Simvastatin Downregulates the SARS-CoV-2-Induced Inflammatory Response and Impairs Viral Infection Through Disruption of Lipid Rafts

Abstract

Coronavirus disease 2019 (COVID-19) is currently a worldwide emergency caused by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). In observational clinical studies, statins have been identified as beneficial to hospitalized patients with COVID-19. However, experimental evidence of underlying statins protection against SARS-CoV-2 remains elusive. Here we reported for the first-time experimental evidence of the protective effects of simvastatin treatment both in vitro and in vivo. We found that treatment with simvastatin significantly reduced the viral replication and lung damage in vivo, delaying SARS-CoV-2-associated physiopathology and mortality in the K18-hACE2-transgenic mice model. Moreover, simvastatin also downregulated the inflammation triggered by SARS-CoV-2 infection in pulmonary tissue and in human neutrophils, peripheral blood monocytes, and lung epithelial Calu-3 cells in vitro, showing its potential to modulate the inflammatory response both at the site of infection and systemically. Additionally, we also observed that simvastatin affected the course of SARS-CoV-2 infection through displacing ACE2 on cell membrane lipid rafts. In conclusion, our results show that simvastatin exhibits early protective effects on SARS-CoV-2 infection by inhibiting virus cell entry and inflammatory cytokine production, through mechanisms at least in part dependent on lipid rafts disruption.

Keywords: COVID-19; SARS-CoV-2; inflammation; lipid rafts; statin.

Figure 1

Effects of simvastatin in physiopathology of SARS-CoV-2 infection in K18-hACE2-transgenic mice. (A–C) K18-hACE2-transgenic mice were initially pretreated with 20 mg/kg of simvastatin 24h and 1h before infection by SARS-CoV-2 gamma strain. After infection, these animals continued to receive treatment daily with 20 mg/kg of simvastatin for six days post-infection. (A) SARS-CoV-2 RNA levels were measured in lungs from mice infected with SARS-CoV-2 treated with Vehicle (Veh) or simvastatin (SimV). (B) Lung damage was evaluated by measuring LDH release in the bronchoalveolar lavage (BAL) from mice infected with SARS-CoV-2 treated with Vehicle (Veh) or simvastatin (SimV). (C) Microphotographs for histology of lung lobe, bronchiole, and alveoli samples from mice infected with SARS-CoV-2 treated with Vehicle (Veh) or simvastatin (SimV). Data are expressed as median with interquartile range; Experiments were performed with 5 mice/group. (D, E) K18-hACE2-transgenic mice were initially pre-treated with 20 mg/kg of simvastatin 24h and 1h before infection by SARS-CoV-2 gamma strain. After infection, these animals continued to receive treatment daily with 20 mg/kg of simvastatin for eleven days post-infection. Weight variation (D), clinical score (E), and Survival (F) were assessed during all days after infection. Weight variation and clinical score data are expressed as means ± SD; Teste t de Student or One-way ANOVA with Dunnett’s post-hoc test, as recommended. *p < 0.05 in comparison to mock, #p < 0.05 comparison to infected untreated (Vehicle - Veh). Survival was statistically assessed by Log-rank (Mantel-Cox) test. (Mock n=5, SARS-CoV-2 + VEH n =8, SARS-CoV-2 + SIMV n = 10).

Figure 2

Effects of simvastatin in inflammatory induced by SARS-CoV-2 in murine lung. K18-hACE2-transgenic mice was initially pre-treated with 20 mg/kg of simvastatin 24h and 1h before infection by SARS-CoV-2 gamma strain. After infection, these animals continued to receive treatment daily with 20 mg/kg of simvastatin for six days post-infection. (A) Total and differential cell counts in bronchoalveolar lavage (BAL) were represented as number of differential cell counts. Total: leukocytes total, Mono: mononuclear leukocytes (Mono), PMN: polymorphonuclear leukocytes. (B) Myeloperoxidase (MPO) activity. The MPO activity in lung tissues were measured by the enzymatic assay. The level of CCL2/MCP1 (C), CCL5/RANTES (D), CXCL1/KC (E), TNF (F), and IL-6 (G) were measured by ELISA. Data are expressed as means ± SEM; One-way ANOVA with Dunnett’s post-hoc test. *p < 0.05, **p < 0.01, ***p < 0.01 in comparison to mock, #p < 0.05 comparison to infected untreated (Vehicle - Veh). Experiments were performed with 3-5 mice/group.

Figure 3

Effects of simvastatin on human neutrophils exposed to SARS-CoV-2. Human neutrophils were treated with simvastatin (10 μM) for 1 h before exposure to inactivated-SARS-CoV-2 (MOI of 0.1 for 3 h) or to PMA (100 nM) for 3 h. (A) Fluorescence analyses of NET after inactivated-SARS-CoV-2 estimulantion (MOI of 0.1) for 3h. NETs were stained with DAPI. Scale bar: 50 μm (B–F) Supernatants were then collected and centrifuged to remove residual neutrophils, and (B) NETs were quantified by Quant-iT PicoGreen. (C) ROS production was evaluated by using DHR (Dihydrorhodamine 1, 2, 3) probe (D–F). Cytokines and chemokines were quantified in the supernatants containing NETs by ELISA. Data are expressed as means ± SD; *, comparison to mock; #, comparison to infected untreated; One-way ANOVA with Dunnett’s post-hoc test; n=5-6.

