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Journal of Neuroinflammation
Published in: Journal of Neuroinflammation 1/2018

Open Access 01-12-2018 | Research

A central role for P2X7 receptors in human microglia

Authors: Laura Janks, Cristian V. R. Sharma, Terrance M. Egan

Published in: Journal of Neuroinflammation | Issue 1/2018

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Abstract

Background

The ATP-gated ionotropic P2X7 receptor (P2X7R) has the unusual ability to function as a small cation channel and a trigger for permeabilization of plasmalemmal membranes. In murine microglia, P2X7R-mediated permeabilization is fundamental to microglial activation, proliferation, and IL-1β release. However, the role of the P2X7R in primary adult human microglia is poorly understood.

Methods

We used patch-clamp electrophysiology to record ATP-gated current in cultured primary human microglia; confocal microscopy to measure membrane blebbing; fluorescence microscopy to demonstrate membrane permeabilization, caspase-1 activation, phosphatidylserine translocation, and phagocytosis; and kit-based assays to measure cytokine levels.

Results

We found that ATP-gated inward currents facilitated with repetitive applications of ATP as expected for current through P2X7Rs and that P2X7R antagonists inhibited these currents. P2X7R antagonists also prevented the ATP-induced uptake of large cationic fluorescent dyes whereas drugs that target pannexin-1 channels had no effect. In contrast, ATP did not induce uptake of anionic dyes. The uptake of cationic dyes was blocked by drugs that target Cl− channels. Finally, we found that ATP activates caspase-1 and inhibits phagocytosis, and these effects are blocked by both P2X7R and Cl− channel antagonists.

