressions and ELISA, malondialdehyde (MDA) and protein (IDO1, AHR, and CYP1A1) levels were determined in the liver samples. Results: A significant three-way interaction of exercise, nettle, and diabetes was observed on the all variables (P< 0.001). In particular, significant increases in blood glucose level (BGL), in gene and protein expression, and in MDA and KYN levels were observed in the liver samples of the D group versus the Ct group (P< 0.05). BGL and liver MDA levels were significantly lower in the D + EndTr and D + NLE groups than that in the D group. However, the D + EndTr + NLE group showed a more significant decrease in these factors (P< 0.05). In addition, liver KYN levels were significantly lower in the EndTr group compared with that in the Ct group as well as in the D + EndTr + NLE and D + EndTr groups compared with that in the D groups (P< 0.05). Whereas both the EndTr and D + NLE groups showed lower Ahr expression and AHR level compared with the Ct and D groups, respectively (P< 0.05), the D + EndTr + NLE group showed a higher significant reduction in the AHR level than the D group (P< 0.05). The Cyp1a1 expression and IDO1 level significantly decreased only in the D + EndTr + NLE group compared to that in the D group (P< 0.05). Conclusion: Overall, this study showed that the combination of EndTr and NLE may synergistically restore the imbalanced IDO1-KYN-AHR pathway in diabetic liver.

Myricetin inhibits interferon-γ-induced PD-L1 and IDO1 expression in lung cancer cells.

Programmed death ligand-1 (PD-L1) and indoleamine 2, 3-dioxygenase 1 (IDO1) are immune checkpoints induced by interferon-γ (IFN-γ) in the tumor microenvironment, leading to immune escape of tumors. Myricetin (MY) is a flavonoid distributed in many edible and medicinal plants. In this study, MY was identified to inhibit IFN-γ-induced PD-L1 expression in human lung cancer cells. It also reduced the expression of IDO1 and the production of kynurenine which is the product catalyzed by IDO1, while didn't show obvious effect on the expression of major histocompatibility complex-I (MHC-I), a crucial molecule for antigen presentation. In addition, the function of T cells was evaluated using a co-culture system consist of lung cancer cells and the Jurkat-PD-1 T cell line overexpressing PD-1. MY restored the survival, proliferation, CD69 expression and interleukin-2 (IL-2) secretion of Jurkat-PD-1 T cells suppressed by IFN-γ-treated lung cancer cells. Mechanistically, IFN-γ up-regulated PD-L1 and IDO1 at the transcriptional level through the JAK-STAT-IRF1 axis, which was targeted and inhibited by MY. Together, our research revealed a new mechanism of MY mediated anti-tumor activity and highlighted the potential implications of MY in tumor immunotherapy.

Kynurenine pathway activation and deviation to anthranilic and kynurenic acid in fibrosing chronic graft-versus-host disease.

Fibrosing chronic graft-versus-host disease (cGVHD) is a debilitating complication of allogeneic stem cell transplantation (alloSCT). A driver of fibrosis is the kynurenine (Kyn) pathway, and Kyn metabolism patterns and cytokines may influence cGVHD severity and manifestation (fibrosing versus gastrointestinal [GI] cGVHD). Using a liquid chromatography-tandem mass spectrometry approach on sera obtained from 425 patients with allografts, we identified high CXCL9, high indoleamine-2,3-dioxygenase (IDO) activity, and an activated Kyn pathway as common characteristics in all cGVHD subtypes. Specific Kyn metabolism patterns could be identified for non-severe cGVHD, severe GI cGVHD, and fibrosing cGVHD, respectively. Specifically, fibrosing cGVHD was associated with a distinct pathway shift toward anthranilic and kynurenic acid, correlating with reduced activity of the vitamin-B2-dependent kynurenine monooxygenase, low vitamin B6, and increased interleukin-18. The Kyn metabolite signature is a candidate biomarker for severe fibrosing cGVHD and provides a rationale for translational trials on prophylactic vitamin B2/B6 supplementation for cGVHD prevention.

Serum Short-Chain Fatty Acids and Associations With Inflammation in Newly Diagnosed Patients With Multiple Sclerosis and Healthy Controls.

