key: cord-1034644-qdrptxsu authors: Zapatero-Belinchón, Francisco J.; Moeller, Rebecca; Lasswitz, Lisa; van Ham, Marco; Becker, Miriam; Brogden, Graham; Rosendal, Ebba; Bi, Wenjie; Carriquí-Madroñal, Belén; Islam, Koushikul; Lenman, Annasara; Gunesch, Antonia P.; Kirui, Jared; Pietschmann, Thomas; Överby, Anna K.; Jänsch, Lothar; Gerold, Gisa title: Fluvastatin mitigates SARS-CoV-2 infection in human lung cells date: 2021-11-18 journal: iScience DOI: 10.1016/j.isci.2021.103469 sha: e655b72f1239cc724bc94a3145a581af6ed2589f doc_id: 1034644 cord_uid: qdrptxsu Clinical data of patients suffering from COVID-19 indicates that statin therapy, used to treat hypercholesterolemia, is associated with a better disease outcome. Whether statins directly affect virus replication or influence the clinical outcome through modulation of immune responses is unknown. We therefore investigated the effect of statins on SARS-CoV-2 infection in human lung cells and found that only fluvastatin inhibited low and high pathogenic coronaviruses in vitro and ex vivo in a dose-dependent manner. Quantitative proteomics revealed that fluvastatin and other tested statins modulated the cholesterol synthesis pathway without altering innate antiviral immune responses in infected lung epithelial cells. However, fluvastatin treatment specifically downregulated proteins that modulate protein translation and viral replication. Collectively, these results support the notion that statin therapy poses no additional risk to individuals exposed to SARS-CoV-2 and that fluvastatin has a moderate beneficial effect on SARS-CoV-2 infection of human lung cells. 2020), a large, observational, multicenter study does not confirm this notion (Mitacchione et al., 2021) . 48 In addition, statins may modulate expression of the angiotensin-converting enzyme 2 (ACE2) 49 Results 78 To assess a possible direct impact of statins on coronavirus infection, we infected Huh7.5 cells 80 expressing a Firefly luciferase (Huh7.5 Fluc) with the low pathogenic human coronavirus 229E 81 (HCoV-229E) in presence of statins. At a concentration of 5 μM, statins reduced infection of HCoV-82 229E moderately between 10 -40%. This inhibition was most pronounced (40%) and statistically 83 significant for fluvastatin (Fig. 1a) . The most commonly prescribed statins simvastatin and atorvastatin 84 reduced HCoV-229E infection up to 10%. Although lovastatin, pravastatin and rosuvastatin reduced 85 HCoV-229E infection up to 15%, 25%, and 30%, respectively, the observations did not reach statistical major cytotoxic effects (Fig. 1b) . Dose-response analysis for rosuvastatin showed a 40% reduction of 91 HCoV-229E infection at 50 µM, which correlated with a decrease in cell viability (Fig. 1c) . We did not 92 observe a dose-dependent reduction of HCoV-229E infection upon simvastatin treatment (Fig. 1d) . 93 To investigate whether statins could modulate infection with highly pathogenic SARS-CoV-2, we 94 pretreated human respiratory epithelial cells with 10 µM fluvastatin, rosuvastatin or simvastatin and 95 infected the cells with SARS-CoV-2 (strain SARS-CoV-2/München-1.2/2020/984,p3) (Wölfel et al., 96 2020) . SARS-CoV-2 genome copy numbers significantly decreased to 60% in the supernatant upon 97 fluvastatin treatment, whereas rosuvastatin or simvastatin treatment did not reduce SARS-CoV-2 98 genome copy numbers (Fig. 1e) . In cell lysates of infected cells treated with fluvastatin, rosuvastatin or 99 simvastatin, a slight reduction of about 15% of SARS-CoV-2 genome copy numbers was detected, but 100 did not reach statistical significance (Fig. 1e) . As we detected a significant decrease in SARS-CoV-2 101 genome copy numbers in supernatants upon fluvastatin treatment, we titrated released infectious SARS-102 CoV-2 particles. Fluvastatin treatment reduced SARS-CoV-2 titers 4-fold compared to DMSO 103 treatment, as determined by TCID50 assay on Vero cells (Fig. 1f) . Collectively, our data highlight that 104 statins do not promote coronavirus infection in human cells as was speculated, but instead selected 105 statins attenuate coronavirus infection in vitro. 106 We next aimed to corroborate our findings for fluvastatin treatment in a model that closely resembles 109 the human respiratory tract. To that end, we differentiated human primary bronchial epithelial cells 110 We next immunostained the HBEC ALI cultures for differentiation markers of ciliated cells (acetylated 127 tubulin, Fig. 2c , yellow) and goblet cells (mucin5AC, Fig. 2c , red) in addition to SARS-CoV-2 128 nucleocapsid protein (SARS-CoV-2 NC) (Fig. 2c, green) , and acquired overview images of all samples 129 ( Fig. S1 ), as well as detailed confocal images of highly infected HBEC ALI cultures (donor #1 and #2; 130 . We observed differences in number of goblet and ciliated cells between the donors, which may 131 reflect donor-dependent cell type variation or slight differences in the differentiation. SARS-CoV-2 132 infection was mainly detected in ciliated cells as reported previously for ALI cultures (Ravindra et al., 133 2020), but also in unstained cell populations, that most possibly represent basal cells or differentiation 134 intermediates as reported for human lung tissues (Hui et al., 2020) . 