The high transmissibility of Severe Acute Respiratory Syndrome–coronavirus 2 (SARS-CoV–2) is due, at least in part, to infectivity in lung type II alveolar cells1 SARS-CoV–2 triggers a wide range of disease phenotypes with severe acute respiratory distress syndrome (ARDS), including interstitial pneumonia2 and viral sepsis3. Here, we tested if the pro-homeostatic lipid mediators, the elovanoids (ELVs)4–7, would block the entrance of the spike (S) protein receptor-binding domain (RBD) that would prompt a protective response against SARS-CoV–2 infection.
Lung alveoli viral attachment through the S protein RBD to ACE2 is followed by proteolytic activation for fusion and viral cell entry8–10. We use human alveolar primary cell cultures (Fig. 1a, and Extended Data Fig. 1). Most of the cells are type II (oil red, specific marker, Fig. 1a, right panel, and Extended Data Fig. 1, bottom panels) and positive to Foxj1, HT2–280 antigen, and β-tubulin IV (Fig. 1a, right panel; Fig. 1g,h and Extended Data Fig. 1). Pneumocytes type II are also mobile, showing lamellae or filopodia positive to HT2–280, a specific type II (Fig. 1b). We exposed these cells to RBD (from S protein)-Alexa 594 for 24 hours. In parallel, we used Nucleocapsid protein N as a specificity control of RBD internalization. In 3D reconstructions of Z-stack images (Imaris software, Bitplane, UK), the lipophilic staining (cell mask) shows a dense membrane above the nuclear zone that is localized close to oil red (Fig. 1a,c i-viii).. A below view (Fig. 1c vi) shows that the RBD protein signal (red) passes through the membrane (white) to the intracellular space surrounding the nucleus (blue), and can also be seen in the above view (Fig. 1c vii,viii).. Herein, we demonstrate for the first time that RBD was shown to be internalized in SARS-CoV–2 since previous work have shown the same for SARS-CoV–111. When IL1β or TNFα was added, the internalized RBD signal was increased (Fig. 1d). In addition, Nucleocapsid (N) protein, a structural viral protein not involved in ACE2 and SARS-CoV–2 interaction12, is at the same level as the control with no protein added that accounted for autofluorescence. RBD was internalized at higher rates than N. In digitalized images, plotted vs. Z-axis in a Z-stack shows the differential position of the N protein versus the RBD with respect to the membrane level (Fig. 1c i-v,ix-xi and Fig. 1d). This observation documents that the N protein remains on the membrane and the extracellular side while the RBD spans intracellularly, passing through to the cytoplasm and demonstrating that RBD internalization is specific and dependent on IL1β and TNFα (Fig. 1d,e).
RBD-Alexa 594 internalization was decreased +/- IL1β + TNFα when ELV-N32 and ELV-N34 were added (Fig. 1f, upper panel). In addition, acetylenic ELV-N32 or ELV-N34 (Extended Data Fig. 2) showed a steep decrease in RBD protein internalization +/- IL1β (Fig. 1f, lower panel). Moreover, the addition of the ELVs precursors 32:6 or 34:6 reduces RBD located below the membrane, suggesting that the pneumocytes convert these precursors into ELVs and thus prevent RBD internalization (Fig. 1f, upper panel).
The reduction in RBD internalization is partially due to a decrease in ACE2 since acetylenic ELV-N32 or ELV-N34 decreases ACE2 mRNA and methyl-ester elovanoids also reduce the protein content in pneumocytes type II (Fig. 1g, plot, and Fig. 1i). In addition, ELV-N32 decreased TMPRSS2 expression (Fig. 1h) in the presence of IL1β (Fig. 1h, plot).
