Lung disease experts at Vanderbilt University Medical Center and their colleagues have determined a key factor as to why COVID-19 appears to infect and sicken adults and older people preferentially while seeming to spare younger children.
The researchers discovered that children have lower levels of an enzyme the virus needs to invade airway epithelial cells in the lung.
Their preliminary findings, has been posted online by the preprint server bioRxiv, suggest that blocking this enzyme potentially could prevent COVID-19 infection in older people.
There is still much to learn about SARS-CoV-2, the one-stranded RNA virus that causes COVID-19, said Jonathan Kropski, MD, assistant professor of Medicine and Cell & Developmental Biology in the Vanderbilt University School of Medicine, who led the study with Jennifer Sucre, MD.
But this much we do know: After a viral particle is inhaled into the lungs, protein “spikes” that stick out of its surface like nail studs in a soccer ball attach to a receptor called ACE2 on the surfaces of certain types of lung cells.
A cellular enzyme called a protease chops up the spike protein, enabling the virus to fuse into the cell membrane and “break into” the cell. Once inside, the virus hijacks the cell’s genetic machinery to make copies of its RNA.
Sucre and Kropski wondered whether levels of ACE2 and the protease, called TMPRSS2, change during lung development. If infants and children express less of these proteins, maybe that’s why they seem to be less vulnerable than older people to severe illness if they are exposed to SARS-CoV-2.
The researchers were well suited to investigate that possibility. As members of the international Human Cell Atlas (HCA) Lung Biological Network, they and their colleagues had built a dataset on lung development in the mouse using a technique called single-cell RNA-sequencing.
The technique can detect the expression of genes in individual cells of tissues such as the lung. In this way the researchers were able to track the expression of genes known to be involved in the body’s response to COVID-19 over time.
They found that while the gene for ACE2 was expressed at low levels in the mouse lung, “TMPRSS2 stood out as having a really striking trajectory of increased expression during development,” said Sucre, assistant professor of Pediatrics and Medicine.
The researchers next applied another technique called RNA in situ hybridization, which uses fluorescent probes to visualize how expression of the TMPRSS2 gene increased over time in specific types of epithelial cells that line the lungs.
With the help of VUMC pathologists, the researchers obtained and analyzed human lung specimens collected from donors of different ages, and confirmed a similar trajectory in TMPRSS2 expression to what they’d found in mice.
Finally the researchers used fluorescent probes to analyze an autopsy specimen from a patient who had died from COVID-19.
They found the virus in three types of cells that express TMPRSS2. In essence, Sucre said, they caught the virus “red-handed,” at the scene of the “crime.”
These findings, the researchers concluded, “underscore the opportunity to consider TMPRSS2 inhibition as a potential therapeutic target for SARS-CoV-2.”
In a previous study of the first SARS virus, which caused a worldwide outbreak in 2002, researchers from Japan reported that a combination of protease inhibitors prevented the virus from entering human bronchial epithelial cells grown in the laboratory.
Kropski said much of the background work for this paper was built upon the collaborative efforts of the Human Cell Atlas (HCA) Lung Biological Network.
The Vanderbilt COVID-19 Consortium Cohort, a multi-disciplinary effort to understand more fully why some people are at greater risk of COVID-19 infection and illness, also aided in the acquisition and analysis of human tissues, he said.
It has been reported that ACE2 and TMPRSS2 are the main cell entry proteins for SARS-CoV-2 and play a critical role in causing COVID-19. To investigate the expression level ofthese SARS-CoV-2 host cell entry genes in lung airway, public gene expression datasets were used.
We have found a differential expression of ACE2 and TMPRSS2 in nasal and bronchial airways relative to age and diseases status. Children were found to have significantly lower expression of COVID-19 receptors in the upper and lower airways (nasal and bronchial).
Moreover, the lung airway expression of both ACE2 and TMPRSS2 was found to be significantly upregulated in smokers compared to non-smokers; and in patients with COPD compared to healthy. No difference was observed in the blood expression levels of ACE2 and TMPRSS2 between children and adults, or in COPD or diabetic patients.
However, a significant increase in blood expression levels of these genes was observed in patients with essential hypertension; while only ACE2 was upregulated in the blood of asthmatic. These results suggest that the observed difference in COVID-19 severity between children and adults could, in part, be attributed to the difference in ACE2 and TMPRSS2 airways tissue expression levels.
