Keeping Neutrophils in Night Mode Could Reduce Post-Heart Attack Inflammation

Heart attacks that occur at night are less severe than those that strike during the day. A new study headed by researchers at the Centro Nacional de Investigaciones Cardiovasculares (CNIC) and at Yale University School of Medicine, has explained why. Andrés Hidalgo, PhD and colleagues found that neutrophils have an internal clock that regulates their aggressiveness throughout the day and determines the extent of damage they cause to the heart after a heart attack.

Studies by the international research team suggest that inhibiting these fluctuations could prevent neutrophils from causing excessive tissue damage during daylight hours, a phenomenon that may underlie the fact that heart attacks in the early morning are more damaging than heart attacks suffered at night.

The researchers also developed a pharmacological strategy—using a drug called ATI2341—that in experimental models blocked the clock in neutrophils, keeping them in a “nighttime” state and thereby reducing their harmful potential during a heart attack. ATI2341 targets a receptor on the surface of neutrophils and switches the cells into the less active mode usually only seen at night.

“Our study demonstrates that pharmacological delivery of a drug that activates a receptor on the surface of neutrophils induces their transition to a night-like, permissive state that alleviates the inflammatory response without interfering with antimicrobial defense,” Hidalgo said. “This therapeutic strategy provides an advantage when compared with other approaches that target neutrophil function or numbers, which compromise the capacity of the host to control infections or to promote wound healing.”

Hidalgo together with colleagues at Yale University School of Medicine, and collaborators, reported on their findings in Journal of Experimental Medicine, in a paper titled “A circadian checkpoint relocates neutrophils to minimize injury.”

The immune system protects the body against microorganisms that cause infection. Because humans are diurnal—active during the day and asleep at night—the likelihood of exposure to pathogens is higher during the day. The immune system therefore adjusts its activity peaks to this circadian rhythm. However, this same defensive response can become harmful. It is well known that in stressful situations such as myocardial infarction, the immune system can cause severe collateral damage to tissues.

Decades of research have shown that almost half of the cardiac damage after a heart attack is caused by neutrophils. Interestingly, this type of inflammatory damage fluctuates naturally throughout the day, suggesting the existence of circadian mechanisms that limit neutrophil activity and protect the body.

Neutrophils provide the first line of defense against microbial infections and tissue injury. However, their efforts to promote inflammation and kill injured or infected cells can result in the death of nearby healthy cells. “This is relevant in the context of both infections and sterile inflammation, conditions in which the affected tissue recruits large numbers of neutrophils that can expand the area of damage by releasing an arsenal of chemicals and cellular components, and induce the death of unaffected neighboring cells,” the authors stated.

These antagonistic effects of immune protection and inflammatory injury have prevented development of effective therapies, “…  because both properties are generally considered inseparable features of neutrophils,” they continued. Studies indicate that neutrophils have an internal clock that makes them more active and prone to cause tissue damage during the daytime. “Intriguingly, inflammatory injury naturally declines at specific times of day, suggesting that circadian mechanisms exist that mitigate the destructive activity of neutrophils and protect the host,” the team pointed out.

This could help explain the long-standing observation that heart attacks in the early morning have more severe consequences than heart attacks at night, because excessive neutrophil activity contributes significantly to the size of myocardial infarcts and long-term reductions in cardiac function. “Myocardial infarction, a leading cause of morbidity and death worldwide, represents a paradigm of collateral tissue injury inflicted by neutrophils and follows marked circadian patterns in severity, both in mice … and in humans,” the investigators stated.

Prior studies had also identified the chemokine receptor CXCR4 as a negative regulator of the clock, and research by Hidalgo and colleagues at Yale University School of Medicine previously found that mice whose neutrophils lack this internal clock do not show daily peaks in neutrophil activity, but are still able to fight off bacterial and fungal infections.

The authors thought it possible that targeting the neutrophil clock could provide what they called “a simple and effective means to blunt the toxic activity of these cells during cardiovascular inflammation without compromising antimicrobial defense.”

Hidalgo added, “We thought that targeting the neutrophil clock could provide a simple and effective means to blunt the toxic activity of these cells during myocardial infarctions, without compromising antimicrobial defense.”

In collaboration with the Multidisciplinary Translational Cardiovascular Research Group at the CNIC, led by Héctor Bueno, MD, PhD, who leads the Multidisciplinary Translational Cardiovascular Research (MTCR) Group at CNIC, the researchers examined data from thousands of patients at Hospital 12 de Octubre. Bueno also leads the Cardiovascular Research Area at the i+12 Research Institute in Hospital Universitario 12 de Octubre. The investigators’ analyses confirmed that lower neutrophil activity at night results in less severe heart attacks during this period.

For their newly reported study Hidalgo and colleagues also performed a series of experiments to confirm that, as in humans, mice suffer greater cardiac tissue damage after a heart attack in the early morning, and that this is due to enhanced neutrophil activity at this time of the day. “We show that the circadian spikes in myocardial injury in mice and humans are mediated by neutrophils and controlled by their intrinsic clock,” the authors wrote.

The team developed a pharmacological strategy to block the molecular clock in neutrophils, reducing their harmful potential during infarction in mouse models. “The compound mimics a factor that the body produces mainly at night,” explains Hidalgo. “In a way, this factor ‘tricks’ neutrophils into thinking it’s nighttime, reducing their toxic activity.”

Treating mice with the CXCR4 agonist ATI2341—which, by inhibiting the neutrophil clock holds cells in their nighttime mode—reduced the amount of myocardial tissue damage after a heart attack and helped to preserve heart function over the following days and weeks.

In their active, daytime mode, neutrophils accumulate around the edge of the initial wound caused by a heart attack, where they are poised to damage neighboring healthy cardiac tissue and extend the size of the injury. In night mode, however, neutrophils accumulate in the center of the initial wound, well away from the surrounding, healthy tissue. Study first author and CNIC investigator Alejandra Aroca-Crevillén, PhD, also highlighted that the observed protection stems from a change in cellular behavior. “At night, neutrophils migrate to the damaged area while sparing healthy tissue. During the day, they lose this directionality and cause more damage to surrounding tissue.”

And reporting in their paper, the team commented, “… CXCR4 agonism relocates neutrophils away from the healthy neighboring tissue, a circadian phenomenon that is naturally active at night and prevents indiscriminate tissue death.”

Experiments confirmed that the agonist had no protective effect in mice with neutrophils in which CXCR4 was deleted, “… indicating that cardiac protection was mediated through specific targeting of CXCR4 in neutrophils,” the investigators noted. The team’s experiments in addition showed that ATI2341 protected mice from several other types of neutrophil-induced tissue damage but, crucially, did not impair the animals’ ability to fend off bacterial and fungal infections. “Thus, the protective effect of CXCR4 agonism during sterile inflammation does not compromise antimicrobial responses, suggesting that targeting this circadian neutrophil checkpoint driven by CXCR4 may be an attractive therapeutic target against the devastating impact of uncontrolled inflammation in human health,” they concluded.

This study is one of the first to harness the circadian rhythms of the immune system to modulate inflammation without compromising infection defense. “We were surprised to find,” added Aroca-Crevillén, “that blocking the neutrophil circadian clock not only protects the heart, but also improves responses to certain microbes and even reduces embolisms associated with sickle cell anemia.”

The authors conclude that the results open the door to new therapies based on chronobiology (the branch of biology that studies how living organisms structure their physiological processes in time), with the potential to protect the heart and other organs from inflammatory damage without weakening the body’s natural defenses.