Resetting Neutrophil Clock Reduces Collateral Damage of Inflammation

Biology runs in a rhythmic oscillation set by an internal clock: Our digestive enzymes peak around mealtimes, our sleep hormones are released at night, and our body temperature drops at daybreak. This is also true of our immune responses, which is why inflammatory injuries, such as heart attacks, are deadlier in the morning than at night.

As the “first-responders” in trauma and injuries, neutrophils—the most abundant type of white blood cell—go to injury sites to kill pathogens and keep the area sterile. But at times, these cells overstay and secrete toxic compounds that damage surrounding healthy cells.

Now, in a study published Dec. 12 in the Journal of Experimental Medicine, Yale School of Medicine (YSM) scientists have discovered the molecular switches that control the internal clock of neutrophils. They also showed that modulating the circadian rhythms of these immune cells in mice could spare healthy tissues from getting damaged during a heart attack.

“Neutrophils were considered these crazy foot soldiers—they don't think, they just destroy. But we realize now that it is not quite the case,” says Andrés Hidalgo, PhD, a professor of immunology at YSM and senior author of the study. “Neutrophils have a sense of time. And they use it to know when they have to be active and where to go.”

In an earlier study, Hidalgo and his team found that the neutrophil clock is run by two molecular switches: the Bmal1 transcription factor protein, which activates neutrophils and is active during the day, and the CXCR4 receptor, which inhibits neutrophils and is active at night.

The Bmal1 gene regulates the circadian oscillation of almost every cell in the body. “When Bmal1 is not functioning properly, the whole organism undergoes some sort of biological jet lag,” Hidalgo says. “You're eating when your genes are not ready to digest, you're resting when you should be active. So, there's a mismatch between functions and the biological clock.”

For the new study, the researchers analyzed the health records of 2,043 individuals who had experienced a heart attack, finding that the outcome of a person’s injury correlated with their neutrophil levels; patients who had a heart attack in the morning, when neutrophil levels are typically high, had worse cardiac injury compared to those who had it at night.

“The idea is that these neutrophils are more active during daytime, so whatever they do, they do more aggressively, causing greater injury to healthy tissue,” Hidalgo says. Therefore, if a heart attack took place when neutrophils were less active, the collateral damage would be much less, he thought.

To test this hypothesis, the researchers induced tissue damage in the hearts of mice whose neutrophils lacked Bmal1. They found that the lack of Bmal1 (the neutrophil activator) led to lower daytime neutrophil activity, which protected the mice from cardiac injuries. Next, the researchers did the same analysis in mice with elevated CXCR4 function (the neutrophil inhibitor) finding they were also protected from cardiac injury.

“So, there are two layers of control,” Hidalgo says.

The researchers used a four-dimensional imaging platform to visualize the shape and behavior of neutrophils inside an inflamed blood vessel. They found that at daytime, neutrophils were sedentary and stuck, flattened against the vessel walls. This behavior is characteristic of an inflammatory condition and has been associated with severe injuries or infections. On the other hand, the neutrophils of mice injured at night resembled uninfected neutrophils; they were motile, able to move away from the area outside the injury.

The researchers also found that neutrophils can be tricked into nighttime behavior using a compound that activates CXCR4, called ATI2341.

“You can see this as a dial,” Hidalgo says. “With ATI2341, you can dial neutrophils from a super-aggressive mode into a less-aggressive mode.”

The added benefit of dialing the neutrophils through CXCR4 is that it doesn’t interfere with the cells’ ability to kill pathogens, thereby preserving their protective nature. This method of protecting the healthy cells could translate to a preventive drug that protects a person from the deadly effects of a heart attack, he adds.

“When we disable the clock, we protect—not as much as nighttime—but we confer sufficient protection to the bystander cells during daytime,” Hidalgo says.

There are remaining questions regarding the limitation of such an intervention. “We want to see if there's any trade-off by dialing down neutrophils,” Hidalgo says. Nonetheless, the implication of controlling neutrophils’ activity and removing the source of stress around injury sites could be significant in preventing severe inflammatory conditions such as cancer, sepsis, and neurodegenerative diseases.

“Circadian regulation is a very important component of immunity,” Hidalgo says. “That’s what is becoming clear here.”