Supplementary Material for: Hierarchical Maturation of Innate Immune Defences in Very Preterm Neonates

<b><i>Background:</i></b> Preterm neonates are highly vulnerable to infection. <b><i>Objectives:</i></b> To investigate the developmental contribution of prematurity, chorioamnionitis and antenatal corticosteroids (ANS) on the maturation of neonatal microbial pathogen recognition responses. <b><i>Methods:</i></b> Using standardized protocols, we assayed multiple inflammatory cytokine responses (IL-1β, IL-6, TNF-α and IL-12/23p40) to three prototypic Toll-like receptor (TLR) agonists, i.e. TLR4 (lipopolysaccharide), TLR5 (flagellin) and TLR7/8 (R848), and to the non-TLR retinoic acid-inducible gene I (RIG-I)-like receptor agonist, in cord blood mononuclear cells from neonates born before 33 weeks of gestation and at term. <b><i>Results:</i></b> TLR responses develop asynchronously in preterm neonates, whereby responses to TLR7/8 were more mature and were followed by the development of TLR4 responses, which were also heterogeneous. Responses to TLR5 were weakest and most immature. Maturity in TLR responses was not influenced by sex. Overall, we detected no significant contribution of ANS and chorioamnionitis to the developmental attenuation of either TLR or RIG-I responses. <b><i>Conclusions:</i></b> The maturation of anti-microbial responses in neonates born early in gestation follows an asynchronous developmental hierarchy independently of an exposure to chorioamnionitis and ANS. Our data provide an immunological basis for the predominance of specific microbial infections in this age group.

**研究背景：** 早产新生儿（preterm neonates）对感染具有极高易感性。
**研究目的：** 探讨早产、绒毛膜羊膜炎（chorioamnionitis）以及产前糖皮质激素（ANS）对新生儿微生物病原体识别应答成熟过程的影响。
**研究方法：** 本研究采用标准化实验方案，以胎龄33周以下及足月新生儿的脐带血单个核细胞（cord blood mononuclear cells）为研究对象，检测其针对三类典型Toll样受体（Toll-like receptor, TLR）激动剂——TLR4（脂多糖lipopolysaccharide）、TLR5（鞭毛蛋白flagellin）与TLR7/8（R848），以及非Toll样受体类视黄酸诱导基因I（RIG-I）样受体激动剂的多种炎性细胞因子应答水平，检测指标包括IL-1β、IL-6、TNF-α及IL-12/23p40。
**研究结果：** 早产新生儿的TLR应答发育呈现异步性特征：针对TLR7/8的应答成熟度更高，随后发育的TLR4应答亦存在异质性；针对TLR5的应答则最为微弱且成熟度最低。TLR应答的成熟度不受性别影响。整体而言，本研究未发现ANS与绒毛膜羊膜炎对TLR或RIG-I应答的发育性衰减存在显著作用。
**研究结论：** 胎龄早期出生的新生儿其抗微生物应答的成熟过程遵循异步发育层级，且该过程不受绒毛膜羊膜炎与ANS暴露的影响。本研究数据为该年龄段新生儿特定微生物感染的高发态势提供了免疫学依据。