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How breathing activates lung defenses against viruses

The lung alveolus chip, shown here, was made by combining microfabrication techniques from the computer industry with modern tissue engineering techniques, human cells, and a vacuum pump.Felice Frankel, Wyss Institute, Harvard University

The COVID-19 pandemic highlighted the need for better experimental models of the lung. Such models are needed to understand disease processes, test existing drugs, and develop new therapies. Lung tissue is exposed to mechanical stress during breathing. These motions are known to affect lung development and function. However, most lung models lack the ability to control breathing movement to study its role in lung diseases.

To address this challenge, a research team led by Dr. Donald Ingber from the Wyss Institute at Harvard University developed the lung alveolus chip, named for the alveoli—the tiny sacs deep in the lung that perform gas exchange. About the size of a memory stick, the chip can support the many cell types in human lung tissue. The cells that line the lung surface grow above a thin, porous membrane. Beneath the membrane grow lung blood vessel cells fed by a continuous flow of liquid. The entire chamber can be stretched and relaxed in cycles to mimic the effects of breathing.

In past work, the team showed that a lung airway chip could be used to mimic viral infection in the human upper airway and quickly identify candidate antiviral drugs. In their new study, they used lung alveolus chips to study how breathing motions affect infection deep in the lung by influenza virus. They used the H3N2 strain, a leading cause of lung infections, hospitalizations, and deaths.

The work was funded in part by NIH’s National Heart, Lung, and Blood Institute (NHLBI). Results were published on April 8, 2022, in Nature Communications.

H3N2 viruses added to the chip caused damage to lung tissue and cell death. In response, the tissue activated injury and repair pathways. Levels of cytokines, which help coordinate the protective immune response and inflammation, rose. Immune cells became better able to adhere to the lower cell level and migrate into the upper one. Notably, the types of cytokines seen differed from those during infection in static 2D cultures.

The team next explored differences in gene activity with or without the physical stresses of breathing. Many genes involved in the protective immune response were more active in the tissue when subjected to the breathing-like motion.

When infections in the lung alveolus chip with or without breathing movements were compared, those with motion had 50% less virus mRNA in the upper layer and 80% lower levels of active virus. When the movement was stopped, the protective immune response was suppressed.

An analysis showed that activity of the gene for a protein called S100A7 was significantly higher when lung chips experienced breathing movements. S100A7, in turn, activated genes involved in the protective immune response, but increased levels of inflammatory cytokines as well.

The team found that both layers of cells in the chip also had high levels of TRPV4, a protein known to be triggered by mechanical forces. When they added a TRPV4 inhibitor, S100A7 and inflammatory cytokines were suppressed. These experiments show that TRPV4 senses breathing movements and activates S100A7, which boosts the immune response. However, too great a response can cause inflammation.

S100A7 is known to activate a receptor called RAGE. Like the TRPV4 inhibitor, a RAGE inhibitor called azeliragon suppressed cytokine production when given before or after infection with H3N2. Drugs that target these pathways might be able to suppress the excessive lung inflammation seen in some influenza or SARS-CoV-2 infections.

“This research demonstrates the importance of breathing motions for human lung function, including immune responses to infection, and shows that our human alveolus chip can be used to model these responses in the deep portions of the lung, where infections are often more severe and lead to hospitalization and death,” says co-first author Dr. Haiqing Bai.

Data from the study were included in an application to the FDA for a clinical trial to test azeliragon in hospitalized COVID-19 patients.

—by Harrison Wein, Ph.D.