Intercellular crosstalk shapes purinergic metabolism and signaling in cancer cells.

CD73-derived adenosine suppresses anti-cancer immunity, and CD73 inhibitors are currently evaluated in several clinical trials. Here, we have assessed enzyme kinetics of all key purinergic ectoenzymes in five cancer cell lines (Hodgkin lymphoma, multiple myeloma, pancreas adenocarcinoma, urinary bladder carcinoma, and glioblastoma) under normoxia and hypoxia. We found that adenosine metabolism varied considerably between individual cancer types. All cell lines investigated exhibited high ecto-adenosine deaminase (ADA) activity, which critically influenced the kinetics of adenosine accumulation. Combining kinetics data with single-cell RNA sequencing data on myeloma and glioblastoma cancerous tissue revealed that purine metabolism is not homogeneously organized, but it differs in a cancer type-specific fashion between malignant cells, stromal cells, and immune cells. Since purine metabolism in cancerous tissue is most likely spatially heterogeneous and differs between the various cell types, diffusion distances in the microenvironment as well as ADA activity may be important variables that influence the level of bioactive adenosine.

CD73 deficiency does not aggravate angiotensin II-induced aortic inflammation in mice.

Vascular inflammation plays a key role in the development of aortic diseases. A potential novel target for treatment might be CD73, an ecto-5'-nucleotidase that generates anti-inflammatory adenosine in the extracellular space. Here, we investigated whether a lack of CD73 results in enhanced aortic inflammation. To this end, angiotensin II was infused into wildtype and CD73-/- mice over 10 days. Before and after infusion, mice were analyzed using magnetic resonance imaging, ultrasound, flow cytometry, and histology. The impact of age and gender was investigated using female and male mice of three and six months of age, respectively. Angiotensin II infusion led to increased immune cell infiltration in both genotypes' aortae, but depletion of CD73 had no impact on immune cell recruitment. These findings were not modified by age or sex. No substantial difference in morphological or functional characteristics could be detected between wildtype and CD73-/- mice. Interestingly, the expression of CD73 on neutrophils decreased significantly in wildtype mice during treatment. In summary, we have found no evidence that CD73 deficiency affects the onset of aortic inflammation. However, as CD73 expression decreased during disease induction, an increase in CD73 by pharmaceutical intervention might result in lower vascular inflammation and less vascular disease.

Mono-ADP-ribosylation sites of human CD73 inhibit its adenosine-generating enzymatic activity.

CD73-derived adenosine plays a major role in damage-induced tissue responses by inhibiting inflammation. Damage-associated stimuli, such as hypoxia and mechanical stress, induce the cellular release of ATP and NAD+ and upregulate the expression of the nucleotide-degrading purinergic ectoenzyme cascade, including adenosine-generating CD73. Extracellular NAD+ also serves as substrate for mono-ADP-ribosylation of cell surface proteins, which in human cells is mediated by ecto-ADP-ribosyltransferase 1 (ARTC1). Here we explored, whether human CD73 enzymatic activity is regulated by mono-ADP-ribosylation, using recombinant human CD73 in the presence of ARTC1 with etheno-labelled NAD+ as substrate. Multi-colour immunoblotting with an anti-etheno-adenosine antibody showed ARTC1-mediated transfer of ADP-ribose together with the etheno label to CD73. HPLC analysis of the enzymatic activity of in vitro-ribosylated CD73 revealed strong inhibition of adenosine generation in comparison to non-ribosylated CD73. Mass spectrometry of in vitro-ribosylated CD73 identified six ribosylation sites. 3D model analysis indicated that three of them (R328, R354, R545) can interfere with CD73 enzymatic activity. Our study identifies human CD73 as target for ARTC1-mediated mono-ADP-ribosylation, which can profoundly modulate its adenosine-generating activity. Thus, in settings with enhanced release of NAD+ as substrate for ARTC1, assessment of CD73 protein expression in human tissues may not be predictive of adenosine formation resulting in anti-inflammatory activity.

Profound inhibition of CD73-dependent formation of anti-inflammatory adenosine in B cells of SLE patients.

