Protease Activated Receptor‐2 (PAR‐2) Antagonist potentially ameliorates the SARS‐CoV‐2 virus‐induced Inflammatory Storm

Abstract

The severe acute respiratory syndrome‐ 2 (SARS‐CoV‐2) caused the coronavirus disease COVID‐19 responsible for the current pandemic presenting a high mortality rate. The development of vaccines provided a transient relief to the social, economic, and health care systems. However, the virus is constantly mutating to survive, generating new pathogenic variants that jeopardize the ongoing efforts and up‐to‐date accomplishments against it. The TMPRSS2 is a crucial player for SARS‐CoV‐2 cell entry. This transmembrane serine protease intervenes with the viral Spike/Angiotensin‐Converting Enzyme 2 (ACE2) host receptor axis, enabling to initiate the SARS‐CoV‐2 entry to target cells. The subsequent viral replication causes pathophysiological changes including a major complication of the COVID‐19 known as the inflammatory storm (IS). This is characterized by an enhanced release of inflammatory modulators leading to a hyper‐inflammation. TMPRSS2 is expressed in the epithelial cells of several tissues, including the epithelium of the respiratory tract, that is the focus of our study. However, this catalytic enzyme shows poor anchoring in the cell membrane and is consequently released into the extracellular space with the potential to activate or cleave neighboring cells dependent on serine proteases. An example of this cellular scenario is represented by the Protease Activated Receptor‐2 (PAR‐2), associated with activation of inflammatory pathways upon cleavage of the N‐terminus by a serine protease. We hypothesized: 1) that TMPRSS2 co‐localizes with PAR‐2 at the cell membrane of target epithelial cells; 2) TMPRSS2 activates PAR‐2 and associated proinflammatory signaling pathways; 3) the SARS‐CoV‐2 chaotic inflammation together with PAR‐2 activation will worsen the resulting IS. Based on our lab prior work on PAR‐2 and its antagonist, we anticipate that by inhibiting PAR‐2 activation, we will observe a decrease in the COVID‐19 associated IS. In this study, we used the MCF‐7 cell line to standardize the protocols, since this cell line expresses both proteins. They are more resilient to adverse conditions in the lab (due to the pandemicconsequences at research labs) and will allow us to establish a proof of concept to be compare with the human primary airway epithelial cells that we will subsequently use. Using MCF‐7 as a model, we established the presence of PAR‐2 and TMPRSS2 via western blot. In addition, by using immuno‐cyto‐fluorescence we confirmed that these two proteins colocalize at the cell membrane. Activation of intracellular calcium‐dependent pathways, detected by intracellular calcium fluorogenic assay, showed that after treating cells with trypsin (Gold Standard for PAR‐2 activation), TMPRSS2, PAR‐2 agonist, and PAR‐2 antagonist, trypsin caused a significant (p < 0.05) increase compared to TMPRSS2 either alone, with the PAR‐2 agonist or the PAR‐2 antagonist with a decreasing of 50 % trypsin activity. Importantly, BrdU assay results showed that PAR‐2 antagonist decreased the proliferation of MCF‐7 cells when compared to trypsin (p < 0.005). Currently, we are working to test our hypothesis with human primary cells from different locations of the respiratory tract and compare the outcomes with the MCF‐7 model. Furthermore, our study will contribute to having a better understanding of PAR‐2 role in SARS‐ CoV‐2 complications.