Over the last two decades, three waves of coronavirus outbreaks among humans has irrupted, Severe Acute Respiratory Syndrome coronavirus (SARS-CoV) in 2002,1 Middle East respiratory syndrome (MERS) in 2012,2 and the latest novel SARS- CoV-2 in December 2019.
The disease caused by these coronaviruses has pneumonia-like symptoms such as fever and dry-cough and leads to progressive respiratory failure, and even death.3 SARS-CoV1 and MERS-CoV 4 caused epidemics in different countries, while the high rate of transmissibility and infectivity of SARS-CoV-2 resulted in a quick escalation of the number of infected cases worldwide, leading to the transformation of this outbreak into a worldwide pandemic (WHO situation report 50, https://www.who.int/docs/default-source/coronaviruse/situation-reports/20200310-sitrep-50-covid-19.pdf?sfvrsn=55e904fb_2).
Causing corona virus disease 19 (COVID-19), as of the preparation of this report, the number of worldwide infections is estimated to be 3,267,184, while the fatality rate reported exceeded 200,000 deaths, according to WHO (WHO situation report 103, https://www.who.int/docs/default-source/coronaviruse/situation-reports/20200502-covid-19-sitrep-103.pdf?sfvrsn=d95e76d8_4).
The new SARS-CoV-2 was found to share 79.6% sequence identity with SARS-CoV.5
Besides, the virus uses the same cell receptor as SARS-CoV, Angiotensin Converting Enzyme 2 (ACE2) to enter the cell. It was also found to need Transmembrane Serine Protease 2 (TMPRSS2) for priming of the viral spike protein.6
The level of expression of the viral receptor (ACE2) and TMPRSS2, especially in the nasal tissue, may be critical for the ability of the virus to transmit and replicate.7
SARS-CoV-2 is believed to have a much higher rate of transmission compared to SARS-CoV and MERS-CoV.8 The majority of people infected were of older ages (approximate age 30-79 range years) and developed mild symptoms; while around 19% developed severe conditions and needed critical care.9
Many of the patients who developed critical conditions had, in fact, chronic diseases such as cardiovascular diseases and diabetes.10 Throughout the three epidemics, low rate of infection was reported in infants and children with mostly a milder disease profile.11-13
The reason behind that is still a mystery.
Several theories has been discussed for why children are less susceptible to SARS-CoV-2 infection,14, 15 and less prone to develop severe conditions following infection with SARS-CoV-2.16
One possibility is their young immune system which could be more efficient in clearing the infection compared to the adults immune system. Another possibility is the presence of cross immunity due to a previous exposure to a milder widespread form of corona viruses.11, 17
However, one possibility could also be that they express lower levels of the viral receptors that could limit transmission to these young population and the development of severe conditions.
Hence, we tested the hypothesis that the expression of viral receptors, ACE2 and TMPRSS2 in children is lower than that in adults. Moreover, since ACE2 is known to be regulated by hypoxia, stress, and several inflammatory mediators,18 we have also tested whether its expression increases due to smoking, and during chronic inflammatory diseases, affecting the lung such as asthma, COPD, and Idiopathic pulmonary fibrosis (IPF); as well as diabetes and hypertension.
In this study, using several public gene-expression data sets, we have showed that SARS-CoV-2 receptor, ACE2, and TMPRSS2 are expressed at significantly higher levels in nasal epithelium compared to blood and saliva; and this expression decreases significantly in lower lung airway tissue.
Importantly, the expression of ACE2 and TMPRSS2 in nasal tissue of children is significantly lower than that of adults. Likewise, the bronchial tissue expression of these genes was also lower in children compared to adults. To the best of our knowledge, this is the first study to report lower ACE2 and TMPRSS2 expression in children nasal and bronchial epithelial cells compared to adults.
The reported rate of SARS-CoV-2 infection and spectrum of symptoms is lower in children than in adults.16 Considering that SARS-CoV-2 cell entry depends on the expression of ACE2 and TMPRSS2 entry genes,6 one can speculate that the transmissibility/clinical manifestations of SARS-CoV-2 could be affected by the levels of ACE2 and TMPRSS2 expression on cell surface.
In fact, by analyzing 7375 contacts, Zhang et al found that children are less susceptible to SARS-CoV-2 infection than adults.15
Therefore, the reduced airway tissue expression of ACE2 and TMPRSS2 reported here may contribute to the lower the risk of infection,14, 15 and the reduced disease severity observed in the younger population.
