Expression Of Ectonucleotidases Research Articles

Endometrial epithelial cell organoids as tools for studying the CD39 family of enzymes and for validating enzyme inhibitors.

Extracellular adenosine triphosphate (ATP) conducts a complex dynamic system of broadly represented cell signaling. Ectonucleotidases are the enzymes with nucleotide hydrolytic ability that regulate ATP levels in physiological and pathological conditions, thus playing a key role in the so-called purinergic signaling. Altered ectonucleotidase expression has been reported in cancer, and the ectonucleoside triphosphate diphosphohydrolase (NTPDase) family of enzymes, with its best-known form NTPDase1 (CD39), is targeted in cancer immunotherapy. The tandem of enzymes CD39-CD73 is responsible for the generation of immunosuppressive adenosine in the tumor microenvironment, and inhibition strategies are of great interest. Organoids have emerged as very convenient models for the study of tumors since they are three-dimensional cultures that retain many of the features of tissue. The present study aims to contribute to improving the methodology and the molecular tools needed for the study of ectonucleotidases in healthy and disease conditions. The study, performed in an endometrial cancer cell model, could be extended to other types of tumors and pathologies in which the purinergic system is involved. We generated organoids from endometrial cancer cells overexpressing NTPDase2 (CD39L1) and NTPDase3 (CD39L3) as fusion proteins with EGFP, and we performed functional assays by adapting in situ cytochemistry protocols. This allowed us to simultaneously detect enzyme activity and protein expression and to demonstrate that organoids can be used to test ectonucleotidase inhibitors-a result that can be used to develop new cancer treatment options.

Curcumin modulates purinergic signaling and inflammatory response in cutaneous metastatic melanoma cells.

Cutaneous melanoma (CM) poses a therapeutic challenge due to its aggressive nature and often limited response to conventional treatments. Exploring novel therapeutic targets is essential, and natural compounds have emerged as potential candidates. This study aimed to elucidate the impact of curcumin, a natural compound known for its anti-inflammatory, antioxidant, and anti-tumor properties, on metastatic melanoma cells, focusing on the purinergic system and immune responses. Human melanoma cell line SK-Mel-28 were exposed to different curcumin concentrations for either 6 or 24h, after which we assessed components related to the purinergic system and the inflammatory cascade. Using RT-qPCR, we assessed the gene expression of CD39 and CD73 ectonucleotidases, as well as adenosine deaminase (ADA). Curcumin effectively downregulated CD39, CD73, and ADA gene expression. Flow cytometry analysis revealed that curcumin significantly reduced CD39 and CD73 protein expression at specific concentrations. Moreover, the A2A receptor's protein expression decreased across all concentrations. Enzymatic activity assays demonstrated that curcumin modulated CD39, CD73, and ADA activities, with effects dependent on concentration and duration of treatment. Extracellular ATP levels increased after 24h of curcumin treatment, emphasizing its role in modulating hydrolytic activity. Curcumin also displayed anti-inflammatory properties by reducing NLRP3 gene expression and impacting the levels of key inflammatory cytokines. In conclusion, this study unveils the potential of curcumin as a promising adjuvant in CM treatment. Curcumin modulates the expression and activity of crucial components of the purinergic system and exhibits anti-inflammatory effects, indicating its potential therapeutic role in combating CM. These findings underscore curcumin's promise and warrant further investigation in preclinical and clinical settings for melanoma management.

The Impact of A3AR Antagonism on the Differential Expression of Chemoresistance-Related Genes in Glioblastoma Stem-like Cells.

