key: cord-0888952-evzft1lt
authors: Calandria, Jorgelina M.; Bhattacharjee, Surjyadipta; Maness, Nicholas J.; Kautzmann, Marie-Audrey I.; Asatryan, Aram; Gordon, William C.; Do, Khanh V.; Jun, Bokkyoo; Mukherjee, Pranab K.; Petasis, Nicos A.; Bazan, Nicolas G.
title: Elovanoids downregulate SARS-CoV-2 cell-entry, canonical mediators and enhance protective signaling in human alveolar cells
date: 2021-06-10
journal: Sci Rep
DOI: 10.1038/s41598-021-91794-z
sha: d2d8eb3e6e51b1011a219f8bfed5a43568696f4c
doc_id: 888952
cord_uid: evzft1lt

The pro-homeostatic lipid mediators elovanoids (ELVs) attenuate cell binding and entrance of the SARS-CoV-2 receptor-binding domain (RBD) as well as of the SARS-CoV-2 virus in human primary alveoli cells in culture. We uncovered that very-long-chain polyunsaturated fatty acid precursors (VLC-PUFA, n-3) activate ELV biosynthesis in lung cells. Both ELVs and their precursors reduce the binding to RBD. ELVs downregulate angiotensin-converting enzyme 2 (ACE2) and enhance the expression of a set of protective proteins hindering cell surface virus binding and upregulating defensive proteins against lung damage. In addition, ELVs and their precursors decreased the signal of spike (S) protein found in SARS-CoV-2 infected cells, suggesting that the lipids curb viral infection. These findings open avenues for potential preventive and disease-modifiable therapeutic approaches for COVID-19.

www.nature.com/scientificreports/ and as ELVs are characterized as pro-homeostatic lipids, we hypothesized that ELVs would block the proinflammatory effect on RBD entrance. 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 protein expression in cells confronted with uncompensated oxidative stress 5-7 , 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 senescence 13 . RNF146 is an E3 ubiquitin-protein ligase that degrades parsylated proteins, thus protecting cells from Parthanatos cell death 14 . PHB (prohibitin type I) functions comprise scaffolding mitochondrial protein, adaptor in membrane signaling, transcriptional co-regulator, and neuroprotection 6 . Bcl-XL and Bcl2 downregulate apoptosis and inflammasome formation 15 . 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. The addition of ELVs and their precursors to the human lung cells in culture for extended periods of time (Extended Data Video 1) did not affect cell survival, in agreement with the fact that the cell possesses the molecular machinery to synthesize them, and thus they are also likely able to degrade them. in the alveolar culture. Pneumocytes type I are reactive to HT1-53 (green), and pneumocytes type II are positive for oil red (red), a marker for the type II lipid/lamellar bodies and for ciliated cell marker Foxj1 (green) which is required for cilia formation and is an early marker of epithelial cell differentiation, recovery, and function. (b) Pneumocytes type II stained positive to HT2-280 labeled (green) type II human lung cells. (c-e) Differential entrance of S protein vs. N in human alveolar cells in culture. Z-stack shows signal distribution (upper panel), and in mobile cells undergoing cell division (lower panel). (c) XZ planes of a Z-stack showing white: membrane, red: RBD tagged with Alexa Fluor 594 and blue nuclei. i through v depict S protein view from XZ plane digitalized of the IMARIS image; vi to viii view from below, above and digitalized showing the internalized protein (white arrows); ix to xi XZ plane depicting the labeled N protein that remains in the surface or within the plasma membrane. Viral Nucleocapsid N protein was tagged with Alexa Fluor-594. XZ plane of a Z-stack image of alveoli culture after 24 h exposure to N protein. 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 expression 16 and the supply to build ELV precursors 7, 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.