Since ELVs stimulate protective proteins expression in cells confronted with uncompensated oxidative stress5–7, we next explored if these lipids under conditions that downregulate canonical SARS-CoV–2 cell-entry mediators in pneumocytes will also activate protective proteins synthesis. We found that ELV heightens the expression of Sirtuin 1 (Fig. 1j), RNF146 (Extended Data Fig. 3a,b), PHB, Bcl-Xl, and Bcl2 (Extended Data Fig. 3c-e). These proteins are involved in pro-homeostatic cellular functions. Sirtuin 1 (Silent information regulator factor 2-related enzyme 1) is a NAD(+)-dependent deacetylase of histone and non-histone proteins and transcription factors, and its regulatory functions target inflammation, aging, mitochondrial biogenesis, and cellular senescence13. RNF146 is an E3 ubiquitin-protein ligase that degrades parsylated proteins, thus protecting cells from Parthanatos cell death14.PHB (prohibitin type I) functions comprise scaffolding mitochondrial protein, adaptor in membrane signaling, transcriptional co-regulator, and neuroprotection6. Bcl-XL and Bcll2 downregulate apoptosis and inflammasome formation15. Our data suggest that, in addition to halting the entrance of the RBD, ELVs in the lung curb cell-damaging/apoptotic events and thus sustains homeostasis by counteracting inflammation over-activation by the formation of protective proteins.
An evolving question prompted by our data is whether alveolar cells in culture can synthesize ELVs. Thus, we incubated human alveolar cells with the precursors VLC-PUFAs (32:6 or 34:6) and then analyzed the products by LC-MS/MS. Interestingly, we found that ELVs are in fact, formed. ELV-N32 was synthesized where the precursor 32:6 was added and not in cells exposed to 34:6. Inversely, ELV-N34 was found in the cultures were 34:6 was added and not in cells exposed to 32:6 (Fig. 2a,c). These results demonstrate that alveolar cells are endowed with pathways for the biosynthesis of ELV-N32 and ELV-N34 (Fig. 2b). We show MS fragmentation for stable derivatives of intermediaries (Fig. 2a-c) as well as of ELVs themselves (Fig. 2a). Moreover, we uncovered that ELVs were actively released from cells to the incubation media, indicating that they act both as autocrine and paracrine mediators.
Our findings contribute to broadening our understanding of the duality of ACE2 in lung function and diseases. In health, ACE2 fosters lung homeostasis by generating Ang-(1–7) and enhancing host defense that would counteract ACE2 virus-induced downregulation of proinflammatory signaling. Herein, we show that ELVs uncover another participant when RBD of the S protein binds to ACE2 and enters alveolar cells in culture. The ELVs are likely part of a fast and coordinated pro-homeostatic inflammatory downregulatory response. To be tested in the future is the prediction that delayed ELV-mediated protective responses would lead to severe lung and systemic inflammation. So direct virus triggered cell damage is critical, but also the activation of the induction of protective proteins. Is diet engaged in building precursors of ELVs in the lung? Diet has been shown to affect ACE2 expression16 and the supply to build ELV precursors7,17. This may contribute to explaining why some patients develop hyper-inflammatory/immune responses and severe disease, but others experience mild or even asymptomatic COVID–19. Questions that remain to be addressed include whether the expression of the protective proteins identified here in the alveoli are activated all at once? Are they coordinated with adaptive immune responses to limit virus spread? Are enzymes for ELV synthesis under tight transcriptional control so that the mediators are expressed at appropriate times and/or levels? To our knowledge, ELVs are the first protective mediators to be identified in the human alveoli confronted with the RBD of the S protein.
Additional research will be needed to elucidate the molecular mechanisms of ACE2 downregulation. Also, the use of the entire S protein instead of the RBD, as in our present study, will provide the connection between cell attachment and cell entrance, as affected by ELVs and VLC-PUFAs, since proteases expression is correlated with ACE2 downregulation. Moreover, the use of the intact virus would offer a direct demonstration of the significance of ELVs. Since the SARS-CoV–2 affects nasal mucosa, GI, the eye, and the nervous system exploring the protective potential of ELVs in other cell types would further expand the scope of our observations beyond the lung. Our results provide a foundation for future research and offer specific mediators for interventions to modify disease risk, progression, and protection of the lung from COVID–19 or other pathologies.