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(The FASEB Journal. 2006;20:1315-1327.)
© 2006 FASEB

LPS regulates proinflammatory gene expression in macrophages by altering histone deacetylase expression

Hnin Thanda Aung*,,{dagger},1, Kate Schroder*,,{dagger},1, Stewart R. Himes*,,{dagger}, Kristian Brion*,,{dagger}, Wendy van Zuylen*,,{dagger}, Angela Trieu*,,{dagger}, Harukazu Suzuki{ddagger}, Yoshihide Hayashizaki{ddagger}, David A. Hume*,,{dagger}, Matthew J. Sweet*,,{dagger} and Timothy Ravasi*,,{dagger},{ddagger},2

* Cooperative Research Centre for Chronic inflammatory Diseases and

{dagger} Australian Research Council Special Research Centre for Functional and Applied Genomics, Institute for Molecular Bioscience, University of Queensland, Australia;

{ddagger} Laboratory for Genome Exploration Research Group, RIKEN Genomic Sciences Center, RIKEN Yokohama Institute, Tsurumi-ku, Yokohama, Japan; and

{dagger} Department of Bioengineering, University of California, San Diego, California, USA

2Correspondence: Department of Bioengineering, University of California, San Diego, CA, USA. E-mail: travasi{at}bioeng.ucsd.edu


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Bacterial LPS triggers dramatic changes in gene expression in macrophages. We show here that LPS regulated several members of the histone deacetylase (HDAC) family at the mRNA level in murine bone marrow-derived macrophages (BMM). LPS transiently repressed, then induced a number of HDACs (Hdac-4, 5, 7) in BMM, whereas Hdac-1 mRNA was induced more rapidly. Treatment of BMM with trichostatin A (TSA), an inhibitor of HDACs, enhanced LPS-induced expression of the Cox-2, Cxcl2, and Ifit2 genes. In the case of Cox-2, this effect was also apparent at the promoter level. Overexpression of Hdac-8 in RAW264 murine macrophages blocked the ability of LPS to induce Cox-2 mRNA. Another class of LPS-inducible genes, which included Ccl2, Ccl7, and Edn1, was suppressed by TSA, an effect most likely mediated by PU.1 degradation. Hence, HDACs act as potent and selective negative regulators of proinflammatory gene expression and act to prevent excessive inflammatory responses in macrophages.—Aung, H. T., Schroder, K., Himes, S. R., Brion, K., van Zuylen, W., Trieu, A., Suzuki, H., Hayashizaki, Y., Hume, D. A., Sweet, M. J., Ravasi, T. LPS regulates proinflammatory gene expression in macrophages by altering histone deacetylase expression.


Key Words: transcriptional regulatory networks • epigenetic • histone code • innate immunity • inflammation


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
TRANSCRIPTIONAL ACTIVATION OF mammalian genes requires chromatin remodeling (1 2 3 4) . Transcriptional activators typically recruit enzymes that modify the tails of histones through acetylation, methylation, and phosphorylation (5 , 6) . Perhaps the most striking demonstration of this process was the recent study of the actions of the estrogen receptor on the model pS2 promoter, which involves cyclical association and dissociation of at least 46 nuclear proteins involved in chromatin remodeling (7) .

As part of the innate immune response, macrophages recognize and are activated by conserved components of microorganisms (pathogen-associated molecular patterns), and respond with a massive alteration in transcriptional output. Expression profiling has revealed thousands of genes that are induced or repressed in macrophages in response to the classical activating agent LPS (8 9 10) . The regulated genes cluster at various locations in the mouse genome, and their expression varies greatly between macrophages from different mouse strains (9) .

The response to LPS is initiated via a signaling complex that includes CD14, toll-like receptor 4 (TLR4), and MD2. Upon activation, adaptor proteins are recruited to the cytoplasmic tail of TLR4, and subsequent signaling generates multiple transcriptional regulators, including NF-{kappa}B, AP1, C/EBP, IRF-3, and numerous other transcription factors (11 12 13 14) . The LPS target genes are induced or repressed in a temporal cascade. Early response genes, including those encoding many proinflammatory cytokines and chemokines, are induced transiently, peaking at ~2–4 h, and are repressed progressively from that time. Other genes are induced at later time points, up to 24 h post-stimulation, and many are targets of inducible transcription factors or of secreted proteins that act in an autocrine manner to elicit secondary signaling cascades (15) . Repression of the earlier response genes is partly irreversible and has been associated with the phenomenon of LPS tolerance (16) .

