The profile of P2X7 receptor expression and properties in polarized primary mouse microglia cultures

Megan Jackson, Robin Wickens, Sarah Bailey, Amanda Mackenzie

Research output: Contribution to journalMeeting abstract

Abstract

Adenosine triphosphate (ATP)-gated P2X7 receptors (P2X7R) play an important role in neuro-inflammation and have been implicated in mood disorders (Sperlágh and Illes, 2014, Trends Pharmacol Sci, 35, 537-547). Microglia mediate brain inflammation where they possess the ability to polarize into M1/M2 phenotypes based on cytokine and toll-like receptor activation. Although a functional role for P2X7R has been demonstrated in lipopolysaccharide (LPS)-treated primary rat microglial cells (Bianco et al., 2006, Journal of Neurochemistry, 99, 745-758), little is known about their expression or function in activated mouse primary microglial cells. This study aims to investigate P2X7R expression and function in M1 polarized, pro-inflammatory, mouse microglia. Primary microglia were obtained from cortices of mixed-gender C57BI/6 mouse pups (P0-P2) using the low trypsinization method (Saura et al., 2003, Glia, 44, 183-189). Cells were plated at 300,000 cells/well; after 21 DIV, astrocytes were removed by incubating with diluted trypsin (0.0625%) and experiments began 24h later. BV-2 cells, (immortalized murine microglia; Blasi et al., 1990, Journal of Neuroimmunology, 27 229-237) and primary microglial cells were treated with either 100 ng/ml LPS (24h) or 100ng/ml LPS plus interferon (IFN)γ (20 ng/ml) (24h) to obtain M1 polarization, confirmed by expression of pro-IL-1β detected by quantitative fluorescent western blot (LICOR) (n=3). P2X7R protein expression was also evaluated by quantitative western blot and compared to β-actin expression. P2X7R activation was measured by uptake of fluorescent ethidium in BV-2 microglia. M1 polarization with LPS+IFNγ down-regulated P2X7R expression in both primary microglia (63.2±29.1% n=3) and BV-2 microglia (87.5±32.8 % n=5) compared with non-polarized controls (M0). LPS alone down-regulated P2X7R expression in primary microglia (62.0± 33.7% n=3) but not BV-2 cells (110.5±36.4% n=5). Extracellular ATP-mediated (>1mM) ethidium uptake in BV-2 M0 microglia (n=3) was inhibited by the selective P2X7R antagonist A740003 (30μM) confirming functional expression of P2X7R. LPS priming +/- IFNγ (24h) did not alter ATP-mediated (1–5 mM) ethidium uptake in BV-2 microglia compared to control conditions (n=3). Understanding changes in microglia leading to pro-inflammatory activated states is important in developing novel treatments that target neuro-inflammation. While there is a trend for down-regulation of P2X7R in activated, polarized M1 mouse primary microglia, there is little evidence of LPS-induced change in P2X7R expression or function in BV-2 cells. This contrasts to findings in rat primary microglia. We are investigating functional activation of P2X7R in primary mouse microglia. RAW is funded by an MRC Industrial Case PhD Studentship
Original languageEnglish
Pages (from-to)A98
JournalJournal of Psychopharmacology
Volume30
Issue numberAbstract Supplement to volume 8
Publication statusPublished - 2016

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Purinergic P2X7 Receptors
Microglia
Lipopolysaccharides
Ethidium
Adenosine Triphosphate
(N-(1-(((cyanoimino)(5-quinolinylamino) methyl) amino)-2,2-dimethylpropyl)-2-(3,4-dimethoxyphenyl)acetamide)
Western Blotting
Inflammation
Neurochemistry
Aptitude
Toll-Like Receptors
Encephalitis

Keywords

  • Depression
  • Neuroinflammation
  • Microglia

Cite this

The profile of P2X7 receptor expression and properties in polarized primary mouse microglia cultures. / Jackson, Megan; Wickens, Robin; Bailey, Sarah; Mackenzie, Amanda.

In: Journal of Psychopharmacology , Vol. 30, No. Abstract Supplement to volume 8, 2016, p. A98.

