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Journal of Neuroimmune Pharmacology

, Volume 14, Issue 2, pp 263–277 | Cite as

P2X7 Receptor Antagonist A804598 Inhibits Inflammation in Brain and Liver in C57BL/6J Mice Exposed to Chronic Ethanol and High Fat Diet

  • Daniel Freire
  • Rachel E. Reyes
  • Ared Baghram
  • Daryl L. Davies
  • Liana AsatryanEmail author
ORIGINAL ARTICLE
  • 346 Downloads

Abstract

Chronic low-grade neuroinflammation is increasingly implicated in organ damage caused by alcohol abuse. Purinergic P2X7 receptors (P2X7Rs) play an important role in the generation of inflammatory responses during a number of CNS pathologies as evidenced from studies using pharmacological inhibition approach. P2X7Rs antagonism has not been tested during chronic alcohol abuse. In the present study, we tested the potential of P2X7R antagonist A804598 to reduce/abolish alcohol-induced neuroinflammation using chronic intragastric ethanol infusion and high-fat diet (Hybrid) in C57BL/6J mice. We have previously demonstrated an increase in neuroinflammatory response in 8 weeks of Hybrid paradigm. In the present study, we found neuroinflammatory response to 4 weeks of Hybrid exposure. A804598 treatment reversed the changes in microglia and astrocytes, reduced/abolished increases in mRNA levels of number of inflammatory markers, including IL-1β, iNOS, CXCR2, and components of inflammatory signaling pathways, such as TLR2, CASP1, NF-kB1 and CREB1, as well in the protein levels of pro-IL-1β and Nf-kB1. The P2X7R antagonist did not affect the increase in mRNA levels of fraktalkine (CX3CL1) and its receptor CX3CR1, an interaction that plays a neuroprotective role in neuron-glia communication. P2X7R antagonism also resulted in reduction of the inflammatory markers but did not alter steatosis in the liver. Taken together, these findings demonstrate how P2X7R antagonism suppresses inflammatory response in brain and liver but does not alter the neuroprotective response caused by Hybrid exposure. Overall, these findings support an important role of P2X7Rs in inflammation in brain and liver caused by combined chronic alcohol and high-fat diet.

Graphical Abstract

Keywords

Intragastric ethanol and high fat diet exposure Purinergic P2X7 receptor pharmacological inhibition Neuroinflammation Liver inflammation and steatosis Immunofluorescence Taqman custom gene expression 

Abbreviations

P2X7R

purinergic P2X7 receptor

BEC

blood ethanol concentration

iG

intragastric

HFD

high fat diet

HCFD

high cholesterol high fat diet

RT-qPCR

reverse transcriptase – quantitative polymerase chain reaction

Gapdh

glyceraldehyde 3-phosphate dehydrogenase

GFAP

glial fibrillary acidic protein

Iba1

ionized calcium binding adaptor molecule 1

Notes

Acknowledgements

We would like to thank the Animal Core of the Southern California Research Center for ALPD and Cirrhosis (Director Dr. H. Tsukamoto) for providing us with the experimental animals, Elliott Cheung for Western blots and Lisa Walter for proof reading the manuscript and valuable recommendations. This work was conducted as partial fulfilment of the requirements for the MS. degree in Pharmaceutical Sciences, University of Southern California (D.F.). Sources of support: NIH/NIAAA AA017243 and Zumberge Individual Research Fund (L.A.), NIH/NIAAA A022448 (D.L.D.), NIH P50AA011999 (H.T.).

Compliance with Ethical Standards

Conflict of Interest

The authors declare that they have no conflict of interest.

