Mannose Binding Lectin (MBL) and MBL Associated Serine Protease-2 (MASP-2) Amplify SARS-CoV-2 Spike Protein Uptake and Epithelial Cell Injury during Cigarette Smoke Exposure

RationaleThe clinical syndrome associated with SARS-CoV2, known as COVID-19 is characterized by a spectrum of hypercoagulability and complement-mediated microvascular injury in severe but also in mild COVID-19 disease(1-4). Studies have demonstrated that lectin complement pathway (driven by MBL/MASP2 complex) is responsible for the complement-mediated injury via MBL binding of the SARS-CoV virion(5) and via deposition of MBL, MASP-2, and C4 seen in the skin and lung specimens of COVID-19 individuals, where SARS-CoV-2 spike protein (SP) co-localized with C4(4, 6). We hypothesize that in smokers, MBL binding to SARS-CoV-2 and MASP-2 cleavage of SP increase viral internalization with subsequent epithelial cell injury and in-situ complement activation.MethodsWe studied ACE-2 expression in lung homogenates of smokers (n=2), mild COPD (n=2), moderate and severe COPD (n=8) by western blotting and qPCR. We used A549 epithelial cells exposed to air control (AC) or CS (10%, 2h) and primary alveolar type 2 (AT2) from smokers and never-smokers to analyze ACE-2 expression by FACS and western blotting, and cell injury by western blotting before and after treatment with his-tagged SARS-CoV-2 SP (15ug/mL, 2h), recombinant human MBL (2ug/mL, 2h), and serum-derived MBL/MASP-2 complex (50% non-heat-inactivated serum). ResultsIn-vivo, CS increases ACE-2 expression in lung homogenates of smokers and COPD patients vs. healthy individuals (p<0.5). Ex-vivo, CS extract increases ACE-2 expression in A549 and AT2 epithelial cells as detected by FACS and western blotting. Moreover CS-exposed A549 epithelial cells demonstrate higher SP - ACE-2 co-localization, especially after treatment with recombinant MBL. In the presence of recombinant MBL and serum-derived MBL/MASP-2 complex we demonstrated higher co-localization of SP with MBL at the plasma membrane and higher expression of cell injury markers (RAGE, cPARP, and p62/LC3B2) of CS-exposed epithelial cells. Interestingly, transitional AT2 from smokers, expressing AT1 (Cav1) and AT2 (Muc1) markers, had the highest ACE-2 membrane expression by FACS vs. transitional AT2 from never-smokers, AT1, AT2 primary human cells. ConclusionsOur results indicate that MBL and MASP-2 of lectin pathway are linked to higher SARS-CoV-2 SP epithelial uptake and injury in smokers and COPD-ers with COVID-19 disease, suggesting that CS-induced airway inflammation and in-situ complement activation increase distal lung ACE-2 expression and AT2 injury significantly tallying airway injury in COVID-19.