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

Juliana K Ariffin, Kaustav das Gupta, Ronan Kapetanovic, Abishek Iyer, Robert C Reid, David P Fairlie, Matthew J Sweet

<figure><img src="image_1.png" /></figure>

## To cite this version:

Juliana K Ariffin, Kaustav das Gupta, Ronan Kapetanovic, Abishek Iyer, Robert C Reid, et al. Histone Deacetylase Inhibitors Promote Mitochondrial Reactive Oxygen Species Production and Bacterial Clearance by Human Macrophages. Antimicrobial Agents and Chemotherapy, 2016, 60, pp.1521 - 1529. 10.1128/aac.01876-15. hal-04352510

HAL Id: hal-04352510

https://hal.inrae.fr/hal-04352510

Submitted on 19 Dec 2023

HAL is a multi-disciplinary open access
archive for the deposit and dissemination of sci-
entific research documents, whether they are pub-
lished or not. The documents may come from
teaching and research institutions in France or
abroad, or from public or private research centers.

L'archive ouverte pluridisciplinaire HAL, est
destinée au dépôt et à la diffusion de documents
scientifiques de niveau recherche, publiés ou non,
émanant des établissements d'enseignement et de
recherche français ou étrangers, des laboratoires
publics ou privés.

<figure><img src="image_2.png" /></figure>

Distributed under a Creative Commons Attribution 4.0 International License
<figure><img src="image_3.png" /></figure>

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

Juliana K. Ariffin, Kaustav das Gupta, Ronan Kapetanovic, Abishek Iyer, Robert C. Reid, David P. Fairlie, Matthew J. Sweet

Institute for Molecular Bioscience, IMB Centre for Inflammation and Disease Research and Australian Infectious Diseases Research Centre, The University of Queensland, Brisbane, Queensland, Australia

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 anti-microbial 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.

Gene expression in eukaryotic cells is controlled by the accessibility of DNA in the form of chromatin wrapped around histone proteins. Histone acetyltransferases (HATs) catalyze acetylation of lysine residues on core histone tails to allow the initiation of transcription, whereas histone deacetylases (HDACs) reverse this process by catalyzing the removal of acetyl groups. Histone deacetylation results in chromatin compaction and the silencing of gene transcription; however, this is a greatly simplified model of gene regulation (1). HDACs are grouped into distinct classes based on sequence homology to yeast proteins. The classical HDACs are subdivided into class I (HDAC1, -2, -3, and -8), class IIa (HDAC4, -5, -7, and -9), class IIb (HDAC6 and -10), and class IV (HDAC11) HDACs. They are dependent on zinc for enzymatic activity and share sequence similarity, and most of these enzymes are inhibited by broad-spectrum HDAC inhibitors (HDACi) such as trichostatin A (TSA) and suberoylanilide hydroxamic acid (SAHA) (2). In contrast, nonclassical class III HDACs, or sirtuins, function through a distinct NAD⁺-dependent mechanism. Although commonly used HDACi such as TSA and SAHA target multiple classical HDACs, isoform-selective HDACi for some individual HDACs have now been described. For example, tubacin and tubastatin A preferentially inhibit HDAC6, while MS-275 is most potent against HDAC1 but also inhibits HDAC2 and HDAC3 (2). HDACi have been found to be effective as anticancer agents, at least for some cancers such as cutaneous T-cell lymphoma, by promoting the expression of tumor suppressor genes and by inducing cell differentiation, growth arrest, and/or apoptosis (3–6). Although HDACs are termed histone deacetylases, they also act on numerous nonhistone proteins,

such as transcription factors, cell cycle proteins, and protein kinases (7). Hence, HDACi have many effects beyond the regulation of epigenetic states and gene expression.

Beyond their anticancer applications, HDACi have been investigated as therapeutic agents in models of acute and chronic inflammatory diseases (3, 8–10). HDACi are reported to inhibit pro-inflammatory cytokine secretion via multiple mechanisms, including inhibiting the transcriptional activity of hypoxia-inducible factor 1-alpha (HIF-1α) (8, 11), impairing recruitment of proinflammatory transcription factors to target promoters (12), reducing the stability of mRNAs encoding inflammatory cytokines (13), and upregulating the expression and activity of the transcriptional repressor complex, Mi-2/NuRD (14). Such studies imply that certain HDACs have proinflammatory functions, and indeed this has been reported for a number of HDACs, for example, HDAC4 (15) and HDAC7 (11). As proinflammatory cytokines play important roles in host defense, it is a distinct possibility that HDACi may, in dampening inflammation, also compromise

Received 14 August 2015 Returned for modification 1 September 2015

Accepted 11 December 2015

Accepted manuscript posted online 28 December 2015

Citation Ariffin JK, das Gupta K, Kapetanovic R, Iyer A, Reid RC, Fairlie DP, Sweet MJ. 2016. Histone deacetylase inhibitors promote mitochondrial reactive oxygen species production and bacterial clearance by human macrophages. Antimicrob Agents Chemother 60:1521–1529. doi:10.1128/AAC.01876-15

Address correspondence to Matthew J. Sweet, m.sweet@imb.uq.edu.au.

Copyright © 2016, American Society for Microbiology. All Rights Reserved.
host defense. Indeed, this is a major limitation of many existing anti-inflammatory agents, including anti-tumor necrosis factor alpha (TNF-α) therapy used to treat patients with rheumatoid arthritis and inflammatory bowel disease (16–18).

