P2X7 Receptor Activation Impairs Exogenous MHC Class I Oligopeptides Presentation in Antigen Presenting Cells

Major histocompatibility complex class I (MHC I) on antigen presenting cells (APCs) is a potent molecule to activate CD8⁺ T cells and initiate immunity. P2X7 receptors (P2X7Rs) are present on the plasma membrane of APCs to sense the extracellular danger signal adenosine-5′-triphosphate (ATP). P2X7R activates the inflammasome and the release of IL-1β in macrophages and other immune cells to initiate the inflammatory response. Here we show that P2X7R stimulation by ATP in APCs decreased the amount of MHC I at the plasma membrane. Specific antagonism or genetic ablation of P2X7R inhibited the effects of ATP on levels of cellular MHC I. Furthermore, P2X7R stimulation was able to inhibit activation of CD8⁺ T cells via specific MHC I-oligopeptide complexes. Our study suggests that P2X7R activation on APCs is a novel inhibitor of adaptive CD8⁺ T cell immunity.     

Extracellular NAD and ATP: Partners in immune cell modulation

Extracellular NAD and ATP exert multiple, partially overlapping effects on immune cells. Catabolism of both nucleotides by extracellular enzymes keeps extracellular concentrations low under steady-state conditions and generates metabolites that are themselves signal transducers. ATP and its metabolites signal through purinergic P2 and P1 receptors, whereas extracellular NAD exerts its effects by serving as a substrate for ADP-ribosyltransferases (ARTs) and NAD glycohydrolases/ADPR cyclases like CD38 and CD157. Both nucleotides activate the P2X7 purinoceptor, although by different mechanisms and with different characteristics. While ATP activates P2X7 directly as a soluble ligand, activation via NAD occurs by ART-dependent ADP-ribosylation of cell surface proteins, providing an immobilised ligand. P2X7 activation by either route leads to phosphatidylserine exposure, shedding of CD62L, and ultimately to cell death. Activation by ATP requires high micromolar concentrations of nucleotide and is readily reversible, whereas NAD-dependent stimulation begins at low micromolar concentrations and is more stable. Under conditions of cell stress or inflammation, ATP and NAD are released into the extracellular space from intracellular stores by lytic and non-lytic mechanisms, and may serve as ‘danger signals–to alert the immune response to tissue damage. Since ART expression is limited to naïve/resting T cells, P2X7-mediated NAD-induced cell death (NICD) specifically targets this cell population. In inflamed tissue, NICD may inhibit bystander activation of unprimed T cells, reducing the risk of autoimmunity. In draining lymph nodes, NICD may eliminate regulatory T cells or provide space for the preferential expansion of primed cells, and thus help to augment an immune response.     

Alternative Splicing of the N-Terminal Cytosolic and Transmembrane Domains of P2X7 Controls Gating of the Ion Channel by ADP-Ribosylation

P2X7 is a homotrimeric ion channel with two transmembrane domains and a large extracellular ATP-binding domain. It plays a key role in the response of immune cells to danger signals released from cells at sites of inflammation. Gating of murine P2X7 can be induced by the soluble ligand ATP, as well as by NAD(+)-dependent ADP-ribosylation of arginine 125, a posttranslational protein modification catalyzed by the toxin-related ecto-enzymes ART2.1 and ART2.2. R125 is located at the edge of the ligand-binding crevice. Recently, an alternative splice variant of P2X7, designated P2X7(k), was discovered that differs from the previously described variant P2X7(a) in the N-terminal 42 amino acid residues composing the first cytosolic domain and most of the Tm1 domain. Here we compare the two splice variants of murine P2X7 with respect to their sensitivities to gating by ADP-ribosylation in transfected HEK cells. Our results show that the P2X7(k) variant is sensitive to activation by ADP-ribosylation whereas the P2X7(a) variant is insensitive, despite higher cell surface expression levels. Interestingly, a single point mutation (R276K) renders the P2X7(a) variant sensitive to activation by ADP-ribosylation. Residue 276 is located at the interface of neighboring subunits approximately halfway between the ADP-ribosylation site and the transmembrane domains. Moreover, we show that naive and regulatory T cells preferentially express the more sensitive P2X7(k) variant, while macrophages preferentially express the P2X7(a) variant. Our results indicate that differential splicing of alternative exons encoding the N-terminal cytosolic and transmembrane domains of P2X7 control the sensitivity of different immune cells to extracellular NAD(+) and ATP.     

