Hard as a Rock: the neutrophil’s role in gallstone generation

It’s incredible how much trouble a small piece of matter can cause the human body when it doesn’t belong there. For example, a traveling blood clot can cause a heart attack. An invading virus can make you fatally ill. And, gallstones can not only cause excruciating abdominal pain, but require serious surgery.

Yet unlike a blood clot or a virus, whose origins are more or less unambiguous, the true mechanics of how gallstones form have remained murky, until now. New research suggests that neutrophils, the foot soldiers of the immune system, are also critical players in precipitating gallstones. 

What do we know about gallstones so far? 

First – let’s paint a clearer picture of what we’re dealing with: what exactly are gallstones? Gallstones are compact deposits that generally form in your gallbladder.  Depending on their size and position, they may not necessarily lead to symptoms, which can include intense pain in the stomach and back, nausea, diarrhea, vomiting – even a fever.  Symptomatic cases almost always result in surgical removal of the gallbladder. A shockingly common problem, gallstones affect 6-8% of Americans, are not only expensive to treat, but lethal if not properly dealt with. 

The process of gallstone genesis actually starts in the liver. Among one of its many functions, the liver produces bile – a  compound that aids in the digestion of fats. This bile is secreted into and stored in the gallbladder, a small organ nestled right under the liver. 

When needed, the gallbladder releases the bile into the small intestine, where it can begin to break down the fats that we consume.  

Among the many constituents of bile (a fluid) are cholesterol and calcium salts – both of which contribute to its valuable ability to digest fat. However, problems arise when the biliary fluid becomes supersaturated with cholesterol and these salts. This supersaturation causes components in the bile to precipitate, or transform from liquid to solid, forming what is affectionately known as biliary sludge. However, the small precipitates present in the sludge are not substantial enough to form larger stones. Rather, these precipitates serve as the “nidus”, or the core, around which a bona fide gallstone can develop.  

But what else? 

So, how do these pebble-like precipitates become full blown gallstones? A study by Muñoz et al. sought to answer this question. They found that neutrophils, immune cells that are acute responders to infection, are critical to gallstone genesis.

Neutrophils are the body’s cellular warriors, licensed to kill unwelcome microbes (like disease causing bacteria) with the help of an impressive arsenal of cellular adaptations. For example, neutrophils can swallow microbes in a process called phagocytosis, or they can secrete potent antimicrobial compounds to kill invaders. 

Perhaps the neutrophil’s most unique adaptation is its ability to release neutrophil extracellular traps, or NETs. In the wake of a microbial threat, neutrophils can actually release their DNA  extracellularly, or outside of the cell, in a fibrous, sticky web – entrapping and suffocating pathogens in this “net”. 

Previous research had shown that cholesterol and calcium salts – the principal components of gallstones – had the power to activate these neutrophils and trigger NET release, or NETosis. The authors thus hypothesized that these NETs were important in the generation of gallstones. 

Testing it out

To investigate their hypothesis, the authors isolated and closely examined human gallstones. In addition to the typical cholesterol and calcium salts, extracellular DNA (ecDNA), as well as neutrophil elastase, were found on the gallstones’ surface. Both ecDNA and elastase (an enzyme that helps destroy pathogenic bacterial membranes) are known constituents of NETs. Their presence strongly argued for neutrophil involvement in gallstone development. 

It has long been thought that the small biliary deposits in sludge grow into gallstones episodically. Think of how cotton candy is made at a carnival. Sugar is quickly heated and cooled into the fine, sugary strands. As you drag the paper cone around the cotton candy machine, the strands wrap around the stick, with the cotton candy increasing in size. 

Like cotton candy, the authors thought perhaps the salts and cholesterol served as the anchoring paper cone, and the NETs, comprised of ecDNA and elastase, were like the fine, cotton candy wisps that eventually aggregated and formed a problematic gallstone. 

To test this hypothesis, isolated gallstones were rotated on a special device in the presence or absence of neutrophils. Gallstones exposed to neutrophils increased in size and had high densities of ecDNA and elastase, whereas those that remained unexposed remained relatively unchanged in size. Moreover, when neutrophils were grown with either calcium or cholesterol in a dish, the neutrophils released sticky NETs, wrapping around the particulates until gallstone-like aggregates were generated. Taken together, these experiments strongly suggest a pivotal role for neutrophil NETs in the development of gallstones from their smaller, calcium-cholesterol precursors (Figure 1).  

Figure 1: NETs are critical in gallstone assembly. Bile can become supersaturated with cholesterol and calcium salts, resulting in small precipitates. These precipitates, upon contact with neutrophils, can trigger NETosis and the release of their ecDNA and elastase. These neutrophil components accumulate around these precipitates, facilitating gallstone assembly and growth.

The group then went on to carry out their experiments in mice. They were particularly interested in studying gallstone formation in one type of mice, called PADI4-deficient mice, that are unable to form neutrophil NETs. The authors were curious to see if mice were capable of forming gallstones in the absence of NETosis. 

In one experiment, normal, wild-type (WT) mice and these PADl4-deficient mice were both fed a cholesterol rich diet (also known as a lithogenic diet) to induce gallstones for six weeks. While gallstones formed in WT mice, the occurrence of gallstones in PADl4-deficient mice was markedly reduced, with the gallstones that did form being significantly smaller than those present in WT mice. This further bolstered the idea that NETs are the principal factor in the enlargement of biliary precipitates into gallstones. Moreover, the results observed with the PADl4-deficient mice suggested to the authors that specifically targeting NET formation by neutrophils could lend itself as possible therapy for gallstones. 

 With this in mind,  the researchers performed another experiment, in which WT mice were treated with a drug able to restrain neutrophil movement and activity, in conjunction with a 6-week, gallstone-inducing, lithogenic diet.  In the drug treated mice, gallstones were less likely to be present and were smaller in size overall than gallstones found in the untreated mice.

What’s next? 

With the newfound knowledge that neutrophil NETs and gallstone genesis are closely intertwined, perhaps alternative methods of treatment can begin development. Currently, gallstone prevention measures include a balanced diet, with particular emphasis on avoiding unnecessary cholesterol. In high-risk patients, preventative medications that can dissolve forming gallstones, or lower the body’s cholesterol, also help. Though more research needs to and should be done, the specific target of neutrophil NET formation through drug intervention looks like a promising and bright path of inquiry.

 

Cover image: “A net” by DBduo Photography is licensed under CC BY-SA 2.0 

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