Previous studies examining the effects of HDACi on innate immune responses in mouse macrophages reported that treatment with broad-spectrum HDACi, TSA, and valproic acid reduced expression of phagocytic receptors, NADPH oxidase subunits, and inducible nitric oxide synthase (19). Consequently, HDACi impaired the phagocytotic activity of mouse bone marrow-derived macrophages and the production of reactive oxygen species (ROS) and nitric oxide. Furthermore, the ability of mouse macrophages to clear *Escherichia coli* and *Staphylococcus aureus* upon challenge was compromised (19). A genome-wide microarray analysis of gene expression profiles from murine macrophages and dendritic cells stimulated with Toll-like receptor (TLR) agonists also revealed that TSA treatment impaired up to 60% of TLR2 and TLR4 target genes, including pattern recognition receptors, cytokines, chemokines, costimulatory molecules, transcription regulators, and kinases, while only 16% of genes were upregulated (14). However, studies on the impacts of HDACi on host immunity have primarily utilized mouse models or mouse cells, and effects on host defense in humans have not been intensively investigated. Although human and mouse innate immune responses are broadly conserved, some phenotypic differences, divergence in gene repertoires, and differential regulation of orthologous genes have been noted between humans and mice (20–23). Here we report that, while long-term treatment of human macrophages with HDACi impairs the phagocytic capacity of human macrophages, acute treatment with HDACi at the time of bacterial infection actually enhances mitochondrial ROS (mitoROS) production from these cells, which correlates with increased antibacterial responses.

## MATERIALS AND METHODS

**Materials.** TSA purified from *Streptomyces* sp. (Sigma-Aldrich) was dissolved in 100% ethanol, stored at 4°C, and diluted in tissue culture medium to be used at the indicated concentrations. SAHA was synthesized in-house, dissolved in dimethyl sulfoxide (DMSO), stored at -20°C, and diluted in tissue culture medium to be used at 10 µM. Tubastatin A (Sigma-Aldrich) and MS-275 (synthesized in-house) were dissolved in DMSO to 30 mM, stored at -20°C, and used at 20 µM. MitoTracker Red CMXRos (Life Technologies) was stored at -20°C and was dissolved in DMSO to 1 mM before further dilution in tissue culture medium for the treatment of cells. Human recombinant gamma interferon (IFN-γ) (R&D Systems) was stored at a concentration of 2.5 µg/ml at -20°C and was diluted in tissue culture medium to a final concentration of 2.5 ng/ml. Cytochalasin D (Life Technologies) was dissolved in DMSO to 2 mM, stored at -20°C, and used at 10 µM. MitoSOX Red mitochondrial superoxide indicator (Life Technologies) was dissolved in DMSO to 5 mM, stored at -20°C, and used at 5 µM. pHrodo Green *E. coli* BioParticles conjugate (Invitrogen) was dissolved in phosphate-buffered saline (PBS) to 1 mg/ml, sonicated for 5 min, vortexed for 1 min, and then stored at -20°C.

**Ethics statement.** Before undertaking the studies described, approval for all experiments using primary human cells was obtained from the Medical Research Ethics Committee of the University of Queensland (approval number 2013001519).

**Cell isolation and culture.** Human monocyte-derived macrophages (HMDM) were prepared from CD14⁺ monocytes from Red Cross blood donors. In brief, CD14⁺ monocytes were differentiated into HMDM by culture with 1 × 10⁴ U/ml colony-stimulating factor 1 (CSF-1) for 6 days,

as previously described (22, 24). On day 6, HMDM were harvested and replated in the presence of CSF-1 for experimentation. THP-1 cells were cultured as previously described (25), differentiated for 48 h with 30 ng/ml phorbol 12-myristate 13-acetate (PMA; Sigma-Aldrich), and rested for 4 h before experimentation. All cells were cultured in an incubator at 37°C with 5% CO₂.

**Bacterial culture.** The *Salmonella enterica* serovar Typhimurium SLI 344 (26) and *E. coli* MG1655 (27) strains were stored in LB broth containing 50% sterile glycerol at -80°C. The appropriate multiplicity of infection (MOI) for infection assays was calculated by spectrophotometry analysis of bacterial cell density from an isolated colony grown overnight in LB broth. Possible effects of HDACi on S. Typhimurium and *E. coli* growth were assessed by spectrophotometric analyses of bacterial cultures grown at 37°C in complete HMDM medium at the indicated time points.

**In vitro infection assays.** Bacterial infection assays were conducted with HMDM and PMA-differentiated THP-1 macrophages to assess intracellular bacterial loads as previously described (28). In brief, cells were cultured at 4 × 10⁵ cells/well in 1 ml antibiotic-free tissue culture medium at 37°C. Then, they were either primed with HDACi for 18 h prior to bacterial challenge (S. Typhimurium or *E. coli*; MOI, 100) or were co-treated with HDACi at the time of infection. At 1 h postinfection, cells were washed with a medium containing 200 µg/ml gentamicin to exclude extracellular bacteria and then maintained in medium containing 20 µg/ml gentamicin. In situations where cells were treated with pharmacological reagents, inhibitors were re-added to the medium containing 20 µg/ml gentamicin and were maintained throughout the incubation period. At appropriate time points, the medium was replaced with 0.01% Triton X-100 in PBS, and cells were lysed by pipetting. Diluted lysates were cultured on agar plates at 37°C overnight, and colony counts were used to assess intracellular bacterial loads. In some experiments, supernatants were harvested at the indicated time points to assess cell death through the release of the metabolic enzyme lactate dehydrogenase (LDH) from dying cells into culture supernatants. LDH release was quantified by measuring LDH in culture supernatants compared to total LDH (LDH present in supernatants from untreated cells following cell lysis with 0.1% Triton X-100), as previously described (25).

**Quantification of phagocytosis.** HMDM and PMA-differentiated THP-1 macrophages were plated at a density of 5 × 10⁵ cells/well, and on the following day, cells were treated with 10 µM cytochalasin D for 30 min at 37°C to inhibit phagocytosis if required and were then treated with 100 µg pHrodo Green *E. coli* BioParticles conjugate with or without HDACi treatment (either priming or cotreatment). Phagocytosis was assessed at 1.5 h posttreatment with pHrodo Green *E. coli* BioParticles conjugate as measured by flow cytometric quantification of fluorescence using an FACSCanto II flow cytometer (BD Bioscience). FACs data were analyzed using FlowJo 7.6.5 software (Tree Star).

