Fetal bovine serum contains biologically available ATP.

ATP is a ubiquitous extracellular messenger released in a wide number of pathophysiological conditions. ATP is known to be present in minute amounts in the extracellular space in healthy tissues and in the blood, and to modulate a multiplicity of cell responses. Cell culture systems are widely used to explore purinergic signaling. We show here that currently used fetal bovine sera contain ATP in the 300-1300 pmol/L range. Serum ATP is associated with albumin as well as with microparticle/microvesicle fraction. Serum microparticles/microvesicles affect in vitro cell responses due to their content of miRNAs, growth factors, and other bioactive molecules. ATP is likely to be one of these bioactive factors found in a variable amount in sera of different commercial sources. ATP in serum supports ATP-dependent biochemical reactions such as the hexokinase-dependent phosphorylation of glucose to glucose 6-phosphate, and affects purinergic signaling. These findings show that cells growing in vitro in serum-supplemented media are exposed to varying levels of extracellular ATP, and thus to varying degrees of purinergic stimulation.

The Coming of Age of the P2X7 Receptor in Diagnostic Medicine.

The discovery of the P2X7 receptor (P2X7R, originally named P2Z) in immune cells, its cloning, and the identification of its role in a multiplicity of immune-mediated diseases raised great hopes for the development of novel and more potent anti-inflammatory medicaments. Unfortunately, such hopes were partially deluded by the unsatisfactory results of most early clinical trials. This failure substantially reduced the interest of the pharmaceutical and biotech industries in the clinical development of P2X7R-targeted therapies. However, recent findings ushered in a second life for the P2X7R in diagnostic medicine. New P2X7R radioligands proved to be very reliable tools for the diagnosis of neuroinflammation in preclinical and clinical studies, and detection and measurement of free P2X7 receptor (or P2X7 subunit) in human blood suggested its potential use as a circulating marker of inflammation. Here we provide a brief review of these novel developments.

The shed P2X7 receptor is an index of adverse clinical outcome in COVID-19 patients.

Introduction: The pathophysiology of the Corona Virus Disease 2019 (COVID-19) is incompletely known. A robust inflammatory response caused by viral replication is a main cause of the acute lung and multiorgan injury observed in critical patients. Inflammasomes are likely players in COVID-19 pathogenesis. The P2X7 receptor (P2X7R), a plasma membrane ATP-gated ion channel, is a main activator of the NLRP3 inflammasome, of the ensuing release of inflammatory cytokines and of cell death by pyroptosis. The P2X7R has been implicated in COVID-19-dependent hyperinflammation and in the associated multiorgan damage. Shed P2X7R (sP2X7R) and shed NLRP3 (sNLRP3) have been detected in plasma and other body fluids, especially during infection and inflammation. Methods: Blood samples from 96 patients with confirmed SARS-CoV-2 infection with various degrees of disease severity were tested at the time of diagnosis at hospital admission. Standard haematological parameters and IL-6, IL-10, IL-1ß, sP2X7R and sNLRP3 levels were measured, compared to reference values, statistically validated, and correlated to clinical outcome. Results: Most COVID-19 patients included in this study had lymphopenia, eosinopenia, neutrophilia, increased inflammatory and coagulation indexes, and augmented sNLRP3, IL-6 and IL-10 levels. Blood concentration of sP2X7R was also increased, and significantly positively correlated with lymphopenia, procalcitonin (PCT), IL-10, and alanine transaminase (ALT). Patients with increased sP2X7R levels at diagnosis also showed fever and respiratory symptoms, were more often transferred to Pneumology division, required mechanical ventilation, and had a higher likelihood to die during hospitalization. Conclusion: Blood sP2X7R was elevated in the early phases of COVID-19 and predicted an adverse clinical outcome. It is suggested that sP2X7R might be a useful marker of disease progression.

Extracellular ATP: A powerful inflammatory mediator in the central nervous system.