135 To validate our findings, we quantified the intracellular infection levels by immunofluorescence staining 136 for SARS-CoV-2 NC on the HBEC ALI cultures at 72 h. Sample integrity was assessed at the same time 137 In summary, these results suggest that fluvastatin treatment has a moderate attenuating effect in SARS-146 CoV-2 infected three-dimensional epithelium cell culture models as seen in the monolayer system. 147 To determine the effects of fluvastatin, rosuvastatin and simvastatin on global cellular processes, (e.g. 149 cholesterol biosynthesis), we pretreated Calu-3 cells with either indicated statins or solvent control 150 followed by mock (uninfected) or SARS-CoV-2 infection. Subsequently, we analyzed cellular 151 proteomes by label free mass spectrometry (Fig. 3a) . We additionally incubated Calu-3 cells with heat-152 inactivated virus to control for changes in the cellular proteome induced by non-infectious particle 153 exposure. All experiments were performed in biological triplicates, generating quantitative information 154 of 27 proteomes in total. In line with our previous experiments, SARS-CoV-2 genome copies decreased 155 in supernatants of fluvastatin treated cells compared to DMSO treated cells (Fig. S2a ). In total, 3229 156 proteins were identified unambiguously by at least one unique tryptic peptide. Global inspection of our 157 proteomic data revealed four cholesterol biosynthesis pathway enzymes that were significantly more 158 abundant (either -log10(p-value) ≥ 2, or log2(fold change) ± 4 and -log10(p-value) ≥ 1.3) upon treatment 159 with fluvastatin, namely 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR), farnesyl-diphosphate 160 farnesyltransferase 1 (FDFT1), squalene monooxygenase (SQLE) and methylsterol monooxygenase 1 161 (MSMO1) (Fig. 3b, In addition to fluvastatin, our initial screen revealed a slight inhibition of HCoV-229E infection 303 by rosuvastatin, pravastatin, and lovastatin ( Fig. 1a) . However, the inhibitory activity of rosuvastatin, 304 the second strongest statin identified, correlated with drug-induced cellular cytotoxicity (Fig. 1c) . Future 305 research could aim to determine the IC50 and CC50 for additional potentially antiviral statins. Our study 306 did not include pitavastatin; a lipophilic, well tolerated statin with a variety of pleotropic effects 307 The authors declare no competing interests. were stained for SARS-CoV-2 nucleocapsid protein for infection (green), mucin5AC for secretory cells 469 (red) and acetylated tubulin for ciliated cells (yellow) and imaged on a scanning confocal microscope. Further information and requests for resources and reagents should be directed to and will be fulfilled 532 by the lead contact, Gisa Gerold (gisa.gerold@tiho-hannover.de). 533 This study did not generate new unique reagents. 535 All data reported in this paper will be shared by the lead contact upon request. 537 This paper does not report original code. 538 Any additional information required to reanalyze the data reported in this paper is available from the 539 lead contact upon request. 540 with 52 μg/ml bovine pituitary extract, 0.5 μg/ml hydrocortisone, 0.5 ng/ml human recombinant 556 epidermal growth factor, 0.5 μg/ml epinephrine, 10 μg/ml transferrin, 5 μg/ml insulin, 50 nM retinoic 557 acid (all from Sigma Aldrich) and PeSt (Danahay et al., 2002) . Cells were maintained submerged for 558 the first 7 days, after which the media was removed from the apical side and cells were grown at air-559 liquid interface for an additional two weeks to reach full differentiation. Media was replaced trice/week 560 with addition of fresh retinoic acid to the media shortly before usage. Differentiation of the HBEC were 561 assessed using light microscopy focusing on epithelial morphology, presence of ciliated cells, and mucus 562 production. Presence of ciliated cells and goblet cells was also determined with IF using antibodies 563 directed against acetylated-tubulin and muc5AC, respectively, as described under IF staining below. 564 2-yl]-2,5 diphenyltetrazolium bromide, 0.5 mg/mL in medium) was added to the cells. The reaction was 624 stopped with DMSO after 1 h and optical density was measured at 570 nm using a plate reader (Synergy 625 2, BioTek ® ). 626 Gothenburg. Viral copy numbers were determined by calibration against a reference standard provided 658 by Sven Reiche (FLI, Germany). A standard curve ranging from 1 to 1 × 10 6 genomes was run together 659 with the SARS CoV-2 samples. 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