Newly published research by investigators at the Children’s Hospital of Philadelphia (CHOP) has started to answer the question of why nonsteroidal anti-inflammatory drugs (NSAIDs) exacerbate gastrointestinal infections by Clostridioides difficile, the leading cause of antibiotic-associated diarrhea worldwide. The newly published in vitro and preclinical in vivo research showed that in addition to the activity of NSAIDs as cyclooxygenase enzyme inhibitors, these drugs disrupt the mitochondria of cells lining the colon, sensitizing them to damage by pathogenic toxins.
“Our work further demonstrates the clinical importance of NSAIDs in patients with C. diff infection and sheds light on why the combination of these two may be so detrimental,” said Joseph P. Zackular, PhD, investigator and assistant professor of pathology and laboratory medicine at Children’s Hospital of Philadelphia. “Our mechanistic findings are a starting point for further research that aims to understand the impact of mitochondrial functions during C. diff infection. These data could also inform how NSAID-mediated mitochondrial uncoupling affects other diseases, such as small intestinal injury, IBD, and colorectal cancer.”
Zackular is senior author of the team’s published paper in Science Advances, titled “Nonsteroidal anti-inflammatory drugs sensitize epithelial cells to Clostridioides difficile toxin–mediated mitochondrial damage.” In their paper the team concluded, “Our results demonstrate that NSAIDs exacerbate CDI by synergizing with C. difficile toxins to damage host cell mitochondria. Together, this work highlights a role for NSAIDs in exacerbating microbial infection in the colon.”
Infection with the bacterium Clostridioides difficile, often referred to as C. difficile or C. diff, is associated with a wide range of symptoms, from mild diarrhea, to complex infection and death. The factors that influence this wide spectrum of clinical outcomes remain largely unclear, but emerging evidence suggests that factors such as diet and pharmaceutical drugs influence both susceptibility to infection and disease progression. “However, we still know very little about the influence of xenobiotics and pharmaceutical drugs on C. difficile infection (CDI),” the authors stated.
NSAIDs are used to treat pain and reduce inflammation, and represent the most widely prescribed drugs worldwide. They work by inhibiting cyclooxygenase (COX) enzymes 1 and 2. Prior studies have shown that NSAIDs such as indomethacin, aspirin, and naproxen, can negatively affect the gut, both in patients with C. difficile infection (CDI) and other conditions, for example, inflammatory bowel disease (IBD). Long-term NSAID use can lead to stomach ulcers and intestinal injuries, such as bleeding and perforation of the intestinal tissue. Researchers hypothesize that this is due to the effects of NSAIDs on the COX enzymes, a process that helps reduce inflammation and pain but also impairs mucosal function in the upper gastrointestinal tract.
NSAIDs do also have off-target effects and have been shown to affect cellular mitochondria by uncoupling cellular mitochondrial functions, although researchers had not explored the mechanism or impact of these off-target effects when it comes to C. difficile infection. “To date, the impact of NSAID-mediated mitochondrial effects on the development of enteropathy independent of COX enzyme inhibition has not been defined,” they wrote. “Moreover, little is known about the role of off-target effects of NSAIDs in the colon, and the role of these effects on infection of the colon has not been explored in-depth.”
To investigate these effects in greater detail, the researchers, led by graduate student Joshua Soto Ocaña, used in vitro and mouse models of C. difficile infection to test the permeability of colonic epithelial cells (CECs) in the presence of the NSAID indomethacin. “Notably, our previous work established that prior exposure to the NSAID, indomethacin, can exacerbate CDI and markedly increase mortality in a mouse model of infection,” they wrote. Through their newly reported work the researchers observed that both indomethacin and C. difficile toxins increased epithelial cell barrier permeability and inflammatory cell death. They also found that the effect was additive: the combined effect on cell permeability of both toxins and indomethacin was increased, compared with each independently, suggesting that NSAIDs and C. difficile work synergistically to increase the virulence of this pathogen. Commenting on data from their in vitro model, the team wrote, “… these data demonstrate that indomethacin sensitizes CECs to C. difficile toxins, and the combination of NSAIDs and C. difficile toxins impairs colonic epithelial cell barrier functions by enhancing cell death, permeability, and inflammation.”
Surprisingly, the researchers found that NSAIDs exacerbate C. difficile infection independent of COX inhibition and instead act through off-target effects on mitochondria. They discovered this by treating colonic epithelial cells with a precursor molecule that is similar in structure to indomethacin but lacks the ability to inhibit the COX enzyme. Not only did their experiments find that this NSAID-like molecule induced cell death, but they also found that adding selective COX inhibitors did not increase cell death, demonstrating that COX enzyme inhibition is not required to induce epithelial cell damage during C. difficile infection and that, instead, this damage occurs through off-target effects of NSAIDs.
To test the role of off-target effects during C. difficile infection, the researchers used mice pretreated either with indomethacin or the NSAID-precursor molecule. When exposed to C. difficile, both groups of mice showed equal enhancement in disease severity and mortality compared with control mice infected with C. difficile only. The researchers observed a similar effect in mice that were pretreated with the NSAID aspirin. “The in vivo impact of NSAIDs was not limited to indomethacin since we observed an increase in mortality and similar C. difficile burdens after pretreatment with the NSAID aspirin.”
To further define the specific mechanisms driving these off-target effects of NSAIDs, researchers looked at mitochondrial functions in colonic epithelial cells in vitro and in mice. They observed that the combination of NSAIDs and C. difficile toxins increased damage to colonic epithelial cell mitochondria and disrupted several important mitochondrial functions. “Mice pretreated with indomethacin and subsequently infected with C. difficile showed increased levels of CECs with damaged mitochondria compared to cells from mice that were treated with indomethacin, C. difficile or cefoperazone only,” the authors stated. “Moreover, we observed that C. difficile, indomethacin, or both in combination equally decreased mitochondrial membrane potential in murine CECs …”
“Our work further demonstrates the clinical importance of NSAIDs in patients with C. diff infection and sheds light on why the combination of these two may be so detrimental,” said senior author Joseph P. Zackular, PhD, Investigator and Assistant Professor of Pathology and Laboratory Medicine at Children’s Hospital of Philadelphia. “Our mechanistic findings are a starting point for further research that aims to understand the impact of mitochondrial functions during C. diff infection. These data could also inform how NSAID-mediated mitochondrial uncoupling affects other diseases, such as small intestinal injury, IBD, and colorectal cancer.”
The authors further commented, “These results highlight an underappreciated role of NSAIDs during GI infection in the colon … Our work sheds light on the clinical importance of NSAIDs in patients with CDI and why the combination of these two may be so detrimental … These findings provide an unexpected framework for how NSAIDs may worsen the clinical outcomes of this important nosocomial infection and provide important insights into the off-target effects of NSAIDs on the colonic epithelium.”
