Negative feedback control of neuronal activity by microglia.

Microglia, the brain's resident macrophages, help to regulate brain function by removing dying neurons, pruning non-functional synapses, and producing ligands that support neuronal survival1. Here we show that microglia are also critical modulators of neuronal activity and associated behavioural responses in mice. Microglia respond to neuronal activation by suppressing neuronal activity, and ablation of microglia amplifies and synchronizes the activity of neurons, leading to seizures. Suppression of neuronal activation by microglia occurs in a highly region-specific fashion and depends on the ability of microglia to sense and catabolize extracellular ATP, which is released upon neuronal activation by neurons and astrocytes. ATP triggers the recruitment of microglial protrusions and is converted by the microglial ATP/ADP hydrolysing ectoenzyme CD39 into AMP; AMP is then converted into adenosine by CD73, which is expressed on microglia as well as other brain cells. Microglial sensing of ATP, the ensuing microglia-dependent production of adenosine, and the adenosine-mediated suppression of neuronal responses via the adenosine receptor A1R are essential for the regulation of neuronal activity and animal behaviour. Our findings suggest that this microglia-driven negative feedback mechanism operates similarly to inhibitory neurons and is essential for protecting the brain from excessive activation in health and disease.

Adenosine Triphosphate Release From Influenza-Infected Lungs Enhances Neutrophil Activation and Promotes Disease Progression.

BACKGROUND: Adenosine triphosphate (ATP) enhances neutrophil responses, but little is known about the role of ATP in influenza infections. METHODS: We used a mouse influenza model to study if ATP release is associated with neutrophil activation and disease progression. RESULTS: Influenza infection increased pulmonary ATP levels 5-fold and plasma ATP levels 3-fold vs healthy mice. Adding ATP at those concentrations to blood from healthy mice primed neutrophils and enhanced CD11b and CD63 expression, CD62L shedding, and reactive oxygen species production in response to formyl peptide receptor stimulation. Influenza infection also primed neutrophils in vivo, resulting in formyl peptide receptor-induced CD11b expression and CD62L shedding up to 3 times higher than that of uninfected mice. In infected mice, large numbers of neutrophils entered the lungs. These cells were significantly more activated than the peripheral neutrophils of infected mice and pulmonary neutrophils of healthy mice. Plasma ATP levels of infected mice and influenza disease progression corresponded with the numbers and activation level of their pulmonary neutrophils. CONCLUSIONS: Findings suggest that ATP release from the lungs of infected mice promotes influenza disease progression by priming peripheral neutrophils, which become strongly activated and cause pulmonary tissue damage after their recruitment to the lungs.

Effects of low-dose acetylsalicylic acid on the inflammatory response to experimental sleep restriction in healthy humans.

