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.

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.

Ethanol extracts from Hemerocallis citrina attenuate the upregulation of proinflammatory cytokines and indoleamine 2,3-dioxygenase in rats.

ETHNOPHARMACOLOGICAL RELEVANCE: Hemerocallis citrina, a traditional herbal medicine, has been used for the improvement of behavioral and emotional status in Eastern-Asia countries. Previous studies in our laboratory demonstrated that ethanol extracts from Hemerocallis citrina (HCE) enhanced monoamines and brain-derived neurotrophic factor (BDNF) in depression-like model of rodents. MATERIALS AND METHODS: The present study extends earlier works on the role of anti-inflammation in regulating the antidepressant-like actions of HCE in rats exposed to chronic unpredictable mild stress (CUMS). Frontal cortex and hippocampal proinflammatory cytokines levels and indoleamine 2,3-dioxygenase (IDO) activity were measured after 4-week HCE treatment in the CUMS an control rats. RESULTS: Chronic administration of HCE reversed the decreased sucrose preference in sucrose preference test. In addition, we also found that HCE inhibited interleukin-1 beta (IL-1ß), interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-α) expression, as well as IDO activity in frontal cortex and hippocampus, which were increased in rats exposed to CUMS. CONCLUSIONS: Combining with our previous studies, our present finding suggests that the anti-inflammatory property of HCE might play a crucial role in its antidepressant-like effect through, at least in part, the restoration or improvement of monoaminergic and neurotrophin systems.

Effects of various phytochemicals on indoleamine 2,3-dioxygenase 1 activity: galanal is a novel, competitive inhibitor of the enzyme.

Indoleamine 2,3-dioxygenase (IDO) 1, that catalyzes the first and rate-limiting step in the degradation of L-tryptophan, has an important immunomodulatory function. The activity of IDO1 increases in various inflammatory diseases, including tumors, autoimmune diseases, and different kinds of inflammation. We evaluated the suppressive effect of plant extracts or phytochemicals on IDO1 induction and activity; sixteen kinds of plants extracts and fourteen kinds of phytochemicals were examined. As a result, the methanol extracts of Myoga flower buds, which are traditional Japanese foods, and labdane-type diterpene galanal derived from Myoga flowers significantly suppressed IDO1 activity. The Lineweaver-Burk plot analysis indicated that galanal is a competitive inhibitor. Galanal attenuated L-kynurenine formation with an IC50 value of 7.7 µM in the assay system using recombinant human IDO1, and an IC50 value of 45 nM in the cell-based assay. Further, mechanistic analysis revealed that galanal interfered with the transcriptional function of the nuclear factor-κB and the interferon-γ signaling pathway. These effects of galanal are important for immune response. Because the inhibitory effect of galanal on IDO1 activity was stronger than that of 1-methyl tryptophan, a tryptophan analog, galanal may have great potential as the novel drug for various immune-related diseases.

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.

The use of nanocomposite coatings with various physicochemical properties in tissue engineering.

We studied the effect of nanocomposite coatings with various physicochemical properties on the structural and functional properties (adhesion potential, phenotype, gene expression) of mesenchymal stem cells. Of all tested nanocoatings (Al2O3, ZrO2, Ta2O5), oxide coating Al2O3 enriched in vitro monolayer bone marrow cell culture with cells carrying mesenchymal stem cells phenotype markers and stimulated expression of ido gene, which can confer new therapeutic potencies to these cells and extend their application in clinical practice.

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.

Oren-gedoku-to and its constituents with therapeutic potential in Alzheimer's disease inhibit indoleamine 2, 3-dioxygenase activity in vitro.