BACKGROUND: Systemic lupus erythematosus (SLE) is a chronic autoimmune disease that leads to a breakdown of tolerance to self-antigens resulting in inflammation and organ damage. The anti-inflammatory activity of CD73-derived adenosine is well documented, however, its role in SLE pathogenesis is unknown. METHODS: Human peripheral blood immune cells were obtained from adult SLE patients (SLE) and healthy controls (HC). Expression and activity of purinergic ectoenzymes were assessed by qRT-PCR, flow cytometry and HPLC. Genes encoding purinergic ectoenzymes in SLE patients were analysed with targeted DNA sequencing. FINDINGS: Among circulating immune cells (both in HC and SLE), CD73 was most highly expressed on B cells, which was mirrored by high enzymatic activity only in HC. CD73 protein molecular weight was unchanged in SLE, however, the enzymatic activity of CD73 on SLE B cells was almost fully abolished. Accordingly, AMP accumulated in cultured SLE B cells. A similar discrepancy between protein expression and enzymatic activity was observed for NAD-degrading CD38 on SLE B cells. No differences were found in the rate of extracellular ATP degradation and expression of CD39, CD203a/c, and CD157. DNA sequencing identified no coding variants in CD73 in SLE patients. INTERPRETATION: We describe a new pathomechanism for SLE, by which inactivation of CD73 on B cells produces less anti-inflammatory adenosine, resulting in immune cell activation. CD73 inactivation was not due to genetic variation but may be related to posttranslational modification. FUNDING: The German Research Council, Medical Faculty of the Heinrich-Heine-University Duesseldorf, Hiller Research Foundation, and Cardiovascular Research Institute Duesseldorf.

Normoxic induction of HIF-1α by adenosine-A2B R signaling in epicardial stromal cells formed after myocardial infarction.

Myocardial infarction (MI) activates the epicardium to form epicardial stromal cells (EpiSC) that reside in the epicardial hypoxic microenvironment. Paracrine factors secreted by EpiSC were shown to modulate the injury response of the post-MI heart and improve cardiac function. We have previously reported that the expression of the angiogenic cytokines vascular endothelial growth factor A (VEGFA) and IL-6 is strongly upregulated in EpiSC by adenosine acting via the A2B receptor (A2B R). Since tissue hypoxia is well known to be a potent stimulus for the generation of extracellular adenosine, the present study explored the crosstalk of A2B R activation and hypoxia-hypoxia-inducible factor 1 alpha (HIF-1α) signaling in cultured EpiSC, isolated from rat hearts 5 days after MI. We found substantial nuclear accumulation of HIF-1α after A2B R activation even in the absence of hypoxia. This normoxic HIF-1α induction was PKC-dependent and involved upregulation of HIF-1α mRNA expression. While the influence of hypoxia on adenosine generation and A2B R signaling was only minor, hypoxia and A2B R activation cumulatively increased VEGFA expression. Normoxic A2B R activation triggered an HIF-1α-associated cell-protective metabolic switch and reduced oxygen consumption. HIF-1α targets and negative regulators PHD2 and PHD3 were only weakly induced by A2B R signaling, which may result in a sustained HIF-1α activity. The A2B R-mediated normoxic HIF-1α induction was also observed in cardiac fibroblasts from healthy mouse hearts, suggesting that this mechanism is also functional in other A2B R-expressing cell types. Altogether, we identified A2B R-mediated HIF-1α induction as novel aspect in the HIF-1α-adenosine crosstalk, which modulates EpiSC activity and can amplify HIF-1α-mediated cardioprotection.

CNP mediated selective toxicity on melanoma cells is accompanied by mitochondrial dysfunction.

Cerium (Ce) oxide nanoparticles (CNP; nanoceria) are reported to have cytotoxic effects on certain cancerous cell lines, while at the same concentration they show no cytotoxicity on normal (healthy) cells. Redox-active CNP exhibit both selective prooxidative as well as antioxidative properties. The former is proposed to be responsible for impairment of tumor growth and invasion and the latter for rescuing normal cells from reactive oxygen species (ROS)-induced damage. Here we address possible underlying mechanisms of prooxidative effects of CNP in a metastatic human melanoma cell line. Malignant melanoma is the most aggressive form of skin cancer, and once it becomes metastatic the prognosis is very poor. We have shown earlier that CNP selectively kill A375 melanoma cells by increasing intracellular ROS levels, whose basic amount is significantly higher than in the normal (healthy) counterpart, the melanocytes. Here we show that CNP initiate a mitochondrial increase of ROS levels accompanied by an increase in mitochondrial thiol oxidation. Furthermore, we observed CNP-induced changes in mitochondrial bioenergetics, dynamics, and cristae morphology demonstrating mitochondrial dysfunction which finally led to tumor cell death. CNP-induced cell death is abolished by administration of PEG-conjugated catalase. Overall, we propose that cerium oxide nanoparticles mediate cell death via hydrogen peroxide production linked to mitochondrial dysfunction.

Genetic profiling and surface proteome analysis of human atrial stromal cells and rat ventricular epicardium-derived cells reveals novel insights into their cardiogenic potential.