P2X ion channel receptors and inflammation

Neuroinflammation limits tissue damage in response to pathogens or injury and promotes repair. There are two stages of inflammation, initiation and resolution. P2X receptors are gaining attention in relation to immunology and inflammation. The P2X7 receptor in particular appears to be an essential immunomodulatory receptor, although P2X1 and P2X4 receptors also appear to be involved. ATP released from damaged or infected cells causes inflammation by release of inflammatory cytokines via P2X7 receptors and acts as a danger signal by occupying upregulated P2X receptors on immune cells to increase immune responses. The purinergic involvement in inflammation is being explored for the development of novel therapeutic strategies.     

P2X7 receptor expression levels determine lethal effects of a purine based danger signal in T lymphocytes

Contact of T lymphocytes with nicotinamide adenine dinucleotide (NAD) or ATP causes cell death that requires expression of purinergic receptor P2X(7) (P2X(7)R). T cell subsets differ in their responses to NAD and ATP, which awaits a mechanistic explanation. Here, we show that sensitivity to ATP correlates with P2X(7)R expression levels in CD4 cells, CD8 cells and CD4(+)CD25(+) cells from both C57BL/6 and BALB/c mice. But P2X(7)R ligands do not only induce cell death but also shedding of CD62L. It is shown here that in CD62L(high) T cells, CD62L shedding correlates with low expression of P2X(7)Rs and lower cell death, whereas in CD62L(low) cells P2X(7)R expression and death are higher. The possibility is therefore investigated that P2X(7)Rs induce T cell activation. Experiments show that spontaneous T cell proliferation is somewhat higher in cells expressing P2X(7)Rs, but this effect we suggest is caused by P2X(7)R expression on accessory cells.     

Activation of P2X(7) receptor by ATP plays an important role in regulating inflammatory responses during acute viral infection

Acute viral infection causes damages to the host due to uncontrolled viral replication but even replication deficient viral vectors can induce systemic inflammatory responses. Indeed, overactive host innate immune responses to viral vectors have led to devastating consequences. Macrophages are important innate immune cells that recognize viruses and induce inflammatory responses at the early stage of infection. However, tissue resident macrophages are not easily activated by the mere presence of virus suggesting that their activation requires additional signals from other cells in the tissue in order to trigger inflammatory responses. Previously, we have shown that the cross-talk between epithelial cells and macrophages generates synergistic inflammatory responses during adenoviral vector infection. Here, we investigated whether ATP is involved in the activation of macrophages to induce inflammatory responses during an acute adenoviral infection. Using a macrophage-epithelial cell co-culture system we demonstrated that ATP signaling through P2X(7) receptor (P2X(7)R) is required for induction of inflammatory mediators. We also showed that ATP-P2X(7)R signaling regulates inflammasome activation as inhibition or deficiency of P2X(7)R as well as caspase-1 significantly reduced IL-1β secretion. Furthermore, we found that intranasal administration of replication deficient adenoviral vectors in mice caused a high mortality in wild-type mice with symptoms of acute respiratory distress syndrome but the mice deficient in P2X(7)R or caspase-1 showed increased survival. In addition, wild-type mice treated with apyrase or inhibitors of P2X(7)R or caspase-1 showed higher rates of survival. The improved survival in the P2X(7)R deficient mice correlated with diminished levels of IL-1β and IL-6 and reduced neutrophil infiltration in the early phase of infection. These results indicate that ATP, released during viral infection, is an important inflammatory regulator that activates the inflammasome pathway and regulates inflammatory responses.     

