Porphyromonas gingivalis bypasses epithelial barrier and modulates fibroblastic inflammatory response in an in vitro 3D spheroid model
Porphyromonas gingivalis bypasses epithelial barrier and modulates fibroblastic inflammatory response in an in vitro 3D spheroid model
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DOI：
10.1038/s41598-018-33267-4
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发表时间：
2018-10-08
影响因子：
4.6
通讯作者：
Huck, Olivier
中科院分区：
文献类型：
--
作者：
Bugueno, Isaac Maximiliano;Batool, Fareeha;Huck, Olivier
牙龈卟啉单胞菌诱导的炎症效应主要在单层培养细胞中研究。本研究的目的是开发一个三维球体模型的牙龈，考虑到上皮细胞成纤维细胞的相互作用。采用悬滴法培养人牙龈上皮细胞（ECs）和人口腔成纤维细胞（FBs），制备三维微组织（MT），通过组织学切片分析其结构，并通过扫描电镜和透射电镜（SEM和TEM）观察细胞间的相互作用。MT被牙龈卟啉单胞菌感染，并通过免疫组织化学和RT-qPCR评估对细胞死亡（Apaf-1、半胱天冬酶-3）、炎性标志物（TNF-α、IL-6、IL-8）和细胞外基质组分（Col-IV、E-钙粘蛋白、整合素β 1）的影响。将结果与实验性牙周炎和人类牙龈活检中原位观察到的结果进行比较。MT表现出明确的空间组织，其中EC被组织在一个外部的细胞多层，而FB构成的核心。MT的感染证明牙龈卟啉单胞菌能够绕过上皮屏障，以到达成纤维细胞核心并诱导球状体结构的解体。在成纤维细胞核心中观察到细胞死亡增加。这种三维模型的发展可能有助于确定EC-FB相互作用对牙周宿主免疫反应的作用，并评估新的治疗方法的疗效。
Porphyromonas gingivalis-induced inflammatory effects are mostly investigated in monolayer cultured cells. The aim of this study was to develop a 3D spheroid model of gingiva to take into account epithelio-fibroblastic interactions. Human gingival epithelial cells (ECs) and human oral fibroblasts (FBs) were cultured by hanging drop method to generate 3D microtissue (MT) whose structure was analyzed on histological sections and the cell-to-cell interactions were observed by scanning and transmission electron microscopy (SEM and TEM). MTs were infected by P. gingivalis and the impact on cell death (Apaf-1, caspase-3), inflammatory markers (TNF-alpha, IL-6, IL-8) and extracellular matrix components (Col-IV, E-cadherin, integrin beta 1) was evaluated by immunohistochemistry and RT-qPCR. Results were compared to those observed in situ in experimental periodontitis and in human gingival biopsies. MTs exhibited a well-defined spatial organization where ECs were organized in an external cellular multilayer, while, FBs constituted the core. The infection of MT demonstrated the ability of P. gingivalis to bypass the epithelial barrier in order to reach the fibroblastic core and induce disorganization of the spheroid structure. An increased cell death was observed in fibroblastic core. The development of such 3D model may be useful to define the role of EC-FB interactions on periodontal host-immune response and to assess the efficacy of new therapeutics.