References

  1. Able SL, Fish RL, Bye H, Booth L, Logan YR, Nathaniel C, Hayter P, Katugampola SD (2011) Receptor localization, native tissue binding and ex vivo occupancy for centrally penetrant P2X7 antagonists in the rat. Br J Pharmacol 162:405–414CrossRefGoogle Scholar
  2. Alfonso-Loeches S, Guerri C (2011) Molecular and behavioral aspects of the actions of alcohol on the adult and developing brain. Crit Rev Clin Lab Sci 48:19–47CrossRefGoogle Scholar
  3. Alfonso-Loeches S, Pascual-Lucas M, Blanco AM, Sanchez-Vera I, Guerri C (2010) Pivotal role of TLR4 receptors in alcohol-induced neuroinflammation and brain damage. J Neurosci 30:8285–8295CrossRefGoogle Scholar
  4. Asatryan L, Khoja S, Rodgers KE, Alkana RL, Tsukamoto H, Davies DL (2015) Chronic ethanol exposure combined with high fat diet up-regulates P2X7 receptors that parallels neuroinflammation and neuronal loss in C57BL/6J mice. J Neuroimmunol 285:169–179CrossRefGoogle Scholar
  5. Asatryan L, Ostrovskaya O, Lieu D, Davies DL (2018) Ethanol differentially modulates P2X4 and P2X7 receptor activity and function in BV2 microglial cells. Neuropharmacol 128:11–21CrossRefGoogle Scholar
  6. Barbera-Cremades M, Gomez AI, Baroja-Mazo A, Martinez-Alarcon L, Martinez CM, de Torre-Minguela C, Pelegrin P (2017) P2X7 receptor induces tumor necrosis factor-alpha converting enzyme activation and release to boost TNF-alpha production. Front Immunol 8:862CrossRefGoogle Scholar
  7. Bartlett R, Stokes L, Sluyter R (2014) The P2X7 receptor channel: recent developments and the use of P2X7 antagonists in models of disease. Pharmacol Rev 66:638–675CrossRefGoogle Scholar
  8. Catanzaro JM, Hueston CM, Deak MM, Deak T (2014) The impact of the P2X7 receptor antagonist A-804598 on neuroimmune and behavioral consequences of stress. Behav Pharmacol 25:582–598Google Scholar
  9. Chatterjee S, Rana R, Corbett J, Kadiiska MB, Goldstein J, Mason RP (2012) P2X7 receptor-NADPH oxidase axis mediates protein radical formation and Kupffer cell activation in carbon tetrachloride-mediated steatohepatitis in obese mice. Free Radic Biol Med 52:1666–1679CrossRefGoogle Scholar
  10. Chavda S, Luthert PJ, Salt TE (2016) P2X7R modulation of visually evoked synaptic responses in the retina. Purinergic Signal 12:611–625CrossRefGoogle Scholar
  11. Chen Q, Wu H, Qin S, Liu C, Chen Y, Yang Y, Xu C (2016) The P2X7 receptor involved in gp120-induced cell injury in BV2 microglia. Inflammation 39:1814–1826CrossRefGoogle Scholar
  12. Chessell IP, Hatcher JP, Bountra C, Michel AD, Hughes JP, Green P, Egerton J, Murfin M, Richardson J, Peck WL (2005) Disruption of the P2X7 purinoceptor gene abolishes chronic inflammatory and neuropathic pain. Pain 114:386–396CrossRefGoogle Scholar
  13. Crews FT, Sarkar DK, Qin L, Zou J, Boyadjieva N, Vetreno RP (2015) Neuroimmune function and the consequences of alcohol exposure. Alcohol Res 37:344–351Google Scholar
  14. Crews FT, Vetreno RP (2014) Neuroimmune basis of alcoholic brain damage. Int Rev Neurobiol 118:315–357CrossRefGoogle Scholar
  15. Crews FT, Vetreno RP (2016) Mechanisms of neuroimmune gene induction in alcoholism. Psychopharmacol (Berl) 233:1543–1557CrossRefGoogle Scholar
  16. Diaz-Hernandez M, Diez-Zaera M, Sanchez-Nogueiro J, Gomez-Villafuertes R, Canals JM, Alberch J, Miras-Portugal MT, Lucas JJ (2009) Altered P2X7-receptor level and function in mouse models of Huntington's disease and therapeutic efficacy of antagonist administration. FASEB J 23:1893–1906CrossRefGoogle Scholar
  17. Donnelly-Roberts DL, Namovic MT, Surber B, Vaidyanathan SX, Perez-Medrano A, Wang Y, Carroll WA, Jarvis MF (2009) [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. Neuropharmacol 56:223–229CrossRefGoogle Scholar
  18. Ferrari D, Pizzirani C, Adinolfi E, Lemoli RM, Curti A, Idzko M, Panther E, Di Virgilio F (2006) The P2X7 receptor: a key player in IL-1 processing and release. J Immunol 176:3877–3883CrossRefGoogle Scholar
  19. Graeber MB (2010) Changing face of microglia. Science 330:783–788CrossRefGoogle Scholar