Given the scale of the transcriptional response to LPS in macrophages, we reasoned there might be global alterations in the expression and/or function of chromatin remodeling genes across the temporal cascade. There have been few published studies of chromatin remodeling in activated macrophages, mostly relating to selected modifications of histones associated with specific promoters (17 18 19 20 21) . In this study, we used expression profiling and quantitative polymerase chain reaction (PCR) to demonstrate that many chromatin and DNA-modifying enzymes are themselves transcriptionally regulated by LPS and that there is a temporal cascade of expression of positive, followed by negative transcriptional regulators. Here we identify LPS-inducible genes that are specifically targeted for inactivation by histone deacetylases, and show that overexpression of Hdac-8 selectively blocked LPS-inducible gene expression. This study represents the first comprehensive analysis of the impact of chromatin remodeling on transcriptional regulation in macrophages, and suggests that regulated histone deacetylation has a global effect on the extent and nature of the inducible transcriptional regulatory cascade in macrophages.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cell culture and reagents
All cells were cultured in RPMI 1640 media supplemented with 10% FCS, 20 U/ml penicillin, 20 µg/ml streptomycin, and 2 mM L-glutamine (complete media) in an incubator at 37°C with 5% CO2. The murine macrophage cell line RAW264 was from the American Type Culture Collection (ATCC) (Manassas, VA, USA). Bone marrow-derived macrophages (BMM) were obtained from femurs of 6- to 8-wk-old C57BL/6J mice as described previously (22) . Briefly, bone marrow cavities were flushed with complete media. Bone marrow cells, recovered by centrifugation at 400 g for 5 min, were cultured for 6 days in complete medium containing 104 U/ml recombinant human colony-stimulating factor 1 (CSF-1) (a gift from Chiron, Emeryville, CA, USA). On day 6, BMM were harvested and seeded at 3 x 106 cells/10 cm tissue culture dish in complete medium + CSF-1. On the following day, BMM were treated with 10 ng/ml LPS (Salmonella minnesota, Sigma-Aldrich, St. Louis, MO, USA) for the indicated times. Trichostatin A (TSA) (Sigma) was dissolved in 100% ethanol, stored at –20°C, and used at a final concentration in cell culture of 0.5 µM unless stated otherwise.

Generation of constructs and stable and transient transfections in RAW264
The pEF6HDAC-8 plasmid was constructed by amplifying the mouse Hdac-8 coding region from mouse macrophage cDNA, using specific primers (Table 1 ) and Pfx polymerase (Invitrogen, Carlsbad, CA, USA). The PCR product was cloned into the mammalian expression plasmid, pEF6/V5-His-TOPO (Invitrogen). The integrity of the coding region was confirmed by DNA sequencing. Protein expression was verified by immunoblotting cell lysates from RAW264 cells transiently transfected with pEF6HDAC-8, with an anti-V5 monoclonal antibody (mAb) (Serotec, Raleigh, NC, USA). The plasmid, pGL2b1.4KbCox2, was made by PCR amplification of a 1.4 kb region upstream of the ATG of Cox-2 from C57Bl6/J genomic DNA using specific primers (Table 1) for insertion into pGL2B (Promega, Madison, WI, USA). RAW264 cells stably expressing Hdac-8 were generated by electroporating 5 x 106 cells in 250 µl complete media, containing 20 mM HEPES and 10 µg of pEF6HDAC-8, at 280 V and 1000 µF capacitance on a Bio-Rad Gene Pulser (Bio-Rad, Hercules, CA, USA). One day post-transfection, cells were selected with 2 µg/ml Blastocidin (Invitrogen). After 10–14 days, stably selected colonies were pooled and screened for Hdac-8 expression by intracellular staining using an antibody (Ab) against the V5 tag, followed by detection with a FACSCalibur flow cytometer (Becton Dickinson, Rockville, MD, USA). RAW264 cells stably transfected with pGL2b1.4KbCox2 were generated identically, except that 1 µg of pNT-neo, which confers resistance to G418, was included in electroporations, and colonies were selected on the basis of resistance to 200 µg/ml G418. RAW264 pools stably transfected with pGL2b1.4KbCox2 were stimulated with LPS for 36 h, lysed, and assayed for luciferase activity according to the manufacturer’s instructions (Roche, Indianapolis, IN, USA). Protein concentration in lysates was determined by bicinchoninic acid (BCA) protein assay (Pierce, Rockford, IL, USA); the level of luciferase activity, calculated as light units/µg of protein assayed, was displayed as relative light units. For transient transfection analysis, electroporations were performed as described above with either 10 µg pGL-2B or 10 µg pGL2b1.4KbCox2. After 24 h cells were stimulated with medium, LPS, TSA, or LPS + TSA for 7 h. Lysates were prepared and assayed for total protein and luciferase activity as described above.


