DISCUSSION
The role of sex-specific factors in disease incidence, pathophysiology
and outcomes are increasingly recognised. Males and females show robust
differences in their susceptibility to autoimmunity, cancers, and at a
less pronounced magnitude, infectious diseases6.
Pathogen-associated damage, including delayed clearance, is associated
with male-biased infectious diseases23. Male neonates,
especially when born extremely preterm (<32 weeks), exhibit
significantly poorer short- and long-term outcomes compared to females
of matched gestational ages and weights1.
Additionally, a significant association between male sex and mortality
among blood culture positive preterm neonates has been
described25. Recent data from our group has
demonstrated GM-CSF, IL-10, IL-1RA and IL-8 increased significantly
following endotoxin stimulation in healthy female neonatal whole blood
but not in males26.
In this pilot study, we assessed the expression levels of CD11b, ER-α,
ER-β and TLR4 surface receptors on CD14+ monocytes in female versus male
umbilical cord blood and adult peripheral blood. Basal cell surface
expression did not differ between male versus female neonates, or
adults. Treating the cells with E2, ICI 182,780 (selective ER
antagonist), and LPS in various combinations did not alter cell surface
expression of CD11b or TLR4 in either adults or neonates or between
sexes. There was a trend towards higher ER-α and ER-β expression in
adult females compared to adult males and treatment with ICI 182,780
alone resulted in significantly higher ER-β expression. We further
demonstrated that E2 attenuates LPS-induced ROS production from UCB
mononuclear cells to a greater extent in females versus male neonates.
This effect was not seen in adults. The data presented here support our
hypothesis that E2 may affect immune function in neonates by potentially
exerting an anti-inflammatory role, however we did not establish clear
effects of E2 treatment on cell surface receptor expression. Based on
these observations we speculate that estrogenic responses could
contribute to sex-specific responses to infection and inflammation.
The neonatal immune system is distinct. It relies largely on innate
mechanisms due to an underdeveloped adaptive system and age-specific
effector functions of immune regulators27. In this
study we were interested in the behaviour of monocytes. Monocytes are
critical cells in the neonatal immune system which originate from
progenitors in the bone marrow and circulate in the bloodstream until
they are attracted to inflammatory signals where they differentiate into
macrophage or dendritic cell populations5. E2 has been
reported to be directly anti-inflammatory in monocyte populations by
reducing IL-8 production and therefore neutrophil recruitment through
ER-dependent mechanisms28. Interestingly, a
bipotential effect of E2 has also been described on human monocytes; at
low doses, levels of pro-inflammatory cytokines IL-1β and TNF-α are
increased, however at high doses of E2 their production is
reduced29. In newborn mononuclear cells, E2 has been
shown to be comparable to hydrocortisone in reducing inflammatory
cytokine production10.
Oxidative stress and inflammation are directly implicated in neonatal
lung disease, brain injury, and retinopathy, amongst other
pathologies30. Diaz-Castro et al. report higher
levels of in vivo biomarkers of oxidative stress and inflammatory
signalling in healthy male neonates compared to females in the immediate
postpartum period, which suggests females may have an improved tolerance
to perinatal oxidative stress16. Estrogens have been
described to have antioxidant properties31,32 and
erythrocyte glutathione peroxidase is positively correlated with serum
estrogens33. Furthermore, E2 upregulates MAP kinases
and NFκB, pathways that lead to upregulation of superoxide dismutase
and glutathione peroxidase gene expression34. In our
study, a main aim was to evaluate on the role of E2 on mitigating
oxidative stress. We saw reduced ROS production from mononuclear cells
from females incubated with E2 and LPS in combination compared to male
neonates. Surprisingly, this effect was not seen in adults, however the
sample size is small.
We did not show major differences in cell surface receptor modulation
with E2, ICI 287,780, or LPS treatment combinations, potentially
indicating that the observed decreased ROS levels in LPS and E2 treated
female versus male monocytes is not ER-mediated. Similar data have been
reported from other groups, suggesting that E2 reduces LPS-induced
cytokine production without modulating monocyte CD11b
expression35. We demonstrated higher ER-β expression
in adult females compared to males with ICI 182,780 treatment alone. The
relevance of this finding to sexual disparities in immune function is
unclear. ICI 182,780 has previously been shown to differentially effect
the expression of ER-α and ER-β in animal models however, suggesting
that these receptors are regulated by different
mechanisms36. Whether this relationship exists in
immune cells is not fully elucidated and addressing these questions are
beyond the scope of this study.
This study has several limitations. Firstly, we acknowledge the results
are marginal in their significance. Also, no differences were detected
between assays performed on term umbilical cord and adult peripheral
blood monocytes. Future work should aim to cover a broader clinical
cohort, including preterm infants. We are also aware that in this study
the entire patient cohort consisted of neonates born by CS without
labour. Since labour promotes LPS-responsiveness37, it
would be interesting to determine whether it also affects estrogenic
responses in an inflammatory context. In addition, UCB was collected for
use in this study. It is increasingly acknowledged that UCB may have
distinct LPS responsiveness compared to whole blood38.
In conclusion, the novel observations from this study regarding E2
reducing LPS-induced ROS production in female versus male UCB
mononuclear cells provide a basis for future mechanistic and
disease-related studies in larger cohorts. Ex vivo hormone
treatment of preterm immune cells stratified by sex coupled with modern
techniques will elucidate further avenues for investigation of
immunomodulation, improve our understanding of perinatal diseases and
identify targets for precision medicine.