apyrase and Sepsis

Sepsis is a life-threatening organ dysfunction caused by a dysregulated inflammatory response to infection. To date, there is no specific treatment established for sepsis. In the extracellular compartment, purines such as adenosine triphosphate (ATP) and adenosine play essential roles in the immune/inflammatory responses during sepsis and septic shock. The balance of extracellular levels among ATP and adenosine is intimately involved in the signals related to immune stimulation/immunosuppression balance. Specialized enzymes, including CD39, CD73, and adenosine deaminase (ADA), are responsible to metabolize ATP to adenosine which will further sensitize the P2 and P1 purinoceptors, respectively. Disruption of the purinergic pathway had been described in the sepsis pathophysiology. Although purinergic signaling has been suggested as a potential target for sepsis treatment, the majority of data available were obtained using pre-clinical approaches. We hypothesized that, as a reflection of deregulation on purinergic signaling, septic patients exhibit differential measurements of serum, neutrophils and monocytes purinergic pathway markers when compared to two types of controls (healthy and ward). It was observed that ATP and ADP serum levels were increased in septic patients, as well as the A2a mRNA expression in neutrophils and monocytes. Both ATPase/ADPase activities were increased during sepsis. Serum ATP and ADP levels, and both ATPase and ADPase activities were associated with the diagnosis of sepsis, representing potential biomarkers candidates. In conclusion, our results advance the translation of purinergic signaling from pre-clinical models into the clinical setting opening opportunities for so much needed new strategies for sepsis and septic shock diagnostics and treatment.

Topics: Adenosine; Adenosine Diphosphate; Adenosine Triphosphatases; Adenosine Triphosphate; Apyrase; Biomarkers; Humans; Sepsis; Shock, Septic

Sepsis results in elevated adenosine in circulation. Extracellular adenosine triggers immunosuppressive signaling via the A2a receptor (A2aR). Sepsis survivors develop persistent immunosuppression with increased risk of recurrent infections. We utilized the cecal ligation and puncture (CLP) model of sepsis and subsequent infection to assess the role of adenosine in post-sepsis immune suppression. A2aR-deficient mice showed improved resistance to post-sepsis infections. Sepsis expanded a subset of CD39

Topics: Adenosine; Animals; Antigens, CD; Apyrase; Cellular Reprogramming; Immune Tolerance; Macrophages; Mice; Plasma Cells; Receptor, Adenosine A2A; Sepsis

Unfettered inflammation is a leading cause of multiple organ failures in sepsis. The anti‑inflammatory role of cluster of differentiation (CD)39 has been previously reported. The present study aimed to investigate the role of unfettered inflammation in sepsis‑induced acute kidney injury (AKI). Lipopolysaccharide (LPS) was introduced to construct a sepsis mouse model. Kidney function and pathological changes in mice were measured at 12, 24 and 48 h. CD39 overexpression and inhibition vectors were transfected into renal tubular epithelial (HK‑2) cells, followed by LPS treatment (10 µg/ml), and the cell viability changes at 24 h after treatment were assessed and the expression of NLR family pyrin domain containing 3 (NLRP3), cleaved caspase‑1 and CD39 were determined by performing ELISAs. Cell apoptosis and reactive oxygen species (ROS) levels were determined by flow cytometry. It was found that after LPS administration, kidney injury was the most serious at 24 h in mice. CD39 overexpression could suppress the upregulation of pro‑inflammatory cytokines induced by LPS treatment. In addition, the cell apoptosis and ROS level exhibited an obvious decrease, while cell viability increased. The NLRP3 expression and activity also showed a great inhibition in CD39‑overexpressed cells. By contrast to CD39 overexpression, CD39 inhibition promoted the activation of the NLRP3 inflammasome. These data indicate the protective role of CD39 in LPS‑induced renal tubular epithelial cell damage through inhibiting NLRP3 inflammasome activation and that CD39 might be a potential therapeutic target in sepsis‑induced AKI.

