AAC Accepted Manuscript Posted Online 28 December 2015 Antimicrob. Agents Chemother. doi:10.1128/AAC.01876-15 Copyright © 2015, American Society for Microbiology. All Rights Reserved.
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Histone deacetylase inhibitors promote mitochondrial reactive oxygen
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species production and bacterial clearance by human macrophages
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Juliana K. Ariffin, Kaustav das Gupta, Ronan Kapetanovic, Abishek Iyer, Robert C.
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Reid, David P. Fairlie, Matthew J. Sweet#
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Institute for Molecular Bioscience, IMB Centre for Inflammation and Disease Research
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and Australian Infectious Diseases Research Centre, The University of Queensland,
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Brisbane, Queensland, 4072 Australia.
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Running Title: HDAC inhibitors boost antimicrobial responses
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#
Address correspondence to Matthew J Sweet,
[email protected].
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Abstract
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Broad-spectrum histone deacetylase inhibitors (HDACi) are used clinically as anti-cancer
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agents, and more isoform-selective HDACi have been sought to modulate other
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conditions, including chronic inflammatory diseases. Mouse studies suggest that HDACi
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down-regulate immune responses and may compromise host defence. However, their
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effects on human macrophage antimicrobial responses are largely unknown. Here we
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show that overnight pre-treatment of human macrophages with HDACi prior to challenge
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with Salmonella enterica serovar Typhimurium (S. Typhimurium) or Escherichia coli (E.
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coli) results in significantly reduced intramacrophage bacterial loads, which likely
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reflects the fact that this treatment regime impairs phagocytosis. In contrast, co-treatment
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of human macrophages with HDACi at the time of bacterial challenge did not impair
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phagocytosis; instead HDACi co-treatment actually promoted clearance of intracellular S.
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Typhimurium and E. coli. Mechanistically, treatment of human macrophages with
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HDACi at the time of bacterial infection enhanced mitochondrial reactive oxygen species
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generation by these cells. The capacity of HDACi to promote clearance of intracellular
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bacteria from human macrophages was abrogated when cells were pre-treated with
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MitoTracker Red CMXRos, which perturbs mitochondrial function. The HDAC6-
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selective inhibitor, tubastatin A, promoted bacterial clearance from human macrophages,
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whereas the class I HDAC inhibitor, MS-275, which inhibits HDAC1-3, had no effect on
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intracellular bacterial loads. These data are consistent with HDAC6, and/or related
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HDACs, constraining mitochondrial reactive oxygen species production from human
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macrophages during bacterial challenge. Our findings suggest that, whereas long-term
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HDACi treatment regimes may potentially compromise host defence, selective HDAC
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inhibitors could have applications in treating acute bacterial infections.
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INTRODUCTION
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Gene expression in eukaryotic cells is controlled by the accessibility of DNA in the form
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of chromatin wrapped around histone proteins. Histone acetyltransferases (HATs)
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catalyze acetylation of lysine residues on core histone tails to allow initiation of
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transcription, whereas histone deacetylases (HDACs) reverse this process by catalyzing
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the removal of acetyl groups. The latter results in chromatin compaction and silencing of
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gene transcription, although this is a greatly simplified model of gene regulation (1).
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HDACs are grouped into distinct classes based on sequence homology to yeast proteins.
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The classical HDACs are sub-divided into Class I (HDAC1, 2, 3, 8), IIa (HDAC4, 5, 7,
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9), IIb (HDAC6, 10) and IV (HDAC11) HDACs. They are dependent on zinc for
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enzymatic activity, share sequence similarity, and most of these enzymes are inhibited by
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broad-spectrum HDAC inhibitors (HDACi) such as trichostatin A (TSA) and
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suberoylanilide hydroxamic acid (SAHA) (2). In contrast, non-classical Class III
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HDACs, or sirtuins, function through a distinct NAD+-dependent mechanism. Although
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commonly used HDACi such as TSA and SAHA target multiple classical HDACs,
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isoform-selective HDACi for some individual HDACs have now been described. For
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example, tubacin and tubastatin A preferentially inhibit HDAC6, whilst MS-275 is most
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potent on HDAC1, but also inhibits HDAC2 and HDAC3 (2). HDACi have been found to
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be effective as anti-cancer agents, at least for some cancers such as cutaneous T cell
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lymphoma, by promoting the expression of tumour suppressor genes and inducing cell
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differentiation, growth arrest and/or apoptosis (3-6). Although HDACs are termed
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‘histone’ deacetylases, they also act on numerous non-histone proteins such as
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transcription factors, cell cycle proteins and protein kinases (7). Hence, HDACi have
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many effects beyond the regulation of epigenetic states and gene expression.
