Choltus Héléna, Lavergne Marilyne, Belville Corinne, Gallot Denis, Minet-Quinard Régine, Durif Julie, Blanchon Loïc, Sapin Vincent
CNRS, INSERM, GReD, Université Clermont Auvergne, Clermont-Ferrand, France.
CHU de Clermont-Ferrand, Obstetrics and Gynecology Department, Clermont-Ferrand, France.
Front Physiol. 2020 Jun 25;11:581. doi: 10.3389/fphys.2020.00581. eCollection 2020.
Sterile inflammation has been shown to play a key role in the rupture of the fetal membranes (FMs). Moreover, an early and exacerbated runaway inflammation can evolve into a preterm premature rupture of membranes and lead to potential preterm birth. In this context, we investigated the receptor for advanced glycation end products (RAGE), an axis implied in physiological sterile inflammation, in conjunction with two major ligands: AGEs and High-Mobility Group Box 1 (HMGB1). Our first objective was to determine the spatiotemporal expression profiles of the different actors of the RAGE-signaling axis in human FMs, including its intracellular adaptors Diaphanous-1 and Myd88. Our second goal was to evaluate the functionality of RAGE signaling in terms of FMs inflammation.
The presence of the actors (RAGE, HMGB1, Myd88, and Diaphanous-1) at the mRNA level was investigated by reverse transcription quantitative polymerase chain reaction (RT-qPCR) in the human amnion and choriodecidua at the three trimesters and at term. Measurements were conducted at two distinct zones: the zone of intact morphology (ZIM) and the zone of altered morphology (ZAM). Then, proteins were quantified using Western blot analysis, and their localization was evaluated by immunofluorescence in term tissues. In addition, pro-inflammatory cytokine secretion was quantified using a Multiplex assay after the treatment of amnion and choriodecidua explants with two RAGE ligands (AGEs and HMGB1) in the absence or presence of a RAGE inhibitor (SAGEs).
The FMs expressed the RAGE-signaling actors throughout pregnancy. At term, RNA and protein overexpression of the RAGE, HMGB1, and Diaphanous-1 were found in the amnion when compared to the choriodecidua, and the RAGE was overexpressed in the ZAM when compared to the ZIM. The two RAGE ligands (AGEs and HMGB1) induced differential cytokine production (IL1β and TNFα) in the amnion and choriodecidua.
Considered together, these results indicate that RAGE signaling is present and functional in human FMs. Our work opens the way to a better understanding of FMs weakening dependent on a RAGE-based sterile inflammation.
无菌性炎症已被证明在胎膜破裂中起关键作用。此外，早期且加剧的失控性炎症可发展为胎膜早破，并导致潜在的早产。在此背景下，我们研究了晚期糖基化终产物受体（RAGE），这是一个参与生理性无菌性炎症的轴，及其两个主要配体：晚期糖基化终产物（AGEs）和高迁移率族蛋白B1（HMGB1）。我们的首要目标是确定RAGE信号轴不同参与者在人胎膜中的时空表达谱，包括其细胞内衔接蛋白Diaphanous-1和髓样分化因子88（Myd88）。我们的第二个目标是从胎膜炎症方面评估RAGE信号的功能。
通过逆转录定量聚合酶链反应（RT-qPCR）研究三个孕期及足月时人羊膜和绒毛膜蜕膜中各参与者（RAGE、HMGB1、Myd88和Diaphanous-1）在mRNA水平的存在情况。在两个不同区域进行测量：形态完整区（ZIM）和形态改变区（ZAM）。然后，使用蛋白质印迹分析对蛋白质进行定量，并通过足月组织中的免疫荧光评估其定位。此外，在用两种RAGE配体（AGEs和HMGB1）处理羊膜和绒毛膜蜕膜外植体后，在有或无RAGE抑制剂（SAGEs）的情况下，使用多重分析法定量促炎细胞因子的分泌。
胎膜在整个孕期均表达RAGE信号参与者。足月时，与绒毛膜蜕膜相比，羊膜中RAGE、HMGB1和Diaphanous-1的RNA和蛋白质表达上调，与ZIM相比，ZAM中RAGE表达上调。两种RAGE配体（AGEs和HMGB1）在羊膜和绒毛膜蜕膜中诱导了不同的细胞因子产生（IL-1β和TNF-α）。
综合考虑，这些结果表明RAGE信号在人胎膜中存在且具有功能。我们的工作为更好地理解依赖基于RAGE的无菌性炎症导致的胎膜弱化开辟了道路。