Conclusions

Our results demonstrate that primary human microglia in culture express functional P2X7Rs that stimulate both ATP-gated cationic currents and uptake of large molecular weight cationic dyes. Importantly, our data demonstrate that hypotheses drawn from work on murine immune cells accurately predict the essential role of P2X7Rs in a number of human innate immune functions such as phagocytosis and caspase-1 activation. Therefore, the P2X7R represents an attractive target for therapeutic intervention in human neuroinflammatory disorders.
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Literature
1.
2.
Arcuri C, Mecca C, Bianchi R, Giambanco I, Donato R. The pathophysiological role of microglia in dynamic surveillance, phagocytosis and structural remodeling of the developing CNS. Front Mol Neurosci. 2017;10:191.PubMedPubMedCentralCrossRef
3.
4.
He J, Liao T, Zhong GX, Zhang JD, Chen YP, Wang Q, Zeng QP. Alzheimer’s disease-like early-phase brain pathogenesis: self-curing amelioration of neurodegeneration from pro-inflammatory ‘wounding’ to anti-inflammatory ‘healing’. Curr Alzheimer Res. 2017;14:1123–35.PubMedCrossRef
5.
Ransohoff RM, Schafer D, Vincent A, Blachere NE, Bar-Or A. Neuroinflammation: ways in which the immune system affects the brain. Neurotherapeutics. 2015;12:896–909.PubMedPubMedCentralCrossRef
6.
Perry VH, Teeling J. Microglia and macrophages of the central nervous system: the contribution of microglia priming and systemic inflammation to chronic neurodegeneration. Semin Immunopathol. 2013;35:601–12.PubMedPubMedCentralCrossRef
7.
8.
Burnstock G. Physiology and pathophysiology of purinergic neurotransmission. Physiol Rev. 2007;87:659–797.PubMedCrossRef
9.
Trautmann A. Extracellular ATP in the immune system: more than just a “danger signal”. Sci Signal. 2009;2:pe6.PubMedCrossRef
10.
Ogura Y, Sutterwala FS, Flavell RA. The inflammasome: first line of the immune response to cell stress. Cell. 2006;126:659–62.PubMedCrossRef
11.
Sutterwala FS, Ogura Y, Szczepanik M, Lara-Tejero M, Lichtenberger GS, Grant EP, Bertin J, Coyle AJ, Galan JE, Askenase PW, Flavell RA. Critical role for NALP3/CIAS1/cryopyrin in innate and adaptive immunity through its regulation of caspase-1. Immunity. 2006;24:317–27.PubMedCrossRef
12.
Di Virgilio F. Liaisons dangereuses: P2X(7) and the inflammasome. Trends Pharmacol Sci. 2007;28:465–72.PubMedCrossRef
13.
14.
Di Virgilio F, Vuerich M. Purinergic signaling in the immune system. Auton Neurosci. 2015;191:117–23.PubMedCrossRef
15.
16.
17.
Gicquel T, Robert S, Loyer P, Victoni T, Bodin A, Ribault C, Gleonnec F, Couillin I, Boichot E, Lagente V. IL-1β production is dependent of the activation of purinergic receptors and NLRP3 pathway in human macrophages. FASEB J. 2015;29:4162–73.PubMedCrossRef
18.
Monif M, Reid CA, Powell KL, Smart ML, Williams DA. The P2X7 receptor drives microglial activation and proliferation: a trophic role for P2X7R pore. J Neurosci. 2009;29:3781–91.PubMedCrossRefPubMedCentral
19.
Ni J, Wang P, Zhang J, Chen W, Gu L. Silencing of the P2X(7) receptor enhances amyloid-beta phagocytosis by microglia. Biochem Biophys Res Commun. 2013;434:363–9.PubMedCrossRef
20.
21.
22.
Suzuki T, Hide I, Ido K, Kohsaka S, Inoue K, Nakata Y. Production and release of neuroprotective tumor necrosis factor by P2X7 receptor-activated microglia. J Neurosci. 2004;24:1–7.PubMedCrossRefPubMedCentral
23.
Shieh CH, Heinrich A, Serchov T, van Calker D, Biber K. P2X7-dependent, but differentially regulated release of IL-6, CCL2, and TNF-alpha in cultured mouse microglia. Glia. 2014;62:592–607.PubMedCrossRef
24.
Murphy N, Lynch MA. Activation of the P2X(7) receptor induces migration of glial cells by inducing cathepsin B degradation of tissue inhibitor of metalloproteinase 1. J Neurochem. 2012;123:761–70.PubMedCrossRef
25.
