Figure 4.

NF-κB and IRF pathways mediate the antagonistic effects of the EP4 receptor in microglial inflammatory responses to oligomeric Aβ₄₂. A, B, Ingenuity Pathway Analysis was performed on the 116 Aβ₄₂-responsive genes for which EP4 agonist treatment opposed the Aβ₄₂ effect (fold change > 1.5 in Aβ₄₂+EP4 agonist vs Aβ₄₂+Veh comparison) in primary microglia cotreated for 6 h with oligomeric Aβ₄₂ (10 μm) and/or the EP4 agonist AE1–329 (100 nm). Simplified diagrams of the two most significantly enriched pathways from these genes, centering on NF-κB and IRF1/IRF7, are displayed. C, GSEA for transcription factor (TF) binding motifs shows positive enrichment for NF-κB and IRF1 binding motifs in the Aβ₄₂ vs Con comparison, and negative enrichment for the same sites in the Aβ₄₂+EP4 agonist versus Aβ₄₂+Veh comparison. D, qRT-PCR demonstrates that EP4 receptor stimulation increases expression of Nurr1 and Nur77, two nuclear receptors that repress NF-κB activity. E, qRT-PCR confirms that EP4 receptor stimulation attenuates expression of IRF1 and IRF7 in response to Aβ₄₂. For each experiment, n = 3 microglia isolations from independent mouse cohorts. *p < 0.05 (Bonferroni multiple comparison test). **p < 0.01 (Bonferroni multiple comparison test). ***p < 0.001 (Bonferroni multiple comparison test). F, Western blot from BV2 microglial cells cotreated for 8 h with Aβ₄₂- (10 μm) and/or the EP4 agonist AE1–329 (100 nm) demonstrates that Aβ₄₂ increases both total and Tyr701-phosphorylated STAT1 levels, whereas EP4 agonist treatment attenuates phospho-STAT1 levels without altering total STAT1 protein level. n = 6 independent samples per group. ***p < 0.001 (Bonferroni multiple comparison test). ns, p > 0.05 (Bonferroni multiple comparison test).