Glioblastoma (GB) is the most aggressive and common primary malignant tumor of the brain and central nervous system. Without treatment, the average patient survival time is about six months, which can be extended to fifteen months with multimodal therapies. The chemoresistance observed in GB is, in part, attributed to the presence of a subpopulation of glioblastoma-like stem cells (GSCs) that are characterized by heightened tumorigenic capacity and chemoresistance. GSCs are situated in hypoxic tumor niches, where they sustain and promote the stem-like phenotype and have also been correlated with high chemoresistance. GSCs have the particularity of generating high levels of extracellular adenosine (ADO), which causes the activation of the A3 adenosine receptor (A3AR) with a consequent increase in the expression and activity of genes related to chemoresistance. Therefore, targeting its components is a promising alternative for treating GB. This analysis determined genes that were up- and downregulated due to A3AR blockades under both normoxic and hypoxic conditions. In addition, possible candidates associated with chemoresistance that were positively regulated by hypoxia and negatively regulated by A3AR blockades in the same condition were analyzed. We detected three potential candidate genes that were regulated by the A3AR antagonist MRS1220 under hypoxic conditions: LIMD1, TRIB2, and TGFB1. Finally, the selected markers were correlated with hypoxia-inducible genes and with the expression of adenosine-producing ectonucleotidases. In conclusion, we detected that hypoxic conditions generate extensive differential gene expression in GSCs, increasing the expression of genes associated with chemoresistance. Furthermore, we observed that MRS1220 could regulate the expression of LIMD1, TRIB2, and TGFB1, which are involved in chemoresistance and correlate with a poor prognosis, hypoxia, and purinergic signaling.

Ectonucleotidase expression on T-helper cells as a response assessment tool to immune-checkpoint therapy in head and neck cancers

Ectonucleotidase expression on T-helper cells as a response assessment tool to immune-checkpoint therapy in head and neck cancers

Altered Expression of Astrocytic ATP Channels and Ectonucleotidases in the Cerebral Cortex and Hippocampus of Chronic Social Defeat Stress-Susceptible BALB/c Mice.

The increasing number of patients with depressive disorder is a serious socioeconomic problem worldwide. Although several therapeutic agents have been developed and used clinically, their effectiveness is insufficient and thus discovery of novel therapeutic targets is desired. Here, focusing on dysregulation of neuronal purinergic signaling in depressive-like behavior, we examined the expression profiles of ATP channels and ectonucleotidases in astrocytes of cerebral cortex and hippocampus of chronic social defeat stress (CSDS)-susceptible BALB/c mice. Mice were exposed to 10-d CSDS, and their astrocytes were obtained using a commercially available kit based on magnetic activated cell sorting technology. In astrocytes derived from cerebral cortex of CSDS-susceptible mice, the expression levels of mRNAs for connexin 43, P2X7 receptors and maxi anion channels were increased, those for connexin 43 and P2X7 receptors being inversely correlated with mouse sociability, and the expression of mRNAs for ecto-nucleoside triphosphate diphosphohydrase 2 and ecto-5'nucleotidase was decreased and increased, respectively. On the other hand, the alteration profiles of ATP channels and ectonucleotidases in hippocampal astrocytes of CSDS-susceptible mice were different from in the case of cortical astrocytes, and there was no significant correlation between expression levels of their mRNAs and mouse sociability. These findings imply that increased expression of ATP channels in cerebral cortex might be involved in the development of reduced sociability in CSDS-subjected BALB/c mice. Together with recent findings, it is suggested that ATP channels expressed by cortical astrocytes might be potential therapeutic targets for depressive disorder.

Relationship between the Gene Expression of Adenosine Kinase Isoforms and the Expression of CD39 and CD73 Ectonucleotidases in Colorectal Cancer.