BACKGROUND: Sleep deficiencies, such as manifested in short sleep duration or insomnia symptoms, are known to increase the risk for multiple disease conditions involving immunopathology. Inflammation is hypothesized to be a mechanism through which deficient sleep acts as a risk factor for these conditions. Thus, one potential way to mitigate negative health consequences associated with deficient sleep is to target inflammation. Few interventional sleep studies investigated whether improving sleep affects inflammatory processes, but results suggest that complementary approaches may be necessary to target inflammation associated with sleep deficiencies. We investigated whether targeting inflammation through low-dose acetylsalicylic acid (ASA, i.e., aspirin) is able to blunt the inflammatory response to experimental sleep restriction. METHODS: 46 healthy participants (19F/27M, age range 19-63 years) were studied in a double-blind randomized placebo-controlled crossover trial with three protocols each consisting of a 14-day at-home monitoring phase followed by an 11-day (10-night) in-laboratory stay (sleep restriction/ASA, sleep restriction/placebo, control sleep/placebo). In the sleep restriction/ASA condition, participants took low-dose ASA (81 mg/day) daily in the evening (22:00) during the at-home phase and the subsequent in-laboratory stay. In the sleep restriction/placebo and control sleep/placebo conditions, participants took placebo daily. Each in-laboratory stay started with 2 nights with a sleep opportunity of 8 h/night (23:00-07:00) for adaptation and baseline measurements. Under the two sleep restriction conditions, participants were exposed to 5 nights of sleep restricted to a sleep opportunity of 4 h/night (03:00-07:00) followed by 3 nights of recovery sleep with a sleep opportunity of 8 h/night. Under the control sleep condition, participants had a sleep opportunity of 8 h/night throughout the in-laboratory stay. During each in-laboratory stay, participants had 3 days of intensive monitoring (at baseline, 5th day of sleep restriction/control sleep, and 2nd day of recovery sleep). Variables, including pro-inflammatory immune cell function, C-reactive protein (CRP), and actigraphy-estimated measures of sleep, were analyzed using generalized linear mixed models. RESULTS: Low-dose ASA administration reduced the interleukin (IL)-6 expression in LPS-stimulated monocytes (p<0.05 for condition*day) and reduced serum CRP levels (p<0.01 for condition) after 5 nights of sleep restriction compared to placebo administration in the sleep restriction condition. Low-dose ASA also reduced the amount of cyclooxygenase (COX)-1/COX-2 double positive cells among LPS-stimulated monocytes after 2 nights of recovery sleep following 5 nights of sleep restriction compared to placebo (p<0.05 for condition). Low-dose ASA further decreased wake after sleep onset (WASO) and increased sleep efficiency (SE) during the first 2 nights of recovery sleep (p<0.001 for condition and condition*day). Baseline comparisons revealed no differences between conditions for all of the investigated variables (p>0.05 for condition). CONCLUSION: This study shows that inflammatory responses to sleep restriction can be reduced by preemptive administration of low-dose ASA. This finding may open new therapeutic approaches to prevent or control inflammation and its consequences in those experiencing sleep deficiencies. TRIAL REGISTRATION: ClinicalTrials.gov NCT03377543.

Impaired ATP hydrolysis in blood plasma contributes to age-related neutrophil dysfunction.

BACKGROUND: The function of polymorphonuclear neutrophils (PMNs) decreases with age, which results in infectious and inflammatory complications in older individuals. The underlying causes are not fully understood. ATP release and autocrine stimulation of purinergic receptors help PMNs combat microbial invaders. Excessive extracellular ATP interferes with these mechanisms and promotes inflammatory PMN responses. Here, we studied whether dysregulated purinergic signaling in PMNs contributes to their dysfunction in older individuals. RESULTS: Bacterial infection of C57BL/6 mice resulted in exaggerated PMN activation that was significantly greater in old mice (64 weeks) than in young animals (10 weeks). In contrast to young animals, old mice were unable to prevent the systemic spread of bacteria, resulting in lethal sepsis and significantly greater mortality in old mice than in their younger counterparts. We found that the ATP levels in the plasma of mice increased with age and that, along with the extracellular accumulation of ATP, the PMNs of old mice became increasingly primed. Stimulation of the formyl peptide receptors of those primed PMNs triggered inflammatory responses that were significantly more pronounced in old mice than in young animals. However, bacterial phagocytosis and killing by PMNs of old mice were significantly lower than that of young mice. These age-dependent PMN dysfunctions correlated with a decrease in the enzymatic activity of plasma ATPases that convert extracellular ATP to adenosine. ATPases depend on divalent metal ions, including Ca2+, Mg2+, and Zn2+, and we found that depletion of these ions blocked the hydrolysis of ATP and the formation of adenosine in human blood, resulting in ATP accumulation and dysregulation of PMN functions equivalent to those observed in response to aging. CONCLUSIONS: Our findings suggest that impaired hydrolysis of plasma ATP dysregulates PMN function in older individuals. We conclude that strategies aimed at restoring plasma ATPase activity may offer novel therapeutic opportunities to reduce immune dysfunction, inflammation, and infectious complications in older patients.

ATP release from influenza-infected lungs enhances neutrophil activation and promotes disease progression.