**Quantification of mitochondrial ROS.** HMDM were cotreated with HDACi and were infected with S. Typhimurium, as above. After 6 h of infection, cells were washed twice with PBS, 5 µM MitoSOX Red mitochondrial superoxide indicator (Life Technologies) was added, and cells were incubated for 10 min at 37°C before mitoROS levels were quantified by flow cytometry.

**Immunoblotting.** Levels of specific proteins were assessed by Western blotting. Briefly, cells were lysed and homogenized in radioimmunoprecipitation assay (RIPA) buffer (50 mM Tris-HCl [pH 7.4], 150 mM NaCl, 0.1% SDS, 1% sodium deoxycholate, and 1% NP-40) supplemented with 1× protease inhibitors (Roche Life Science). Cell lysates were resolved on precast 4% to 20% gradient SDS-PAGE gels (Bio-Rad) or on 4% to 12% Bis-Tris NuPAGE gels (Invitrogen) and were separated by electrophoresis and then transferred onto nitrocellulose membranes (Bio-Rad). Membranes were probed using anti-acetyl histone H3 (Cell Signaling), anti-acetyl-α-tubulin (Cell Signaling), or anti-GAPDH (anti-glyceraldehyde-3-phosphate dehydrogenase) (Trevigen) antibodies and were then developed by chemiluminescence (Amersham ECL Plus; GE Healthcare).
**Statistics.** For data combined from 3 or more independent experiments, statistical analyses were performed using Prism 6 software (Graph-Pad) with error bars indicating the standard error of the mean (SEM). For data sets with 3 or more variables, a one-way analysis of variance (ANOVA) was performed followed by Dunnett's multiple comparison test. Data with confidence values of ≥95% (*P* < 0.05) were considered statistically significant.

## RESULTS

**HDACi reduce intracellular bacterial loads within human macrophages.** We first investigated the potential impact of treatment with HDACi on the antimicrobial activity of human macrophages that were infected with either S. Typhimurium or *E. coli*. HMDM and PMA-differentiated THP-1 macrophages were treated with the broad-spectrum HDACi SAHA 18 h prior to infection (priming) or at the time of infection (cotreatment) in order to determine whether potential effects on antimicrobial responses were time dependent. Cells were also primed with IFN-γ as a positive control for priming of antimicrobial responses. Priming or cotreating cells with SAHA significantly reduced intracellular bacterial loads of S. Typhimurium and *E. coli* at 24 h postinfection in HMDM (Fig. 1A and B) and PMA-differentiated THP-1 macrophages (Fig. 1C and D). As expected, IFN-γ priming also reduced intracellular bacterial loads within HMDM. The SAHA-mediated reduction in intracellular bacterial loads may simply reflect the direct killing of bacteria or the effects on macrophage viability of this compound. To exclude these possibilities, we next assessed bacterial growth in the presence of an even higher concentration of SAHA than that which was used in infection assays as well as the release of LDH in cell supernatants following the infection of human macrophages with S. Typhimurium. SAHA treatment did not affect either S. Typhimurium growth (Fig. 1E) or the release of LDH from S. Typhimurium-infected macrophages that had been primed overnight with SAHA (Fig. 1F). Thus, the SAHA-mediated reduction in intramacrophage bacterial loads was not due to the direct killing of bacteria or to reduced macrophage viability. These data also highlight that human macrophages are resistant to cell death triggered by HDACi.

**Priming but not cotreatment of human macrophages with HDACi at the time of infection reduces phagocytosis in human macrophages.** Another potential explanation for the HDACi-mediated reduction in intracellular bacteria (Fig. 1A to D) is impaired phagocytic uptake rather than enhanced antimicrobial responses. Since treatment with HDACi was previously shown to impair phagocytosis in mouse macrophages (19), this was a distinct possibility. We therefore examined the effects of SAHA as well as another broad-spectrum HDACi, TSA, on phagocytosis in human macrophages. TSA (1 µM) and SAHA (10 µM) induced approximately equal levels of hyperacetylation of histone H3 protein (a hallmark of HDAC inhibition) (Fig. 2A), and thus these concentrations were used for subsequent experiments. To assess phagocytosis, the uptake of pHrodo Green *E. coli* BioParticles was measured by flow cytometry in HMDM and PMA-differentiated THP-1 macrophages that were primed or cotreated with either TSA or SAHA. The actin polymerization inhibitor cytochalasin D was used as a positive control for inhibition of phagocytosis (29). Priming, but not cotreatment, with either TSA or SAHA reduced phagocytosis by HMDM (Fig. 2B) and PMA-differentiated THP-1 cells (Fig. 2C) as quantified by fluorescence of phagocytosed pHrodo Green *E. coli*. In contrast, priming with IFN-γ had no

<figure><img src="image_4.png" /><figcaption>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.</figcaption></figure>

effect on phagocytosis in these assays. Thus, the effect of IFN-γ priming in reducing intramacrophage bacterial loads (Fig. 1) is consistent with its well-known capacity to boost macrophage antimicrobial responses. Collectively, the above data (Fig. 1 and 2) suggest that cotreatment of human macrophages with HDACi at the time of infection does not impair bacterial uptake but promotes bacterial clearance. To more directly address this, HMDM from matched donors were infected with live *E. coli* or S. Typhimurium isolates and were cotreated with either TSA or SAHA.
<figure><img src="image_5.png" /><figcaption>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.</figcaption></figure>

Cotreatment of human macrophages with HDACi at the time of infection reduced intracellular bacterial loads at 24 h, whereas these treatments had no effect at 1 h postinfection (Fig. 2D and E). Moreover, a direct comparison of HDACi priming versus cotreatment on bacterial loads at 1 h postinfection in PMA-differentiated THP-1 macrophages also showed that cotreatment with HDACi did not result in a reduction of bacterial loads at this acute time

point, whereas priming with HDACi did (Fig. 2F and G). In total, these findings show that overnight priming with HDACi impairs macrophage phagocytosis resulting in reduced intracellular bacterial loads, whereas cotreatment with HDACi does not impair phagocytosis but rather promotes bacterial clearance by human macrophages. We therefore focused subsequent efforts on understanding how cotreatment of human macrophages with
<figure><img src="image_6.png" /><figcaption>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.</figcaption></figure>

HDACi promotes antimicrobial responses. These studies concentrated on the professional intramacrophage pathogen S. Typhimurium, given the clinical significance of devising strategies to clear intracellular reservoirs of this pathogen.

