Knockout Mice for the Study of Coronavirus Infections

The recent emergence of the novel, pathogenic SARS-coronavirus 2 (SARS-CoV-2, COVID-19), and its rapid international spread pose a global health emergency. The SARS-CoV-2 virus belongs to the family of coronaviridae viruses, which are enveloped, positive-sense, single-stranded RNA viruses. Entry into human (mammalian) cells depends on binding of the viral spike (S) proteins to human cellular receptors and on S protein priming by host cell proteases. Patients with COVID-19 present with many symptoms and predominantly suffer from respiratory and pulmonary complications¹. A serious complication of COVID-19, and especially amongst a large percentage of COVID-19 deaths, is acute respiratory distress syndrome (ARDS)². This is the most severe form of clinical acute lung injury and has a very high mortality rate (30-60%)³. Increasing publications reveal that SARS-CoV-2 uses the SARS-CoV receptor ACE2 for cellular entry and the serine protease TMPRSS2 for S protein priming^4-6.

Genetically Engineered Mouse Models for SARS Research

Genetically engineered mouse models (GEMs) have been instructive for the development of novel treatments and vaccines for SARS and are also expected to be valuable in researching COVID-19 treatments. GEM knockout (KO) mouse models of angiotensin-converting enzyme (Ace2)^7,8 and transmembrane protease serine-type 2 (Tmprss2)⁹ were instrumental in demonstrating the critical roles that these proteins play in SARS infection and in the development of pulmonary symptoms. The SARS S protein binds to the ACE2 receptor, which is further primed by TMPRSS2 within human cells^10,11. Ace2 Knockout mice have been critical in SARS and ARDS research in demonstrating that the binding of SARS viral S protein to ACE2 in mice down-regulates ACE2 expression and that the loss of ACE2 expression is associated with severe lung failure¹². Likewise, Tmprss2 KO mice have been utilized in SARS research to demonstrate the involvement of Tmprss2 in SARS-CoV entry into cells, and its inhibition may constitute a treatment/prophylaxis mechanism. Emerging literature supports the utility of these Ace2 and Tmprss2 GEM models for contemporaneous studies into COVID-19 disease pathogenesis and therapeutics ^13,14.

The Angiotensin-Converting Enzyme 2 (ACE2) Knockout Mouse

Figure Legend: Graphical abstract illustrating how the S protein of the SARS-CoV-2 virus binds to the SARS-CoV receptor ACE2 in order to enter human host cells, which is followed by priming by TMPRSS2. Therefore, antibodies against the SARS-CoV S protein or inhibitors of TMPRSS2 may offer protection against COVID-19 pathologies⁴.

Mice genetically deficient for Angiotensin-Converting Enzyme 2 (Ace2) have been used for the study of ACE2 in lung diseases such as ARDS and SARS, as well as other pathologies associated with cardiovascular and renal diseases¹¹. ACE2 is an enzymatic member of the carboxypeptidases, and functions within the renin-angiotensin system (RAS) to catalytically cleave a single residue from either ANG I or ANG II to produce either Angiotensin(1-9) or Angiotensin(1-7) respectively. The catalytic products exhibit negative feedback onto their parent substrates (Angiotensin I and Angiotensin II, respectively)¹⁵. Therefore, ACE2 functions as a key negative regulator of the RAS, which has critical roles in maintaining homeostasis of blood pressure, as well as salt and fluid balance in the human body. This has been reported to play a beneficial role in many diseases such as hypertension, diabetes, and cardiovascular disease, where its expression is decreased ¹⁵.

The Ace2 KO mouse in lung disease research

The role of ACE2 in the pathogenesis of acute lung injury such as ARDS was demonstrated by utilizing Ace2 KO and wild type (WT) mice in experimental models (namely acid-aspiration-induced ARDS, endotoxin-induced ARDS, and peritoneal sepsis-induced ARDS) that mimic the common lung pathology observed in several human diseases. Ace2 KO mice demonstrated severe disease compared to WT mice, with enhanced vascular permeability, increased lung edema, neutrophil accumulation, and worsened lung function⁸. These symptoms of acute lung injury were improved by treatment with catalytically active recombinant ACE2 protein in both WT and Ace2 KO mice. These results show that the loss of ACE2 is essential for disease pathogenesis, demonstrating a critical protective role of ACE2 in acute lung injury⁷.

