FABP4-mediated lipid droplet formation in Streptococcus uberis-infected macrophages supports host defence

Figures

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  Figure 1. S. uberis infection leads to LD increases in macrophages. RAW264.7 cells were infected with S. uberis at an MOI of 10 and incubated at 37 ℃ for 3 h as described in the “Materials and methods”. A BODIPY 493/503 staining for LD visualization (green) increased in S. uberis infection. RAW264.7 cells were fixed with 4% paraformaldehyde and stained with BODIPY. Uninfected cells were incubated under identical conditions. Images are representative of n = 10 samples/group. Scale bar, 20 µm. B Intracellular LD content was evaluated by staining cells (10 000/sample) with BODIPY 493/503, followed by analysis with CellQuest Pro acquisition software and FlowJo software. Data are presented as geometric mean fluorescence intensity (geoMFI). The dotted line indicates background fluorescence in uninfected cells. Experiment B is representative of 3 independent experiments. Data are presented as the means ± SEMs (n = 6, unless otherwise indicated). *(P < 0.05) = significantly different between the indicated groups by unpaired t test.

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  Figure 2. Increased fatty acid synthesis in macrophages caused by S. uberis. A Relative mRNA expression levels related to lipid synthesis in RAW264.7 cells during S. uberis invasion. Heatmap (on the left) and statistical chart (on the right). B Acox1 and Ehhadh mRNA expression relative to that of fatty acid β oxidation. C Cd36 mRNA relative expression related to fatty acid uptake. D–F NEFA, TG and TC contents in RAW264.7 cells during S. uberis challenge. Data are representative of 3 independent experiments. Data are presented as the means ± SEMs (n = 6, unless otherwise indicated). *(P < 0.05) = significantly different between the indicated groups by unpaired t test.

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  Figure 3. Modulating lipid metabolism influences LD levels in macrophages. RAW264.7 cells were treated with 10 µM C75 for 24 h to inhibit fatty acid synthesis, incubated with 360 µM oleic acid for 24 h, and infected with S. uberis in mid-exponential phase (MOI = 10) for 3 h at 37 ℃. A BODIPY 493/503 staining for LD visualization (green) in different groups. Images are representative of n = 10 samples/group. Scale bar, 20 µm. B, C Flow cytometry of BODIPY-stained RAW264.7 cells. Left: Representative histograms. Right: Mean fluorescence intensities from 3 independent experiments. The dotted line indicates background fluorescence in uninfected cells. Data are presented as the means ± SEMs (n = 6). *(P < 0.05) = significantly different between the indicated groups by one-way ANOVA. D–F NEFA, TG and TC contents in RAW264.7 cells during S. uberis challenge. Experiments B–F were conducted in triplicate. Data are presented as the means ± SEMs (n = 6). *(P < 0.05) = significantly different between the indicated groups by one-way ANOVA.

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  Figure 4. Effect of LD levels on bacterial load in macrophages. RAW264.7 cells were treated with 10 µM C75 for 24 h to inhibit fatty acid synthesis, incubated with 360 µM oleic acid for 24 h, and then infected with S. uberis in mid-exponential phase (MOI = 10) for 3 h at 37 ℃. A Viable bacteria enumerated as colony-forming units (CFU) on THB agar (left). CFUs were counted by the spread plate method after incubation for 12 h at 37 °C. The efficacy of C75 and oleic acid on the elimination of S. uberis was confirmed by culturing mMEC lysates on THB agar plates. The number of S. uberis colonies in cells (right). Data are presented as the means ± SDs (n = 6). *(P < 0.05) = significantly different between the indicated groups by one-way ANOVA. B, C Representative flow cytometry histogram of the FLI-H channel at 3 hpi in the FITC-d-Lys-S. uberis-infected RAW264.7 cells of the indicated groups. The dotted line indicates background fluorescence in the uninfected cells. Data are presented as the means ± SEMs (n = 6). *(P < 0.05) = significantly different between the indicated groups by one-way ANOVA. Experiments were conducted in triplicate.

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  Figure 5. LD increases in macrophages cause increased inflammation and cell damage. RAW264.7 cells were treated with 10 µM C75 for 24 h to inhibit fatty acid synthesis and then incubated with 360 µM oleic acid for 24 h. The cells were then infected with S. uberis in mid-exponential phase (MOI = 10) for 3 h at 37 ℃. A–C Tnf, Il1b, and Il6 mRNA expression. D Nos2 and Cxcl9 mRNA expression relative to inflammation. E, F NAG and LDH activity in RAW2647 cells. All experiments were repeated 3 times. Data are presented as the means ± SEMs (n = 6). *(P < 0.05) = significantly different between the indicated groups by one-way ANOVA.

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  Figure 6. FABP4 is involved in LD increases during S. uberis infection. RAW264.7 cells were treated with 10 µM C75 for 24 h to inhibit fatty acid synthesis and then incubated with 360 µM oleic acid for 24 h. S. uberis in mid-exponential phase (MOI = 10) was added for 3 h at 37 ℃. A Relative Fabp4 mRNA expression. B The protein expression levels of FABP4 with or without C75 or oleic acid during S. uberis infection were determined by Western blots in RAW264.7 cells. For quantitative analysis, bands were evaluated densitometrically with ImageJ analyser software normalized for β-actin density. C Immunofluorescence and LD staining were performed by FABP4 (red) and BODIPY (green) in the control group and stimulated S. uberis groups as well as the chemical pretreatment groups. Images are representative of n = 10 samples/group. Scale bar, 50 µm. Experiments A–G were repeated 3 times. Data are presented as the means ± SEMs (n = 6, unless otherwise indicated). *(P < 0.05) = significantly different between the indicated groups by one-way ANOVA.

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  Figure 7. Inhibiting FABP4 expression leads to reduced LDs and increased bacterial burden. RAW264.7 cells were treated with 40 µM BMS309403 for 24 h or transfected with 50 nM siFabp4 for 24 h at 37 ℃ using Lipofectamine 3000 reagent (Invitrogen) to inhibit FABP4 expression prior to infection with S. uberis in mid-exponential phase (MOI = 10) for 3 h at 37 ℃. A Fabp4 mRNA expression in the control and interference groups during S. uberis infection. B Protein expression levels of FABP4 with or without BMS309403 or siFabp4 during S. uberis infection were determined by Western blots of RAW264.7 cells. For quantitative analysis, bands were evaluated densitometrically with ImageJ analyser software normalized for β-actin density. C Relative mRNA expression of genes related to the synthesis of fatty acids. D Flow cytometry of BODIPY-stained RAW264.7 cells. Left: Representative histograms. Right: Mean fluorescence intensities from 3 independent experiments. The dotted line indicates background fluorescence in uninfected cells. Data are presented as the means ± SEMs (n = 6). *(P < 0.05) = significantly different between the indicated groups by one-way ANOVA. E Immunofluorescence and LD staining was…

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  Figure 8. Model depicting the role of FABP4 mediated lipid droplets in macrophages after S. uberis infection