Histone Deacetylase Inhibitors Promote Mitochondrial Reactive Oxygen Species Production and Bacterial Clearance by Human Macrophages

Abstract

Broad-spectrum histone deacetylase inhibitors (HDACi) are used clinically as anticancer agents, and more isoform-selective HDACi have been sought to modulate other conditions, including chronic inflammatory diseases. Mouse studies suggest that HDACi downregulate immune responses and may compromise host defense. However, their effects on human macrophage antimicrobial responses are largely unknown. Here, we show that overnight pretreatment of human macrophages with HDACi prior to challenge with Salmonella enterica serovar Typhimurium or Escherichia coli results in significantly reduced intramacrophage bacterial loads, which likely reflect the fact that this treatment regime impairs phagocytosis. In contrast, cotreatment of human macrophages with HDACi at the time of bacterial challenge did not impair phagocytosis; instead, HDACi cotreatment actually promoted clearance of intracellular S. Typhimurium and E. coli. Mechanistically, treatment of human macrophages with HDACi at the time of bacterial infection enhanced mitochondrial reactive oxygen species generation by these cells. The capacity of HDACi to promote the clearance of intracellular bacteria from human macrophages was abrogated when cells were pretreated with MitoTracker Red CMXRos, which perturbs mitochondrial function. The HDAC6-selective inhibitor tubastatin A promoted bacterial clearance from human macrophages, whereas the class I HDAC inhibitor MS-275, which inhibits HDAC1 to -3, had no effect on intracellular bacterial loads. These data are consistent with HDAC6 and/or related HDACs constraining mitochondrial reactive oxygen species production from human macrophages during bacterial challenge. Our findings suggest that, whereas long-term HDACi treatment regimes may potentially compromise host defense, selective HDAC inhibitors may have applications in treating acute bacterial infections.

FIG 1

Priming or cotreatment of primary human macrophages with SAHA reduces intracellular bacterial loads of E. coli and S. Typhimurium. Human macrophages were primed with either 10 μM SAHA or 5 ng/ml IFN-γ for 18 h and were then infected with S. Typhimurium or E. coli at an MOI of 100. Alternatively, cells were cotreated with 10 μM SAHA at the time of infection. (A to D) Intracellular survival of S. Typhimurium (A and C) and E. coli (B and D) was assessed at 24 h postinfection in HMDM (A and B) and PMA-differentiated THP-1 macrophages (C and D). Data (mean + SEM) are combined from 3 or 4 independent experiments and are presented as fold change relative to untreated controls. ***, P value of <0.001; ****, P value of <0.0001 by one-way ANOVA followed by Dunnett's multiple comparison test. p indicates overnight priming with HDACi, and co indicates cotreatment with HDACi at the time of infection. (E) S. Typhimurium was cultured with or without 30 μM SAHA for 7 h. Data (mean + SEM) are combined from 3 independent experiments. (F) Cell death was quantified by assessing LDH release into culture supernatants from HMDM primed with 10 μM SAHA or 5 ng/ml IFN-γ for 18 h prior to infection with S. Typhimurium for 24 h. Data (mean + SEM) are combined from 4 independent experiments.

FIG 2

Priming, but not cotreatment, of primary human macrophages with HDAC inhibitors impairs phagocytosis of E. coli. (A) HMDM were treated with 1 μM TSA or 10 μM SAHA for 6 h, after which cell lysates were prepared and histone H3 hyperacetylation was assessed by Western blotting. Data show immunoblots from one experiment and are representative of 3 independent experiments. Phagocytic capacity of HMDM (B) and PMA-differentiated THP-1 cells (C) after priming or cotreatment with 1 μM TSA or 10 μM SAHA was measured by uptake of fluorescent pHrodo Green E. coli BioParticles. For cytochalasin D treatment (10 μM), cells were treated 30 min prior to treatment with E. coli BioParticles. Intracellular bacterial loads of E. coli (D) and S. Typhimurium (E) in HMDM derived from the same donors as in the phagocytosis assay at 1 and 24 h postinfection are shown after cotreatment with 1 μM TSA or 10 μM SAHA. Bacterial loads of E. coli (F) and S. Typhimurium (G) in PMA-differentiated THP-1 macrophages at 1 h postinfection, after either cotreatment or overnight priming with 1 μM TSA or 10 μM SAHA, are shown. Data (mean + SEM) (B to G) are combined from 3 or 4 independent experiments and are presented as fold change relative to untreated controls. **, P value of <0.01; ***, P value of <0.001; ****, P value of <0.0001 by one-way ANOVA followed by Dunnett's multiple comparison test. p indicates overnight priming with HDACi, and co indicates cotreatment with HDACi at the time of infection.

FIG 3

Cotreatment of HMDM with either TSA or SAHA at the time of infection with S. Typhimurium enhances mitochondrial ROS generation. HMDM were cotreated with 1 μM TSA or 10 μM SAHA during infection with S. Typhimurium at an MOI of 100. At 6 h postinfection, MitoSOX Red was added to cells, and mitochondrial ROS generation (median fluorescence intensity) was measured by flow cytometry. Data (mean + SEM, combined from 3 independent experiments) represent fold change relative to untreated cells stained with MitoSOX Red. *, P value of <0.05; **, P value of <0.01 by one-way ANOVA followed by Dunnett's multiple comparison test.

FIG 4

Treatment with MitoTracker Red CMXRos prevents HDACi-mediated clearance of S. Typhimurium from HMDM. HMDM were cotreated with 1 μM TSA in the presence or absence of 100 or 200 nM MitoTracker CMXRos during infection with S. Typhimurium at an MOI of 100. Intracellular survival was assessed at 8 h (A) and 24 h (B) postinfection. Data (mean + SEM) are combined from 3 independent experiments. ****, P value of <0.0001 by one-way ANOVA followed by Dunnett's multiple comparison test. (C) Cell death was assessed by quantifying LDH release from 24-h culture supernatants. Data (mean + SEM) are combined from 4 independent experiments.

FIG 5

Cotreatment of human macrophages with tubastatin A at the time of infection with S. Typhimurium enhances bacterial clearance. (A) α-Tubulin hyperacetylation was assessed in HMDM treated with 1 μM TSA, 20 μM tubastatin A, or 20 μM MS-275 for 6 h. Data show immunoblots from one experiment and are representative of 3 independent experiments. Intracellular bacterial loads of S. Typhimurium in HMDM (B) and PMA-differentiated THP-1 (D) were determined in cells cotreated with either 1 μM TSA, 1 to 20 μM tubastatin A, or 1 to 20 μM MS-275 during infection (MOI, 100). Intracellular survival was assessed at 24 h postinfection. Data (mean + SEM) are combined from 4 independent experiments. Cell death in HMDM (C) and PMA-differentiated THP-1 (E) was assessed by quantifying LDH release from 24-h culture supernatants. Data (mean + SEM) are combined from 3 independent experiments.