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.Background: 表皮葡萄球菌（Staphylococcus epidermidis, SE）是引发早产婴儿晚发性败血症的主要医院获得性致病菌。近期研究表明，产后表皮葡萄球菌感染与短期神经发育结局相关。然而，产后SE感染对长期神经可塑性及认知功能的潜在影响——这类功能对早期脑损伤较为敏感——仍有待阐明。鉴于此，我们利用新生小鼠模型探究了产后SE感染对识别记忆功能的影响。 Methods: 出生后第4天，雌雄C57Bl/6小鼠分别腹腔注射3.5×10^7菌落形成单位的SE或无菌生理盐水。出生后第45天（±5天），采用新物体识别实验（Novel Object Recognition Test, NORT）评估小鼠的识别记忆功能。新物体识别实验结束后，收集小鼠脑组织开展神经可塑性分析，包括神经元成熟度评估、突触可塑性的三维分析以及海马组织神经生长因子（Nerve growth factor, NGF）的表达水平检测。 Results: 产后SE感染可对识别记忆与海马神经可塑性产生长期的性别特异性影响。雌性SE感染小鼠的识别记忆能力显著增强，而雄性小鼠在新生期感染SE后，其识别记忆未出现明显变化。在细胞层面，SE感染后雌雄小鼠的齿状回中双皮质素阳性（doublecortin-positive, DCX⁺）神经元数量均显著减少，提示神经可塑性受损。但雄性小鼠的树突棘密度（尤其是未成熟细树突棘）有所增加，且树突棘的空间排布出现紊乱，而雌性小鼠的树突棘无明显变化。值得注意的是，SE感染会升高雄性小鼠的海马NGF表达水平，但雌性小鼠未出现该变化，这表明性别特异性的分子应答或为神经可塑性与认知结局差异的成因。 Conclusion: 本研究证实，产后SE感染可诱导识别记忆出现长期且性别特异性的改变。早期免疫激活会破坏海马神经可塑性，雄性小鼠的易感性更强。上述研究结果表明，早产婴儿在新生儿期遭遇免疫挑战后，其神经发育轨迹存在性别差异，该发现有助于理解性别特异性的认知结局。