Studies in mice by Harvard Medical School (HMS) researchers suggest how a class of regulatory T cells (Tregs) that are produced in the gut also play a role in repairing injured muscles and mending damaged livers.
The research found that gut microbiota fuel the production of, and act as the training camp for, a class of immune cells—RORγ+ Tregs—that are recruited to heal muscle injury. The study findings also indicated that these same gut immune cells help repair injured fatty livers. The collective results suggest that these Tregs act as immune healers that go on patrol around the body and respond to distress signals from distant sites of injury.
The new insights point to a broader role for gut immune cells—which have long been known as guardians of intestinal health—in repairing damage outside of the gut environment. The findings support a growing body of evidence demonstrating the importance of gut microbiota in regulating various physiologic functions beyond the gut, and suggest that gut immune cells may have a wider role in taming inflammation and healing damage that extends beyond the intestines.
The team cautions that their findings are based on experiments in mice and remain to be replicated in larger animals and in humans. However, they do raise interesting possibilities about harnessing the power of gut microbes to enhance recovery from injury. Another possibility, said research co-lead Diane Mathis, PhD, professor of immunology at the Blavatnik Institute at HMS, is the potential that the new findings could inform the design of treatments for fatty liver disease, a common condition in which accumulation of fat in the liver leads to liver cell damage and death.
“Our observations indicate that gut microbes drive the production of a class of regulatory T cells that are constantly exiting the gut and act as sentries that sense damage at distant sites in the body and then act as emissaries to repair that damage,” said Mathis, who is senior author of the team’s study published in Immunology and titled, “The gut microbiota promotes distal tissue regeneration via RORγ+ regulatory T cell emissaries.”
The human immune system is incredibly versatile. Among its most skilled multitaskers are T cells, known for their roles in everything from fighting infection to reining in inflammation to killing nascent tumors. Tregs are highly specialized. They reside in various organs, where they control local inflammation and regulate organ-specific immunity.
“Foxp3-expressing regulatory T cells are a subset of CD4+ T lymphocytes key for maintaining immunological tolerance,” the authors noted. A category of specialized “‘tissue-Treg cells’’ cells are found in non-lymphoid tissues, including the skeletal muscle, skin, and the colon. These tissue-Tregs communicate with a wide range of immunological and non-immunological cells in their microenvironment and exert diverse functions, the team continued, “and thereby guard tissue homeostasis in various contexts, such as regulating local and systemic metabolism, enforcing tolerance to the microbiota, and regulating tissue regeneration in response to injury.”
The researchers were already familiar with the type of Tregs normally found in the colon. These cells play an important role in maintaining gut health, such as protecting the body from food allergens, autoimmune conditions like colitis, and even colon cancer. “The function of RORγ+ Treg cells has so far been linked primarily to mucosal health as they play an indispensable role in maintaining intestinal tolerance, with their loss resulting in increased incidences of colitis, colon cancer, and food allergies,” the authors explained. Scientists also understood that gut microbes act as regulators of gut immunity by controlling the production of Tregs, but there was little evidence that intestinal Tregs could affect tissues and processes beyond the gut.
So, when carrying out routine cataloging of various immune cells in different organs, the researchers were puzzled to come across gut Tregs intermingled with muscle cells. “While surveying Treg cell heterogeneity in non-lymphoid tissues, we noticed a population of RORγ+ Treg cells that accrued in regenerating skeletal muscle early after acute injury,” they wrote. The majority of Treg cells are generated in the thymus, but such intestinal RORγ+ Treg cells are mostly induced locally, in the gut, in response to microbial or food antigens. “I stumbled upon some cells that looked very similar and had all the same features of Tregs that derive from the gut,” said study first author Bola Hanna, PhD, a research fellow in immunology at HMS. “This caught our attention because we know these cells are produced in the gut and are shaped by the microbiota.”
Colonic Tregs had been rarely found outside of the small and large intestines, so the researchers queried why muscle should contain this type of immune cell. They first had to verify their unusual observation and confirm that the Tregs they’d found in the muscle tissue really were colonic Tregs. To do so, the scientists analyzed the cells’ molecular signatures. This confirmed that the Tregs they found in the muscle were, indeed, colonic-like Tregs. “In brief, multiple experimental approaches provided data arguing that muscle RORγ+ Treg cells were engendered in the colon,” they stated. “Muscle and colon RORγ+ Treg cells shared transcriptional particularities and TCR specificities.
The scientists tagged colonic Tregs with light and followed them as they made their way around the bodies of mice. The results showed the light-tagged cells left the gut environment and migrated to other parts of the animals’ bodies. “Both adoptive transfer and photo-tagging experiments evidenced colon to muscle cell flow.” The team also looked at the Tregs’ surface receptors for antigens, a kind of unique barcode that marks each cell. “The immune cells we had found in the muscle shared the same barcodes with the equivalent Treg cells in the gut,” Hanna said.