Tumor cells have the capacity to create an adenosine-rich immunosuppressive environment, which can interfere with antitumor immunotherapy. Approaches are currently being developed with a view to suppressing the production of adenosine or its signals. Such approaches include the use of antibodies to inhibit CD39, CD73, and adenosine-receptor antagonists. However, the abundance of enzymatic pathways that control the ATP-adenosine balance, as well as the still poorly understood intracellular adenosine regulation, makes the hoped-for success unlikely. In the present study, the enzyme adenosine kinase (ADK) needed to convert adenosine to adenosine monophosphate, thereby regulating its levels, was investigated. To do so, peripheral blood samples from patients with colorectal cancer (CRC) (n = 31) were collected with blood samples from healthy donors (n = 17) used as controls. ADK gene expression levels and those of its long (ADK-L) and short (ADK-S) isoforms were measured. The relationship between the levels of ADK gene expression and that of CD39, CD73, and A2aR genes was analyzed. It turned out that in the group of CRC patients (stages III-IV), the level of ADK-L mRNA was lower (p < 0.0011) when compared to that of the control. For the first time, an average correlation was found between the level of expression of CD39 and ADK-S (r = -0.468 at p = 0.043) and between CD73 and ADK-L (r = 0.518 at p = 0.0232) in CRC patients. Flow cytometry was used to assess the content of CD39/CD73-expressing CD8+, CD4+ and Treg lymphocytes, as well as their relationship with the level of ADK gene expression in CRC patients. But no significant correlations were found.

Macrophages Provide Essential Support for Erythropoiesis, and Extracellular ATP Contributes to a Erythropoiesis-Supportive Microenvironment during Repeated Psychological Stress.

Psychological stress is a significant contributor to various chronic diseases and affects multiple physiological processes including erythropoiesis. This study aimed to examine the tissue-specific contributions of macrophages and extracellular ATP, as a signal of disturbed tissue homeostasis, to erythropoiesis under conditions of repeated psychological stress. Adult male BALB/c mice were subjected to 2 h daily restraint stress for seven consecutive days. Clodronate-liposomes were used to deplete resident macrophages from the bone marrow and spleen two days prior to the first restraint procedure, as well as newly recruited macrophages, every third day for the duration of the experiment. Repeated stress induced a considerable increase in the number of erythroid progenitor cells as well as in the percentage of CD71+/Ter119+ and CD71-/Ter119+ cells in the bone marrow and spleen. Macrophage depletion completely abolished the stimulative effect of repeated stress on immature erythroid cells, and prevented stress-induced increases in ATP levels, P2X7 receptor (P2X7R) expression, and ectonucleotidase CD39 activity and expression in the bone marrow and spleen. The obtained results demonstrate the stimulative effects of repeated stress on erythroid cells, extracellular ATP levels, P2X7R expression, CD39 activity and expression within the bone marrow and spleen, as well as the essential role of macrophages in stress-induced changes.

Improving combination therapies: targeting A2B-adenosine receptor to modulate metabolic tumor microenvironment and immunosuppression.

We investigated the role of A2B-adenosine receptor in regulating immunosuppressive metabolic stress in the tumor microenvironment. Novel A2B-adenosine receptor antagonist PBF-1129 was tested for antitumor activity in mice and evaluated for safety and immunologic efficacy in a phase I clinical trial of patients with non-small cell lung cancer. The antitumor efficacy of A2B-adenosine receptor antagonists and their impact on the metabolic and immune tumor microenvironment were evaluated in lung, melanoma, colon, breast, and epidermal growth factor receptor-inducible transgenic cancer models. Employing electron paramagnetic resonance, we assessed changes in tumor microenvironment metabolic parameters, including pO2, pH, and inorganic phosphate, during tumor growth and evaluated the immunologic effects of PBF-1129, including its pharmacokinetics, safety, and toxicity, in patients with non-small cell lung cancer. Levels of metabolic stress correlated with tumor growth, metastasis, and immunosuppression. Tumor interstitial inorganic phosphate emerged as a correlative and cumulative measure of tumor microenvironment stress and immunosuppression. A2B-adenosine receptor inhibition alleviated metabolic stress, downregulated expression of adenosine-generating ectonucleotidases, increased expression of adenosine deaminase, decreased tumor growth and metastasis, increased interferon γ production, and enhanced the efficacy of antitumor therapies following combination regimens in animal models (anti-programmed cell death 1 protein vs anti-programmed cell death 1 protein plus PBF-1129 treatment hazard ratio = 11.74 [95% confidence interval = 3.35 to 41.13], n = 10, P < .001, 2-sided F test). In patients with non-small cell lung cancer, PBF-1129 was well tolerated, with no dose-limiting toxicities; demonstrated pharmacologic efficacy; modulated the adenosine generation system; and improved antitumor immunity. Data identify A2B-adenosine receptor as a valuable therapeutic target to modify metabolic and immune tumor microenvironment to reduce immunosuppression, enhance the efficacy of immunotherapies, and support clinical application of PBF-1129 in combination therapies.

