Differential Effects of TNF (TNFSF2) and IFN-γ on Intestinal Epithelial Cell Morphogenesis and Barrier Function in Three-Dimensional Culture

BackgroundThe cytokines TNF (TNFSF2) and IFNγ are important mediators of inflammatory bowel diseases and contribute to enhanced intestinal epithelial permeability by stimulating apoptosis and/or disrupting tight junctions. Apoptosis and tight junctions are also important for epithelial tissue morphogenesis, but the effect of TNF and IFNγ on the process of intestinal epithelial morphogenesis is unknown.
Methods/Principal FindingsWe have employed a three-dimensional cell culture system, reproducing in vivo-like multicellular organization of intestinal epithelial cells, to study the effect of TNF on intestinal epithelial morphogenesis and permeability. We show that human intestinal epithelial cells in three-dimensional culture assembled into luminal spheres consisting of a single layer of cells with structural, internal, and planar cell polarity. Exposure of preformed luminal spheres to TNF or IFNγ enhanced paracellular permeability, but via distinctive mechanisms. Thus, while both TNF and IFNγ, albeit in a distinguishable manner, induced the displacement of selected tight junction proteins, only TNF increased paracellular permeability via caspase-driven apoptosis and cell shedding. Infliximab and adalumimab inhibited these effects of TNF. Moreover, we demonstrate that TNF via its stimulatory effect on apoptosis fundamentally alters the process of intestinal epithelial morphogenesis, which contributes to the de novo generation of intestinal epithelial monolayers with increased permeability. Also IFNγ contributes to the de novo formation of monolayers with increased permeability, but in a manner that does not involve apoptosis.
ConclusionsOur study provides an optimized 3D model system for the integrated analysis of (real-time) intestinal epithelial paracellular permeability and morphogenesis, and reveals apoptosis as a pivotal mechanism underlying the enhanced permeability and altered morphogenesis in response to TNF, but not IFNγ.

背景
细胞因子TNF（TNFSF2）与IFNγ是炎症性肠病的重要介导因子，可通过诱导细胞凋亡及/或破坏紧密连接（tight junctions）增强肠上皮通透性。细胞凋亡与紧密连接在上皮组织形态发生过程中同样发挥关键作用，但目前尚不清楚TNF与IFNγ对肠上皮形态发生过程的具体影响。
方法/主要结果
本研究采用可模拟体内肠上皮细胞多细胞组织结构的三维细胞培养系统，探究TNF对肠上皮形态发生及通透性的影响。研究发现，三维培养中的人肠上皮细胞可组装形成具有结构极性、内部极性及平面细胞极性（planar cell polarity）的单层细胞腔面球体。将已形成的腔面球体暴露于TNF或IFNγ后，细胞旁通透性显著升高，但二者的作用机制存在差异。具体而言，尽管TNF与IFNγ均可诱导特定紧密连接蛋白的移位，但二者的作用模式存在明显区别：仅TNF可通过半胱天冬酶（caspase）介导的细胞凋亡与细胞脱落途径提高细胞旁通透性。英夫利昔单抗（Infliximab）与阿达木单抗（adalumimab）可抑制TNF的上述作用。此外，本研究证实TNF可通过调控细胞凋亡从根本上改变肠上皮形态发生过程，进而促使通透性升高的肠上皮单层结构从头生成。IFNγ同样可促使通透性升高的单层结构从头形成，但其作用机制不涉及细胞凋亡。
结论
本研究构建了可用于（实时）整合分析肠上皮细胞旁通透性与形态发生的优化三维模型系统，并揭示细胞凋亡是TNF诱导肠上皮通透性升高及形态发生改变的关键机制，而该机制并不适用于IFNγ。