The Ace2 KO Mouse in Studies of Infectious Respiratory Disease and SARS

The ACE2 protein has been demonstrated to be an essential receptor for SARS infections in vivo by binding to the SARS-CoV S protein, which promotes syncytia formation in the lungs of diseased humans^12,16, in mice in vivo⁸, and similarly for SARS-CoV-2 in vitro^4-6. The Ace2 KO mouse was used in a mouse-adapted SARS-CoV infection model and found to be resistant to viral infection¹². No lung histology from Ace2 KO mice challenged with SARS-CoV showed signs of inflammation¹², whereas some (but not all) SARS-infected WT mice displayed mild inflammation with leukocyte infiltration^8,17,18. These results and additional publications demonstrated the validity of Ace2 KO mouse in evaluating a recombinant ACE2 protein as a treatment to block the spread of SARS and to protect SARS patients from developing lung failure^7,8.

Thus the Ace2 KO mouse can be used:

• to investigate the pathophysiological role of ACE2 in SARS infections and other emerging infectious diseases that affect the lungs
• to study the role of acute lung injury such as ARDS
• to interrogate the relevance of ACE2 as a target for therapeutic intervention

The Transmembrane Protease Serine Type 2 (TMPRSS2) Knockout Mouse

Mice genetically deficient for Transmembrane Protease Serine Type 2 (Tmprss2) have been used to study the role of TMPRSS2 during coronavirus infection in vivo⁹. The human TMPRSS2 gene encodes for an androgen-regulated type II transmembrane serine protease (TTSP), which is highly expressed in normal human prostate epithelium and has been implicated in prostate carcinogenesis ¹⁹. TMPRSS2 (also known as epitheliasin) is also highly expressed in the epithelial tissues, including those lining the upper respiratory airways, bronchi, and lungs in humans²⁰. There is related interest in TMPRSS2 is due to its ability to cleave coronavirus spike proteins to induce virus-cell membrane fusion at the cell surface and facilitate rapid early viral entry into the host cell^21-23. Thus, substrate analogue inhibitors of TMPRSS2's active sites^24,25 are increasingly investigated as potential therapeutic targets for coronaviral infections as well as influenza viral infections^10,13,26.

The role of Tmprss2 KO mouse in SARS research

The Tmprss2 KO mouse was used in a mouse-adapted SARS-CoV infection model and exhibited no loss of body weight and weak pro-inflammatory responses upon viral infection when compared to identically infected WT mice that developed signs of weight loss and acute pneumonia⁹. The loss of Tmprss2 restricted the primary sites of viral infection within the lungs, diminished viral replication (especially within the bronchioles), viral dissemination within the respiratory airways, and was accompanied by less severe immunopathology in the Tmprss2 KO as compared to the WT mouse. However, the Tmprss2 KO mouse exhibited weakened or delayed inflammatory chemokine and cytokine responses that are mediated by Toll-like receptor 3 (TLR3) when intra-nasally stimulated with poly(I·C), which is a synthetic analogue of double-stranded RNA in order to assess the effect of Tmprss2 on the innate immune responses⁹. These results demonstrate that Tmprss2 plays a crucial role in viral spread within the airway of murine models infected by SARS-CoV and in the resulting pulmonary pathology and systemic immunopathology. Additional findings suggest that Tmprss2 may contribute to more severe or rapid immunopathology in WT mice by increasing the levels of inflammatory cytokines and chemokines after TLR3 stimulation.

Thus Tmprss2 KO mouse can be used in the research of SARS and COVID-19 infections:^10,13,26

• to investigate the pathophysiological role of TMPRSS2
• to interrogate TMPRSS2 as a potential therapeutic target

Summary

GEMs have been instrumental in the development of novel treatments and vaccines for infectious disease, and they are expected to play a similar role in the fight against the recent global SARS-CoV-2 (COVID-19) pandemic. Ace2 and Tmprss2 KO mice have been integral in informing our understanding of the underlying pathogenesis of clinical symptoms due to coronaviral infections. Therefore, these two GEMs may aid researchers in investigating the role of ACE2 and TMPRSS2 in SARS-CoV-2 infection, as well as assessing these proteins as targets for therapeutic intervention.