Multiple sclerosis (MS) is a chronic immune-mediated disease characterized by demyelination and neuroaxonal damage in the central nervous system. The etiology is complex and is still not fully understood. Accumulating evidence suggests that our gut microbiota and its metabolites influence the MS pathogenesis. Short-chain fatty acids (SCFAs), such as acetate, propionate and butyrate, are metabolites produced by gut microbiota through fermentation of indigestible carbohydrates. SCFAs and kynurenine metabolites have been shown to have important immunomodulatory properties, and propionate supplementation in MS patients has been associated with long-term clinical improvement. However, the underlying mechanisms of action and its importance in MS remain incompletely understood. We analyzed serum levels of SCFAs and performed targeted metabolomics in relation to biomarkers of inflammation, and clinical and MRI measures in newly diagnosed patients with relapsing-remitting MS before their first disease modifying therapy and healthy controls (HCs). We demonstrated that serum acetate levels were nominally reduced in MS patients compared with HCs. The ratios of acetate/butyrate and acetate/(propionate + butyrate) were significantly lower in MS patients in a multivariate analysis (orthogonal partial least squares discriminant analysis; OPLS-DA). The mentioned ratios and acetate levels correlated negatively with the pro-inflammatory biomarker IFNG, indicating an inverse relation between acetate and inflammation. In contrast, the proportion of butyrate was found higher in MS patients in the multivariate analysis, and both butyrate and valerate correlated positively with proinflammatory cytokines (IFNG and TNF), suggesting complex bidirectional regulatory properties of SCFAs. Branched SCFAs were inversely correlated with clinical disability, at a nominal significance level. Otherwise SCFAs did not correlate with clinical variables or MRI measures. There were signs of an alteration of the kynurenine pathway in MS, and butyrate was positively correlated with the immunomodulatory metabolite 3-hydroxyanthranilic acid. Other variables that influenced the separation between MS and HCs were NfL, ARG1 and IL1R1, D-ribose 5-phosphate, pantothenic acid and D-glucuronic acid. In conclusion, we provide novel results in this rapidly evolving field, emphasizing the complexity of the interactions between SCFAs and inflammation; therefore, further studies are required to clarify these issues before supplementation of SCFAs can be widely recommended.

Indoleamine 2,3-Dioxygenase Activity-Induced Acceleration of Tumor Growth, and Protein Kinases-Related Novel Therapeutics Regimens.

Indoleamine 2,3-dioxygenase (IDO) is overexpressed in response to interferon-gamma (IFN-γ). IDO-mediated degradation of tryptophan (Trp) along the kynurenine (Kyn) pathway by immune cells is associated with the anti-microbial, and anti-tumor defense mechanisms. In contrast, IDO is constitutively expressed by various tumors and creates an immunosuppressive microenvironment around the tumor tissue both by depletion of the essential amino acid Trp and by formation of Kyn, which is immunosuppressive metabolite of Trp. IDO may activate its own expression in human cancer cells via an autocrine aryl hydrocarbon receptor (AhR)- interleukin 6 (IL-6)-signal transducer and activator of transcription 3 (STAT3) signaling loop. Although IDO is not a unique marker, in many clinical trials serum IDO activity is suggested to be an important parameter in the pathogenesis of cancer development and growth. Measuring IDO activity in serum seems to be an indicator of cancer growth rate, however, it is controversial whether this approach can be used as a reliable guide in cancer patients treated with IDO inhibitors. Thus, IDO immunostaining is strongly recommended for the identification of higher IDO producing tumors, and IDO inhibitors should be included in post-operative complementary therapy in IDO positive cancer cases only. Novel therapies that target the IDO pathway cover checkpoint protein kinases related combination regimens. Currently, multi-modal therapies combining IDO inhibitors and checkpoint kinase blockers in addition to T regulatory (Treg) cell-modifying treatments seem promising.

AhR and IDO1 in pathogenesis of Covid-19 and the "Systemic AhR Activation Syndrome:" a translational review and therapeutic perspectives.