To determine the effect of the lipids in viral infection to the alveolar cells, we exposed them to the SARS-CoV-2 to record impedance at confluence, taking images every hour for visual confirmation. We observed no changes in impedance when comparing no-virus exposure with two different Median Tissue Culture Infectious Dose (TCID50), 1000 and 10,000. The time/image composites (depicted in Extended Data Video 2) show that as soon as infected cells are lifted, the neighboring cells fill the gap resulting in no difference in impedance. In light of this observation, we infected human alveolar cells with SARS-CoV-2 for 3 days and immunostained them using anti-Spike protein (Fig. 3a) . The infection resulted in an intracellular Spike protein signal (Fig. 3b) . The addition of ELV-N32 methyl-ester, ELV-N34 sodium salt, and both acetylenic ELVs and their precursors, VLC-PUFAs 32:6 and 34:6, remarkably decreased the signal of S protein in the cells, suggesting that the lipids prevented the viral infection of the alveolar cells. The VLC-PUFAs may exert this protective effect by the synthesis of ELVs or by modifying the cell surface membrane lipidome remodeling that would disrupt tetraspaninmembrane microdomains 18, 19 to contribute to blocking SARS-CoV-2 virus cell binding and entrance, and in addition, perturb endosome formation hindering virus replication. www.nature.com/scientificreports/ Additional research will be needed to elucidate the molecular mechanisms of ACE2 downregulation. 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.

We have used primary cell cultures of human alveoli, which consist of a mixture of ciliated cells, club cells, type I pneumocytes, and type II pneumocytes (PromoCell, HSAEpC). We have characterized the histology and immunocytochemistry in these primary cultures (Fig. 1a,b,g,h) . We performed all the experiments in 48 wells with passage 4 cells seeded at 15,000 cells per cm 2 density. The cells were incubated to confluency and maintained in the proprietary Medium provided by Promocell with the addition of Pen/Strep and exposed to 0.5 μg of tagged protein per well for 24 h in the presence or absence of 10 ng/ml IL1β (PeProTech Inc., Rocky Hill, NJ Cat# 200-01B) and/or 10 ng/ml TNFα (Cell Sciences Inc., Newburyport MA. Cat# CRH520B). After this period, cells were incubated 10 min with 1/1000 cell mask (Thermo Scientific cat# C37608) medium and fixed using PFA 4%. After fixation, nuclei were stained with 10 μg/ml Hoechst 33342 (Thermo Scientific cat#H3570). To characterize the cell types in culture after fixation, we performed immunocytochemistry using the following primary antibodies: HT1-53, a marker of pneumocytes type I, and HT2-280 (Terrace Biotech cat#HT1-53 and HT2-280); Foxj1 (Santa Cruz Biotech, sc-53139) and β-Tubulin IV marker of pneumocytes type II (Abcam cat# ab179509). ACE2 (Santa Cruz Biotech, sc-390851) and TMPRSS2 (Abcam cat #Ab109131) were used for Immunostain the two mentioned proteins in cell culture.

ACE2 protein abundance using Jess technology. The western assay was performed using a Jess Protein Simple system (San Jose, CA, USA) following the manufacturer's protocol. Briefly, samples were lysed with RIPA buffer containing a protease inhibitor cocktail (Sigma, Cat. P8340). Soluble protein concentration was determined by BCA assay (Thermo Fisher Scientific, Cat. 23225) and 0.4 µg used/reaction. Samples were heated at 95 °C/5 min, and 3 µL of each sample were loaded. The 12-230 kDa cartridge (Protein Simple-#SM-W004) was used. Primary antibodies were diluted in antibody diluent 2 buffer (Protein Simple, #042-203), and the working solution of secondary antibodies was provided by the company (Protein Simple, #042-206). For data analysis, the area of spectra that matched the molecular weight of the target protein was used (Fig. 1j) . We used the anti-ACE2 antibody from Abcam (cat# ab108252) in a concentration of 1 μg/ml. The standardization was performed using total protein stain and using an anti-GAPDH antibody from Santa Cruz Biotech (cat# Sc-25778).

Quantification of RNF-146. Western blot was performed from human primary alveolar cells, as described in Calandria et al., 2015 (18) . Briefly, cell lysates were produced using RIPA buffer supplemented with protease inhibitor cocktail (Sigma, cat# P8340.St Louis MO). Total protein (30 mg) was mixed with Laemmli buffer containing DTT and loaded in Novex 4-12% precast gels and ran in X-Cell running system at 120 V for 1.5 h. The transference was performed using the Trans Blot Turbo dry transferring system (Bio-Rad, Hercules CA) on low fluorescent background PVDF membranes (GE Healthcare, Piscataway NJ). Membranes were incubated with the corresponding primary antibodies overnight. Primary antibodies used RNF-146 (UC Davis/NIH-Neuromab Lab Facility, cat #75-233) and GAPDH (Satnta Cruz Biotech Cat# sc-47724). After this period, the membranes were incubated with fluorescent-tagged secondary antibodies (GE healthcare, cat# PA45011) for 1 h and imaged. Data was acquired using ChemiDoc MP (Biorad). Densitometric analysis was performed using ImageLab 6.0.1. (Biorad).

