A non-reversible state of epithelial to mesenchymal transition (EMT) at term accumulates proinflammatory mesenchymal cells and predisposes fetal membrane to weakening prior to delivery at term. We investigated the induction of EMT in amnion epithelial cells (AEC) in response to inflammation and infection associated with spontaneous preterm birth (SPTB). For this, membranes from SPTB were screened for EMT markers. Primary AEC in culture were treated with TNF-α (10 and 50 ng/mL) and LPS (50 and 100 ng/mL) for 72 h. Cell shape index (SI) was determined based on morphological shift (microscopy followed by ImageJ software analysis). Immunocytochemistry and Western blot assessed changes in epithelial markers (cytokeratin-18 and E-cadherin) and mesenchymal markers (vimentin and N-cadherin). Involvement of transforming growth factor beta (TGF-β) in EMT induction and EMT associated inflammation was tested using specific markers (Western blot) and by measuring MMP9 (ELISA), respectively. We report that PTB is associated with fetal membrane EMT. TNF-α produced dose- and time-dependent induction of EMT; within 24 h by 50 ng/mL and after 72 h by 10 ng/mL. AEC showed mesenchymal morphology, lower E-cadherin, higher vimentin and N-cadherin and higher MMP9 compared to control. TNF-α-induced EMT was not associated with canonical TGF-β pathway. LPS, regardless of dose or time, did not induce EMT in AEC. We conclude that PTB with intact membranes is associated with EMT. Our data suggest that inflammation, but not infection, is associated with non-canonical activation of EMT and inflammation that can predispose membrane to undergo weakening.

中文翻译：

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炎症，但不是感染，在人羊膜上皮细胞中诱导 EMT

足月时上皮向间充质转化 (EMT) 的不可逆状态会积聚促炎间充质细胞，并使胎膜在足月分娩前容易变弱。我们研究了羊膜上皮细胞 (AEC) 中 EMT 的诱导，以响应与自发性早产 (SPTB) 相关的炎症和感染。为此，针对 EMT 标记筛选了来自 SPTB 的膜。培养物中的原代 AEC 用 TNF-α（10 和 50 ng/mL）和 LPS（50 和 100 ng/mL）处理 72 小时。细胞形状指数 (SI) 基于形态变化（显微镜检查，然后是 ImageJ 软件分析）确定。免疫细胞化学和蛋白质印迹评估了上皮标志物（细胞角蛋白 18 和 E-钙粘蛋白）和间充质标志物（波形蛋白和 N-钙粘蛋白）的变化。分别使用特异性标记物（蛋白质印迹）和测量 MMP9（ELISA）来测试转化生长因子 β（TGF-β）在 EMT 诱导和 EMT 相关炎症中的参与。我们报告 PTB 与胎膜 EMT 相关。TNF-α 产生 EMT 的剂量和时间依赖性诱导；24 小时内降低 50 ng/mL，72 小时后降低 10 ng/mL。与对照相比，AEC 显示出间充质形态、较低的 E-钙粘蛋白、较高的波形蛋白和 N-钙粘蛋白以及较高的 MMP9。TNF-α 诱导的 EMT 与典型的 TGF-β 通路无关。无论剂量或时间如何，LPS 都不会在 AEC 中诱导 EMT。我们得出结论，具有完整膜的 PTB 与 EMT 相关。我们的数据表明，炎症，而不是感染，与 EMT 的非典型激活和炎症有关，这可能使膜易于减弱。