Covid-19 is the acute illness caused by SARS-CoV-2 with initial clinical symptoms such as cough, fever, malaise, headache, and anosmia. After entry into cells, corona viruses (CoV) activate aryl hydrocarbon receptors (AhRs) by an indoleamine 2,3-dioxygenase (IDO1)-independent mechanism, bypassing the IDO1-kynurenine-AhR pathway. The IDO1-kynurenine-AhR signaling pathway is used by multiple viral, microbial and parasitic pathogens to activate AhRs and to establish infections. AhRs enhance their own activity through an IDO1-AhR-IDO1 positive feedback loop prolonging activation induced by pathogens. Direct activation of AhRs by CoV induces immediate and simultaneous up-regulation of diverse AhR-dependent downstream effectors, and this, in turn, results in a "Systemic AhR Activation Syndrome" (SAAS) consisting of inflammation, thromboembolism, and fibrosis, culminating in multiple organ injuries, and death. Activation of AhRs by CoV may lead to diverse sets of phenotypic disease pictures depending on time after infection, overall state of health, hormonal balance, age, gender, comorbidities, but also diet and environmental factors modulating AhRs. We hypothesize that elimination of factors known to up-regulate AhRs, or implementation of measures known to down-regulate AhRs, should decrease severity of infection. Although therapies selectively down-regulating both AhR and IDO1 are currently lacking, medications in clinical use such as dexamethasone may down-regulate both AhR and IDO1 genes, as calcitriol/vitamin D3 may down-regulate the AhR gene, and tocopherol/vitamin E may down-regulate the IDO1 gene. Supplementation of calcitriol should therefore be subjected to epidemiological studies and tested in prospective trials for prevention of CoV infections, as should tocopherol, whereas dexamethasone could be tried in interventional trials. Because lack of physical exercise activates AhRs via the IDO1-kynurenine-AhR signaling pathway increasing risk of infection, physical exercise should be encouraged during quarantines and stay-at-home orders during pandemic outbreaks. Understanding which factors affect gene expression of both AhR and IDO1 may help in designing therapies to prevent and treat humans suffering from Covid-19.

Prunella vulgaris L. attenuates experimental autoimmune thyroiditis by inducing indoleamine 2,3-dioxygenase 1 expression and regulatory T cell expansion.

BACKGROUND: Prunella vulgaris L. (P. vulgaris) has traditionally been used to treat swelling and inflammation of the thyroid gland. This study aimed to evaluate the effects of P. vulgaris on experimental autoimmune thyroiditis (EAT) and explore the roles of indoleamine 2,3-dioxygenase 1 (IDO1) and regulatory T cells (Tregs) in these P. vulgaris-mediated effects. METHODS: The main bioactive compounds in P. vulgaris were analysed by high-performance liquid chromatography. An EAT model was established by immunization of Lewis rats with thyroglobulin via subcutaneous injection. Thyroid volume was assessed by ultrasound, and lymphatic infiltration in the thyroid was evaluated by haematoxylin and eosin staining. The serum levels of thyroglobulin antibody (TgAb) and cytokines were measured by indirect enzyme-linked immunosorbent assay. The percentage of CD4+CD25+Foxp3+ Tregs was detected by flow cytometry. The mRNA and protein levels of IDO1 were measured by qRT-PCR and Western blotting, respectively. The levels of tryptophan (Trp) and kynurenine (Kyn) in serum and faecal samples were assessed with a fluorometric kit and spectrophotometry. RESULTS: The main bioactive compound in P. vulgaris was rosmarinic acid. The TgAb level and thyroid volume in EAT rats were significantly decreased after administration of P. vulgaris (P < 0.01). The inflammation score in EAT rats that were administered P. vulgaris was significantly lower than that in the EAT controls (P < 0.01). In addition, P. vulgaris promoted the expansion of splenic Tregs and increased the production of IL-10 and TGF-ß (P < 0.01) in EAT rats. Moreover, P. vulgaris induced IDO1 mRNA and protein expression in the spleen and intestine in P. vulgaris-treated EAT rats (P < 0.01). Finally, Trp levels were reduced and Kyn levels and the Kyn/Trp ratio were increased in the serum of P. vulgaris-treated EAT rats. CONCLUSION: We were the first to demonstrate the role of IDO1-induced Treg expansion in P. vulgaris-mediated attenuation of EAT. Our study provides insight into the immunopathogenesis of autoimmune thyroiditis and shows the potential therapeutic value of P. vulgaris.

Antidepressant-Like Effect and Mechanism of Action of Honokiol on the Mouse Lipopolysaccharide (LPS) Depression Model.

