Impact of Diverse Ion Channels on Regulatory T Cell Functions.

The population of regulatory T cells (Tregs) is critical for immunological self-tolerance and homeostasis. Proper ion regulation contributes to Treg lineage identity, regulation, and effector function. Identified ion channels include Ca2+ release-activated Ca2+, transient receptor potential, P2X, volume-regulated anion and K+ channels Kv1.3 and KCa3.1. Ion channel modulation represents a promising therapeutic approach for the treatment of autoimmune diseases such as rheumatoid arthritis and multiple sclerosis. This review summarizes studies with gene-targeted mice and pharmacological modulators affecting Treg number and function. Furthermore, participation of ion channels is illustrated and the power of future research possibilities is discussed.

Identification of a subset of immunosuppressive P2RX1-negative neutrophils in pancreatic cancer liver metastasis.

The immunosuppressive microenvironment that is shaped by hepatic metastatic pancreatic ductal adenocarcinoma (PDAC) is essential for tumor cell evasion of immune destruction. Neutrophils are important components of the metastatic tumor microenvironment and exhibit heterogeneity. However, the specific phenotypes, functions and regulatory mechanisms of neutrophils in PDAC liver metastases remain unknown. Here, we show that a subset of P2RX1-negative neutrophils accumulate in clinical and murine PDAC liver metastases. RNA sequencing of murine PDAC liver metastasis-infiltrated neutrophils show that P2RX1-deficient neutrophils express increased levels of immunosuppressive molecules, including PD-L1, and have enhanced mitochondrial metabolism. Mechanistically, the transcription factor Nrf2 is upregulated in P2RX1-deficient neutrophils and associated with PD-L1 expression and metabolic reprogramming. An anti-PD-1 neutralizing antibody is sufficient to compromise the immunosuppressive effects of P2RX1-deficient neutrophils on OVA-activated OT1 CD8+ T cells. Therefore, our study uncovers a mechanism by which metastatic PDAC tumors evade antitumor immunity by accumulating a subset of immunosuppressive P2RX1-negative neutrophils.

P2X receptor agonist enhances tumor-specific CTL responses through CD70+ DC-mediated Th17 induction.

Extracellular ATP is known to promote Th17 cell differentiation in the intestinal lamina propria by stimulating CD70+CD11clow dendritic cells (DCs) via P2X receptors (P2XRs). Recent studies have also shown that Th17 cells enhance antitumor immunity by directly promoting proliferation of cytotoxic T lymphocytes (CTLs). These finding led us to test a P2XR agonist, αß-methylene ATP (αß-ATP), as a mucosal vaccine adjuvant to promote CTL responses through Th17 induction. We demonstrated that (i) CD70+CD11clow DCs were present in the nasal lamina propria and expressed P2X1R, P2X2R and P2X4R; (ii) CD70+CD11clow DCs isolated from the nasal lamina propria enhanced Th17 cell differentiation of cocultured splenic CD4+ T cells upon stimulation with αß-ATP; (iii) mice intranasally immunized with ovalbumin (OVA) and αß-ATP had increased OVA-specific Th17 cells and CTLs in the nasal lamina propria and regional lymph nodes; (iv) mice intranasally immunized with OVA and αß-ATP also had elevated resistance to E.G7-OVA tumor growth compared with those intranasally immunized with OVA alone; (v) suramin, a broad-range inhibitor of P2 receptors, suppressed the increases of OVA-specific Th17 cells and CTLs in mice intranasally immunized with OVA and αß-ATP; and (vi) suramin also abrogated the enhanced antitumor immunity of mice intranasally immunized with OVA and αß-ATP against E.G7-OVA. Collectively, αß-ATP may be a promising mucosal adjuvant that promotes antigen-specific CTL responses via CD70+CD11clow DC-mediated Th17 induction.

P2X receptors in cancer growth and progression.

It is increasingly appreciated that ion channels have a crucial role in tumors, either as promoters of cancer cell growth, or modulators of immune cell functions, or both. Among ion channels, P2X receptors have a special status because they are gated by ATP, a common and abundant component of the tumor microenvironment. Furthermore, one P2X receptor, i.e. P2X7, may also function as a conduit for ATP release, thus fuelling the increased extracellular ATP level in the tumor interstitium. These findings show that P2X receptors and extracellular ATP are indissoluble partners and key regulators of tumor growth, and suggest the exploitation of the extracellular ATP-P2X partnership to develop innovative therapeutic approaches to cancer.

