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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.

The P2X7 receptor (P2X7R) is attracting increasing attention for its involvement in cancer. Several recent studies have shown a crucial role of P2X7R in tumour cell growth, angiogenesis and invasiveness. In this study, we investigated the role of the two known human P2X7R functional splice variants, the full length P2X7RA and the truncated P2X7RB, in osteosarcoma cell growth. Immunohistochemical analysis of a tissue array of human osteosarcomas showed that forty-four, of a total fifty-four tumours (81.4%), stained positive for both P2X7RA and B, thirty-one (57.4%) were positive using an anti-P2X7RA antibody, whereas fifteen of the total number (27.7%) expressed only P2X7RB. P2X7RB positive tumours showed increased cell density, at the expense of extracellular matrix. The human osteosarcoma cell line Te85, which lacks endogenous P2X7R expression, was stably transfected with either P2X7RA, P2X7RB, or both. Receptor expression was a powerful stimulus for cell growth, the most efficient growth-promoting isoform being P2X7RB alone. Growth stimulation was matched by increased Ca(2+) mobilization and enhanced NFATc1 activity. Te85 P2X7RA+B cells presented pore formation as well as spontaneous extracellular ATP release. The ATP release was sustained in all clones by P2X7R agonist (BzATP) and reduced following P2X7R antagonist (A740003) application. BzATP also increased cell growth and activated NFATc1 levels. On the other hand cyclosporin A (CSA) affected both NFATc1 activation and cell growth, definitively linking P2X7R stimulation to NFATc1 and cell proliferation. All transfected clones also showed reduced RANK-L expression, and an overall decreased RANK-L/OPG ratio. Mineralization was increased in Te85 P2X7RA+B cells while it was significantly diminished in Te85 P2X7RB clones, in agreement with immunohistochemical results. In summary, our data show that the majority of human osteosarcomas express P2X7RA and B and suggest that expression of either isoform is differently coupled to cell growth or activity.

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. 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-1β, sP2X7R and sNLRP3 levels were measured, compared to reference values, statistically validated, and correlated to clinical outcome. 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. 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.

P2X7R is an extracellular ATP-gated receptor involved in inflammatory and autoimmune processes mainly acting through NLPR3-inflammasome activation and IL-1β 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-1β 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. 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-1β 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 [Ca ] increments using Fura-2-AM fluorescent probe; (3) isolation of monocytes/macrophages and assessment of IL-1β and IL-6 release following stimulation with lipopolysaccharide (LPS) and BzATP, either separately or in combination. Plasma IL-1β 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-1β 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 [Ca ]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. 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.

Purinergic signalling plays a crucial role in immunity and autoimmunity. Among purinergic receptors, the P2X7 receptor (P2X7R) has an undisputed role as it is expressed to high level by immune cells, triggers cytokine release and modulates immune cell differentiation. In this review, we focus on evidence supporting a possible role of the P2X7R in the pathogenesis of systemic lupus erythematosus (SLE).

The P2X7 receptor (P2X7R) is a key pro-inflammatory plasma membrane receptor responsible for NLRP3 inflammasome activation and IL-1β 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 ( = 26) in healthy subjects, from 33.1 to 484.0 ng/L, mean ± SE 114.78 ± 12.22 ( = 45) in patients with CRP <3 mg/L, and from 63.65 to 1092.3 ng/L, mean ± SE 204.2 ± 30.94 ( = 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.

Microparticles (MPs) are secreted by all cells, where they play a key role in intercellular communication, differentiation, inflammation, and cell energy transfer. P2X7 receptor (P2X7R) activation by extracellular ATP (eATP) causes a large MP release and affects their contents in a cell-specific fashion. We investigated MP release and functional impact in microglial cells from P2X7R-WT or P2X7R-KO mice, as well as mouse microglial cell lines characterized for high (N13-P2X7RHigh) or low (N13-P2X7RLow) P2X7R expression. P2X7R stimulation promoted release of a mixed MP population enriched with naked mitochondria. Released mitochondria were taken up and incorporated into the mitochondrial network of the recipient cells in a P2X7R-dependent fashion. NLRP3 and the P2X7R itself were also delivered to the recipient cells. Microparticle transfer increased the energy level of the recipient cells and conferred a pro-inflammatory phenotype. These data show that the P2X7R is a master regulator of intercellular organelle and MP trafficking in immune cells. Graphical Abstract Graphical Abstract