Epigenetic regulation of innate immune memory in microglia

Microglia are the tissue-resident macrophages of the CNS. They originate in the yolk sac, colonize the CNS during embryonic development and form a self-sustaining population with limited turnover. A consequence of their relative slow turnover is that microglia can serve as a long-term memory for inflammatory or neurodegenerative events. We characterized the epigenomes and transcriptomes of microglia exposed to different stimuli; an endotoxin challenge (LPS) and genotoxic stress (DNA repair deficiency-induced accelerated aging). Whereas the enrichment of permissive epigenetic marks at enhancer regions explains training (hyperresponsiveness) of primed microglia to LPS challenge, the tolerized response of microglia seems to be regulated by loss of permissive epigenetic marks. Here, we identify that inflammatory stimuli and accelerated aging because of genotoxic stress activate distinct gene networks. These gene networks and associated biological processes are partially overlapping, which is likely driven by specific transcription factor networks, resulting in altered epigenetic signatures and distinct functional (desensitized vs. primed) microglia phenotypes. We performed RNA, ATAC, and ChIP-sequencing on acutely isolated, FACS-purified microglia from mice that were either recurrently treated with LPS with a 1-month interval, or from Ercc1Δ/ko mice that were stimulated with LPS near the end of their lifespan at 10-12 weeks of age. For the tolerance model, we analyzed four treatment groups: the controls that were treated with PBS twice (PBS-PBS), mice that were treated with LPS and after 1 month with PBS (LPS-PBS) to investigate desensitization, mice treated with PBS followed by LPS after 1 month to determine the acute response to LPS (PBS-LPS) and mice that were treated with LPS twice with a 1-month interval between challenges (LPS-LPS) to identify the tolerant response. For the microglia priming model, both the Ercc1Δ/ko mice and their control littermates (Ercc1wt/Δ, Ercc1wt/ko or Ercc1wt/wt) were treated with PBS (WT-PBS, KO-PBS) to identify priming effects or with LPS (WT-LPS, KO-LPS) to identify training. For RNA-seq, microglia from 3 individual mice per condition were collected. For ChIP-seq, chromatin of 5 mice per experimental group was pooled and subjected to ChIP. For ATAC-seq, microglia from 2 mice per experimental group were pooled and subjected to ATAC. RNA-seq and ATAC-seq was performed for all four experimental groups in both the tolerance and training model. ChIP-seq was performed for all four experimental group in the tolerance model, but only for PBS-treated Ercc1Δ/ko and control mice. In case of the tolerance model, we used antibodies targeting H3K4me3 and H3K27Ac to identify transcription start sites (TSSs) and enhancers of actively transcribed genes, respectively. In Ercc1Δ/ko mice, we also analyzed H3K4me3 and H3K27ac, and additionally H3K4me1 which together with H3K27ac marks active enhancers and the Polycomb-regulated H3K27me3 associated with transcriptional repression.