Abstract：
Shiga toxin (Stx) is produced by some pathogenic strains of Escherichia coli. To study the impact of Stx on the human intestine, we utilized human intestinal organoids and human intestinal enteroids grown as human intestinal enteroid monolayers (HIEMs) in transwells. To establish optimal experimental conditions, HIEMs were grown with or without mesenchymal cells added to the basolateral wells to recapitulate the interactions between the intestinal epithelium and the underlying mesenchyme. Monolayer barrier integrity was determined through transepithelial electrical resistance (TEER) readings. Apical saline was used on the apical surface since growth medium caused uneven development of the TEER. The medium used for epithelial cells contains added growth factors, while the mesenchymal medium lacks these growth factors. We have shown that mesenchymal cells can maintain the epithelial monolayer in the medium lacking growth factors, suggesting they produce these factors. Furthermore, growth factors produced by mesenchymal cells need to build up in the medium over time, since daily medium changes were not as effective as medium changes performed every 3 days. We have also shown that addition of growth factors is toxic to mesenchymal cells. Epithelial cells were more resistant to Stx2 than the mesenchymal cells, and mesenchymal cells contributed to epithelial cell death. Epithelial cells tolerated luminal exposure better than basolateral exposure. These studies demonstrate the importance of understanding tissue interactions in a disease state when using in vitro and in vitro models.
OBJECTIVE: These studies have cemented the need for complex cell culture models when studying host-pathogen interactions. Common animal models such as mice are resistant to E. coli O157:H7 infections and intestinal delivery of Stx2, while humans appear to be sensitive to both. It has been proposed that in humans, shiga toxin-producing E. coli-mediated intestinal damage destroys the intestinal barrier and allows basolateral access to Stx2. In mice, there is no epithelial damage; therefore, they are resistant to epithelial delivery of Stx2 while remaining sensitive to Stx2 injection. Our studies show that like mice, the human epithelial layer is quite resistant to Stx2, and it is the sensitivity of the mesenchymal cells that kills the epithelial cells. We have shown that Stx2 is transported through the intact epithelium without causing damage to the resistant epithelial layer. Understanding tissue interactions during infections is therefore critical in determining the effects of pathogens on human tissues.
OBJECTIVE: These studies have cemented the need for complex cell culture models when studying host-pathogen interactions. Common animal models such as mice are resistant to E. coli O157:H7 infections and intestinal delivery of Stx2, while humans appear to be sensitive to both. It has been proposed that in humans, shiga toxin-producing E. coli-mediated intestinal damage destroys the intestinal barrier and allows basolateral access to Stx2. In mice, there is no epithelial damage; therefore, they are resistant to epithelial delivery of Stx2 while remaining sensitive to Stx2 injection. Our studies show that like mice, the human epithelial layer is quite resistant to Stx2, and it is the sensitivity of the mesenchymal cells that kills the epithelial cells. We have shown that Stx2 is transported through the intact epithelium without causing damage to the resistant epithelial layer. Understanding tissue interactions during infections is therefore critical in determining the effects of pathogens on human tissues.
摘要：
志贺毒素(Stx)是由一些致病性大肠杆菌菌株产生的。为了研究Stx对人体肠道的影响，我们利用了人类肠道类器官和人类肠道类肠样细胞（HIEM）。为了建立最佳的实验条件，HIEM在有或没有添加到基底外侧孔中的间充质细胞的情况下生长，以概括肠上皮与下层间充质之间的相互作用。通过跨上皮电阻(TEER)读数确定单层屏障完整性。由于生长培养基引起TEER的不均匀发育,因此在顶表面上使用顶盐水。用于上皮细胞的培养基含有添加的生长因子，而间充质培养基缺乏这些生长因子。我们已经证明，间充质细胞可以在缺乏生长因子的培养基中维持上皮单层，表明它们产生了这些因素。此外，间充质细胞产生的生长因子需要随着时间的推移在培养基中积累，因为每天更换培养基不如每3天更换培养基有效。我们还表明，添加生长因子对间充质细胞具有毒性。上皮细胞对Stx2的抗性高于间充质细胞，间充质细胞导致上皮细胞死亡。上皮细胞耐受腔暴露优于基底外侧暴露。这些研究证明了当使用体外和体外模型时理解疾病状态中的组织相互作用的重要性。
目的：这些研究表明，在研究宿主-病原体相互作用时，需要复杂的细胞培养模型。常见的动物模型如小鼠对大肠杆菌O157：H7感染和肠道递送Stx2具有抗性，而人类似乎对两者都敏感。有人提出，在人类中，产生志贺毒素的大肠杆菌介导的肠损伤破坏了肠屏障，并允许基底外侧进入Stx2。在老鼠身上,没有上皮损伤；因此，它们对Stx2的上皮递送有抗性，而对Stx2注射仍然敏感。我们的研究表明，像老鼠一样，人的上皮层对Stx2具有相当的抗性，是间充质细胞的敏感性杀死了上皮细胞。我们已经表明，Stx2通过完整的上皮运输，而不会对抗性上皮层造成损害。因此，了解感染过程中的组织相互作用对于确定病原体对人体组织的影响至关重要。
目的：这些研究表明，在研究宿主-病原体相互作用时，需要复杂的细胞培养模型。常见的动物模型如小鼠对大肠杆菌O157：H7感染和肠道递送Stx2具有抗性，而人类似乎对两者都敏感。有人提出，在人类中，产生志贺毒素的大肠杆菌介导的肠损伤破坏了肠屏障，并允许基底外侧进入Stx2。在老鼠身上,没有上皮损伤；因此，它们对Stx2的上皮递送有抗性，而对Stx2注射仍然敏感。我们的研究表明，像老鼠一样，人的上皮层对Stx2具有相当的抗性，是间充质细胞的敏感性杀死了上皮细胞。我们已经表明，Stx2通过完整的上皮运输，而不会对抗性上皮层造成损害。因此，了解感染过程中的组织相互作用对于确定病原体对人体组织的影响至关重要。