  20. He J, Crews FT (2008) Increased MCP-1 and microglia in various regions of the human alcoholic brain. Exp Neurol 210:349–358CrossRefGoogle Scholar
  21. Idzko M, Ferrari D, Eltzschig HK (2014) Nucleotide signalling during inflammation. Nature 509:310–317CrossRefGoogle Scholar
  22. Jung YH, Kim YO, Han JH, Kim YC, Yoon MH (2017) Isobolographic analysis of drug combinations with intrathecal BRL52537 (kappa-opioid agonist), Pregabalin (Calcium Channel modulator), AF 353 (P2X3 receptor antagonist), and A804598 (P2X7 receptor antagonist) in neuropathic rats. Anesth Analg 125:670–677CrossRefGoogle Scholar
  23. Kane CJ, Phelan KD, Douglas JC, Wagoner G, Johnson JW, Xu J, Phelan PS, Drew PD (2014) Effects of ethanol on immune response in the brain: region-specific changes in adolescent versus adult mice. Alcohol Clin Exp Res 38:384–391CrossRefGoogle Scholar
  24. Karmakar M, Katsnelson MA, Dubyak GR, Pearlman E (2016) Neutrophil P2X7 receptors mediate NLRP3 inflammasome-dependent IL-1beta secretion in response to ATP. Nat Commun 7:10555CrossRefGoogle Scholar
  25. Khakh BS, North RA (2006) P2X receptors as cell-surface ATP sensors in health and disease. Nature 442:527–532CrossRefGoogle Scholar
  26. Kneer K, Green MB, Meyer J, Rich CB, Minns MS, Trinkaus-Randall V (2018) High fat diet induces pre-type 2 diabetes with regional changes in corneal sensory nerves and altered P2X7 expression and localization. Exp Eye Res 175:44–55CrossRefGoogle Scholar
  27. Lazaro R, Wu R, Lee S, Zhu NL, Chen CL, French SW, Xu J, Machida K, Tsukamoto H (2015) Osteopontin deficiency does not prevent but promotes alcoholic neutrophilic hepatitis in mice. Hepatol 61:129–140CrossRefGoogle Scholar
  28. Liu X, Zhao Z, Ji R, Zhu J, Sui QQ, Knight GE, Burnstock G, He C, Yuan H, Xiang Z (2017) Inhibition of P2X7 receptors improves outcomes after traumatic brain injury in rats. Purinergic Signal 13:529–544CrossRefGoogle Scholar
  29. Lovinger DM (2008) Communication networks in the brain: neurons, receptors, neurotransmitters. and alcohol Alcohol Res Health 31:196–214Google Scholar
  30. Marshall SA, McClain JA, Kelso ML, Hopkins DM, Pauly JR, Nixon K (2013) Microglia activation is not equivalent to neuroinflammation in alcohol-induced neurodegeneration: the importance of mciroglia phenotype. Neurobiol Dis 54:239–251CrossRefGoogle Scholar
  31. Matute C, Torre I, Perez-Cerda F, Perez-Samartin A, Alberdi E, Etxebarria E, Arranz AM, Ravid R, Rodriguez-Antiguedad A, Sanchez-Gomez M, Domercq M (2007) P2X(7) receptor blockade prevents ATP excitotoxicity in oligodendrocytes and ameliorates experimental autoimmune encephalomyelitis. J Neurosci 27:9525–9533CrossRefGoogle Scholar
  32. McClain JA, Morris SA, Deeny MA, Marshall SA, Hayes DM, Kiser ZM, Nixon K (2011) Adolescent binge alcohol exposure induces long-lasting partial activation of microglia. Brain Behav Immun 25(Suppl 1):S120–S128CrossRefGoogle Scholar
  33. McLarnon JG, Ryu JK, Walker DG, Choi HB (2006) Upregulated expression of purinergic P2X7 receptor in Alzheimer disease and amyloid-[beta] peptide-treated microglia and in peptide-injected rat hippocampus. J Neuropathol Exp Neurol 65:1090–1097CrossRefGoogle Scholar
  34. Monif M, Burnstock G, Williams DA (2010) Microglia: proliferation and activation driven by the P2X7 receptor. Int J Biochem Cell Biol 42:1753–1756CrossRefGoogle Scholar
  35. North RA, Jarvis MF (2013) P2X receptors as drug targets. Mol Pharmacol 83:759–769CrossRefGoogle Scholar
  36. Pandolfi JB, Ferraro AA, Sananez I, Gancedo MC, Baz P, Billordo LA, Fainboim L, Arruvito L (2016) ATP-induced inflammation drives tissue-resident Th17 cells in metabolically unhealthy obesity. J Immunol 196:3287–3296CrossRefGoogle Scholar
  37. Panenka W, Jijon H, Herx LM, Armstrong JN, Feighan D, Wei T, Yong VW, Ransohoff RM, MacVicar BA (2001) P2X7-like receptor activation in astrocytes increases chemokine monocyte chemoattractant protein-1 expression via mitogen-activated protein kinase. J Neurosci 21:7135–7142CrossRefGoogle Scholar
