Cytocentric Visionaries: Dr. David Gozal, MD
Part 1: COVID-19, ACE2, and the Circadian Clock
Alicia Henn, PhD MBA, Chief Scientific Officer, BioSpherix
David Gozal, MD, MBA, PhD (Hon) is the Marie M. and Harry L. Smith Endowed Chair and Chairperson of the Department of Child Health, and Pediatrician-in-Chief, MU Women’s and Children’s Hospital, both at the University of Missouri School of Medicine. He is an expert in pediatric sleep disorders and the effects of hypoxia resulting from sleep apnea.
Here, Alicia talks with Dr. Gozal about his recent publication in the European Respiratory Journal, “Putative contributions of circadian clock and sleep in the context of SARS-CoV-2 infection”, published in April of this year.  This interview was edited for length and clarity.
Thank you for joining me today. We see you as a Cytocentric Visionary because you are bringing to the attention of the research community critical factors like the Circadian Cycle that need to be considered for cells in research. We read with great interest your recent editorial in the European Respiratory Journal about the circadian clock and SARS-CoV-2 infection. What made you focus on this aspect of COVID-19?
It's not as if this is new. The quality of sleep and alignment of the circadian clock with all the zeitgebers that drive our circadian rhythmicity is fundamentally important to a variety of functions in our body. We’ve known that for quite some time. Three years ago the people that discovered the fundamental machinery that governs the circadian clock in every living organism received the Nobel Prize.
There's been a substantial amount of work on how people that work in night shifts, for example, are susceptible to a variety of diseases that involve the immune system. We were the first to describe that patients with sleep apnea were at higher risk of developing cancers of certain types and that the cancer was more aggressive. We were the first to characterize the gene expression patterns in children with sleep apnea. The major hub recruited was the inflammatory response. So we were the first to propose, now 20 years ago, that sleep apnea was a low-grade systemic inflammatory disease.
We also know from work from others that if you don't sleep well before you go for your flu shot the efficacy is not very good. People who do shift work, even if they get the vaccine for flu or for other viruses, seem to be more susceptible to failure of those vaccines. Statistics of epidemiology show that.
On one side we know that the circadian clock is fundamentally important to a variety of functions, including the immune function. We know that the rhythmicity of natural killer cells, or innate immunity, is regulated by the clock. We know that disruption of the clock can lead to changes in immune function and may lead to disease such as cardiovascular disease. It affects cancer, it affects metabolic function. All of these result from changes in the way that we regulate the inflammatory process within those tissues. So that's one side of the Circadian Clock.
The second is sleep per se. If we sleep better, we have more functional innate response, but at the same time less recruitment. Even if you're prone to develop inflammation, with good restorative sleep we have a dampening of the inflammation. This is nothing new. So that's the setting in which SARS-CoV-2 appeared.
In your paper you were able to connect sleep disruption and changes in ACE2 expression?
We know that ACE2 is a very regulated gene and downstream protein expression and function. If you look at the gene promoter region, it has elements that relate to inflammation, it has elements that relate to hypoxia, it has elements that relate to the circadian clock. BMAL1 , a critical regulatory gene of the circadian clock system is a very important regulator for ACE2 expression. People have looked at this. Even at the level of the central clock located in the preoptic area of the brain, if you disrupt ACE2 expression, clock function will be modified and affect cardiovascular functions differently - so ACE2 has multiple functions. So, from that perspective, it seems very reasonable to assume that clock changes could alter the expression of ACE2 in any cell of the body where ACE2 is normally expressed and functional. Therefore, if it alters the expression of ACE2, let's say through normal moment to moment changes in the circadian system, you might be differentially susceptible at different times of the day in normal conditions to COVID-19 infection or transmission.