**HDACi promote mitochondrial ROS release from human macrophages.** Previous studies reported that treatment of breast cancer cells and human monocyte lymphoma U937 cells with various HDACi, including TSA, resulted in a loss of mitochondrial membrane potential and enhanced mitoROS production (30, 31). In addition, mitoROS was previously shown to play an important role in the clearance of S. Typhimurium, at least in mouse macrophages (32). Thus, we examined whether HDACi enhanced mitoROS production by HMDM upon bacterial challenge with S. Typhimurium. Generation of mitoROS in HMDM cotreated with TSA or SAHA during infection with S. Typhimurium was quantified by flow cytometric detection of MitoSOX Red fluorescence. S. Typhimurium infection, which triggers multiple TLRs, resulted in a modest increase in mitoROS production from HMDM at 6 h postinfection (Fig. 3), which is in accordance with a previous report that TLR activation triggers mitoROS production in mouse macrophages at this time point (32). Whereas HDACi alone did not promote mitoROS production from these cells, TSA and SAHA significantly enhanced S. Typhimurium-induced mitoROS production. Thus, cotreatment of human macrophages with HDACi during bacterial infection with S. Typhimurium increases mitoROS levels. To determine whether enhanced mitoROS production was likely to be causally related to HDACi-mediated bacterial clearance, we used a pharmacological approach. The mitochondrial stain MitoTracker Red CMXRos is reported to bind irreversibly to the polarized mitochondrial membrane (33, 34) and is thus predicted to impair the generation of mitoROS. We found that CMXRos blocked HDACi-mediated bacterial clearance (Fig. 4A and B) without having pronounced effects on macrophage viability (Fig. 4C). Although CMXRos-mediated inhibition of mitoROS production was not assessed because this stain

interferes with this assay due to spectral overlap, these data implicate enhanced mitoROS production as a likely mechanism accounting for HDACi-mediated clearance of intracellular bacteria.

**A specific inhibitor of HDAC6 reduces S. Typhimurium survival in human macrophages.** A recent study showed that the HDAC6 inhibitor tubastatin A improved survival and reduced bacterial loads in the blood in a mouse cecal ligation and puncture

<figure><img src="image_7.png" /></figure>

<figure><img src="image_8.png" /></figure>

<figure><img src="image_9.png" /><figcaption>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.</figcaption></figure>
<figure><img src="image_10.png" /><figcaption>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.</figcaption></figure>

model of sepsis, whereas this effect was not observed with the class I HDACi MS-275 (35). To determine whether HDACi-mediated antimicrobial responses in human macrophages are likely linked to HDAC6 inhibition, we treated HMDM and PMA-differentiated THP-1 macrophages with the HDAC6-selective inhibitor tubastatin A, and for comparison, we also used MS-275, which inhibits the class I HDACs, HDAC1 to -3 (2). As expected, treatment of HMDM with TSA (1 µM) or tubastatin A (20 µM), but not with MS-275 (20 µM), resulted in hyperacetylation of α-tubulin (Fig. 5A), which is a hallmark of HDAC6 inhibition (36). Cotreatment with tubastatin A significantly reduced intracellular bacterial loads of S. Typhimurium in HMDM (Fig. 5B) and PMA-differentiated THP-1 macrophages (Fig. 5D), with 20 µM tubastatin A having similar efficacy to 1 µM TSA in THP-1 cells. In contrast, cotreatment with MS-275 had no effect on intracellular bacterial loads. Neither tubastatin A nor MS-275 affected cell viability of S. Typhimurium-infected macrophages as assessed by the release of LDH (Fig. 5C and E). These data therefore suggest

that HDAC6, but not HDAC1, HDAC2, or HDAC3, constrains infection-induced mitoROS production and that selective inhibition of HDAC6 may promote mitoROS and boost clearance of intracellular bacteria by human macrophages. However, the fact that tubastatin A had effects similar to those of TSA on tubulin hyperacetylation (Fig. 5A), and yet was less effective than TSA in promoting bacterial clearance from HMDM (Fig. 5B), suggests that multiple HDACs may be involved in constraining human macrophage antimicrobial responses.

## DISCUSSION

HDACi are known to exert direct antimicrobial effects against some pathogens, such as *Plasmodium* species (37), and to reactivate latent HIV reservoirs to enable viral clearance (38, 39). As such, HDACi are being pursued as potential therapeutic agents in these infectious disease contexts. Although some mouse studies have suggested that HDACi may compromise host defense against bacterial and fungal pathogens (14, 19), very few studies have
examined the regulation of human macrophage antimicrobial re-
sponses by HDACi. Given some differences in innate immune
pathways between human and mouse, particularly in relation to
host defense (20, 22), we examined the effects of HDACi on hu-
man macrophage responses to bacterial challenge in this study.

Cotreatment of primary human macrophages or PMA-differ-
entiated THP-1 macrophages with HDACi reduced intracellular
loads of *E. coli* and S. Typhimurium at 24 h postinfection (Fig. 1A
to D) without affecting macrophage viability (Fig. 5C and E), mac-
rophage phagocytic activity (Fig. 2B to G), or bacterial growth
(Fig. 1E). Our findings are thus consistent with HDACs constrain-
ing human macrophage host defense responses upon bacterial
challenge, such that HDACi promote the clearance of intracellular
bacteria. Similar conclusions were recently reached using Ana-
plasma phagocytophilum (40) and Mycobacterium infection mod-
els in THP-1 cells (41). In our studies, we found that cotreatment
with broad-spectrum HDACi enhances S. Typhimurium-induc-
ible mitoROS levels in HMDM (Fig. 3), while treatment of these
cells with MitoTracker Red CMXRos, which is predicted to inhibit
mitoROS production, abrogates the ability of HDACi to promote
bacterial clearance (Fig. 4). Enhanced antimicrobial responses
were also observed in human macrophages upon cotreatment
with the HDAC6-selective inhibitor, tubastatin A, but not with the
HDAC1-selective inhibitor MS-275, also known as Entinostat
(Fig. 5). Collectively, these data are consistent with inhibition of
HDAC6 and/or related HDACs, enhancing human macrophage
antimicrobial responses likely via enhanced mitoROS production.

