Proinflammatory Pulmonary Effects of SARS-CoV-2 Main Protease in Mice and Human Bronchial Epithelial Cells
Caceres, A; Jabba, SV; Jordt, SE
Published in: American Journal of Respiratory and Critical Care Medicine
Rationale: The SARS-CoV-2 main protease (Mpro, also known as 3CLpro or Nsp5), is a critical enzyme in the viral replication process, cleaving viral nonstructural polypeptides into individual functional proteins, and playing a vital role in SARS-CoV-2 viral particle formation. While not routinely measured in patient samples, Mpro protein has been detected in nasopharyngeal swabs, bronchoalveolar lavage fluid, blood, and saliva, suggesting that the protease is released from virus-infected cells. The presence of viral RNA in patient fluid samples indirectly suggests that viral proteins, including Mpro, are also released by infected host-cells. Additionally, Mpro has been found to not only process viral proteins, but also cut host proteins. Some studies have detected Mpro-mediated cleavage products in patient samples, indicating that the protease is present and active during the course of infection. Proteases are essential for normal lung function, but their dysregulation can lead to a variety of lung diseases characterized by inflammation, tissue destruction, and impaired lung function. Here we hypothesize that Mpro, like other proteases, can activate inflammatory mechanisms and promote lung remodeling. Methods: We performed in vivo intranasal instillations of Mpro in mice and evaluated lung inflammation and immune response by qPCR, Flow Cytometry and BALF analysis. We also exposed human bronchial epithelial cells BEAS-2B to Mpro in vitro and evaluated its effects on gene expression of inflammatory markers. Results: Mice exposed intranasally to Mpro developed lung inflammation with significant neutrophilia, high protein extravasation, and a marked immune response. BEAS-2B cells incubated with different concentrations of Mpro also showed an increased expression of inflammatory markers. Cells exposed to heat-inactivated Mpro or Mpro inhibited by nirmatrelvir showed a diminished but not abolished response, which may suggest the participation of other pathways besides the protease activity alone. Conclusions: The presence of free Mpro in COVID-19 patients’ lungs may trigger an inflammatory response that might exacerbate pulmonary damage and contribute to respiratory symptoms. Our findings demonstrate that the administration of Mpro protein by itself, without the presence of SARS-CoV-2 virus, is capable to induce an inflammatory response in mouse lungs and in human bronchial epithelial cells, and that this response is ameliorated, but not abolished, by Mpro heat-inactivation or inhibition. This suggests that Mpro activates additional pro-inflammatory pathways independent from its proteolytic activity. These observations may open the door to potential treatments targeting Mpro besides protease inhibitors.
