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Isolation of Cortical Microglia with Preserved Immunophenotype and Functionality From Murine Neonates
Published on: January 30, 2014
The role of microglial P2X7: modulation of cell death and cytokine release
Yingbo He1, Natalie Taylor2, Lawrence Fourgeaud2
1Janssen Research & Development, LLC., Neuroscience Drug Discovery, 3210 Merryfield Row, San Diego, CA, 92121, USA. yhe50@its.jnj.com.
Background:
ATP-gated P2X7 is a non-selective cation channel, which participates in a wide range of cellular functions as well as pathophysiological processes including neuropathic pain, immune response, and neuroinflammation. Despite its abundant expression in microglia, the role of P2X7 in neuroinflammation still remains unclear.
Methods:
Primary microglia were isolated from cortices of P0-2 C57BL/6 wild-type or P2X7 knockout (P2X7-/-) mouse pups. Lipopolysaccharide, lipopolysaccharide plus IFNγ, or IL4 plus IL13 were used to polarize microglia to pro-inflammatory or anti-inflammatory states. P2rx7 expression level in resting or activated mouse and human microglia was measured by RNA-sequencing and quantitative real-time PCR. Microglial cell death was measured by cell counting kit-8 and immunocytochemistry, and microglial secretion in wild-type or P2X7-/- microglia was examined by Luminex multiplex assay or ELISA using P2X7 agonist BzATP or P2X7 antagonist A-804598. P2X7 signaling was analyzed by Western blot.
Results:
First, we confirmed that P2rx7 is constitutively expressed in mouse and human primary microglia. Moreover, P2rx7 mRNA level was downregulated in mouse microglia under both pro- and anti-inflammatory conditions. Second, P2X7 agonist BzATP caused cell death of mouse microglia, while this effect was suppressed either by P2X7 knockout or by A-804598 under both basal and pro-inflammatory conditions, which suggests the mediating role of P2X7 in BzATP-induced microglial cell death. Third, BzATP-induced release of IL1 family cytokines including IL1α, IL1β, and IL18 was blocked in P2X7-/- microglia or by A-804598 in pro-inflammatory microglia, while the release of other cytokines/chemokines was independent of P2X7 activation. These findings support the specific role of P2X7 in IL1 family cytokine release. Finally, P2X7 activation was discovered to be linked to AKT and ERK pathways, which may be the underlying mechanism of P2X7 functions in microglia.
Conclusions:
These results reveal that P2X7 mediates BzATP-induced microglial cell death and specific release of IL1 family cytokines, indicating the important role of P2X7 in neuroinflammation and implying the potential of targeting P2X7 for the treatment of neuroinflammatory disorders.
Insights
The P2X7 receptor mediates microglial cell death and IL1 family cytokine release, playing a key role in neuroinflammation. Targeting P2X7 offers potential for treating neuroinflammatory disorders.
Area of Science:
- Neuroscience
- Immunology
Background:
- ATP-gated P2X7 channels are involved in neuropathic pain, immune response, and neuroinflammation.
- P2X7 is highly expressed in microglia, but its specific role in neuroinflammation is unclear.
Purpose of the Study:
- To investigate the role of P2X7 in microglial activation and neuroinflammation.
- To elucidate the mechanisms underlying P2X7-mediated effects in microglia.
Main Methods:
- Primary microglia from wild-type and P2X7 knockout mice were used.
- Microglia were polarized to pro- or anti-inflammatory states.
- P2X7 expression, cell death, cytokine release, and signaling pathways were analyzed.
Main Results:
- P2X7 expression was confirmed in mouse and human microglia and downregulated under inflammatory conditions.
- P2X7 activation by BzATP induced microglial cell death and IL1 family cytokine release (IL1α, IL1β, IL18).
- These effects were blocked by P2X7 knockout or antagonist A-804598, and linked to AKT and ERK pathways.
Conclusions:
- P2X7 mediates BzATP-induced microglial cell death and specific IL1 family cytokine release.
- P2X7 plays a significant role in neuroinflammation.
- Targeting P2X7 presents a potential therapeutic strategy for neuroinflammatory disorders.
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