Nucleotides play a crucial role in extracellular signaling across species boundaries. All the three kingdoms of life (Bacteria, Archea and Eukariota) are responsive to extracellular ATP (eATP) and many release this and other nucleotides. Thus, eATP fulfills different functions, many related to danger-sensing or avoidance reactions. Basically all living organisms have evolved sensors for eATP and other nucleotides with very different affinity and selectivity, thus conferring a remarkable plasticity to this signaling system. Likewise, different intracellular transduction systems were associated during evolution to different receptors for eATP. In mammalian evolution, control of intracellular ATP (iATP) and eATP homeostasis has been closely intertwined with that of Ca2+, whether in the extracellular milieu or in the cytoplasm, establishing an inverse reciprocal relationship, i.e. high extracellular Ca2+ levels are associated to negligible eATP, while low intracellular Ca2+ levels are associated to high eATP concentrations. This inverse relationship is crucial for the messenger functions of both molecules. Extracellular ATP is sensed by specific plasma membrane receptors of widely different affinity named P2 receptors (P2Rs) of which 17 subtypes are known. This confers a remarkable plasticity to P2R signaling. The central nervous system (CNS) is a privileged site for purinergic signaling as all brain cell types express P2Rs. Accruing evidence suggests that eATP, in addition to participating in synaptic transmission, also plays a crucial homeostatic role by fine tuning microglia, astroglia and oligodendroglia responses. Drugs modulating the eATP concentration in the CNS are likely to be the new frontier in the therapy of neuroinflammation. This article is part of the Special Issue on 'Purinergic Signaling: 50 years'.

Modulation of Cell Energy Metabolism by the P2X7 Receptor.

For many years the P2X7 receptor (P2X7R) was considered the prototypic cytolytic receptor due to its ability to cause dramatic changes in plasma membrane permeability, eventually leading to cell death. However, later studies revealed that controlled P2X7R activation has beneficial effects on cell metabolism and nowadays our perception of the physiological role of this receptor has radically changed. Some of the biochemical pathways underlying the trophic effect of the P2X7R are being unveiled, thus disclosing an unanticipated role of P2X7Rs in mitochondrial and glycolytic metabolism. We provide here an update of the effects of the P2X7R on cell energy metabolism.

Signalling by extracellular nucleotides in health and disease.

Nucleotides are released from all cells through regulated pathways or as a result of plasma membrane damage or cell death. Outside the cell, nucleotides act as signalling molecules triggering multiple responses via specific plasma membrane receptors of the P2 family. In the nervous system, purinergic signalling has a key function in neurotransmission. Outside the nervous system, purinergic signalling is one of the major modulators of basal tissue homeostasis, while its dysregulation contributes to the pathogenesis of various disease, including inflammation and cancer. Pre-clinical and clinical evidence shows that selective P2 agonists or antagonists are effective treatments for many pathologies, thus highlighting the relevance of extracellular nucleotides and P2 receptors as therapeutic targets.

Extracellular ATP is increased by release of ATP-loaded microparticles triggered by nutrient deprivation.

Rationale: Caloric restriction improves the efficacy of anti-cancer therapy. This effect is largely dependent on the increase of the extracellular ATP concentration in the tumor microenvironment (TME). Pathways for ATP release triggered by nutrient deprivation are largely unknown. Methods: The extracellular ATP (eATP) concentration was in vivo measured in the tumor microenvironment of B16F10-inoculated C57Bl/6 mice with the pmeLuc probe. Alternatively, the pmeLuc-TG-mouse was used. Caloric restriction was in vivo induced with hydroxycitrate (HC). B16F10 melanoma cells or CT26 colon carcinoma cells were in vitro exposed to serum starvation to mimic nutrient deprivation. Energy metabolism was monitored by Seahorse. Microparticle release was measured by ultracentrifugation and by Nanosight. Results: Nutrient deprivation increases eATP release despite the dramatic inhibition of intracellular energy synthesis. Under these conditions oxidative phosphorylation was dramatically impaired, mitochondria fragmented and glycolysis and lactic acid release were enhanced. Nutrient deprivation stimulated a P2X7-dependent release of ATP-loaded, mitochondria-containing, microparticles as well as of naked mitochondria. Conclusions: Nutrient deprivation promotes a striking accumulation of eATP paralleled by a large release of ATP-laden microparticles and of naked mitochondria. This is likely to be a main mechanism driving the accumulation of eATP into the TME.