In light of the potentially increased transmissibility of SARS-Cov-2 in smokers and adults with other comorbidities,10, 19, 20 ACE2 and TMPRSS2 lung airway expression was found to be upregulated upon smoking and in COPD patients, and slightly increased in asthmatics.
Of all the chronic lung diseases tested, smokers and COPD had the highest increase in these genes compared to healthy lung tissue. This indicated that smoking could be the reason behind the increased expression observed in COPD, and not the inflammatory mediators.
In fact, while preparing this manuscript, Brake et al. reported an increase in ACE2 in the lung airways of smokers using immunohistochemistry.21
Hypoxia is known to regulate ACE2 expression 18,22 which could explain the mechanism by which smoking increase these receptors. The increased expression of ACE2 in smokers may, hence, justify the observed increase in rate of infection and severity of disease observed in smokers compared to non-smokers.23
No difference in receptors gene expression was observed in lung tissue of patients with idiopathic pulmonary fibrosis, nor with sarcoidosis or scleroderma-associated interstitial lung disease.
In addition, no difference in the blood levels of ACE2 and TMPRSS2 expression between children and adults was observed. However, a significant level in blood expression of ACE2 and TMPRSS2 observed for patients with hypertension; while only ACE2 was upregulated in the blood of asthmatics.
This may explain the increased risk of these patients for SARS-CoV-2 infection. The absence of difference in blood expression levels of patients with chronic diseases such as diabetes does not exclude the possibility of increased expression of these entry genes in lung tissue of these patients, however, we had no available data to investigate that. It would, hence, be interesting to determine the lung expression levels of SARS-CoV-2 receptors in these patients.
The increased expression of ACE2 and TMPRSS2 in upper respiratory tract compared to blood and lower respiratory tract could contribute to the increased replication of SARS-CoV-2 in this tissue. Recently, Zou et al reported that SARS- CoV-2 viral load is higher in nasal compared to throat swabs.7
Based on our finding, the reason for that could be the higher expression of ACE2 and TMPRSS2 in nasal compared to throat swaps. In fact, the level of these genes in saliva was significantly lower than that of nasal epithelium (Figure S2D).
Interestingly, TMPRSS2 was found to be expressed in nasal and bronchial epithelial cells along with ACE2. SARS-CoV-2 needs this host serine protease for spike protein priming, allowing for viral fusion and cellular enter.24, 25
These findings of co-expression of ACE2 as well as TMPRSS2 on the nasal epithelial cells might provide further insights into the reason for increased infectivity and transmissibility of SARS-CoV-2 and may pave the way for novel therapeutic interventions.
It is worth mentioning that serine protease inhibitors such as camostat mesylate which blocks TMPRSS2 activity could be used for treatment of SARS-CoV-2-infected patients,6, 26 or as a preventive approach especially for patients identified as high risk based on ACE2 nasal screening.
Of note, infecting human airway epithelial cells with SARS-CoV was found to downregulate ACE2 and TMPRSS2 expression compared to the mock-infected cells. This was in line with a previous similar observation.6, 27
The fact that the level of expression of ACE2 is not increased following infection indicate that the baseline level of ACE2 may play a critical role in determining the level of diseases severity.
Moreover, a dramatic decrease in lung tissue expression of ACE2 was shown to be associated with acute respiratory distress syndrome (ARDS).28 The downregulation of ACE2 expression observed with SARS-CoV infection, would, hence, play a role in the development of ARDS, a common clinical phenotype for patients with COVID-19.
Our results are mainly based on public gene expression datasets. It is warrant that further histological methods and protein validation are performed to confirm our findings.
The observed differential level of expression of ACE2 and TMPRSS2 may contribute, at least partially, to our understanding of why elderly, and patients with other comorbidities, are prone to develop a more severe disease compared to children and younger population.
More information: Bryce A. Schuler et al. Age-related expression of SARS-CoV-2 priming protease TMPRSS2 in the developing lung, (2020). DOI: 10.1101/2020.05.22.111187
1. Kuiken, T, Fouchier, RAM, Schutten, M, Rimmelzwaan, GF, Van Amerongen, G, van Riel, D, Laman, JD, de Jong, T, van Doornum, G, and Lim, W (2003). Newly discovered coronavirus as the primary cause of severe acute respiratory syndrome. The Lancet 362: 263-270.
2. Zaki, AM, Van Boheemen, S, Bestebroer, TM, Osterhaus, ADME, and Fouchier, RAM (2012). Isolation of a novel coronavirus from a man with pneumonia in Saudi Arabia. 366 N Engl J Med 367: 1814-1820.
