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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 171

stimulation with LPS, triggering nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB)
inflammatory signaling cascades, increase of protein levels of the proinflammatory factors NOS2 (the
enzyme that catalyzes the production of NO) and cyclooxygenase 2b (COX2), and secretion of TNFα.
Importantly, they are also responsive to the anti-inflammatory effects of interleukin 4 and increasing levels
of Arginase 1, demonstrating that SIM-A9 cells can switch between pro- and anti-inflammatory states.
Finally, they also exhibit phagocytic uptake of bacterial particles and fluorescently-labeled amyloid-β (Aβ).

Additionally, we chose LPS as the immune stimulant because: (1) intraperitoneal and/or intracranial
administration of LPS in mice led to increased microglial activation, neuroinflammation, neuronal
[8]
loss including loss of dopaminergic neurons in the substantia nigra in a mouse model of PD , and
[36]
cognitive and neurological deficits ; (2) aged individuals show increased systemic levels of LPS in the
[38]
[37]
bloodstream , which are associated with increased inflammation and microglial activation ; and (3) in
humans, TLR4 activation is linked to age-related pathologies such as PD and AD [39-41] ; thus, LPS serves as a
relevant aging-related physiological immune stimulant.
KD of Phf15 resulted in a significant reduction in Phf15 mRNA transcript levels of 52% and 60% for cell
lines shPhf15-1 and shPhf15-2, respectively [Figure 2A], as well as significantly increased mRNA expression
of Tnfα, a proinflammatory cytokine, after KD with shPhf15-2 at 0, 1, 6, and 12 h after LPS stimulation
[Figure 2B].

Similarly, mRNA levels of Nos2 were significantly elevated at 1, 6, and 12 h post stimulation for shPhf15-2 and
0, 6, and 12 h for shPhf15-1 [Figure 2D]. Overall, our experiments show that ~50%-60% KD, the equivalent of
a “heterozygous” condition, results in increased expression of proinflammatory mediators over a 12-h time
course that resolves and falls below control levels by 24 h after immune stimulation. Importantly, microglial
inflammatory function was elevated in the absence of immune stimulation (0 h time point, Figure 2B and D,
and no stimulation condition, Figure 2C and E), suggesting a loss of repressive mechanisms that inhibit basal
state inflammatory gene transcription.

We repeated the immune activation time course experiments in Phf15 KD cells using two separate immune
stimulants specific to two distinct Toll-like receptors to test the pathway specificity of the inflammatory
response: CpG ODN, a synthetic bacterial and viral DNA mimic that targets TLR9, and Poly(I:C), a
synthetic viral dsRNA mimic that targets TLR3. While TLR4 uses both the myeloid differentiation primary
response 88 (MyD88) and TIR-domain-containing adapter-inducing interferon-β (TRIF) downstream
adapters to transduce its inflammatory cascade, TLR9 and TLR3 utilize MyD88 and TRIF, respectively
[Supplementary Figure 1] [42,43] .

Immune stimulation with CpG ODN and Poly(I:C) both yielded similar results to those obtained with LPS
stimulation [Supplementary Figures 2 and 3, respectively], denoting no adapter selectivity and confirming
that Phf15 antagonizes inflammatory gene expression downstream of both the MyD88 and TRIF signaling
pathways.

Genetic deletion of Phf15 increases the magnitude and prolongs the duration of the microglial
inflammatory response
Since our KD strategy resulted in ~50% reduction in Phf15 mRNA expression, we next performed CRISPR/
Cas9-mediated genetic deletion of Phf15 in SIM-A9 microglial cells followed by immune activation with
LPS. KO of Phf15 [Figure 3A] resulted in significantly increased LPS-induced expression of Tnf α [Figure 3B],
IL-1 β [Figure 3D], and Nos2, albeit to a lesser extent [Figure 3F], over a 24-h time course. Importantly, mRNA
levels of both Tnf α and IL-1 β remained elevated at 24 h compared to control cells, denoting a prolonged
inflammatory response and failure to return to steady-state. mRNA expression of Nos2 showed a significant