Bartlett R, Yerbury JJ, Sluyter R. P2X7 receptor activation induces reactive oxygen species formation and cell death in murine EOC13 microglia. Mediat Inflamm. 2013;2013:271813.CrossRef
26.
He Y, Taylor N, Fourgeaud L, Bhattacharya A. The role of microglial P2X7: modulation of cell death and cytokine release. J Neuroinflammation. 2017;14:135.PubMedPubMedCentralCrossRef
27.
Roger S, Pelegrin P, Surprenant A. Facilitation of P2X7 receptor currents and membrane blebbing via constitutive and dynamic calmodulin binding. J Neurosci. 2008;28:6393–401.PubMedCrossRefPubMedCentral
28.
Yan Z, Khadra A, Li S, Tomic M, Sherman A, Stojilkovic SS. Experimental characterization and mathematical modeling of P2X7 receptor channel gating. J Neurosci. 2010;30:14213–24.PubMedPubMedCentralCrossRef
29.
Edelstein A, Amodaj N, Hoover K, Vale R, Stuurman N. Computer control of microscopes using microManager. Curr Protoc Mol Biol. 2010;Chapter 14:Unit14–20.PubMed
30.
31.
Crain JM, Nikodemova M, Watters JJ. Expression of P2 nucleotide receptors varies with age and sex in murine brain microglia. J Neuroinflammation. 2009;6:24.PubMedPubMedCentralCrossRef
32.
33.
34.
Rassendren F, Buell GN, Virginio C, Collo G, North RA, Surprenant A. The permeabilizing ATP receptor, P2X7. Cloning and expression of a human cDNA. J Biol Chem. 1997;272:5482–6.PubMedCrossRef
35.
Liang X, Samways DS, Wolf K, Bowles EA, Richards JP, Bruno J, Dutertre S, DiPaolo RJ, Egan TM. Quantifying Ca2+ current and permeability in ATP-gated P2X7 receptors. J Biol Chem. 2015;290:7930–42.PubMedCrossRef
36.
Nelson DW, Gregg RJ, Kort ME, Perez-Medrano A, Voight EA, Wang Y, Grayson G, Namovic MT, Donnelly-Roberts DL, Niforatos W, et al. Structure-activity relationship studies on a series of novel, substituted 1-benzyl-5-phenyltetrazole P2X7 antagonists. J Med Chem. 2006;49:3659–66.PubMedCrossRef
37.
Donnelly-Roberts DL, Namovic MT, Surber B, Vaidyanathan SX, Perez-Medrano A, Wang Y, Carroll WA, Jarvis MF. [3H]A-804598 ([3H]2-cyano-1-[(1S)-1-phenylethyl]-3-quinolin-5-ylguanidine) is a novel, potent, and selective antagonist radioligand for P2X7 receptors. Neuropharmacology. 2009;56:223–9.PubMedCrossRef
38.
Ase AR, Honson NS, Zaghdane H, Pfeifer TA, Seguela P. Identification and characterization of a selective allosteric antagonist of human P2X4 receptor channels. Mol Pharmacol. 2015;87:606–16.PubMedCrossRef
39.
Khakh BS, Proctor WR, Dunwiddie TV, Labarca C, Lester HA. Allosteric control of gating and kinetics at P2X(4) receptor channels. J Neurosci. 1999;19:7289–99.PubMedCrossRefPubMedCentral
40.
Norenberg W, Sobottka H, Hempel C, Plotz T, Fischer W, Schmalzing G, Schaefer M. Positive allosteric modulation by ivermectin of human but not murine P2X7 receptors. Br J Pharmacol. 2012;167:48–66.PubMedPubMedCentralCrossRef
41.
Charles AC, Brennan KC. Calcium homeostasis in glia. San Diego: Academic Press; 2009.CrossRef
42.
Raouf R, Chabot-Dore AJ, Ase AR, Blais D, Seguela P. Differential regulation of microglial P2X4 and P2X7 ATP receptors following LPS-induced activation. Neuropharmacology. 2007;53:496–504.PubMedCrossRef
43.
Toulme E, Garcia A, Samways D, Egan TM, Carson MJ, Khakh BS. P2X4 receptors in activated C8-B4 cells of cerebellar microglial origin. J Gen Physiol. 2010;135:333–53.PubMedPubMedCentralCrossRef
44.
Shi J, Zhao Y, Wang Y, Gao W, Ding J, Li P, Hu L, Shao F. Inflammatory caspases are innate immune receptors for intracellular LPS. Nature. 2014;514:187–92.CrossRefPubMed
45.
Kim M, Jiang LH, Wilson HL, North RA, Surprenant A. Proteomic and functional evidence for a P2X(7) receptor signalling complex. EMBO J. 2001;20:6347–58.PubMedPubMedCentralCrossRef
46.
Gu BJ, Saunders BM, Petrou S, Wiley JS. P2X(7) is a scavenger receptor for apoptotic cells in the absence of its ligand, extracellular ATP. J Immunol. 2011;187:2365–75.PubMedCrossRef
47.
Gu BJ, Saunders BM, Jursik C, Wiley JS. The P2X7-nonmuscle myosin membrane complex regulates phagocytosis of nonopsonized particles and bacteria by a pathway attenuated by extracellular ATP. Blood. 2010;115:1621–31.PubMedCrossRef
