Millions of children in low- and middle-income countries suffer from environmental enteric dysfunction (EED), a chronic inflammatory bowel disease that is the second leading cause of death in children under five. EED is a devastating condition that is associated with malnutrition, retarded growth, and poor cognitive development, which permanently affects patients ’quality of life. In addition, oral vaccines are less effective in children with EED, making them vulnerable to diseases that could otherwise be prevented. While some cases of EED can be treated simply by improving a patient’s diet, better nutrition does not help all children. Lack of adequate nutrients and exposure to contaminated water and food contribute to EED, but the underlying mechanism of the disease is still unknown.
Now, a team of researchers from Harvard University’s Wyss Institute has created an in vitro human model of EED in an intestinal microengineering device, providing a window into the complex interaction between malnutrition and the genetic factors that drive it. the disease. Its EED chips summarize various features of EED found in biopsies of human patients, such as inflammation, intestinal barrier dysfunction, reduced nutrient absorption, and atrophy of the villi (small projections). similar to hair) in intestinal cells.
They also found that deprivation of healthy intestinal chips of two crucial nutrients: niacinamide (a vitamin) and tryptophan (an essential amino acid) caused morphological, functional, and genetic changes similar to those found in patients with EED, suggesting that the your model could be used. to identify and test the effects of possible treatments.
Functionally, there is something very wrong with these children’s digestive system and their ability to absorb nutrients and fight infections, which cannot be cured simply by giving them the nutrients they are missing from their diet. Our DEE model allowed us to decipher what has happened to the gut, both physically and genetically, which so dramatically affects its normal function in patients with DEE. “
Amir Bein, RD, Ph.D., co-first author, former senior postdoctoral researcher at the Wyss Institute who is now the Vice President of Biology at Quris Technologies
The research is published today in Nature Biomedical Engineering.
Modeling a complex disease on a chip
The EED Chip project arose from conversations between the founding director of the Wyss Institute, Donald Ingber, MD, Ph.D. and the Bill and Melinda Gates Foundation, which has a well-established interest in supporting research to understand and treat enteric diseases. Recognizing that there had been no in vitro EED studies to study its molecular mechanisms, a Wyss team of more than 20 people set about creating an EED model using their Human Organ Chip technology developed in the laboratory. ‘Ingber.
They began with the Wyss Institute’s Intestine Chips, first developed in 2012, which consist of parallel hollow microfluidic channels running through a flexible polymeric material. One channel is lined with human intestinal epithelial cells, while the other canal is lined with human blood vessel cells. A medium that mimics blood flows through the blood vessel chip to keep cells alive, and a permeable membrane between the two channels allows nutrients and chemical signals to travel between the two tissues. To replicate EED in these chips, the researchers aligned the epithelial canal of intestinal chips with cells derived from surgical biopsies of patients with EED, which the Gates Foundation helped obtain from Aga Khan University in Pakistan. They also created Healthy Chips lined with intestinal epithelial cells from healthy children for comparison.
“Because EED is so common in parts of the world where malnutrition and poor sanitation are big problems, it wasn’t clear what role, if any, genetics or epigenetics have in the disease. We knew there was there was a group of patients who just do it. ”“ They don’t respond to nutrition and I thought their gut cells might respond differently to malnutrition compared to the gut cells of healthy children, ”he said. co-author Cicely Fadel, MD, Ph.D., assistant neonatologist at Beth Israel. Deaconess Medical Center, pediatric instructor at Harvard Medical School (HMS) and former clinical researcher working with Ingber at the Wyss Institute. She and her co-authors sought to solve this mystery by studying the patterns of gene expression in the cells of their EED chips versus healthy chips.
The team found that 287 genes from the EED chips showed different levels of expression. Among them were genes associated with inflammation, intestinal lesions, and inter-cell junctions. When they compared the genetic profile of EED chips with a clinical genetic signature of patients with EED whose disease was not resolved by a nutritional intervention, they found some overlap between the genes on their chips and the genes in the samples.