Research output: Contribution to journalMeeting abstract

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title = "The profile of P2X7 receptor expression and properties in polarized primary mouse microglia cultures",
abstract = "Adenosine triphosphate (ATP)-gated P2X7 receptors (P2X7R) play an important role in neuro-inflammation and have been implicated in mood disorders (Sperl{\'a}gh and Illes, 2014, Trends Pharmacol Sci, 35, 537-547). Microglia mediate brain inflammation where they possess the ability to polarize into M1/M2 phenotypes based on cytokine and toll-like receptor activation. Although a functional role for P2X7R has been demonstrated in lipopolysaccharide (LPS)-treated primary rat microglial cells (Bianco et al., 2006, Journal of Neurochemistry, 99, 745-758), little is known about their expression or function in activated mouse primary microglial cells. This study aims to investigate P2X7R expression and function in M1 polarized, pro-inflammatory, mouse microglia. Primary microglia were obtained from cortices of mixed-gender C57BI/6 mouse pups (P0-P2) using the low trypsinization method (Saura et al., 2003, Glia, 44, 183-189). Cells were plated at 300,000 cells/well; after 21 DIV, astrocytes were removed by incubating with diluted trypsin (0.0625{\%}) and experiments began 24h later. BV-2 cells, (immortalized murine microglia; Blasi et al., 1990, Journal of Neuroimmunology, 27 229-237) and primary microglial cells were treated with either 100 ng/ml LPS (24h) or 100ng/ml LPS plus interferon (IFN)γ (20 ng/ml) (24h) to obtain M1 polarization, confirmed by expression of pro-IL-1β detected by quantitative fluorescent western blot (LICOR) (n=3). P2X7R protein expression was also evaluated by quantitative western blot and compared to β-actin expression. P2X7R activation was measured by uptake of fluorescent ethidium in BV-2 microglia. M1 polarization with LPS+IFNγ down-regulated P2X7R expression in both primary microglia (63.2±29.1{\%} n=3) and BV-2 microglia (87.5±32.8 {\%} n=5) compared with non-polarized controls (M0). LPS alone down-regulated P2X7R expression in primary microglia (62.0± 33.7{\%} n=3) but not BV-2 cells (110.5±36.4{\%} n=5). Extracellular ATP-mediated (>1mM) ethidium uptake in BV-2 M0 microglia (n=3) was inhibited by the selective P2X7R antagonist A740003 (30μM) confirming functional expression of P2X7R. LPS priming +/- IFNγ (24h) did not alter ATP-mediated (1–5 mM) ethidium uptake in BV-2 microglia compared to control conditions (n=3). Understanding changes in microglia leading to pro-inflammatory activated states is important in developing novel treatments that target neuro-inflammation. While there is a trend for down-regulation of P2X7R in activated, polarized M1 mouse primary microglia, there is little evidence of LPS-induced change in P2X7R expression or function in BV-2 cells. This contrasts to findings in rat primary microglia. We are investigating functional activation of P2X7R in primary mouse microglia. RAW is funded by an MRC Industrial Case PhD Studentship",
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T1 - The profile of P2X7 receptor expression and properties in polarized primary mouse microglia cultures

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AU - Wickens, Robin

AU - Bailey, Sarah

AU - Mackenzie, Amanda

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N2 - Adenosine triphosphate (ATP)-gated P2X7 receptors (P2X7R) play an important role in neuro-inflammation and have been implicated in mood disorders (Sperlágh and Illes, 2014, Trends Pharmacol Sci, 35, 537-547). Microglia mediate brain inflammation where they possess the ability to polarize into M1/M2 phenotypes based on cytokine and toll-like receptor activation. Although a functional role for P2X7R has been demonstrated in lipopolysaccharide (LPS)-treated primary rat microglial cells (Bianco et al., 2006, Journal of Neurochemistry, 99, 745-758), little is known about their expression or function in activated mouse primary microglial cells. This study aims to investigate P2X7R expression and function in M1 polarized, pro-inflammatory, mouse microglia. Primary microglia were obtained from cortices of mixed-gender C57BI/6 mouse pups (P0-P2) using the low trypsinization method (Saura et al., 2003, Glia, 44, 183-189). Cells were plated at 300,000 cells/well; after 21 DIV, astrocytes were removed by incubating with diluted trypsin (0.0625%) and experiments began 24h later. BV-2 cells, (immortalized murine microglia; Blasi et al., 1990, Journal of Neuroimmunology, 27 229-237) and primary microglial cells were treated with either 100 ng/ml LPS (24h) or 100ng/ml LPS plus interferon (IFN)γ (20 ng/ml) (24h) to obtain M1 polarization, confirmed by expression of pro-IL-1β detected by quantitative fluorescent western blot (LICOR) (n=3). P2X7R protein expression was also evaluated by quantitative western blot and compared to β-actin expression. P2X7R activation was measured by uptake of fluorescent ethidium in BV-2 microglia. M1 polarization with LPS+IFNγ down-regulated P2X7R expression in both primary microglia (63.2±29.1% n=3) and BV-2 microglia (87.5±32.8 % n=5) compared with non-polarized controls (M0). LPS alone down-regulated P2X7R expression in primary microglia (62.0± 33.7% n=3) but not BV-2 cells (110.5±36.4% n=5). Extracellular ATP-mediated (>1mM) ethidium uptake in BV-2 M0 microglia (n=3) was inhibited by the selective P2X7R antagonist A740003 (30μM) confirming functional expression of P2X7R. LPS priming +/- IFNγ (24h) did not alter ATP-mediated (1–5 mM) ethidium uptake in BV-2 microglia compared to control conditions (n=3). Understanding changes in microglia leading to pro-inflammatory activated states is important in developing novel treatments that target neuro-inflammation. While there is a trend for down-regulation of P2X7R in activated, polarized M1 mouse primary microglia, there is little evidence of LPS-induced change in P2X7R expression or function in BV-2 cells. This contrasts to findings in rat primary microglia. We are investigating functional activation of P2X7R in primary mouse microglia. RAW is funded by an MRC Industrial Case PhD Studentship

