On a chip, researchers simulate a complex paediatric ailment.

On a chip, researchers simulate a complex paediatric ailment.

Environmental enteric dysfunction (EED), a chronic inflammatory disease of the gut that is the second greatest cause of death in children under the age of five, affects millions of children in low- and middle-income countries. EED is a fatal disorder that impairs patients' quality of life in the long run by causing malnutrition, stunted growth, and poor cognitive development. Additionally, oral immunizations are less effective in EED patients, leaving them more susceptible to infections that might otherwise be preventable. Better nutrition doesn't assist all kids, even if some cases of EED can be treated by merely altering a patient's diet. EED is exacerbated by a deficiency in minerals and exposure to tainted water and food, but its exact cause is yet unknown.

An in vitro human model of EED has now been developed by a team of researchers at the Wyss Institute at Harvard University in a microengineered Intestine Chip device, opening a view into the intricate interplay between starvation and the genetic causes causing the disease. Their EED Chips replicate a number of EED symptoms seen in human patient biopsies, including inflammation, impaired intestinal barrier function, decreased food absorption, and atrophy of the villi (tiny hair-like projections on intestinal cells).

Additionally, they discovered that depriving healthy Intestine Chips of two essential nutrients, tryptophan and niacinamide, led to morphological, functional, and genetic changes that were similar to those observed in EED patients. This finding suggests that their model could be used to determine and test the effects of potential treatments.

These children's digestive systems and their capacity to absorb nutrients and fight infections are severely compromised, and this is a condition that cannot be cured by merely supplementing their diets with the nutrients they are deficient in. We were able to understand what had happened to the gut physically and genetically to have such a severe impact on its normal function in EED patients thanks to our EED model.

Former Senior Postdoctoral Research Fellow at the Wyss Institute and co-first author Amir Bein, R.D., Ph.D., is now the VP of Biology at Quris Technologies.

The study was released in Nature Biomedical Engineering today.

On-chip modelling of a complex disease

The Bill and Melinda Gates Foundation, which has a well-established interest in financing research to study and treat enteric disorders, and Donald Ingber, M.D., Ph.D., the founding director of the Wyss Institute, had discussions that led to the creation of the EED Chip project. A Wyss team of more than 20 people set out to create a model of EED using its Human Organ Chip technology, which was developed in Ingber's lab, after realising that there had been no in vitro investigations of EED to examine its molecular causes.

They began with the 2012-first-released Intestine Chips from the Wyss Institute, which are flexible polymer materials with parallel hollow microfluidic channels inside of them. Human intestinal epithelial cells line one of the channels, whereas human blood vessel cells line the other. To keep the cells alive, a medium similar to blood is pumped via the blood vessel chip. A permeable membrane separating the two channels allows nutrients and chemical signals to pass between the two tissues. The Gates Foundation assisted in obtaining cells from surgical biopsies of EED patients from Aga Khan University in Pakistan, which the researchers used to fill the epithelial channel of the Intestine Chips to reproduce the disease. Additionally, they produced Healthy Chips encased in

healthy children's intestinal epithelial cells for comparison.

"It was unclear what, if any, impact genetics or epigenetics play in the disease because EED is so common in regions of the world where malnutrition and poor sanitation are major issues. We hypothesised that intestinal cells from those patients might react differently to malnutrition than intestinal cells from healthy children because we were aware that some patients simply don't respond to nutrition "According to co-first author Cicely Fadel, M.D., Ph.D., a former clinical fellow who worked with Ingber at the Wyss Institute and is currently an attending neonatologist at Beth Israel Deaconess Medical Center, HMS instructor in paediatrics. By examining the gene, she and her co-authors hoped to unravel that riddle.

their Healthy Chips versus EED Chips cell expression profiles.

287 genes in the EED Chips displayed various levels of expression, the research team discovered. Among them were genes linked to cellular connections, intestinal damage, and inflammation. They discovered some overlap between the genes on their chips and the genes in the samples when they compared the genetic profile of the EED Chips with a clinical genetic signature from EED patients whose illness was not improved by nutritional intervention.

They next altered the medium that was giving nutrients to their chips by removing niacinamide and tryptophan, both of which are essential for good growth in children and maintenance of health in adults, to imitate the malnutrition that many EED patients face.

