DNA double-strand breaks (DSBs) and blocked replication forks resulting from bulky adducts and inhibitors of replication activate the DNA damage response (DDR), a signaling pathway marked by phosphorylation of histone 2AX (H2AX). The phosphorylated form, γH2AX, accumulates at the site of the damage and can be visualized as foci by immunocytochemistry. The objective of this study was to assess if γH2AX is a reliable biomarker for genotoxic exposures. To this end, we selected 14 well-known genotoxic compounds and compared them with 10 nongenotoxic chemicals, using CHO-9 cells because they are well characterized as to DNA repair and DDR. We quantified γH2AX foci manually and automatically. In addition, total γH2AX activation was determined by flow cytometry. For all chemicals the cytotoxic dose response was assayed by a metabolic cytotoxicity assay. We show that (1) all genotoxic agents induced γH2AX dose-dependently whereas nongenotoxic agents do not; (2) γH2AX was observed for genotoxicants in the cytotoxic dose range, revealing a correlation between cytotoxicity and γH2AX for genotoxic agents; for nongenotoxic agents cytotoxicity was not related to γH2AX; (3) manual scoring of γH2AX and automated scoring provided comparable results, the automated scoring was faster and investigator independent; (4) data obtained by foci counting and flow cytometry showed a high correlation, suggesting that γH2AX scoring by flow cytometry has the potential for high-throughput analysis. However, the microscopic evaluation can provide additional information as to foci size, distribution, colocalization and background staining; (5) γH2AX foci were colocalized with 53BP1 and Rad51, supporting the notion that they represent true DSBs. Collectively, the automated analysis of γH2AX foci allows for rapid determination of genetic damage in mammalian cells. The data revealed that the induction of γH2AX by genotoxicants is related to loss of viability and support γH2AX as a reliable bio-indicator for pretoxic DNA damage.

Tryptophan metabolism is a key process that shapes the immunosuppressive tumor microenvironment. The two rate-limiting enzymes that mediate tryptophan depletion, indoleamine-2,3-dioxygenase (IDO) and tryptophan-2,3-dioxygenase (TDO), have moved into the focus of research and inhibitors targeting IDO and TDO have entered clinical trials. Local tryptophan depletion is generally viewed as the crucial immunosuppressive mechanism. In T cells, the kinase general control non-derepressible 2 (GCN2) has been identified as a molecular sensor of tryptophan deprivation. GCN2 activation by tryptophan depletion induces apoptosis and mitigates T cell proliferation. Here, we investigated whether GCN2 attenuates tumor rejection in experimental B16 melanoma using T cell-specific knockout mice. Our data demonstrate that GCN2 in T cells did not affect immunity to B16 tumors even when animals were treated with antibodies targeting cytotoxic T lymphocyte antigen-4 (CTLA4). GCN2-deficient gp100 TCR-transgenic T cells were equally effective as wild-type pmel T cells against gp100-expressing B16 melanomas after adoptive transfer and gp100 peptide vaccination. Even augmentation of tumoral tryptophan metabolism in B16 tumors by lentiviral overexpression of did not differentially affect GCN2-proficient vs. GCN2-deficient T cells . Importantly, GCN2 target genes were not upregulated in tumor-infiltrating T cells. MALDI-TOF MS imaging of B16 melanomas demonstrated maintenance of intratumoral tryptophan levels despite high tryptophan turnover, which prohibits a drop in tryptophan sufficient to activate GCN2 in tumor-infiltrating T cells. In conclusion, our results do not suggest that suppression of antitumor immune responses by tryptophan metabolism is driven by local tryptophan depletion and subsequent GCN2-mediated T cell anergy.

Tryptophan (Trp) metabolism is an important target in immuno-oncology as it represents a powerful immunosuppressive mechanism hijacked by tumors for protection against immune destruction. However, it remains unclear how tumor cells can proliferate while degrading the essential amino acid Trp. Trp is incorporated into proteins after it is attached to its tRNA by tryptophanyl-tRNA synthestases. As the tryptophanyl-tRNA synthestases compete for Trp with the Trp-catabolizing enzymes, the balance between these enzymes will determine whether Trp is used for protein synthesis or is degraded. In human cancers expression of the Trp-degrading enzymes indoleamine-2,3-dioxygenase-1 (IDO1) and tryptophan-2,3-dioxygenase (TDO2) was positively associated with the expression of the tryptophanyl-tRNA synthestase WARS. One mechanism underlying the association between IDO1 and WARS identified in this study is their joint induction by IFNγ released from tumor-infiltrating T cells. Moreover, we show here that IDO1- and TDO2-mediated Trp deprivation upregulates WARS expression by activating the general control non-derepressible-2 (GCN2) kinase, leading to phosphorylation of the eukaryotic translation initiation factor 2α (eIF2α) and induction of activating transcription factor 4 (ATF4). Trp deprivation induced cytoplasmic WARS expression but did not increase nuclear or extracellular WARS levels. GCN2 protected the cells against the effects of Trp starvation and enabled them to quickly make use of Trp for proliferation once it was replenished. Computational modeling of Trp metabolism revealed that Trp deficiency shifted Trp flux towards WARS and protein synthesis. Our data therefore suggest that the upregulation of WARS via IFNγ and/or GCN2-peIF2α-ATF4 signaling protects Trp-degrading cancer cells from excessive intracellular Trp depletion.

