Expression kinetics of HDAC transcripts in primary human macrophages following Mtb infection. Mϕ1 and Mϕ2 derived from 4 different donors were mock infected or infected with Mtb for 1 h at MOI 10. Transcript levels of HDAC1-11 were determined in triplicate using qRT-PCR before (0 h baseline) and 4 and 24 h post-infection. Data was normalized to GAPDH (ΔCt) and mean expression levels of triplicate samples were calculated for each donor. (A) Heatmap displaying median log₂ fold changes (FC) of HDAC1-11 expression levels (pooled data of 4 donors) in response to Mtb infection compared to their respective baseline controls. (B) Dot plots displaying log₂ FC expression levels of HDAC1, 3, 5, 7, 10, and 11 in response to Mtb infection compared to their respective baseline controls, calculated using the 2^−ΔΔCT formula. Each dot represents a single donor. Horizontal lines indicate median log₂ FC values of all 4 donors and whiskers represent 95% confidence intervals. Statistically significant differences compared to uninfected controls were tested using a paired sample t-test (*p < 0.05, **p < 0.01).

HDAC inhibitors decrease Mtb survival in human macrophages in a host-directed manner. (A) Schematic outline of the experimental setup used in (B). (B) Mϕ1 and Mϕ2 macrophages derived from 11 different donors were infected with Mtb for 1 h at MOI 10 and treated with TMP195 (10 μM), TMP269 (10 μM), TSA (100 nM), or DMSO at equal v/v for 48 h post-infection. 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 11 donors and whiskers represent 95% confidence intervals. Statistically significant differences compared to DMSO were tested using a RM one-way ANOVA (**p < 0.01, ****p < 0.0001). (C) Thirteen days treatment of a Mtb broth culture in the presence of 10 μM TMP195, TMP269, or TSA. Rifampicin (20 μg/ml) was used as a positive control. Bars depict the mean bacterial density at 550 nm ± standard deviation of six replicates from a representative experiment out of two independent experiments. The bacterial load is expressed as a percentage of the DMSO control value. Statistically significant difference compared to DMSO was tested using a one-way ANOVA with Dunnett's multiple test correction (****p < 0.0001).

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).

Zebrafish embryos exposed to HDAC inhibitors display reduced bacterial burden. (A) Outline of the experimental setup used in (B,C). (B) Zebrafish embryos were at the 20 somite stage [19 h post fertilization (hpf)], exposed to TMP195 (10 μM), TSA (30 nM), or DMSO at equal v/v for 24 h. Embryos were subsequently infected by microinjection of 250–300 CFU Mycobacterium marinum (Mmar) expressing Wasabi fluorescent protein, into the Duct of Cuvier. Shown are representative infected larvae of each treatment group imaged for Mmar fluorescence at 3 days post infection. Scale bar indicates 500 μm. (C) Indicated is bacterial burden of all zebrafish larvae (left) or each independent experiment (right). Graphs represent zebrafish larvae from different parent couples used in 3 independent experiments, and treated with TMP195 (n = 92), TSA (n = 90), or DMSO at equal v/v (n = 102) expressed as a percentage of the DMSO control. Each dot indicates an embryo with horizontal line and whiskers in red representing median with 95% confidence intervals (left) or each dot indicates the mean of a single experiment with floating bars representing min. to max. while the horizontal line represents the mean of the 3 independent experiments (***p < 0.001; ****p < 0.0001).

Exposure to low concentrations HDAC inhibitors during monocyte differentiation alters the cytokine/chemokine response of Mϕ1 and Mϕ2 upon Mtb infection. (A) Heat map displaying median cytokine/chemokine expression levels (of 4 different donors) in supernatants of standardly differentiated Mϕ1 and Mϕ2 24 h following Mtb or mock infection. Each row represents the relative expression of the indicated cytokine/chemokine using a white to red color scale. Of the 41 analytes measured, only cytokines/chemokines that changed in at least 3 out of 4 donors and exhibited a minimal median log₂ fold change (FC) of 0.5 in a single comparison are shown. (B) Heat map displaying median log₂ FC of cytokine/chemokine levels in supernatants of Mϕ1 and Mϕ2 of 4 different donors. In this experimental setup monocytes were exposed to TMP195 (300 nM), TMP269 (300 nM), TSA (30 nM), or DMSO at equal v/v during differentiation toward Mϕ1 and Mϕ2. Gray color depicts cytokine/chemokine levels that were detected above the linear range of the assay. (C) Experimental setup as in (A). Variable Importance in Projection (VIP) scores of the first x-variate were extracted from each PLS-DA analysis and cytokine values ≥1 were considered relevant. In parallel, Kendall's tau-b correlation coefficients were calculated for each cytokine. Coefficients between 0–0.33, 0.33–0.67, and 0.67–1 were considered to have a weak, moderate and strong correlation, respectively. Every dot represents a cytokine/chemokine. Cytokines/chemokines with a VIP score >1 and demonstrating at least a moderate correlation are annotated in black. Annotated cytokines that were produced below or equal to a median concentration of 40 pg/ml are depicted by a diamond. (D) Experimental setup as in (B). VIP scores and Kendall's tau-b correlation coefficients calculations as in (C).