Previous studies with mouse macrophages have shown that
mitochondria are recruited to phagosomes downstream of TLR1,
TLR2, and TLR4 activation, leading to the release of mitoROS and
to the enhanced killing of S. Typhimurium (32). Inhibition of
HDAC activity has also been reported to increase the generation of
mitoROS in other cellular contexts. For example, treatment of the
human breast cancer cell lines MDA-MB-231 and MCF-7 with
TSA led to cell death, and this was dependent on mitoROS induc-
tion through impairment of the mitochondrial respiratory chain
(31). The HDACi spiruchostatins A and B were also shown to
stimulate the production of mitoROS, contributing to the apop-
tosis of human monocytic lymphoma U937 cells (30). Although
we found that HDACi boosted S. Typhimurium-inducible mi-
toROS generation (Fig. 3), these agents did not enhance cell death
in human macrophages challenged with this pathogen (Fig. 1F
and 5C and E). Thus, human macrophages appear to be somewhat
resistant to mitoROS-initiated cell death. However, our finding
that treatment with the MitoTracker Red CMXRos stain blocked
the ability of broad-spectrum HDACi to reduce bacterial loads
within HMDM (Fig. 4) is consistent with a causal role of HDACi-
induced mitoROS in the clearance of intracellular bacteria. Due to
its cationic lipophilic structure, this stain passively distributes into
the mitochondrial matrix to cause monovalent substitution of
thiols, preventing changes in the mitochondrial membrane poten-
tial (33, 34), which is linked to the generation of mitoROS (42).
However, further mechanistic studies are required to definitively
link the effects of HDACi on mitoROS production to bacterial
clearance. Enhanced antimicrobial activity was also observed
upon cotreatment with the selective HDAC6 inhibitor tubastatin
A but not with MS-275 (Fig. 5B and D), which inhibits HDAC1 to
-3. This finding is in keeping with a previous study that showed
that an HDAC6 inhibitor improved survival and reduced bacte-
remia in a murine sepsis model (35). Moreover, previous studies

have linked HDAC6 to the control of mitochondrial function (43, 44), and a very recent study reported that HDAC6 inhibition disrupted mitochondrial membrane potential and enhanced ROS production in melanoma cells (45). Collectively, these studies suggest that HDAC6 may have a regulatory role in controlling mitoROS production in infected macrophages such that inhibition of HDAC6 increases mitoROS concentrations to facilitate bacterial clearance. In contrast, on the basis of inhibitor studies (Fig. 5B and D), HDAC1 to -3 (class I HDACs) are unlikely to exert such effects.

Prior studies have reported that priming of mouse bone mar-
row-derived macrophages with TSA or another broad-spectrum
HDACi, valproic acid, reduced the uptake of *E. coli* and *Staphylo-
coccus aureus* and impaired the expression of phagocytic and op-
sonophagocytic receptors in these cells (19). Further, the authors
also showed that priming with HDACi reduced cellular ROS pro-
duction at 30 min post bacterial challenge as well as inducible
nitric oxide production, leading to impaired bacterial killing by
mouse macrophages. Similarly, mouse macrophages and den-
dritic cells pretreated with SAHA, valproic acid, or TSA had im-
paired antimicrobial responses to *E. coli*, *S. aureus*, and *Candida
albicans*, and in vivo pretreatment with HDACi increased suscep-
tibility to bacterial and fungal infections in mouse models (14).
These effects were linked to the downregulation of host defense
genes such as pattern recognition receptors and costimulatory
molecules as well as to the repression of macrophage cytokine
production through the Mi-2/NuRD complex (14). We similarly
found that overnight pretreatment of human macrophages with
broad-spectrum HDACi impaired phagocytic responses (Fig. 2B,
C, F, and G); however, we did not investigate whether HDACi
priming suppressed specific host defense genes in these cells.
Overall, these findings may raise some concerns about potential
applications for broad-spectrum HDACi, particularly for long-
term treatment regimes. However, clinical evidence linking
HDACi to infectious disease susceptibility is rather limited so far.
Indeed, systemic-onset juvenile idiopathic arthritis patients
treated for 12 weeks with the HDACi givinostat concomitantly
with glucocorticoids were not reported to display enhanced sus-
ceptibility to infections (46). Moreover, it is quite possible that the
anti-inflammatory effects of HDACi may actually reduce the im-
munopathology that is associated with many infectious diseases.

Nonetheless, concerns remain about the potential for broad-
spectrum HDACi to increase susceptibility to infectious diseases
by reducing phagocytosis and/or inflammation-mediated im-
mune cell recruitment. This highlights the need for studies with
selective HDACi, which may offer new opportunities to promote
antibacterial responses without compromising host defense. In-
deed, a very recent study showed that the HDAC6-selective inhib-
itor tubastatin A promotes bacterial clearance, reduces inflamma-
tory cytokine production, and improves survival in a mouse cecal
ligation puncture model of sepsis (35). This exciting finding raises
the possibility that pharmacological agents targeting HDAC6 may
have dual activities as anti-infective agents in promoting innate
immune-mediated bacterial clearance and in reducing immuno-
pathology caused by excessive inflammation. In this regard, bac-
terial and viral infections are well known to exacerbate inflamma-
tion and increase disease severity in various chronic conditions,
particularly respiratory diseases (47). Moreover, in mouse mod-
els, allergic airway disease is associated with impaired host defense
responses against bacterial pathogens such as *Haemophilus influ-
enzae (48). The potential to reduce inflammation and boost host defense with HDACi may thus be particularly attractive in the context of new therapies for inflammatory diseases of the airways. However, the fact that loss of HDAC2 expression and function has been associated with excessive inflammation and corticosteroid resistance in chronic lung diseases (49) again highlights the need for studies with selective HDACi in this context. Future studies assessing the efficacy of selective inhibitors of HDAC6, as well as other HDACs, in models of infection-mediated exacerbation of inflammatory diseases are thus clearly warranted. More specifically, short-term HDACi treatment may be predicted to reduce infection-induced exacerbations of inflammatory diseases.

