Abstract Body: Introduction
The fundamental roles of chromatin biology and epigenetics in progression of diseases have been increasingly recognized. Some genes escaping from X chromosome inactivation (XCI) impact neuroinflammation through epigenetic regulation. Our previous studies have suggested that the X escapee genes Kdm6a and Kdm5c were involved in microglial activation after stroke in aged mice. However, the underlying mechanisms remain elusive.
Hypothesis: We hypothesized that Kdm6a/5c demethylate H3K27Me3/H3K4Me3 in microglia respectively, and mediate the transcription of interferon regulatory factor 5 (IRF5) and IRF4, leading to microglial pro-inflammatory responses and exacerbated stroke injury.
Methods
Aged (17–20 months) Kdm6a/5c microglial conditional knockout (CKO) female mice (one allele of the gene) were subjected to a 60-min middle cerebral artery occlusion (MCAO). Gene floxed females (two alleles) and males (one allele) were included as controls. Infarct volume and behavioral deficits were quantified 3 days after stroke. Immune responses including microglial activation and infiltration of peripheral leukocytes in the ischemic brain were assessed by flow cytometry. Epigenetic modification of IRF5/4 by Kdm6a/5c were investigated by CUT&RUN ChIP assay.
Results
The demethylation of H3K27Me3 by Kdm6a, increased IRF5 transcription; meanwhile Kdm5c demethylated H3K4Me3 to repress IRF5 (IRF5 ratio H3K27Me1/H3K27Me3 in fl/y vs. fl/fl, p = 0.0331; fl/fl vs. CKO, p = 0.0082, Figure 1A). Both Kdm6a^fl/fl and Kdm5c^fl/fl mice had worse stroke outcomes compared to fl/y and CKO mice (Figure 1B). Gene floxed females showed more robust expression of CD68 in microglia (Kdm6a fl/y vs. fl/fl, p < 0.0001), elevated brain and plasma levels of IL-1β or TNF-α, after stroke (Figure 1C, 1D).
Conclusion
We concluded that IRF5 signaling plays a critical role in mediating the deleterious effect of Kdm6a; whereas Kdm5c’s effect is independent of IRF5.
Keywords: Stroke, Aging, Kdm6a/5c, Microglia, Epigenetics, Ischemia, IRF