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

Figure 5

Fisetin recovers phagocytosis defect mediated by CFTR inhibition. (a) RAW cells were pretreated with CFTR172 inhibitor and/or fisetin (20 μM) for 8 hrs followed by infection with PA01-GFP for 3 hrs at a MOI of 10. After infection, the cells were washed twice with PBS and observed using bright-field and fluorescence microscopy (scale bar, 70 μm). The fluorescent images were used to count the number of macrophages infected (intracellular bacteria) using the ImageJ software. The data shows that the inhibition of CFTR impairs phagocytosis shown by a significantly lower number of intracellular bacteria, which was significantly recovered by fisetin treatment. (b) The data from (a) represented as the mean ± SEM of percentage of macrophages infected, , . (c) A bacterial survival assay was performed using the cellular media (100 μl) from experimental groups in A, which were plated on 2% LB agar plates and incubated for 24 hrs at 37°C. The colony-forming unit bacterial counts show that CFTR inhibition results in significantly higher numbers of CFUs representing impaired phagocytosis, which was significantly recovered by fisetin treatment. The data represents mean ± SEM of CFUs, , . (d) RAW cells were treated with CSE and/or fisetin (20 μM) for 8 hrs. After treatment, the total protein lysate was isolated and analyzed by immunoblotting for changes in the expression of CFTR and p62 expression. The Western blot analysis shows that membrane CFTR expression (C-band) was significantly decreased by CSE exposure, which was significantly recovered upon fisetin treatment. β-Actin was used as a loading control. (e) Densitometry analysis of CFTR and p62 expression was normalized to β-actin. Data represent in each group, and error bars depict mean ± SEM, . Thus, data shows that CFTR inhibition affects the phagocytic ability of macrophages. Moreover, fisetin shows the ability to correct this CFTR-mediated phagocytic defect in RAW cells.

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