Epicardium-derived cells (EPDC) and atrial stromal cells (ASC) display cardio-regenerative potential, but the molecular details are still unexplored. Signals which induce activation, migration and differentiation of these cells are largely unknown. Here we have isolated rat ventricular EPDC and rat/human ASC and performed genetic and proteomic profiling. EPDC and ASC expressed epicardial/mesenchymal markers (WT-1, Tbx18, CD73, CD90, CD44, CD105), cardiac markers (Gata4, Tbx5, troponin T) and also contained phosphocreatine. We used cell surface biotinylation to isolate plasma membrane proteins of rEPDC and hASC, Nano-liquid chromatography with subsequent mass spectrometry and bioinformatics analysis identified 396 rat and 239 human plasma membrane proteins with 149 overlapping proteins. Functional GO-term analysis revealed several significantly enriched categories related to extracellular matrix (ECM), cell migration/differentiation, immunology or angiogenesis. We identified receptors for ephrin and growth factors (IGF, PDGF, EGF, anthrax toxin) known to be involved in cardiac repair and regeneration. Functional category enrichment identified clusters around integrins, PI3K/Akt-signaling and various cardiomyopathies. Our study indicates that EPDC and ASC have a similar molecular phenotype related to cardiac healing/regeneration. The cell surface proteome repository will help to further unravel the molecular details of their cardio-regenerative potential and their role in cardiac diseases.

CD73 on T Cells Orchestrates Cardiac Wound Healing After Myocardial Infarction by Purinergic Metabolic Reprogramming.

BACKGROUND: T cells are required for proper healing after myocardial infarction. The mechanism of their beneficial action, however, is unknown. The proinflammatory danger signal ATP, released from damaged cells, is degraded by the ectonucleotidases CD39 and CD73 to the anti-inflammatory mediator adenosine. Here, we investigate the contribution of CD73-derived adenosine produced by T cells to cardiac remodeling after ischemia/reperfusion and define its mechanism of action. METHODS: Myocardial ischemia (50 minutes followed by reperfusion) was induced in global CD73-/- and CD4-CD73-/- mice. Tissue injury, T-cell purinergic signaling, cytokines, and cardiac function (magnetic resonance tomography at 9.4 T over 4 weeks) were analyzed. RESULTS: Changes in functional parameters of CD4-CD73-/- mice were identical to those in global CD73 knockouts (KOs). T cells infiltrating the injured heart significantly upregulated at the gene (quantitative polymerase chain reaction) and protein (enzymatic activity) levels critical transporters and enzymes (connexin43, connexin37, pannexin-1, equilibrative nucleoside transporter 1, CD39, CD73, ecto-nucleotide pyrophosphatase/phosphodiesterases 1 and 3, CD157, CD38) for the accelerated release and hydrolysis of ATP, cAMP, AMP, and NAD to adenosine. It is surprising that a lack of CD39 on T cells (from CD39-/- mice) did not alter ATP hydrolysis and very likely involves pyrophosphatases (ecto-nucleotide pyrophosphatase/phosphodiesterases 1 and 3). Circulating T cells predominantly expressed A2a receptor (A2aR) transcripts. After myocardial infarction, A2b receptor (A2bR) transcription was induced in both T cells and myeloid cells in the heart. Thus, A2aR and A2bR signaling may contribute to myocardial responses after myocardial infarction. In the case of T cells, this was associated with an accelerated secretion of proinflammatory and profibrotic cytokines (interleukin-2, interferon-γ, and interleukin-17) when CD73 was lacking. Cytokine production by T cells from peripheral lymph nodes was inhibited by A2aR activation (CGS-21680). The A2bR agonist BAY 60-6583 showed off-target effects. The adenosine receptor agonist NECA inhibited interferon-γ and stimulated interleukin-6 production, each of which was antagonized by a specific A2bR antagonist (PSB-603). CONCLUSIONS: This work demonstrates that CD73 on T cells plays a crucial role in the cardiac wound healing process after myocardial infarction. The underlying mechanism involves a profound increase in the hydrolysis of ATP/NAD and AMP, resulting primarily from the upregulation of pyrophosphatases and CD73. We also define A2bR/A2aR-mediated autacoid feedback inhibition of proinflammatory/profibrotic cytokines by T cell-derived CD73.

Adenosine signalling mediates the anti-inflammatory effects of the COX-2 inhibitor nimesulide.

