Obesity Exacerbates Coxsackievirus Infection via Lipid-Induced Mitochondrial Reactive Oxygen Species Generation

Figures

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  Figure 1. HFD-induced obesity increased mortality and morbidity in CVB3-infected mice. Eight-week-old male C57BL/6 mice were divided into three groups (n = 10 per group), and each group of mice was fed a RD for 4 wk, HFD for 2 wk, or HFD for 4 wk. (A) Survival in the CVB3-infected group was monitored for 7 days after intraperitoneal CVB3 (1×106 pfu/mouse) injection. (B) The body temperature of mice was measured for 1–3 days post-infection. (C) Normalized expression of the positive-strand and negative-strand CVB3 gene (5′-UTR) was determined by real-time PCR 1–3 days post-infection. Data are expressed as means±SEMs. NS, not significant. *p<0.05, **p<0.01, and ***p<0.001.

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  Figure 2. HFD-fed obese mice showed accelerated acute inflammation and lipotoxicity in WAT following CVB3 infection. Groups of mice were fed a RD or HFD for 4 wk and intraperitoneally infected with CVB3 (1×106 pfu/mouse). (A) Representative images of WAT at 1–3 days post-infection. Lipocytoxicity of WAT as indicated by red arrows. (B) Representative H&E staining results for WAT. (C) Immunohistochemical staining for F4/80 expression in WAT at 3 days post-infection. (D) eWAT was surgically removed (eWATx) from obese mice fed an HFD for 4 wk, and the mice were intraperitoneally infected with CVB3 (1×106 pfu/mouse). Survival of mice was monitored for 7 days following CVB3 infection (n = 5/group). Data are expressed as means±SEMs. *p<0.05 and **p<0.01.

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  Figure 3. OA increased CVB3 replication in vitro. (A) Vero cells were pretreated for 24 h with 50 or 100 µM OA noncovalently conjugated with 0.1% BSA and infected with CVB3 at a MOI of 10 for 24 h. Normalized CVB3 gene expression in Vero cells was determined by real-time PCR. (B) Vero cells were treated with the indicated concentrations of OA with the virus for 48 h. (C) Vero cells were treated with the indicated concentrations of OA without the virus for 48 h. Viability of Vero cells was measured using SRB assays. (D) HeLa cells were pretreated for 24 h with 50 or 100 µM OA noncovalently conjugated with 0.1% BSA and infected with CVB3 at an MOI of 10 for 24 h. Normalized expression of the CVB3 gene in HeLa cells was determined by real-time PCR. (E) Vero cells were pretreated for 24 h with 100 µM OA noncovalently conjugated with 0.1% BSA and were infected with CVB3 at an MOI of 10 for 24 h. Representative images stained with anti-CVB3 Ab (green). All figures are representative examples of experiments performed in triplicate. Data are expressed as means±SEMs. Veh, vehicle; NS, not significant; MOI, multiplicity of infection. *p<0.05, and ***p<0.001.

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  Figure 4. CVB3-induced cytotoxicity enhanced by OA and a HFD did not recover by valsartan treatment. (A) HeLa cells were treated with 0.1% BSA, 50 µM OA in 0.1% BSA, or 100 µM OA in 0.1% BSA for 24 h. The expression of CAR and β-actin was determined from cell lysates using western blot analysis. (B) Mice were fed a RD or HFD for 4 wk, and the expression of CAR protein in the heart, liver, pancreas, and WAT of mice was determined by western blot analysis. (C) Vero cells were infected with CVB3 at a MOI of 10 and treated with the indicated concentrations of valsartan for 48 h. Cell viability was measured using SRB assays. (D) Vero cells were pretreated with 100 µM OA in 0.1% BSA for 24 h and subsequently infected with CVB3 at an MOI of 10 in the presence of 250 or 500 μM valsartan for 48 h. All figures are representative examples of experiments performed in triplicate. Data are expressed as means±SEMs. NS, not significant; Veh, vehicle; MOI, multiplicity of infection. *p<0.05, **p<0.01, and ***p<0.001.

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  Figure 5. RO formation induced by CVB3 infection was correlated with OA treatment conditions. Vero cells were pretreated with 100 µM OA in 0.1% BSA for 24 h and infected with CVB3 at a MOI of 10 for 12 h. (A) Formation of MVBs (red arrow) was evaluated using transmission electron microscopy. (B-D) Mitochondrial length (C) and number of mitochondria (D) were quantitatively analyzed in Vero cells as indicated. (E) Cells were subsequently immunostained for detection of TOM20 (green), PDH2/3 bp (red), and nuclei (blue). The numbers of TOM20+ MDVs (white arrow) or PDH+ MDVs (red arrow) per cell were quantified using immunofluorescence imaging. Data are expressed as means±SEMs. NS, not significant; Veh, vehicle; MOI, multiplicity of infection. *p<0.05, **p<0.01, and ***p<0.001.

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  Figure 6. Inhibition of mitochondrial ROS reduced CVB3 replication. (A) Vero cells were pretreated with 100 µM OA in 0.1% BSA for 24 h and infected with CVB3 at a MOI of 10 for 1 h. (A) Cells were stained with 5 µM MitoSOX and analyzed by flow cytometry. (B, C) Vero cells were pretreated for 24 h with 100 µM OA in 0.1% BSA and infected with CVB3 (MOI 10) in the presence or absence of 50 µM Mito Tempo for 48 h. (B) Normalized CVB3 gene expression was determined by real-time PCR. (C) Cell viability was measured using SRB assays. All figures are representative examples of experiments performed in triplicate. Data are expressed as means±SEMs. MOI, multiplicity of infection. *p<0.05, **p<0.01, and ***p<0.001.

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  Figure 7. MitoQ had therapeutic effects on obese mice infected with CVB3. Mice were fed a HFD for 4 wk. Mice were infected with CVB3 at 1×106 pfu/mouse, and 2 mg/kg MitoQ was intraperitoneally administered. (A) Survival was monitored for 14 days (n=10/group). (B) Representative histology staining of WAT 3 days post-infection. (C) Normalized CVB3 gene expression in the pancreas was determined by PCRRT-PCR 3 days post-infection. Data are expressed as means±SEMs. NS, not significant; Veh, vehicle. **p<0.01, and ***p<0.001.