Activation of the damage-associated molecular pattern receptor P2X7 induces interleukin-1B release from canine monocytes

P2X7, a damage-associated molecular pattern receptor and adenosine 5\u27-triphosphate (ATP)-gated cation channel, plays an important role in the activation of the NALP3 inflammasome and subsequent release of interleukin (IL)-1β from human monocytes; however its role in monocytes from other species including the dog remains poorly defined. This study investigated the role of P2X7 in canine monocytes, including its role in IL-1β release. A fixed-time flow cytometric assay demonstrated that activation of P2X7 by extracellular ATP induces the uptake of the organic cation, YO-PRO-12+, into peripheral blood monocytes from various dog breeds, a process impaired by the specific P2X7 antagonist, A438079. Moreover, in five different breeds, relative P2X7 function in monocytes was about half that of peripheral blood T cells but similar to that of peripheral blood B cells. Reverse transcription-PCR demonstrated the presence of P2X7, NALP3, caspase-1 and IL-1β in LPSprimed canine monocytes. Immunoblotting confirmed the presence of P2X7 in LPS-primed canine monocytes. Finally, extracellular ATP induced YO-PRO-12+ uptake into and IL-1β release from these cells, with both processes impaired by A438079. These results demonstrate that P2X7 activation induces the uptake of organic cations into and the release of IL-1β from canine monocytes. These findings indicate that P2X7 may play an important role in IL-1β-dependent processes in dogs

R270C polymorphism leads to loss of function of the canine P2X7 receptor

The relative function of the P2X7 receptor, an ATP-gated ion channel, varies between humans due to polymorphisms in the P2RX7 gene. This study aimed to assess the functional impact of P2X7 variation in a random sample of the canine population. Blood and genomic DNA were obtained from 69 dogs selected as representatives of a cross section of different breeds. P2X7 function was determined by flow cytometric measurements of dye uptake and patch-clamp measurements of inward currents. P2X7 expression was determined by immunoblotting and immunocytochemistry. Sequencing was used to identify P2RX7 gene polymorphisms. P2X7 was cloned from an English springer spaniel, and point mutations were introduced into this receptor by site-directed mutagenesis. The relative function of P2X7 on monocytes varied between individual dogs. The canine P2RX7 gene encoded four missense polymorphisms: F103L and P452S, found in heterozygous and homozygous dosage, and R270C and R365Q, found only in heterozygous dosage. Moreover, R270C and R365Q were associated with the cocker spaniel and Labrador retriever, respectively. F103L, R270C, and R365Q but not P452S corresponded to decreased P2X7 function in monocytes but did not explain the majority of differences in P2X7 function between dogs, indicating that other factors contribute to this variability. Heterologous expression of site-directed mutants of P2X7 in human embryonic kidney-293 cells indicated that the R270C mutant was nonfunctional, the F103L and R365Q mutants had partly reduced function, and the P452S mutant functioned normally. Taken together, these data highlight that a R270C polymorphism has major functional impact on canine P2X7

Probenecid Blocks Human P2X7 Receptor-Induced Dye Uptake via a Pannexin-1 Independent Mechanism

P2X7 is a ligand-gated ion channel which is activated by ATP and displays secondary permeability characteristics. The mechanism of development of the secondary permeability pathway is currently unclear, although a role for the hemichannel protein pannexin-1 has been suggested. In this study we investigated the role of pannexin-1 in P2X7-induced dye uptake and ATP-induced IL-1β secretion from human monocytes. We found no pharmacological evidence for involvement of pannexin-1 in P2X7-mediated dye uptake in transfected HEK-293 cells with no inhibition seen for carbenoxolone and the pannexin-1 mimetic inhibitory peptide, 10Panx1. However, we found that probenecid inhibited P2X7-induced cationic and anionic dye uptake in stably transfected human P2X7 HEK-293 cells. An IC50 value of 203 μM was calculated for blockade of ATP-induced responses at human P2X7. Probenecid also reduced dye uptake and IL-1β secretion from human CD14+ monocytes whereas carbenoxolone and 10Panx1 showed no inhibitory effect. Patch clamp and calcium indicator experiments revealed that probenecid directly blocks the human P2X7 receptor