BACKGROUND: ATP enhances neutrophil responses, but little is known about the role of ATP in influenza infections. METHODS: We used a mouse influenza model to study if ATP release is associated with neutrophil activation and disease progression. RESULTS: Influenza infection increased pulmonary ATP levels 5-fold and plasma ATP levels 3-fold over the levels in healthy mice. Adding ATP at those concentrations to blood from healthy mice primed their neutrophils and enhanced CD11b and CD63 expression, CD62L shedding, and reactive oxygen species production in response to formyl peptide receptor (FPR) stimulation. Influenza infection also primed neutrophils in vivo, resulting in FPR-induced CD11b expression and CD62L shedding up to 3-times higher than that of uninfected mice. In infected mice, large numbers of neutrophils entered the lungs. These cells were significantly more activated than peripheral neutrophils of infected and pulmonary neutrophils of healthy mice. Plasma ATP levels of infected mice and influenza disease progression corresponded with the numbers and activation level of their pulmonary neutrophils. CONCLUSION: Our findings suggest that ATP release from the lungs of infected mice promotes influenza disease progression by priming peripheral neutrophils that become strongly activated and cause pulmonary tissue damage after their recruitment to the lungs.

Extracellular mitochondria drive CD8 T cell dysfunction in trauma by upregulating CD39.

RATIONALE: The increased mortality and morbidity seen in critically injured patients appears associated with systemic inflammatory response syndrome (SIRS) and immune dysfunction, which ultimately predisposes to infection. Mitochondria released by injury could generate danger molecules, for example, ATP, which in turn would be rapidly scavenged by ectonucleotidases, expressed on regulatory immune cells. OBJECTIVE: To determine the association between circulating mitochondria, purinergic signalling and immune dysfunction after trauma. METHODS: We tested the impact of hepatocyte-derived free mitochondria on blood-derived and lung-derived CD8 T cells in vitro and in experimental mouse models in vivo. In parallel, immune phenotypic analyses were conducted on blood-derived CD8 T cells obtained from trauma patients. RESULTS: Isolated intact mitochondria are functional and generate ATP ex vivo. Extracellular mitochondria perturb CD8+ T cells in co-culture, inducing select features of immune exhaustion in vitro. These effects are modulated by scavenging ATP, modelled by addition of apyrase in vitro. Injection of intact mitochondria into recipient mice markedly upregulates the ectonucleotidase CD39, and other immune checkpoint markers in circulating CD8+ T cells. We note that mice injected with mitochondria, prior to instilling bacteria into the lung, exhibit more severe lung injury, characterised by elevated neutrophil influx and by changes in CD8+ T cell cytotoxic capacity. Importantly, the development of SIRS in injured humans, is likewise associated with disordered purinergic signalling and CD8 T cell dysfunction. CONCLUSION: These studies in experimental models and in a cohort of trauma patients reveal important associations between extracellular mitochondria, aberrant purinergic signalling and immune dysfunction. These pathogenic factors with immune exhaustion are linked to SIRS and could be targeted therapeutically.

ATP breakdown in plasma of children limits the antimicrobial effectiveness of their neutrophils.