Epidermal melanocytes metabolize extracellular nucleotides by purinergic enzymes.

The human epidermal melanocyte (hEM) are melanin-producing cells that provide skin pigmentation and protection against ultraviolet radiation. Although purinergic signaling is involved in skin biology and pathology, the presence of NTPDase members, as well as the rate of nucleotides degradation by melanocytes were not described yet. Therefore, in this study, we analyzed the expression of ectonucleotidases in hEM derived from discarded foreskin of male patients. The expression of purinergic enzymes was confirmed by mRNA and flow cytometry. Among the ectonucleotidases, ectonucleoside triphosphate diphosphohydrolase1 (NTPDase1) and ecto-5´-nucleotidase were the ectoenzymes with higher expressions. The hydrolysis rate for ATP, ADP, and AMP was low in comparison to other primary cells already investigated. The amount of ATP in the culture medium was increased after a scratch wound and decreased to basal levels in 48 h, while the NTPDase1 and P2X7 expressions increased. Therefore, it is possible to suggest that after cell injury, the ATP released by hEM into the extracellular space will be hydrolyzed by ectonucleotidases as the NTPDase1 that will control the levels of nucleotides in the skin micro-environment.

Adenosine pathway regulates inflammation during Plasmodium vivax infection.

Plasmodium spp. infection triggers the production of inflammatory cytokines that are essential for parasite control, and conversely responsible for symptoms of malaria. Monocytes play a role in host defense against Plasmodium vivax infection and represent the main source of inflammatory cytokines and reactive oxygen species. The anti-inflammatory cytokine IL-10 is a key regulator preventing exacerbated inflammatory responses. Studies suggested that different clinical presentations of malaria are strongly associated with an imbalance in the production of inflammatory and anti-inflammatory cytokines. A convenience sampling of peripheral blood mononuclear cells from Plasmodium vivax-infected patients and healthy donors were tested for the characterization of cytokine and adenosine production and the expression of ectonucleotidases and purinergic receptors. Here we show that despite a strong inflammatory response, monocytes also bear a modulatory role during malaria. High levels of IL-10 are produced during P. vivax infection and its production can be triggered in monocytes by P. vivax-infected reticulocytes. Monocytes express high levels of ectonucleotidases, indicating their important role in extracellular ATP modulation and consequently in adenosine production. Plasmatic levels of adenosine are not altered in patients experiencing acute malaria; however, their monocyte subsets displayed an increased expression of P1 purinergic receptors. In addition, adenosine decreases Tumor Necrosis Factor production by monocytes, which was partially abolished with the blockage of the A2a receptor. Monocytes have a dual role, attempting to control both the P. vivax infection and the inflammatory response. Purinergic receptor modulators emerge as an untapped approach to ameliorate clinical malaria.

