PGE₂ Inhibits IL-10 Production via EP2-Mediated β-Arrestin Signaling in Neuroinflammatory Condition

Regulatory mechanisms of the expression of interleukin-10 (IL-10) in brain inflammatory conditions remain elusive. To address this issue, we used multiple primary brain cell cultures to study the expression of IL-10 in lipopolysaccharide (LPS)-elicited inflammatory conditions. In neuron–glia cultures, LPS triggered well-orchestrated expression of various immune factors in the following order: tumor necrosis factor-α (TNF-α), cyclooxygenase-2 (COX-2), prostaglandin E2 (PGE₂), and lastly IL-10, and these inflammatory mediators were mainly produced from microglia. While exogenous application of individual earlier-released pro-inflammatory factors (e.g., TNF-α, IL-1β, or PGE₂) failed to induce IL-10 expression, removal of LPS from the cultures showed the requirement of continuing presence of LPS for IL-10 expression. Interestingly, genetic disruption of tnf-α, its receptors tnf-r1/r2, and cox-2 and pharmacological inhibition of COX-2 activity enhanced LPS-induced IL-10 production in microglia, which suggests negative regulation of IL-10 induction by the earlier-released TNF-α and PGE₂. Further studies showed that negative regulation of IL-10 production by TNF-α is mediated by PGE₂. Mechanistic studies indicated that PGE₂-elicited suppression of IL-10 induction was eliminated by genetic disruption of the PGE₂ receptor EP2 and was mimicked by the specific agonist for the EP2, butaprost, but not agonists for the other three EP receptors. Inhibition of cAMP-dependent signal transduction failed to affect PGE₂-mediated inhibition of IL-10 production, suggesting that a G protein-independent pathway was involved. Indeed, deficiency in β-arrestin-1 or β-arrestin-2 abolished PGE₂-elicited suppression of IL-10 production. In conclusion, we have demonstrated that COX-2-derived PGE₂ inhibits IL-10 expression in brain microglia through a novel EP2- and β-arrestin-dependent signaling pathway.