Pulmonary Infection with Hypervirulent Mycobacteria Reveals a Crucial Role for the P2X7 Receptor in Aggressive Forms of Tuberculosis

The purinergic P2X7 receptor (P2X7R) is a sensor of extracellular ATP, a damage-associated molecule that is released from necrotic cells and that induces pro-inflammatory cytokine production and cell death. To investigate whether the innate immune response to damage signals could contribute to the development of pulmonary necrotic lesions in severe forms of tuberculosis, disease progression was examined in C57BL/6 and P2X7R^−/− mice that were intratracheally infected with highly virulent mycobacterial strains (Mycobacterium tuberculosis strain 1471 of the Beijing genotype family and Mycobacterium bovis strain MP287/03). The low-dose infection of C57BL/6 mice with bacteria of these strains caused the rapid development of extensive granulomatous pneumonia with necrotic areas, intense bacillus dissemination and anticipated animal death. In contrast, in P2X7R^−/− mice, the lung pathology presented with moderate infiltrates of mononuclear leukocytes without visible signs of necrosis; the disease attenuation was accompanied by a delay in mortality. In vitro, the hypervirulent mycobacteria grew rapidly inside macrophages and induced death by a P2X7R-dependent mechanism that facilitated the release of bacilli. Furthermore, these bacteria were resistant to the protective mechanisms elicited in macrophages following extracellular ATP stimulation. Based on this study, we propose that the rapid intracellular growth of hypervirulent mycobacteria results in massive macrophage damage. The ATP released by damaged cells engages P2X7R and accelerates the necrotic death of infected macrophages and the release of bacilli. This vicious cycle exacerbates pneumonia and lung necrosis by promoting widespread cell destruction and bacillus dissemination. These findings suggest the use of drugs that have been designed to inhibit the P2X7R as a new therapeutic approach to treat the aggressive forms of tuberculosis.     

Extracellular ATP exerts opposite effects on activated and regulatory CD4+ T cells via purinergic P2 receptor activation

It has been reported that ATP inhibits or stimulates lymphoid cell proliferation depending on the cellular subset analyzed. In this study, we show that ATP exerts strikingly opposite effects on anti-CD3/CD28-activated and regulatory CD4(+) T cells (T(regs)), based on nucleotide concentration. We demonstrate that physiological concentrations of extracellular ATP (1-50 nM) do not affect activated CD4(+) T cells and T(regs). Conversely, higher ATP concentrations have a bimodal effect on activated CD4(+) T cells. Whereas 250 nM ATP stimulates proliferation, cytokine release, expression of adhesion molecules, and adhesion, 1 mM ATP induces apoptosis and inhibits activated CD4(+) T cell functions. The expression analysis and pharmacological profile of purinergic P2 receptors for extracellular nucleotides suggest that activated CD4(+) T cells are induced to apoptosis via the upregulation and engagement of P2X7R and P2X4R. On the contrary, 1 mM ATP enhances proliferation, adhesion, migration, via P2Y2R activation, and immunosuppressive ability of T(regs). Similar results were obtained when activated CD4(+) T cells and T(regs) were exposed to ATP released by necrotized leukemic cells. Taken together, our results show that different concentrations of extracellular ATP modulate CD4(+) T cells according to their activated/regulatory status. Because extracellular ATP concentration highly increases in fast-growing tumors or hyperinflamed tissues, the manipulation of purinergic signaling might represent a new therapeutic target to shift the balance between activated CD4(+) T cells and T(regs).     

ATP/P2X7 axis modulates myeloid-derived suppressor cell functions in neuroblastoma microenvironment

