Transcriptional regulation of homeostatic and disease‐associated‐microglial genes by IRF1, LXRβ, and CEBPα

Abstract

Microglia transform from homeostatic to disease‐associated‐microglia (DAM) profiles in neurodegeneration. Within DAM, we recently identified distinct pro‐inflammatory and anti‐inflammatory sub‐profiles although transcriptional regulators of homeostatic and distinct DAM profiles remain unclear. Informed by these studies, we nominated CEBPα, IRF1, and LXRβ as likely regulators of homeostatic, pro‐inflammatory and anti‐inflammatory DAM states and performed in‐vitro siRNA studies in primary microglia to identify roles of each transcriptional factor (TF) in regulating microglial activation, using an integrated transcriptomics, bioinformatics and experimental validation approach. Efficient (>70%) silencing of TFs in microglia revealed reciprocal regulation between each TF specifically following pro‐inflammatory activation. Neuroinflammatory transcriptomic profiling of microglia coupled with qPCR validation revealed distinct gene clusters with unique patterns of regulation by each TF, which were independent of LPS stimulation. While all three TFs (especially IRF1 and LXRβ) positively regulated core DAM genes (Apoe, Axl, Clec7a, Tyrobp, and Trem2) as well as homeostatic and pro‐inflammatory DAM genes, LPS, and IFNγ increased pro‐inflammatory DAM but suppressed homeostatic and anti‐inflammatory DAM gene expression via an Erk1/2‐dependent signaling pathway. IRF1 and LXRβ silencing suppressed microglial phagocytic activity for polystyrene microspheres as well as fAβ42 while IRF1 silencing strongly suppressed production of pro‐inflammatory cytokines in response to LPS. Our studies reveal complex transcriptional regulation of homeostatic and DAM profiles whereby IRF1, LXRβ, and CEBPα positively regulate both pro‐ and anti‐inflammatory DAM genes while activating stimuli independently augment pro‐inflammatory DAM responses and suppress homeostatic and anti‐inflammatory responses via Erk signaling. This framework can guide development of therapeutic immuno‐modulatory strategies for neurodegeneration.

Microglial NMDA receptors drive pro‐inflammatory responses via PARP‐1/TRMP2 signaling

“…Intriguingly, ERK1/2 was recently shown to mediate microglial phenotype that increases pro‐inflammatory while suppressing anti‐inflammatory and homeostatic genes in disease associated‐microglia (DAM; Gao et al, ), supporting our notion of microglial polarization to extended pro‐inflammatory phenotype. Our cytokine data demonstrates that activation of PARP‐1/TRPM2 axis promotes pro‐inflammatory cytokines (ICAM1, IL‐1α, IL‐7, GM‐CSF, M‐CSF, MIG), without promoting anti‐inflammatory cytokines (IL‐10, IL‐13, MIP1α, MIP1γ, PF‐4) in order to retain homeostasis.…”

Microglial ERK signaling is a critical regulator of pro-inflammatory immune responses in Alzheimer’s disease

“…In particular, ERK was found to positively regulate several canonical DAM genes, including Ccl3, Ch25h, Spp1, Igf1, and Itgax (Supplemental Figure S2) and pro-inflammatory DAM genes, including Ccl2, Irf1, Tnf, Slamf9, and Cd69 (Supplemental Figure S3). We also calculated a synthetic eigenvalue to represent overall expression of homeostatic, anti-inflammatory DAM and pro-inflammatory DAM profiles across each experimental condition using previously identified hub genes (Kme>0.75) of each microglial profile that were present in our Nanostring dataset (Dai et al, 2018; Gao et al, 2019). We confirmed that inhibition of ERK activity suppresses homeostatic, anti-inflammatory as well as pro-inflammatory DAM gene expression while IFNγ increases pro-inflammatory DAM and suppresses anti-inflammatory DAM gene expression (Figure 4d).…”

“…Primary microglia cultures were prepared from P0-3 post-natal mouse brains as previously described (Gao et al, 2019). Briefly, brain tissue was harvested from C57BL/6J mice (P0 to P3) and digested with Trypsin for 15 min.…”

“…The effect of ERK inhibition and inflammatory stimulation on microglial phagocytosis of fluorescent fibrillar amyloid-β 42 (fAβ42) was determined through flow cytometry as previously described (Gao et al, 2019). Briefly, fAβ42 peptide conjugated to HiLyte Fluor 488 was mixed with 1% NH 4 OH and diluted in 1xPBS to achieve a concentration of 100µM, after which it was incubated in the dark for 6 days at RT.…”

“…qRT-PCR validation studies were performed using RNA isolated from primary microglia that were cultured in the presence or absence of ERK inhibitor and IFNγ to mirror the conditions used in NanoString studies, using protocols described previously (Gao et al, 2019). RNA was isolated from microglia, and RNA concentration and purity were determined using the NanoDrop 2000 spectrophotometer.…”