There is growing evidence that neuroinflammation is closely linked to depression. Honokiol, a biologically active substance extracted from Magnolia officinalis, which is widely used in traditional Chinese medicine, has been shown to exert significant anti-inflammatory effects and improve depression-like behavior caused by inflammation. However, the specific mechanism of action of this activity is still unclear. In this study, the lipopolysaccharide (LPS) mouse model was used to study the effect of honokiol on depression-like behavior induced by LPS in mice and its potential mechanism. A single administration of LPS (1 mg/kg, intraperitoneal injection) increased the immobility time in the forced swimming test (FST) and tail suspension test (TST), without affecting autonomous activity. Pretreatment with honokiol (10 mg/kg, oral administration) for 11 consecutive days significantly improved the immobility time of depressed mice in the FST and TST experiments. Moreover, honokiol ameliorated LPS-induced NF-κB activation in the hippocampus and significantly reduced the levels of the pro-inflammatory cytokines; tumor necrosis factor α (TNF-α), interleukin 1ß (IL-1ß), and interferon γ (IFN-γ). In addition, honokiol inhibited LPS-induced indoleamine 2,3-dioxygenase (IDO) activation and quinolinic acid (a toxic product) increase and reduced the level of free calcium in brain tissue, thereby inhibiting calcium overload. In summary, our results indicate that the anti-depressant-like effects of honokiol are mediated by its anti-inflammatory effects. Honokiol may inhibit the LPS-induced neuroinflammatory response through the NF-κB signaling pathway, reducing the levels of related pro-inflammatory cytokines, and furthermore, this may affect tryptophan metabolism and increase neuroprotective metabolites.

Feiji Recipe inhibits the growth of lung cancer by modulating T-cell immunity through indoleamine-2,3-dioxygenase pathway in an orthotopic implantation model.

OBJECTIVE: Escape from the body's immune response is a basic characteristic of lung cancer, and indoleamine-2,3-dioxygenase (IDO) plays a key role in mediating immune escape of non-small-cell lung cancer, which leads to recurrence and metastasis. Feiji Recipe, a compound Chinese herbal medicine, has the effect of stabilizing lesions and prolonging survival in patients with lung cancer. The purpose of this study was to investigate the mechanisms underlying the anticancer properties of Feiji Recipe. METHODS: An orthotopic transplant model of mouse Lewis lung cancer, with stable expression of IDO gene, was established in C57BL/6 mice. Optical imaging was used to observe the effects of Feiji Recipe in the treatment of lung cancer in vivo. The effects of Feiji Recipe on the proliferation of mouse Lewis lung cancer cell line 2LL, 2LL-enhanced green fluorescent protein (2LL-EGFP) and 2LL-EGFP-IDO were investigated, and the apoptosis of T-cells was examined by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide using flow cytometry. Chemical composition of Feiji Recipe was validated by high-performance liquid chromatography. RESULTS: Compared to the control group, the survival of animals treated with Feiji Recipe was significantly prolonged (P = 0.0074), and the IDO protein level decreased (P = 0.0072); moreover, the percentages of CD4+CD25+ T-cells and Foxp3+ T-cells were significantly decreased (P < 0.05). The molecular mechanism of Feiji Recipe against lung cancer may relate to the regulation of immune cells, such as T-cells and regulatory T-cells. CONCLUSION: The molecular mechanism of Feiji Recipe in treatment of lung cancer is to restore the function of T-cells in the cancer microenvironment through interfering with the IDO pathway.

Regulation of the kynurenine metabolism pathway by Xiaoyao San and the underlying effect in the hippocampus of the depressed rat.