Tumor microenvironment of solid tumors is characterized by a strikingly high concentration of adenosine and ATP. Physiological significance of this biochemical feature is unknown, but it has been suggested that it may affect infiltrating immune cell responses and tumor progression. There is increasing awareness that many of the effects of extracellular ATP on tumor and inflammatory cells are mediated by the P2X7 receptor (P2X7R). Aim of this study was to investigate whether: (i) extracellular ATP is a component of neuroblastoma (NB) microenvironment, (ii) myeloid-derived suppressor cells (MDSCs) express functional P2X7R and (iii) the ATP/P2X7R axis modulates MDSC functions. Our results show that extracellular ATP was detected in NB microenvironment in amounts that increased in parallel with tumor progression. The percentage of CD11b⁺/Gr-1⁺ cells was higher in NB-bearing mice compared with healthy animals. Within the CD11b/Gr-1⁺ population, monocytic MDSCs (M-MDSCs) produced higher levels of reactive oxygen species (ROS), arginase-1 (ARG-1), transforming growth factor-β1 (TGF-β1) and stimulated more potently in vivo tumor growth, as compared with granulocytic MDSCs (G-MDSCs). P2X7R of M-MDSCs was localized at the plasma membrane, coupled to increased functionality, upregulation of ARG-1, TGF-β1 and ROS. Quite surprisingly, the P2X7R in primary MDSCs as well as in the MSC-1 and MSC-2 lines was uncoupled from cytotoxicity. This study describes a novel scenario in which MDSC immunosuppressive functions are modulated by the ATP-enriched tumor microenvironment.     

P2X7 receptor regulation of non‐classical secretion from immune effector cells

P2X7 receptors (P2X7R) are extracellular ATP‐gated ion channels expressed in the immune effector cells that carry out critical protective responses during the early phases of microbial infection or acute tissue trauma. P2X7R‐positive cells include monocytes, macrophages, dendritic cells and T cells. Given its presence in all host and pathogen cell types, ATP can be readily released into extracellular compartments at local sites of tissue damage and microbial invasion. Thus, extracellular ATP and its target receptors on host effector cells can be considered as additional elements of the innate immune system. In this regard, stimulation of P2X7R rapidly triggers a key step of the inflammatory response: induction of NLRP3/caspase‐1 inflammasome signalling complexes that drive the proteolytic maturation and secretion of the proinflammatory cytokines interleukin‐1β (IL‐1β) and interleukin‐18 (IL‐18). IL‐1β (and IL‐18) lacks a signal sequence for compartmentation within the Golgi and classical secretory vesicles and the proIL‐1β precursor accumulates within the cytosol following translation on free ribosomes. Thus, ATP‐induced accumulation of the mature IL‐1β cytokine within extracellular compartments requires non‐classical mechanisms of export from the cytosolic compartment. Five proposed mechanisms include: (i) exocytosis of secretory lysosomes that accumulate cytosolic IL‐1β via undefined protein transporters; (ii) release of membrane‐delimited microvesicles derived from plasma membrane blebs formed by evaginationsof the surface membrane that entrap cytosolic IL‐β; (iii) release of membrane‐delimited exosomes secondary to the exocytosis of multivesicular bodies formed by invaginations of recycling endosomes that entrap cytosolic IL‐β; (iv) exocytosis of autophagosomes or autophagolysosomes that accumulate cytosolic IL‐1β via entrapment during formation of the initial autophagic isolation membrane or omegasome and (v) direct release of cytosolic IL‐1β secondary to regulated cell death by pyroptosis or necroptosis. These mechanisms are not mutually exclusive and may represent engagement of parallel or intersecting membrane trafficking responses to P2X7R activation.     

P2 Receptors of microglia: sensors for danger signals in the CNS

Following tissue damage or invasion by pathogens a number of soluble signals are generated to alert the immune system of the impending danger and initiate inflammation. Some danger signals are released from injured or dying cells. Once released, danger signals activate a autocrine/paracrine network that recruits inflammatory cells, stimulates cytokine production, promotes dendritic cell maturations and increases the antigen (Ag) presenting efficiency. These events also occurs in the central nervous system (CNS) where cytokines and cytokine-releasing cells have a central role in spreading inflammation. P2 receptors of microglia are the focus of increasing interest, especially after they were shown to mediate chemotaxis, cytokine release and cell death in microglia. We propose that P2 receptors may function in microglia as sensors of the ATP/UTP concentration in the pericellular space, and therefore as sensors of danger signals in the CNS. Furthermore, microglia itself can release ATP when stimulated by inflammatory stimuli. Thus extracellular nucleotides may be included in the family of the early inflammatory mediators acting via P2 receptors to spread inflammation in the CNS.
References 1. Ferrari D., Villalba M., Chiozzi P., Falzoni S., Ricciardi-Castagnoli P. and Di Virgilio F. (1996) Mouse microglia cells express a plasma membrane pore gated by extracellular ATP. J. Immunol. 156 , 1531–1539.
2. Ferrari D., Chiozzi P., Falzoni S., Hanau S. and Di Virgilio F. (1997) Purinergic modulation of interleukin-1B release from microglia cells stimulated with bacterial endotoxin. J. Exp. Med. 185 , 579–582.     

