Up in Smoke with COVID-19

Image credit: “Person Smoking Cigarette” by fotografierende is under the Pexels License.

This article was reviewed by Dr. Phil Lange, one of our subject matter experts.

This study was conducted by researchers from Cold Spring Harbor Laboratory.

The paper we’ll be demystifying can be found here, if you’d like to follow along!

TL:DR; Researchers compared the lungs of smokers and non-smokers, finding higher levels in smokers of ACE2, a key protein used by SARS-CoV-2 to enter cells. They also identified larger populations of lung cells that produce ACE2 in the lungs of smokers. This points to smoking as a compounding factor in infection with SARS-CoV-2.

With COVID-19 in the air, we can sometimes forget that cigarette smoke has been hiding out in the atmosphere for far longer. A known agent in lung cancer, we’ve all been educated on the dangers of smoking and second-hand smoke in school PSAs and haunting commercials in the 2000s. But in light of the recent crisis, smoking may have found a new way into the spotlight.

Going for a double-act, smoking has been suspected to partner with SARS-CoV-2 infection to increase its risk of infection and severity. Researchers at Cold Spring Harbor Laboratory in New York tackled this problem by examining how smoking affects a protein called ACE2. A lynchpin of SARS-CoV-2 infection, ACE2 is the binding spot for the virus, providing an entry point into cells. A “receptor” protein, it sits embedded in the membrane of many types of cells, some of which are found in the lungs.

To examine the levels of ACE2 in tissues, researchers measured the expression of its corresponding RNA. In a cell, the gene is copied as a strand of RNA, which is then used as the instructions to produce the corresponding protein. A technique called a quantitative real-time polymerase chain reaction (qRT-PCR) was used to analyze ACE2 RNA.

Researchers made sure that ACE2 RNA and protein levels are strongly correlated by performing three tests. Cancer cells were tested using RNA-seq, another method of measuring RNA, and mass spectrometry, a technique that identifies proteins by their weight and charge. Normal cells were tested by measuring the number of antibodies bound to ACE2 in a technique called immunohistochemistry. Then, seven different human cell lines were compared with qRT-PCR and Western Blotting, another technique to quantify proteins. All three tests showed ACE2 RNA and protein levels to be well correlated.

Knowing this, they first checked if age or sex affected ACE2 levels in the lungs, as both factor into SARS-CoV-2 infection. The lungs of aging mice and rats were found to have no significant difference in ACE2 based on age or sex. Looking to humans, researchers tested lungs tissue from the Genotype-Tissue Expression Project (GTEx), The Cancer Genome Atlas (TCGA), and organ donors. Like the rodents, ACE2 levels were same between the sexes, as well as the young and old.

The researchers started investigating how smoking could affect ACE2 levels by measuring gene expression levels in mouse lungs. These mice were exposed to diluted cigarette smoke for five months, for 2, 3, or 4 hours daily. These chain-smoking rodents showed a proportional increase in ACE2 levels, the highest being an 80% rise in ACE2 in the 4-hour group. Next, the researchers went to humans, assessing lung epithelial cells from current smokers and non-smokers taken by fiberoptic bronchoscopy, a procedure where a long camera-mounted probe is snaked down the airways. Tissue from current smokers had ~30-55% higher level of ACE2 than non-smokers. In fact, patients who smoked over 80 pack years had a 100% increase in ACE2 levels compared to those smoking under 20 pack years. Bear in mind, a pack year is the equivalent of 1 pack per day for 1 year and can be divided into 2 packs per day for 6 months, or 3 packs for 4 months.

But can you turn over a new physiological leaf? The researchers investigated patients who had quit smoking for a least a year, finding a ~40% decrease in ACE2 levels. Out of all genes affected by quitting smoking, ACE2 was in the top 5%. As such, while smoking increases ACE2 levels in the lungs, the elevation can be reversed.

Smoking is also known to increase levels of an enzyme named Cathepsin B, the backup protein for TMPRSS2. Classified as a “serine protease,” the purpose of TMPRSS2 is to cut proteins. SARS-CoV-2 uses TMPRSS2 to cut the virus’ spike (S) protein, activating it the protein to facilitate infection. When TMPRSS2 is absent in a cell, SARS-CoV-2 uses Cathepsin B. Therefore, not only does smoking increase levels of the SARS-CoV-2 receptor, but also an enzyme needed for its infection.