Mitochondrial P2X7 Receptor Localization Modulates Energy Metabolism Enhancing Physical Performance.

Basal expression of the P2X7 receptor (P2X7R) improves mitochondrial metabolism, Adenosine 5'-triphosphate (ATP) synthesis, and overall fitness of immune and non-immune cells. We investigated P2X7R contribution to energy metabolism and subcellular localization in fibroblasts (mouse embryo fibroblasts and HEK293 human fibroblasts), mouse microglia (primary brain microglia, and the N13 microglia cell line), and heart tissue. The P2X7R localizes to mitochondria, and its lack (1) decreases basal respiratory rate, ATP-coupled respiration, maximal uncoupled respiration, resting mitochondrial potential, mitochondrial matrix Ca2+ level, (2) modifies expression pattern of oxidative phosphorylation enzymes, and (3) severely affects cardiac performance. Hearts from P2rx7-deleted versus wild-type mice are larger, heart mitochondria smaller, and stroke volume, ejection fraction, fractional shortening, and cardiac output, are significantly decreased. Accordingly, the physical fitness of P2X7R-null mice is severely reduced. Thus, the P2X7R is a key modulator of mitochondrial energy metabolism and a determinant of physical fitness.

The P2X7 Receptor Is Overexpressed in the Lesional Skin of Subjects Affected by Hidradenitis Suppurativa: A Preliminary Study.

BACKGROUND: P2X receptors (P2XRs) are plasma membrane channels involved in the modulation of immune responses. The role of the P2X7 receptor (P2X7R) has never been investigated in hidradenitis suppurativa (HS), which is a recurrent skin disease characterized by inflammatory nodules, scarring, and suppuration. OBJECTIVE: Our aim was to investigate by immunohistochemistry (IHC) P2X7R, NLRP3 (NOD-like receptor family, pyrin domain-containing 3), and interleukin-1ß (IL-1ß) expression in HS lesions compared to healthy control (HC) skin. METHOD: The intensity of IHC immunostaining was semi-quantitatively graded for keratinocytes, neutrophils, lymphocytes, and monocytes. Statistical significance was assessed by the Mann-Whitney U test, Cohen's κ coefficient, and χ2 test. RESULTS: A total of 59 samples, 31 from HS and 28 from HC, were collected and analysed. In skin keratinocytes, lymphocytes, and monocytes, but not in neutrophils, P2X7R and NLRP3 protein expression was significantly increased in HS versus the HC group. IL-1ß protein expression was also higher in HS versus the HC group both in skin keratinocytes and in the inflammatory infiltrate. Cohen's κ correlation coefficients for the expression of P2X7R versus NLRP3 or IL-1ß in skin keratinocytes were significant (κ = 0.43 and 0.34, respectively). The same association between P2X7R and NLRP3 or IL-1ß was confirmed by χ2 tests. CONCLUSION: P2X7R, NLRP3, and IL-1ß are overexpressed, and therefore the entire P2X7R/NLRP3/IL-1ß pro-inflammatory axis is likely overactive in the skin of HS patients. This observation might provide clues to the pathogenesis of this disease and suggest novel therapies and markers of disease activity.

Ectonucleotidases in Acute and Chronic Inflammation.

