Abstract TP99: Hypoxia Drives Anti-Inflammatory Polarization of Microglia Through the JMJD3-IRF4 Axis

Abstract

Background: Microglia, as the innate immune cells of the central nervous system, are poised to be the first responders of the immune system to ischemic stroke. In response to an injury or stimulus, microglia polarize from a resting to a pro- or anti-inflammatory phenotype. We have previously shown that hypoxia, such as that in the setting of ischemic stroke, acts as a stimulus itself to polarize microglia to an anti-inflammatory phenotype. Additionally, we have shown that Jumonji Domain Containing 3 (JMJD3) is a histone demethylase that is important for anti-inflammatory polarization of microglia. JMJD3 functions to demethylate trimethylated histone H3 lysine 27 (H3K27me3) at the Irf4 promoter to monomethylation marks (H3K27me1), resulting in transcriptional activation of Irf4 and downstream expression of anti-inflammatory genes. Hypothesis: Thus, we hypothesized that hypoxia drives microglial anti-inflammatory polarization through the JMJD3-IRF4 axis. Methods: We cultured male and female primary microglia from neonatal P0.5-P3 C57Bl/6 mice, inhibited JMJD3 with GSK-J4 (Cayman Chemical) or DMSO control, and stimulated cultures with oxygen glucose deprivation (OGD). After 8hr OGD, we isolated RNA (RNEasy Micro Plus kit, Qiagen) and performed down-stream real-time PCR array analysis (PrimePCR Arrays, Bio-Rad) or protein (RIPA Buffer, Thermo Fisher) for Western blot analysis (Bio-Rad) for JMJD3, histone H3 (Abcam), H3K27me1, and H3K27me3 (Millipore). Results: We found that OGD induced up-regulation anti-inflammatory genes, notably Csf1 , Cntf , Gdf15 , Il1rn , Irf4 , Mif , and Vegfa while down-regulating pro-inflammatory genes Tnfa , Il1b , and Il6 (n=4, p<0.05). Additionally, we found that Kdm6b (the gene encoding JMJD3) was up-regulated following 8hr OGD, suggesting JMJD3 is involved in hypoxia-induced anti-inflammatory microglial phenotype polarization. Pharmacological inhibition of JMJD3 with GSK-J4 prevented demethylation of H3K27me3 at the Irf4 promoter and blocked hypoxia-induced up-regulation of Irf4 and abrogated hypoxia-induced anti-inflammatory gene up-regulation. Conclusion: In conclusion, our data suggests that hypoxia polarizes microglia to an anti-inflammatory phenotype through the JMJD3-IRF4 axis.