In summary, the findings herein reveal that HDACs constrain infection-induced mitoROS production from human macrophages and that inhibition of these enzymes provides a means of enhancing macrophage antimicrobial responses. HDAC-selective inhibitors may provide new prospects as anti-infective agents, and since broad-spectrum HDACi are already approved for clinical use in cancer, there is clear potential for rapid translation of other HDACi into the clinic.

## ACKNOWLEDGMENTS

We thank the Australian Red Cross Blood Service for providing buffy coats used for the isolation of primary human monocytes, as well as Daniel Hohenhaus for editorial assistance.

## FUNDING INFORMATION

The National Health and Medical Research Council of Australia (NHMRC) provided funding under grants APP1047921 and APP1030169. M.J.S. is supported by an NHMRC Senior Research Fellowship (APP1107914). D.P.F. is supported by an NHMRC Senior Principal Research Fellowship (1027369). A.I. is supported by a University of Queensland Postdoctoral Fellowship and a University of Queensland Early Career Grant. R.K. is supported by an Australian Research Council Discovery Early Career Researcher Award (DE1310470). We thank the Australian Research Council for a Centre of Excellence in Advanced Molecular Imaging grant (CE140100011). The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

## REFERENCES

1. Minucci S, Pelicci PG. 2006. Histone deacetylase inhibitors and the promise of epigenetic (and more) treatments for cancer. Nat Rev Cancer 6:38–51. http://dx.doi.org/10.1038/nrc1779.

2. Gupta P, Reid RC, Iyer A, Sweet MJ, Fairlie DP. 2012. Towards isozyme-selective HDAC inhibitors for interrogating disease. Curr Top Med Chem 12:1479–1499. http://dx.doi.org/10.2174/156802612802652420.

3. Falkenberg KJ, Johnstone RW. 2014. Histone deacetylases and their inhibitors in cancer, neurological diseases and immune disorders. Nat Rev Drug Discov 13:673–691. http://dx.doi.org/10.1038/nrd4360.

4. Li Z, Zhu WG. 2014. Targeting histone deacetylases for cancer therapy: from molecular mechanisms to clinical implications. Int J Biol Sci 10:757–770. http://dx.doi.org/10.7150/ijbs.9067.

5. Mai A, Massa S, Rotili D, Cerbara I, Valente S, Pezzi R, Simeoni S, Ragno R. 2005. Histone deacetylation in epigenetics: an attractive target for anticancer therapy. Med Res Rev 25:261–309. http://dx.doi.org/10.1002/med.20024.

6. Zhang J, Zhong Q. 2014. Histone deacetylase inhibitors and cell death. Cell Mol Life Sci 71:3885–3901. http://dx.doi.org/10.1007/s00018-014-1656-6.

7. Choudhary C, Kumar C, Gnad F, Nielsen ML, Rehman M, Walther TC, Olsen JV, Mann M. 2009. Lysine acetylation targets protein complexes and co-regulates major cellular functions. Science 325:834–840. http://dx.doi.org/10.1126/science.1175371.

8. Shakespear MR, Halili MA, Irvine KM, Fairlie DP, Sweet MJ. 2011. Histone deacetylases as regulators of inflammation and immunity. Trends Immunol 32:335–343. http://dx.doi.org/10.1016/j.it.2011.04.001.

9. Sweet MJ, Shakespear MR, Kamal NA, Fairlie DP. 2012. HDAC inhibitors: modulating leukocyte differentiation, survival, proliferation and inflammation. Immunol Cell Biol 90:14–22. http://dx.doi.org/10.1038/icb.2011.88.

10. Halili MA, Andrews MR, Sweet MJ, Fairlie DP. 2009. Histone deacetylase inhibitors in inflammatory disease. Curr Top Med Chem 9:309–319. http://dx.doi.org/10.2174/156802609788085250.

11. Shakespear MR, Hohenhaus DM, Kelly GM, Kamal NA, Gupta P, Labzin LI, Schroder K, Garceau V, Barbero S, Iyer A, Hume DA, Reid RC, Irvine KM, Fairlie DP, Sweet MJ. 2013. Histone deacetylase 7 promotes Toll-like receptor 4-dependent proinflammatory gene expression in macrophages. J Biol Chem 288:25362–25374. http://dx.doi.org/10.1074/jbc.M113.496281.

12. Bode KA, Schroder K, Hume DA, Ravasi T, Heeg K, Sweet MJ, Dalpke AH. 2007. Histone deacetylase inhibitors decrease Toll-like receptor-mediated activation of proinflammatory gene expression by impairing transcription factor recruitment. Immunology 122:596–606. http://dx.doi.org/10.1111/j.1365-2567.2007.02678.x.

13. Grabiec AM, Korchynskyi O, Tak PP, Reedquist KA. 2012. Histone deacetylase inhibitors suppress rheumatoid arthritis fibroblast-like synoviocyte and macrophage IL-6 production by accelerating mRNA decay. Ann Rheum Dis 71:424–431. http://dx.doi.org/10.1136/ard.2011.154211.

14. Roger T, Lugrin J, Le Roy D, Goy G, Mombelli M, Koessler T, Ding XC, Chanson A-L, Reymond MK, Miconnet I, Schrenzel J, François P, Calandra T. 2011. Histone deacetylase inhibitors impair innate immune responses to Toll-like receptor agonists and to infection. Blood 117:1205–1217. http://dx.doi.org/10.1182/blood-2010-05-284711.

