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

Figure 2

CS exposure impairs bacterial clearance by macrophages that promote bacterial survival. (a) RAW cells were pretreated with fisetin (20 μM) and or CSE (5%) for 8 hrs. Following treatment, the cells were infected with PA01-GFP for 3 hrs at a MOI of 10. After infection, the cells were washed twice with sterile PBS, followed by bright-field and fluorescence microscopy (scale bar, 100 μm). These florescent images were utilized to quantify the number of infected cells (intracellular bacteria) using the ImageJ software. The data shows that CSE treatment significantly impairs bacterial clearance, indicated by a decrease in the number of intracellular bacteria, which was significantly recovered by fisetin. (b) The data from images shown in (a) are represented here as mean ± SEM of percentage of macrophages infected, , ; . (c) The cell culture media (100 μl) from the experimental groups shown in (a) were spread on 2% LB agar plates and incubated for 24 hrs at 37°C. The number of colony-forming units (CFU) was counted to quantify the number of extracellular bacteria as a representation of bacterial survival. CSE treatment resulted in significantly increased bacterial survival that was controlled by fisetin treatment. Data represents mean ± SEM of CFUs, , . (d) Flow cytometry results of RAW cells treated with the same groups as shown in (a) showed the CS-induced phagocytic defect as well as verify fisetin’s ability to recover CSE-impaired macrophage phagocytic function. (e) The flow cytometry data expressed as mean of positive GFP florescent phagocytic cells; shown as mean ± SEM, , ; . (f) Bacterial survival assay of cell culture media (100 μl) from flow cytometry experiment showed a significant increase in the number of CFUs after CSE treatment, which was significantly reduced by fisetin. The data is representative of mean ± SEM of CFUs, , ; . This data suggests that CS exposure impairs phagocytosis in murine macrophages that can be restored by autophagy induction.

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