P2X7 Integrates PI3K/AKT and AMPK-PRAS40-mTOR Signaling Pathways to Mediate Tumor Cell Death

Background

Extracellular adenosine triphosphate (ATP) functions as a novel danger signal that boosts antitumor immunity and can also directly kill tumor cells. We have previously reported that chronic exposure of tumor cells to ATP provokes P2X7-mediated tumor cell death, by as yet incompletely defined molecular mechanisms.

Methodology/Principal Findings

Here, we show that acute exposure of tumor cells to ATP results in rapid cytotoxic effects impacting several aspects of cell growth/survival, leading to inhibition of tumor growth in vitro and in vivo. Using agonist and antagonist studies together with generation of P2X7 deficient tumor cell lines by lentiviral shRNA delivery system, we confirm P2X7 to be the central control node transmitting extracellular ATP signals. We identify that downstream intracellular signaling regulatory networks implicate two signaling pathways: the known P2X7-PI3K/AKT axis and remarkably a novel P2X7-AMPK-PRAS40-mTOR axis. When exposed to high levels of extracellular ATP, these two signaling axes perturb the balance between growth and autophagy, thereby promoting tumor cell death.

Conclusions

Our study defines novel molecular mechanisms underpinning the antitumor actions of P2X7 and provides a further rationale for purine-based drugs in targeted cancer therapy.     

Secretion of intracellular IL-1 receptor antagonist (type 1) is dependent on P2X7 receptor activation

Inflammatory mechanisms are critical in the arterial response to injury. Both IL-1 and the naturally occurring inhibitor of IL-1, IL-1R antagonist (IL-1ra), are expressed in the arterial wall, and in particular in the endothelium. Previous studies suggest that endothelial cells only make the intracellular type I isoform of IL-1ra (icIL-1ra1), an isoform known to lack a secretory signal peptide. It is unclear how icIL-1ra is released from the endothelial cell to act as an antagonist on cell surface IL-1 type I receptors. IL-1beta, which also lacks a secretory signal peptide, may be released by ATP stimulation of the P2X(7)R. Therefore, we examined whether icIL-1ra1 release occurs in an analogous manner, using both the mouse macrophage cell line RAW264.7 and HUVECs. P2X(7)R activation caused icIL-1ra1 release from LPS-primed RAW264.7 macrophages and from HUVECs. This release was inhibited in the absence of extracellular calcium, and attenuated by preincubation with oxidized ATP, KN62, and apyrase. Endogenous ATP release, which also facilitated release of icIL-1ra1, was detected during LPS treatment of both RAW264.7 macrophages and HUVECs. Annexin V assays showed that ATP stimulation resulted in a rapid phosphatidylserine (PS) exposure on the cell surface of RAW264.7 macrophages, and that PS-exposed microvesicles contained icIL-1ra1. However, PS flip and microvesicle shedding was not apparent in ATP-treated HUVECs. These data support a general role for the P2X(7)R in the release of leaderless cytokines into the extracellular medium, and indicate how icIL-1ra1 may act upon its extracellular target, the IL-1R.     

Radioligands targeting purinergic P2X7 receptor

The purinergic P2X7 receptor (P2X7R) is an adenosine triphosphate (ATP) ligand-gated cationic channel receptor. P2X7R is closely associated with various inflammatory, immune, cancer, neurological, musculoskeletal and cardiovascular disorders. P2X7R is an interesting therapeutic target as well as molecular imaging target. This brief digest highlights the radioligands targeting P2X7R recently developed in drug discovery and molecular imaging agent development.     