A well-known traditional Chinese medicinal prescription, Oren-gedoku-to (OGT), has been used in clinical therapies for many types of dementia in China and Japan. Additionally, it ameliorates the age-related deterioration of learning and memory in an Alzheimer's disease (AD) rat model. Indoleamine 2, 3-dioxygenase (IDO-1) is the first and rate-limiting enzyme in the kynurenine pathway of tryptophan catabolism, which ultimately leads to the production of the excitotoxin quinolinic acid (QUIN). IDO-1 has recently been established as one of the key players involved in the pathogenesis of AD. OGT is indicated to prevent cholinergic dysfunction and reduce oxidative stress; however, the exact mechanism underlying its ability to improve cognitive ability remains elusive. Here we present a novel mechanism of OGT's therapeutic potential in AD. We demonstrated that OGT significantly inhibited recombinant human IDO-1 (rhIDO-1) activity in vitro, and its four main constituents (i.e., berberine, palmatine, jatrorrhizine, and baicalein) were potent IDO-1 inhibitors. IC50 values, obtained from a cell-based assay, of HEK 293 cells and an enzymatic assay were much lower than the most commonly used IDO-1 inhibitor, 1-methyl tryptophan (1-MT). Berberine was the best inhibitor and had IC50 values of 7 µM (cell-based assay) and 9.3 µM (enzymatic assay). Jatrorrhizine and palmatine exhibited irreversible inhibition of rhIDO-1, whereas berberine and baicalein behaved as uncompetitive, reversible inhibitors with Ki values of 8 µM and 215 µM, respectively. In conclusion, constituents of OGT show strong IDO-1 inhibitory activity and may have significant therapeutic potential for AD.

Zinc protoporphyrin IX stimulates tumor immunity by disrupting the immunosuppressive enzyme indoleamine 2,3-dioxygenase.

The tryptophan catabolic enzyme indoleamine 2,3-dioxygenase (IDO) has emerged as an important driver of immune escape in a growing number of cancers and cancer-associated chronic infections. In this study, we define novel immunotherapeutic applications for the heme precursor compound zinc protoporphyrin IX (ZnPP) based on our discovery that it is a potent small-molecule inhibitor of IDO. Inhibitory activity was determined using in vitro and in-cell enzyme assays as well as a novel in vivo pharmacodynamic system. An irreversible mechanism of inhibition was documented, consistent with competition for heme binding in newly synthesized cellular protein. siRNA methodology and an IDO-deficient mouse strain were used to verify the specificity of ZnPP as an IDO inhibitor. In a preclinical model of melanoma, ZnPP displayed antitumor properties that relied on T-cell function and IDO integrity. ZnPP also phenocopied the known antitumor properties of IDO inhibitors in preclinical models of skin and breast carcinoma. Our results suggest clinical evaluation of ZnPP as an adjuvant immunochemotherapy in chronic infections and cancers in which there is emerging recognition of a pathophysiologic role for IDO dysregulation.

Expression profiling identifies genes that predict recurrence of breast cancer after adjuvant CMF-based chemotherapy.

Cyclophosphamide, methotrexate and 5-fluorouracile (CMF)-based chemotherapy for adjuvant treatment of breast cancer reduces the risk of relapse. In this exploratory study, we tested the feasibility of identifying molecular markers of recurrence in CMF-treated patients. Using Affymetrix U133A GeneChips, RNA samples from 19 patients with primary breast cancer who had been uniformly treated with adjuvant CMF chemotherapy were analyzed. Two supervised class prediction approaches were used to identify gene markers that can best discriminate between patients who would experience relapse and patients who would remain disease-free. An additional independent validation set of 51 patients and 21 genes were analyzed by quantitative RT-PCR. Applying different algorithms to evaluate our microarray data, we identified two gene expression signatures of 21 and 12 genes containing eight overlapping genes, that predict recurrence in 19 cases with high accuracy (94%). Quantitative RT-PCR demonstrated that six genes from the combined signatures (CXCL9, ITSN2, GNAI2, H2AFX, INDO, and MGC10986) were significantly differentially expressed in the recurrence versus the non-recurrence group of the 19 cases and the independent breast cancer patient cohort (n = 51) treated with CMF. High expression levels of CXCL9, ITSN2, and GNAI2 were associated with prolonged disease-free survival (DFS) (P = 0.029, 0.018 and 0.032, respectively). When patients were stratified by combined CXCL9/ITSN2 or CXCL9/FLJ22028 tumor levels, they exhibited significantly different disease-free survival curves (P = 0.0073 and P = 0.005, respectively). Finally, the CXCL9/ITSN2 and CXCL9/FLJ22028 ratio was an independent prognostic factor (P = 0.034 and P = 0.003, respectively) for DFS by multivariate Cox analysis in the 70-patient cohort. Our data highlight the feasibility of a prognostic assay that is applicable to therapeutic decision-making for breast cancer. Whether the biomarker profile is chemotherapy-specific or whether it is a more general indicator of bad prognosis of breast cancer patients remains to be explored.