Ectonucleotidases are extracellular enzymes with a pivotal role in inflammation that hydrolyse extracellular purine and pyrimidine nucleotides, e.g., ATP, UTP, ADP, UDP, AMP and NAD+. Ectonucleotidases, expressed by virtually all cell types, immune cells included, either as plasma membrane-associated or secreted enzymes, are classified into four main families: 1) nucleoside triphosphate diphosphohydrolases (NTPDases), 2) nicotinamide adenine dinucleotide glycohydrolase (NAD glycohydrolase/ADP-ribosyl cyclase/cyclic ADP-ribose hydrolase 1), 3) ecto-5'-nucleotidase (NT5E), and 4) ecto-nucleotide pyrophosphatase/phosphodiesterases (NPPs). Concentration of ATP, UTP and NAD+ can be increased in the extracellular space thanks to un-regulated, e.g., cell damage or cell death, or regulated processes. Regulated processes include secretory exocytosis, connexin or pannexin hemichannels, ATP binding cassette (ABC) transporters, calcium homeostasis modulator (CALMH) channels, the ATP-gated P2X7 receptor, maxi-anion channels (MACs) and volume regulated ion channels (VRACs). Hydrolysis of extracellular purine nucleotides generates adenosine, an important immunosuppressant. Extracellular nucleotides and nucleosides initiate or dampen inflammation via P2 and P1 receptors, respectively. All these agents, depending on their level of expression or activation and on the agonist concentration, are potent modulators of inflammation and key promoters of host defences, immune cells activation, pathogen clearance, tissue repair and regeneration. Thus, their knowledge is of great importance for a full understanding of the pathophysiology of acute and chronic inflammatory diseases. A selection of these pathologies will be briefly discussed here.

Extreme thrombocytosis in systemic juvenile idiopathic arthritis. A case report.

BACKGROUND: Systemic onset juvenile idiopathic arthritis (SoJIA) is a rare inflammatory disorder characterized by remitting fevers, evanescent rash, generalized lymphadenopathy, hepatomegaly/splenomegaly, and/or serositis. CASE PRESENTATION: Here we report the case of a 5 years-old girl with SoJIA complicated by severe thrombocytosis. Treatment with the Interleukin-1ß (IL-1ß) receptor antagonist Anakinra caused a fast reduction of blood platelets and of the associated systemic inflammatory response. Measurement of IL-1ß, IL-6 and Tpo plasma levels at different time points confirmed the etiopathogenetic role of IL-1ß in causing the thrombocytosis, while Tpo did not appear to be involved and this explains the excellent response to treatment with Anakinra. CONCLUSION: The excellent response to treatment with the IL-1ß receptor antagonist, suggests a key pathogenic role of IL-1ß in thrombocytosis as well as in the associated systemic symptoms of inflammation.

P2X7 Receptor Expression in Patients With Serositis Related to Systemic Lupus Erythematosus.

Introduction: P2X7R is an extracellular ATP-gated receptor involved in inflammatory and autoimmune processes mainly acting through NLPR3-inflammasome activation and IL-1ß release, also implicated in lymphocyte proliferation and cellular apoptosis. Several observations from animal models and patients' studies highlight a possible link between P2X7R-NLRP3 axis and Systemic Lupus Erythematosus (SLE) pathogenesis. The P2X7R-inflammasome axis in addition to the direct production of IL-1ß and IL-18, indirectly mediates the release of other cytokines implicated in the pathogenesis of SLE, such as IL-6. The aim of this study was to investigate the role of P2X7R and NLRP3-inflammasome in SLE. Methods: Forty-eight SLE patients, 16 with (SLE-S) and 32 without (SLE-NS) history of serositis, and 20 healthy control (HC) subjects were enrolled. Demographic, clinical, and therapeutic data were collected. IL-1ß and IL-6 plasma levels were evaluated by ELISA. Peripheral blood mononuclear cells (PBMCs) were isolated from venous blood by Ficoll gradient sedimentation and employed as follows: (1) evaluation of P2X7R and NLRP3 expression by RT-PCR; (2) determination of P2X7R activity as Benzoyl ATP (BzATP)-induced [Ca2+]i increments using Fura-2-AM fluorescent probe; (3) isolation of monocytes/macrophages and assessment of in vitro IL-1ß and IL-6 release following stimulation with lipopolysaccharide (LPS) and BzATP, either separately or in combination. Results: Plasma IL-1ß levels were unmodified in SLE respect to HC whereas IL-6 levels were higher in SLE than in HC, resulting significantly increased in SLE-S. Macrophages isolated from SLE patients released lower quantities of IL-1ß after stimulation with BzATP, whereas IL-6 release was significantly augmented in SLE-NS respect to both HC and SLE-S after all types of stimulation. The [Ca2+]i increase following BzATP stimulation was significantly lower in PBMCs from SLE patients than in PBMCs from HC. RT-PCR showed significantly reduced P2X7R and significantly augmented NLRP3 expression in PBMCs from SLE patients. Conclusion: Our data indicate reduced P2X7R expression and function in SLE patients compared with HC and, conversely, increased IL-6 signaling. The possible consequences of reduced P2X7R, mainly on cytokines network deregulation and lymphocyte proliferation, will be further investigated as well as the role of IL-6 as a possible therapeutic target especially in lupus serositis.

