Macrophages exposed during differentiation to low concentrations of HDAC inhibitors are more bactericidal. (A) Outline of the experimental setup used in (B–D). (B) Monocytes derived from 7 different donors were differentiated toward Mϕ1 and Mϕ2 while being exposed to TMP195 (300 nM), TMP269 (300 nM), TSA (30 nM), or DMSO at equal v/v for 6 days. Differentiated Mϕ1 and Mϕ2 were subsequently infected with Mtb for 1 h at MOI 10 and incubated for 48 h post-infection until readout. Dots represent the mean of 3 CFU assay replicates of a single donor expressed as a percentage of the DMSO control. Horizontal lines indicate median CFU values of all 7 donors and whiskers represent 95% confidence intervals. Statistically significant differences compared to DMSO were tested using a RM one-way ANOVA (*p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001). (C) Cell viability measurement of Mtb-infected Mϕ1 and Mϕ2 (experimental setup as in B). Dots represent the mean of 3 cell viability assay replicates of a single donor expressed as a percentage of the DMSO control. Bars indicate median values of all 3 donors and whiskers represent 95% confidence intervals. Statistically significant differences compared to DMSO were tested using a RM one-way ANOVA. (D) Phagocytic capacity of Mϕ1 and Mϕ2 was evaluated by flow-cytometry using fluorescent beads (experimental setup as in B). Macrophages were categorized into populations containing either 0, 1, 2, or 3+ beads. Bars depict mean ± standard deviation of 3 replicates. Data shown is 1 representative donor out of 4. Statistically significant differences compared to DMSO were tested using a one-way ANOVA with Dunnett's multiple test correction (*p < 0.05; **p < 0.01; ****p < 0.0001).
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