Our lungs have over 30 different types of cells, with epithelial cells being the most relevant to this study. These cells make up the surface of the lungs, giving them immediate contact with smoke. Researchers investigated exactly which of these cells express ACE2 to get a better picture of SARS-CoV-2 infection using RNA-Seq on single mouse cells, finding ACE2 to expressed solely by the epithelium. Specifically, ACE2 was found in the goblet and club cells of the upper respiratory epithelium, cells that generate protective fluids. ACE2 was also produced by LAMP3+ alveolar type 2 cells of the lower respiratory epithelium, which regulate fluid balance in the lungs and can transform into gas-exchanging alveolar type 1 cells after an injury.

Abstract art in a cross-section of the lung epithelium. Image credit: “File:Lung epithelium 80294-2.6.jpg” by Louisa Howard and Michael Binder has been released into the public domain.

In humans, this analysis also found ACE2 almost entirely in the epithelium, again in LAMP3+ alveolar type 2 cells and MUC5AC+ goblet and club cells. Unique to humans was ACE2 expression in FOXJ1+ ciliated cells from the upper respiratory epithelium, which use their microscopic “hairs”, called cilia, to brush inhaled particles out of the airways. The MUC5AC+ goblet and club cells expressed the most ACE2, followed by the FOXJ1+ ciliated cells and other secretory cells. This links ACE2 with mucus generation and foreign matter response.     

There are reports that smoking increases the number of goblet cells, as the body needs to make more mucus to protect the respiratory tract. Known as secretory cell hyperplasia, the researchers checked for evidence of this increase in the upper airways of smokers. They found a large population of basal cells, epithelial stem cells that can differentiate into populations of secretory and ciliated cells.

Oddly, FOXJ1+ ciliated cells were decreased in smokers, likely because exposure of smoke prevents the formation of cilia. The rest of the aforementioned cells increased in frequency in smokers, with ACE2+ cells having both a higher population and level of expression. Researchers confirmed these cell counts by finding signature genes for each cell type in similar proportions.

To simulate the effects in real time, the researchers made a model of the lung epithelium by growing mouse tracheal cells and primary human lung cells by the surface of a liquid medium, exposed to air, in a system called an air-liquid interface (ALI). As they tracked its growth to different types of cells, such as goblet and club cells, they noticed a rise in ACE2. Examining the results of cigarette exposure, they found the same rise in ACE2 expression and number of goblet and club cells, and lack of ciliated cells. In essence, the researchers demonstrated the cigarette smoke increases the amount of ACE2 expression and ACE2-expressing cells.

That being said, there may be less direct links between ACE2 and smoking, so the researchers at Cold Spring Harbor looked into ACE2 expression in various other lung diseases. Those strongly associated with smoking, such as chronic obstructive pulmonary disease (COPD) and idiopathic pulmonary fibrosis (IPF), displayed an increase in ACE2 expression. Furthermore, both diseases are also risk factors for severe COVID-19. For asthma and various other lung conditions, however, ACE2 expression remained unaffected. This means that ACE2 elevation isn’t universal to all lung disease, only those tightly linked to cigarette exposure.

Another link between SARS-CoV-2 and smoking is inflammation; cigarette smoke is inflammatory, and inflammation during SARS-CoV-2 infection is a leading cause of mortality, in a phenomenon known as a “cytokine storm.” As expected at this point, inflammatory signals can increase ACE2 expression in the epithelium of the respiratory system.  

The researchers investigated this process by growing cells from the trachea and smaller airways, exposing them to different compounds generated by the body during an inflammatory response. They discovered that ACE2 expressed rose from exposure to interferons, proteins that signal the immune system to act in the presence of viruses, like an alarm bell. Interferons IFN-α, IFN-β heightened ACE2 expression in all cells, while only the tracheal cells were also sensitive to IFN-γ.

This project isn’t without its limitations, of course. For example, other studies have found changes in ACE2 expression due to age, although differences may be too small for RNA-seq detection. Additionally, we’re assuming a rise in ACE2 expression increases ACE2 protein available for SARS-CoV-2, and that it will increase the severity of the infection. However, there may be methods of regulating ACE2 in human lungs yet to be investigated. Likewise, while ACE2 is essential for infection, ACE2-deficient mice have been shown to be prone to lung illnesses, providing an added wrinkle in the relationship between ACE2 and infection severity.

Regardless of this uncertainty, smoking is always bad for your health! We don’t need a virus to know that. But perhaps it’ll help clear the air.

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