An international research team headed by scientists at the University of Cambridge has grown hundreds of intestinal epithelial organoids (IEOs), or ‘mini-guts’ to help understand the role that epigenetics may play in Crohn’s disease (CD). Organoids are 3D cell cultures that mimic key functions of a particular organ, in this case the gut epithelium.

Comparative studies with IEOs derived from CD patient’s mucosal stem cells, and those from healthy controls, identified stable DNA methylation (DNAm) changes leading to increased MHC-I expression in the intestinal epithelium of the CD patients, which resulted in functional effects and inflammation. The research also demonstrated that the lab grown IEOs retain patient-specific CD-associated DNAm changes, further validating their use as clinically relevant research tools.

The researchers say the mini-guts could in future be used to identify the best treatment for an individual patient, allowing for more precise and personalized treatments. Matthias Zilbauer, professor of pediatric gastroenterology at the University of Cambridge and Cambridge University Hospitals NHS Foundation Trust (CUH), commented, “This is the first time where anyone has been able to show that stable epigenetic changes can explain what is wrong in the gut epithelium in patients with Crohn’s disease.”

Zilbauer is senior author of the team’s published paper in Gut, in a paper titled “Patient-derived organoid biobank identifies epigenetic dysregulation of intestinal epithelial MHC-I as a novel mechanism in severe Crohn’s disease.” In their paper the team wrote, “Our study has identified epigenetically regulated intestinal epithelial MHC-I as a novel mechanism in CD pathogenesis.”

Crohn’s disease is a form of inflammatory bowel disease (IBD) characterized by inflammation of the digestive tract. The disease affects around one in 350 people in the U.K., with one in four presenting before the age of 18, the team noted. Even at its least severe CD can cause symptoms that have a major impact on quality of life, including stomach pain, diarrhea, weight loss and fatigue. It can also lead to the need for hospitalization, extensive surgery, and exposure to toxic drugs.

While there is some evidence that an individual is at greater risk of developing Crohn’s disease if a first-degree relative also has the condition, the authors further pointed out that there has been only limited success in identifying genetic risk factors. And while epigenetic mechanisms, including DNA methylation, have been proposed to play a key role in Crohn’s disease (CD) pathogenesis, “… specific cell types and pathways affected as well as their potential impact on disease phenotype and outcome remain unknown.”

IBD, and in particular Crohn’s disease, is characterized by persistent chronic relapsing mucosal inflammation in specific gut segments, the authors continued. Even after successful treatment to resolve mucosal inflammation, relapses in inflammation tend to recur in the same anatomical locations. “This phenomenon suggests the presence of stable molecular changes leading to altered function in local tissue-specific, resident cell types,” the team noted But while altered function of the intestinal epithelium has been implicated in CD pathogenesis, they noted, “… underlying mechanisms remain ill defined.”

One of the main obstacles preventing scientists from better understanding the role of intestinal epithelial cell (IEC)-specific mechanisms in Crohn’s disease in humans has been the lack of patient-derived experimental models. Zilbauer commented, “The number of cases of Crohn’s disease and IBD are rising dramatically worldwide, particularly amongst younger children, but despite decades of research, no one knows what causes it. Part of the problem is that it’s been difficult to model the disease. We’ve had to rely mainly on studies in mice, but these are limited in what they can tell us about the disease in people.”

For their newly reported studies the researchers used cells from inflamed guts, donated by 160 patients at CUH to grow more than 300 IEOs in the lab to help them better understand the condition. Samples were donated by patients with Crohn’s disease and ulcerative colitis, and by patients unaffected by IBD.

“The organoids that we’ve generated are primarily from children and adolescents,” said professor Zilbauer. “They’ve essentially given us pieces of their bowel to help with our research. Crohn’s can be a severe condition to have to deal with at any age, but without our volunteers’ bravery and support, we would not be able to make such discoveries as this.”

The researchers carried out genome-wide molecular profiling, including DNAm, bulk, and single-cell RNA sequencing of the IEOs, alongside studies to investigate the functional role of disease-associated DNAm changes. Their results showed that gut epithelia from Crohn’s disease patients have different epigenetic patterns on their DNA when compared with healthy controls. The results highlighted epigenetic changes relevant to the MHC-I pathway, a mechanism by which immune cells recognize antigens. The findings indicated that the epithelial cells forming the inner lining of the gut in Crohn’s disease patients have an increased activity of MHC-I, which can lead to inflammation in specific parts of the gut.  “Genome-wide molecular profiling as well as functional analyses of over 300 patient-derived intestinal organoids has identified epigenetically regulated major histocompatibility complex (MHC)-I as a novel mechanism in CD,” they wrote. “… observed correlation between IEO-derived MHC-I DNAm signatures and disease severity highlights the major potential for these molecular signatures as clinical biomarkers.”

The epigenetic modifications were found to be very stable, which may explain why even after treatment, when a patient appears to be healed, inflammation can return after several months, indicating that drugs are treating the symptoms, not the underlying cause. Professor Zilbauer, a researcher at the Stem Cell Institute at the University of Cambridge, further commented, “What we saw was that not only were the epigenetic changes different in Crohn’s disease, but there was a correlation between these changes and the severity of the disease. Every patient’s disease course is different, and these changes help explain why —not every organoid had the same epigenetic changes.”

The researchers say the organoids could be used to evaluate how effective new treatments are  on the lining of the gut in Crohn’s disease. Importantly, the team stated, “… our study demonstrates that IEOs faithfully retain patient-specific, CD-associated DNAm changes in culture, further validating them as powerful translational research tools.”

The technology may help to tailor treatments to individual patients. Co-author Robert Heuschkel, MD, consultant pediatric gastroenterologist at CUH and lead of the pediatric IBD service, said, “At the moment, we have no way of knowing which treatment will work best for a patient. Even those treatments we currently have only work in around half of our patients and become less effective over time. It’s a huge problem. In future, you could imagine taking cells from a particular patient, growing their organoid, testing different drugs on the organoid, and saying, ‘OK, this is the drug that works for this person.”

The authors added, “The availability of a large living organoid biobank, containing over 300 well-characterized lines, provides unique opportunities for the development of novel treatments specifically targeting intestinal epithelial MHC-I.”

Epigenetic changes are programmed into our cells very early on during fetal development. They are influenced by environmental factors, which may include exposure to infections or antibiotics—or even lack of exposure to infection, the so-called ‘hygiene hypothesis’ that says we are not exposed to sufficient microbes for our immune systems to properly develop. The researchers say this may offer one possible explanation for how the epigenetic changes that lead to Crohn’s disease occur in the first place.

“Although the cause of stable DNAm changes in the intestinal epithelium of patients with CD remains speculative, our findings provide further evidence to support the emerging concept of inflammatory memory and trained innate immunity,” the authors also pointed out. “Specifically, exposure to environmental triggers, occurring at critical periods during development, can lead to enduring epigenetic changes that determine cellular function.”

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