The development of rheumatoid arthritis (RA) is linked to functional changes in synovial fibroblasts (SF) and local infiltration of T lymphocytes. Fibroblasts possess the capacity to suppress T cell responses, although the molecular mechanisms of this suppression remain incompletely understood. In this study, we aimed to define the mechanisms by which noninflammatory SF modulate Th cell responses and to determine the immunosuppressive efficacy of RASF. Hence, the influence of SF from osteoarthritis or RA patients on total Th cells or different Th cell subsets of healthy donors was analyzed in vitro. We show that SF strongly suppressed the proliferation of Th cells and the secretion of IFN-γ in a cell contact-independent manner. In cocultures of SF and Th cells, tryptophan was completely depleted within a few days, resulting in eukaryotic initiation factor 2α phosphorylation, TCRζ-chain downregulation, and proliferation arrest. Blocking IDO1 activity completely restored Th cell proliferation, but not IFN-γ production. Interestingly, only the proliferation of Th1 cells, but not of Th2 or Th17 cells, was affected. Finally, RASF had a significantly lower IDO1 expression and a weaker Th cell suppressive capacity compared with osteoarthritis SF. We postulate that the suppression of Th cell growth by SF through tryptophan catabolism may play an important role in preventing inappropriate Th cell responses under normal conditions. However, expansion of Th17 cells that do not induce IDO1-mediated suppression and the reduced capacity of RASF to restrict Th cell proliferation through tryptophan metabolism may support the initiation and propagation of synovitis in RA patients.

Kynurenine formation by tryptophan-catabolic indoleamine-2,3-dioxygenase 1 (IDO1) plays a key role in tumor immune evasion and inhibition of IDO1 is efficacious in preclinical models of breast cancer. As the response of breast cancer to immune checkpoint inhibitors may be limited, a better understanding of the expression of additional targetable immunomodulatory pathways is of importance. We therefore investigated the regulation of IDO1 expression in different breast cancer subtypes. We identified estrogen receptor α (ER) as a negative regulator of IDO1 expression. Serum kynurenine levels as well as tumoral IDO1 expression were lower in patients with ER-positive than ER-negative tumors and an inverse relationship between and estrogen receptor mRNA was observed across 14 breast cancer data sets. Analysis of whole genome bisulfite sequencing, 450k, MassARRAY and pyrosequencing data revealed that the promoter is hypermethylated in ER-positive compared with ER-negative breast cancer. Reduced induction of IDO1 was also observed in human ER-positive breast cancer cell lines. IDO1 induction was enhanced upon DNA demethylation in ER-positive but not in ER-negative cells and methylation of an promoter construct reduced expression, suggesting that enhanced methylation of the promoter suppresses IDO1 in ER-positive breast cancer. The association of ER overexpression with epigenetic downregulation of IDO1 appears to be a particular feature of breast cancer as was not suppressed by promoter hypermethylation in the presence of high ER expression in cervical or endometrial cancer.

Objective The development of RA is linked to local infiltration of immune cells and to changes in the phenotype of synovial fibroblasts. Synovial fibroblasts possess the capacity to suppress T cell responses through indoleamine 2, 3-dioxygenase 1 (IDO1)-mediated tryptophan metabolism. However, synovial fibroblasts from RA patients are restricted in this immune-modulatory function. Moreover, hypoxic conditions are detected within synovial tissues of RA patients, with oxygen tensions of only 3.2% O2. This study aims at investigating the effects of hypoxia on the interaction between T cells and synovial fibroblasts, particularly on the T cell-suppressive capacities of synovial fibroblasts. Methods Synovial fibroblasts were cultured with Th cells under normoxic and hypoxic conditions (3% O2). Th cell proliferation was detected by flow cytometry. Tryptophan and kynurenine amounts were measured by HPLC. IDO1 expression and signal transducer and activator of transcription 1 (STAT1) phosphorylation were quantified by real-time PCR or western blot, and cytokine secretion by ELISA. Results Hypoxic conditions strongly diminished the Th cell-suppressive capacities of both OA synovial fibroblasts and RA synovial fibroblasts. Accordingly, IDO1 mRNA and protein expression, STAT1 phosphorylation and tryptophan metabolism were greatly reduced in OA synovial fibroblasts by hypoxia. MMP-3, IL-6, IL-10 and IFNγ secretion were significantly decreased under hypoxia in synovial fibroblast-Th cell co-cultures, while IL-17A levels were elevated. Supplementation with IFNγ, a well-known inducer of IDO1 expression, could rescue neither IDO1 expression nor Th cell suppression under hypoxic conditions. Conclusion Hypoxia strongly affected the crosstalk between synovial fibroblasts and Th cells. By reducing the efficiency of synovial fibroblasts to restrict Th cell proliferation and by increasing the expression of IL-17A, hypoxia might have implications on the pathophysiology of RA.