AB - Adenosine triphosphate (ATP)-gated P2X7 receptors (P2X7R) play an important role in neuro-inflammation and have been implicated in mood disorders (Sperlágh and Illes, 2014, Trends Pharmacol Sci, 35, 537-547). Microglia mediate brain inflammation where they possess the ability to polarize into M1/M2 phenotypes based on cytokine and toll-like receptor activation. Although a functional role for P2X7R has been demonstrated in lipopolysaccharide (LPS)-treated primary rat microglial cells (Bianco et al., 2006, Journal of Neurochemistry, 99, 745-758), little is known about their expression or function in activated mouse primary microglial cells. This study aims to investigate P2X7R expression and function in M1 polarized, pro-inflammatory, mouse microglia. Primary microglia were obtained from cortices of mixed-gender C57BI/6 mouse pups (P0-P2) using the low trypsinization method (Saura et al., 2003, Glia, 44, 183-189). Cells were plated at 300,000 cells/well; after 21 DIV, astrocytes were removed by incubating with diluted trypsin (0.0625%) and experiments began 24h later. BV-2 cells, (immortalized murine microglia; Blasi et al., 1990, Journal of Neuroimmunology, 27 229-237) and primary microglial cells were treated with either 100 ng/ml LPS (24h) or 100ng/ml LPS plus interferon (IFN)γ (20 ng/ml) (24h) to obtain M1 polarization, confirmed by expression of pro-IL-1β detected by quantitative fluorescent western blot (LICOR) (n=3). P2X7R protein expression was also evaluated by quantitative western blot and compared to β-actin expression. P2X7R activation was measured by uptake of fluorescent ethidium in BV-2 microglia. M1 polarization with LPS+IFNγ down-regulated P2X7R expression in both primary microglia (63.2±29.1% n=3) and BV-2 microglia (87.5±32.8 % n=5) compared with non-polarized controls (M0). LPS alone down-regulated P2X7R expression in primary microglia (62.0± 33.7% n=3) but not BV-2 cells (110.5±36.4% n=5). Extracellular ATP-mediated (>1mM) ethidium uptake in BV-2 M0 microglia (n=3) was inhibited by the selective P2X7R antagonist A740003 (30μM) confirming functional expression of P2X7R. LPS priming +/- IFNγ (24h) did not alter ATP-mediated (1–5 mM) ethidium uptake in BV-2 microglia compared to control conditions (n=3). Understanding changes in microglia leading to pro-inflammatory activated states is important in developing novel treatments that target neuro-inflammation. While there is a trend for down-regulation of P2X7R in activated, polarized M1 mouse primary microglia, there is little evidence of LPS-induced change in P2X7R expression or function in BV-2 cells. This contrasts to findings in rat primary microglia. We are investigating functional activation of P2X7R in primary mouse microglia. RAW is funded by an MRC Industrial Case PhD Studentship

KW - Depression

KW - Neuroinflammation

KW - Microglia

M3 - Meeting abstract

VL - 30

SP - A98

JO - Journal of Psychopharmacology

JF - Journal of Psychopharmacology

SN - 0269-8811

IS - Abstract Supplement to volume 8

ER -