48.
Gu BJ, Rathsam C, Stokes L, McGeachie AB, Wiley JS. Extracellular ATP dissociates nonmuscle myosin from P2X(7) complex: this dissociation regulates P2X(7) pore formation. Am J Phys Cell Phys. 2009;297:C430–9.CrossRef
49.
Sluyter R, Shemon AN, Wiley JS. Glu496 to Ala polymorphism in the P2X7 receptor impairs ATP-induced IL-1 beta release from human monocytes. J Immunol. 2004;172:3399–405.PubMedCrossRef
50.
Sluyter R, Dalitz JG, Wiley JS. P2X7 receptor polymorphism impairs extracellular adenosine 5′-triphosphate-induced interleukin-18 release from human monocytes. Genes Immun. 2004;5:588–91.PubMedCrossRef
51.
Ou A, Gu BJ, Wiley JS. The scavenger activity of the human P2X7 receptor differs from P2X7 pore function by insensitivity to antagonists, genetic variation and sodium concentration: relevance to inflammatory brain diseases. Biochim Biophys Acta. 2018;1864:1051–9.CrossRef
52.
Bianco F, Pravettoni E, Colombo A, Schenk U, Moller T, Matteoli M, Verderio C. Astrocyte-derived ATP induces vesicle shedding and IL-1 beta release from microglia. J Immunol. 2005;174:7268–77.PubMedCrossRef
53.
Wei L, Caseley E, Li D, Jiang LH. ATP-induced P2X receptor-dependent large pore formation: how much do we know? Front Pharmacol. 2016;7:5.PubMedPubMedCentral
54.
Adinolfi E, Giuliani AL, De Marchi E, Pegoraro A, Orioli E, Di Virgilio F. The P2X7 receptor: a main player in inflammation. Biochem Pharmacol. 2017;151:234–244.PubMedCrossRef
55.
Cankurtaran-Sayar S, Sayar K, Ugur M. P2X7 receptor activates multiple selective dye-permeation pathways in RAW 264.7 and human embryonic kidney 293 cells. Mol Pharmacol. 2009;76:1323–32.PubMedCrossRef
56.
Schachter J, Motta AP, de Souza Zamorano A, da Silva-Souza HA, Guimaraes MZ, Persechini PM. ATP-induced P2X7-associated uptake of large molecules involves distinct mechanisms for cations and anions in macrophages. J Cell Sci. 2008;121:3261–70.PubMedCrossRef
57.
Steinberg TH, Newman AS, Swanson JA, Silverstein SC. ATP4- permeabilizes the plasma membrane of mouse macrophages to fluorescent dyes. J Biol Chem. 1987;262:8884–8.PubMed
58.
59.
Schilling WP, Wasylyna T, Dubyak GR, Humphreys BD, Sinkins WG. Maitotoxin and P2Z/P2X(7) purinergic receptor stimulation activate a common cytolytic pore. Am J Phys. 1999;277:C766–76.CrossRef
60.
Faria RX, Defarias FP, Alves LA. Are second messengers crucial for opening the pore associated with P2X7 receptor? Am J Phys Cell Phys. 2005;288:C260–71.CrossRef
61.
Jiang LH, Rassendren F, Mackenzie A, Zhang YH, Surprenant A, North RA. N-methyl-D-glucamine and propidium dyes utilize different permeation pathways at rat P2X(7) receptors. Am J Phys Cell Phys. 2005;289:C1295–302.CrossRef
62.
Locovei S, Scemes E, Qiu F, Spray DC, Dahl G. Pannexin1 is part of the pore forming unit of the P2X(7) receptor death complex. FEBS Lett. 2007;581:483–8.PubMedPubMedCentralCrossRef
63.
Bernier LP, Ase AR, Boue-Grabot E, Seguela P. P2X4 receptor channels form large noncytolytic pores in resting and activated microglia. Glia. 2012;60:728–37.PubMedCrossRef
64.
Pelegrin P, Surprenant A. The P2X(7) receptor-pannexin connection to dye uptake and IL-1beta release. Purinergic Signal. 2009;5.2:129–137.
65.
Ousingsawat J, Wanitchakool P, Kmit A, Romao AM, Jantarajit W, Schreiber R, Kunzelmann K. Anoctamin 6 mediates effects essential for innate immunity downstream of P2X7 receptors in macrophages. Nat Commun. 2015;6:6245.PubMedCrossRef
66.
Yaron JR, Gangaraju S, Rao MY, Kong X, Zhang L, Su F, Tian Y, Glenn HL, Meldrum DR. K(+) regulates Ca(2+) to drive inflammasome signaling: dynamic visualization of ion flux in live cells. Cell Death Dis. 2015;6:e1954.PubMedPubMedCentralCrossRef
67.
Stokes L, Layhadi JA, Bibic L, Dhuna K, Fountain SJ. P2X4 receptor function in the nervous system and current breakthroughs in pharmacology. Front Pharmacol. 2017;8:291.
68.
