Scientists take a trip to the lungs of mice infected with the flu

In the 1966 novel, Fantastic Voyage, written by biochemist and author Isaac Asimov, a team of people miniaturizes to travel through the body of a scientist and save him from a blood clot in his brain .

For Yoshihiro Kawaoka, a virologist and influenza expert at the University of Wisconsin-Madison, the recent discovery of a true active influenza infection in the lungs of living mice reminds of this science fiction play of 50 years, which has also been adapted film.

Publication today (June 25, 2018) in the Proceedings of the National Academy of SciencesKawaoka and his team describe a new tool that they call FluVision, which allows them to witness an influenza infection in a live animal in action. Plus, it offers a window onto a world that no one has ever seen before, allowing scientists to observe and better understand what happens when a virus infects the lungs and that the body reacts.

"Now we can see in the real-time body of animals infected with viruses," says UW-Madison professor of pathobiological sciences at the UW-Madison School of Veterinary Medicine. "It's like we can shrink and enter the body."

In doing so, scientists have documented the differences in the action of two different strains of influenza, have seen influenza viruses spread to the lungs, showed a reduction in the speed of blood flow in the infected areas of the lungs, monitored activation and immune behavior. cells called neutrophils, and revealed some of the damage that can be caused by infection with a highly pathogenic strain of influenza.

Notably, infection with a highly pathogenic influenza strain – the "bird flu," H5N1 – occurs faster and causes more damage than infection with a more gentle and adapted human H1N1 strain to the mouse. Pathogenicity refers to the ability of a virus to cause disease.

To microscopically scan inside the lungs of living mice, the Kawaoka team had to overcome several challenges. The first was to find a technology that allows them to see through the lungs. Another group was pioneering with an approach called two – photon excitation microscopy, and the Kawaoka team adapted it for its study.

The team had to build a system allowing it to work with influenza viruses at a high level of biosecurity – level three biosecurity – while allowing technicians to access the laser source needed to see the objects in the field. Interest in the lungs. Lead author Hiroshi Ueki helped design a system in which the laser is located outside the high-containment laboratory space, through a small glass window up to the microscope inside the laboratory, all built on physically separate stabilized platforms.

The Kawaoka team also had to create fluorescently labeled viruses that could be used to infect mice and visualized with the laser under the microscope, but which also functioned similarly to viruses found in nature. They call Color-flu technology.

In addition, researchers had to develop a method to keep a portion of the lung motionless while the mouse was breathing so that they could obtain high quality images and videos. The team had a small, custom-made device called a chest window, which, according to Kawaoka, was patented, which uses a vacuum to stabilize a small portion of the lung exposed surgically during imaging.

For the study, the researchers infected mice with fluorescently labeled H5N1 or H1N1. Two days after the infection, they could see lung cells infected with viral particles. The number of these cells reached its maximum three days after infection and was higher in H5N1-infected lungs.

Blood flow into the capillaries of the lungs infected with influenza slowed after infection by one or the other virus, but to a lesser extent in mice infected with the H1N1 virus. This suggests that viruses affect the vascular system before causing lung damage.

The lungs of mice infected with the H5N1 virus also "leaked" two days after exposure to the virus, the contents of the capillaries penetrating the tiny air sacs of the lungs, called alveoli. This was also associated with an increase in the number of dead cells in the lungs.

"It is clear that something is wrong with the pulmonary capillaries," says Kawaoka, who is also a professor at the University of Tokyo, where the work was done. "The reason we see this leak is that the junctions between the endothelial cells (which make up the vessels in the lungs) loosen up for whatever reason we have documented for the first time."

The study of the mechanisms of infection can be a kind of attempt at chicken and egg, because once the infection started, the body's response triggers a cascade of actions which can also cause some of the damage associated with the pathogen. Some of them, like the pulmonary leak observed by the Kawaoka team, can help the body react, he says. This relationship between the virus and the host is what motivates most of Kawaoka's curiosity.

His team chose to look at immune cells called neutrophils, one of the body's first lines of defense. Their action can cause inflammation. In H5N1-infected mice, neutrophils were recruited to the lungs on the first day after exposure, becoming six times more common. They doubled in the lungs of mice infected with the H1N1 virus.

After the number of cells infected with influenza peaked on the third day, the number of neutrophils decreased, but those who remained behaved differently from neutrophils in healthy mice.

The team discovered that neutrophils had two types of movements: slow and fast. In influenza-infected lungs, neutrophils that remained after the peak day showed a decrease in fast movements and spent more time moving slowly, as they were looking for infected cells.

"We do not yet know why and what they do," says Kawaoka, but, he notes, it's the first time this behavior is documented. And for him, it is a motivation to dig even deeper, and to adapt the technology for other respiratory viruses.

"We see the mechanisms of the immune system at work," he says. "These are the things you discover and it's exciting, but now we have to understand what's going on."

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More information:
Hiroshi Ueki et al., "In Vivo Imaging of Pathophysiological Changes and Neutrophil Dynamics in the Lungs of Influenza Virus-infected Mice" PNAS (2018). www.pnas.org/cgi/doi/10.1073/pnas.1806265115