Neutrophils (PMNs) require extracellular ATP and adenosine (ADO) to fight bacterial infections, which often have life-threatening consequences in pediatric patients. We wondered whether the ATP and ADO levels in the plasma of children change with age and if these changes influence the antimicrobial efficacy of the PMNs of these children. We measured plasma concentrations of ATP and ADO and the activities of the enzymes responsible for the breakdown of these mediators in plasma samples from healthy children and adolescents (n = 45) ranging in age from 0.2 to 15 years. In addition, using blood samples of these individuals, we compared how effective their PMNs were in the phagocytosis of bacteria. In an experimental sepsis model with young (10 days) and adolescent mice (10 weeks), we studied how age influenced the resilience of these animals to bacterial infections and whether addition of ATP could improve the antimicrobial capacity of their PMNs. We found that plasma ATP levels correlated with age and were significantly lower in infants (< 1 year) than in adolescents (12-15 years). In addition, we observed significantly higher plasma ATPase and adenosine deaminase activities in children (< 12 years) when compared to the adolescent population. The activities of these ATP and ADO breakdown processes correlated inversely with age and with the ability of PMNs to phagocytize bacteria. Similar to their human counterparts, young mice also had significantly lower plasma ATP levels when compared to adolescent animals. In addition, we found that mortality of young mice after bacterial infection was significantly higher than that of adolescent mice. Moreover, bacterial phagocytosis by PMNs of young mice was weaker when compared to that of older mice. Finally, we found that ATP supplementation could recover bacterial phagocytosis of young mice to levels similar to those of adolescent mice. Our findings suggest that rapid ATP hydrolysis in the plasma of young children lowers the antimicrobial functions of their PMNs and that this may contribute to the vulnerability of pediatric patients to bacterial infections.

Optimized HPLC method to elucidate the complex purinergic signaling dynamics that regulate ATP, ADP, AMP, and adenosine levels in human blood.

ATP released into the bloodstream regulates immune responses and other physiological functions. Excessive accumulation of extracellular ATP interferes with these functions, and elevated plasma ATP levels could indicate infections and other pathological disorders. However, there is considerable disagreement about what constitutes normal plasma ATP levels. Therefore, we optimized a method to accurately assess ATP concentrations in blood. We found that rapid chilling of heparinized blood samples is essential to preserve in vivo ATP levels and that differential centrifugation minimizes inadvertent ATP release due to cell damage and mechanical stress. Plasma samples were stabilized with perchloric acid, etheno-derivatized, and delipidated for sensitive analysis of ATP and related compounds using high-performance liquid chromatography (HPLC) and fluorescence detection. We measured 33 ± 20 nM ATP, 90 ± 45 nM ADP, 100 ± 55 nM AMP, and 81 ± 51 nM adenosine in the blood of healthy human adults (n = 10). In critically ill patients, ATP levels were 6 times higher than in healthy subjects. The anticoagulant greatly affected results. ATP levels were nearly 8 times higher in EDTA plasma than in heparin plasma, while AMP levels were 3 times lower and adenosine was entirely absent in EDTA plasma. If EDTA blood was not immediately chilled, ATP, ADP, and AMP levels continued to rise, which indicates that EDTA interferes with the endogenous mechanisms that regulate plasma adenylate levels. Our optimized method eliminates artifacts that prevent accurate determination of plasma adenylates and will be useful for studying mechanisms that regulate adenylate levels and for monitoring of pathological processes in patients with infections and other diseases.

Structural and functional characterization of engineered bifunctional fusion proteins of CD39 and CD73 ectonucleotidases.

Extracellular diphosphate and triphosphate nucleotides are released from activated or injured cells to trigger vascular and immune P2 purinergic receptors, provoking inflammation and vascular thrombosis. These metabokines are scavenged by ectonucleoside triphosphate diphosphohydrolase-1 (E-NTPDase1 or CD39). Further degradation of the monophosphate nucleoside end products occurs by surface ecto-5'-nucleotidase (NMPase) or CD73. These ectoenzymatic processes work in tandem to promote adenosinergic responses, which are immunosuppressive and antithrombotic. These homeostatic ectoenzymatic mechanisms are lost in the setting of oxidative stress, which exacerbates inflammatory processes. We have engineered bifunctional enzymes made up from ectodomains (ECDs) of CD39 and CD73 within a single polypeptide. Human alkaline phosphatase-ectodomain (ALP-ECD) and human acid phosphatase-ectodomain (HAP-ECD) fusion proteins were also generated, characterized, and compared with these CD39-ECD, CD73-ECD, and bifunctional fusion proteins. Through the application of colorimetrical functional assays and high-performance liquid chromatography kinetic assays, we demonstrate that the bifunctional ectoenzymes express high levels of CD39-like NTPDase activity and CD73-like NMPase activity. Chimeric CD39-CD73-ECD proteins were superior in converting triphosphate and diphosphate nucleotides into nucleosides when compared with ALP-ECD and HAP-ECD. We also note a pH sensitivity difference between the bifunctional fusion proteins and parental fusions, as well as ectoenzymatic property distinctions. Intriguingly, these innovative reagents decreased platelet activation to exogenous agonists in vitro. We propose that these chimeric fusion proteins could serve as therapeutic agents in inflammatory diseases, acting to scavenge proinflammatory ATP and also generate anti-inflammatory adenosine.