69.
Bohlen CJ, Bennett FC, Tucker AF, Collins HY, Mulinyawe SB, Barres BA. Diverse requirements for microglial survival, specification, and function revealed by defined-medium cultures. Neuron. 2017;94:759–773.e8.PubMedPubMedCentralCrossRef
70.
Gosselin D, Skola D, Coufal NG, Holtman IR, Schlachetzki JCM, Sajti E, Jaeger BN, O'Connor C, Fitzpatrick C, Pasillas MP, et al. An environment-dependent transcriptional network specifies human microglia identity. Science. 2017;356.6344:eaal3222.PubMedPubMedCentralCrossRef
71.
Mizee MR, Miedema SS, van der Poel M, Adelia, Schuurman KG, van Strien ME, Melief J, Smolders J, Hendrickx DA, Heutinck KM, et al. Isolation of primary microglia from the human post-mortem brain: effects of ante- and post-mortem variables. Acta Neuropathol Commun. 2017;5:16.PubMedPubMedCentralCrossRef
72.
73.
Di Virgilio F, Schmalzing G, Markwardt F. The elusive P2X7 macropore. Trends Cell Biol. 2018;28.5:392-404.
74.
Migita K, Haines WR, Voigt MM, Egan TM. Polar residues of the second transmembrane domain influence cation permeability of the ATP-gated P2X(2) receptor. J Biol Chem. 2001;276:30934–41.PubMedCrossRef
75.
Harkat M, Peverini L, Cerdan AH, Dunning K, Beudez J, Martz A, Calimet N, Specht A, Cecchini M, Chataigneau T, Grutter T. On the permeation of large organic cations through the pore of ATP-gated P2X receptors. Proc Natl Acad Sci U S A. 2017;114:E3786–95.PubMedPubMedCentralCrossRef
76.
Karasawa A, Michalski K, Mikhelzon P, Kawate T. The P2X7 receptor forms a dye-permeable pore independent of its intracellular domain but dependent on membrane lipid composition. Elife. 2017;6:e31186.
77.
Li M, Toombes GE, Silberberg SD, Swartz KJ. Physical basis of apparent pore dilation of ATP-activated P2X receptor channels. Nat Neurosci. 2015;18:1577–83.PubMedPubMedCentralCrossRef
78.
Pelegrin P, Surprenant A. Pannexin-1 mediates large pore formation and interleukin-1beta release by the ATP-gated P2X7 receptor. EMBO J. 2006;25:5071–82.PubMedPubMedCentralCrossRef
79.
Domercq M, Vazquez-Villoldo N, Matute C. Neurotransmitter signaling in the pathophysiology of microglia. Front Cell Neurosci. 2013;7:49.PubMedPubMedCentral
80.
El Ouaaliti M, Seil M, Dehaye JP. Activation of calcium-insensitive phospholipase A(2) (iPLA(2)) by P2X(7) receptors in murine peritoneal macrophages. Prostaglandins Other Lipid Mediat. 2012;99:116–23.PubMedCrossRef
81.
Schreiber R, Ousingsawat J, Wanitchakool P, Sirianant L, Benedetto R, Reiss K, Kunzelmann K. Regulation of TMEM16A/ANO1 and TMEM16F/ANO6 ion currents and phospholipid scrambling by Ca(2+) and plasma membrane lipid. J Physiol. 2018;596:217–29.PubMedCrossRef
82.
Wiley JS, Gu BJ. A new role for the P2X7 receptor: a scavenger receptor for bacteria and apoptotic cells in the absence of serum and extracellular ATP. Purinergic Signal. 2012;8:579–86.PubMedPubMedCentralCrossRef
83.
84.
85.
Puren AJ, Fantuzzi G, Dinarello CA. Gene expression, synthesis, and secretion of interleukin 18 and interleukin 1beta are differentially regulated in human blood mononuclear cells and mouse spleen cells. Proc Natl Acad Sci U S A. 1999;96:2256–61.PubMedPubMedCentralCrossRef
86.
Aglietti RA, Dueber EC. Recent insights into the molecular mechanisms underlying pyroptosis and gasdermin family functions. Trends Immunol. 2017;38:261–71.PubMedCrossRef
87.
Monif M, Reid CA, Powell KL, Drummond KJ, O'Brien TJ, Williams DA. Interleukin-1beta has trophic effects in microglia and its release is mediated by P2X7R pore. J Neuroinflammation. 2016;13:173.PubMedPubMedCentralCrossRef
Metadata
Title
A central role for P2X7 receptors in human microglia
Authors
Laura Janks
Cristian V. R. Sharma
Terrance M. Egan
Publication date
01-12-2018
Publisher
BioMed Central
Published in
Journal of Neuroinflammation / Issue 1/2018
Electronic ISSN: 1742-2094
DOI
https://doi.org/10.1186/s12974-018-1353-8

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