Possibility number two: You are socially jet-lagged and therefore you are going to change your clock. That clock is going then to change the levels of ACE2 expression. This might happen at different times than the usual times they might have happened in the normal clock. By having this regulation of the clock being altered, you might have higher levels of ACE2 at a time that you're in contact with people. So rather than have the highest levels at midnight, for example, you might have ACE2 expression highest at two o'clock in the afternoon. That could be a time that you are at work, in the streets going shopping, or whatever. If there is a little group of viruses in the air in those droplets and if you have a much higher level of ACE2, they may bind to it. This could therefore make you more susceptible to it.
And there are underlying conditions to think about too?
We know for example that obesity shifts the clock. We know that disorders such as sleep disorders and lung disorders/hypoxia and cardiac failure all have an effect on the clock. So, it
could very well be that the regulation of the clock within the cells might be affected by underlying conditions that then leads to differential expression of ACE2.
So is it true? I don't know. We will need to do a very thorough study. For example, a study that would be very interesting would be of the people who have been infected with COVID-19. What was the proportion of shift workers relative to the population in the same area who get infected? And if hey were infected, will shift workers suffer from more severe disease? But the problem is we don't do universal sampling and so all these studies are going to be fraught by sampling bias. But the reality is that because if you work in shifts, then some people might be more susceptible to COVID-19 infection.
These are all hypotheses, but nonetheless they are very viable on the basis of the biology we know. We have a figure in the paper in the European Respiratory Journal that shows all the pathways that the clock could potentially affect not only the expression of ACE2 but also the processing and the replication of the virus within the cells.
What considerations should researchers take for in vivo or in vitro studies on COVID?
The advantage of using animal models is that we can preserve in a very standardized fashion the regulation of most of the circadian clock elements. However, very few investigators actually document those and that's one of the things that would be very important to do. For example, when do you inoculate the virus? At what level should you do a circadian inoculum and see whether the transmission is different? That could potentially open ways to understand better high-risk from lower risk populations.
Even if you do it in cells, you need to know whether they have been standardized for their circadian and intrinsic clocks. They are oscillators and you need to understand what they do. If two labs are conducting the same experiment and one decided to inject the virus in the morning and the other in the afternoon, at different times of the cycle of the clock in those cells, you may have very different results. So now you have dissonant findings, when in fact all they represent is clock related misalignment in the experiments.
So how important is it for researchers to properly control oxygen levels for cells when they're trying to do in vitro studies for underlying conditions?
It’s critically important and there are two reasons. One is most of the cells that we grow we really don't know what the optimal level of oxygen needs to be for these cells, what is the normoxia for these cells. Most of the time what we do is generate hyperoxic environments.
In room air?
What we call room air may not truly be normoxia for a cell. That cell may need less oxygen than that. I think that because of the tradition of how we started the process of cell culture, we think if we live in room air then that means cells are happy in room air. Maybe this is not their natural environment and we have created a different cell than the one that we thought we were studying because we have been growing it in an atmosphere that would constitute relative hyperoxia.
The second part is we change the levels of oxygen of these cells in order to mimic diseases. We do this routinely in order to create either sustained or intermittent hypoxia, an area of research that we've been actively pursuing.
This is Part One of a Two Part Series.
In part 2, we talk with Dr. Gozal to learn about Prone Positioning, and Chronoadequacy.
If you would like to be featured in our Cytocentric Visionary Series, contact us. We would love to hear about your work.
Meira, E.C.M., M. Miyazawa, and D. Gozal, Putative contributions of circadian clock and sleep in the context of SARS-CoV-2 infection. Eur Respir J, 2020: p. 2001023.
About the Author
Alicia D Henn, PhD, MBA
Alicia Henn has been the Chief Scientific Officer of BioSpherix, Ltd for two years. Previously, she was a researcher at the Center for Biodefense Immune Modeling in Rochester, NY. Alicia obtained her PhD in molecular pharmacology and cancer therapeutics from Roswell Park Cancer Institute in Buffalo, NY and her MBA from the Simon School at University of Rochester in Rochester, NY.