The Danger Signal Extracellular ATP Is Involved in the Immunomediated Damage of α-Sarcoglycan-Deficient Muscular Dystrophy.

In muscular dystrophies, muscle membrane fragility results in a tissue-specific increase of danger-associated molecular pattern molecules (DAMPs) and infiltration of inflammatory cells. The DAMP extracellular ATP (eATP) released by dying myofibers steadily activates muscle and immune purinergic receptors exerting dual negative effects: a direct damage linked to altered intracellular calcium homeostasis in muscle cells and an indirect toxicity through the triggering of the immune response and inhibition of regulatory T cells. Accordingly, pharmacologic and genetic inhibition of eATP signaling improves the phenotype in models of chronic inflammatory diseases. In α-sarcoglycanopathy, eATP effects may be further amplified because α-sarcoglycan extracellular domain binds eATP and displays an ecto-ATPase activity, thus controlling eATP concentration at the cell surface and attenuating the magnitude and/or the duration of eATP-induced signals. Herein, we show that in vivo blockade of the eATP/P2X purinergic pathway by a broad-spectrum P2X receptor-antagonist delayed the progression of the dystrophic phenotype in α-sarcoglycan-null mice. eATP blockade dampened the muscular inflammatory response and enhanced the recruitment of forkhead box protein P3-positive immunosuppressive regulatory CD4+ T cells. The improvement of the inflammatory features was associated with increased strength, reduced necrosis, and limited expression of profibrotic factors, suggesting that pharmacologic purinergic antagonism, altering the innate and adaptive immune component in muscle infiltrates, might provide a therapeutic approach to slow disease progression in α-sarcoglycanopathy.

Mitochondria regulate neutrophil activation by generating ATP for autocrine purinergic signaling.

Polymorphonuclear neutrophils (PMNs) form the first line of defense against invading microorganisms. We have shown previously that ATP release and autocrine purinergic signaling via P2Y2 receptors are essential for PMN activation. Here we show that mitochondria provide the ATP that initiates PMN activation. Stimulation of formyl peptide receptors increases the mitochondrial membrane potential (Δψm) and triggers a rapid burst of ATP release from PMNs. This burst of ATP release can be blocked by inhibitors of mitochondrial ATP production and requires an initial formyl peptide receptor-induced Ca(2+) signal that triggers mitochondrial activation. The burst of ATP release generated by the mitochondria fuels a first phase of purinergic signaling that boosts Ca(2+) signaling, amplifies mitochondrial ATP production, and initiates functional PMN responses. Cells then switch to glycolytic ATP production, which fuels a second round of purinergic signaling that sustains Ca(2+) signaling via P2X receptor-mediated Ca(2+) influx and maintains functional PMN responses such as oxidative burst, degranulation, and phagocytosis.

Transcript analysis of P2X receptors in PBMCs of chronic HCV patients: an insight into antiviral treatment response and HCV-induced pathogenesis.

BACKGROUND: After invasion of hepatocytes and immune cells, hepatitis C virus has the ability to escape from the host immune system, leading to the progression of disease into chronic infection with associated liver morbidities. Adenosine 5'triphosphate (ATP) is released in most of the pathological events from the affected cells and acts as a signaling molecule by binding to P2X receptors expressed on the host's immune cells and activates the immune system for pro-inflammatory response. Therefore, the present study was designed to analyze the transcript expression of the ionotropic purinergic P2X receptors on peripheral blood mononuclear cells (PBMCs) of chronic HCV patients to have study the immune responses mediated by P2X receptors in chronic HCV infections. METHODS: PBMCs were isolated from the collected blood samples. Transcript analysis of P2X receptors in PBMCs was done. The identity of amplified product was confirmed by sequencing PCR, while the quantification of the transcript expression was done by real time PCR. The relative expression of the P2X receptors was analyzed by unpaired Student's t test using GraphPad Prims 5 software. RESULTS: We found that out of seven isoforms of P2X receptors, P2X1, P2X4, P2X5, and P2X7 receptors are expressed on the PBMCs. P2X1 and P2X7 are significantly upregulated in treatment-naïve chronic HCV patients by 2.2- and 2.5-fold, respectively. However, only P2X7 expression is found increased by 2.7-fold in patients achieving sustained virological response (SVR) after antiviral treatment compared to healthy controls. The expression of P2X receptors remained unaltered in chronic HCV patients not responding to the treatment. CONCLUSION: The present study confirms the significant involvement of P2X receptors in the immune responses mediated by the PBMCs in the chronic HCV infection, which should be further investigated to devise strategies to augment the immune system against this chronic viral disease.

