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Muroy et al. Neuroimmunol Neuroinflammation 2020;7:166-82 I http://dx.doi.org/10.20517/2347-8659.2020.16 Page 179
mechanisms that can remove transcription factors such as NF-κB from inflammatory gene promoters,
effectively blocking expression of secondary response genes, that is, genes which require chromatin-
modification as well as protein synthesis for their induction (e.g., Nos2 and ISGs) [8,16,17] . Timely resolution
of an inflammatory response is crucial in order to limit cellular and tissue damage caused by prolonged
or chronic inflammation. Our results suggest that Phf15 may be involved in regulating all three of the
abovementioned mechanisms.

However, how might Phf15 be involved in regulating transcriptional repression of the inflammatory
response in microglia? PHF15 was first described in embryonic stem cells as an E3 ligase that directly
targets Lysine-specific demethylase 1 (LSD1, Kdm1a) - a key demethylase of histone 3 lysine 4 - for
[22]
degradation . LSD1 has been identified as a member of the CoREST co-repressor complex [59,60] , which is
[8]
required for transcriptional repression of inflammation in microglia . We therefore initially hypothesized
that increased levels of Phf15 upon aging might lead to decreased levels of LSD1 and increased microglial
inflammatory output. Our results, however, demonstrate that Phf15 itself inhibits microglial inflammatory
function; thus, its purported mechanism for inhibition is likely not via degradation of LSD1.

Interestingly, the global transcriptional changes caused by Phf15 deletion are highly similar to previously reported
[63]
age-associated transcriptional changes in microglia [9,61,62] . In particular, a study by Deczkowska et al. , found
“immune system process” and specifically “response to virus” among the most highly upregulated biological
categories for differentially expressed genes in microglia of young (2-month old) versus aged (22-month old)
[62]
mice, consistent with our results in Phf15 KO microglia. Notably, a study by Hammond et al. , which used
single-cell RNAseq to look at microglia profiles throughout the mouse lifespan, found subpopulations in
aged (P540) mouse brains which were largely: (1) inflammatory, that is, they upregulated IL-1 β, Tnf α, and
other cytokines; or (2) IFN-I-responsive, upregulating Irf7 and ISGs, particularly Ifit3, Isg15, Oasl2, interferon
induced transmembrane protein 3 (Ifitm3), and receptor transporter protein 4 (Rtp4), compared to younger
[64]
adult (P100) brains. Similarly, a recent study from the Tabula Muris Consortium , which produced a single-
cell transcriptomic atlas of 23 tissues and organs across the Mus musculus life span, confirmed that microglia
in the aged (P540 and P720) brain are enriched for IFN-I-responsive genes and upregulate a similar set of
genes including Ifit3, Irf7, Isg15, Oasl2, Ifitm3, and Rtp4. The genes upregulated by the interferon-responsive
microglia clusters in both studies are highly similar to those upregulated in our Phf15 KO cells under basal
[23]
conditions [see Figure 5A and C]. Because ISGs can modulate inflammation , it is possible that interferon-
responsive microglia could play a role in contributing to the inflammatory signature found in the aged
brain. Interestingly, among the set of downregulated genes in Phf15 KO cells at baseline and 6 h after LPS
stimulation is myocyte enhancer factor 2C (Mef2C). MEF2C is an important checkpoint inhibitor that
restrains microglial activation in response to proinflammatory insults and is lost in brain aging via IFN-I
mediated downregulation [63,65] . Thus, an increase in Phf15 expression in microglia during healthy aging could
putatively work to counteract not only microglial activation but increased IFN-I in the aged brain as well.

Notably, a recent study by Readhead et al. found that several virus species are commonly present in
[66]
the aged human brain. Among them, human herpesvirus 6A and 7 (HHV-6A and HHV-7) were highly
upregulated in the brain of AD patients and were found to modulate host genes associated with AD risk, for
example amyloid precursor protein (APP) processing. APP is the precursor molecule whose proteolysis forms
Aβ and formation of Aβ plaques has long been thought of as the driving force behind Alzheimer’s disease .
[67]
[68]
Aβ has more recently been found to have antimicrobial properties , conferring increased resistance against
infection from both bacteria and viruses . App is among the significantly upregulated genes under basal
[69]
conditions in our Phf15 KO cells (log -fold change = 1.492 and P adj < 0.0001; see Figure 5A). Upregulation of
2
App due to loss of Phf15 in mouse microglia is thus consistent with our data showing Phf15 regulation of the
antiviral microglial response.