Extracellular adenosine formation from ATP is controlled by ecto-nucleoside triphosphate diphosphohydrolase (E-NTPDase/CD39) and ecto-5'-nucleotidase (e-5NT/CD73); the latter converts AMP to adenosine and inorganic phosphate, representing the rate limiting step controlling the ratio between extracellular ATP and adenosine. Evidence that cellular expression and activity of CD39 and CD73 may be subject to changes under pathophysiological conditions has identified this pathway as an endogenous modulator in several diseases and was shown to be involved in the molecular mechanism of drugs, such as methotrexate, salicylates , interferon-ß. We evaluated whether CD73/adenosine/A2A signalling pathway is involved in nimesulide anti-inflammatory effect, in vivo and in vitro. We found that the adenosine A2A agonist, 4-[2-[[6-amino-9-(N-ethyl-ß-d-ribofuranuronamidosyl)-9H-purin-2-yl]amino]ethyl]benzenepropanoic acid hydrochloride (CGS21680, 2mg/kg ip.), inhibited carrageenan-induced rat paw oedema and the effect was reversed by co-administration of the A2A antagonist -(2-[7-amino-2-[2-furyl][1,2,4]triazolo[2,3-a][1,3,5]triazin-5-yl-amino]ethyl)phenol (ZM241385; 3mg/kg i.p.). Nimesulide (5mg/kg i.p.) anti-inflammatory effect was inhibited by pre-treatment with ZM241385 (3mg/kg i.p.) and by local administration of the CD73 inhibitor, adenosine 5'-(α,ß-methylene)diphosphate (APCP; 400µg/paw). Furthermore, we found increased activity of 5'-nucleotidase/CD73 in paws and plasma of nimesulide treated rats, 4h following oedema induction. In vitro, the inhibitory effect of nimesulide on nitrite and prostaglandin E2 production by lipopolysaccharide-activated J774 cell line was reversed by ZM241385 and APCP. Furthermore, nimesulide increased CD73 activity in J774 macrophages while it did not inhibit nitrite accumulation by lipopolysaccharide-activated SiRNA CD73 silenced J774 macrophages. Our data demonstrate that the anti-inflammatory effect of nimesulide in part is mediated by CD73-derived adenosine acting on A2A receptors.

IL-21 is a central memory T cell-associated cytokine that inhibits the generation of pathogenic Th1/17 effector cells.

IL-21 promotes Th17 differentiation, and Th17 cells that upregulate T-bet, IFN-γ, and GM-CSF drive experimental autoimmune diseases in mice. Anti-IL-21 treatment of autoimmune patients is therefore a therapeutic option, but the role of IL-21 in human T cell differentiation is incompletely understood. IL-21 was produced at high levels by human CD4(+) central memory T cells, suggesting that it is associated with early T cell differentiation. Consistently, it was inhibited by forced expression of T-bet or RORC2, the lineage-defining transcription factors of Th1 and Th17 effector cells, respectively. Although IL-21 was efficiently induced by IL-12 in naive CD4(+) T cells, it inhibited the generation of Th1 effector cells in a negative feedback loop. IL-21 was also induced by IL-6 and promoted Th17 differentiation, but it was not absolutely required. Importantly, however, IL-21 promoted IL-10 secretion but inhibited IFN-γ and GM-CSF production in developing Th17 cells, and consequently prevented the generation of polyfunctional Th1/17 effector cells. Moreover, in Th17 memory cells, IL-21 selectively inhibited T-bet upregulation and GM-CSF production. In summary, IL-21 is a central memory T cell-associated cytokine that promotes Th17 differentiation and IL-10 production, but inhibits the generation of potentially pathogenic Th1/17 effector cells. These findings shed new light on the role of IL-21 in T cell differentiation, and have relevant implications for anti-IL-21 therapy of autoimmune diseases.

Purinergic signaling on leukocytes infiltrating the LPS-injured lung.

Extracellular nucleotides and nucleosides have been implicated as important signaling molecules in the pathogenesis of acute lung injury (ALI). While adenosine is known to inhibit T cell activation, little information is available as to ATP and NAD degrading enzymes, the expression of ATP and adenosine receptors/transporters in different T cell subsets. ALI was induced by challenging mice with intra-tracheal instillation of 60 µl (3 µg/g) LPS. After 3 d and 7 d blood, lung tissue and bronchoalveolar lavage was collected and immune cells were analyzed using flow cytometry. The transcriptional phenotype of T helper cells, cytotoxic and regulatory T cells sorted by FACS was assessed by measuring the expression profile of 28 genes related to purinergic signaling using TaqMan Array Micro Fluidic Cards. Catabolism of ATP, NAD and cAMP by activated CD4+ T cells was evaluated by HPLC. CD73 was found to be highly abundant on lymphoid cells with little abundance on myeloid cells, while the opposite was true for CD39. After ALI, the abundance of CD39 and CD73 significantly increased on all T cell subsets derived from lung tissue and bronchoalveolar space. Expression analysis in T cell subsets of the lung revealed ATP (Cd39, Cd73) and NAD (Cd38, Cd157, Cd296, Pc-1) degrading enzymes. However, only transcription of Cd38, Cd39, Cd73, Ent1 and A2a receptor was significantly upregulated after ALI in T helper cells. CD4+ T cells from injured lung rapidly metabolized extracellular ATP to AMP and adenosine but not NAD or cAMP. These findings show that lung T cells--the dominant cell fraction in the later phase of ALI--exhibit a unique expression pattern of purinergic signaling molecules. Adenosine is formed by T cells at an enhanced rate from ATP but not from NAD and together with upregulated A2a receptor is likely to modulate the healing process after acute lung injury.