Next, the researchers investigated whether these cells played a role in muscle regeneration. In one experiment, they showed that mice genetically modified to lack this class of colonic Tregs showed markedly slower rates of muscle recovery. Taking a closer look at the healing process, the researchers found that these animals had higher levels of inflammation in injured muscle tissue. And when the mice did eventually heal, they developed muscle scarring, or fibrosis, a sign of poor muscle repair.
To determine whether gut microbes fuelled the production of colonic Tregs to heal muscle tissue the researchers fed mice antibiotics to deplete their beneficial gut bacteria. These antibiotic-treated mice had a harder time recovering from muscle injury. But when their gut microbiota were restored, the animals’ ability to heal their muscles also improved. Further experiments demonstrated that colonic Tregs helped the muscle healing process by suppressing an inflammatory signal called IL-17. Their analyses indicated that there is a direct, time-dependent impact of IL-17A on muscle satellite cells (MuScs)—these cells are a population of quiescent stem cells that are activated to proliferate in response to muscle injury—such that prolonged exposure to IL-17A impedes their differentiation. So, lowering levels of this signal during a precise time window moderated the inflammatory response and helped stop inflammation when it was no longer needed for the healing process. “Given the critical role of RORg+ Treg cells in the temporal regulation of IL-17A production in regenerating muscle, timely recruitment of RORγ+ Treg cells at early stages of regeneration appears to be crucial for shielding differentiating MuSCs from IL-17A, thereby promoting return to homeostasis,” they further pointed out.
“When muscles are healing, you need a certain dose of inflammation within a certain time frame,” Hanna said. “And in the absence of these gut-derived regulatory T cells, we found that the degree of inflammation gets higher and extends for longer, and you end up having inferior repair.”
Next, the researchers wanted to see whether gut immune cells played a similar damage-repair role more generally. “Our observation of a population of RORγ+ Treg cells in regenerating skeletal muscle prompted us to investigate whether intestinal RORγ+ Treg cells can drive tissue homeostasis at distal sites, thereby constituting a mode of cross-tissue communication coordinated by microbiota: Treg cell interactions,” they noted.
To answer that question, they looked for traces of gut Treg presence in various organs including the liver, kidneys, and spleen. All of these organs contained intestinal Tregs, but at lower levels than was seen in injured muscles. To determine whether intestinal Tregs would increase in response to injury in these organs, the researchers induced fatty liver disease in a group of mice. Fatty liver disease—marked by abnormal accumulation of fat in the liver—can lead to liver scarring, cell death, and organ damage.
The researchers’ experiments showed that mice with fatty livers had notably higher levels of colonic Tregs than mice with healthy livers—an observation that affirmed the role of gut Tregs in controlling inflammation outside the intestines.
Moreover, mice that had fatty livers and were also genetically engineered to lack gut Tregs had markedly worse outcomes from their disease, showing worse liver scarring. This finding affirmed the protective role of gut Treg cells in reducing inflammation and scarring in fatty liver disease, the team concluded.
The study highlights an important interplay between the gut microbes and the immune system, highlighting the versatile role that gut bacteria can play in affecting immune function outside the gut. As the team concluded, “Here, we demonstrated that the impact of colonic RORγ+ Treg cells extended far beyond their site of generation: they formed a specialized pool of gut-trained regulators that could be rapidly mobilized in response to tissue emergencies at non-mucosal sites. Equipped with a distinct set of TCRs, homing receptors, and effector molecules, these cells orchestrated effective tissue regeneration and return to homeostasis.”
The results also underscore the importance of maintaining a healthy gut microbiota, the team suggested. One interesting question the study raises is the timing of antibiotic (Abx) treatment in people with musculoskeletal injuries, given the drugs’ potential to impede the healing response by disrupting the gut microbiota. “It is well known that antibiotics can eradicate beneficial gut microbes as collateral damage of their main function, which is to kill harmful bacteria,” Mathis said. “Our results further underscore the importance of judicious antibiotic use, which is important for many reasons that go well beyond muscle recovery.” As the authors stated, “Our data suggest that regulation of IL-17-mediated inflammation by microbiota-dependent RORγ+ Treg cells may be a general axis in play across multiple extra-gut sites. Thus, dysbiotic gut conditions capable of inhibiting the generation or export of RORγ+ Treg cells are likely to have systemic impacts on tissue homeostasis at multiple extra-gut sites. The potential for such detrimental outcomes advocates for judicious use of Abxs after tissue injury.”
If affirmed in subsequent research, the results could also inform the design of new treatments using beneficial microbes to promote healing of fatty livers or injured skeletal muscle. More broadly, the authors added, the findings raise the possibility that gut immune cells may be involved in healing damage in various other organs throughout the body—a question they plan to explore in their subsequent research. In certain contexts, they stated, “modulating RORγ+ Treg cells via diet or personalized probiotics could prove to be potent therapeutic add-ons.”