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  		Table 1. List of primer sequences used for conventional PCR, real-time PCR, PCR cloning, and gel shift assay

Quantitative real-time PCR
Total RNA was extracted from BMM using RNAeasy Midi columns (Qiagen, Chatsworth, CA, USA) following the manufacturer’s protocol. Total RNA (1 µg) from each sample was treated with DNase I (Ambion, Austin, TX, USA) and reverse transcribed using a 17-mer oligo(dT) and the Superscript III RNase H reverse transcriptase kit or Superscript III First-Strand Synthesis System for RT-PCR (Invitrogen), according to the manufacturer’s instructions. Negative control samples (no first-strand synthesis) were prepared by performing reverse transcription reactions in the absence of reverse transcriptase. Quantitative real-time PCR was carried out with cDNA using the SYBR Green kit (Applied Biosystems, Foster City, CA, USA), gene-specific primers and an ABI Prism 7000 Sequence Detection System (Applied Biosystems). The threshold cycle (Ct) value was calculated from amplification plots, and gene expression was normalized using the Ct of the housekeeping gene hypoxanthine guanine phosphoribosyl transferase (hprt). Gene-specific primer pairs were designed using Primer3 software (http://frodo.wi.mit.edu/cgi-bin/primer3/primer3_www.cgi), with an optimal primer size of 20 bases, amplicon size of 100 bp, and annealing temperature of 65°C. All primer sequences are displayed in Table 1 .

Immunoblotting
BMM were treated with 10 ng/ml of LPS and/or TSA, as required, before either isolation of nuclei and extraction of protein or collection of whole cell extract according to established protocols (23) . The concentration of protein in the extracts was determined by BCA protein assay according to the manufacturer’s protocol (Pierce). Samples (5 µg protein) were resolved on precast 12% polyacrylamide gels (Invitrogen) before blotting onto methanol-activated immobilon-P PDVF membranes (Millipore, Billerica, MA, USA). Blots were blocked and probed with specific antibodies to PU.1 (Santa Cruz, Biotechnology, Santa Cruz, CA, USA), ERK-1/2 (Cell Signaling Technology, Beverly, MA, USA), Cox-2 (Cell Signaling Technology), or STAT-1 (Cell Signaling Technology). Bands were visualized using a horseradish peroxidase-conjugated anti-rabbit IgG Ab (Cell Signaling Technology) and chemiluminescence (Amersham Biosciences, Piscataway, NJ, USA). Signals on X-ray film were scanned using a Bio-Rad GS 800 densitometer.

Endothelin-1 ELISA
BMM were treated with medium, TSA (500 nM), LPS (10 ng/ml), or TSA+LPS over a 24 h time course. Levels of endothelin-1 in BMM culture supernatants were determined using an endothelin-1 ELISA kit (R&D Systems, Minneapolis, MN, USA), as per the instructions of the manufacturer.

Chromatin immunoprecipitation (ChIP) assay
Cross-linking was performed by adding 1% formaldehyde directly into tissue culture dishes containing 3 x 106 BMM at different time points after LPS treatment, followed by incubation at room temperature for 10 min. Cells were washed twice with PBS, collected, and pelleted by centrifugation at 400 g for 5 min. Nuclei, isolated as described previously (24) , were resuspended in sonication buffer (containing 50 mM Tris-HCl, pH 8.1, 10 mM EDTA, 1% SDS, and protease inhibitors) and incubated on ice for 10 min to lyse the nuclei. Nuclear extracts were then sonicated to obtain ~200-1000 bp fragments of chromatin. Immunoprecipitation was carried out according to the protocol provided by Upstate Biotechnology (Lake Placid, NY, USA). Briefly, chromatin was diluted 10-fold in ChIP dilution buffer. A small amount of chromatin was kept aside at this step to be used as input control in subsequent PCR reactions. Antibodies antiacetyl-histone H4 (K12) (Abcam), anti-RNA-polymerase II, and antihistone-H4 (Upstate) were incubated with diluted chromatin at 4°C overnight. Immunoprecipitations were also carried out without Ab (no Ab controls). Protein A Sepharose (Amersham Biosciences), blocked with sheared salmon sperm DNA, was used to collect Ab-chromatin complexes. Immune complexes were then washed once with low salt immune complex wash buffer (containing 0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl, pH 8.1, 150 mM NaCl), once with high salt immune complex wash buffer (containing 0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl, pH 8.1500 mM NaCl), once with LiCl immune complex wash buffer (0.25M LiCl, 1% IGEPAL-CA630, 1% deoxycholic acid, 1 mM EDTA, 10 mM Tris-HCl, pH 8.1), and twice with sterile TE buffer. The chromatin (histone-DNA complexes) were eluted with freshly prepared elution buffer (containing 1% SDS and 0.1M NaHCO3), followed by reverse cross-linking with 0.3M NaCl at 65°C for 4–5 h. DNA was then recovered by phenol-chloroform extraction and ethanol precipitation, followed by PCR amplification, by using primers listed in Table 1 . The PCR conditions used were initial denaturation at 95°C for 5 min, followed by 30 cycles of 95°C (30 s), 58°C (30 s), and 72°C (30 s). Alternatively, recovered DNA was subjected to real-time PCR as described above.