Figure 4

Effects of simvastatin on human monocytes infected by SARS-CoV-2. Monocytes from healthy donors were pre-treated or not with simvastatin (10 µM) then infected for 1 hour by SARS-CoV-2 h (MOI of 0.01). After removal of the virus inoculum, simvastatin was added again to the pre-treated group and added to infected cells as a posttreatment, at same concentration, for 24 hours (A) (The image was created with BioRender.com). The levels of TNF-α (B), IL-8 (C), IL-6 (D) and INF-α (E) were measured in culture supernatants by ELISA. (F) Cellular viability was analyzed by measuring LDH release in the supernatants of infected cells pre-treated or posttreated with simvastatin compared to the supernatant of uninfected cells or nontreated SARS-CoV-2-infected cells. Data are expressed as means ± SEM; #, comparison to mock; *, comparison to infected untreated; One-way ANOVA with Dunnett’s post-hoc test; n=3-6.

Figure 5

Effects of simvastatin on SARS-CoV-2 induced cytokine production in Calu-3. Calu-3 cells pre-treated or not with simvastatin were infected by SARS-CoV-2 with a MOI of 0.01 for 1 hours. After that, the viral inoculum was removed and simvastatin was replaced both in pretreatment and posttreatment group, for 48 hours. The levels of IL-6 (A, D), IL-8 (B, E) and TNF-α (C, F) were measured in culture supernatants by ELISA. Data represent mean ± SD; #, comparison to Mock; *, comparison to infected untreated; One-way ANOVA with Dunnett’s post-hoc test. n=4.

Figure 6

Effects of simvastatin on SARS-CoV-2 infection in Calu-3. Calu-3 were pre-treated with different concentrations of simvastatin (0.01, 0.1, 1, 10, 25 and 50 μM) for 24 hours before the infection with SARS-CoV-2 (MOI of 0.01 for 1h, in the absence of the drug). After infection, simvastatin was added again for additional 48 hours of incubation. Posttreatment refers to simvastatin treatment for 48 hour after infection only. Viral replication was determined in the cell-free supernatant by plaque assay. (A, B) Viral replication in cells (A) pre-treated or (B) posttreated only with simvastatin. (C, D) Cellular viability was analyzed by measuring LDH release in the supernatants of infected cells pre-treated (C) or posttreated (D) with simvastatin compared to the supernatant of uninfected cells or nontreated SARS-CoV-2-infected cells. Data are expressed a mean ± SD; #, comparison to Mock; *, comparison to infected untreated; One-way ANOVA with Dunnett`s post-hoc test. n=4.

Figure 7

Simvastatin affects the expression level and location of ACE2, as well as adsorption and entry of SARS-CoV-2. Calu-3 were pre-treated or not with simvastatin 25 µM for 24h and then infected by SARS-CoV-2 with MOI of 0.1 at 4°C for 1h for viral adsorption assay (A) and at 4°C for 1h plus 1h at 37°C for viral internalization assay (B). (C) Calu-3 pre-treated or not with simvastatin were infected by SARS-CoV-2 with a MOI of 0.01 for 1 hours. Thereafter, the inoculum was removed, and simvastatin were added again both in pretreatment and posttreatment group, for 24-hour incubation. Cell lysates were collected for the detection of ACE2 and TMPRSS2 by Western blotting. GAPDH levels were used for control of protein loading. (D) Immunofluorescence analyses of Calu-3 after SARS-CoV-2 infection with MOI of 0.01 for 48h. ACE2 was detected by indirect immunofluorescence (Green), nuclei were stained with DAPI (Blue). Scale bar: 20 µm. (E) Calu-3 cells treated or not for 24h with 25 mM simvastatin were lysed in cold Triton X-100-containing buffer and solubilized proteins were separated by ultracentrifugation on discontinuous sucrose density gradients. Detection of proteins was determined by Western blotting with specific antibodies. Caveolin-1 and flotillin-1 were used as markers of detergent-insoluble fractions. Data are expressed as mean ± SD; *, comparison to infected untreated, Paired t-test. n=3-4.

Figure 8

Simvastatin have prophylactic effect against COVID-19. Simvastatin pretreatment significantly reduced the viral replication and lung damage in vivo, delaying SARS-CoV-2-associated physiopathology and mortality in the K18-hACE2-transgenic mice model. In vitro models, Simvastatin treatment downregulated key pro-inflammatory cytokines triggered by SARS-CoV-2 infection in human neutrophils, peripheral blood monocytes and lung epithelial Calu-3 cells, showing the potential to modulate the inflammatory response both at the site of infection and systemically. Additionally, Simvastatin pretreatment affects the course of SARS-CoV-2 infection by inhibiting virus entry through mechanisms of ACE2 displacement of rafts. The image was created with BioRender.com.