An immune cell that helps set the daily rhythms of the digestive system has been identified by researchers at Washington University School of Medicine in St. Louis. The findings open the door to new treatments for digestive ailments targeting such cells. [Qianli Wang]

Two independent sets of research published in Science Immunology have demonstrated how a subset of immune cells in the gut, known as group 3 innate lymphoid cells (ILC3), are tuned to the body’s daily circadian clock as they sense and interact with gut microbes. Collective results from the research indicate that specific “clock genes” that are expressed in synchrony with specific times of day could represent targets for countering the negative effects of poor or erratic sleep schedules on gastric health.

One set of studies, reported by scientists at Washington University School of Medicine in St. Louis, found that ILC3 cells are responsible for keeping the intestine operating in a normal, healthy manner. The research, described in a paper titled, “Circadian rhythm-dependent and circadian rhythm-independent impacts of the molecular clock on type 3 innate lymphoid cells,” showed how these clock genes are highly active in ILC3 cells, and that ILC3 cell production of immune molecules including IL-22 and IL-17 stays in tune with clock gene activity. “It’s become increasingly clear that the disruptions of circadian rhythms so common in modern life—shift work, jet lag, chronic sleep deprivation—have harmful effects on people’s health, but we still don’t know a lot about how exactly sleep disruptions cause these problems,” commented senior study author Marco Colonna, MD, the Robert Rock Belliveau, MD, professor of pathology and a professor of medicine at Washington University School of Medicine in St. Louis. “What we’ve found here is that circadian rhythms directly affect the function of immune cells in the gut, which could help explain some of the health issues we see, such as inflammatory bowel disease and metabolic syndrome.”

The research reported by Fei Teng, PhD, a postdoctoral associate, and colleagues at Cornell University, similarly demonstrated that intestinal ILC3s activity is directed by molecular instructions that are involved in circadian clock control. Their studies, in addition, demonstrated altered circadian gene expression in ILC3s of patients with inflammatory bowel disease (IBD). The researchers stated that the collective findings suggested that “… circadian regulation is essential for the homeostasis of ILC3s in the presence of a complex intestinal microbiota … These findings reveal that circadian regulation critically orchestrates ILC3 homeostasis in the gut and suggest that disruption in this pathway drives the impaired ILC3 responses in the inflamed intestine of patients with IBD. ” The paper by Teng et al. is titled, “A circadian clock is essential for homeostasis of group 3 innate lymphoid cells in the gut.”

The papers in Science Immunology are published less than a month after researchers at the Champalimaud Centre for the Unknown in Lisbon reported on their studies demonstrating the effects of disrupting clock genes on gut ILC3 cell function and localization.

Many people have fairly regular daytime and nighttime routines, and digestive tract function similarly follows a daily rhythm. Food is digested and nutrients are absorbed during waking hours, and worn out cells are replaced during sleep. However, shift work and jet lag can disrupt normal sleep times and throw our digestive rhythms out of kilter, which has been linked with an increased risk of intestinal infections, and disorders including obesity, inflammatory bowel disease, and colorectal cancer. The mammalian circadian clock plays important roles in regulating multiple immune cell functions,” Teng and colleagues commented. “However, very little is known about the circadian clock and ILC3s.”

ILC3 cells in the gut play a role in maintaining gastrointestinal equilibrium by helping to bolster the barrier between the normal microbial communities that live in our gut, and the cells that make up the intestine. ILC3 cells also produce immune molecules and help to keep the gastrointestinal system in immune balance, which includes retaining the ability to fight invading pathogens. “The mammalian gastrointestinal tract is continuously exposed to both pathogenic and commensal microbes, and the immune system must carefully regulate immunity, inflammation, and tolerance at this site to maintain tissue homeostasis,” Teng et al. wrote. “Innate lymphoid cells (ILCs) have emerged as critical regulators of these processes … In particular, group 3 ILCs (ILC3s) play numerous roles in regulating intestinal health through production of interleukin-22 (IL-22) as well as their ability to control adaptive immune responses to dietary antigens or the intestinal microbiota.”