The P2X7 purinergic receptor in dogs

The P2X7 receptor is an adenosine 5’-triphosphate (ATP)-gated ion channel expressed on the cell-surface of many cell types, including cells of haematopoietic origin and epithelial cells. Upon prolonged activation by extracellular ATP, P2X7 has the ability to form pores permeable to large organic cations such as ethidium+ and YO-PRO-12+. P2X7 activation leads to the stimulation of the NALP3 inflammasome and the subsequent release of the proinflammatory cytokines interleukin (IL)-1β and IL-18. The first aim of this study was to confirm the presence or absence of P2X7 and NALP3-related components in Madin-Darby canine kidney (MDCK) epithelial cells and lipopolysaccharide (LPS)-primed canine monocytes. Reverse transcriptase (RT)-PCR detected the presence of P2X7, NALP3, caspase-1, IL-1β and IL-18 mRNA in MDCK cells and monocytes. The identity of each transcript, except IL-1β due to its small size, was confirmed by sequencing. This data suggests that MDCK cells will provide a useful model cell line to study the role of P2X7 and NALP3-related components in kidney epithelial cells and renal disorders. Moreover, these results indirectly support our previous observations that P2X7 activation induces IL-1β from LPS-primed canine monocytes. The second aim of this study was to determine if ATP or the Toll-like receptor (TLR) ligands, LPS or lipoteichoic acid (LTA), induce IL-1β release from MDCK cells and in whole canine blood. ELISA measurements failed to detect IL-1β release from MDCK cells despite examining various combinations of ATP, LPS and/or nigericin (which induces IL-1β release independently of P2X7). Further studies are required however to determine whether MDCK cells can release IL-1β. In contrast to MDCK cells, LPS and ATP but not LTA induced IL-1β release in whole blood. Moreover, ATP-induced IL-1β release in whole blood required priming with LPS. A role for P2X7 in LPS- and ATP-induced IL-1β release in blood however could not be established, as the P2X7 antagonist AZ10606120 failed to impair IL-1β release. Further studies are required to determine if either or both of these processes require P2X7 activation. The third aim of this study was to sequence and pharmacologically characterise a recombinant canine P2X7 receptor cloned from an English Springer Spaniel. Sequencing of the cloned receptor revealed two nonsynonymous (missense) single nucleotide polymorphisms (SNPs): Leu440Phe and Pro452Ser. Immunoblotting confirmed the expression of P2X7 protein in canine P2X7-transfected HEK 293 cells. Moreover, flow cytometric measurements demonstrated that ATP induced ethidium+ (314 Da) uptake into P2X7-transfected but not mock-transfected HEK 293 cells in a time-dependent manner. The P2X7 agonists ATP, 3’-O-(4-benzoyl)benzoyl ATP (BzATP) and adenosine 5’-O-(3-thiotriphosphate) (ATPγS) induced ethidium+ uptake into P2X7-transfected HEK 293 cells in a concentration-dependent manner with EC50 values of 253 μM, 13 μM and 438 μM respectively. In contrast, adenosine 5’-diphosphate (ADP), uridine 5’-triphosphate (UTP) and α,β-methylene ATP (α,β-meATP) failed to induce ethidium+ uptake into these cells. The P2X7 antagonists A438079, AZ10606120, AZ11645373, BBG and KN-62 completely impaired ATP-induced ethidium+ uptake into P2X7-transfected HEK 293 cells with IC50 values of 190 nM, 11 nM, 7 nM, 1110 nM and 19 nM respectively. Finally, ATP induced YO-PRO-12+ (375 Da) and propidium2+ (415 Da) uptake into P2X7-transfected cells. Collectively, the recombinant canine P2X7 receptor demonstrated similar characteristics to that of native canine and recombinant human P2X7. Thus, P2X7 drugs developed to block P2X7 in humans may also be of therapeutic value in dogs. The last aim of this thesis was to confirm if the relative monocyte P2X7 function varies between dogs and to determine if this variation is due to SNPs in the P2RX7 gene. Flow cytometric measurements of ATP-induced YO-PRO-12+ uptake into peripheral blood canine monocytes confirmed that the relative P2X7 function varied between dogs and within breeds. Flow cytometric measurements also demonstrated that the relative P2X7 function of human monocytes varies between subjects and over time. Amplification and sequencing of the canine P2RX7 gene of 19 dogs and MDCK cells identified four non-synonymous SNPs: Phe103Leu (exon 3), Arg270Cys (exon 8), Arg365Gln (exon 11), and Pro452Ser (exon 13). The dogs for which we had both functional data and genomic DNA (n = 62) were screened for the above SNPs and the resulting genotypes were compared to relative P2X7 function. Three of the SNPs (Phe103Leu, Arg365Gln and Pro452Ser) did not correspond with a change in P2X7 function. In contrast, the Arg270Cys SNP was associated with a loss-of-function in P2X7, however this finding was restricted to one dog, as well as MDCK cells which have low P2X7 function. Future studies using mutant P2X7 receptors, obtained by site-directed mutagenesis, are required to explore if any of these SNPs alter P2X7 function. This thesis forms a part of an ongoing study investigating the role of canine P2X7 in inflammation and immunity. The confirmation of P2X7 in canine monocytes and MDCK cells, and the identification of SNPs in the canine P2RX7 gene will provide future opportunities to investigate the role of the P2X7 receptor and its gene in canine health and disease

