Viral infection of human Lung Chips: It's all the RAGE

A human Lung Alveolus Chip is used to identify RAGE as a central regulator of innate immunity in response to viral infection
Viral infection of human Lung Chips: It's all the RAGE
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Two years into the COVID-19 pandemic, we are armed with more tools than ever before. Yet, still thousands of people are dying every day due to the disease-causing SARS-CoV-2 virus. It has become clear that vaccines and antiviral drugs, such as Paxlovid, are not enough to contain this disease. This is because people do not usually die due to viral replication, but because of the 'cytokine storm' that drives uncontrolled inflammation in the distal portion of the lung. To meet this challenge, we urgently need treatments that can dampen these exuberant host immune responses that are triggered by viral infection.

         Unfortunately, current drug development processes, which rely largely on animal-based preclinical models, are not well suited for this task. This is because animals are usually not the natural hosts of virus strains that infect humans. Perhaps more importantly, the responses in animals can be very different from those in humans. Recognizing the potential for pandemic respiratory viruses to emerge in the future and the limitations of existing animal models, the NIH funded work in our laboratory beginning in 2017 to explore whether our organ-on-a-chip (Organ Chip) microfluidic culture models of human lung airway and alveolus could be leveraged to study infection by potential pandemic respiratory viruses and to identify treatments that target the host rather than the virus itself; however, at that time, the focus was on influenza.

         In 2021 we published an article in Nature Biomedical Engineering that described how influenza virus infection can be modeled in our human lung Airway Chip, as well as how we pivoted to model infection by a pseudotyped SARS-CoV-2 virus when the COVID-19 pandemic emerged in early 2020. That effort enabled us to rapidly repurpose an existing drug (amodiaquine), which is currently in clinical trials for COVID-19 across multiple sites in Africa, as described in our Nature Biomedical Engineering article in 2021. However, we also had been exploring use of our human Lung Alveolus Chip to study infection in the alveoli of the distal lung because that is where the virus inflicts the greatest damage, which leads to pneumonia, hospitalization, and death. In fact, when we carried out RNAseq analysis, we found that infection of our Alveolus Chips with influenza H3N2 that causes pneumonia in humans produced changes in gene expression that more closely resembled those induced by SARS-CoV-2 than infection by more closely related coronaviruses (e.g., OC43).

         So to pursue this challenge, we infected our 'breathing' human Lung Alveolus Chip with H3N2 virus. This chip is lined by primary human lung alveolar epithelial cells cultured under an air-liquid interface interfaced with human lung microvascular endothelial cells exposed to dynamic fluid flow while the entire alveolar-capillary interface experiences cyclic mechanical deformations to mimic rhythmic breathing motions. This is done by applying rhythmical suction to hollow side chambers adjacent to the cell-lined fluidic channels with the device that is made from a flexible, optically clear silicone rubber.  

         To our surprise, when we carried out control studies without breathing motions, we discovered that viral infection rates almost doubled compared to chips that experienced physiological breathing motions. We then carried out RNAseq studies to analyze the molecular mechanism responsible for this response. It turns out that the lung epithelial and endothelial cells increase the expression of a gene called S100A7 when they are stretched during breathing. S100A7 encodes a protein that binds to the receptor for glycation end products (RAGE) that activates an innate immune response including the production of protective type I/III interferons that help to inhibit virus infection. However, when we applied pathological levels of mechanical strain to these chips to mimic hyperextension (e.g., due to ventilator-induced injury), we observe hyperactivation of this pathway leading to great increases in pro-inflammatory cytokines. In addition, we found that influenza virus infection itself can induce the expression of several genes in the S100 family, which also bind to RAGE, as well as hyperactivation of this pathway. It was now clear to us that RAGE might be a central player that controls the innate immune response in the lung.       

         We soon realized that although there are many differences between infections produced by influenza and SARS-CoV-2 viruses, there are also many similarities in the host responses, especially when the infection reaches the distal lung alveolus. For example, many genes in the S100 family are also among those most highly induced by SARS-CoV-2 infection. In fact, clinical studies suggest that levels of these proteins in blood correlate directly with severity. This made us realize that this could be a convergence point where our knowledge and models could be used to confront the COVID-19 pandemic. We therefore reached out to obtain inhibitors of RAGE to test this idea. One of the inhibitors that seemed especially promising called azeliragon, is an oral drug that was found to be extremely safe and well-tolerated when tested in over 2,000 patients in a Phase 3 clinical trial for Alzheimer’s disease.  Remarkably, we found that azeliragon effectively prevented activation of the inflammatory response in response to hyper-extension or infection by influenza virus. It also produced synergistic effects in reducing inflammatory cytokine production when combined with molnupiravir, another antiviral medication that is used to treat both influenza and SARS-CoV-2 virus infections. Based on the potential importance of these findings, we reached out to the Cantex Pharmaceuticals which owns the rights to azeliragon and share our findings with the company. This led Cantex to submit an investigational new drug (IND) application to the FDA to initiate a Phase 2 COVID-19 trial using azeliragon, and our data were included in this application along with an extensive summary of the literature supporting the key role that RAGE may play in COVID-19.

         Over the last decade, we have witnessed the rapid development of the Organ Chip field, both in academia and industry, and multiple demonstrations of the ability of this technology to effectively mimic human organ-level physiology and pathophysiology in vitro. However, our discovery of the vital role of RAGE in lung inflammation following viral infection using human Organ Chips, and the rapid repurposing of this compound as a potential host-targeted therapeutic for viral infections has opened up a new stage for the use of this technology in drug development. We believe that we are now at a tipping point where Organ Chips will be increasingly employed as preclinical drug development tools to enable identification of novel therapeutic targets that would be impossible to identify using existing animal models.