ETHNOPHARMACOLOGICAL RELEVANCE: Xiaoyao San (XYS) is a classic Chinese herbal formula for treatment of depression. The present study aimed to investigate the antidepressant effects of XYS in a rat model of chronic unpredictable mild stress (CUMS) and the underlying mechanisms. MATERIALS AND METHODS: A CUMS rat model of depression was established via 4 weeks of unpredictable stimulation. Then the rats were orally administered paroxetine and XYS for 2 weeks with continued stress. Behavioral assessments, including an open field test (OFT), sucrose preference test (SPT) and forced swim test (FST), were conducted to evaluate the antidepressant effects of XYS. The concentrations in rat plasma of tryptophan (Trp) and its metabolic products, including kynurenine (Kyn) and quinolinic acid (QUIN), were determined using high performance liquid chromatography tandem mass spectrometry with electrochemical detection (HPLC-MS/MS). The mRNA and protein levels in rat hippocampus of depression-related brain derived neurotrophic factor (BDNF), cyclic AMP response element binding protein (CREB) and nerve cell adhesion molecule (NCAM) were determined by real-time qPCR and Western blot, respectively. Enzyme Linked Immunosorbent Assay (ELISA) was used to detect the activities of indoleamine 2,3-dioxygenase (IDO) and kynurenine-3-monooxygenase (KMO) in rat plasma. RESULTS: The results showed that a successful CUMS rat model was established through 4 weeks of continuous unpredictable stimulation, as indicated by the significant decrease in locomotor activity and increase in immobility time in the OFT, reduction in body weight and food intake etc. Compared with the normal group, the concentrations of Kyn and QUIN had significantly (p < 0.05) decreased at day 28 in the control group, but then improved after drug treatment with paroxetine and XYS. There were no obvious changes in the activities of IDO and KMO. Compared with the normal group, the mRNA of NCAM, CREB and BDNF were significantly down-regulated (p < 0.001) in the control group, BDNF gene was up-regulated by paroxetine or XYS treatment, NCAM and CREB gene did not change in XYS group, protein expressions of BDNF and CREB were significantly increased, and NCAM was significantly reduced (p < 0.05). CONCLUSIONS: XYS reversed the abnormalities of the tryptophan-kynurenine metabolic pathways in depressed rats and achieved an excellent antidepressant effect. Its direct impact may be observed as changes in biological indicators in rat hippocampus tissue.

Rhesus Macaque Myeloid-Derived Suppressor Cells Demonstrate T Cell Inhibitory Functions and Are Transiently Increased after Vaccination.

Myeloid-derived suppressor cells (MDSCs) are major regulators of T cell responses in several pathological conditions. Whether MDSCs increase and influence T cell responses in temporary inflammation, such as after vaccine administration, is unknown. Using the rhesus macaque model, which is critical for late-stage vaccine testing, we demonstrate that monocytic (M)-MDSCs and polymorphonuclear (PMN)-MDSCs can be detected using several of the markers used in humans. However, whereas rhesus M-MDSCs lacked expression of CD33, PMN-MDSCs were identified as CD33+ low-density neutrophils. Importantly, both M-MDSCs and PMN-MDSCs showed suppression of T cell proliferation in vitro. The frequency of circulating MDSCs rapidly and transiently increased 24 h after vaccine administration. M-MDSCs infiltrated the vaccine injection site, but not vaccine-draining lymph nodes. This was accompanied by upregulation of genes relevant to MDSCs such as arginase-1, IDO1, PDL1, and IL-10 at the injection site. MDSCs may therefore play a role in locally maintaining immune balance during vaccine-induced inflammation.

Xenotransplantation of interferon-gamma-pretreated clumps of a human mesenchymal stem cell/extracellular matrix complex induces mouse calvarial bone regeneration.

BACKGROUND: Three-dimensional cultured clumps of a mesenchymal stem cell (MSC)/extracellular matrix (ECM) complex (C-MSC) consists of cells and self-produced ECM. C-MSC can regulate the cellular function in vitro and induce successful bone regeneration using ECM as a cell scaffold. Potentiating the immunomodulatory capacity of C-MSCs, which can ameliorate the allo-specific immune response, may be helpful in developing beneficial "off-the-shelf" cell therapy for tissue regeneration. It is well reported that interferon (IFN)-γ stimulates the immunosuppressive properties of MSC via upregulation of the immunomodulatory enzyme IDO. Therefore, the aim of this study was to investigate the effect of IFN-γ on the immunomodulatory capacity of C-MSC in vitro and to test the bone regenerative activity of C-MSC or IFN-γ-pretreated C-MSC (C-MSCγ) xenografts in a mice calvarial defect model. METHODS: Human bone marrow-derived MSCs were seeded at a density of 2.0 × 105 cells/well into 24-well plates and cultured with growth medium supplemented with 50 µg/mL L-ascorbic acid for 4 days. To obtain C-MSC, confluent cells that had formed on the cellular sheet were scratched using a micropipette tip and were then torn off. The cellular sheet was rolled to make a round clump of cells. C-MSC was stimulated with IFN-γ and IDO expression, immunosuppressive capacity, and immunophenotype were evaluated in vitro. Moreover, C-MSC or C-MSCγ was xenotransplanted into immunocompetent or immunodeficient mice calvarial defect models without artificial scaffold, respectively. RESULTS: IFN-γ stimulated IDO expression in C-MSC. C-MSCγ, but not C-MSC, attenuated CD3/CD28-induced T cell proliferation and its suppressive effect was reversed by an IDO inhibitor. C-MSCγ showed upregulation of HLA-DR expression, but its co-stimulatory molecule, CD86, was not detected. Xenotransplantation of C-MSCγ into immunocompetent mice calvarial defect induced bone regeneration, whereas C-MSC xenograft failed and induced T cell infiltration in the grafted area. On the other hand, both C-MSC and C-MSCγ xenotransplantation into immunodeficient mice caused bone regeneration. CONCLUSIONS: Xenotransplantation of C-MSCγ, which exerts immunomodulatory properties via the upregulation of IDO activity in vitro, may attenuate xenoreactive host immune response, and thereby induce bone regeneration in mice. Accordingly, C-MSCγ may constitute a promising novel allograft cell therapy for bone regeneration.

