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

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