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Beyond their anti-cancer applications, HDACi have been investigated as therapeutic
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agents in models of acute and chronic inflammatory diseases (3, 8-10). HDACi are
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reported to inhibit pro-inflammatory cytokine secretion via multiple mechanisms, for
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example inhibiting the transcriptional activity of HIF-1α (8, 11), impairing recruitment of
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pro-inflammatory transcription factors to target promoters (12), reducing the stability of
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mRNAs encoding inflammatory cytokines (13), and upregulating the expression and
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activity of the transcriptional repressor complex, Mi-2/NuRD (14). Such studies imply
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that certain HDACs have pro-inflammatory functions, and indeed this has been reported
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for a number of HDACs, for example HDAC4 (15) and HDAC7 (11). As pro-
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inflammatory cytokines play important roles in host defense, it is a distinct possibility
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that HDACi could, in dampening inflammation, also compromise host defence. Indeed,
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this is a major limitation of many existing anti-inflammatory agents, including anti-TNFα
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therapy used to treat patients with rheumatoid arthritis and inflammatory bowel disease
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(16-18).
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Previous studies examining the effects of HDACi on innate immune responses in mouse
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macrophages reported that treatment with the broad spectrum HDACi, TSA and valproic
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acid, reduced expression of phagocytic receptors, NADPH oxidase subunits and inducible
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nitric oxide synthase (19). Consequently, HDACi impaired phagocytotic activity of
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mouse bone marrow-derived macrophages, as well as production of reactive oxygen
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species (ROS) and nitric oxide. Furthermore, the ability of mouse macrophages to clear
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Escherichia coli (E. coli) and Staphylococcus aureus upon challenge was compromised
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(19). A genome-wide microarray analysis of gene expression profiles from murine
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macrophages and dendritic cells stimulated with toll-like receptor (TLR) agonists also
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revealed that TSA treatment impaired up to 60% of TLR2 and TLR4 target genes,
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including
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molecules, transcription regulators and kinases, while only 16% of genes were
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upregulated (14). However, studies on the impacts of HDACi on host immunity have
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primarily utilized mouse models or mouse cells, and effects on host defence in humans
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have not been intensively investigated. Although human and mouse innate immune
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responses are broadly conserved, some phenotypic differences, divergence in gene
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repertoires and differential regulation of orthologous genes have been noted between
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humans and mice (20-23). Here we report that, whilst long-term treatment of human
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macrophages with HDACi impairs the phagocytic capacity of human macrophages, acute
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treatment with HDACi at the time of bacterial infection actually enhances mitochondrial
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ROS production from these cells, which correlated with increased antibacterial responses.
pattern
recognition
receptors,
cytokines,
chemokines,
co-stimulatory
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MATERIALS AND METHODS
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Materials. TSA purified from Streptomyces sp. (Sigma Aldrich) was dissolved in 100%
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ethanol, stored at 4˚C, and diluted in tissue culture media to be used at the indicated
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concentrations. SAHA was synthesized in-house, dissolved in DMSO, stored at -20˚C,
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and diluted in tissue culture media to be used at 10 µM. Tubastatin A (Sigma Aldrich)
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and MS-275 (synthesised in-house) were dissolved in DMSO to 30 mM, stored at -20˚C
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and used at 20 µM. MitoTracker® Red CMXRos (Life Technologies) was stored at -20˚C
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and dissolved in DMSO to 1 mM before further dilution in tissue culture media for
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treatment of cells. Human recombinant IFN-γ (R&D Systems) was stored at a
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concentration of 2.5 µg/mL at -20˚C and diluted in tissue culture media to a final
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concentration of 2.5 ng/mL. Cytochalasin D (Life Technologies) was dissolved in DMSO
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to 2 mM, stored at -20˚C, and used at 10 µM. MitoSOXTM Red mitochondrial superoxide
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indicator (Life Technologies) was dissolved in DMSO to 5 mM, stored at -20˚C, and used
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at 5 µM. pHrodo® Green E. coli BioParticles® Conjugate (Invitrogen) was dissolved in
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PBS to 1 mg/ml, sonicated for 5 minutes, vortexed for 1 minute, then stored at -20˚C.
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Ethics statement. Before undertaking the studies described, approval for all experiments
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using primary human cells was obtained from the Medical Research Ethics Committee of
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the University of Queensland (Approval number 2013001519).
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Cell isolation and culture. Human monocyte-derived macrophages (HMDM) were
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prepared from CD14+ monocytes from Red Cross blood donors. In brief, CD14+
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monocytes were differentiated into HMDM, by culture with 1x104 U/mL CSF-1 for 6
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days, as previously described (22, 24). On day 6, HMDM were harvested and re-plated,
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in the presence of CSF-1, for experimentation. THP-1 cells were cultured as previously
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described (25), differentiated for 48 h with 30 ng/mL phorbol 12-myristate 13-acetate
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(PMA, Sigma Aldrich) and rested for 4 h before experimentation. All cells were cultured
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in an incubator at 37˚C with 5% CO2.
