ABOVE: Colored scanning electron micrograph of segmented filamentous bacteria (green) attached to the mouse intestinal wall at 6 AM (left) and 6 PM (right).

Microbes in a mouse’s gut synchronize the animal’s immune responses to its daily activity patterns, according to a study published yesterday (July 28) in Cell. Certain intestinal bacteria that are sensitive to the timing of mouse feeding drive rhythmic signaling in the innate immune system, researchers found, such that immune activity peaks when the mouse is most likely to come into contact with pathogens: when it’s eating and running about.

The findings identify the microbiota as an important link between an animal’s circadian clock and its immune system, says Christoph Thaiss, a microbiologist at the University of Pennsylvania who was not involved in the work. 

“In a way, this was a long-awaited finding,” he says. While researchers knew that circadian rhythms in feeding could affect the activity of the gut microbiota, and also that some gut microbes influence immune signaling, “it was not known whether the relationship between the circadian clock and the microbiome . . . would affect components of the gut immune system. That’s exactly what they show here.”

See “Microbes Shape Circadian Rhythms in the Mouse Gut

To establish these links between circadian rhythms, the microbiota, and host immunity, study coauthor John Brooks, a postdoc in Lora Hooper’s lab at the University of Texas Southwestern Medical Center in Dallas, started out by searching for any antimicrobial proteins—peptides secreted as part of the innate immune response—whose production in the mouse intestine showed rhythmicity. 

He identified multiple proteins that were present at higher levels at the beginning of the night, when mice become active and start eating, than at the start of day, when they typically rest. Brooks was especially interested in the antibacterial peptide REG3G: this protein reliably cycled across the 24-hour cycle in regular mice, but not in germ-free mice, suggesting the microbiota was necessary to maintain the rhythm.

Sifting through images of mouse intestines for clues as to what might be driving the REG3G cycles, Brooks, Hooper, and colleagues noticed segmented filamentous bacteria (SFB) clinging to the gut wall. These bacteria were already known to influence immune signaling, Brooks says, but what the researchers hadn’t realized before was that their behavior changed across the 24-hour cycle, with more bacteria attached to the gut epithelium at the onset of night than at the onset of day.

These antimicrobial proteins are energetically expensive to produce, so you want to produce them when they’re absolutely required.

—John Brooks, UT Southwestern Medical Center

Inoculating germ-free mice with SFB caused REG3G to start cycling, they found, while SFB in mice lacking REG3G still showed rhythmic patterns in their attachment to the gut wall, suggesting it was indeed the bacteria that were driving REG3G cycles, and not the other way around. In another series of mouse experiments, the team connected the dots between bacterial attachment and REG3G cycles, showing that attachment triggers a “surprisingly complicated” cascade of immune signaling via cytokines and other proteins, culminating in a boost in levels of the antimicrobial protein, Hooper tells The Scientist.

Based on previous research by Thaiss and others, Brooks says he suspected that the SFB in a mouse’s gut were getting their own rhythms from the animal’s feeding patterns, which are regulated by the mammalian circadian clock. Sure enough, mice engineered to lack certain circadian clock proteins ate more randomly and didn’t show the same cyclic patterns of bacterial attachment to the gut wall; mice that fasted for 24 hours also lost their SFB rhythms. When the researchers fed mice only during the day, meanwhile, the SFB cycles—and all the associated downstream effects on the immune system—shifted by 12 hours. 

Together, the results suggest that the mouse gut evolved to anticipate the riskiest time periods in the day and to prep immune defenses accordingly, Brooks says. “These antimicrobial proteins are energetically expensive to produce, so you want to produce them when they’re absolutely required,” he explains. “This work kind of ties the production of these antimicrobial proteins to the times in which you’re most likely to acquire a foodborne pathogen.”

Interested in whether these immune cycles might have practical consequences for mouse health, the team monitored the animals’ susceptibility to infection with a pathogen, Salmonella Typhimurium, at various times throughout the 24-hour cycle. 

S. Typhimurium isn’t your typical bacterium, Brooks notes, because it’s better at infecting mice when the gut is inflamed. Accordingly, mice’s resistance to the bug varied as expected across the 24-hour cycle: animals infected at the onset of night, when SFB attachment and antimicrobial proteins levels were high, got sicker than animals infected during the day. 

The team used Salmonella for this experiment because it’s one of the few pathogens that easily infect mice even when the animals haven’t been pretreated with antibiotics—something the researchers worried would disrupt the microbiota cycles they’d observed, Brooks explains. For Salmonella, the immune boost actually favors the pathogen, but in many cases the elevated immune activity probably protects the mouse from infection, Hooper says. 

Listeria bacteria, for example, would likely show the opposite pattern from Salmonella, Hooper says. “We weren’t able to test Listeria directly because of the antibiotic issue, but our expectation there would be that the susceptibility to Listeria would go down when antimicrobial protein expression was high.”

Thaiss says he was particularly pleased to see the inclusion of the Salmonella experiment. “This was a big question in the field—whether the known circadian properties of the microbiome and the known circadian properties of the host . . . come together” to provide an evolutionary benefit for host defense against intestinal infections, he says. The demonstration that infection susceptibility varies across the day suggests that this is indeed the case.

It’s not yet clear how this kind of microbiota-driven immune cycling might play out in humans, says Julie Gibbs, a chronobiologist at the University of Manchester in the UK who was not involved in the work; SFB are present in people, but whether they attach rhythmically to the gut wall is unknown. Nevertheless, she says, the team’s findings hint at one possible explanation for a well-known association in human health: the link between disrupted sleep and disease risk.

“We know that shift working is generally bad for our health and has a negative impact on the immune system and risks of developing inflammatory diseases,” Gibbs says. In addition, “we know that shift work is associated with disordered eating. So I think it’s of interest for shift workers if this disordered eating may be affecting the rhythmic microbiome, which may be having knock on effects on our immune system and immunity.”