  38. Pascual M, Balino P, Aragon CM, Guerri C (2015) Cytokines and chemokines as biomarkers of ethanol-induced neuroinflammation and anxiety-related behavior: role of TLR4 and TLR2. Neuropharmacol 89:352–359CrossRefGoogle Scholar
  39. Peng W, Cotrina ML, Han X, Yu H, Bekar L, Blum L, Takano T, Tian GF, Goldman SA, Nedergaard M (2009) Systemic administration of an antagonist of the ATP-sensitive receptor P2X7 improves recovery after spinal cord injury. PNAS USA 106:12489–12493CrossRefGoogle Scholar
  40. Sacks JJ, Gonzales KR, Bouchery EE, Tomedi LE, Brewer RD (2015) 2010 national and state costs of excessive alcohol consumption. Am J Prev Med 49:e73–e79CrossRefGoogle Scholar
  41. Schmittgen TD, Livak KJ (2008) Analyzing real-time PCR data by the comparative C(T) method. Nat Protoc 3:1101–1108CrossRefGoogle Scholar
  42. Sheridan GK, Murphy KJ (2013) Neuron-glia crosstalk in health and disease: fractalkine and CX3CR1 take Centre stage. Open Biol 3:130181CrossRefGoogle Scholar
  43. Skaper SD, Debetto P, Giusti P (2010) The P2X7 purinergic receptor: from physiology to neurological disorders. FASEB J 24:337–345CrossRefGoogle Scholar
  44. Stahre M, Roeber J, Kanny D, Brewer RD, Zhang X (2014) Contribution of excessive alcohol consumption to deaths and years of potential life lost in the United States. Prev Chronic Dis 11:E109CrossRefGoogle Scholar
  45. Suzuki T, Hide I, Ido K, Kohsaka S, Inoue K, Nakata Y (2004) Production and release of neuroprotective tumor necrosis factor by P2X7 receptor-activated microglia. J Neurosci 24:1–7CrossRefGoogle Scholar
  46. Szabo G, Lippai D (2014) Converging actions of alcohol on liver and brain immune signaling. Int Rev Neurobiol 118:359–380CrossRefGoogle Scholar
  47. Takenouchi T, Sekiyama K, Sekigawa A, Fujita M, Waragai M, Sugama S, Iwamaru Y, Kitani H, Hashimoto M (2010) P2X7 receptor signaling pathway as a therapeutic target for neurodegenerative diseases. Arch Immunol Therap Exp 58:91–96CrossRefGoogle Scholar
  48. Takenouchi T, Sugama S, Iwamaru Y, Hashimoto M, Kitani H (2009) Modulation of the ATP-induced release and processing of IL-1beta in microglial cells. Crit Rev Immunol 29:335–345CrossRefGoogle Scholar
  49. Tewari M, Seth P (2015) Emerging role of P2X7 receptors in CNS health and disease. Ageing Res Rev 24:328–342CrossRefGoogle Scholar
  50. Ueno A, Lazaro R, Wang PY, Higashiyama R, Machida K, Tsukamoto H (2012) Mouse intragastric infusion (iG) model. Nat Protoc 7:771–781CrossRefGoogle Scholar
  51. Vengeliene V, Bilbao A, Molander A, Spanagel R (2008) Neuropharmacology of alcohol addiction. Br J Pharmacol 154:299–315CrossRefGoogle Scholar
  52. Vetreno RP, Crews FT (2014) Current hypotheses on the mechanisms of alcoholism. Handb Clin Neurol 125:477–497CrossRefGoogle Scholar
  53. Volonte C, Apolloni S, Carri MT, D'Ambrosi N (2011) ALS: focus on purinergic signalling. Pharmacol Ther 132:111–122CrossRefGoogle Scholar
  54. Volonte C, Apolloni S, Skaper SD, Burnstock G (2012) P2X7 receptors: channels, pores and more. CNS & neurological disorders drug targets 11:705–721CrossRefGoogle Scholar
  55. Wieser V, Adolph TE, Enrich B, Kuliopulos A, Kaser A, Tilg H, Kaneider NC (2017) Reversal of murine alcoholic steatohepatitis by pepducin-based functional blockade of interleukin-8 receptors. Gut 66:930–938CrossRefGoogle Scholar
  56. Xu J, Lai KK, Verlinsky A, Lugea A, French SW, Cooper MP, Ji C, Tsukamoto H (2011) Synergistic steatohepatitis by moderate obesity and alcohol in mice despite increased adiponectin and p-AMPK. J Hepatol 55:673–682CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2018

Authors and Affiliations

  1. 1.Department of Pharmacology and Pharmaceutical Sciences, School of PharmacyUniversity of Southern CaliforniaLos AngelesUSA
  2. 2.Department of NeurologyKeck School of MedicineLos AngelesUSA
  3. 3.Titus Family Department of Clinical Pharmacy, School of PharmacyUniversity of Southern CaliforniaLos AngelesUSA

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