BackgroundThe pathogenesis of rheumatoid arthritis (RA) is linked to functional changes in synovial fibroblasts (SF) and local infiltration of T lymphocytes. Increased synovial inflammation is also associated with a hypoxic joint microenvironment. Oxygen levels in the joints of RA patients are significantly decreased compared to those of osteoarthritis (OA) patients with values of about 22.5mmHg corresponding to ambient oxygen tensions of 3.2%. So far, little is known about the effects of hypoxia on the interaction between fibroblasts and T lymphocytes and its implications on the pathophysiology of RA.ObjectivesThe aim of this study was to compare the influence of SF from RA versus OA patients on T helper (Th) cell responses both under normoxic and hypoxic conditions.MethodsSF were isolated from synovectomy tissues of OA or RA patients, Th cells were isolated from peripheral blood of RA patients or healthy donors. Cell cultures were performed under normoxic or hypoxic (3% O2) conditions. Th cell proliferation was determined by PKH26 labelling and flow cytometry. Cytokine secretion was quantified by ELISA. Indoleamine 2,3-dioxygenase 1 (IDO1) expression was analysed by Western Blot and expression of enzymes of the kynurenine pathway by real-time PCR. Tryptophan/ kynurenine levels in culture supernatants were quantified by HPLC.ResultsSF strongly inhibited the proliferation of co-cultured Th cells. Tryptophan was completely depleted within a few days in co-cultures of SF and Th cells, resulting in eukaryotic initiation factor (eIF)2α phosphorylation, TCRζ-chain down-regulation and proliferation arrest. Blocking of IDO1 completely restored Th cell proliferation, indicating that SF suppressed the proliferation of Th cells through IDO1-mediated tryptophan catabolism. Interestingly, RASF showed a significantly lower IDO1 expression, tryptophan metabolism and a weaker Th cell suppressive capacity compared to OASF. Under hypoxic conditions, the secretion of IFNg, the expression of IDO1, the tryptophan metabolism and the Th cell suppressive capacity of both OASF and RASF were significantly reduced.ConclusionsSF suppressed Th cell growth through IDO1-mediated tryptophan catabolism. This mechanism may play an important role in preventing inappropriate Th cell responses under normal conditions. The reduced tryptophan metabolism under hypoxia together with the inferior efficiency of RASF to restrict T cell proliferation likely supports the development of synovitis in RA.Disclosure of InterestNone declared

BackgroundThe development of rheumatoid arthritis (RA) is linked to changes in synovial fibroblasts’ (SF) phenotype and local infiltration of immune cells. In addition, hypoxic conditions are detected within the inflamed joints of RA patients. The oxygen level in RA joints is only about 22,5mmHg corresponding to ambient oxygen tensions of 3.2%. Right now there is not much known about the effect of hypoxia on the interaction between fibroblasts and immune cells and its implications on the pathophysiology of RA.Materials and methodsSF from synovectomy tissues of patients with osteoarthritis (OA) and RA were cultured in vitro. CD4-positive T cells were isolated from peripheral blood. The interaction between both cell types was examined through in vitro co-cultures both under normoxic and hypoxic (3%O2) conditions. The proliferation of Th cells was detected by PKH26 labelling and flow cytometry. Secretion of cytokines was quantified by ELISA. Indoleamine 2,3-dioxygenase 1 (IDO1) expression was measured by real-time PCR and Western Blot. Tryptophan amounts were detected by HPLC. IDO1 activity was blocked by 1-l-methyl-tryprophan (1-L-MT).ResultsSF strongly inhibit the proliferation of co-cultured Th cells. However, RASF have a significantly weaker T cell suppressive capacity compared to OASF. The inhibition of T cell proliferation – both via OASF and RASF - is diminished through hypoxia. Blockade of IDO1 completely restores Th cell proliferation, indicating that the suppression of Th cell proliferation by SF is mediated by tryptophan metabolism. Accordingly, in the supernatants of co-cultures with OASF, the amount of tryptophan is highly reduced, whereas in co-cultures with RASF more tryptophan is left. Under hypoxic conditions the tryptophan metabolism as well as the mRNA expression and protein levels of IDO1 are reduced. IFNγ levels are decreased, IL-6 and IL-8 expression are unaffected by hypoxia.ConclusionsThe suppression of activated T cells by SF through tryptophan metabolism may participate in the prevention of inappropriate T cell responses under normal conditions. The inferior efficiency of RASF to restrict T cell proliferation and the reduced tryptophan metabolism under hypoxia likely support the development of synovitis in RA.