Neutrophils Shown to Play the Key Role in Gallstone Formation

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Neutrophil Elastase on Human Gallstones
Neutrophil elastase (green) and DNA (red) co-localize on the surfaces of human gallstones (yellow). Scale bar 4mm. [Munoz et al./Immunity]

Sticky meshworks of DNA and proteins extruded by neutrophils act as the glue that binds together calcium and cholesterol crystals during gallstone formation, according to scientists in Germany who published a study (“Neutrophil Extracellular Traps Initiate Gallstone Formation”) in Immunity. Both genetic and pharmacological approaches that inhibited the formation of these so-called neutrophil extracellular traps (NETs) reduced the formation and growth of gallstones in mice.

“The presence of gallstones (cholelithiasis) is a highly prevalent and severe disease and one of the leading causes of hospital admissions worldwide. Due to its substantial health impact, we investigated the biological mechanisms that lead to the formation and growth of gallstones. We show that gallstone assembly essentially requires neutrophil extracellular traps. We found consistent evidence for the presence of NETs in human and murine gallstones and describe an immune-mediated process requiring activation of the innate immune system for the formation and growth of gallstones,” the investigators wrote.

Neutrophil Extracellular Traps
This image shows how neutrophil’s extracellular traps glue together gallstone gravel (left) and the crystalline gallstone building blocks cholesterol (middle) and calcium carbonate (right). Scale bar 20 μm. [Munoz et al./Immunity]

“Targeting NET formation via inhibition of peptidyl arginine deiminase type 4 or abrogation of reactive oxygen species (ROS) production, as well as damping of neutrophils by metoprolol, effectively inhibit gallstone formation in vivo. Our results show that after the physicochemical process of crystal formation, NETs foster their assembly into larger aggregates and finally gallstones. These insights provide a feasible therapeutic concept to prevent cholelithiasis in patients at risk.”

“Neutrophils have long been considered the first line of defense against infection and have been shown to generate NETs that entangle and kill pathogens,” said senior study author Martin Herrmann, MD, PhD, an immunologist at Universitätsklinikum Erlangen. “Here, we provide additional evidence for the double-edged-sword nature of these NETs by showing that they play an important role in the assembly and growth of gallstones. Targeting neutrophils and NET formation may become an attractive instrument to prevent gallstones in high-risk populations.”

Gallstones are hard, pebble-like pieces of material that may be as small as a grain of sand or as large as a golf ball. They form in a pear-shaped organ called the gallbladder, which releases bile to the small intestine through the bile ducts during meals to help break down fat. Although most people with gallstones do not have symptoms, they can cause abdominal pain, nausea, and vomiting, and they are a leading cause of hospital admissions worldwide. Surgery to remove the gallbladder is one of the most common operations performed on adults in the United States. Although medicines do exist, they may require months or years of treatment to break up all gallstones.

One hurdle limiting the development of new treatments is that scientists do not yet fully understand exactly how gallstones form. It has been known for decades that the precipitation of cholesterol and calcium salts in the bile is a prerequisite for gallstone formation. But it has not been clear what causes calcium and cholesterol crystals to stick together during gallstone assembly and growth.

A clue to this mystery came while the team was examining biliary sludge, small stones in the bile—extracted from human patients receiving stents. They observed large aggregates of DNA, as well as robust activity of neutrophil elastase. The researchers also observed DNA patches and high neutrophil elastase activity on the surfaces of larger human gallstones. Together, these molecules are telltale signs of NETs, web-like structures extruded by neutrophils that help protect against infection but have also been implicated in autoimmune and inflammatory disorders. To determine what triggered NET formation, the investigators cultured cholesterol crystals with human neutrophils and observed the release of DNA from these cells.

To test whether NETs play a role in gallstone growth, the researchers mounted human gallstones to rotating and shaking devices and spun them in the presence or absence of neutrophil suspensions. In the presence of neutrophils, the gallstones rapidly collected DNA patches and neutrophil elastase on their surfaces. Additional experiments showed that NETs aggregated cholesterol and calcium crystals to form gallstones, and the DNA wrapped around the crystals and pulled them together to form larger stones.

DNA Patches on Gallstones
DNA patches on the surfaces of human gallstones. Scale bar 1 cm. [Munoz et al./Immunity]

To further examine the role of NETs and neutrophils in gallstone development, the researchers fed mice a cholesterol-rich diet that induced gallstone formation. The number and size of gallstones were smaller in mice with genetic defects that inhibited NET formation, and the size of gallstones was also smaller in mice with fewer neutrophils. In addition, the number and size of gallstones were reduced in mice whose diets were supplemented with either a compound that inhibits protein-arginine deiminase 4 (PAD4), an enzyme involved in NET formation, or the beta blocker metoprolol, which interferes with neutrophil migration and is also widely used for the treatment of high blood pressure and chest pain. Treatment with the PAD4-inhibiting compound or metoprolol also completely blocked the additional growth of pre-existing gallstones.

The authors say that the identification of neutrophils and NETs as culprits in gallstone formation and growth opens new avenues for therapeutic interventions.

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