Discovery and evaluation of inhibitors to the immunosuppressive enzyme indoleamine 2,3-dioxygenase 1 (IDO1): Probing the active site-inhibitor interactions.

High expression of the immunosuppressive enzyme, indoleamine 2,3-dioxygenase 1 (IDO1) for a broad range of malignancies is associated with poor patient prognosis, and the enzyme is a validated target for cancer intervention. To identify novel IDO1 inhibitors suitable for drug development, 1597 compounds in the National Cancer Institute Diversity Set III library were tested for inhibitory activity against recombinant human IDO1. We retrieved 35 hits that inhibited IDO1 activity >50% at 20 µM. Five structural filters and the PubChem Bioassay database were used to guide the selection of five inhibitors with IC50 between 3 and 12 µM for subsequent experimental evaluation. A pyrimidinone scaffold emerged as being the most promising. It showed excellent cell penetration, negligible cytotoxicity and passed four out of the five structural filters applied. To evaluate the importance of Ser167 and Cys129 residues in the IDO1 active site for inhibitor binding, the entire NCI library was subsequently screened against alanine-replacement mutant enzymes of these two residues. The results established that Ser167 but not Cys129 is important for inhibitory activity of a broad range of IDO1 inhibitors. Structure-activity-relationship studies proposed substituents interacting with Ser167 on four investigated IDO1 inhibitors. Three of these four Ser167 interactions associated with an increased IDO1 inhibition and were correctly predicted by molecular docking supporting Ser167 as an important mediator of potency for IDO1 inhibitors.

Effect of Coffee in Lipopolysaccharide-Induced Indoleamine 2,3-Dioxygenase Activation and Depressive-like Behavior in Mice.

Research has identified a potential inverse correlation between coffee consumption and the risk of depression. The aim of this study was to investigate the effects of caffeinated coffee on lipopolysaccharide-induced depressive-like behaviors and inflammatory biomarkers in an in vivo model of depression in a C57BL/6J mouse model. The behavioral studies showed that caffeinated coffee decreased immobility time in both the tail suspension test (caffeinated coffee 56.60 ± 9.17; p < 0.0001) and the forced swimming test (caffeinated coffee 28.80 ± 5.93; p < 0.0001), suggesting antidepressant-like activity. The effects of caffeinated coffee on the inflammatory biomarkers associated with depression supported the results observed in the behavioral studies. Statistically significant decreases in indoleamine 2,3-dioxygenase activity (p < 0.001) and the neopterin/biopterin ratio (p < 0.001) were observed in animals pretreated with caffeinated coffee 24 h post-lipopolysaccharide exposure in comparison to the lipopolysaccharide control group. In conclusion, this study has provided evidence to suggest that caffeinated coffee has antidepressant-like activities; however, further studies are required to fully investigate these effects.

Zinc Induces Dendritic Cell Tolerogenic Phenotype and Skews Regulatory T Cell-Th17 Balance.