Purinergic P2Y2 receptors modulate endothelial sprouting.

Purinergic P2 receptors are critical regulators of several functions within the vascular system, including platelet aggregation, vascular inflammation, and vascular tone. However, a role for ATP release and P2Y receptor signalling in angiogenesis remains poorly defined. Here, we demonstrate that blood vessel growth is controlled by P2Y2 receptors. Endothelial sprouting and vascular tube formation were significantly dependent on P2Y2 expression and inhibition of P2Y2 using a selective antagonist blocked microvascular network generation. Mechanistically, overexpression of P2Y2 in endothelial cells induced the expression of the proangiogenic molecules CXCR4, CD34, and angiopoietin-2, while expression of VEGFR-2 was decreased. Interestingly, elevated P2Y2 expression caused constitutive phosphorylation of ERK1/2 and VEGFR-2. However, stimulation of cells with the P2Y2 agonist UTP did not influence sprouting unless P2Y2 was constitutively expressed. Finally, inhibition of VEGFR-2 impaired spontaneous vascular network formation induced by P2Y2 overexpression. Our data suggest that P2Y2 receptors have an essential function in angiogenesis, and that P2Y2 receptors present a therapeutic target to regulate blood vessel growth.

RIG-I and TLR4 responses and adverse outcomes in pediatric influenza-related critical illness.

BACKGROUND: Decreased TNF-α production in whole blood after ex vivo LPS stimulation indicates suppression of the Toll-like receptor (TLR)4 pathway. This is associated with increased mortality in pediatric influenza critical illness. Whether antiviral immune signaling pathways are also suppressed in these patients is unclear. OBJECTIVES: We sought to evaluate suppression of the TLR4 and the antiviral retinoic acid-inducible gene-I (RIG-I) pathways with clinical outcomes in children with severe influenza infection. METHODS: In this 24-center, prospective, observational cohort study of children with confirmed influenza infection, blood was collected within 72 hours of intensive care unit admission. Ex vivo whole blood stimulations were performed with matched controls using the viral ligand polyinosinic-polycytidylic acid-low-molecular-weight/LyoVec and LPS to evaluate IFN-α and TNF-α production capacities (RIG-I and TLR4 pathways, respectively). RESULTS: Suppression of either IFN-α or TNF-α production capacity was associated with longer duration of mechanical ventilation and hospitalization, and increased organ dysfunction. Children with suppression of both RIG-I and TLR4 pathways (n = 33 of 103 [32%]) were more likely to have prolonged (≥7 days) multiple-organ dysfunction syndrome (30.3% vs 8.6%; P = .004) or prolonged hypoxemic respiratory failure (39.4% vs 11.4%; P = .001) compared with those with single- or no pathway suppression. CONCLUSIONS: Suppression of both RIG-I and TLR4 signaling pathways, essential for respective antiviral and antibacterial responses, is common in previously immunocompetent children with influenza-related critical illness and is associated with bacterial coinfection and adverse outcomes. Prospective testing of both pathways may aid in risk-stratification and in immune monitoring.

Lipopolysaccharide suppresses T cells by generating extracellular ATP that impairs their mitochondrial function via P2Y11 receptors.

