Inhibition of TNFalpha Release by Purine Nucleosides in a Human Monocyte-derived Microglial Model Exhibits Apparent Insensitivity to Adenosine, P2X, and P2Y Receptor Antagonism
Omar Qureshi1, Jamie Cowley1, Arshpreet Kaur1, Ashley Pegg1, Leila Sallie1, Ines Morano1, Neale Harrison1, Hujo Chan1, Zhi Li1, Emma Welsh1, Mark Bryant1, Catherine Brady1, John Gordon1, Nicholas Barnes1
1Celentyx Ltd
Objective:

To understand the impact of purinergic signaling on the regulation of pro-inflammatory cytokine release in human microglia.

Background:

Genetic evidence has associated microglial associated genes (e.g. TREM2, CD33, ABCA7 and CR1) with neurological disease implicating neuroinflammation as a driver of pathogenesis. Activation of anti-inflammatory pathways may potentially bring therapeutic benefit. The present study investigated the impact of purinergic agonists and antagonists on TNFa release from human microglia derived from monocytes (iMDM).

Design/Methods:

Human microglia (iMDM) were differentiated from human peripheral blood monocytes by culture in a cocktail of CNS-associated cytokines over 5-10 days. The immuno-phenotype of the iMDM versus the originating monocytes were assessed by immuno-cytochemistry. iMDM were primed with LPS before stimulation of purinergic receptors with selected agonists and antagonists to investigate cytokine release.

Results:

Although high concentrations of ATP are generally associated with pro-inflammatory (e.g. IL-1β, IL-18) cytokine release through activation of the P2X7 receptor, a reduction in LPS-evoked TNFa release was also evident. The more P2X7 receptor-selective agonist BzATP evoked a similar reduction in LPS-evoked TNFα release although this was not reversed by selective P2X7 or P2Y receptor antagonists. Adenosine evoked a similar anti-inflammatory effect, which was blocked by adenosine receptor antagonists. However adenosine receptor antagonists did not prevent the anti-inflammatory effect upon TNFα release by high concentrations of ATP or BzATP. High concentrations of ATP and BzATP also resulted in a sensitivity of TNFα release to NLRP3 inflammasome inhibition suggesting potential cross-talk between the two seemingly disparate pathways. These findings suggest a potential anti-inflammatory role of the P2X7 receptor that is insensitive to the small molecule P2X7 receptor antagonists used in this study.

Conclusions:

These findings suggest a previously undescribed pathway regulating TNFα release that may have the potential for therapeutic targeting in neurological disease involving neuoinflammation.

10.1212/WNL.0000000000206094