Zinc (Zn) is an essential metal for development and maintenance of both the innate and adaptive compartments of the immune system. Zn homeostasis impacts maturation of dendritic cells (DCs) that are important in shaping T cell responses. The mechanisms by which Zn regulates the tolerogenic phenotype of DCs remain largely unknown. In this study, we investigated the effect of Zn on DC phenotype and the generation of Foxp3(+) regulatory T cells (Tregs) using a model of Histoplasma capsulatum fungal infection. Exposure of bone marrow-derived DCs to Zn in vitro induced a tolerogenic phenotype by diminishing surface MHC class II (MHCII) and promoting the tolerogenic markers, programmed death-ligand (PD-L)1, PD-L2, and the tryptophan degrading enzyme, IDO. Zn triggered tryptophan degradation by IDO and kynurenine production by DCs and strongly suppressed the proinflammatory response to stimulation by TLR ligands. In vivo, Zn supplementation and subsequent H. capsulatum infection supressed MHCII on DCs, enhanced PD-L1 and PD-L2 expression on MHCII(lo) DCs, and skewed the Treg-Th17 balance in favor of Foxp3(+) Tregs while decreasing Th17 cells. Thus, Zn shapes the tolerogenic potential of DCs in vitro and in vivo and promotes Tregs during fungal infection.

Ganoderic acid Me induces the apoptosis of competent T cells and increases the proportion of Treg cells through enhancing the expression and activation of indoleamine 2,3-dioxygenase in mouse lewis lung cancer cells.

The indoleamine 2,3-dioxygenase-(IDO-) mediated microenvironment plays an important role in tumor immune escape. It is known that ganoderic acid Me can enhance IFN-γ expression and IDO is preferentially induced by IFN-γ. However, whether GA-Me can induce IDO expression has not been clarified yet. We established stable clones of IDO-overexpressing 2 LL cells (2LL-EGFP-IDO). After co-culturing with IDO expressing or control vector-transfected 2LL-EGFP cells, T cell apoptosis was determined and the proportion of the regulatory T cells (Tregs) and CD8+ T cell subset was measured. The total cellular protein samples of 2 LL-EGFP-IDO cells were isolated for detecting JAK-STAT1 signalling pathway. Co-culture supernatants were used to detect amino acids and cytokines. IDO transfected 2 LL cells yielded high level of IDO enzymatic activity, resulting in complete depletion of tryptophan from the culture medium. We found that apoptosis occurred in T cells after cocultured with IDO+2LL cells and the proportion of CD4+CD25+ cells and FoxP3+ cells increased while CD8+ cells decreased. The specific inhibitor of IDO, 1-D-MT and GA-Me efficiently enhanced T cell apoptosis, increased Tregs, and reduced CD8+ T cells in vitro. Increased expression of IDO, p-JAK1 and p-STAT1 were confirmed by Western blot analysis. The levels of IFN-γ, IL-10, LDH and kynurenine in co-culture supernatant correspondingly increased, while tryptophan reduced. These results suggest that GA-Me contributing to IDO helps to create a tolerogenic milieu in lung tumors by directly inducing T cell apoptosis, restraining CD8+ T cell activation, and enhancing Treg-mediated immunosuppression.

Inhibition of indoleamine 2,3-dioxygenase by stereoisomers of 1-methyl tryptophan in an experimental graft-versus-tumor model.

Indoleamine 2,3-dioxygenase (IDO) is a rate-limiting enzyme in tryptophan catabolism that plays an important role in the induction of immune tolerance. Its role in graft-versus-tumor effect after allogeneic stem cell transplantation (allo-SCT) remains unclear. Using a murine graft-versus-tumor model of reduced-intensity allo-HSCT followed by donor leukocyte infusion (DLI), we examined the role of IDO inhibition. Two stereoisomers of 1-methyl tryptophan (1-MT), a small-molecule inhibitor of IDO, reduced the growth of inoculated tumor in the mice that received DLI and had higher expression of IDO1 and IFNγ. However, L-1MT, but not D-1MT, mitigated tumor growth in mice that did not receive DLI and did not express IDO1 and IFNγ. Accordingly, both stereoisomers reduced plasma kynurenine concentrations early after DLI and enhanced in vitro cytotoxic lymphocyte function after allogeneic mixed lymphocyte reaction. Furthermore, L-1MT was more efficient in causing direct cytotoxic effects than D-1MT. Our results suggest that IDO inhibition can benefit anti-tumor therapy in the setting of reduced-intensity allo-SCT using DLI.

Peripheral tumors alter neuroinflammatory responses to lipopolysaccharide in female rats.

