Gallstones, those hard masses of excess cholesterol and calcium crystals lodged in the gallbladder that can cause considerable pain, may have immune cells called neutrophils as their partners in crime. Researchers have found that gallstones also contain neutrophil extracellular traps (NETs)—sticky clumps of DNA and protein—that hold the crystals together like a spider web, they report August 15 in Immunity. Additionally, using molecules that inhibit the formation of these NETs, the scientists prevented the formation and growth of gallstones in mice, suggesting they could be a target for pharmaceutical interventions.
“This group has been highlighting aggregated NETs in various conditions,” Paul Kubes, an immunologist at the University of Calgary who was not involved in the study, tells The Scientist in an email. “But this is the first pathogenic role for these aggregated NETs.”
Another uninvolved researcher, David Wang, who studies gallstones at the Albert Einstein College of Medicine, suggests that the results “cannot clearly show” the authors’ claims. He says the immunologists didn’t use best practices for studying gallstone formation, calling some of the researchers’ methods “inappropriate.”
About 1 million people are diagnosed with gallstones every year. Often, they cause no symptoms, but sometimes they can be extremely painful and lead to serious complications or even death. There are drugs that can treat gallstones, but their efficacy is limited, and most patients who need treatment—about a quarter of those diagnosed—end up requiring surgery.
Researchers have long known that gallstones contain hardened cholesterol and calcium salts, but it’s still a mystery why these crystals end up sticking together to form stones that can reach the size of golf balls.
Martin Herrmann, an immunologist at Universitätsklinikum Erlangen, and his team were studying NETs for several years, when he began to suspect that they played a role in gallstone formation. NETs are known to function within the immune system to trap pathogens and prevent their spread between cells. It’s thought that they may also play a role in inflammatory diseases.
First, the team collected samples of biliary “sludge,” or gallbladder fluid containing small stones, from humans receiving stents to reduce blockage in the gallbladder. Under a fluorescent microscope, the group found robust activity of neutrophil elastase, an enzyme found in NETs and a sign that NETs were forming. They confirmed these observations in samples of larger gallstones, which they then mounted on devices that shook and spun them in the presence or absence of neutrophils, which contain the DNA strands that forms NETs. When the neutrophils were present, DNA and elastase stuck to stones’ surfaces where they could collect more cholesterol and calcium crystals.
The paper has substantial translational value, but “although the elastase suggests some evidence that neutrophils were infiltrating and presumably [forming NETs],” says Kubes, “I would have liked to have seen clear evidence of neutrophils forming NETs.”
Hoping to understand what the neutrophil elastase was doing in the gallstones—and if inhibiting NETs could help treat or prevent gallstones—the researchers turned to mice, half of which were genetically engineered to prevent the formation of NETs. All of the mice ate a high cholesterol diet to encourage gallstone formation for nine weeks before the researchers analyzed their gallbladders. While the organs of both sets of mice contained some small, crystallized cholesterol, only the wildtype mice had developed stones, suggesting that the NETs are required for gallstones to grow.
Their final goal was to test a strategy to treat gallstones that had already formed by inhibiting NETs. They fed mice a high-cholesterol diet for six weeks so they would develop gallstones. Then, the scientists treated the mice with the beta blocker metoprolol, which is occasionally used to treat gallstones, a compound that inhibits NET formation (GSK484), or a control for two more weeks. Only the gallstones in the control mice continued to grow after week six, while the stones in the other mice decreased in size.
But, Wang says, the imaging techniques the researchers used were insufficient. “Because phase contrast and polarizing light microscopy were not used . . . real gallstones cannot be clearly identified” in the mice in this study.
Herrmann, a basic scientist, is moving on to test the role of NETs in other diseases, and says he welcomes the feedback from gallstone experts on techniques to study the crystallization of calcium salts and cholesterol in body fluids.
If the NET-gallstone link were to be pursued for clinical applications, larger human trials will be needed to determine whether inhibiting NET formation will be useful. “Being able to stop gall stone production is interesting,” Kubes says, “and if there is some degradation of the gall stones, that would give a non-invasive way of treating this problem.”
L.E. Muñoz et al., “Neutrophil extracellular traps initiate gallstone formation,” Immunity, doi:10.1016/ j.immuni.2019.07.002, 2019.
Emma Yasinski is a Florida-based freelance reporter. Follow her on Twitter @EmmaYas24.