T cell suppression contributes to immune dysfunction in sepsis. However, the underlying mechanisms are not well-defined. Here, we show that exposure of human peripheral blood mononuclear cells to bacterial lipopolysaccharide (LPS) can rapidly and dose-dependently suppress interleukin-2 (IL-2) production and T cell proliferation. We also report that these effects depend on monocytes. LPS did not prevent the interaction of monocytes with T cells, nor did it induce programmed cell death protein 1 (PD-1) signaling that causes T cell suppression. Instead, we found that LPS stimulation of monocytes led to the accumulation of extracellular ATP that impaired mitochondrial function, cell migration, IL-2 production, and T cell proliferation. Mechanistically, LPS-induced ATP accumulation exerted these suppressive effects on T cells by activating the purinergic receptor P2Y11 on the cell surface of T cells. T cell functions could be partially restored by enzymatic removal of extracellular ATP or pharmacological blocking of P2Y11 receptors. Plasma samples obtained from sepsis patients had similar suppressive effects on T cells from healthy subjects. Our findings suggest that LPS and ATP accumulation in the circulation of sepsis patients suppresses T cells by promoting inappropriate P2Y11 receptor stimulation that impairs T cell metabolism and functions. We conclude that inhibition of LPS-induced ATP release, removal of excessive extracellular ATP, or P2Y11 receptor antagonists may be potential therapeutic strategies to prevent T cell suppression and restore host immune function in sepsis.

Autocrine stimulation of P2Y1 receptors is part of the purinergic signaling mechanism that regulates T cell activation.

Previous studies have shown that T cell receptor (TCR) and CD28 coreceptor stimulation involves rapid ATP release, autocrine purinergic feedback via P2X receptors, and mitochondrial ATP synthesis that promote T cell activation. Here, we show that ADP formation and autocrine stimulation of P2Y1 receptors are also involved in these purinergic signaling mechanisms. Primary human CD4 T cells and the human Jurkat CD4 T cell line express P2Y1 receptors. The expression of this receptor increases following T cell stimulation. Inhibition of P2Y1 receptors impairs the activation of mitochondria, as assessed by mitochondrial Ca2+ uptake, and reduces cytosolic Ca2+ signaling in response to TCR/CD28 stimulation. We found that the addition of exogenous ADP or overexpression of P2Y1 receptors significantly increased IL-2 mRNA transcription in response to TCR/CD28 stimulation. Conversely, antagonists or silencing of P2Y1 receptors reduced IL-2 mRNA transcription and attenuated T cell functions. We conclude that P2Y1 and P2X receptors have non-redundant, synergistic functions in the regulation of T cell activation. P2Y1 receptors may represent potential therapeutic targets to modulate T cell function in inflammation and host defense.

Adenosine Triphosphate Release is Required for Toll-Like Receptor-Induced Monocyte/Macrophage Activation, Inflammasome Signaling, Interleukin-1ß Production, and the Host Immune Response to Infection.

OBJECTIVES: Monocytes and macrophages produce interleukin-1ß by inflammasome activation which involves adenosine triphosphate release, pannexin-1 channels, and P2X7 receptors. However, interleukin-1ß can also be produced in an inflammasome-independent fashion. Here we studied if this mechanism also involves adenosine triphosphate signaling and how it contributes to inflammasome activation. DESIGN: In vitro studies with human cells and randomized animal experiments. SETTING: Preclinical academic research laboratory. SUBJECTS: Wild-type C57BL/6 and pannexin-1 knockout mice, healthy human subjects for cell isolation. INTERVENTIONS: Human monocytes and U937 macrophages were treated with different inhibitors to study how purinergic signaling contributes to toll-like receptor-induced cell activation and interleukin-1ß production. Wild-type and pannexin-1 knockout mice were subjected to cecal ligation and puncture to study the role of purinergic signaling in interleukin-1ß production and host immune defense. MEASUREMENTS AND MAIN RESULTS: Toll-like receptor agonists triggered mitochondrial adenosine triphosphate production and adenosine triphosphate release within seconds. Inhibition of mitochondria, adenosine triphosphate release, or P2 receptors blocked p38 mitogen-activated protein kinase and caspase-1 activation and interleukin-1ß secretion. Mice lacking pannexin-1 failed to activate monocytes, to produce interleukin-1ß, and to effectively clear bacteria following cecal ligation and puncture. CONCLUSIONS: Purinergic signaling has two separate roles in monocyte/macrophage activation, namely to facilitate the initial detection of danger signals via toll-like receptors and subsequently to regulate nucleotide-binding oligomerization domain, leucine rich repeat and pyrin domain containing 3 inflammasome activation. Further dissection of these mechanisms may reveal novel therapeutic targets for immunomodulation in critical care patients.

