<scp>SARS</scp> ‐ <scp>CoV</scp> ‐2 Nucleocapsid Protein Does Not Induce Inflammation in Endothelial Cells or Monocytes

The nucleocapsid (N) protein of SARS-CoV-2 is critical for viral replication and genome packaging. However, whether it induces inflammation through endothelial cell and monocyte activation remains controversial. One aspect that has been overlooked is the consideration of residual endotoxins in recombinant proteins when conducting immune response studies. We aimed to assess whether N-protein induces an inflammatory response in mouse and human microvascular endothelial cells (MEC and HMEC) and THP-1 cells, independent of endotoxin contamination. MEC, HMEC, and THP-1 cells were treated with vehicle, lipopolysaccharide (LPS), or recombinant N-protein in the presence or absence of the LPS-neutralizing agent polymyxin B. The inflammatory response was assessed through quantification of mRNA and protein levels of inflammatory markers and monocyte adhesion to endothelial cells. In MEC, treatment with N-protein resulted in at least a 15-fold increase in inflammatory marker levels. Similarly, in THP-1 cells, N-protein caused a significant increase in mRNA inflammatory marker levels, which were brought back to control levels with polymyxin B. LPS or N-protein alone resulted in a tripling of monocyte adhesion to MEC, and this was reduced to control levels with polymyxin B. In addition, in HMEC, endotoxin-depleted N-protein did not cause a significant increase in inflammatory marker levels. In conclusion, our data show that N-protein does not induce inflammation in endothelial cells or monocytes. Some of its observed inflammatory effects may be due to endotoxin contamination, suggesting that it does not directly induce inflammation.