Intestinal tissue response to Shiga toxin exposure

Abstract

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. IMPORTANCE 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.