Résumé
Background: There is a paucity of therapies for acute lung injury (ALI) induced by respiratory viruses. A previously demonstrated key mechanism of ALI, particularly in the setting of severe acute respiratory syndrome coronavirus infections, has been ascribed to decreased cell surface angiotensin converting enzyme 2 (ACE2) leading to increased circulating levels of angiotensin II (Ang2). In turn, supraphysiological Ang2 levels trigger a cascade of events that culminates with endothelial injury in the systemic circulation via acid sphingomyelinase (ASMase) activation. ASMase has been implicated in several models of ALI, but its specific involvement in Ang2-induced ALI is unknown. ASMase hydrolyzes sphingomyelin to pro-apoptotic, edemagenic ceramide, which can be metabolized to endothelial-protective sphingosine-1-phosphate (S1P). Therefore, the ratio of ceramide/S1P can determine endothelial cell fate and lung vascular permeability. We hypothesized that ceramide levels are increased relative to S1P in mice with Ang2-induced ALI. Methods: Following a published protocol of Ang2-induced ALI (Wu et al, 2017), we delivered Ang2 via osmotic pumps (1 ug/kg/min, 7 days;Ang2-mice), using saline (sham) or untreated C57BL/6 mice as controls. We evaluated pulmonary function (FlexiVent);albumin, IgM (ELISA), and inflammatory cell abundance in bronchoalveolar lavage fluid (BALF);and lung parenchyma inflammation and fibrosis (Ashcroft score) on H/E-stained lungs. Sphingolipid levels in lungs and plasma were measured by tandem liquid chromatography/mass spectrometry. Results: Inspiratory capacity, lung compliance, and body weight all decreased in Ang2-mice (by 13-14%, p<0.05 each) compared to sham. Lung pressure-volume loops exhibited a right-shift in Ang2- vs. sham or untreated mice. There was no significant change in BALF albumin, IgM, or inflammatory cells, or in lung histology inflammation or fibrosis scores in Ang2-mice. Compared to sham, S1P levels were significantly increased in plasma and unlavaged lung in Ang2-mice, decreasing ceramide/S1P ratios (from 3.1 to 2.0, and 26 to 20, respectively, p<0.05 each). Conclusions: Sustained subacute systemic elevations of Ang2 increased lung stiffness, but did not cause severe ALI in mice. Lung and circulatory elevations of S1P but not ceramide may have protected against lung edema and inflammatory injury. Although the cause of increased lung stiffness in this model remains to be elucidated, it is notable that chronic (months) supraphysiological elevations of either Ang2 or S1P have been associated with lung fibrosis. In conclusion, a second-hit injury may be necessary to augment the susceptibility of murine lung to Ang2-induced endothelial damage and inflammation relevant to coronavirus.
Résumé
Rationale. Coronavirus disease 2019 (COVID-19), caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is the third leading cause of death in the United States. While many risk factors for severe COVID-19 are emerging, the effects by which other inhalational exposures affect susceptibility are not well defined. Patients with COVID-19 demonstrate high rates of co-infection with respiratory viruses, including influenza A (IAV). When infected with IAV, human small airway epithelial cells (SAEC) exhibit increased abundance of angiotensin-converting enzyme 2 (ACE2), the primary receptor for SARS-CoV-2. However, it remains unknown if this effect increases the risk for COVID-19. Similarly, there are conflicting reports of the effect of e-cigarette (E-cig) vaping on COVID-19 manifestations. We hypothesized that exposures to IAV or E-cig increase the severity of SARS-CoV-2 infection. Methods. Golden Syrian hamsters (male and female) were exposed to E-cig vapor via nebulization for 5d. IAV was administered intranasally once on day 6 (A/California/07/2009 H1N1, 106 PFU/hamster). On day 3 post-IAV infection, SARSCoV- 2 was administered intranasally (WA01;104 PFU/hamster). On day 7 post-SARS-CoV-2 infection animals were sacrificed, bronchoalveolar lavage fluid (BALF) cell differentials were obtained, and inflated lung sections were stained and scored for immunohistology. Lung RNA was quantified for ACE2, TMPRSS2, STAT1, CXCL10, IFN-gamma, gene expression using RT-qPCR. Results: SARS-CoV-2 infection caused progressive weight loss that was less pronounced in animals pre-infected with IAV. SARS-CoV-2 titers from nasal swabs peaked at day 2 in both groups. IAV pre-infection reduced PMN and eosinophils in the BALF, and the overall inflammatory cell infiltration in the lung parenchyma of SARS-CoV-2-infected animals. IAV pre-infection reduced lung levels of STAT1, CXCL10 (2.5-fold;p<0.01), CCL5, and IFN-gamma in SARS-CoV-2-infected animals compared to animals that were only infected with SARS-CoV-2. Pre-exposure to E-cig worsened the SARS-CoV-2-induced weight loss in female animals only. E-cig pre-exposure increased lymphocytes and decreased PMN and eosinophils in the BALF compared to animals that were only infected with SARS-CoV-2. E-cig pre-exposure increased lung levels of STAT1, CXCL10 (2.5-fold;p<0.05), CCL5, and IFN-gamma in SARS-CoV-2-infected animals compared to animals that were only infected with SARS-CoV-2. Conclusion: Pre-infection with IAV resulted in decreased inflammatory response to SARS-CoV-2 infection. In contrast, pre-exposure to E-cig vaping increased the severity of the inflammatory response to SARS-CoV-2 with notable differences between sexes. Whereas anti-viral priming effects of prior viral infection are well described, the mechanisms that explain the worsening effects of E-cig on SARS-CoV-2 outcomes remain unknown.
Résumé
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.