P2X7 receptor activation mediates organic cation uptake into human myeloid leukaemic KG-1 cells

The P2X7 purinergic receptor is an ATP-gated cation channel with an emerging role in neoplasia. In this study we demonstrate that the human KG-1 cell line, a model of acute myelogenous leukaemia, expresses functional P2X7. RT-PCR and immunochemical techniques demonstrated the presence of P2X7 mRNA and protein respectively in KG-l cells, as well as in positive control multiple myeloma RPMI 8226 cells. Flow cytometric measurements demonstrated that ATP induced ethidium(+) uptake into KG-l cells suspended in sucrose medium (EC(50) of ∼3 μM), but not into cells in NaCl medium. In contrast, ATP induced ethidium(+) uptake into RPMI 8226 cells suspended in either sucrose or NaCl medium (EC(50) of ∼3 or ∼99 μM, respectively), as well as into RPMI 8226 cells in KCl medium (EC(50) of ∼18 μM). BzATP and to a lesser extent ATPγS and αβ-methylene ATP, but not ADP or UTP, also induced ethidium(+) uptake into KG-1 cells. ATP-induced ethidium(+) uptake was completely impaired by the P2X7 antagonists, AZ10606120 and A-438079. ATP-induced ethidium(+) uptake was also impaired by probenecid but not by carbenoxolone, both pannexin-1 antagonists. ATP induced YO-PRO-1(2+) and propidium(2+) uptake into KG-1 cells. Finally, sequencing of full-length P2X7 cDNA identified several single nucleotide polymorphisms (SNPs) in KG-1 cells including H155Y, A348T, T357S and Q460R. RPMI 8226 cells contained A348T, A433V and H521Q SNPs. In conclusion, the KG-1 cell line expresses functional P2X7. This cell line may help elucidate the signalling pathways involved in P2X7-induced survival and invasiveness of myeloid leukaemic cells

Functional expression of the damage-associated molecular pattern receptor P2X7 on canine kidney epithelial cells

Epithelial cells are important in inflammation and immunity. In this study, we examined if Madin-Darby canine kidney (MDCK) epithelial cells express functional P2X7 receptors, which bind the damage-associated molecular pattern extracellular adenosine 5\u27-triphosphate (ATP). Reverse transcription (RT)-PCR and immunoblotting revealed the expression of P2X7 in MDCK cells. A flow cytometric assay demonstrated that ATP or 2\u27(3\u27)-O-(4-benzoylbenzoyl)ATP induced ethidium(+) uptake into MDCK cells, and that this process was impaired by the P2X7 antagonists KN-62 and A438079. RT-PCR also demonstrated the presence of Toll-like receptor 4, NALP3, caspase-1, interleukin-1β and interleukin-18 in MDCK cells, as well as in positive control LPS-primed canine monocytes. In conclusion, the MDCK epithelial cell line expresses functional P2X7, as well as Toll-like receptor 4 and molecules associated with the NALP3 inflammasome. This cell line may help elucidate the role of these molecules in kidney epithelial cells and renal disorders in dogs and humans