The potential of P2X7 receptors as a therapeutic target,including inflammation and tumour progression

Seven P2X ion channel nucleotide receptor subtypes have been cloned and characterised. P2X7 receptors (P2X7R) are unusual in that there are extra amino acids in the intracellular C terminus. Low concentrations of ATP open cation channels sometimes leading to cell proliferation, whereas high concentrations of ATP open large pores that release inflammatory cytokines and can lead to apoptotic cell death. Since many diseases involve inflammation and immune responses, and the P2X7R regulates inflammation, there has been recent interest in the pathophysiological roles of P2X7R and the potential of P2X7R antagonists to treat a variety of diseases. These include neurodegenerative diseases, psychiatric disorders, epilepsy and a number of diseases of peripheral organs, including the cardiovascular, airways, kidney, liver, bladder, skin and musculoskeletal. The potential of P2X7R drugs to treat tumour progression is discussed.     

P2 Receptors of microglia: sensors for danger signals in the CNS

Following tissue damage or invasion by pathogens a number of soluble signals are generated to alert the immune system of the impending danger and initiate inflammation. Some danger signals are released from injured or dying cells. Once released, danger signals activate a autocrine/paracrine network that recruits inflammatory cells, stimulates cytokine production, promotes dendritic cell maturations and increases the antigen (Ag) presenting efficiency. These events also occurs in the central nervous system (CNS) where cytokines and cytokine‐releasing cells have a central role in spreading inflammation. P2 receptors of microglia are the focus of increasing interest, especially after they were shown to mediate chemotaxis, cytokine release and cell death in microglia. We propose that P2 receptors may function in microglia as sensors of the ATP/UTP concentration in the pericellular space, and therefore as sensors of danger signals in the CNS. Furthermore, microglia itself can release ATP when stimulated by inflammatory stimuli. Thus extracellular nucleotides may be included in the family of the early inflammatory mediators acting via P2 receptors to spread inflammation in the CNS. References
1. Ferrari D., Villalba M., Chiozzi P., Falzoni S., Ricciardi‐Castagnoli P. and Di Virgilio F. (1996) Mouse microglia cells express a plasma membrane pore gated by extracellular ATP. J. Immunol. 156 , 1531–1539. 2. Ferrari D., Chiozzi P., Falzoni S., Hanau S. and Di Virgilio F. (1997) Purinergic modulation of interleukin‐1B release from microglia cells stimulated with bacterial endotoxin. J. Exp. Med. 185 , 579–582.     

P2X7 receptor-mediated purinergic signaling promotes liver injury in acetaminophen hepatotoxicity in mice

Inflammation contributes to liver injury in acetaminophen (APAP) hepatotoxicity in mice and is triggered by stimulation of immune cells. The purinergic receptor P2X7 is upstream of the nod-like receptor family, pryin domain containing-3 (NLRP3) inflammasome in immune cells and is activated by ATP and NAD that serve as damage-associated molecular patterns. APAP hepatotoxicity was assessed in mice genetically deficient in P2X7, the key inflammatory receptor for nucleotides (P2X7-/-), and in wild-type mice. P2X7-/- mice had significantly decreased APAP-induced liver necrosis. In addition, APAP-poisoned mice were treated with the specific P2X7 antagonist A438079 or etheno-NAD, a competitive antagonist of NAD. Pre- or posttreatment with A438079 significantly decreased APAP-induced necrosis and hemorrhage in APAP liver injury in wild-type but not P2X7-/- mice. Pretreatment with etheno-NAD also significantly decreased APAP-induced necrosis and hemorrhage in APAP liver injury. In addition, APAP toxicity in mice lacking the plasma membrane ecto-NTPDase CD39 (CD39-/-) that metabolizes ATP was examined in parallel with the use of soluble apyrase to deplete extracellular ATP in wild-type mice. CD39-/- mice had increased APAP-induced hemorrhage and mortality, whereas apyrase also decreased APAP-induced mortality. Kupffer cells were treated with extracellular ATP to assess P2X7-dependent inflammasome activation. P2X7 was required for ATP-stimulated IL-1β release. In conclusion, P2X7 and exposure to the ligands ATP and NAD are required for manifestations of APAP-induced hepatotoxicity.