15. Wang B, Liu TY, Lai CH, Rao YH, Choi MC, Chi JT, Dai JW, Rathmell JC, Yao TP. 2014. Glycolysis-dependent histone deacetylase 4 degradation regulates inflammatory cytokine production. Mol Biol Cell 25:3300–3307. http://dx.doi.org/10.1091/mbc.E13-12-0757.

16. Bongartz T, Sutton AJ, Sweeting MJ, Buchan I, Matteson EL, Montori V. 2006. Anti-TNF antibody therapy in rheumatoid arthritis and the risk of serious infections and malignancies: systematic review and meta-analysis of rare harmful effects in randomized controlled trials. JAMA 295:2275–2285. http://dx.doi.org/10.1001/jama.295.19.2275.

17. Galloway JB, Hyrich KL, Mercer LK, Dixon WG, Fu B, Ustianowski AP, Watson KD, Lunt M, Symmons DPM. 2011. Anti-TNF therapy is associated with an increased risk of serious infections in patients with rheumatoid arthritis especially in the first 6 months of treatment: updated results from the British Society for Rheumatology Biologics Register with special emphasis on risks in the elderly. Rheumatology 50:124–131. http://dx.doi.org/10.1093/rheumatology/keq242.

18. Keane J, Gershon S, Wise RP, Mirabile-Levens E, Kasznica J, Schwieterman WD, Siegel JN, Braun MM. 2001. Tuberculosis associated with infliximab, a tumor necrosis factor alpha-neutralizing agent. N Engl J Med 345:1098–1104. http://dx.doi.org/10.1056/NEJMoa011110.

19. Mombelli M, Lugrin J, Rubino I, Chanson A-L, Giddey M, Calandra T, Roger T. 2011. Histone deacetylase inhibitors impair antibacterial defenses of macrophages. J Infect Dis 204:1367–1374. http://dx.doi.org/10.1093/infdis/jir553.

20. Ariffin JK, Sweet MJ. 2013. Differences in the repertoire, regulation and function of Toll-like receptors and inflammasome-forming Nod-like receptors between human and mouse. Curr Opin Microbiol 16:303–310. http://dx.doi.org/10.1016/j.mib.2013.03.002.

21. Mestas J, Hughes CCW. 2004. Of mice and not men: differences between mouse and human immunology. J Immunol 172:2731–2738. http://dx.doi.org/10.4049/jimmunol.172.5.2731.

22. Schroder K, Irvine KM, Taylor MS, Bokil NJ, Le Cao KA, Masterman KA, Labzin LI, Semple CA, Kapetanovic R, Fairbairn L, Akalin A, Faulkner GJ, Baillie JK, Gongora M, Daub CO, Kawaji H, McLachlan GJ, Goldman N, Grimmond SM, Carninci P, Suzuki H, Hayashizaki Y, Lenhard B, Hume DA, Sweet MJ. 2012. Conservation and divergence in Toll-like receptor 4-regulated gene expression in primary human versus mouse macrophages. Proc Natl Acad Sci U S A 109:E944–E953. http://dx.doi.org/10.1073/pnas.1110156109.

23. Shay T, Jojic V, Zuk O, Rothamel K, Puyraimond-Zemmour D, Feng T, Wakamatsu E, Benoist C, Koller D, Regev A. 2013. Conservation and divergence in the transcriptional programs of the human and mouse immune systems. Proc Natl Acad Sci U S A 110:2946–2951. http://dx.doi.org/10.1073/pnas.1222738110.
24. Irvine KM, Andrews MR, Fernandez-Rojo MA, Schroder K, Burns CJ, Su S, Wilks AF, Parton RG, Hume DA, Sweet MJ. 2009. Colony-stimulating factor-1 (CSF-1) delivers a proatherogenic signal to human macrophages. J Leukoc Biol 85:278-288. http://dx.doi.org/10.1189/jlb .0808497.

25. Schaale K, Peters KM, Murthy AM, Fritzsche AK, Phan MD, Totsika M, Robertson AAB, Nichols KB, Cooper MA, Stacey KJ, Ulett GC, Schroder K, Schembri MA, Sweet MJ. 2016. Strain- and host species-specific inflammasome activation, IL-1β release, and cell death in macrophages infected with uropathogenic *Escherichia coli*. Mucosal Immunol 9:124-136. http://dx.doi.org/10.1038/mi.2015.44.

26. Hoiseth SK, Stocker BA. 1981. Aromatic-dependent *Salmonella* Typhi-murium are non-virulent and effective as live vaccines. Nature 291:238-239. http://dx.doi.org/10.1038/291238a0.

27. Blattner FR, Plunkett G, III, Bloch CA, Perna NT, Burland V, Riley M, Collado-Vides J, Glasner JD, Rode CK, Mayhew GF, Gregor J, Davis NW, Kirkpatrick HA, Goeden MA, Rose DJ, Mau B, Shao Y. 1997. The complete genome sequence of *Escherichia coli* K-12. Science 277:1453-1462. http://dx.doi.org/10.1126/science.277.5331.1453.

28. Bokil NJ, Totsika M, Carey AJ, Stacey KJ, Hancock V, Saunders BM, Ravasi T, Ulett GC, Schembri Ma, Sweet MJ. 2011. Intramacrophage survival of uropathogenic *Escherichia coli*: differences between diverse clinical isolates and between mouse and human macrophages. Immuno-biology 216:1164-1171. http://dx.doi.org/10.1016/j.imbio.2011.05.011.

29. Mimura N, Asano A. 1976. Synergistic effect of colchicine and cytochalasin D on phagocytosis by peritoneal macrophages. Nature 261:319-321. http://dx.doi.org/10.1038/261319a0.

