Human TLR2 Stable Cell Line-HEK293T

Mounting evidence suggests that endothelial cell activation plays a central role in the pathogenesis of acute respiratory distress syndrome (ARDS) and multiple organ failure in patients with COVID-19. Here, researchers screened SARS-CoV-2 viral proteins that effectively activate human endothelial cells to elucidate the molecular mechanisms of endothelial cell activation. The study found that the SARS-CoV-2 nucleocapsid protein (NP) significantly activates human endothelial cells through the Toll-like receptor 2 (TLR2)/NF-κB and mitogen-activated protein kinase (MAPK) signaling pathways. Furthermore, by screening a natural microbial compound library containing 154 natural compounds, simvastatin was identified as a potent inhibitor of NP-induced endothelial cell activation. Notably, although the N protein sequence of coronaviruses is highly conserved, only the SARS-CoV-2 NP protein can induce endothelial cell activation. NP proteins from other coronaviruses (such as SARS-CoV, MERS-CoV, HUB1-CoV, and H1N1 influenza A virus) do not activate endothelial cells. These findings are consistent with clinical studies showing widespread endotheliitis and organ damage in patients with severe COVID-19. In summary, this study delves into SARS-CoV-2-induced vascular lesions and coagulation disorders, suggesting that the FDA-approved lipid-lowering drug simvastatin may help prevent the pathogenesis of COVID-19 and improve patient outcomes.

The TLR2 antagonist (CU-CPT22) significantly inhibited NP-induced expression of ICAM-1 and VCAM-1 in human endothelial cells, suggesting that the N protein may bind to TLR2, triggering the activation of NF-κB and MAPK, thereby inducing endothelial cell activation (Figure 1a). CU-CPT22 dose-dependently inhibited NP-induced expression of ICAM-1 and VCAM-1 (Figure 1d). To further confirm that the N protein can activate TLR2, researchers treated wild-type 293T cells (non-TLR2 expressing) and TLR2-overexpressing 293T cells with or without the N protein. As shown in Figure 1e, the N protein did not induce phosphorylation of JNK and p38 in wild-type 293T cells. However, the N protein significantly induced phosphorylation of JNK and p38 in TLR2-overexpressing 293T cells. Finally, pretreatment with IKK, JNK, and p38 inhibitors completely blocked NP-induced expression of ICAM-1 and VCAM-1 (Figure 1a). These results indicate that the N protein activates endothelial cells through the TLR2-mediated NF-κB and MAPK signaling pathways.

Figure 1. N protein induced endothelial cell activation via the TLR2-mediated signaling pathway. (Qian Y, et al., 2021)