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

Figure 3

Autophagy inhibition impairs phagocytosis in murine macrophages. (a) RAW cells were pretreated with fisetin (20 μM) and chloroquine (60 μM) for 8 hrs. Then, the cells were infected with PA01-GFP for 3 hrs at a MOI of 10. After infection, the cells were washed twice with PBS followed by bright-field and fluorescence microscopy (scale bar, 70 μm). ImageJ software was utilized to count the number of infected cells (intracellular bacteria). Data shows that chloroquine treatment impairs phagocytosis as indicated by the observations of significantly lower numbers of intracellular bacteria in chloroquine-treated cells, while fisetin demonstrated the ability to recover phagocytosis shown by a significant increase in the number of intracellular bacteria. (b) The data shown in (a) demonstrated the mean ± SEM of percentage of macrophages infected, , ; . (c) The media (100 μl) from the experimental groups in (a) were spread on 2% LB agar plates and incubated for 24 hrs at 37°C, and the number of CFUs was counted to quantify the number of extracellular bacteria as a representation of survival. Data suggests that autophagy inhibition leads to the impairment of PA01-GFP clearance, as extracellular bacterial survival was significantly higher in cells treated with chloroquine, an autophagy inhibitor. Moreover, fisetin significantly reduces the bacterial survival as anticipated. Data represents mean ± SEM of CFUs, , and verifies that autophagy inhibition leads to the impairment of phagocytosis in RAW cells.

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