Leaky Gut Syndrome: A Tale Of Two Mice Pairs

Mass General researchers reverse gut permeability and chronic inflammation in “zonulin mice.”
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When BBC Horizon filmmaker Tristan Quinn contacted the Center for Celiac Research and Treatment to interview physician-scientist Dr. Alessio Fasano, it seemed like a pretty straightforward request. Quinn wanted Fasano’s expert opinion on the gluten-free diet for a documentary on clean eating. As communications director for the center at Massachusetts General Hospital (MGH), all I had to do was set up the date, alert the MGH Public Affairs Office, help the crew scout out a good location, and escort them around on the day of the shoot. Or so I thought.

Quinn and one of his associates had already interviewed Fasano by phone several times. They seemed to have a clear understanding of his area of expertise: gluten and the effect it can have on the human body, particularly when someone has been diagnosed with celiac disease. Along with his two-man camera crew, Quinn planned to film the world-renowned expert on gluten-related disorders in his natural habitat of the laboratory at the Mucosal Immunology and Biology Research Center in Charlestown, Mass. They wanted to know what happens — physiologically speaking — when someone eats gluten, a complex protein found in wheat, rye and barley.

Fasano had already told the BBC team about zonulin, a substance that regulates intestinal permeability. Zonulin controls the openings between the epithelial cells lining the human intestine. The cells help to maintain a healthy balance between absorbing nutrients and keeping unwanted microorganisms (bad bacteria!) and other substances from getting into the intestine. Higher zonulin levels can lead to increased intestinal permeability, also called “leaky gut.”

When someone eats gluten, certain proteins in gluten activate zonulin release, which opens the “tight junctions” between the epithelial cells. For most individuals, it’s a temporary condition, says Fasano, and the immune system takes care of “mopping up any damage.” For people with celiac disease, increased zonulin can mean those tight junctions remain open. The increased intestinal permeability, also called “leaky gut,” can lead to malabsorption of nutrients and the intestinal damage that characterizes celiac disease.

Filming Lab Mice?

Two days before the film date, filmmaker Quinn sent me a message asking about the “zonulin-type mice” that Fasano had described over the phone. Zonulin-type mice have two copies of the gene related to a high production of zonulin. Quinn wanted to film the zonulin-type mice and the laboratory’s normal mice, which have zero copies of the zonulin gene.

I spent the next 36 hours in conversations with the animal manager of our lab, the head veterinarian in charge of animal control and welfare, the head of public affairs, and Fasano. We ultimately received approval to film four mice: one breeding pair genetically engineered with two zonulin genes and one wild-type or “normal” breeding pair, which had no zonulin genes.

The long day of filming resulted in final footage of several minutes of Fasano enthusiastically explaining intestinal permeability, including shots of the two pairs of mice running around in their cages. Fasano emphasized that unless you are genetically susceptible to a gluten-related disorder, eating gluten is not a problem for most people—or mice.

As agreed upon beforehand, Quinn sent the footage of the mice for review and approval by MGH’s head veterinarian, our lab manager and the public affairs office. Approval granted, our zonulin-type mice premiered in “Clean Eating – The Dirty Truth,” a documentary that aired in the United Kingdom on BBC Horizon in January 2017. But the real story behind the mice, now primed for increased intestinal permeability and a disposition to chronic inflammatory disease, had begun nearly 17 years earlier, in a disappointing search for a cholera vaccine...

A Lucky Discovery

Enterotoxins are poisonous substances that affect the intestines, such as those causing food poisoning or cholera. Early in his career as a pediatric gastroenterologist and researcher, Fasano liked to investigate deadly enterotoxins like Shigella and vibrio cholerae. While trying to develop a vaccine to fight cholera, his group discovered a new enterotoxin. In an act of scientific serendipity, Fasano’s group learned that this new enterotoxin could open the tight junctions between intestinal epithelial cells.

Fasano consequently shifted his research to study the complex molecular pathway involved in intestinal permeability. Following the path of this new enterotoxin, in 2000, he and his team discovered zonulin, which he defines as “the human protein master regulator of intestinal permeability in health and disease.”

Since its discovery, zonulin has been the subject of numerous studies that implicate increased intestinal permeability with chronic inflammatory disease, including allergic, autoimmune and metabolic disorders. Fasano prefers the term “increased intestinal permeability” to leaky gut or leaky gut syndrome.

“The term ‘leaky gut’ has been embraced by some non-mainstream practitioners as the cause of everything that is wrong with a person,” says Fasano. “For this reason, leaky gut syndrome, or the condition caused by increased intestinal permeability, has been vilified by the medical profession.” No matter what the terminology, Fasano and other researchers postulate that this impaired gut barrier function, along with genetic factors and environmental triggers, plays a key role in the development of several chronic inflammatory diseases, including celiac disease.

Findings from the paper by Fasano and Craig Sturgeon, PhD, released April 19, 2017, in the Annals of the New York Academy of Science, definitively link zonulin to the development of an inflammatory disease. After treating both zonulin-type mice and normal mice with an agent that produces inflammation in the colon, they measured intestinal permeability. Results show that the zonulin-type mice had a much higher rate of intestinal permeability as well as increased illness and mortality.

