The lungs’ constant pattern of stretching and relaxing that accompanies breathing does more than allow for basic lung functioning—it generates immune responses against invading viruses. Using a human lung chip that replicates the structures and functions of the alveolus, a research team discovered that applying mechanical forces that mimic breathing motions suppresses influenza virus replication by activating protective innate immune responses. They also identified several drugs that reduced the production of inflammatory cytokines in infected Alveolus Chips, which could be useful in treating excessive inflammation in the lung.

Haiqing Bai, PhD, brings his experience studying diseases that affect the human lung’s air sacs, or “alveoli” to his research on organ chips. [Wyss Institute at Harvard University]
“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,” said Haiqing Bai, PhD, a Wyss technology development fellow at the Institute. “This model can also be used for preclinical drug testing to ensure that candidate drugs actually reduce infection and inflammation in functional human lung tissue.”

This study is published in Nature Communications in the paper, “Mechanical control of innate immune responses against viral infection revealed in a human lung alveolus chip.”

Bai recreated a flu infection in an Alveolus Chip so that the team could study how these deep lung spaces mount immune responses against viral invaders. Bai and his team first lined the two parallel microfluidic channels of an organ chip with different types of living human cells—alveolar lung cells in the upper channel and lung blood vessel cells in the lower channel—to recreate the interface between human air sacs and their blood-transporting capillaries. To mimic the conditions that alveoli experience in the human lung, the channel lined by alveolar cells was filled with air while the blood vessel channel was perfused with a flowing culture medium containing nutrients that are normally delivered via the blood. The channels were separated by a porous membrane that allowed molecules to flow between them.

The Human Alveolus Chip contains hollow side channels that allow suction to be applied to the chips, applying cyclic strain that mimics the motions of normal human breathing (left). A permeable membrane separates human alevolus cells in the upper channel from human blood vessel cells in the lower channel, allowing them to exchange molecular signals (right). [Wyss Institute at Harvard University]
When the team infected the Alveolus Chips with H3N2 influenza by introducing the virus into the air channel, they observed the development of several known hallmarks of influenza infection, including the breakdown of junctions between cells, a 25% increase in cell death, and the initiation of cellular repair programs. Infection also led to much higher levels of multiple inflammatory cytokines in the blood vessel channel including type III interferon (IFN-III), a natural defense against viral infection that is also activated in in vivo flu infection studies.

In addition, the blood vessel cells of infected chips expressed higher levels of adhesion molecules, which allowed immune cells including B cells, T cells, and monocytes in the perfusion medium to attach to the blood vessel walls to help combat the infection. These results confirmed that the Alveolus Chip was mounting an immune response against H3N2 that recapitulated what happens in the lung of human patients infected with flu virus.

The team then carried out the same experiment without mechanical breathing motions. To their surprise, chips exposed to breathing motions ​​had 50% less viral mRNA in their alveolar channels and a significant reduction in inflammatory cytokine levels compared to static chips. Genetic analysis revealed that the mechanical strain had activated molecular pathways related to immune defense and multiple antiviral genes, and these activations were reversed when the cyclical stretching was stopped.

These immunofluorescence micrographs (at different magnifications) show the 3D cellular structure that develops within the alveolar channel and mimics the microstructure of human alveoli. [Wyss Institute at Harvard University]
“This was our most unexpected finding—that mechanical stresses alone can generate an innate immune response in the lung,” said Longlong Si, PhD, a former Wyss technology development fellow who is now a professor at the Shenzhen Institute of Advanced Technology in China.

Knowing that sometimes the lungs experience greater than 5% strain, such as in chronic obstructive pulmonary disorder (COPD) or when patients are put on mechanical ventilators, the scientists increased the strain to 10% to see what would happen. The higher strain caused an increase in innate immune response genes and processes, including several inflammatory cytokines.

“Because the higher strain level resulted in greater cytokine production, it might explain why patients with lung conditions like COPD suffer from chronic inflammation, and why patients who are put on high-volume ventilators sometimes experience ventilator-induced lung injury,” Si explained.

The scientists then went a step further, comparing the RNA present in cells within strained vs. static Alveolus Chips to see if they could pinpoint how the breathing motions were generating an immune response. They identified a calcium-binding protein, S100A7, that was not detected in static chips but highly expressed in strained chips, suggesting that its production was induced by mechanical stretching. They also found that increased expression of S100A7 upregulated many other genes involved in the innate immune response, including multiple inflammatory cytokines.

S100A7 is one of several related molecules known to bind to a protein on cells’ membranes called the receptor for advanced glycation end products (RAGE). RAGE is more highly expressed in the lung than in any other organ in the human body, and has been implicated as a major inflammatory mediator in several lung diseases. The drug azeliragon is a known inhibitor of RAGE, so the scientists perfused azeliragon through the blood vessel channel of strained Alveolus Chips for 48 hours before infecting the chips with H3N2 virus. This pretreatment prevented the cytokine-storm-like response that they had observed in untreated chips.

Based on this result, the team then infected strained Alveolus Chips with H3N2 and administered azeliragon at its therapeutic dose two hours after infection. This approach significantly blocked the production of inflammatory cytokines—an effect that was further enhanced when they added the antiviral drug molnupiravir (which was recently approved for patients with COVID-19) to the treatment regimen.

These results caught the eye of Cantex Pharmaceuticals, which owns patent rights to azeliragon and was interested in using it to treat inflammatory diseases. Based in part on the Wyss team’s work in Alveolus Chips, Cantex licensed azeliragon for the treatment of COVID-19 and other inflammatory lung diseases in early 2022. Given the drug’s excellent safety record in previous Phase III clinical trials, the company has applied for FDA approval to start a Phase II trial in patients with COVID-19 patients, and plans to follow with additional Phase II trials for other diseases including COPD and steroid-resistant asthma.

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