30. Rehman MU, Jawaid P, Yoshihisa Y, Li P, Zhao QL, Narita K, Katoh T, Kondo T, Shimizu T. 2014. Spiruchostatin A and B, novel histone deacetylase inhibitors, induce apoptosis through reactive oxygen species-mitochondria pathway in human lymphoma U937 cells. Chem Biol Interact 221:24-34. http://dx.doi.org/10.1016/j.cbi.2014.07.004.

31. Sun S, Han Y, Liu J, Fang Y, Tian Y, Zhou J, Ma D, Wu P. 2014. Trichostatin A targets the mitochondrial respiratory chain, increasing mitochondrial reactive oxygen species production to trigger apoptosis in human breast cancer cells. PLoS One 9:e91610. http://dx.doi.org/10.1371 /journal.pone.0091610.

32. West PA, Brodsky IE, Rahner C, Woo DK, Erdjument-Bromage H, Tempst P, Walsh MC, Choi Y, Shadel GS, Ghosh S. 2011. TLR signalling augments macrophage bactericidal activity through mitochondrial ROS. Nature 472:476-480. http://dx.doi.org/10.1038/nature09973.

33. Kroemer G, Zamzami N, Susin SA. 1997. Mitochondrial control of apoptosis. Immunol Today 18:44-51. http://dx.doi.org/10.1016/S0167 -5699(97)80014-X.

34. Marchetti P, Decaudin D, Macho A, Zamzami N, Hirsch T, Susin SA, Kroemer G. 1997. Redox regulation of apoptosis: impact of thiol oxidation status on mitochondrial function. Eur J Immunol 27:289-296. http: //dx.doi.org/10.1002/eji.1830270142.

35. Li Y, Zhao T, Liu B, Halaweish I, Mazitschek R, Duan X, Alam HB. 2015. Inhibition of histone deacetylase 6 improves long-term survival in a lethal septic model. J Trauma Acute Care Surg 78:378-385. http://dx.doi .org/10.1097/TA.0000000000000510.

36. Zhao Z, Xu H, Gong W. 2010. Histone deacetylase 6 (HDAC6) is an independent deacetylase for alpha-tubulin. Protein Pept Lett 17:555-558. http://dx.doi.org/10.1747/092986610791112620.

37. Andrews KT, Tran TN, Fairlie DP. 2012. Towards histone deacetylase inhibitors as new antimalarial drugs. Curr Pharm Des 18:3467-3479.

38. Wightman F, Ellenberg P, Churchill M, Lewin SR. 2012. HDAC inhibitors in HIV. Immunol Cell Biol 90:47-54. http://dx.doi.org/10.1038/icb .2011.95.

39. Margolis DM. 2011. Histone deacetylase inhibitors and HIV latency. Curr Opin HIV AIDS 6:25-29. http://dx.doi.org/10.1097/COH .0b013e328341242d.

40. Garcia-Garcia JC, Barat NC, Trembley SJ, Dumler JS. 2009. Epigenetic silencing of host cell defense genes enhances intracellular survival of the rickettsial pathogen *Anaplasma phagocytophilum*. PLoS Pathog 5:e1000488. http://dx.doi.org/10.1371/journal.ppat.1000488.

41. Wang Y, Curry HM, Zwilling BS, Lafuse WP. 2005. Mycobacteria inhibition of IFN-gamma induced HLA-DR gene expression by up-regulating histone deacetylation at the promoter region in human THP-1 monocytic cells. J Immunol 174:5687-5694. http://dx.doi.org/10.4049 /jimmunol.174.9.5687.

42. Go Y-M, Park H, Koval M, Orr M, Reed M, Liang Y, Smith D, Pohl J, Jones DP. 2010. A key role for mitochondria in endothelial signaling by plasma cysteine/cystine redox potential. Free Radic Biol Med 48:275-283. http://dx.doi.org/10.1016/j.freeradbiomed.2009.10.050.

43. Chen J, Shi X, Padmanabhan R, Wang Q, Wu Z, Stevenson SC, Hild M, Garza D, Li H. 2008. Identification of novel modulators of mitochondrial function by a genome-wide RNAi screen in *Drosophila melanogaster*. Genome Res 18:123-136.

44. Kamemura K, Ogawa M, Ohkubo S, Ohtsuka Y, Shitara Y, Komiya T, Maeda S, Ito A, Yoshida M. 2012. Depression of mitochondrial metabolism by downregulation of cytoplasmic deacetylase, HDAC6. FEBS Lett 586:1379-1383. http://dx.doi.org/10.1016/j.febslet.2012.03.060.

45. Bai J, Lei Y, An GL, He L. 2015. Down-regulation of deacetylase HDAC6 inhibits the melanoma cell line A375.S2 growth through ROS-dependent mitochondrial pathway. PLoS One 10:e0121247. http://dx.doi.org/10 .1371/journal.pone.0121247.

46. Vojinovic J, Damjanov N, D'Urzo C, Furlan A, Susic G, Pasic S, Iagaru N, Stefan M, Dinarello CA. 2011. Safety and efficacy of an oral histone deacetylase inhibitor in systemic-onset juvenile idiopathic arthritis. Arthritis Rheum 63:1452-1458. http://dx.doi.org/10.1002/art.30238.

47. Starkey MR, Jarnicki AG, Essilfie AT, Gellatly SL, Kim RY, Brown AC, Foster PS, Horvat JC, Hansbro PM. 2013. Murine models of infectious exacerbations of airway inflammation. Curr Opin Pharmacol 13:337-344. http://dx.doi.org/10.1016/j.coph.2013.03.005.

48. Essilfie AT, Simpson JL, Dunkley ML, Morgan LC, Oliver BG, Gibson PG, Foster PS, Hansbro PM. 2012. Combined *Haemophilus influenzae* respiratory infection and allergic airways disease drives chronic infection and features of neutrophilic asthma. Thorax 67:588-599. http://dx.doi .org/10.1136/thoraxjnl-2011-200160.

49. Barnes PJ, Adcock IM, Ito K. 2005. Histone acetylation and deacetylation: importance in inflammatory lung diseases. Eur Respir J 25:552-563. http://dx.doi.org/10.1183/09031936.05.00117504.