Tryptophanyl-tRNA synthetase mediates high-affinity tryptophan uptake into human cells.

The tryptophan (Trp) transport system has a high affinity and selectivity toward Trp, and has been reported to exist in both human and mouse macrophages. Although this system is highly expressed in interferon-γ (IFN-γ)-treated cells and indoleamine 2,3-dioxygenase 1 (IDO1)-expressing cells, its identity remains incompletely understood. Tryptophanyl-tRNA synthetase (TrpRS) is also highly expressed in IFN-γ-treated cells and also has high affinity and selectivity for Trp. Here, we investigated the effects of human TrpRS expression on Trp uptake into IFN-γ-treated human THP-1 monocytes or HeLa cells. Inhibition of human TrpRS expression by TrpRS-specific siRNAs decreased and overexpression of TrpRS increased Trp uptake into the cells. Of note, the TrpRS-mediated uptake system had more than hundred-fold higher affinity for Trp than the known System L amino acid transporter, promoted uptake of low Trp concentrations, and had very high Trp selectivity. Moreover, site-directed mutagenesis experiments indicated that Trp- and ATP-binding sites, but not tRNA-binding sites, in TrpRS are essential for TrpRS-mediated Trp uptake into the human cells. We further demonstrate that the addition of purified TrpRS to cell culture medium increases Trp uptake into cells. Taken together, our results reveal that TrpRS plays an important role in high-affinity Trp uptake into human cells.

Platelet-activating Factor Mediates Endotoxin Tolerance by Regulating Indoleamine 2,3-Dioxygenase-dependent Expression of the Suppressor of Cytokine Signaling 3.

Indoleamine 2,3-dioxygenase (IDO) mediates immune tolerance, and suppressor of cytokine signaling 3 (SOCS3) negatively regulates the JAK/STAT signal transduction pathway. We determined previously that platelet-activating factor (PAF) protects mice against LPS-induced endotoxic shock, but its detailed mechanism of action was unknown. We performed survival experiments in IDO+/+ and IDO-/- mice using an LPS-induced endotoxemia model and rated organ injury (neutrophil infiltration and liver function). Using ELISA and Western blotting, we also investigated the mechanism of PAF-mediated endotoxin tolerance during endotoxemia. PAF-mediated endotoxin tolerance was dependent on IDO in vivo and in vitro and was not observed in IDO-/- mice. JAK/STAT signaling, crucial for SOCS3 expression, was also impaired in the absence of IDO. In an IDO- and STAT-dependent manner, PAF mediated a decrease in IL-12 and a dramatic increase in IL-10 and reduced mouse mortality. In addition, PAF attenuated LPS-mediated neutrophil infiltration into the lungs and interactions between neutrophil-like (THP-1) and endothelial cells (human umbilical vein endothelial cells). These results indicate that PAF-mediated endotoxin tolerance is initiated via IDO- and JAK/STAT-dependent expression of SOCS3. Our study has revealed a novel tolerogenic mechanism of IDO action and an important association between IDO and SOCS3 with respect to endotoxin tolerance.

Signal Transducer and Activator of Transcription 1 Plays a Pivotal Role in RET/PTC3 Oncogene-induced Expression of Indoleamine 2,3-Dioxygenase 1.

Indoleamine 2,3-dioxygenase 1 (IDO1) is a single chain oxidoreductase that catalyzes tryptophan degradation to kynurenine. In cancer, it exerts an immunosuppressive function as part of an acquired mechanism of immune escape. Recently, we demonstrated that IDO1 expression is significantly higher in all thyroid cancer histotypes compared with normal thyroid and that its expression levels correlate with T regulatory (Treg) lymphocyte densities in the tumor microenvironment. BRAFV600E- and RET/PTC3-expressing PcCL3 cells were used as cellular models for the evaluation of IDO1 expression in thyroid carcinoma cells and for the study of involved signal transduction pathways. BRAFV600E-expressing PcCL3 cells did not show IDO1 expression. Conversely, RET/PTC3-expressing cells were characterized by a high IDO1 expression. Moreover, we found that, the STAT1-IRF1 pathway was instrumental for IDO1 expression in RET/PTC3 expressing cells. In detail, RET/PTC3 induced STAT1 overexpression and phosphorylation at Ser-727 and Tyr-701. STAT1 transcriptional regulation appeared to require activation of the canonical NF-κB pathway. Conversely, activation of the MAPK and PI3K-AKT pathways primarily regulated Ser-727 phosphorylation, whereas a physical interaction between RET/PTC3 and STAT1, followed by a direct tyrosine phosphorylation event, was necessary for STAT1 Tyr-701 phosphorylation. These data provide the first evidence of a direct link between IDO1 expression and the oncogenic activation of RET in thyroid carcinoma and describe the involved signal transduction pathways. Moreover, they suggest possible novel molecular targets for the abrogation of tumor microenvironment immunosuppression. The detection of those targets is becoming increasingly important to yield the full function of novel immune checkpoint inhibitors.

Glycogen Synthase Kinase-3ß (GSK-3ß) Inhibition Enhances Dendritic Cell-based Cancer Vaccine Potency via Suppression of Interferon-γ-induced Indoleamine 2,3-Dioxygenase Expression.

