In ancient medicine, body fluids, or humors, were associated with personality types. For example, “black bile” favored a melancholic (placid) temperament, and “yellow bile,” a choleric (fiery) temperament. All this seems quaint today. Modern medicine tells us that bile, a greenish-brown secretion of the liver and gallbladder, dissolves fats in the small intestine. So, that’s it for humorism. Or is it? New research suggests that bile’s predominant components, the bile acids, indirectly influence temperament after all—not human temperament, but the temperament of immune cells.

According to two recent studies, both from Harvard Medical School (HMS), bile acids play a role in immunity and inflammation. These studies, which were conducted in mice, show that bile acids promote the differentiation and activity of several types of T cells involved in regulating inflammation and linked to intestinal inflammatory conditions. These studies also reveal that gut microbes are critical for converting bile acids into immune-signaling molecules.

The newfound connection between bile and immunity/inflammation suggests new therapeutic approaches. For example, regulatory pathways could be targeted to modulate intestinal inflammation, a process that underlies the development of inflammatory bowel disease (IBD) and other autoimmune conditions.

The first study, led by immunologist Jun Huh, PhD, assistant professor of immunology in the Blavatnik Institute at HMS, produced findings that appeared in Nature, in an article titled, “Bile acid metabolites control TH17 and Treg cell differentiation.” According to this article, bile acids exert their immune-modulating effect by interacting with immune cells in the gut.

Once bile acids leave the gallbladder and complete their fat-dissolving duties, they make their way down the digestive tract where they are modified into immune-regulatory molecules by gut bacteria. The modified bile acids then activate two class of immune cells: regulatory T cells (Treg cells) and effector helper T cells, specifically TH17, each responsible for modulating immune response by either curbing or promoting inflammation.

Under normal conditions, the levels of proinflammatory TH17 cells and anti-inflammatory Treg cells balance each other, maintaining a degree of protection against pathogens without causing too much tissue-damaging inflammation. These cells play a key role in the context of intestinal infection. TH17 cells ignite inflammation to quell the infection, while Treg cells curb inflammation once the threat has subsided. Unrestrained, the activity of TH17 can also lead to aberrant inflammation that promotes autoimmune disease and damages the intestine.

In their experiments, the researchers used undifferentiated, or naïve, mouse T cells, and exposed them to various bile acid metabolites one at a time. The experiments showed that two separate bile acid molecules exerted different effects on T cells.

“Here we screen a library of bile acid metabolites and identify two distinct derivatives of lithocholic acid (LCA), 3-oxoLCA and isoalloLCA, as T cell regulators in mice,” the Nature article indicated. “3-OxoLCA inhibited the differentiation of TH17 cells by directly binding to the key transcription factor retinoid-related orphan receptor-γt (RORγt), and isoalloLCA increased the differentiation of Treg cells through the production of mitochondrial reactive oxygen species (mitoROS), which led to increased expression of FOXP3.”

When the researchers administered each molecule to mice, they observed the animals’ TH17 and Treg cells fell and rose, accordingly. Additionally, the researchers found that the two bile acid byproducts are also present in human stool, including stools of people with IBD—a finding that suggests the same mechanism is at play in humans.

“Our findings identify an important regulatory mechanism in gut immunity, showing that microbes in our intestines can modify bile acids and turn them into regulators of inflammation,” said Huh.

If affirmed in further studies, the results can inform the development of small-molecule therapies that target Treg and TH17 cells as a way to control inflammation and treat autoimmune diseases affecting the gut.

The second study, led by Dennis Kasper, PhD, professor of immunology in the Blavatnik Institute at HMS and the William Ellery Channing professor of medicine at HMS and Brigham and Women’s Hospital, produced findings that appeared in Nature, in an article titled, “Microbial bile acid metabolites modulate gut RORγ+ regulatory T cell homeostasis.” This article focused on a subset of inflammation-taming Treg cells that arise in the colon as a result of exposure to gut microbes. In contrast, most other immune cells originate in the thymus.

Low levels of colonic Treg cells have been linked to the development of autoimmune conditions such as IBD and Crohn’s disease.

Kasper organized experiments demonstrating that gut microbes and diet work in concert to modify bile acids, which in turn affect the levels of colonic Treg cells in mice. The experiments also show that low levels of Treg cells induced by lack of bile acids or deficiency in bile acid sensors makes animals prone to developing inflammatory colitis—a condition that mimics human IBD.

To test the hypothesis that gut bacteria convert food-derived bile acids produced in response to food into immune signaling molecules, the researchers silenced bile acid-converting genes in various gut microbes and then put both the modified and nonmodified microbes in mice specially bred to have germ-free guts. Animals whose guts were populated by microbes without bile acid-converting genes had notably lower levels of Treg cells. The researchers then fed animals either nutrient-rich meals or minimal food.

Animals with normal microbe populations in their guts that were receiving minimal food had lower levels of colonic Treg cells and lower bile acid levels than mice eating rich food. Yet animals with germ-free guts receiving rich food also had low levels of Treg cells—a finding which shows that both gut microbes and food-derived bile acids are required to modulate immune cell levels.

To test whether bile acids are directly involved in immune cell regulation, the researchers then mixed various bile acid molecules with the drinking water of animals that had low Treg cell levels and minimal diets. Several weeks later, these animals had an increase in the levels of inflammation-curbing Treg cells.

In a final step, the researchers gave three groups of mice a compound that induces colitis. One group was fed a minimal diet, another group received nutrient-rich meals and a third group received minimal food and drank water supplemented with bile acid molecules. As expected, only mice fed minimal diets not supplemented by bile acid molecules developed colitis. The experiment confirmed that bile acids play a critical role in Treg cell regulation, intestinal inflammation, and colitis risk.

“By comparing mouse colonic tissue expression of various bile acid receptors, we found that nuclear receptors, especially vitamin D receptor (VDR), were more abundant,” the Nature article noted. “Mechanistically, it is intriguing to speculate that the nuclear receptor VDR—a colonic Treg cell-preferring transcription factor—may modulate colonic Treg cell homeostasis by coordinating bile acid signals with transcription factor activity.

“An understanding of the molecular mechanisms underlying the regulation of colonic Treg cells by this biliary network between hosts and their associated microorganisms will be valuable in improving therapy for human gastrointestinal inflammatory disorders.”

“Our results,” said Kasper, “demonstrate an elegant three-way interaction between gut microbes, bile acids, and the immune system. Importantly, our work suggests it is plausible to think of harnessing certain gut bacteria as a way to modulate disease risk.”

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