SARS-CoV-2-infected adipocytes drive adipose inflammation and hepatocyte lipid accumulation

Figures

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  Figure 1. SARS-CoV-2 triggers adipocyte hypertrophy with a proinflammatory phenotype. (A) Schematic representation of the experimental timeline. Cell-surface ACE2 expression was measured in adipocytes. Adipocytes were infected with SARS-CoV-2, and viral replication was assessed at 1, 2, and 3 DPI by RT-qPCR and viral titration. At 2 DPI, IL-6 levels were measured in culture supernatants by ELISA, FASN and ATGL expression were quantified by RT-qPCR, and triglyceride and glycerol levels were measured in cell lysates and culture supernatants, respectively. (B) Kinetics of SARS-CoV-2 (Wh) replication in adipocytes infected at a multiplicity of infection (MOI) of 0.01. SARS-CoV-2 RNA levels in culture supernatants were quantified by RT-qPCR and expressed as copies/mL. (C) SARS-CoV-2 titers in adipocyte culture supernatants, measured as plaque-forming units per milliliter (PFU/mL). (D) Evaluation of ACE2 surface expression in adipocytes and Huh7.5 (positive control) measured through flow cytometry (E). Representative histograms obtained by flow cytometry illustrating ACE2 expression as depicted in (D). (F) Representative images of lipid droplets stained with Bodipy 493/503. (G–I) Quantification…

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  Figure 2. Soluble mediators from SARS-CoV-2–infected adipocytes promote lipid droplet accumulation in hepatocytes. (A) Schematic representation of the experimental timeline schedule. Adipocytes were infected, and supernatants were collected at 2 DPI. These culture supernatants were then used to stimulate hepatocytes. Lipid droplets were assessed 2 DPS, and viral replication was measured at 1, 2, 3, and 6 DPS. (B) Representative images of lipid droplets from hepatocytes stained with Bodipy 493/503. (C, D) Quantification of lipid droplets shown in panel B: (C) mean fluorescence intensity (MFI) (W = 0.9345, p = 0.6149; W = 0.8024, p = 0.0617; W = 0.9961, p = 0.9988) and (D) lipid droplet area (W = 0.8614, p = 0.1501; W = 0.9772, p = 0.09; W = 0.9354, p = 0.08). (E) SARS-CoV-2 (Wh) replication in hepatocytes stimulated with culture supernatants from infected adipocytes. Non-infected (NI). Days post-infection (DPI). Days post-stimulation (DPS). Culture supernatants (CS). Non-significant (NS). Scale bar: 25 µm. Data are presented as mean ± SD from six independent biological replicates for panels C and D, and three independent biological replicates for panel E, each performed in technical…

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  Figure 3. Spike neutralization abrogates lipid droplet accumulation in hepatocytes exposed to supernatants from SARS-CoV-2–infected adipocytes. (A) Schematic representation of the experimental timeline. Adipocytes were infected, and supernatants were collected at 2 DPI. These culture supernatants were then used to stimulate hepatocytes in the presence of either an anti-Spike (anti-S) antibody or an irrelevant control antibody against snakebite venom (anti-SB). Lipid droplets were assessed at 2 DPS, and viral replication was measured at 1, 2, 3, and 6 DPS. (B) Representative images of lipid droplets in hepatocytes stimulated with culture supernatants from SARS-CoV-2–infected adipocytes pre-incubated for 1 h at 37 °C with either an anti-S antibody or an irrelevant control antibody against anti-SB. Lipid droplets were stained with Bodipy 493/503. (C, D) Quantification of lipid droplets shown in panel B: (C) mean fluorescence intensity (W = 0.8619, p = 0.2671; W = 0.8965, p = 0.4141; W = 0.9987, p = 0.9960; W = 0.9818, p = 0.9127; W = 0.7895, p = 0.0846; W = 0.8844, p = 0.3579; W = 0.9024, p = 0.4431; W = 0.9437, p = 0.6770; W = 0.8294, p = 0.1663; W = 0.9381, p = 0.6431; W = 0.9466, p =…

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  Figure 4. UV-C inactivation abolishes the ability of supernatants from SARS-CoV-2–infected adipocytes to induce lipid droplet accumulation in hepatocytes. (A) Schematic representation of the experimental timeline. Adipocytes were infected, and supernatants were collected at 2 DPI. The culture supernatants were then UV-C inactivated and used to stimulate hepatocytes. Lipid droplets were assessed 2 DPS, and viral replication was measured at 1, 2, 3, and 6 DPS (B) Representative images of lipid droplets in hepatocytes stimulated with UV-C–inactivated culture supernatants from SARS-CoV-2 (Wuhan strain)–infected adipocytes. Lipid droplets were stained with Bodipy 493/503. (C, D) Quantification of lipid droplets shown in panel B: (C) mean fluorescence intensity (W = 0.8119, p = 0.1252; W = 0.8119, p = 0.1253; W = 0.9720, p = 0.8541; W = 0.9398, p = 0.6532; W = 0.7816, p = 0.0731; W = 0.9529, p = 0.7346; W = 0.9198, p = 0.5360) and (D) lipid droplet area (W = 0.8594, p = 0.0671; W = 0.8627, p = 0,1567; W = 0.8493, p = 0,0718; W = 0.9176, p = 0.0846; W = 0.8705, p = 0.0692; W = 0.9397, p = 0.0862; W = 0.8933, p = 0.1451). (E) Infectivity of the UV-C–inactivated culture supernatants was assessed by…