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. 2022 Feb 16;12:819554. doi: 10.3389/fcimb.2022.819554

Figure 2.

Figure 2

CFTR is essential for maintaining autophago-lysosomal acidity in macrophages and responds to CFTR modulators. (A) Mean fluorescence intensity (MFI) of LysoSensor Green DND-189 in wild-type (WT), F508del, and cftr−/− macrophages either non-infected (NT) or treated with Baf-A1. Data show MFI normalized to the number of cells. Data represent mean ± SEM (n = 4 biological replicates). Statistical analysis was performed using two-way ANOVA; *, p ≤ 0.05; **, p ≤ 0.01; ***, p ≤ 0.001; ns, non-significant. (B) Corresponding lysosomal pH values in WT, F508del, and cftr−/− macrophages stained with LysoSensor Green DND-189. Data represent MFI normalized to number of cells and plotted to the pH calibration curve. Data represent mean ± SEM (n = 3 biological replicates). Statistical analysis was performed using two-way ANOVA; *, p ≤ 0.05; **, p ≤ 0.01. (C) MFI of LysoSensor Green DND-189 in F508del macrophages treated with 10 µM of Teza, 5 µM of Iva, or both for 24 h. Data represent mean ± SEM (n = 3 biological replicates). Statistical analysis was done using one-way ANOVA; *, p ≤ 0.05. (D) Corresponding lysosomal pH values in F508del macrophages NT or treated with the indicated CFTR modulators for 24 h. Data represent mean ± SEM (n = 3 biological replicates). Statistical analysis was performed using one-way ANOVA; ns, non-significant. (E) WT or F508del CFTR macrophages were seeded in coverslips, and F508del macrophages were treated with 10 μM of Teza and 5 μM of Iva for 24 h. Representative current traces for CFTR chloride channel conductance in WT and F508del macrophages are shown as basal state and after the addition of a cocktail (shown as forskolin) containing 1 μM of forskolin, 10 μM of cAMP, 100 μM of IBMX, and 2 mM of ATP. Step protocol consisted of 400-ms voltage from −80 to +80 mV from a holding potential of −40 mV. The traces show the basal and activated CFTR current in WT, F508del CFTR, WT Teza+Iva, and F508del CFTR Teza+ Iva macrophages (n = 3 biological replicates).