Treating the airways of mice with an experimental drug that activates the repair pathway protected their airways from damage after infection with influenza or the virus that causes COVID-19. Conversely, animals in which the pathway was blocked experienced much more severe damage to their airways.
Activating the signaling pathway initiated by airway or pancreatic neuroendocrine cells in a similar way in humans might enhance the ability of firefighters and those with respiratory illnesses to avoid permanent lung damage, the researchers believe. They also suspect it could help prevent people with metabolic syndrome from progressing to diabetes.
“This whole signaling cascade both protects and regenerates vulnerable cells in the airway and the pancreas,” said Philip Beachy, PhD, professor of urology and of developmental biology. “If this circuit is disrupted, the damage is much worse — specialized airway cells are lost, and the stem cells can’t divide to repair the damage. We think it’s likely to be important in many other tissues in the body.”
Although the study was conducted in mice, there are tantalizing clues of a similar pathway in humans: People treated with a cancer drug that blocks the pathway are twice as likely as their peers to develop diabetes after their treatment.
“The association is highly significant and gives us early hints that activating this pathway might be protective for people with metabolic syndrome who are beginning to lose beta cell function,” Beachy said.
Beachy, who is the Ernest and Amelia Gallo Professor and a member of the Stanford Stem Cell Institute, is the senior author of the research, which was published online June 9 in Cell. Research scientist William Kong, PhD, is the study’s first author.
Neuroendocrine cells are less than 1% of the total number of cells in the cells that line the airway, which is made up of a type of tissue called epithelium. Some of them cluster together in what are called neuroepithelial bodies and play an important role in sensing oxygen levels and modulating immune responses in the lungs. But others, especially those in the tracheal airway, are solitary, nestled alone among other types of epithelial cells. It’s not been clear until now exactly what function these solitary neuroendocrine cells perform.
The hedgehog family
Beachy’s laboratory has focused on the function of a protein family called Hedgehog proteins since Beachy identified the first member in fruit flies in 1992. Members of the family are best known for their critical function in embryo patterning in early development, but they also aid in the rejuvenation of many types of tissue. Desert hedgehog is one of the least studied of the three family members (the others are Sonic hedgehog and Indian hedgehog).
Previous work in Beachy’s lab showed that stem cells in the epithelial lining of the bladder respond to a signal cascade initiated by Sonic hedgehog to regenerate the bladder lining after bacterial infection. They wondered if hedgehog proteins were involved in the repair of damage in other epithelial tissues like the airway.
When Kong used a technique called bulk RNA sequencing to search for genetic messages encoding any of the hedgehog family members in the cells of the trachea, they detected a faint signal for the Desert hedgehog protein, but not for the other two family members. When they engineered mice in a way that caused cells expressing the Desert hedgehog protein to become fluorescent, they saw that the solitary neuroendocrine cells were making the Desert hedgehog protein.
Further research showed that the Desert hedgehog protein leaves the epithelium and travels into the layer of tissue beneath the epithelium, called the mesenchyme. There, it triggers cells to begin producing another protein called Gli1. When the airway cells sense damage, Gli1 induces the expression of a protein called IL-6 that triggers stem cells in the epithelium called basal cells to begin dividing and specializing to repair the damage.
This crosstalk between tissue layers, which the researchers call epithelial-mesenchymal feedback, protects and regenerates specialized cells in the lung epithelium, including multi-ciliated cells that use their feathery arms to sweep particles and viruses out of the lungs and secretory cells that make mucus to trap unwanted invaders. In the absence of these cells, viruses and toxins can penetrate much more deeply into the lungs.
Protective process
The entire process happens within hours of toxin exposure in a coordinated cascade that eventually includes even non-Gli1-expressing cells of the airway.
“At each stage, the signal is amplified until the entire trachea is impacted,” Beachy said. “This rapid response not only protects the epithelial cells from dying but it also activates a regenerative response.”
The consequences of impeding this protective message are severe.
“If this signal cascade is disrupted, the damage is much worse. Ciliated and secretory cells are lost, and the basal cells don’t divide. In fact, it’s all they can do to stretch out and try to cover the injured area,” Kong said.
Both Desert hedgehog and Gli1 are critical to the repair process. Mice unable to produce Desert hedgehog or Gli1 were much more sensitive to exposure to sulfur dioxide gas, which is an environmental pollutant and mimics the damage inflicted by other inhaled toxins. While control mice lost 85% of ciliated cells and 41% of secretory cells within 24 hours, mice lacking the protein lost 96% of ciliated cells and 88% of secretory cells during the same time.
Activating the hedgehog signaling pathway with a small molecule dramatically increased cell survival after sulfur dioxide exposure: 66% of ciliated cells and 82% of secretory cells survived in treated animals, versus 9.7% of ciliated cells and 43% of secretory cells in control animals.
Kong next tested the effect of the Desert hedgehog pathway activation on mice infected with influenza and the virus that causes COVID-19. Although no mice unable to make Gli survived more than five days after infection with influenza, all mice treated with the small molecule activator survived at least eight days after infection. Mice infected with the virus that causes COVID-19 that were unable to activate the Desert hedgehog pathway suffered extensive loss of ciliated cells in the airway.
Finally, the researchers turned their attention to the pancreas, which has a similar tissue organization as the airway. They found that the insulin-producing beta cells, which are a type of neuroendocrine cell, also make Desert hedgehog and that the organ exhibits the same epithelial-mesenchymal feedback loop with IL-6 to protect the vulnerable cells.
The researchers are now exploring whether and how the hedgehog pathway could be activated in humans to prevent lung damage in people exposed to airborne toxins or who are at risk for diabetes.
“We have reasons to think it might not be a good idea to activate the hedgehog pathway long term,” Beachy said. “We are considering how to stimulate the pathway in a targeted way, either delivering it to the airway with an aerosol or targeting it to the pancreas. And we have early hints it might be possible.”
Researchers from the University of California, San Francisco, contributed to the study.
The study was funded by the National Institutes of Health (grants R01CA157877, R01DC016892 and U19AI35990), the Department of Defense, the Chan Zuckerberg Biohub, the Roddenberry Foundation, P. and E. Taft, Schmidt Futures, and the James B. Pendelton Charitable Trust.
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