Lymphocytes from B-acute lymphoblastic leukemia patients present differential regulation of the adenosinergic axis depending on risk stratification

PurposeAlthough risk-stratified chemotherapy regimens improve B-cell acute lymphoblastic leukemia (B-ALL) clinical outcome, relapse occurs in a significant number of cases. The identification of new therapeutic targets as well as prognostic and diagnostic biomarkers can improve B-ALL patients' clinical outcomes. Purinergic signaling is an important pathway in cancer progression, however the expression of ectonucleotidases and their impact on immune cells in B-ALL lacks exploration. We aimed to analyze the expression of ectonucleotidases in B-ALL patients’ lymphocyte subpopulations.MethodsPeripheral blood samples from 15 patients diagnosed with B-ALL were analyzed. Flow cytometry was used to analyze cellularity, expression level of CD38, CD39, and CD73, and frequency of {mathrm{CD}38}^{+}{/mathrm{CD}73}^{+}, and {mathrm{CD}39}^{+}{/mathrm{CD}73}^{+} in lymphocyte subpopulations. Plasma was used for cytokines (by CBA kit) and adenine nucleosides/nucleotides detection (by HPLC).ResultsComparing B-ALL patients to health donors, we observed an increase of CD4 + and CD8 + T-cells. In addition, a decrease in CD38 expression in B and Treg subpopulations and an increase in CD39+ CD73+ frequency in Breg and CD8+ T-cells. Analyzing cytokines and adenine nucleosides/nucleotides, we found a decrease in TNF, IL-1β, and ADO concentrations, together with an increase in AMP in B-ALL patients' plasma.ConclusionAs immunomodulators, the expression of ectonucleotidases might be associated with activation states, as well as the abundance of different cellular subsets. We observed a pro-tumor immunity expression profile in B-ALL patients at diagnosis, being associated with cell exhaustion and immune evasion in B-ALL.

Регуляторные Т-клетки и T-хелперы 17-го типа с экспрессией эктонуклеотидаз CD39 и CD73 при тяжелой механической травме у детей

Frequent resulting disability and case mortality support the urgency of investigation of the immune response mechanisms triggered by severe injury (SI) in children. This study aimed to determine the informative immunological criteria of traumatic injury severity and prognosis in children (n = 43) based on the assessment of expression of CD39 and CD73 ectonucleotidase in populations of regulatory T cells (Treg, CD4+CD127lowCD25high) and T-helper 17 cells (Th17, CD4+CD161+CD3+) in SI cases grouped by the outcome (favorable (SIfav, n = 24), unfavorable (SIunfav, n = 17) and lethal (n = 2)). With the help of flow cytometry, we identified a pronounced decrease in the absolute number of Treg and Th17, as well as Treg and Th17 expressing CD39 and CD73, in the early post-traumatic period. In the SIfav and SIunfav groups the relative number of Treg and Th17 cells expressing CD39 differed significantly (p &lt;0.05); it was substantially higher form the first to the third day post injury in the SIunfav group. The level of Treg CD39 (44.4%) is a premise for an unfavorable outcome in children surviving an SI. In fatality cases, we registered extremely low ectonucleotidase expression rates: CD39+Treg — 9.52% (9.52–13.75) and CD39+Th17 — 0.92% (0.74–1.1). In the SIunfav group, the intensity of fluorescence (FL) of CD39 on Treg cells in the early post-traumatic period was higher than seen in the SIfav group. The threshold value for the average fluorescence intensity (FL) of CD39 on Treg was 8.25 c.u. In fatality cases, the Treg CD39 FL values were extremely low: 3.95 c.u. (3.7–4.67). The results of the study indicate that in children, the expression of CD39 and CD73 in Treg and Th17 populations is significantly associated with the severity of injury and outcome of the traumatic disease.

Dysfunctional purinergic signaling correlates with disease severity in COVID-19 patients.