3. Weiss, P, and Murdoch, DR (2020). Clinical course and mortality risk of severe COVID-19. The Lancet 395: 1014-1015.
4 de Groot, RJ, Baker, SC, Baric, RS, Brown, CS, Drosten, C, Enjuanes, L, Fouchier, RAM, Galiano, M, Gorbalenya, AE, and Memish, ZA (2013). Commentary: Middle East respiratory syndrome coronavirus (MERS-CoV): announcement of the Coronavirus Study Group. J Virol 87: 7790-7792.
5. Zhou, P, Yang, X-L, Wang, X-G, Hu, B, Zhang, L, Zhang, W, Si, H-R, Zhu, Y, Li, B, Huang, C-L, et al. (2020). A pneumonia outbreak associated with a new coronavirus of probable bat origin. Nature 579: 270-273.
6. Hoffmann, M, Kleine-Weber, H, Schroeder, S, Krüger, N, Herrler, T, Erichsen, S, Schiergens, TS, Herrler, G, Wu, N-H, Nitsche, A, et al. (2020). SARS-CoV-2 Cell Entry Depends on ACE2 and TMPRSS2 and Is Blocked by a Clinically Proven Protease 379 Inhibitor. Cell 181: 271-280.e278.
7. Zou, L, Ruan, F, Huang, M, Liang, L, Huang, H, Hong, Z, Yu, J, Kang, M, Song, Y, Xia, J,et al. (2020). SARS-CoV-2 Viral Load in Upper Respiratory Specimens of Infected Patients. N Engl J Med 382: 1177-1179.
8. Ghinai, I, McPherson, TD, Hunter, JC, Kirking, HL, Christiansen, D, Joshi, K, Rubin, R,Morales-Estrada, S, Black, SR, Pacilli, M, et al. (2020). First known person-to-person transmission of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in the 386 USA. The Lancet 395: 1137-1144.
9. Wu, Z, and McGoogan, JM (2020). Characteristics of and Important Lessons From the Coronavirus Disease 2019 (COVID-19) Outbreak in China: Summary of a Report of 72 314 Cases From the Chinese Center for Disease Control and Prevention. JAMA 323: 1239-1242.
10. Yang, X, Yu, Y, Xu, J, Shu, H, Xia, J, Liu, H, Wu, Y, Zhang, L, Yu, Z, Fang, M, et al. (2020). Clinical course and outcomes of critically ill patients with SARS-CoV-2 pneumonia in Wuhan, China: a single-centered, retrospective, observational study. Lancet Respir Med 8: 475-481.
11. Van Bever, HP, Chng, SY, and Goh, DY (2004). Childhood severe acute respiratory syndrome, coronavirus infections and asthma. Pediatr Allergy Immunol 15: 206-209.
12. Wei, M, Yuan, J, Liu, Y, Fu, T, Yu, X, and Zhang, Z-J (2020). Novel coronavirus infection in hospitalized infants under 1 year of age in China. JAMA.
13. Dong, Y, Mo, X, Hu, Y, Qi, X, Jiang, F, Jiang, Z, and Tong, S (2020). Epidemiology of COVID-19 Among Children in China. Pediatrics: e20200702.
14. Li, W, Zhang, B, Lu, J, Liu, S, Chang, Z, Cao, P, Liu, X, Zhang, P, Ling, Y, Tao, K, et al. (2020). The characteristics of household transmission of COVID-19. Clin Infect Dis 10.1093/cid/ciaa450.
15. Zhang, J, Litvinova, M, Liang, Y, Wang, Y, Wang, W, Zhao, S, Wu, Q, Merler, S, Viboud,C, Vespignani, A, et al. (2020). Changes in contact patterns shape the dynamics of the COVID-19 outbreak in China. Science: eabb8001.
16. Lu, X, Zhang, L, Du, H, Zhang, J, Li, YY, Qu, J, Zhang, W, Wang, Y, Bao, S, Li, Y, et al.(2020). SARS-CoV-2 Infection in Children. N Engl J Med 382: 1663-1665.
17. Weng, N-p (2006). Aging of the immune system: how much can the adaptive immune system adapt? Immunity 24: 495-499.
18. Clarke, NE, Belyaev, ND, Lambert, DW, and Turner, AJ (2014). Epigenetic regulation of angiotensin-converting enzyme 2 (ACE2) by SIRT1 under conditions of cell energy stress. Clin Sci 126: 507-516.