Topics: Acute Kidney Injury; Animals; Antigens, CD; Apoptosis; Apyrase; Cell Survival; Cytokines; Disease Models, Animal; Epithelial Cells; Inflammasomes; Inflammation; Kidney Tubules; Male; Mice; Mice, Inbred C57BL; NLR Family, Pyrin Domain-Containing 3 Protein; Reactive Oxygen Species; Sepsis; Signal Transduction

The severity of sepsis can be linked to excessive inflammatory responses resulting in hepatic injury. P2X7 receptor activation by extracellular ATP (eATP) exacerbates inflammation by augmenting cytokine production; while CD39 (ENTPD1) scavenges eATP to generate adenosine, thereby limiting P2X7 activation and resulting in A. Sepsis was induced by cecal ligation and puncture in C57BL/6 wild-type (WT) and CD39. CD39 expression in macrophages limits ATP-P2X7 receptor pro-inflammatory signaling. P2X7 receptor paradoxically boosts CD39 activity. Inhibition and/or deletion of P2X7 receptor in LPS-primed macrophages attenuates cytokine production and inflammatory signaling as well as preventing ATP-induced increases in CD39 activity. Septic CD39. CD39 attenuates sepsis-associated liver injury by scavenging eATP and ultimately generating adenosine. We propose boosting of CD39 would suppress P2X7 responses and trigger adenosinergic signaling to limit systemic inflammation and restore liver homeostasis during the acute phase of sepsis. Lay summary: CD39 expression in macrophages limits P2X7-mediated pro-inflammatory responses, scavenging extracellular ATP and ultimately generating adenosine. CD39 genetic deletion exacerbates sepsis-induced experimental liver injury. Combinations of a P2X7 antagonist and adenosine A

Topics: Adenosine A2 Receptor Agonists; Adenosine Triphosphate; Animals; Antigens, CD; Apyrase; Cytokines; Disease Models, Animal; Interleukin-1beta; Liver; Macrophages; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; NF-kappa B; Purinergic P2X Receptor Antagonists; Receptors, Purinergic P2X7; Sepsis; Signal Transduction; STAT3 Transcription Factor

Sepsis remains an unresolved clinical problem. Therapeutic strategies focusing on inhibition of neutrophils (polymorphonuclear neutrophils) have failed, which indicates that a more detailed understanding of the underlying pathophysiology of sepsis is required. Polymorphonuclear neutrophil activation and chemotaxis require cellular adenosine triphosphate release via pannexin-1 channels that fuel autocrine feedback via purinergic receptors. In the current study, we examined the roles of endogenous and systemic adenosine triphosphate on polymorphonuclear neutrophil activation and host defense in sepsis.. Prospective randomized animal investigation and in vitro studies.. Preclinical academic research laboratory.. Wild-type C57BL/6 mice, pannexin-1 knockout mice, and healthy human subjects used to obtain polymorphonuclear neutrophils for in vitro studies.. Wild-type and pannexin-1 knockout mice were treated with suramin or apyrase to block the endogenous or systemic effects of adenosine triphosphate. Mice were subjected to cecal ligation and puncture and polymorphonuclear neutrophil activation (CD11b integrin expression), organ (liver) injury (plasma aspartate aminotransferase), bacterial spread, and survival were monitored. Human polymorphonuclear neutrophils were used to study the effect of systemic adenosine triphosphate and apyrase on chemotaxis.. Inhibiting endogenous adenosine triphosphate reduced polymorphonuclear neutrophil activation and organ injury, but increased the spread of bacteria and mortality in sepsis. By contrast, removal of systemic adenosine triphosphate improved bacterial clearance and survival in sepsis by improving polymorphonuclear neutrophil chemotaxis.. Systemic adenosine triphosphate impairs polymorphonuclear neutrophil functions by disrupting the endogenous purinergic signaling mechanisms that regulate cell activation and chemotaxis. Removal of systemic adenosine triphosphate improves polymorphonuclear neutrophil function and host defenses, making this a promising new treatment strategy for sepsis.

