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.