Supplementary Material for: Olfactomedin-4-Positive Neutrophils in Neonates: Link to Systemic Inflammation and Bronchopulmonary Dysplasia

<b><i>Introduction:</i></b> Little is known about the interplay between neutrophil heterogeneity in neonates in health and disease states. Olfactomedin-4 (OLFM4) marks a subset of neutrophils that have been described in adults and pediatric patients but not neonates, and this subset is thought to play a role in modulating the host inflammatory response. <b><i>Methods:</i></b> This is a prospective cohort of neonates who were born between June 2020 and December 2021 at the University of Cincinnati Medical Center NICU. Olfactomedin-4-positive (OLFM4+) neutrophils were identified in the peripheral blood using flow cytometry. <b><i>Results:</i></b> OLFM4+ neutrophil percentage was not correlated with gestational age or developmental age. Neonates with sepsis had a higher percentage than those without the condition, 66.9% (IQR 24.3–76.9%) versus 21.5% (IQR 10.6–34.7%), respectively, <i>p</i> = 0.0003. At birth, a high percentage of OLFM4+ neutrophils was associated with severe chorioamnionitis at 49.1% (IQR 28.2–61.5%) compared to those without it at 13.7% (IQR 7.7–26.3%), <i>p</i> &lt; 0.0001. Among neonates without sepsis, the percentages of OLFM4+ neutrophils were lower in the BPD/early death group compared to those without BPD, 11.8% (IQR 6.3–29.0%) versus 32.5% (IQR 18.5–46.1%), <i>p</i> = 0.003, and this retained significance in a multiple logistic regression model that included gestational age, birthweight, and race. <b><i>Conclusion:</i></b> This is the first study describing OLFM4+ neutrophils in neonates and it shows that this neutrophil subpopulation is not influenced by gestational age but is elevated in inflammatory conditions such as sepsis and severe chorioamnionitis, and lower percentage at birth is associated with developing bronchopulmonary dysplasia.

**引言：** 目前关于健康与疾病状态下新生儿中性粒细胞异质性之间的相互作用尚不清楚。嗅素蛋白4（Olfactomedin-4, OLFM4）可标记一类中性粒细胞亚群，该亚群在成人及儿科患者中已有报道，但尚未见新生儿群体的相关描述，且被认为在调节宿主炎症反应中发挥作用。
**方法：** 本研究为前瞻性队列研究，纳入2020年6月至2021年12月间于辛辛那提大学医学中心新生儿重症监护病房（NICU）收治的新生儿。采用流式细胞术在外周血中鉴定嗅素蛋白4阳性（OLFM4+）中性粒细胞。
**结果：** OLFM4+中性粒细胞占比与胎龄及发育年龄均无相关性。脓毒症新生儿的OLFM4+中性粒细胞占比显著高于非脓毒症新生儿，分别为66.9%（四分位间距24.3%~76.9%）与21.5%（四分位间距10.6%~34.7%），P=0.0003。出生时较高的OLFM4+中性粒细胞占比与重度绒毛膜羊膜炎相关：合并重度绒毛膜羊膜炎的新生儿该占比为49.1%（四分位间距28.2%~61.5%），未合并者为13.7%（四分位间距7.7%~26.3%），P<0.0001。在非脓毒症新生儿中，支气管肺发育不良（bronchopulmonary dysplasia, BPD）/早期死亡组的OLFM4+中性粒细胞占比显著低于非BPD组，分别为11.8%（四分位间距6.3%~29.0%）与32.5%（四分位间距18.5%~46.1%），P=0.003；且在校正胎龄、出生体重及种族的多因素logistic回归分析中，该差异仍具有统计学意义。
**结论：** 本研究为首个针对新生儿OLFM4+中性粒细胞的报道，结果显示该中性粒细胞亚群不受胎龄影响，但在脓毒症、重度绒毛膜羊膜炎等炎症性疾病中表达升高；且出生时较低的OLFM4+中性粒细胞占比与支气管肺发育不良的发生风险相关。