Fig 1.
SARS-CoV-2 infection modulates the lipid metabolism in human monocytes.
(A and C) LDs were captured by fluorescent microscopy after Oil Red O staining (Red) and nuclei stained with DAPI (Blue). (A) Representative images of monocytes from COVID-19 patients and health volunteers. (C) Representative images of human monocytes obtained from PBMC infected by SARS-CoV-2 with MOI of 0.01 for 24 hours. Scale bar 20μm. (B and D) LDs were evaluated by ImageJ software analysis by the measurement of the fluorescent area. (E) Representative scheme of the increase of proteins associated with lipid metabolism by SARS-CoV-2 infection in monocyte can regulate the lipid droplet formation. (F) Monocytes were infected by SARS-CoV-2 with MOI of 0.01 during 24h. Cell lysates were collected for the detection of CD36, PPAR-γ, SREBP-1, DGAT-1 by Western blotting. β-actin levels were used for control of protein loading. (G) Densitometric evaluation of data of panel 1F. Data are expressed as mean ± SEM of five healthy volunteers (HV) and six COVID-19 patients for ex vivo experiments and three healthy donors for LDs staining and western blot. *p < 0.05 versus health volunteers or uninfected cells.
Fig 2.
The A922500 inhibits lipid droplet biogenesis induced by SARS-CoV-2 in human pulmonary cells and monocytes.
Human pulmonary cell (A549 cell line) and monocytes were pre-treated with DGAT-1 inhibitor A922500 for 2 hours before the infection with SARS-CoV-2 at MOI of 0.01 during 24h in monocytes and 48h in A549 cell line in the presence of the inhibitor. (A) LDs were captured by fluorescent microscopy after Oil Red O staining (Red) and nuclei stained with DAPI (Blue). Scale bar 20μm. (B and C) LDs were evaluated by ImageJ software analysis by the measurement of the fluorescent area of LDs from (B) A549 cells and (C) human monocytes pre-treated with A922500 using different concentrations (0.1, 1 and 10μM). Data are expressed as mean ± SEM obtained in four independent experiments and three independent donors. *p < 0.05 versus uninfected cells and #p < 0.05 versus infected cells.
Fig 3.
DGAT-1 Inhibitor A922500 decreases the pro-inflammatory profile and cell death induced by SARS-CoV-2 infection and reduces the viral load in human monocyte.
Monocytes were pre-treated with DGAT-1 inhibitor A922500 in different concentrations (0.1, 1 and 10μM) for 2 hours before the infection with SARS-CoV-2 with MOI of 0.01 during 24h in presence of the inhibitor. (A) Cell death was measured in the supernatant by LDH activity fold change in relation to the uninfected cell. (B) Viral load by qPCR. Monocytes of each sample were counted for normalization. (C) Images of phase contrast from monocytes. Scale bar 20μm. (D-F) The inflammatory cytokines were measured in supernatants by ELISA (D) leukotrienes: CysLT and LTB4, (E) chemokines: IL-8 and CXCL10, (F) inflammatory cytokines: IL-6, TNF-α and IL-10. Data are expressed as mean ± SEM obtained in three independent donors for viral replication, leukotrienes and the group A922500 alone, and seven independent donors for the other groups. * p <0.05 versus uninfected cells and #p <0.05 versus infected cells.
Fig 4.
Lipid droplets are necessary for SARS-CoV-2 replication in VERO E6.
VERO E6 were pre-treated with DGAT-1 inhibitor A922500 with different concentrations (0.1, 1, 10 and 50μM) for 2 hours before the infection with SARS-CoV-2 with MOI of 0.01 for 24h in presence of the inhibitor. (A) Viral replication was determined by Plaque assay. (B) Representative Plaque assay. (C-E) Immunofluorescence analyses of VERO E6 after SARS-CoV-2 infection with MOI of 0.01 for 48h. (C) The virus was detected by indirect immunofluorescence using convalescent donor serum (Red or white) or (E) the double strain RNA was detected by indirect immunofluorescence by J2 antibody (Red), the lipid droplets were stained with BODIPY 493/503 (Green) and nuclei stained with DAPI (Blue). (C’ and E’) Representative zoom images. Data are expressed of four independent experiments for SARS-CoV-2 replication and three for immunofluorescent analyses. #p <0.05 versus infected cells. Scale bar 20μm.
Fig 5.
SARS-CoV-2 is associated with lipid droplet in Vero E6.
Ultrastructural analyses by transmission electron microscopy of (A) uninfected Vero E6 Cell and (B-F) Vero E6 cells 48 hours post infection by SARS-CoV-2 (MOI 0.01). Arrows (virus particles), lipid droplet (LD), mitochondria (M), nucleus (N).