19. Guan, W-j, Ni, Z-y, Hu, Y, Liang, W-h, Ou, C-q, He, J-x, Liu, L, Shan, H, Lei, C-l, and Hui,DSC (2020). Clinical characteristics of coronavirus disease 2019 in China. N Engl J Med.
20. Zhang, Jj, Dong, X, Cao, YY, Yuan, Yd, Yang, Yb, Yan, Yq, Akdis, CA, and Gao, Yd (2020). Clinical characteristics of 140 patients infected by SARS-CoV-2 in Wuhan,China. Allergy.
21. Brake, SJ, Barnsley, K, Lu, W, McAlinden, KD, Eapen, MS, and Sohal, SS (2020). Smoking Upregulates Angiotensin-Converting Enzyme-2 Receptor: A PotentialAdhesion Site for Novel Coronavirus SARS-CoV-2 (Covid-19). Journal of Clinical Medicine 9: 841.
22. Joshi, S, Wollenzien, H, Leclerc, E, and Jarajapu, YPR (2019). Hypoxic regulation of angiotensin-converting enzyme 2 and Mas receptor in human CD34+ cells. J Cell Physiol 234: 20420-20431.
23. Cai, H (2020). Sex difference and smoking predisposition in patients with COVID-19. Lancet Respir Med 8: e20.
24. Hoffmann, M, Kleine-Weber, H, Schroeder, S, Krüger, N, Herrler, T, Erichsen, S,Schiergens, TS, Herrler, G, Wu, N-H, Nitsche, A, et al. SARS-CoV-2 Cell Entry Depends on ACE2 and TMPRSS2 and Is Blocked by a Clinically Proven Protease Inhibitor. Cell.
25. Zhou, P, Yang, X-L, Wang, X-G, Hu, B, Zhang, L, Zhang, W, Si, H-R, Zhu, Y, Li, B, and Huang, C-L (2020). A pneumonia outbreak associated with a new coronavirus of probable bat origin. Nature: 1-4.
26. Matsuyama, S, Nagata, N, Shirato, K, Kawase, M, Takeda, M, and Taguchi, F (2010). Efficient Activation of the Severe Acute Respiratory Syndrome Coronavirus Spike Protein by the Transmembrane Protease TMPRSS2. J Virol 84: 12658.
27. Haga, S, Yamamoto, N, Nakai-Murakami, C, Osawa, Y, Tokunaga, K, Sata, T, Yamamoto, N, Sasazuki, T, and Ishizaka, Y (2008). Modulation of TNF-alpha-converting enzyme by the spike protein of SARS-CoV and ACE2 induces TNF-alpha production and facilitates viral entry. Proc Natl Acad Sci U S A 105: 7809-7814.
28. Reddy, R, Asante, I, Liu, S, Parikh, P, Liebler, J, Borok, Z, Rodgers, K, Baydur, A, and Louie, SG (2019). Circulating angiotensin peptides levels in Acute Respiratory Distress Syndrome correlate with clinical outcomes: A pilot study. PLoS One 14.
29. Fang, L, Karakiulakis, G, and Roth, M (2020). Are patients with hypertension and diabetes mellitus at increased risk for COVID-19 infection? Lancet Respir Med 8: e21.
30. The Tumor Analysis Best Practices Working, G (2004). Expression profiling — best practices for data generation and interpretation in clinical trials. Nature Reviews Genetics 5: 229-237.
31. Galamb, O, Sipos, F, Spisak, S, Galamb, B, Krenacs, T, Valcz, G, Tulassay, Z, and Molnar, B (2009). Potential biomarkers of colorectal adenoma-dysplasia-carcinoma progression: mRNA expression profiling and in situ protein detection on TMAs reveal 15 sequentially upregulated and 2 downregulated genes. Cell Oncol 31: 19-29.
32. Hughey, JJ, and Butte, AJ (2015). Robust meta-analysis of gene expression using the elastic net. Nucleic Acids Res 43: e79-e79.
33. Ritchie, ME, Phipson, B, Wu, DI, Hu, Y, Law, CW, Shi, W, and Smyth, GK (2015). Limma powers differential expression analyses for RNA-sequencing and microarray studies. Nucleic Acids Res 43: e47-e47.
34. Dudoit, S, Yang, YH, Callow, MJ, and Speed, TP (2002). Statistical methods for identifying differentially expressed genes in replicated cDNA microarray experiments. Statistica sinica: 111-139. 462
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