The result was impressive.

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The Healthy Chips showed 690 genes with distinct expression patterns when they were cultivated with nutrient shortage compared to their counterparts who received adequate nutrition, while the EED Chips showed an astounding 969 genes with different expression patterns. In the EED Chips cultivated with nutritional shortage, six of the top ten genes that were increased in the clinical EED gene signature from patient biopsies were also upregulated.

"It was quite amazing to see that the signature on our nutrient-deficient EED Chip matched the signature on patients. By employing the same genetic pathways that are active in people with EED, we are not only able to reproduce the appearance and function of the EED intestinal tract. This makes it possible to test medications and other therapies on the EED Chip and obtain results that may be comparable to those seen in patients "Fadel added.

contrasting nature versus nurture

In order to pinpoint precisely which variations between the EED Chips and Healthy Chips were brought on by nutritional shortage alone as opposed to innate gene expression differences, the scientists subsequently examined the chips from a variety of viewpoints.

The Healthy and EED Chips appeared to be similarly affected by several dietary deficiencies. Both varieties of chips showed a dramatic reduction in the growth of the villus-like structures normally present on their surfaces, as well as an upregulation of specific genetic pathways linked to the production of inflammatory chemokines and responses to amino acid starvation. They also produced a much thinner mucus layer. Both types of chips developed "leaky" cell connections that allowed fluids to flow through and changed how efficiently they absorbed fatty acids.

However, the EED Chips showed certain distinct variations in how they responded to nutritional deprivation, all of which matched characteristics found in EED biopsies from humans. Downregulation of genetic pathways linked to both of these processes was seen in the EED Chips, which is consistent with the diminished development of the intestines' interior surfaces (known as the brush boundary) and defective cell growth in EED patients. Paneth cell markers were decreased in the EED Chips, and Paneth cells, which assist in regulating the microbiota in the gut, are known to be deficient in EED patients. Additionally downregulated were amino acid transporters.

In comparison to Healthy Chips cultivated under the identical conditions, the scientists discovered that EED Chips produced lower amounts of inflammatory cytokines. However, when both chips were exposed to nutritional insufficiency, the EED Chips considerably outproduced the Healthy Chips in terms of cytokine production. EED patients with gut chronic inflammation may not be able to ingest enough calories to sustain their tissues and promote their growth, which can result in stunting. Inflamed tissue takes more calories to maintain and regenerate. The intestine's capacity to handle oral vaccinations may be hampered by this inflammation.

EED chips demonstrated a decreased capacity to absorb those nutrients in the epithelial channel and transfer them into the vascular channel when compared to the Healthy Chips, even when the entire spectrum of nutrients was available.

function was degraded by design.

"One of the major contributions of this study to EED research and treatment efforts is that we were able to specifically attribute various cellular responses to nutritional deficiency, genetic changes in the intestinal cells, or a combination of both," said Bein. These distinctions were not possible to make in clinical studies or animal models. "The additional exposure to starvation was required since the EED gene signature alone wasn't sufficient to fully recreate EED in our chips. This suggests that nutritional insufficiency itself interferes with the digestion of nutrients, setting off a feedback loop that exacerbates nutritional absorption in EED patients."

The team is still researching EED using their model, and they intend to add immune cells to further explore inflammation, how it interacts with diet, and how it affects the body's reaction to vaccinations. In order to research how changes to the microbiome can affect the condition, they are also aiming to include a microbiome from EED patients onto the chips.

"A slide presentation featuring actual kids who have EED has opened each regular meeting our team has had with the Gates Foundation regarding this initiative over the years. They are the Wyss Institute's clients; their patients inspire us to work hard, frequently for years, to develop answers to challenging challenges that can significantly raise their quality of life. Once we've found that answer, we won't stop looking "said Ingber, who is also the Hansjörg Wyss Professor of Bioinspired Engineering at Harvard's John A. Paulson School of Engineering and Applied Sciences and the Judah Folkman Professor of Vascular Biology at HMS and Boston Children's Hospital (BCH), among other positions (SEAS).

Biologically Inspired Design, Wyss Institute