There are 100 million neurons scattered along the gastrointestinal tract that are directly in the line of fire of gut infections. While damage to these neurons by intestinal pathogens could potentially lead to long-term GI disease, research by Rockefeller University scientists has shown that mice infected with bacteria or parasites develop a unique form of tolerance that is unlike that of a textbook immune response. The studies demonstrated how gut macrophages respond to prior insult by shielding enteric neurons, preventing them from dying off when future pathogens strike.

These findings may ultimately have clinical implications for conditions such as irritable bowel syndrome, which have been linked to the runaway death of intestinal neurons. “We’re describing a sort of innate memory that persists after the primary infection is gone,” said Daniel Mucida, PhD, professor, Rockefeller University. “This tolerance does not exist to kill future pathogens, but to deal with the damage that infection causes—preserving the number of neurons in the intestine.”

Mucida and colleagues reported on their findings in Cell, in a paper titled, “Enteric pathogens induce tissue tolerance and prevent neuronal loss,” in which they concluded, “Our observations demonstrate how certain enteric infections induce durable beneficial effects utilizing pathways distinct from classical immunological memory.”

Known as the body’s “second brain,” the enteric nervous system houses the largest depot of neurons and glia outside of the brain itself. The GI tract’s own nervous system exists more or less autonomously, without significant input from the brain. It controls the movement of nutrients and waste by fiat, coordinating local fluid exchange and blood flow with authority not seen anywhere else in the peripheral nervous system. But if enough of those neurons die, the GI tract spirals out of control. As the authors further noted, “The GI tract needs to simultaneously generate tolerance to harmless or beneficial dietary and microbial antigens and resistance to pathogen invasion. Additionally, disease tolerance to pathogen- and inflammation-induced tissue damage must operate in the intestine to maintain homeostasis.” This is particularly true for cells, such as enteric neurons, which have limited proliferative or regenerative capacity.

Mucida and colleagues reported last year that gut infections in mice can kill the rodents’ enteric neurons, with disastrous consequences for gut motility. At the time, the researchers noted that the symptoms of IBS closely mirror what one might expect to see when enteric neurons die en masse—raising the possibility that otherwise minor gut infections might be decimating enteric neurons in some people more than others, leading to constipation and other unexplained GI conditions.

The researchers wondered whether the body has some mechanism for preventing neuronal loss following infection. The authors commented, “Whether a state of tolerance can be induced after exposure to pathogens, preventing cumulative neuronal loss and functional changes, is still not known.”

In previous work, the lab had, however, demonstrated that macrophages in the gut produce specialized molecules that prevent neurons from dying in response to stress. And so a hypothesis began to take shape. “We knew that enteric infections cause neuronal loss, and we knew that macrophages prevent neuronal cell death,” Mucida said. “We wondered whether we were really looking at a single pathway. Does a prior infection activate these macrophages to protect the neurons in future infections?”

To investigate this further, first author Tomasz Ahrends, PhD, and lab colleagues infected mice with a non-lethal strain of Salmonella, a standard bacterial source of food poisoning. The scientists confirmed that the mice cleared the infection in about a week, losing a number of enteric neurons along the way. They then infected those same mice with another comparable foodborne bacterium. This time, the mice suffered no further loss of enteric neurons, suggesting that the first infection had created a tolerance mechanism that prevented neuronal loss. “These results point to development of tissue or disease tolerance postbacterial infection, which prevented neuronal loss to subsequent infection with a different pathogen,” the scientists wrote.

They then evaluated whether infection by parasites generated the same protection. “Next, we asked whether infection-induced neuroprotection could be induced by other pathogens. We aimed to determine whether helminths, which co-evolved with mammals and typically induce a very distinct immune response to bacterial pathogens, could prevent neuronal loss,” the team noted.

“In contrast to pathogenic bacteria, some parasites like helminths have learned to live within us without causing excessive harm to the tissue,” Ahrends said. This family of parasites, which includes flukes, tapeworms, and nematodes, infect in a way that is more subtle than highly hostile bacteria. But they also induced even greater, and more far-reaching, protection.

But the pathways leading to protection were different in response to bacterial, or helminth infection. During a primary bacterial infection, Mucida found, neurons call out to macrophages, which rush to the area and protect its vulnerable cells from future attacks. When a helminth insinuates itself into the gut, however, it is T cells that recruit the macrophages, sending them to even distant parts of the intestine to ensure that the whole gamut of enteric neurons are shielded from future harm.

“We found that following helminth or bacterial infection, and via distinct pathways, muscularis macrophages (MMs) acquire tissue-protective qualities and prevent neuronal loss during subsequent challenge with an unrelated pathogen,” the investigators wrote. “Bacteria-induced neuroprotection relied on activation of gut-projecting sympathetic neurons and signaling via ß 2-adrenergic receptors (ß2AR) on MMs. In contrast, helminth-mediated neuroprotection was dependent on T cells and systemic production of interleukin (IL)-4 and IL-13 by eosinophils, which induced arginase-expressing MMs that prevented neuronal loss from an unrelated infection located in a different intestinal region.” So, bacterial and helminth infections were both leading to protection of enteric neurons, albeit via different mechanisms. “The mechanistic studies described here indicate that the intestinal tissue co-opted pathways induced by distinct pathogens into a convergent tissue macrophage phenotype that mediates enteric neuronal protection, aiding host fitness via intestinal motility and energy balance.”

Macrophages (green) surrounding enteric neurons (red). [Laboratory of Mucosal Immunology at Rockefeller University]
Next, Ahrends repeated the experiments in mice from a pet store. “Animals in the wild have likely had some of these infections already,” he pointed out. “We would expect a pre-set tolerance to neuronal loss.” And that’s what the investigators found. The pet shop animals suffered no neuronal loss following experimentally induced infection. “They had a lot of helminths in general,” Mucida noted. “The parasitic infections were doing their jobs, preventing the neuronal losses that we have seen in isolated animals in the lab.” The authors stated, “Based on intestinal surface area and average neuronal densities, estimated total number of enteric neurons in the ileum is comparable between SPF [specific-pathogen-free] and pet store mice, suggesting that free-living animals might maintain the state of tissue tolerance due to constant exposure to different pathogens.”

Mucida is now hoping to determine the precise impact of neuronal loss in the GI tract. “We’ve observed that animals consume more calories without gaining more weight after neuronal loss,” he stated. “This may mean that the loss of enteric neurons is also impacting the absorption of nutrients, metabolic and caloric intake. There may be more consequences of neuronal loss than we expected,” he added.

Mucida believes that the reported research could contribute to a more complete understanding of the underlying causes of IBS and related conditions. “One speculation is that the number of enteric neurons throughout your life is set by early childhood infections, which prevent you from losing neurons after every subsequent infection.” People who for some reason do not develop tolerance may continue to lose enteric neurons throughout their life with every subsequent infection. Future studies will explore alternative methods of protecting enteric neurons, hopefully paving the way for therapies.