“This is the first time that we have been able to mechanistically link a zonulin-dependent modulation of gut permeability and enhanced antigen trafficking (a foreign substance producing an immune response) to the development of an inflammatory disease,” says Fasano. The data presented by Fasano, Sturgeon and Jinggang Lan, PhD, lines up with their hypothesis that the presence of the zonulin gene isn’t enough to cause disease. But add two copies of the zonulin gene to a pro-inflammatory stimuli, such as a colitis-inducing treatment, and it increases morbidity and mortality through loss of the intestinal barrier function and increased antigen trafficking.

“An even more remarkable finding,” adds Fasano, “is that adding a zonulin inhibitor to the drinking water of the mice completely returned the zonulin mice, which had previously shown chronic inflammation, to an inflammatory rate similar to normal mice—and it completed prevented mortality in the zonulin mice.” The zonulin inhibitor they used was larazotide acetate, or AT1001, a therapeutic agent for celiac disease developed by Fasano while at the University of Maryland School of Medicine in the 1990s.

Fasano was once scientific advisor to the startup company that developed larazotide acetate, Alba Therapeutics Corporation of Baltimore, MD. He is no longer involved in the enterprise, which has been taken over by Innovate Biopharmaceuticals of Raleigh, N.C. The zonulin-blocking agent, renamed INN-202 by Innovate, is scheduled to go into Phase III clinical trials sometime in 2017, according to the company website. Given a “path forward” agreement from the Food and Drug Administration, it could be the first drug approved for treatment for people with celiac disease. Fasano still owns stock in Alba Therapeutics, Inc.

Making Mice at Home

Sturgeon, co-author of the April 2017 paper and a graduate student who relocated from Maryland to MGH with Fasano in 2013, has his own tale to tell about zonulin mice. “We lucked into finding this animal model,” he says. “We got them from a group in Israel that created them to study the effect of haptoglobin-2 and heart function.” Zonulin is a precursor of haptoglobin-2, a protein that binds hemoglobin to prevent oxidative stress. The three original mice pairs came from the lab of Andrew Levy, MD, PhD, professor of cardiovascular biology at the Rappaport Family Institute for Research in the Medical Sciences in Haifa, Israel.

“When Alessio discovered pre-habtoglobin-2 (zonulin) in 2000, we thought these mice would make a useful tool for studying zonulin in animal models,” says Sturgeon. “And, as my boss likes to say, it was serendipity that led us to the zonulin mouse model.”

Fasano confirms that serendipity is indeed his favorite word, but it took a lot of perseverance on his part and Sturgeon’s to take the zonulin mice through more than ten years of research to the results recently published. The research project, which examined altered gut permeability and increased illness and fatality in mice treated with a colitis-inducing substance, began as part of Sturgeon’s dissertation for his PhD from the University of Maryland.

“I was lucky to develop this project along with Alessio, but we had a lot of hiccups,” says Sturgeon. One of the biggest hurdles happened in June 2011, when researchers couldn’t figure out why the first set of animals didn’t get sick from the colitis-inducing substance. They eventually realized that the mouse colony had a parasitic nematode infection. Sturgeon was in the nearby hospital having surgery on his neck when colleagues from his research group discovered the nematodes in the mice.

“Alessio came to the hospital and told me that the mice had worms—that was a rough day. We couldn’t get the infestation cleared up enough to meet university standards, so we had to euthanize the whole colony,” says Sturgeon. “We got another set, three breeding pairs, from Andrew Levy to create enough mice for experiments and breeding.” After a few more rounds of experiments in Maryland, Sturgeon and the mice were ready to move to the former shipbuilding facility at the Charlestown Navy Yard when Fasano moved his research enterprise from Baltimore to Boston.

“Mice are finicky,” says Sturgeon. “Changes in their environment can cause changes in their phenotype (observable traits and characteristics), especially in animals with disease. Also, we couldn’t combine data and analyze data from two different animal facilities. Because we didn’t have enough data from Maryland for a full research project at that point, basically we had to start again at MGH. We asked Andrew’s group to ship a couple more breeding pairs, then we went through the regulatory approvals for MGH and started the project all over.”

Linking Leaky Gut to Zonulin Gene

Eventually Sturgeon and Fasano began to see the clear difference they describe in their paper. When Sturgeon performed an intestinal permeability test in the live mice, he found increased gut permeability at baseline in the zonulin mice, before they had been treated with the agent to induce colitis.

“That was an ‘aha’ moment,” says Sturgeon. “Before that, we had seen a clear phenotypic difference in the two types of mice after colitis treatment, but we were having trouble linking it to changes in permeability.” The researchers also found that the mice with zonulin genes got much sicker much more quickly and died at a faster rate than the mice without the zonulin genes.

The researchers note that a key roadblock to studying intestinal permeability in the pathogenesis of chronic inflammatory disease to date has been the lack of a good animal model. Fasano and Sturgeon are enthusiastic about using the mice to study the role of zonulin-mediated intestinal permeability in a variety of chronic inflammatory diseases. “Use of these mice will allow us insight into specific mechanisms as to how zonulin-dependent increased intestinal permeability can affect disease, disease onset, clinical severity, clinical outcomes and even possible prevention,” says Sturgeon.

“We know zonulin expression is not sufficient to instigate disease and we know it’s not necessary for disease, but it’s associated with a lot of disease,” says Sturgeon. “And where there’s smoke, there’s normally a fire close by.”

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