Ectonucleotidases modulate inflammatory responses by balancing extracellular ATP and adenosine (ADO) and might be involved in COVID-19 immunopathogenesis. Here, we explored the contribution of extracellular nucleotide metabolism to COVID-19 severity in mild and severe cases of the disease. We verified that the gene expression of ectonucleotidases is reduced in the whole blood of patients with COVID-19 and is negatively correlated to levels of CRP, an inflammatory marker of disease severity. In line with these findings, COVID-19 patients present higher ATP levels in plasma and reduced levels of ADO when compared to healthy controls. Cell type-specific analysis revealed higher frequencies of CD39+ T cells in severely ill patients, while CD4+ and CD8+ expressing CD73 are reduced in this same group. The frequency of B cells CD39+CD73+ is also decreased during acute COVID-19. Interestingly, B cells from COVID-19 patients showed a reduced capacity to hydrolyze ATP into ADP and ADO. Furthermore, impaired expression of ADO receptors and a compromised activation of its signaling pathway is observed in COVID-19 patients. The presence of ADO in vitro, however, suppressed inflammatory responses triggered in patients’ cells. In summary, our findings support the idea that alterations in the metabolism of extracellular purines contribute to immune dysregulation during COVID-19, possibly favoring disease severity, and suggest that ADO may be a therapeutic approach for the disease.

Expression of CD39 and CD73 ectonucleotidases in CD4+ lymphocyte populations in healthy children

Purinergic system plays an important role in functional regulation of immune system. Extracellular ATP belongs to non-infectious danger signals (DAMP), has a pro-inflammatory effect and modulates the immune response. The end product of ATP breakdown, adenosine, plays an important role in limiting the inflammatory response. The activity of ectonucleotidase CD39 and CD73 supports the balance of ATP and adenosine at the site of inflammation. CD39 and CD73 expression is characterized by high variability. From the literature data, appropriate studies were carried out either in transgenic mice, or with adult healthy donors. The number of cells expressing CD39 and CD73 in T lymphocyte populations has not been evaluated in healthy children. Hence, our aim was to study the features of CD39 and CD73 expression in various subpopulations of CD4+ lymphocytes in apparently healthy children of different ages.&#x0D; We examined 45 healthy children aged 3.7 to 17.5 years (Me (Q0.25-Q0.75) 12.4 (10-16.1). The numbers of CD4+ cells, regulatory T lymphocytes (Treg CD4+CD25highCD127low), activated T helpers (Tact CD4+CD25+CD127high), and Th17 lymphocytes (CD4+CD161+CD3+) expressing CD39 and CD73 were evaluated by the flow cytometry.&#x0D; The number of cells expressing CD39 and CD73 depends on specific cell subpopulation. The highest content of CD39+ cells was observed in Tregs, and maximal amounts of CD73+ cells were found among Tact subset. In the Th17 lymphocyte subpopulation, there was no significant difference between the number of cells expressing CD39 and CD73. E have also shown an increase in the relative number of Th17 cells expressing CD73, along with age-dependent decrease in the relative number of Tact cells expressing CD39. An age-dependent decrease in absolute values with age was revealed for Treg, CD39+Treg, CD73+Treg, CD39+Th17, thus being consistent with age-related decrease in absolute numbers of lymphocytes and CD4+ cells.&#x0D; The obtained data concerning specific pattern of ectonucleotidases expression in functionally different populations of CD4+ lymphocytes should be taken into account when studying children with immune-mediated diseases from different age groups.

Emerging roles of purinergic signaling in anti-cancer therapy resistance.

Cancer is a complex disease with a rapid growing incidence and often characterized by a poor prognosis. Although impressive advances have been made in cancer treatments, resistance to therapy remains a critical obstacle for the improvement of patients outcome. Current treatment approaches as chemo-, radio-, and immuno-therapy deeply affect the tumor microenvironment (TME), inducing an extensive selective pressure on cancer cells through the activation of the immune system, the induction of cell death and the release of inflammatory and damage-associated molecular patterns (DAMPS), including nucleosides (adenosine) and nucleotides (ATP and ADP). To survive in this hostile environment, resistant cells engage a variety of mitigation pathways related to metabolism, DNA repair, stemness, inflammation and resistance to apoptosis. In this context, purinergic signaling exerts a pivotal role being involved in mitochondrial function, stemness, inflammation and cancer development. The activity of ATP and adenosine released in the TME depend upon the repertoire of purinergic P2 and adenosine receptors engaged, as well as, by the expression of ectonucleotidases (CD39 and CD73) on tumor, immune and stromal cells. Besides its well established role in the pathogenesis of several tumors and in host–tumor interaction, purinergic signaling has been recently shown to be profoundly involved in the development of therapy resistance. In this review we summarize the current advances on the role of purinergic signaling in response and resistance to anti-cancer therapies, also describing the translational applications of combining conventional anticancer interventions with therapies targeting purinergic signaling.

