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Notably, primary cultures of immature microglia from female, but not male, whole mouse brain at PND 3
[52]
have been shown to exhibit increased expression of proinflammatory cytokines, such as IL-6 and IL-1β .
While indo was originally administered in the present study to inhibit the masculinizing effect of microglia-
secreted PGE2 to elucidate its role in the sex-biased epigenetic modulation in developing males, the anti-
inflammatory mechanism of action also inhibited pro-inflammatory cytokines like IL-1β. Thus, indo-
related suppression of these pro-inflammatory signals critical for postnatal female microglial development
also underscored the role of not only PGE2, but pro-inflammatory cytokines as well, in the epigenetic
regulation of microglia in males and females, respectively. While suppression of PGE2 via indo exposure
did not detectably “feminize” the vulnerability of male microglia to heightened neuroimmune epigenetic
modification, indo exposure in females did reveal how timely modulation of microglia phenotype during a
critical window of postnatal development significantly altered certain epigenetic cascades in neuroimmune
development. For example, the significant and persistent upregulation of miR-132 in indo-exposed Tg
females at PND 10 [Figure 3C] and PND 21 [Figure 4C] implies the presence of heightened sensitivity to
cytokine-mediated miR-132 epigenetic reprogramming associated with neurite sprouting, synaptogenesis,
and neurotransmission. Although miR-132 is upregulated for the first 2-4 weeks of postnatal development
[27]
during a period of NMDA-dependent synaptic pruning , additional upregulation may disrupt the
miR-132-promoted positive feedback loop in NMDA depolarization, while interfering in GABAergic
[53]
interneuron maturation . These data further corroborate the heightened GxE-related vulnerability and
female bias for the epigenetic reprogramming of microglia during this postnatal window.

Further, given reports that the majority of microRNAs only persist for about five days without external
[23]
stimulus , this upregulation of microRNA profiles persisting until or occurring at PND 21 would suggest
the continuation of an atypical epigenetic stimulus despite the cessation of exposure. In further validation
of the GxE female-specific vulnerability, postnatal exposures increased miR-124 at PND 10 in WT females
[Figure 3A] but PND 21 was rectified via significant downregulation with Pb or indo exposure [Figure 4A].
This was in contrast to Tg females exposed to Pb, in which upregulation of miR-124 did not occur until
PND 21 [Figure 4C], indicating that a rectifying epigenetic signal for Tg miR-124 was not present for a
similarly-vulnerable population of immature female microglia as with WT females despite a parallel trend
for significant reductions in hippocampal DAP12 expression [Figure 2]. The current study is limited by
a hypothesis-driven bias in the neurimmiRs selected for analysis in our GxE model for AD, and future
analyses might benefit from wider microarrays to further characterize this window of heightened sensitivity
to epigenetic reprogramming for female neuroimmune development. While little is known regarding
this subtle and life-long maladaptive neuroimmune phenotype, the data presented here demonstrate that
changes to the epigenetic landscape are detectable early in postnatal development and are significantly
biased by sexual dimorphism for neuroimmune development during this time.

As hypothesized, our study demonstrated a clear female bias in the exacerbating effect of GxE interactions
in early epigenetic regulation promoting later life maladaptibility and neuroimmune dysfunction,
[4,5]
which we have previously correlated with heightened susceptibility to AD . We have also shown that,
although the most detrimental phenotype occurred in Pb-exposed Tg females, there was evidence to
suggest that this DOAD model may be useful in investigating toxicant- and timing-specific windows of
vulnerabilities for other sex-biased adult diseases. Ultimately, investigation of these early perturbations to
epigenetic regulation of cellular phenotype could reveal thresholds of adaptability consequent to atypical
developmental conditions, and potential identification of biomarkers for susceptibility.

DECLARATIONS
Acknowledgments
The authors would like to acknowledge Ishmael Gomez for his assistance with portions of immuno-
histochemistry. We would also like to acknowledge Dr. Qun Lu and the Harriet and John Wooten