Circadian Biology of Lung Fibrosis and Relation to Sleep

The genes and proteins that regulate circadian rhythms may not work properly in the lungs of those with idiopathic pulmonary fibrosis (IPF), a new research study suggests. Supporting the link between circadian rhythms and pulmonary fibrosis, the study also found that people who regularly sleep less than 4 hours or 11 or more hours per day are more likely to have the disease. 

The study, “The circadian clock protein REVERBα inhibits pulmonary fibrosis development,” was published in the journal Proceedings of the National Academy of Sciences.

Many biological processes cycle over about 24 hours, and these cycles are known as circadian rhythms. Most people are aware of circadian rhythms in terms of the entire body. For example, it is the circadian rhythm of the entire body that helps the body eat and sleep on a regular cycle.

Researchers have discovered in the past two decades that every individual organ in the body has its own circadian rhythm as well. For example, the lungs have a circadian rhythm in which lung function is highest during the late afternoon, getting us ready for increased physical activity. At night, our breathing slows down to help cool down the body and allow us to sleep better. 

Circadian rhythms in organs are controlled by some of the cells that make up those organs. These cells are called oscillatory cells. Over 24 hours, specialized proteins within oscillatory cells go through a cycle of being present at high levels and then tapering off to low levels. These proteins are called circadian clock proteins. The repeated change from high levels of circadian clock proteins to low levels of these proteins over 24 hours is called a circadian oscillation. There are over a dozen genes that control the circadian oscillations in oscillatory cells.

In healthy lungs, oscillatory cells are found in two places. The first place is in the bronchioles, which are the small tubes that allow air to flow to the tiny air sacs (alveoli) in the lungs. The second place is in the alveoli, where oxygen is delivered to the blood and carbon dioxide is taken out of the blood. Oscillatory cells in the alveoli make up only a small percentage of the overall number of cells in the alveoli.

In this new study, researchers discovered that one of the circadian clock proteins was acting differently in mice lungs with fibrosis (scarring) than it does in healthy mice lungs. This protein, called PER2, was present in scar tissue cells. In those cells, the levels of PER2 cycled at higher high levels and lower low levels than seen in normal, healthy lung oscillatory cells. In addition, the PER2 levels moved up and down in a less coordinated way in the scar tissue cells when compared to the well-synchronized movements seen in healthy lung oscillatory cells. 

When researchers did more experiments, they discovered that a type of cell called fibroblasts, were causing the irregular circadian cycling. Fibroblasts are cells that invade the alveoli of lungs with fibrosis (scarring). Looking closely at the fibroblasts, researchers found that another circadian clock protein, called REVERBα, did not seem to be working properly. 

The researchers next asked the question: does disrupting the normal function of the circadian clock protein REVERBα in fibroblast cells lead to the formation of too much fibrosis (scarring) in the lungs? 

To answer the question, the researchers used several tactics. The first tactic used mice. Pulmonary fibrosis was started using chemicals in normal mice and in mice engineered to have non-working REVERBα. The mice engineered to have non-working REVERBα had much more fibrosis (scarring) in their lungs than the normal mice did. This shows that healthy, functional REVERBα is important to keep fibrosis in the lungs in check and under control.

Taking it a step further, the researchers engineered different types of mice that had non-working REVERBα only in certain types of lung cells. The difference in fibrosis severity was only seen when REVERBα was not working specifically in fibroblasts. No differences in fibrosis severity were seen when REVERBα was not working in other types of cells in the lung. This suggests that healthy, functional REVERBα is important to keep fibroblast cells from making too much scar tissue.

The second tactic used lung cells grown in the laboratory in petri dishes. Cells with non-working REVERBα produced more integrinβ1, a protein involved in fibrosis. Studying those cells closely allowed the researchers to also determine that REVERBα requires a helper protein called TBPL1 to work properly. Together, these experiments support the idea that the circadian clock proteins REVERBα and TBPL1 play a role in keeping fibroblasts in the lungs from causing too much fibrosis (scarring). When these proteins are not functioning properly, fibrosis may happen at levels that are too high, leading to disease.

Following this, the researchers took a third tactic to link the circadian clock with pulmonary fibrosis in humans. They looked at sleep duration data (that is, how long people sleep on average each night) from over 500,000 people in the U.K. Biobank. They found that people who report they regularly sleep 4 hours or less in a day doubled their chance of having pulmonary fibrosis while those sleeping 11 hours or longer in a day tripled their chance of having the disease, compared to those sleeping about 7 hours per day. People reporting that they slept about 7 hours per day had the lowest risk of developing pulmonary fibrosis. This suggests that a smoothly running circadian clock, as evidenced by regular sleep of about 7 hours per night, is associated with a lower risk of developing pulmonary fibrosis. 

The last tactic to link the circadian clock with pulmonary fibrosis in humans was to study genetic data from people with IPF using the Lung Genomics Research Consortium. The researchers in this study examined the genes that control the regular expressions of circadian clock proteins REVERBα and TBPL1 and found them to be disordered in tissue from the lungs of people with IPF.

Dr. John Blaikley from The University of Manchester, who led the project said: “Pulmonary fibrosis is a devastating condition which is incurable at present. Therefore, the discovery that the body clock is potentially a key player potentially opens new ways to treat or prevent the condition. More work will need to be done around studying the association between pulmonary fibrosis and sleep duration to establish both causation and reproducibility. If these results are confirmed, then sleeping for the optimal time may reduce the impact of this devastating disease.”

Dr. Peter Cunningham, joint lead author on the paper, said: “The discovery that the [circadian] clock plays a role in fibrosis suggests that altering these oscillations could become an important therapeutic approach.”

Reference

Cunningham PS, Meijer P, Nazgiewicz A, et al. The circadian clock protein REVERBα inhibits pulmonary fibrosis development. Proc Natl Acad Sci U S A. 2020;117(2):1139–1147. doi:10.1073/pnas.1912109117