Is the regulation by miRNAs of NTPDase1 and ecto-5'-nucleotidase genes involved with the different profiles of breast cancer subtypes?

Breast cancer (BC) is a public health problem worldwide, causing suffering and premature death among women. As a heterogeneous disease, BC-specific diagnosis and treatment are challenging. Ectonucleotidases are related to tumor development and their expression may vary among BC. miRNAs may participate in epigenetic events and may regulate ectonucleotidases in BC. This study aimed to evaluate the expression of ectonucleotidases according to BC subtypes and to predict if there is post-transcriptional regulation of them by miRNAs. MCF 10A (non-tumorigenic), MCF7 (luminal BC), and MDA-MB-231 (triple-negative BC - TNBC) breast cell lines were used and ENTPD1 (the gene encoding for NTPDase1) and NT5E (the gene encoding for ecto-5'-nucleotidase) gene expression was determined. Interestingly, the expression of ENTPD1 was only observed in MCF7 and NT5E was lower in MCF7 compared to MDA-MB-231 cell line. ATP, ADP, and AMP hydrolysis were observed on the surface of all cell lines, being higher in MDA-MB-231. Like qPCR, the activity of AMP hydrolysis was also lower in the MCF7 cells, which may represent a striking feature of this BC subtype. In silico analyses confirmed that the miRNAs miR-101-3p, miR-141-3p, and miR-340-5p were higher expressed in MCF7 cells and targeted NT5E mRNA. Altogether, data suggest that the regulation of NT5E by miRNAs in MCF7 lineage may direct the molecular profile of luminal BC. Thus, we suggest that the roles of ecto-5'-nucleotidase and the aforementioned miRNAs must be unraveled in TNBC to be possibly defined as diagnostic and therapeutic targets.

Breast Cancer Cell-Derived Adenosine Enhances Generation and Suppressor Function of Human Adaptive Regulatory T Cells.

Introduction: Adaptive regulatory T cells (Tr1) are induced in the periphery by environmental stimuli. CD73 expression and adenosine (ADO) production by tumor cells may influence Tr1 generation and their immunosuppressive activity. Material and Methods: Tr1 were generated in co-cultures of CD4+CD25neg T cells, autologous immature dendritic cells (iDC), and irradiated ADO-producing CD73+ or non-producing CD73neg breast cancer (BrCa) cell lines (TU). The expression of ectonucleotidases and other surface markers on Tr1 was determined by flow cytometry. Tr1-mediated suppression of proliferation was evaluated in CFSE-based assays. Luciferase-based ATP detection assays and mass spectrometry were used to measure ATP hydrolysis and ADO levels. Cytokine levels were measured by ELISA or Luminex. CD73 expression on tumor cells or T cells in TU tissues was assessed by immunofluorescence. Results: CD73+ TU induced higher numbers of Tr1 cells (p < 0.01) than CD73neg TU. Tr1TU73+ hydrolyzed more exogenous ATP, produced more ADO, and mediated higher suppression than Tr1TU73neg (p < 0.05 for all). ARL67156, an ectonucleotidase inhibitor, and ZM241385, A2A receptor antagonist, reduced suppression of proliferation mediated by Tr1TU73+ cells (p < 0.01). Basal-like primary BrCa cells expressed higher levels of ectonucleotidases and induced more Tr1 than less aggressive primary luminal-like BrCa. Conclusion: BrCa producing ADO (CD73+ TU) favor the induction of Tr1, which expresses CD39 and CD73, hydrolyzes ATP to ADO, and effectively suppresses anti-tumor immunity.

