Supplementary Material for: Immune Synaptopathy and Sex Differences in Recognition Memory from Neonatal Staphylococcus epidermidis Infection

Background: Staphylococcus epidermidis (SE) is a predominant hospital-acquired bacterium leading to late-onset sepsis in preterm infants. Recent findings have suggested that postnatal S. epidermidis infection is associated with short-term neurodevelopmental consequences. However, the potential effects of postnatal SE infection on long-term neuronal plasticity and cognitive functions, which are sensitive to early-life brain insults, remain unclear. In light of these findings, we investigated the effects of postnatal SE infection on recognition memory function using a neonatal mouse model. Methods: On postnatal day 4, male and female C57Bl/6 mice were injected intraperitoneally with either 3.5 × 107 colony-forming units of SE or sterile saline. On postnatal day 45 (± 5 days), the mice were subjected to the Novel Object Recognition Test (NORT) to assess recognition memory function. Following NORT, the brains of the mice were collected for neuronal plasticity analyses by considering maturation of neurons and 3-D analysis of synaptic plasticity and hippocampal, measuring the Nerve growth factor (NGF) expression. Results: Postnatal SE infection induced long-term, sex-specific effects on recognition memory and hippocampal neuroplasticity. Female SE-infected mice showed enhanced recognition memory, whereas males showed no significant difference in the recognition memory after neonatal SE infection. At the cellular level, both sexes displayed a significant decrease in doublecortin-positive (DCX⁺) neurons in the dentate gyrus after SE infection, indicating impaired neuroplasticity. However, male mice showed increased spine density, particularly of immature thin spines and disrupted spatial organization of spines, while females demonstrated no change in spines. Notably, SE infection elevated hippocampal NGF expression in males, but not in females, suggesting sex-specific molecular responses that may contribute to the observed differences in neuroplasticity and cognitive outcomes. Conclusion: This study demonstrates that postnatal SE infection induces long-lasting, sex-specific changes in recognition memory. Early-life immune activation disrupted hippocampal neuroplasticity, with males showing greater vulnerability. These findings indicate distinct neurodevelopmental trajectories shaped by neonatal immune challenges in preterm infants, with implications for understanding sex-specific cognitive outcomes.