Systemic Adenosine Triphosphate Impairs Neutrophil Chemotaxis and Host Defense in Sepsis.

OBJECTIVE: 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. DESIGN: Prospective randomized animal investigation and in vitro studies. SETTING: Preclinical academic research laboratory. SUBJECTS: Wild-type C57BL/6 mice, pannexin-1 knockout mice, and healthy human subjects used to obtain polymorphonuclear neutrophils for in vitro studies. INTERVENTIONS: 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. MEASUREMENTS AND MAIN RESULTS: 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. CONCLUSIONS: 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.

CD39 Expression Identifies Terminally Exhausted CD8+ T Cells.

Exhausted T cells express multiple co-inhibitory molecules that impair their function and limit immunity to chronic viral infection. Defining novel markers of exhaustion is important both for identifying and potentially reversing T cell exhaustion. Herein, we show that the ectonucleotidse CD39 is a marker of exhausted CD8+ T cells. CD8+ T cells specific for HCV or HIV express high levels of CD39, but those specific for EBV and CMV do not. CD39 expressed by CD8+ T cells in chronic infection is enzymatically active, co-expressed with PD-1, marks cells with a transcriptional signature of T cell exhaustion and correlates with viral load in HIV and HCV. In the mouse model of chronic Lymphocytic Choriomeningitis Virus infection, virus-specific CD8+ T cells contain a population of CD39high CD8+ T cells that is absent in functional memory cells elicited by acute infection. This CD39high CD8+ T cell population is enriched for cells with the phenotypic and functional profile of terminal exhaustion. These findings provide a new marker of T cell exhaustion, and implicate the purinergic pathway in the regulation of T cell exhaustion.

Mitochondrial Dysfunction, Depleted Purinergic Signaling, and Defective T Cell Vigilance and Immune Defense.

T cell suppression in sepsis is a well-known phenomenon; however, the underlying mechanisms are not fully understood. Previous studies have shown that T cell stimulation up-regulates mitochondrial adenosine triphosphate (ATP) production to fuel purinergic signaling mechanisms necessary for adequate T cell responses. Here we show that basal mitochondrial ATP production, ATP release, and stimulation of P2X1 receptors represent a standby purinergic signaling mechanism that is necessary for antigen recognition. Inhibition of this process impairs T cell vigilance and the ability of T cells to trigger T cell activation, up-regulate mitochondrial ATP production, and stimulate P2X4 and P2X7 receptors that elicit interleukin 2 production and T cell proliferation. T cells of patients with sepsis lack this standby purinergic signaling system owing to defects in mitochondrial function, ATP release, and calcium signaling. These defects impair antigen recognition and T cell function and are correlated with sepsis severity. Pharmacological targeting of these defects may improve T cell function and reduce the risk of sepsis.

Cutting off the power: inhibition of leukemia cell growth by pausing basal ATP release and P2X receptor signaling?