Immunoregulation through extracellular nucleotides.

Extracellular ATP (eATP), the most abundant among nucleotides, can act as a mediator during inflammatory responses by binding to plasmamembrane P2 purinergic receptors, which are widely expressed on cells of the immune system. eATP is generally considered as a classical danger signal, which stimulates immune responses in the presence of tissue damage. Converging evidence from several studies using murine models of chronic inflammation have supported this hypothesis; however, the role of eATP in the regulation of human immune function appears to be more complex. Chronic stimulation with micromolar eATP concentrations inhibits the proliferation of T and NK lymphocytes and enhances the capacity of dendritic cells to promote tolerance. The effect of eATP depends on multiple factors, such as the extent of stimulation, eATP concentration, presence/absence of other mediators in the microenvironment, and pattern of P2 receptor engagement. Small but significant differences in the pattern of P2 receptor expression in mice and humans confer the diverse capacities of ATP in regulating the immune response. Such diversity, which is often overlooked, should therefore be carefully considered when evaluating the role of eATP in human inflammatory and autoimmune diseases.

Purinergic receptor agonists modulate phagocytosis and clearance of apoptotic cells in macrophages.

Phagocytosis plays an important role in controlling inflammation and antigen cross-presentation through the uptake of apoptotic bodies from dying cells. As dying cells are known to release nucleotides and other "danger signals", we investigated whether extracellular nucleotides may affect phagocytosis through binding to P2 purinergic receptors on phagocytic cells. We here show that the purinergic receptor agonists, ATP, ADP, α,ß-methylene ATP (α,ß-meATP), 3'-O-(4-benzoyl)benzoyl ATP, UTP and UDP, increased phagocytosis of latex beads, and some of them increased endocytosis and/or macropinocytosis of dextran by macrophages. The enhanced phagocytosis could be inhibited by pre-treatment with the P2X and P2Y antagonists, pyridoxal-phosphate-6-azophenyl-2',4'-disulphonic acid and suramin, and the P2Y1-selective antagonist, MRS2179. The nucleotides induced upregulation in macrophages of the ß2 integrin CD11b/CD18 (Mac-1) and the vitronectin receptor (α(v)ß3, CD51/CD61), both of which are involved in recognition and internalization of apoptotic cells. In addition, ATP and α,ß-meATP increased adhesion of apoptotic cells to macrophages, both in vitro and in vivo, and α,ß-meATP had a small effect on adhesion of necrotic cells. The nucleotides had no effect on adhesion of viable cells. We propose that engagement of the P2 receptors (P2X1, or P2X3) by extracellular nucleotides released from dying cells increases the ability of macrophages to bind apoptotic bodies, thus enhancing their ability to internalize and present antigens from the dying cells.

ATP secreted by endothelial cells blocks CX3CL 1-elicited natural killer cell chemotaxis and cytotoxicity via P2Y11 receptor activation.

Endothelial cells (ECs) represent a major source of actively secreted adenosine triphosphate (ATP). Natural killer (NK) cells can mediate vascular injury in several pathologic conditions, including cytomegalovirus infection and vascular leak syndrome. We studied NK-cell expression of P2 receptors and the role of these nucleotide receptors in the regulation of endothelial-NK cell cross-talk. NK cells from healthy subjects expressed P2Y(1,2,4,6,11,12,13,14) and P2X(1,4,5,6,7) receptors. NK cells stimulated with ATP, but not uridine triphosphate, increased intracellular Ca²(+) and chemokinesis. Moreover, ATP, but not uridine triphosphate, inhibited NK chemotaxis in response to CX3CL1, whereas chemotaxis to CXCL12 was increased. CX3CL1 elicited killing of human umbilical vein ECs and human coronary artery ECs by NK cells. However, in the presence of ATP, CX3CL1 failed to stimulate killing of ECs. Such inhibitory effect was lost on exogenous addition of the ATP-hydrolyzing enzyme apyrase or by pharmacologic inhibition of the P2Y11R, and correlated with increased intracellular cyclic adenosine monophosphate concentrations induced by ATP or other P2Y11R agonists, including NAD(+). Extracellular ATP regulates NK-cell cytotoxicity via P2Y11R activation, protecting ECs from CX3CL1-elicited NK cell-mediated killing. These findings point out the P2Y11R as a potential target for pharmacologic intervention aimed at reducing NK-mediated vascular injury.