References

1. Fauci, A. S.; Lane, H. C.; Redfield, R. R. Covid-19 - Navigating the Uncharted. The New England Journal of Medicine. 2020.
2. Matthay, M. A.; Aldrich, J. M.; Gotts, J. E. Treatment for Severe Acute Respiratory Distress Syndrome from COVID-19. Lancet Respir. Med. 2020.
3. Ware, L. B.; Matthay, M. A. The Acute Respiratory Distress Syndrome. The New England Journal of Medicine. 2000.
4. Hoffmann, M.; Kleine-Weber, H.; Schroeder, S.; Krüger, N.; Herrler, T.; Erichsen, S.; Schiergens, T. S.; Herrler, G.; Wu, N. H.; Nitsche, A.; Müller, M. A.; Drosten, C.; Pöhlmann, S. SARS-CoV-2 Cell Entry Depends on ACE2 and TMPRSS2 and Is Blocked by a Clinically Proven Protease Inhibitor. Cell 2020.
5. Shang, J.; Ye, G.; Shi, K.; Wan, Y.; Luo, C.; Aihara, H.; Geng, Q.; Auerbach, A.; Li, F. Structural Basis of Receptor Recognition by SARS-CoV-2. Nature 2020.
6. Yan, R.; Zhang, Y.; Li, Y.; Xia, L.; Guo, Y.; Zhou, Q. Structural Basis for the Recognition of SARS-CoV-2 by Full-Length Human ACE2. Science (80-. ). 2020.
7. Imai, Y.; Kuba, K.; Rao, S.; Huan, Y.; Guo, F.; Guan, B.; Yang, P.; Sarao, R.; Wada, T.; Leong-Poi, H.; Crackower, M. A.; Fukamizu, A.; Hui, C. C.; Hein, L.; Uhlig, S.; Slutsky, A. S.; Jiang, C.; Penninger, J. M. Angiotensin-Converting Enzyme 2 Protects from Severe Acute Lung Failure. Nature 2005.
8. Kuba, K.; Imai, Y.; Rao, S.; Gao, H.; Guo, F.; Guan, B.; Huan, Y.; Yang, P.; Zhang, Y.; Deng, W.; Bao, L.; Zhang, B.; Liu, G.; Wang, Z.; Chappell, M.; Liu, Y.; Zheng, D.; Leibbrandt, A.; Wada, T.; Slutsky, A. S.; Liu, D.; Qin, C.; Jiang, C.; Penninger, J. M. A Crucial Role of Angiotensin Converting Enzyme 2 (ACE2) in SARS Coronavirus-Induced Lung Injury. Nat. Med. 2005.
9. Iwata-Yoshikawa, N.; Okamura, T.; Shimizu, Y.; Hasegawa, H.; Takeda, M.; Nagata, N. TMPRSS2 Contributes to Virus Spread and Immunopathology in the Airways of Murine Models after Coronavirus Infection. J. Virol. 2019.
10. Laporte, M.; Naesens, L. Airway Proteases: An Emerging Drug Target for Influenza and Other Respiratory Virus Infections. Current Opinion in Virology. 2017.
11. Kuba, K.; Imai, Y.; Rao, S.; Jiang, C.; Penninger, J. M. Lessons from SARS: Control of Acute Lung Failure by the SARS Receptor ACE2. Journal of Molecular Medicine. 2006.
12. Li, W.; Moore, M. J.; Vasllieva, N.; Sui, J.; Wong, S. K.; Berne, M. A.; Somasundaran, M.; Sullivan, J. L.; Luzuriaga, K.; Greeneugh, T. C.; Choe, H.; Farzan, M. Angiotensin-Converting Enzyme 2 Is a Functional Receptor for the SARS Coronavirus. Nature 2003.
13. Wang, X.; Dhindsa, R. S.; Povysil, G.; Zoghbi, A.; Joshua, E.; Hostyk, J. A.; Goldstein, D. B. Transcriptional Inhibition of Host Viral Entry Proteins as a Therapeutic Strategy for SARS-CoV-2. Preprints 2020.
14. Bao, L.; Deng, W.; Huang, B.; Gao, H.; Liu, J.; Ren, L.; Wei, Q.; Yu, P.; Xu, Y.; Qi, F.; Qu, Y.; Li, F.; Lv, Q.; Wang, W.; Xue, J.; Gong, S.; Liu, M.; Wang, G.; Wang, S.; Song, Z.; Zhao, L.; Liu, P.; Zhao, L.; Ye, F.; Wang, H.; Zhou, W.; Zhu, N.; Zhen, W.; Yu, H.; Zhang, X.; Guo, L.; Chen, L.; Wang, C.; Wang, Y.; Wang, X.; Xiao, Y.; Sun, Q.; Liu, H.; Zhu, F.; Ma, C.; Yan, L.; Yang, M.; Han, J.; Xu, W.; Tan, W.; Peng, X.; Jin, Q.; Wu, G.; Qin, C. The Pathogenicity of SARS-CoV-2 in HACE2 Transgenic Mice. bioRxiv 2020.