T cells respond to antigen stimulation with the rapid release of cellular ATP, which stimulates an autocrine feedback mechanism that regulates calcium influx through P2X receptors. This autocrine purinergic feedback mechanism plays an essential role in the activation of T cells resulting in cell proliferation and clonal expansion. We recently reported that increases in mitochondrial ATP production drive this stimulation-induced purinergic signaling mechanism but that low-level mitochondrial ATP production fuels basal T cell functions required to maintain vigilance of unstimulated T cells. Here we studied whether defects in these purinergic signaling mechanisms are involved in the unwanted proliferation of leukemia T cells. We found that acute leukemia T cells (Jurkat) possess a larger number and more active mitochondria than their healthy counterparts. Jurkat cells have higher intracellular ATP concentrations and generat more extracellular ATP than unstimulated T cells from healthy donors. As a result, increased purinergic signaling through P2X1 and P2X7 receptors elevates baseline levels of cytosolic Ca(2+) in Jurkat cells. We found that pharmacological inhibition of this basal purinergic signaling mechanism decreases mitochondrial activity, Ca(2+) signaling, and cell proliferation. Similar results were seen in the leukemic cell lines THP-1, U-937, and HL-60. Combined treatment with inhibitors of P2X1 or P2X7 receptors and the chemotherapeutic agent 6-mercaptopurine completely blocked Jurkat cell proliferation. Our results demonstrate that increased mitochondrial metabolism promotes autocrine purinergic signaling and uncontrolled proliferation of leukemia cells. These findings suggest that deranged purinergic signaling can result in T cell malignancy and that therapeutic targeting aimed at purinergic signaling is a potential strategy to combat T cell leukemia.

Adenosine arrests breast cancer cell motility by A3 receptor stimulation.

In neutrophils, adenosine triphosphate (ATP) release and autocrine purinergic signaling regulate coordinated cell motility during chemotaxis. Here, we studied whether similar mechanisms regulate the motility of breast cancer cells. While neutrophils and benign human mammary epithelial cells (HMEC) form a single leading edge, MDA-MB-231 breast cancer cells possess multiple leading edges enriched with A3 adenosine receptors. Compared to HMEC, MDA-MB-231 cells overexpress the ectonucleotidases ENPP1 and CD73, which convert extracellular ATP released by the cells to adenosine that stimulates A3 receptors and promotes cell migration with frequent directional changes. However, exogenous adenosine added to breast cancer cells or the A3 receptor agonist IB-MECA dose-dependently arrested cell motility by simultaneous stimulation of multiple leading edges, doubling cell surface areas and significantly reducing migration velocity by up to 75 %. We conclude that MDA-MB-231 cells, HMEC, and neutrophils differ in the purinergic signaling mechanisms that regulate their motility patterns and that the subcellular distribution of A3 adenosine receptors in MDA-MB-231 breast cancer cells contributes to dysfunctional cell motility. These findings imply that purinergic signaling mechanisms may be potential therapeutic targets to interfere with the motility of breast cancer cells in order to reduce the spread of cancer cells and the risk of metastasis.

Circulating mitochondrial DAMPs cause inflammatory responses to injury.

Injury causes a systemic inflammatory response syndrome (SIRS) that is clinically much like sepsis. Microbial pathogen-associated molecular patterns (PAMPs) activate innate immunocytes through pattern recognition receptors. Similarly, cellular injury can release endogenous 'damage'-associated molecular patterns (DAMPs) that activate innate immunity. Mitochondria are evolutionary endosymbionts that were derived from bacteria and so might bear bacterial molecular motifs. Here we show that injury releases mitochondrial DAMPs (MTDs) into the circulation with functionally important immune consequences. MTDs include formyl peptides and mitochondrial DNA. These activate human polymorphonuclear neutrophils (PMNs) through formyl peptide receptor-1 and Toll-like receptor (TLR) 9, respectively. MTDs promote PMN Ca(2+) flux and phosphorylation of mitogen-activated protein (MAP) kinases, thus leading to PMN migration and degranulation in vitro and in vivo. Circulating MTDs can elicit neutrophil-mediated organ injury. Cellular disruption by trauma releases mitochondrial DAMPs with evolutionarily conserved similarities to bacterial PAMPs into the circulation. These signal through innate immune pathways identical to those activated in sepsis to create a sepsis-like state. The release of such mitochondrial 'enemies within' by cellular injury is a key link between trauma, inflammation and SIRS.