SARS-CoV-2, S1 Subunit Protein (RBD), and Recombinant SARS-CoV-2 Nucleocapsid Protein from Raybiotech (Peachtree Corners GA, cat# 230-30162-1000 and 230-30164-500 correspondingly). The proteins were labeled using Alexa Fluor™ 546 Protein Labeling Kit from Thermo Scientific (cat# A10237) following manufacturer directions except for the RBD that was purified from the dye with Amicon-Ultra 10K cutoff filters (Merck, Millipore cat#UFC201024) instead of the column provided by the kit has a restrictive MW of 50KD (the recombinant RBD protein was 25KDa). The recovery of the protein and labeling efficiency was measured using nanodrop and was about 80% recovery and 0.02 dye molecules per aminoacid. We added 0.5 μg per well of protein.

of Alexa 594-conjugated RBD domain belonging to the SARS-CoV-2 virus Spike protein (Raybiotech. Cat. 230-30162-1000) was incubated with human alveolar cells for 24 h. After this period, Cell Mask (Thermofisher Scientific, cat#C37608) was added to a final concentration of 1 in 1000 and left in the incubator for 10 min. The cells were then fixed with 4% Paraformaldehyde in PBS, washed three times, and Nucblue (Thermofisher Scientific cat# R37606) was added for nuclear staining. The images were taken using an Olympus FluoView 3000 laser confocal microscope as z-stacks with a fixed step size of 1.6 μm. The cero was registered for each well specifying − 30 and 30 μm as the lower and upper limit in the Z-axes. Using the Z-drift compensation system, 9 blind positions were set up per well, and the image acquisition was performed automatically. The pictures were processed using Imaris 9.5.1 software (Bitplane) to render the 3D image and assess the position of the different surfaces (elements rendered with the surface function) along the Z-axes. The portion of the tagged protein that was internalized was 

The human primary alveolar cells were preincubated with 1 uM of ELV-32 Me, ELV-34 Na, ELV-32 AC, ELV-34 AC or their precursors VLC-PUFA 32:6 or 34:6 for 24 h before infection. To infect primary culture cells in chamber slides, a stock of SARS-CoV-2 (USA-WA1/2020 isolate, accession MN985325) was diluted to the appropriate concentration using serum-free DMEM and overlaid on the cells. Briefly, 10,000 TCID50 of virus was added at a volume of 100ul per well and allowed to incubate for 1 h at 37C, with gentle rocking every 15 min. After one hour, the inoculum was removed and replaced with media containing a fresh batch of the indicated lipids. Slides were returned to the incubator and monitored for 3 days. After this period, the media was removed and replaced with 2% paraformaldehyde and left overnight at room temperature to fix the cells and inactivate the virus, facilitating the transfer of the slides from BSL3 to BSL2. The next day, the PFA was replaced with PBS, and the cells were immunostained using antibody anti-Spike protein from Abcam (cat#ab273433), counterstained, and imaged as described above. eSight assay. To measure the effects of the lipids on SARS-CoV-2 infection, we used real-time cell analysis (RTCA) to measure impedance with the Agilent eSight system. The day before the assay was initiated, the alveolar cells were at confluence and exposed to 1 uM of ELV-32 Me, ELV-34 Na, ELV-32 AC, ELV-34 AC or their precursors, VLC-PUFA 32:6 or 34:6, followed by incubation in Agilent e-plates. The virus was incubated with the cells at 37C for 1 h. After that, the virus was removed, and the medium was replaced by lipid-containing media. The plates were loaded on the eSight instrument. Impedance was measured every 15 min, and an image was taken every hour for 125 total hrs. The data was analyzed using Agilent RTCA Pro software. 

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The authors are grateful to an Institutional Grant from the School of Medicine, LSUHSC (to N.G.B.). 

The authors declare no competing interests.

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