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Bacterial culture. The Salmonella enterica serovar Typhimurium (S. Typhimurium) SLI
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344 (26) and E. coli MG1655 (27) strains were stored in LB broth containing 50% sterile
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glycerol at -80˚C. The appropriate multiplicity of infection (MOI) for infection assays
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was calculated by spectrophotometry analysis of bacteria cell density from an isolated
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colony grown overnight in LB broth. Possible effects of HDACi on S. Typhimurium and
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E. coli growth were assessed by spectrophotometric analysis of bacterial cultures grown
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at 37˚C in complete HMDM media at the indicated time points.
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In vitro infection assays. Bacterial infection assays were conducted in HMDM and
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PMA-differentiated THP-1 macrophages to assess intracellular bacterial loads, as
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previously described (28). In brief, cells were cultured at 4x105 cells/well in 1 mL
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antibiotic-free tissue culture media at 37˚C, then either primed with HDACi for 18 h prior
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to bacterial challenge (S. Typhimurium or E. coli; MOI 100), or co-treated with HDACi
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at the time of infection. At 1 h post-infection, cells were washed with media containing
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200 µg/mL gentamicin to exclude extracellular bacteria, then maintained in media
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containing 20 µg/mL gentamicin. In situations where cells were treated with
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pharmacological reagents, inhibitors were re-added to the media containing 20 µg/mL
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gentamicin and maintained throughout the incubation period. At appropriate time points,
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media were replaced with 0.01% Triton X-100 in PBS and cells were lysed by pipetting.
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Diluted lysates were cultured on agar plates at 37˚C overnight, and colony counts were
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used to assess intracellular bacterial loads. In some experiments, supernatants were
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harvested at the indicated time points to assess cell death through the release of the
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metabolic enzyme, lactate dehydrogenase (LDH), from dying cells into culture
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supernatants. LDH release was quantified by measuring LDH in culture supernatants in
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comparison to total LDH (LDH present in supernatants from untreated cells following
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cell lysis with 0.1% Triton X-100), as previously described (25).
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Quantification of phagocytosis. HMDM and PMA-differentiated THP-1 macrophages
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were plated at a density of 5x105 cells/well, and on the following day, cells were treated
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with 10 µM Cytochalasin D for 30 min at 37˚C to inhibit phagocytosis if required, then
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treated with 100 µg pHrodo Green E. coli BioParticles® Conjugate with or without
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HDACi treatment (either priming or co-treatment). Phagocytosis was assessed at 1.5 h
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post-treatment with pHrodo Green E. coli BioParticles® Conjugate, as measured by flow
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cytometric quantification of fluorescence using a FACs Canto II Flow Cytometer (BD
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Bioscience). FACs data was analysed using FlowJo 7.6.5 software (Tree Star).
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Quantification of mitochondrial ROS (mitoROS). HMDM were co-treated with
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HDACi and infected with S. Typhimurium, as above. After 6 h of infection, cells were
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washed twice with PBS, 5 µM MitoSOXTM Red Mitochondrial Superoxide Indicator
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(Life Technologies) was added, and cells were incubated for 10 min at 37˚C, before
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mitoROS levels were quantified by flow cytometry.
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Immunoblotting. Levels of specific proteins were assessed by Western Blot. Briefly,
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cells were lysed and homogenised in RIPA buffer (50 mM Tris-HCl pH 7.4, 150 mM
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NaCl, 0.1% SDS, 1% sodium deoxycholate, 1% NP-40) supplemented with 1 x protease
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inhibitors (Roche Life Science). Cell lysates were resolved on pre-cast 4-20% gradient
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SDS-PAGE gels (Bio-Rad) or 4-12% Bis-Tris NuPAGE gels (Invitrogen), separated by
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electrophoresis, and then transferred onto nitrocellulose membranes (Bio-Rad).
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Membranes were probed using either anti-acetyl histone H3 (Cell Signaling), anti-acetyl-
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α-tubulin (Cell Signaling) or anti-GAPDH (Trevigen) antibodies, then developed by
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chemiluminescence (Amersham ECL Plus, GE Healthcare).
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Statistics. For data combined from 3 or more independent experiments, statistical
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analyses were performed using Prism 6 software (Graphpad), with error bars indicating
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standard error of the mean. For data sets with 3 or more variables, a one-way ANOVA
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analysis was performed, followed by Dunnett’s multiple comparison test. Data with
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confidence values of ≥ 95% (p