Multi-omics approach reveals dysregulated genes during hESCs neuronal differentiation exposure to paracetamol

Summary: Prenatal paracetamol exposure has been associated with neurodevelopmental outcomes in childhood. Pharmacoepigenetic studies show differences in cord blood DNA methylation between unexposed and paracetamol-exposed neonates, however, causality and impact of long-term prenatal paracetamol exposure on brain development remain unclear. Using a multi-omics approach, we investigated the effects of paracetamol on an in vitro model of early human neurodevelopment. We exposed human embryonic stem cells undergoing neuronal differentiation with paracetamol concentrations corresponding to maternal therapeutic doses. Single-cell RNA-seq and ATAC-seq integration identified paracetamol-induced chromatin opening changes linked to gene expression. Differentially methylated and/or expressed genes were involved in neurotransmission and cell fate determination trajectories. Some genes involved in neuronal injury and development-specific pathways, such as KCNE3, overlapped with differentially methylated genes previously identified in cord blood associated with prenatal paracetamol exposure. Our data suggest that paracetamol may play a causal role in impaired neurodevelopment

Probenecid but not pannexin-1 blockers reduce P2X7-induced IL-1β secretion from human monocytes and J774 macrophages.

<p>Magnetically isolated CD14⁺ human monocytes were primed with LPS (100 ng/ml) for 4 hours and stimulated with 3 mM ATP for 15 minutes. After stimulation supernatants were removed and tested for IL-1β. (A) representative data from one donor, (B) shows pooled data from 6 donors expressed as % of ATP control. Carbenoxolone (CBX) was used at 50 μM, probenecid (PRO) at 1 mM and 2.5 mM, ¹⁰Panx1 at 100 μM, and A-438079 at 10 μM. (C) The J774 mouse macrophage cell line was primed with LPS (1 μg/ml) and stimulated with ATP (5 mM) for 20 minutes in physiological saline. P2X7 selective antagonist AZ10606120 (AZ106) was used at 10 μM. Mean data from three independent experiments is shown. ATP-induced IL-1β released into supernatant was measured by specific ELISAs for human and mouse IL-1β respectively. Error bars represent S.E.M and * represents P<0.05.</p

Probenecid inhibits ATP-induced dye uptake in HEK-hP2X7 cells.

<p>(A) Ethidium⁺ uptake was induced in HEK-hP2X7 expressing cells by the addition of 1 mM ATP (as denoted by the arrow) in low divalent physiological solution. Control ethidium uptake to ATP is shown in black squares and ethidium uptake in the presence of 2.5 mM probenecid (pre-incubated for 10 minutes at 37°C) is shown in open circles. (B) Concentration response curves for probenecid on human P2X7 dye uptake responses induced by 0.2 mM or 1 mM ATP. Data was calculated at 300 second timepoint as baseline corrected endpoint data and normalised to the ATP control. Data was fit using a normalised response variable slope in GraphPad Prism. (C) Concentration response curves for agonist ATP in the presence of increasing concentrations of probenecid (0.5 mM, 1 mM and 5 mM) showing a rightward shift.</p

Probenecid blocks P2X7-mediated calcium influx and inward currents in HEK-hP2X7 cells.

<p>HEK-hP2X7 cells were loaded with 1 μM Fluo-4 for 30 minutes and calcium responses recorded at room temperature (26°C) using a fluorescent plate reader. ATP (1 mM) was injected and fluorescence recorded at 520 nm in the absence of inhibitors or in the presence of 1 mM probenecid (purple) or 10 μM A-438079 (blue). (B) Mean data from three independent calcium experiments. Sustained portion of the calcium response was measured and calculated as % of ATP control. Probenecid reduced response to 63±3.4% of control whereas A-438079 completely abolished the sustained calcium response. (C) Inward currents through wild-type human P2X7 receptors expressed in HEK-293 cells were recorded using whole cell patch clamp at room temperature. Membrane was clamped at −60 mV and ATP (1 mM in low divalent solution) was applied using a fast-flow delivery system. Black bars indicate ATP exposure (5 seconds). The initial ATP response was measured then the cell was exposed to probenecid for 2 minutes before re-challenge with ATP in the continued presence of probenecid.</p