Extracellular ATP hydrolysis in Caco-2 human intestinal cell line

Extracellular nucleotides and nucleosides activate signaling pathways that play major roles in the physiology and pathophysiology of the gastrointestinal tract. Ectonucleotidases hydrolyze extracellular nucleotides and thus regulate ligand exposure to purinergic receptors. In this study, we investigated the expression, localization and activities of ectonucleotidases using Caco-2 cells, a model of human intestinal epithelial cells. In addition, by studying ATP release and the rates of extracellular ATP (eATP) hydrolysis, we analyzed the contribution of these processes to the regulation of eATP in these cells. Results show that Caco-2 cells regulate the metabolism of eATP and by-products by ecto-nucleoside triphosphate diphosphohydrolase-1 and -2, a neutral ecto-phosphatase and ecto-5′-nucleotidase. All these ectoenzymes were kinetically characterized using intact cells, and their presence confirmed by denatured and native gels, western blot and cytoimmunofluorescence techniques.In addition, regulation of eATP was studied by monitoring the dynamic balance between intracellular ATP release and ectoATPase activity. Following mechanical and hypotonic stimuli, Caco-2 cells triggered a strong but transient release of intracellular ATP, with almost no energy cost, leading to a steep increase of eATP concentration, which was later reduced by ectoATPase activity. A data-driven algorithm allowed quantifying and predicting the rates of ATP release and ATP consumption contributing to the dynamic accumulation of ATP at the cell surface.

Conversion of extracellular ATP into adenosine: a master switch in renal health and disease.

ATP and its ultimate degradation product adenosine are potent extracellular signalling molecules that elicit a variety of pathophysiological functions in the kidney through the activation of P2 and P1 purinergic receptors, respectively. Extracellular purines can modulate immune responses, balancing inflammatory processes and immunosuppression; indeed, alterations in extracellular nucleotide and adenosine signalling determine outcomes of inflammation and healing processes. The functional activities of ectonucleotidases such as CD39 and CD73, which hydrolyse pro-inflammatory ATP to generate immunosuppressive adenosine, are therefore pivotal in acute inflammation. Protracted inflammation may result in aberrant adenosinergic signalling, which serves to sustain inflammasome activation and worsen fibrotic reactions. Alterations in the expression of ectonucleotidases on various immune cells, such as regulatory T cells and macrophages, as well as components of the renal vasculature, control purinergic receptor-mediated effects on target tissues within the kidney. The role of CD39 as a rheostat that can have an impact on purinergic signalling in both acute and chronic inflammation is increasingly supported by the literature, as detailed in this Review. Better understanding of these purinergic processes and development of novel drugs targeting these pathways could lead to effective therapies for the management of acute and chronic kidney disease.

Human platelets express functional ectonucleotidases that restrict platelet activation signaling

Platelets play central role in thrombosis and haemostasis. Platelets store adenine nucleotides in their dense granules, which are released upon agonist-stimulation. Level of these nucleotides in extracellular fluid is regulated by activities of ectonucleotidases such as ectonucleoside triphosphate diphosphohydrolase-1 (CD39) and ecto-5′-nucleotidase (CD73) expressed on platelet surface. Here we demonstrate that, expression of surface-bound ectonucleotidases rose significantly in platelets, concomitant with upregulation of their enzymatic activities, when cells were stimulated with thrombin. Interestingly, inhibition of CD73 in thrombin-treated platelets led to enhanced tyrosine phosphorylation of proteins and rise in intracellular free calcium, [Ca2+]i, thus signifying the inhibitory role of the ectonucleotidase on agonist-mediated platelet signaling.