Cancer is associated with an increased prevalence of depression. Peripheral tumors induce inflammatory cytokine production in the brain and depressive-like behaviors. Mounting evidence indicates that cytokines are part of a pathway by which peripheral inflammation causes depression. Neuroinflammatory responses to immune challenges can be exacerbated (primed) by prior immunological activation associated with aging, early-life infection, and drug exposure. This experiment tested the hypothesis that peripheral tumors likewise induce neuroinflammatory sensitization or priming. Female rats with chemically-induced mammary carcinomas were injected with either saline or lipopolysaccharide (LPS, 250µg/kg; i.p.), and expression of mRNAs involved in the pathway linking inflammation and depression (interleukin-1beta [Il-1ß], CD11b, IκBα, indolamine 2,3-deoxygenase [Ido]) was quantified by qPCR in the hippocampus, hypothalamus, and frontal cortex, 4 or 24h post-treatment. In the absence of LPS, hippocampal Il-1ß and CD11b mRNA expression were elevated in tumor-bearing rats, whereas Ido expression was reduced. Moreover, in saline-treated rats basal hypothalamic Il-1ß and CD11b expression were positively correlated with tumor weight; heavier tumors, in turn, were characterized by more inflammatory, necrotic, and granulation tissue. Tumors exacerbated CNS proinflammatory gene expression in response to LPS: CD11b was greater in hippocampus and frontal cortex of tumor-bearing relative to tumor-free rats, IκBα was greater in hippocampus, and Ido was greater in hypothalamus. Greater neuroinflammatory responses in tumor-bearing rats were accompanied by attenuated body weight gain post-LPS. The data indicate that neuroinflammatory pathways are potentiated, or primed, in tumor-bearing rats, which may exacerbate future negative behavioral consequences.

Topical application of soluble CD83 induces IDO-mediated immune modulation, increases Foxp3+ T cells, and prolongs allogeneic corneal graft survival.

Modulation of immune responses is one of the main research aims in transplant immunology. In this study, we investigate the local immunomodulatory properties of soluble CD83 (sCD83) at the graft-host interface using the high-risk corneal transplantation model. In this model, which mimics the inflammatory status and the preexisting vascularization of high-risk patients undergoing corneal transplantation, allogeneic donor corneas are transplanted onto sCD83-treated recipient animals. This model allows the direct and precise application of the immune modulator at the transplantation side. Interestingly, sCD83 was able to prolong graft survival after systemic application as well as after topical application, which is therapeutically more relevant. The therapeutic effect was accompanied by an increase in the frequency of regulatory T cells and was mediated by the immune-regulatory enzyme IDO and TGF-ß. In vitro, sCD83 induced long-term IDO expression in both conventional and plasmacytoid dendritic cells via autocrine or paracrine production of TGF-ß, a cytokine previously shown to be an essential mediator of IDO-dependent, long-term tolerance. These findings open new treatment avenues for local immune modulation after organ and tissue transplantation.

Cardiac and gastrointestinal liabilities caused by deficiency in the immune modulatory enzyme indoleamine 2,3-dioxygenase.

Indoleamine 2,3-dioxygenase (IDO) modifies adaptive immunity, in part by determining the character of inflammatory responses in the tissue microenvironment. Small molecule inhibitors of IDO are being developed to treat cancer, chronic infections and other diseases, so the systemic effects of IDO disruption on inflammatory phenomena may influence the design and conduct of early phase clinical investigations of this new class of therapeutic agents. Here, we report cardiac and gastrointestinal phenotypes observed in IDO deficient mice that warrant consideration in planned assessments of the safety risks involved in clinical development of IDO inhibitors. Calcification of the cardiac endometrium proximal to the right ventricle was a sexually dimorphic strain-specific phenotype with ~30% penetrance in BALB/c mice lacking IDO. Administration of complete Freund's adjuvant containing Toll-like receptor ligands known to induce IDO caused acute pancreatitis in IDO deficient mice, with implications for the design of planned combination studies of IDO inhibitors with cancer vaccines. In an established model of hyperlipidemia, IDO deficiency caused a dramatic elevation in levels of serum triglycerides. In the large intestine, IDO loss only slightly increased sensitivity to induction of acute colitis, but it markedly elevated tumor incidence, multiplicity and staging during inflammatory colon carcinogenesis. Together, our findings suggest potential cardiac and gastrointestinal risks of IDO inhibitors that should be monitored in patients as this new class of drugs enter early clinical development.