Then, to mimic the malnutrition experienced by many patients with EED, they changed the medium that supplied nutrients to their chips by eliminating niacinamide and tryptophan, both crucial to the healthy growth of children and maintaining the health of adults.
The effect was shocking.
When grown with a nutrient deficiency, Healthy Chips showed 690 genes with different expression patterns compared to their full-nutrition counterparts, and in EED Chips, a whopping 969 genes had different expression patterns. . Six of the top ten genes that were regulated in the clinical signature of the EED gene from patient biopsies were also regularized in nutrient-deficient cultured EED chips.
“The agreement between our nutrient-deficient EED chip signature and the signature found in human patients was really exciting. Not only are we able to recreate the shape and intestinal function of the EED, but we’re also doing it. “Using the same genetic pathways that operate in human patients. This opens up the possibility that we can test drugs and other treatments on the EED chip and get a response that could be similar to what would be seen in patients,” Fadel said.
Mock Nature Vs. nourish
The scientists then analyzed the chips from multiple angles to determine exactly what differences between EED chips and healthy chips were caused only by nutrient deficiency versus the differences inherent in gene expression.
Some effects of nutritional deficiency appeared to affect healthy chips and EEDs alike. Both types of chips showed a regulation of certain genetic pathways associated with the production of inflammatory chemokines and amino acid starvation responses, drastically reduced the growth of hair-like structures normally found on their surfaces and went produce a much thinner layer of mucus. Both types of chips became “leaked” as the bonds between their cells were disrupted and allowed fluids to seep in and showed changes in the absorption of fatty acids.
But EED chips showed some unique differences in their responses to nutritional deficiency, all of which match the traits observed in human EED biopsies. The intestines of patients with EED have reduced development of their internal surfaces (known as brush edge) and impaired cell growth, and downward regulation of the genetic pathways associated with both processes in the EED chips. Paneth cells, which help regulate the microbiome in the gut, are known to be depleted in patients with EED, and markers of Paneth cells were reduced in EED chips. Amino acid transporters were also regulated downward.
The team found that EED chips produced lower levels of inflammatory cytokines compared to healthy chips grown under the same conditions. But once both chips were exposed to nutritional deficiency, EED chips produced significantly more cytokines compared to healthy chips. Inflamed tissue requires more calories to maintain and renew itself, and patients with EED with chronic intestinal inflammation may not be able to consume enough calories to maintain their tissues and to support their growth, contributing to growth retardation. This inflammation can also reduce the gut’s ability to process oral vaccines.
Even when the full range of nutrients was available, EED chips showed a reduced ability to absorb these nutrients into the epithelial canal and transfer them to the vascular canal compared to healthy chips, suggesting in addition to function of the EED tissue was inherently compromised.
“One of the main contributions of this study to EED research and treatment efforts is that we were able to attribute various cellular responses specifically to nutritional deficiency, genetic changes in intestinal cells, or a combination of both, distinctions that have not been possible to make in clinical studies or animal models, “Bein said. “Signing the EED gene alone was not enough to fully replicate the EED on our chips; additional exposure to malnutrition was necessary. This implies that nutritional deficiency in itself alters nutrient processing, which which creates a feedback loop that further worsens the nutritional uptake of patients with EED. “
The team is still studying EED with its model and plans to incorporate immune cells to further study inflammation and its interaction with nutrition, as well as how it affects the body’s response to vaccines. They are also working to add a microbiome of EED patients to the chips to study how changes in the microbiome can affect the disease.
“Every regular meeting our team has had with the Gates Foundation about this project over the years has begun with a slide show of real children suffering from EED. These children are our clients: patients drive everything we do to the Wyss Institute., and motivate us to work hard, often for years, to create solutions to difficult problems that can drastically improve their quality of life. We will not rest until we find that solution, “he said …