The P2X7 Receptor Is Shed Into Circulation: Correlation With C-Reactive Protein Levels.

The P2X7 receptor (P2X7R) is a key pro-inflammatory plasma membrane receptor responsible for NLRP3 inflammasome activation and IL-1ß release. Various inflammatory plasma membrane receptors (e.g., IL-1 type I receptor, TNF type I and II receptors, IL-2 receptor) are shed under different pathophysiological conditions. In the present study, we show that the full length P2X7R is released into circulation in patients as well as in healthy subjects. Blood levels of shed P2X7R (sP2X7R) correlate to those of the inflammatory marker C reactive protein (CRP). Blood sP2X7R ranged from 16.74 to 82.17 ng/L, mean ± SE 40.97 ± 3.82 (n = 26) in healthy subjects, from 33.1 to 484.0 ng/L, mean ± SE 114.78 ± 12.22 (n = 45) in patients with CRP <3 mg/L, and from 63.65 to 1092.3 ng/L, mean ± SE 204.2 ± 30.94 (n = 42) in patients with CRP >3 mg/L. sP2X7R in plasma was largely associated to microvesicles/microparticles. Peripheral blood monocytes from healthy subjects released sP2X7R upon stimulation with the semi-selective P2X7R agonist benzoyl ATP. These data show that the P2X7R can be released into circulation, and that its blood levels increase in various disease conditions.

Amyloid ß-dependent mitochondrial toxicity in mouse microglia requires P2X7 receptor expression and is prevented by nimodipine.

Previous data from our laboratory show that expression of the P2X7 receptor (P2X7R) is needed for amyloid ß (Aß)-stimulated microglia activation and IL-1ß release in vitro and in vivo. We also showed that Aß-dependent stimulation is inhibited by the dihydropyridine nimodipine at an intracellular site distal to the P2X7R. In the present study, we used the N13 microglia cell line and mouse primary microglia from wt and P2rx7-deleted mice to test the effect of nimodipine on amyloid ß (Aß)-dependent NLRP3 inflammasome expression and function, and on mitochondrial energy metabolism. Our data show that in microglia Aß causes P2X7R-dependent a) NFκB activation; b) NLRP3 inflammasome expression and function; c) mitochondria toxicity; and these changes are fully inhibited by nimodipine. Our study shows that nimodipine is a powerful blocker of cell damage caused by monomeric and oligomeric Aß, points to the mitochondria as a crucial target, and underlines the permissive role of the P2X7R.

Extracellular nucleotides and nucleosides as signalling molecules.

