Nicotine and cotinine enhance SARS-CoV-2 entry through distinct but complementary mechanisms in human respiratory epithelial cells.

Previous epidemiological studies have shown that E-cigarette and tobacco users are more likely to develop COVID-19 symptoms than non-users. To investigate the underlying mechanisms, we examined the effects of nicotine, the major component of tobacco and E-cigarette, and its major metabolite, cotinine, on the susceptibility of human respiratory epithelial cells to SARS-CoV-2 infection. We found that pre-treatment with nicotine and cotinine significantly and additively enhanced viral infection. While nicotine increased the expression of the viral receptor ACE2 and the serine protease TMPRSS2, cotinine upregulated cysteine protease cathepsin B and promoted viral spike protein cleavage. These findings suggest that nicotine and cotinine enhance SARS-CoV-2 infection at the cell entry stage through distinct mechanisms. Using a SARS-CoV-2 pseudovirus system, we further investigated the effects and mechanisms of nicotine and cotinine on viral entry. We found that both compounds enhanced pseudovirus infection, but with different time courses. Nicotine's effect correlated with the upregulation of ACE2 and TMPRSS2, whereas cotinine's effect corresponded with increased cathepsin B and viral spike protein cleavage. The cathepsin B inhibitor E64d completely abolished cotinine-enhanced viral spike protein cleavage and viral entry. In contrast, ACE2 and TMPRSS2 inhibitors (chloromethylketone and camostat) had limited effects on viral spike protein cleavage and only partially reduced the viral entry enhancement induced by nicotine and cotinine. These results indicate that nicotine promotes virus-receptor binding and cell entry, while cotinine facilitates viral entry through cathepsin B-mediated spike protein cleavage.IMPORTANCEThis study highlights the potential risks of tobacco and E-cigarette use in increasing susceptibility to COVID-19. We show that the major neurostimulant in tobacco and E-cigarette, nicotine, and its metabolite, cotinine, additively enhance SARS-CoV-2 infection in human respiratory epithelial cells by promoting viral entry. Specifically, nicotine and cotinine upregulate the expression of distinct, yet complementary sets of key cellular proteins required for viral entry. These findings suggest that both tobacco smoking and E-cigarette vaping may exacerbate COVID-19 infection rates and severity and provide new insights into how nicotine and cotinine contribute to viral susceptibility. This research underscores the need for public health measures to address the heightened risk posed by tobacco smoking and E-cigarette vaping to encounter the SARS-CoV-2 infection.