Indoleamine 2,3-dioxygenase (IDO) functions as a crucial mediator of tumor-mediated immune tolerance by causing T-cell suppression via tryptophan starvation in a tumor environment. Glycogen synthase kinase-3ß (GSK-3ß) is also involved in immune and anti-tumor responses. However, the relativity of these proteins has not been as well defined. Here, we found that GSK-3ß-dependent IDO expression in the dendritic cell (DC) plays a role in anti-tumor activity via the regulation of CD8(+) T-cell polarization and cytotoxic T lymphocyte activity. By the inhibition of GSK-3ß, attenuated IDO expression and impaired JAK1/2-Stat signaling crucial for IDO expression were observed. Protein kinase Cδ (PKCδ) activity and the interaction between JAK1/2 and Stat3, which are important for IDO expression, were also reduced by GSK-3ß inhibition. CD8(+) T-cell proliferation mediated by OVA-pulsed DC was blocked by interferon (IFN)-γ-induced IDO expression via GSK-3ß activity. Specific cytotoxic T lymphocyte activity mediated by OVA-pulsed DC against OVA-expressing EG7 thymoma cells but not OVA-nonexpressing EL4 thymoma cells was also attenuated by the expressed IDO via IFN-γ-induced activation of GSK-3ß. Furthermore, tumor growth that was suppressed with OVA-pulsed DC vaccination was restored by IDO-expressing DC via IFN-γ-induced activation of GSK-3ß in an OVA-expressing murine EG7 thymoma model. Taken together, DC-based immune response mediated by interferon-γ-induced IDO expression via GSK-3ß activity not only regulates CD8(+) T-cell proliferation and cytotoxic T lymphocyte activity but also modulates OVA-pulsed DC vaccination against EG7 thymoma.

Identification and Characterization of a Novel Dual Inhibitor of Indoleamine 2,3-dioxygenase 1 and Tryptophan 2,3-dioxygenase.

Kynurenine (Kyn), a metabolite of tryptophan (Trp), is a key regulator of mammal immune responses such as cancer immune tolerance. Indoleamine-2,3-dioxygenase (IDO) and tryptophan-2,3-dioxygenase (TDO) are main enzymes regulating the first and rate-limiting step of the Kyn pathway. To identify new small molecule inhibitors of TDO, we selected A172 glioblastoma cell line constitutively expressed TDO. Characterization of this cell line using kinase inhibitor library resulted in identification of MEK/ERK pathway-dependent TDO expression. After knowing the properties for TDO expression, we further proceeded to screen chemical library for TDO inhibitors. We previously determined that S-benzylisothiourea derivatives are enzymatic inhibitors of indoleamine 2,3-dioxygenase 1 (IDO1) and suggested that the isothiourea moiety could be an important pharmacophore for binding to heme. Based on this premise, we screened an in-house library composed of various isothiourea derivatives and identified a bisisothiourea derivative, PVZB3001, as an inhibitor of TDO. Interestingly, PVZB3001 also inhibited the enzymatic activity of IDO1 in both cell-based and cell-free assays but did not inhibit other heme enzymes. Molecular docking studies suggested the importance of isothiourea moieties at the ortho position of the phenyl ring for the inhibition of catalytic activity. PVZB3001 showed competitive inhibition against TDO, and this was supported by the docking simulation. PVZB3001 recovered natural killer (NK) cell viability and functions by inhibiting Kyn accumulation in conditioned medium of both IDO1- and TDO-expressing cells. Furthermore, oral administration of IDO1-overexpressing tumor-bearing mice with PVZB3001 significantly inhibited tumor growth. Thus, we identified a novel selective dual inhibitor of IDO1 and TDO using the Kyn production assay with a glioblastoma cell line. This inhibitor could be a useful pharmacological tool for modulating the Kyn pathway in a variety of experimental systems.

Regulation of kynurenine biosynthesis during influenza virus infection.

Influenza A viruses (IAVs) remain serious threats to public health because of the shortage of effective means of control. Developing more effective virus control modalities requires better understanding of virus-host interactions. It has previously been shown that IAV induces the production of kynurenine, which suppresses T-cell responses, enhances pain hypersensitivity and disturbs behaviour in infected animals. However, the regulation of kynurenine biosynthesis during IAV infection remains elusive. Here we showed that IAV infection induced expression of interferons (IFNs), which upregulated production of indoleamine-2,3-dioxygenase (IDO1), which catalysed the kynurenine biosynthesis. Furthermore, IAV attenuated the IDO1 expression and the production of kynurenine through its NS1 protein. Interestingly, inhibition of viral replication prior to IFN induction limited IDO1 expression, while inhibition after did not. Finally, we showed that kynurenine biosynthesis was activated in macrophages in response to other stimuli, such as influenza B virus, herpes simplex virus 1 and 2 as well as bacterial lipopolysaccharides. Thus, the tight regulation of the kynurenine biosynthesis by host cell and, perhaps, pathogen might be a basic signature of a wide range of host-pathogen interactions, which should be taken into account during development of novel antiviral and antibacterial drugs.