Topics: Adenosine Triphosphate; Animals; Apyrase; Chemotaxis, Leukocyte; Humans; Mice, Inbred C57BL; Mice, Knockout; Neutrophil Activation; Neutrophils; Sepsis; Suramin

Sepsis results in unfettered inflammation, tissue damage, and multiple organ failure. Diffuse brain dysfunction and neurological manifestations secondary to sepsis are termed sepsis-associated encephalopathy (SAE). Extracellular nucleotides, proinflammatory cytokines, and oxidative stress reactions are associated with delirium and brain injury, and might be linked to the pathophysiology of SAE. P2X7 receptor activation by extracellular ATP leads to maturation and release of IL-1β by immune cells, which stimulates the production of oxygen reactive species. Hence, we sought to investigate the role of purinergic signaling by P2X7 in a model of sepsis. We also determined how this process is regulated by the ectonucleotidase CD39, a scavenger of extracellular nucleotides. Wild type (WT), P2X7 receptor (P2X7

Topics: Animals; Antigens, CD; Apyrase; Brain; Catalase; Cytokines; Inflammation; Male; Mice; Mice, Knockout; Oxidative Stress; Receptors, Purinergic P2X7; Sepsis; Signal Transduction; Superoxide Dismutase

The number of regulatory T cells (Treg cells) and the expression of ectonucleoside triphosphate diphosphohydrolase 1 (ENTPD1; also known as CD39) and 5'-ectonucleotidase (NT5E; also known as CD73) on the Treg cell surface are increased during sepsis. In this study, to determine the factors leading to the high expression of CD39 and CD73, and the regulation of the CD39/CD73/adenosine pathway in Treg cells under septic conditions, we constructed a mouse model of sepsis and separated the Treg cells using a flow cytometer. The Treg cells isolated from the peritoneal lavage and splenocytes of the mice were treated with adenosine or the specific adenosine A2A receptor agonist, CGS21680, and were transfected with specific siRNA targeting E2F transcription factor 1 (E2F-1) or cyclic adenosine monophosphate (cAMP) response element-binding protein (CREB), which are predicted transcription regulatory factors of CD39 or CD73. The regulatory relationships among these factors were then determined by western blot analysis and dual-luciferase reporter assay. In addition, changes in adenosine metabolism were measured in the treated cells. The results revealed that adenosine and CGS21680 significantly upregulated CD39 and CD73 expression (P<0.01). E2F-1 and CREB induced CD39 and CD73 expression, and were upregulated by adenosine and CGS21680. Adenosine triphosphate (ATP) hydrolysis and adenosine generation were inhibited by the knockdown of E2F-1 or CREB, and were accelerated in the presence of CGS21680. Based on these results, it can be inferred that adenosine, the adenosine A2A receptor agonist, E2F-1 and CREB are the possible factors contributing to the high expression of CD39 and CD73 on the Treg cell surface during sepsis. Adenosine and its A2A receptor agonist served as the signal transducer factors of the CD39/CD73/adenosine pathway, accelerating adenosine generation. Our study may benefit further research on adenosine metabolism for the treatment of sepsis.

Topics: 5'-Nucleotidase; Adenosine; Adenosine A2 Receptor Agonists; Adenosine Monophosphate; Adenosine Triphosphate; Animals; Antigens, CD; Apyrase; Blotting, Western; Cells, Cultured; Culture Media, Conditioned; Cyclic AMP Response Element-Binding Protein; E2F1 Transcription Factor; Mice, Inbred BALB C; Phenethylamines; Receptor, Adenosine A2A; RNA Interference; Sepsis; T-Lymphocytes, Regulatory; Up-Regulation

Sepsis remains the leading cause of morbidity and mortality in critically ill patients. Excessive inflammation is a major cause of organ failure and mortality in sepsis. Ectonucleoside triphosphate diphosphohydrolase 1, ENTPDase1 (CD39) is a cell surface nucleotide-metabolizing enzyme, which degrades the extracellular purines ATP and ADP, thereby regulating purinergic receptor signaling. Although the role of purinergic receptor signaling in regulating inflammation and sepsis has been addressed previously, the role of CD39 in regulating the host's response to sepsis is unknown. We found that the CD39 mimic apyrase (250 U/kg) decreased and knockout or pharmacologic blockade with sodium polyoxotungstate (5 mg/kg; IC50 ≈ 10 μM) of CD39 increased mortality of mice with polymicrobial sepsis induced by cecal ligation and puncture. CD39 decreased inflammation, organ damage, immune cell apoptosis, and bacterial load. Use of bone marrow chimeric mice revealed that CD39 expression on myeloid cells decreases inflammation in septic mice. CD39 expression is upregulated during sepsis in mice, as well as in both murine and human macrophages stimulated with Escherichia coli. Moreover, E. coli increases CD39 promoter activity in macrophages. Altogether, these data indicate CD39 as an evolutionarily conserved inducible protective pathway during sepsis. We propose CD39 as a novel therapeutic target in the management of sepsis.