The innate immune response to adjuvants dictates the adaptive immune response to autoantigens.

To elucidate the role of innate immunity in susceptibility to the animal model of multiple sclerosis, experimental autoimmune encephalomyelitis (EAE), we induced EAE by immunization with spinal cord homogenate (SCH) plus complete Freund adjuvant or carbonyl iron in 3 inbred rat strains. Lewis are considered "susceptible," PVG/c-Rt7a (PVG) as "semisusceptible," and Brown Norway (BN) as "resistant" to EAE. Immunization with SCH-carbonyl iron resulted in clinical disease in all 3 strains, but the pathologic features of EAE in the resistant BN and the semisusceptible PVG rats differed from those in the Lewis and PVG model of EAE induced with SCH-complete Freund adjuvant. In BN and PVG rats, there were numerous inflammatory lesions with prominent involvement of microglia and, to a lesser extent, perivascular macrophages. These data suggest that different levels of activation of the innate immune system by different adjuvants determine whether EAE will or will not develop. Accordingly, the widely accepted scale of susceptibility to EAE development (Lewis > PVG > BN) should be revised because it does not take into account the important contribution of the composition of the adjuvant to the quality and quantity of the innate immune response and, consequently, to the generation and extent of the pathogenic T-cell-mediated, that is, adaptive, autoimmune disease.

Herpes simplex virus co-infection-induced Chlamydia trachomatis persistence is not mediated by any known persistence inducer or anti-chlamydial pathway.

Several inducers of chlamydial persistence have been described, including interferon-gamma (IFN-gamma), IFN-alpha, IFN-beta, and tumour necrosis factor-alpha (TNF-alpha) exposure, and iron, amino acid or glucose deprivation. A tissue-culture model of Chlamydia trachomatis/herpes simplex virus type-2 (HSV-2) co-infection indicates that viral co-infection stimulates the formation of persistent chlamydiae. This study was designed to ascertain whether co-infection-induced persistence is mediated by a previously characterized mechanism. Luminex assays indicate that IFN-gamma, IFN-alpha, and TNF-alpha are not released from co-infected cells. Semiquantitative RT-PCR studies demonstrate that IFN-beta, IFN-gamma, indoleamine 2,3-dioxygenase, lymphotoxin-alpha and inducible nitric oxide synthase are not expressed during co-infection. These data indicate that viral-induced persistence is not stimulated by any persistence-associated cytokine. Supplementation of co-infected cells with excess amino acids, iron-saturated holotransferrin, glucose or a combination of amino acids and iron does not restore chlamydial infectivity, demonstrating that HSV-2-induced persistence is not mediated by depletion of these nutrients. Finally, inclusions within co-infected cells continue to enlarge and incorporate C(6)-NBD-ceramide, indicating that HSV-2 co-infection does not inhibit vesicular transport to the developing inclusion. Collectively these data demonstrate that co-infection-induced persistence is not mediated by any currently characterized persistence inducer or anti-chlamydial pathway. Previous studies indicate that HSV-2 attachment and/or entry into the host cell is sufficient for stimulating chlamydial persistence, suggesting that viral attachment and/or entry may trigger a novel host pathway which restricts chlamydial development.