Researchers in Colonna’s lab have been studying ILC3 for years, and when the team’s Qianli Wang, and Michelle Robinette, MD, PhD, noticed that clock genes were highly activated in ILC3 cells, they reasoned that the cells might link circadian rhythms to the gut’s immune system. “If ILC3 cells are attuned to circadian rhythms, they can anticipate when nutrition is going to arrive in the intestine, which is also when dangerous bacteria might accidentally be consumed and arrive in the gut, too,” said Wang, who is first author on the team’s published paper. “For optimal functioning, the gut needs to be prepared for these daily rhythms, and these cells play a pivotal role in that process.”

When the Colonna lab researchers examined ILC3 cells taken from mouse intestines at six-hour intervals, they found that their clock gene activity varied predictably over the course of a day, and that expression of the cells’ genes for immune molecules tracked in parallel with clock genes. When the team then put a group of mice on a light-dark schedule similar to one experienced by a shift worker—an eight-hour change in the light-dark cycle every two days—the animals’ ILC3 cells stopped functioning normally, and only produced low levels of immune molecules when stimulated to respond to an infection. In addition, mice engineered to lack the clock protein REV-ERBα failed to develop normal numbers of ILC3 cells. “I think it’s fair to say that ILC3 is under circadian rhythm regulation and certain key circadian genes are crucial for ILC3 cells to develop and function,” Wang said.

Wang and Colonna reasoned that reductions in ILC3 cell numbers or changes in ILC3 function could affect the body’s ability to fight intestinal infections. Subsequent studies showed that in comparison with control mice, animals engineered to lack the clock protein REV-ERBα failed to mount an effective response against Clostridium difficile infection (CDI), a bacterium that can cause severe diarrhea in people. The animals’ REV-ERBα-deficient ILC3 cells produced more IL-17 and less IL-22, and the pathogenic bacteria were able to spread in the body. “Previous reports have suggested that IL-17A drives inflammatory responses in CDI and implicated increased IL-17A in poorer disease outcomes,” the team wrote. “Therefore, hyper-responsive IL-17 secretion by REV-ERBα-deficient ILC3s may contribute to more severe inflammation and bacterial burden.”

The scientists said their results also hint at potential therapeutic targets. “Given the role of REV-ERBα as developmental regulator of ILC3s that we report here, and the availability of REV-ERBα agonists and antagonists, REV-ERBα could prove an impactful target for therapeutic interventions aimed at modulating ILC3s in pathology.”

“The emerging relevance of the circadian regulation in gut health is likely to impact medical and hospital practice,” Colonna said. “I think we will have to start taking circadian rhythms of the gut cells into consideration when choosing optimal timing for nutritional and pharmacological interventions.” The equilibrium of the gut is upset by disruptions to circadian rhythms, Wang added. “ILC3 cells are so important to gut equilibrium that we may be able to counter some of these disruptions by targeting clock genes in ILC3 cells.”

The studies by Teng et al. similarly showed that deleting the clock proteins REV-ERBα and BMAL1 in mice resulted in dramatically reduced numbers of gut ILC3s. They also found that using antibiotics to deplete the intestinal microbiome of these mice restored the activity of BMAL1-deficient ILC3s, further indicating a potential link between circadian rhythms in microbes and ILC3 activation. “Depletion of the microbiota with antibiotics partially reduced the hyperactivation of BMAL1-deficient ILC3s and restored cellular homeostasis in the intestine,” the team wrote.

Additional human studies by Teng’s group showed that ILC3s isolated from the inflamed gut of patients with inflammatory bowel disease (IBD) expressed lower levels of clock proteins than ILC3 from the non-inflamed gut in the same patients. This suggested that chronic inflammation and disease may be linked with altered circadian gene regulation that can lead to less protective ILC3s. “… it has become increasingly appreciated that there are certain signatures of immune responses and therapies that exert day and night differences (termed as chrono-immunotherapy),” the Cornell University researchers continued. “Therefore, our critical finding that ILC3s in the inflamed intestine of patients with IBD exhibit altered circadian gene expression suggests that this is an important pathway in human health and disease and may hold a key for developing novel strategies to boost ILC3 responses in the context of impaired intestinal homeostasis or microbial dysbiosis.”

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