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Page 178 Muroy et al. Neuroimmunol Neuroinflammation 2020;7:166-82 I http://dx.doi.org/10.20517/2347-8659.2020.16

Taken together, our RNA-seq results confirm that Phf15 is a repressor of microglial inflammatory gene
expression, regulating the antiviral responses - specifically, IFN-I-dependent responses - as well as
processes related to proinflammatory cytokine production and release.

DISCUSSION
Our results show that Phf15 inhibits microglial expression of proinflammatory mediators under basal and
signal-dependent activation, regulating both the magnitude and duration of the inflammatory response.
Genetic deletion of PhfF15 in a microglial cell line followed by stimulation with LPS led to an exaggerated
proinflammatory response with increased production of Tnf α, IL-1 β, and Nos2 over a time course of 24 h.
Importantly, levels of proinflammatory factors remained elevated at 24 h, demonstrating a sustained and
prolonged response. Consistent with our LPS stimulation of TLR4 results, similar results were obtained
after TLR9 and TLR3 activation, confirming that Phf15 is a general negative regulator and controls both
the MyD88 and TRIF downstream signal transduction pathways [Supplementary Figure 1]. Overexpression
of Phf15 showed a dampened microglial inflammatory response, highlighting a reciprocal response
phenotype that further supports our loss-of-function results.

Prolonged inflammation can damage surrounding healthy tissue, eventually resulting in neuronal
degeneration and loss, and negatively affecting brain function. For example, levels of TNFα are seen to
rapidly rise in experimental models of PD and are highly toxic to dopaminergic neurons [13,14,49] . Similarly,
high levels of TNFα are a hallmark of PD in humans [50-52] . Additionally, both TNFα and IL-1β are involved
in maintaining proper synaptic plasticity at physiological levels [53,54] and overproduction of these cytokines
can result in neuronal death via excitotoxicity and cognitive dysfunction [55,56] .

Our studies further demonstrate that Phf15 can regulate both basal and signal-dependent microglial
inflammatory gene expression. KD and KO of Phf15 in microglial cell lines resulted in significantly increased
levels of proinflammatory cytokine gene expression - without stimulation and after immune activation -
while OE had the reverse effect. The inflammatory response is a tightly controlled process in immune cells
in order to protect against unintended damage to healthy tissue. Even in aged microglia, where production
and secretion of proinflammatory mediators is generally increased, this process is dependent upon
treatment with immune stimulants [9,57,58] . Increased proinflammatory cytokine gene expression without
stimulation denotes constitutive or “leaky” expression of inflammatory mediators, simulating a state of
low-grade but constant activation. Similarly, hyperresponsiveness to immune stimuli combined with a
lack of resolution of the inflammatory response can lead to a state of chronic inflammation. All three can
trigger pathological chronic inflammation in the brain, which is detrimental to brain function.

Importantly, distinct molecular mechanisms regulate transcriptional control of different phases
(“modules”) of the inflammatory response and it is noteworthy that Phf15 might be involved in regulating
several of these. Basal inflammatory function, for example, is generally regulated by co-repressors such as
nuclear receptor co-repressor (NCOR), silencing mediator of retinoid and thyroid receptors (SMRT), and
RE1 silencing transcription factor (REST) co-repressor 1 (RCOR1 or CoREST) that block poised promoters
from active transcription, preventing “leaky” expression of primary response genes (e.g., TNFα, Type I
[17]
IFNs, IL-1β, etc.) (for review, see ). Significantly increased inflammatory gene transcription under baseline
conditions, as observed in our Phf15 KD and KO experiments, suggests a loss of this repressive mechanism.

After stimulation by an activating signal, additional mechanisms can maintain quiescence by restraining
active transcription. For example, nuclear receptors such as peroxisome proliferator-activated receptor-γ,
glucocorticoid receptor, and liver X receptors can inhibit the signal-activated exchange of co-repressors
for co-activators at poised promoters, inhibiting the initiation of transcription [15,17] . Lastly, several
mechanisms regulate resolution of inflammation at the transcriptional level, including transrepression