15. Tikellis, C.; Thomas, M. C. Angiotensin-Converting Enzyme 2 (ACE2) Is a Key Modulator of the Renin Angiotensin System in Health and Disease. International Journal of Peptides. 2012.
16. Hamming, I.; Timens, W.; Bulthuis, M. L. C.; Lely, A. T.; Navis, G. J.; van Goor, H. Tissue Distribution of ACE2 Protein, the Functional Receptor for SARS Coronavirus. A First Step in Understanding SARS Pathogenesis. J. Pathol. 2004.
17. Yang, Z. Y.; Kong, W. P.; Huang, Y.; Roberts, A.; Murphy, B. R.; Subbarao, K.; Nabel, G. J. A DNA Vaccine Induces SARS Coronavirus Neutralization and Protective Immunity in Mice. Nature 2004.
18. Hogan, R. J.; Gao, G.; Rowe, T.; Bell, P.; Flieder, D.; Paragas, J.; Kobinger, G. P.; Wivel, N. A.; Crystal, R. G.; Boyer, J.; Feldmann, H.; Voss, T. G.; Wilson, J. M. Resolution of Primary Severe Acute Respiratory Syndrome-Associated Coronavirus Infection Requires Stat1. J. Virol. 2004.
19. Jacquinet, E.; Rao, N. V.; Rao, G. V.; Zhengming, W.; Albertine, K. H.; Hoidal, J. R. Cloning and Characterization of the CDNA and Gene for Human Epitheliasin. Eur. J. Biochem. 2001.
20. Bugge, T. H.; Antalis, T. M.; Wu, Q. Type II Transmembrane Serine Proteases. Journal of Biological Chemistry. 2009.
21. Matsuyama, S.; Nagata, N.; Shirato, K.; Kawase, M.; Takeda, M.; Taguchi, F. Efficient Activation of the Severe Acute Respiratory Syndrome Coronavirus Spike Protein by the Transmembrane Protease TMPRSS2. J. Virol. 2010.
22. Bertram, S.; Dijkman, R.; Habjan, M.; Heurich, A.; Gierer, S.; Glowacka, I.; Welsch, K.; Winkler, M.; Schneider, H.; Hofmann-Winkler, H.; Thiel, V.; Pohlmann, S. TMPRSS2 Activates the Human Coronavirus 229E for Cathepsin-Independent Host Cell Entry and Is Expressed in Viral Target Cells in the Respiratory Epithelium. J. Virol. 2013.
23. Gierer, S.; Bertram, S.; Kaup, F.; Wrensch, F.; Heurich, A.; Kramer-Kuhl, A.; Welsch, K.; Winkler, M.; Meyer, B.; Drosten, C.; Dittmer, U.; von Hahn, T.; Simmons, G.; Hofmann, H.; Pohlmann, S. The Spike Protein of the Emerging Betacoronavirus EMC Uses a Novel Coronavirus Receptor for Entry, Can Be Activated by TMPRSS2, and Is Targeted by Neutralizing Antibodies. J. Virol. 2013.
24. Meyer, D.; Sielaff, F.; Hammami, M.; Böttcher-Friebertshäuser, E.; Garten, W.; Steinmetzer, T. Identification of the First Synthetic Inhibitors of the Type II Transmembrane Serine Protease TMPRSS2 Suitable for Inhibition of Influenza Virus Activation. Biochem. J. 2013.
25. Lucas, J. M.; Heinlein, C.; Kim, T.; Hernandez, S. A.; Malik, M. S.; True, L. D.; Morrissey, C.; Corey, E.; Montgomery, B.; Mostaghel, E.; Clegg, N.; Coleman, I.; Brown, C. M.; Schneider, E. L.; Craik, C.; Simon, J. A.; Bedalov, A.; Nelson, P. S. The Androgen-Regulated Protease TMPRSS2 Activates a Proteolytic Cascade Involving Components of the Tumor Microenvironment and Promotes Prostate Cancer Metastasis. Cancer Discov. 2014.
26. Shin, W. J.; Seong, B. L. Type II Transmembrane Serine Proteases as Potential Target for Anti-Influenza Drug Discovery. Expert Opinion on Drug Discovery. 2017.