Topics: 5'-Nucleotidase; Animals; Antigens, CD; Apyrase; Chemokines; Cytokines; Escherichia coli; Humans; Inflammation; Interleukin-10; Interleukin-12; Macrophages; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Promoter Regions, Genetic; Sepsis; Transplantation Chimera

Sepsis encompasses two phases, the 'hyper'-reactive phase and the 'hypo'-reactive phase. The initial inflammatory stage is quickly counterbalanced by an anti-inflammatory response, which compromises the immune system, leading to immune suppression. Regulatory T cells (Tregs) have been implicated in the pathogenesis of sepsis by inducing immunosuppression; however, the role of CD39(+) Tregs in the process of sepsis is uncertain. This study investigated the dynamic levels of CD39(+) Tregs and their phenotypic change in sepsis.. Fourteen patients with systemic inflammatory response syndrome (SIRS), 42 patients with sepsis, and 14 healthy controls were enrolled. Sequential blood samples were used to analyze the numbers of CD39(+) Tregs and their phenotypic changes. Survival at 28 days was used to evaluate the capacity of CD39(+) Treg levels to predict mortality in sepsis patients.. Sepsis patients displayed a high percentage (3.13%, 1.46%, and 0.35%, respectively) and mean fluorescence intensity (MFI) (59.65, 29.7, and 24.3, respectively) of CD39(+) Tregs compared with SIRS patients and healthy subjects. High-level expression of CD39(+) Tregs was correlated with the severity of sepsis, which was reflected by the sepsis-related organ failure assessment score (r=0.322 and r=0.31, respectively). In addition, the expression of CD39(+) Tregs was associated with survival of sepsis patients (p<0.01). By receiver-operating characteristic (ROC) curve analysis, the percentage and MFI of CD39(+) Tregs showed similar sensitivities and specificities to predict mortality (74.2% and 85.1%, and 73.9% and 84.1%, respectively). Using Kaplan-Meier curves to assess the impact of CD39(+) Tregs percentage and MFI on overall survival, we found that a high CD39(+) Tregs percentage (p<0.001; >4.1%) and MFI (p<0.001; >49.2) were significantly associated with mortality. Phenotypically, CD39(+) Tregs from sepsis patients showed high expression of CD38 and PD-1 (p<0.01 and p<0.01 respectively).. Increased expression of CD39(+) Tregs was associated with a poor prognosis for sepsis patients, which suggests that CD39(+) Treg levels could be used as a biomarker to predict the outcome of sepsis patients.

Topics: Adult; Aged; Antigens, CD; Apyrase; Female; Humans; Male; Middle Aged; Sepsis; Survival Analysis; Systemic Inflammatory Response Syndrome; T-Lymphocytes, Regulatory

We investigated in rats induced to sepsis the activity of ectonucleoside triphosphate diphosphohydrolase (NTPDase; CD39; E.C. 3.6.1.5), an enzyme involved in the modulation of immune responses. After 12 hours of surgery, lymphocytes were isolated from blood and NTPDase activity was determined. It was also performed the histology of kidney, liver, and lung. The results demonstrated an increase in the hydrolysis of adenosine-5'-triphosphate (ATP) (P < 0.01), but no changes regarding adenosine-5'-monophosphate (ADP) hydrolysis (P > 0.05). Histological analysis showed several morphological changes in the septic group, such as vascular congestion, necrosis, and infiltration of mononuclear cells. It is known that the intracellular milieu contains much more ATP nucleotides than the extracellular. In this context, the increased ATPasic activity was probably induced as a dynamic response to clean up the elevated ATP levels resulting from cellular death.

Topics: Adenosine Diphosphate; Adenosine Triphosphate; Animals; Antigens, CD; Apyrase; Cell Death; Cell Proliferation; Disease Models, Animal; Female; Hydrolysis; Immune System; Leukocytes, Mononuclear; Lymphocytes; Male; Nucleotides; Rats; Rats, Wistar; Sepsis; Tissue Distribution