The study explored new territory in Shiga toxin research, enabled by the use of human intestinal organoids (HIOs), reported lead author Suman Pradhan, PhD, of the University of Cincinnati, and colleagues.
Each year, Shiga toxin–producing Escherichia coli infections cause approximately 3 million cases of bloody diarrheal disease, with about 4,000 of those patients developing the life-threatening complication of hemolytic uremic syndrome (HUS), the investigators wrote in Cellular and Molecular Gastroenterology and Hepatology.
But little is known about the underlying biological processes driving Shiga-induced disease.
“Developing effective interventions for disease resulting from Shiga toxin is exacerbated by a lack of tractable model systems,” the investigators wrote. “Mice do not develop the symptoms characteristic of HUS, and the murine intestinal tract is resistant to Shiga toxin.”
To overcome this obstacle, Dr. Pradhan and colleagues turned to HIOs, which are grown in culture by directing differentiation of pluripotent stem cells. HIOs represent the small bowel, complete with a lumen surrounded by epithelial and mesenchymal layers that include typical cell types, such as goblet cells and myofibroblasts. The model is made more realistic by transplantation into mice, where it grows under the kidney capsule to form crypts, structured villi, and proliferating progenitor zones. And HIOs grown with neuronal precursors develop an enteric nervous system, complete with functional peristalsis.
For the present study, the investigators evaluated the effect of Shiga toxin on HIOs both in culture and after transplantation into mice.
First, they demonstrated that HIOs in culture expressed glycolipid Gb3, the Shiga toxin receptor. “Reports regarding expression of glycolipid Gb3 ... on human intestine have been inconsistent,” the investigators noted. “For negative reports, the inability to detect Gb3 could be owing to technical limitations.”
Next, Dr. Pradhan and colleagues showed that HIOs were susceptible to Shiga toxin whether it be delivered lumenally or basolaterally, which respectively represent intestinal exposure and exposure via circulating toxin or after breakdown of the epithelial barrier. Leakage from the lumen was observed with both Shiga toxin 1 (Stx1) and 2 (Stx2). Subsequent testing involved only Stx2, as this form is more relevant to human disease.
In addition to lumenal leakage, Stx2 exposure caused significant transcriptional up-regulation of multiple gene families, including those involved in cellular transport and metabolic processes. Increased expression also was observed for epithelial structural proteins, lineage-specific proteins, factors involved in mucus layer formation and stabilization, and cytokines interleukin-18 and CCL15.
In both epithelial and mesenchymal layers, transcriptional changes were accompanied by cellular necrosis and apoptosis, and, to a greater degree with interstitial exposure, cellular proliferation.
With lumenal exposure, mesenchymal necrosis was observed before loss of epithelial barrier function, indicating toxin access to mesenchymal cells. This phenomenon was explained by transcytosis, which the investigators observed in two-dimensional monolayers of enteroids grown in Transwells.
“[Shiga toxin] was transferred from the apical to the basolateral surface in the absence of loss of epithelial barrier function,” the investigators wrote, noting that this finding explains how Shiga toxin can quickly access the circulatory system, and from there damage the kidneys and brain, as seen in cases of HUS.
Mice with transplanted HIOs, and those receiving HIOs with an enteric nervous system (HIO + ENS), lost weight when organoids were injected with 10 ng of Stx2. Mice with HIO + ENS transplants developed more severe responses, prompting closer analysis.
Postmortem histologic examination of HIO + ENS transplants revealed epithelial damage and blood accumulation in the mesenchyme and villi. Additional staining showed signs of apoptosis and mesenchymal-epithelial transition.
Dr. Pradham and colleagues suggested that their findings could inform therapeutic research.
“If preventing cellular death is to be an effective intervention, it is likely that both necrosis and apoptosis need to be targeted,” the investigators wrote.
More generally, the study supports the use of HIOs as a disease model for future investigations.
“The advent of stem cell–derived human tissue models, both in vitro and in vivo, has a tremendous potential to increase our understanding of Shiga toxin disease and lead to development of therapeutic interventions,” the investigators concluded.
The study was funded by the National Institutes of Health, the Center for Clinical and Translational Science, the National Institute of Diabetes and Digestive and Kidney Diseases, and others. The investigators disclosed no conflicts of interest.
SOURCE: Pradhan S et al. Cell Mol Gastroenterol Hepatol. 2020 Mar 5.