Extracellular nucleotides, mainly ATP, but also ADP, UTP, UDP and UDP-sugars, adenosine, and adenine base participate in the "purinergic signalling" pathway, an ubiquitous system of cell-to-cell communication. Fundamental pathophysiological processes such as tissue homeostasis, wound healing, neurodegeneration, immunity, inflammation and cancer are modulated by purinergic signalling. Nucleotides can be released from cells via unspecific or specific mechanisms. A non-regulated nucleotide release can occur from damaged or dying cells, whereas exocytotic granules, plasma membrane-derived microvesicles, membrane channels (connexins, pannexins, calcium homeostasis modulator (CALHM) channels and P2X7 receptor) or specific ATP binding cassette (ABC) transporters are involved in the controlled release. Four families of specific receptors, i.e. nucleotide P2X and P2Y receptors, adenosine P1 receptors, and the adenine-selective P0 receptor, and several ecto- nucleotidases are essential components of the "purinergic signalling" pathway. Thanks to the activity of ecto-nucleotidases, ATP (and possibly other nucleotides) are degraded into additional messenger molecules with specific action. The final biological effects depend on the type and amount of released nucleotides, their modification by ecto-nucleotidases, and their possible cellular re-uptake. Overall, these processes confer a remarkable level of selectivity and plasticity to purinergic signalling that makes this network one of the most relevant extracellular messenger systems in higher organisms.

The P2X7 receptor: A main player in inflammation.

Damage associated molecular patterns (DAMPs) are intracellular molecules released from infected or injured cells to activate inflammatory and reparatory responses. One of the most ancient and conserved DAMPs is extracellular ATP that exerts its phlogistic activity mainly through activation of the P2X7 receptor (P2X7R). The P2X7R is an ATP gated ion channel, expressed by most immune cells, including the monocyte-derived cell lineages, T and B lymphocytes and their precursors. Here we give an overview of recent and established literature on the role of P2X7R in septic and sterile inflammation. P2X7R ability in restraining intracellular bacteria and parasite infection by modulation of the immune response are described, with particular focus on Mycobacteria and Plasmodium. Emerging literature on the role of P2X7 in viral infections such as HIV-1 is also briefly covered. Finally, we describe the numerous intracellular pathways related to inflammation and activated by the P2X7R, including the NLRP3 inflammasome, NF-kB, NFAT, GSK3ß and VEGF, and discuss the involvement of P2X7R in chronic diseases. The possible therapeutic applications of P2X7R antagonists are also described.

The P2X7 Receptor in Infection and Inflammation.

Adenosine triphosphate (ATP) accumulates at sites of tissue injury and inflammation. Effects of extracellular ATP are mediated by plasma membrane receptors named P2 receptors (P2Rs). The P2R most involved in inflammation and immunity is the P2X7 receptor (P2X7R), expressed by virtually all cells of innate and adaptive immunity. P2X7R mediates NLRP3 inflammasome activation, cytokine and chemokine release, T lymphocyte survival and differentiation, transcription factor activation, and cell death. Ten human P2RX7 gene splice variants and several SNPs that produce complex haplotypes are known. The P2X7R is a potent stimulant of inflammation and immunity and a promoter of cancer cell growth. This makes P2X7R an appealing target for anti-inflammatory and anti-cancer therapy. However, an in-depth knowledge of its structure and of the associated signal transduction mechanisms is needed for an effective therapeutic development.

The P2X7 Receptor-Interleukin-1 Liaison.

Interleukin-1ß (IL-1ß) plays a central role in stimulation of innate immune system and inflammation and in several chronic inflammatory diseases. These include rare hereditary conditions, e.g., auto-inflammatory syndromes, as well as common pathologies, such as type II diabetes, gout and atherosclerosis. A better understanding of IL-1ß synthesis and release is particularly relevant for the design of novel anti-inflammatory drugs. One of the molecules mainly involved in IL-1ß maturation is the P2X7 receptor (P2X7R), an ATP-gated ion channel that chiefly acts through the recruitment of the NLRP3 inflammasome-caspase-1 complex. In this review, we will summarize evidence supporting the key role of the P2X7R in IL-1ß production, with special emphasis on the mechanism of release, a process that is still a matter of controversy. Four different models have been proposed: (i) exocytosis via secretory lysosomes, (ii) microvesicles shedding from plasma membrane, (iii) release of exosomes, and (iv) passive efflux across a leaky plasma membrane during pyroptotic cell death. All these models involve the P2X7R.