FIG 1

In vitro identification of tamoxifen as a novel repurposed host-directed therapeutic. (A and B) CFU assay of Mφ1 (left) and Mφ2 (right) macrophages infected with H37Rv-Mtb (A) or Stm (B) and treated with 10 μM tamoxifen (TAM) or control (CTRL; DMSO at equal volume) for 24 h. Each dot represents a single donor (8 and 9 donors for Mφ1 and Mφ2 macrophages, respectively, in A, and 6 donors in B) and depicts the mean of 3 or 4 replicates. Statistical significance was tested using by Wilcoxon matched-pairs signed-rank test. (C and D) H37Rv-Mtb growth (C) or Stm growth (D) in liquid culture during treatment with 10, 20, 40, or 80 μM tamoxifen or control (DMSO at equal volume [10 μM is shown]) up to the assay endpoint day 14 (C) or overnight (D). Rifampicin (20 μg/mL) (C) or gentamicin (50 μg/mL) (D) was used as a positive control for growth inhibition. Each line depicts the mean ± standard deviation of three replicates. The experiment shown is a representative of two (C) or three (D) independent experiments. Statistical significance of treatment versus control treatment was tested by two-way ANOVA with Dunnett’s multiple-comparison test. (E) CFU assay of Mφ2 macrophages infected with MDR-Mtb strain Dutch outbreak 2003-1128 (left) or Mtb Beijing strain 16319 (right) and treated with 10 μM tamoxifen or control (DMSO at equal volume) for 24 h. Each dot represents a single donor (6 donors in total) and depicts the mean of three replicates. Statistical significance was tested using a ratio paired t test. (F and G) CFU assay of Mφ2 macrophages infected with H37Rv-Mtb and treated for 24 h with 10 μM tamoxifen or control (DMSO at equal volume) in combination with suboptimal doses of 0.05 μg/mL rifampicin (F) or 0.4 μg/mL isoniazid (G). Each bar depicts the mean ± standard deviation of three replicates from a representative donor (out of 4 donors tested in F and 3 donors in G) expressed as a percentage of the control treatment in the absence of antibiotics. Bars with solid colors represent tamoxifen or control treatment only, and bars with patterns represent the combination with antibiotic. Statistical significance was tested by two-way ANOVA with Tukey’s multiple-comparison test comparing tamoxifen treatment (in the absence or presence of antibiotics) to the corresponding control treatment. (H) LDH release assay of Mφ1 (left) and Mφ2 (right) macrophages infected with H37Rv-Mtb and treated with 10 μM tamoxifen or control (DMSO at equal volume) for 24 h. Each dot represents a single donor (5 and 6 donors for Mφ1 and Mφ2 macrophages, respectively) and depicts the mean of three replicates. Dotted lines indicate the control set at 100%, and median values + 95% confidence intervals are shown for every condition. Statistical significance was tested using a ratio paired t test; *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.