U of T researchers unveil new preclinical insights into why males are more likely to have severe COVID-19

A new study from a team of Emerging and Pandemic Infections Consortium (EPIC) members has uncovered the biological reasons underlying sex differences in COVID-19 outcomes and is offering a promising new strategy to prevent illness.

Early on during the pandemic, clinicians quickly noticed that males were more likely than females to be hospitalized or admitted to the ICU or to die from COVID-19 despite having similar infection rates. This pattern held true across all age groups and in countries around the world.

“COVID-19 severity and mortality are much higher in males than in females, but the reasons for this remain poorly understood,” says Haibo Zhang, senior author of the study and a professor of anesthesiology and pain medicine, critical care medicine and physiology at the University of Toronto’s Temerty Faculty of Medicine.

“That was the driving force for our work.”

The research, conducted in mice and published in the journal iScience, points to the ACE2 protein as a key contributor to differences in COVID-19 outcomes between males and females.

Located on the cell’s outer surface, ACE2 play an important role in controlling blood pressure and inflammation and protecting organs from damage caused by excess inflammation. During a SARS-CoV-2 infection, the coronavirus spike protein locks on to ACE2 to enter the cell.

The gene encoding the ACE2 protein is located on the X chromosome, which means that females have two copies of the gene and males only have one.

In times of health, the extra copy of ACE2 doesn’t appear to make a difference — Zhang and his team found similar levels of ACE2 protein in healthy male and female mice.

In contrast, they observed a dramatic decrease in ACE2 in male mice following a SARS-CoV-2 infection while levels remained consistent in females, suggesting that the additional copy of ACE2 on the X chromosome is helping to compensate and maintain high protein levels in female mice.

The changes in ACE2 levels were also correlated with a drop in estrogen hormone signalling in male mice, which could also contribute to the sex-specific differences in COVID-19 outcomes.

To test whether low levels of ACE2 was responsible for the more severe outcomes seen in male mice with COVID-19, the researchers devised a therapeutic approach using an inhaler to deliver lab-made ACE2 proteins directly into the lungs. Male mice that were received a daily puff of ACE2 after SARS-CoV-2 infection had less virus in their lungs, less lung injury and higher levels of estrogen signalling.

Together, these results paint a clearer picture of how the extra copy of the ACE2 gene and higher estrogen levels in female mice work together to protect them from experiencing more severe COVID-19.

“A common misconception is that an increased presence of ACE2 receptors would result in a higher infection rate,” says Zhang, who is also a staff scientist in the Keenan Research Centre for Biomedical Science of St. Michael’s Hospital, a site of Unity Health Toronto.

“However, the enhanced activation of ACE2 in females actually serves as a compensatory mechanism during infection that’s aimed at safeguarding the lungs and other vital organs from potential damage.”

In males who lack the second copy of the gene, much of the existing ACE2 gets co-opted by SARS-CoV-2 during an infection. As a result, that there is not enough of the protein to fulfil its usual functions of tamping down inflammation and preventing organ damage.

The extra dose of ACE2 delivered by inhaler serve as decoys to glob onto the coronavirus, thereby preventing it from entering cells while also keeping the native ACE2 proteins free to exert its protective effects.

Beyond the thrill of discovery, Zhang is excited by the potential implications of these findings, which are the first to demonstrate the effectiveness of inhaling ACE2, on preventing and treating COVID-19 in humans.

He imagines a scenario where people who are entering high-risk situations — boarding an airplane or attending a large in-person conference, for example — might take a puff of ACE2 to protect their lungs from the virus. Similarly, the treatment could also be given to people after infection to reduce the risk of hospitalization and death.

“By using the inhaler, ACE2 remains in the lungs at a sustained, low concentration over an extended period, where it can neutralize the virus even before it enters into our cells. We anticipate that our research will motivate individuals to contemplate this faster and more efficacious strategy for both prevention and treatment of COVID-19 in humans,” says Zhang.

The study was a collaborative effort between Zhang and fellow EPIC members Samira Mubareka, Theo Moraes and Mingyao Liu, with much of the work undertaken in the Toronto High Containment Facility (THCF), which is the only containment level 3 research lab in the Greater Toronto Area and the largest in the province.

Having access to the THCF allowed Zhang and his team to pivot quickly during the early months of the pandemic and apply their expertise in lung physiology and disease to answering rapidly emerging questions about COVID-19.

Jady Liang, the co-lead author of the new study, had just started her PhD with Zhang when the pandemic started. She recalls the stress and intensity of training and working in the THCF during that time but credits EPIC staff and other THCF users with helping her become comfortable with the processes and protocols.

“It was a lot of hard work from everyone on the team during the pandemic, especially during the first wave,” says Liang, who is now a fourth year PhD student in the department of physiology.

“We need a lot of people with expertise in different fields to work together so that we can advance and be prepared for the next pandemic.”

EPIC is a partnership between the University of Toronto and five research partners — The Hospital for Sick Children (SickKids) Research Institute, Lunenfeld-Tanenbaum Research Institute at Sinai Health, Sunnybrook Research Institute, Unity Health Toronto and the University Health Network. EPIC brings together researchers from different disciplines to facilitate an integrated and innovative response to high-risk, high-burden infectious diseases.