Cigarette Smoke Exposure Inhibits Bacterial Killing via TFEB-Mediated Autophagy Impairment and Resulting Phagocytosis Defect

Figure 4

TFEB knockdown impairs phagocytosis in murine macrophages. (a) RAW cells were transfected with TFEB-Mission™ shRNA for 24 hrs. Following transfection, the cells were pretreated with fisetin (20 μM) for 8 hrs. Afterwards, cells were infected with PA01-GFP for 3 hrs at a MOI of 10. Following infection, the cells were washed twice with PBS followed by bright-field and fluorescence microscopy (scale bar, 70 μm). The florescent images were utilized to count the number of infected cells (intracellular bacteria) using ImageJ software. The data shows that the bacterial clearance is significantly inhibited as indicated by the significantly lower number of intracellular bacteria in TFEB knockdown cells. Meanwhile, fisetin significantly restores bacterial clearance, indicated by an increase in the number of intracellular bacteria. (b) Data from A, represented as percentage of macrophages infected, which are shown as mean ± SEM, , . (c) A bacterial survival assay was performed by spreading the cell culture media (100 μl) from the experimental groups in (a), on 2% LB agar plates that were incubated for 24 hrs at 37°C. CFUs were counted to quantify the extracellular bacteria survival. This data suggests that decreased TFEB expression significantly increases bacterial survival due to impaired phagocytosis. Moreover, fisetin was able to restore phagocytosis as determined by significantly fewer CFUs. The data represents mean ± SEM of CFUs, , ; .

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