Nuclear extract preparation and gel shift assays
The preparation of nuclear extracts as well as the method used for gel shift assays has been described (25) . The probes used for gel shift assays (shown in Table 1 ) were end-labeled using T4 polynucleotide kinase and [{gamma}-32P]-ATP, and separated on NAP-5 columns (Amersham Biosciences). Nuclear proteins bound to radiolabeled probe were resolved using 8% discontinuous polyacrylamide gel electrophoresis. Gels were dried onto 3M paper and visualized by exposure to X-ray film.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Chromatin remodeling on macrophage activation with LPS
An earlier study from our group documented the time course of LPS stimulation of murine BMM, using RIKEN full-length mouse cDNA microarrays (9) . In BMM from C57Bl6/J mice, ~30% of genes were regulated by LPS at some time over a 21 h time course, indicating that LPS has a global effect on gene expression in macrophages (9) . Many transcripts that encode proteins associated with chromatin remodeling were also acutely modulated by LPS (data not shown). Broadly speaking, between 2 and 7 h, when the majority of LPS-inducible genes reach peak expression, there was an up-regulation of transcripts encoding nuclear proteins associated with transcriptional activation and "opening up" of chromatin. In particular, transcripts encoding members of the histone acetyltransferase complex, CBP, p300, Cited-2, and P/CAF (17 ,19 ,26) , were transiently up-regulated on LPS stimulation as assessed by real-time PCR (Fig. 1 ), consistent with a previous report (26) . Whereas CBP, p300, and P/CAF expression peaked 7 h after stimulation, Cited-2 (CBP/p300-interacting transactivator with ED-rich tail 2) peaked at 2 h. Combined with the scale of the transcriptional response to LPS, these data led us to the hypothesis that the transcriptional activation that occurs early in the LPS response is supported by increased production of positive regulators. This pattern may be amplified by a coordinate decrease in mRNA encoding negative regulators such Sirt 1, 2, and 3 and Ncor, which was also evident in our analysis (data not shown).


Figure 1
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  		Figure 1. Regulation of components of the histone acetyltransferase complex by LPS in BMM. BMM were stimulated with LPS (10 ng/ml) for 0, 2, 7, or 21 h. Levels of p300, P/CAF, Cited-2, and CBP mRNAs were quantified by real-time PCR. Data points represent the average of triplicate determinations ± SD.

Expression and regulation of histone deacetylases in macrophages
Upon long-term treatment of BMM with LPS (21 h), the transcriptional profile returned to a state that was similar but not identical to unstimulated cells (9) . Members of the HDAC family repress transcription through histone modification (27 28 29 30 31) . We hypothesized that regulation of HDAC mRNA expression by LPS might contribute to the early induction and subsequent repression of proinflammatory gene expression. We therefore assessed mRNA levels for HDAC members in BMM over an LPS time course (Fig. 2 ). Most of the HDACs analyzed were expressed at very low levels in BMM relative to hprt. Hdac-1, at least, is known to be expressed and functional in macrophages (32) , so the mRNA level does not necessarily correlate with protein level or function. As predicted, LPS transiently repressed expression of several of the HDACs (Hdac-4, 5, 6, and 7) at time points (4, 6 h) that correlated with maximal activation of proinflammatory gene expression. By 24 h after LPS treatment, Hdac-4, 5, and 7 mRNA levels had returned to greater than those present in unstimulated cells. In contrast, induction of Hdac-1 mRNA by LPS was more rapid and peaked at 8 h post-LPS stimulation, suggesting it may play a divergent role from other HDACs in regulating LPS-inducible gene expression in macrophages.


Figure 2
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  		Figure 2. Regulation of HDAC mRNA expression by LPS in BMM. BMM were stimulated with LPS (10 ng/ml) for 0, 2, 4, 6, 8, or 24 h. RNA and cDNA was prepared, and Hdac-1, 3, 4, 5, 6, 7, 8, 9, 10, and 11 mRNA levels were quantitated by real-time PCR. Data points represent the average of triplicate determinations ± SD. Similar results were obtained in 3 independent experiments.

The histone deacetylase inhibitor, trichostatin A, differentially regulates induction of subclasses of LPS-responsive genes in macrophages
The expression analysis described above indicated that the transient repression followed by the late induction of HDACs might serve to allow for activation and subsequent down-regulation of proinflammatory gene expression. To determine whether HDACs do indeed act to switch off LPS-inducible gene expression and to identify the specific genes that HDACs target, we performed expression array profiling using microarray chips enriched for LPS-inducible transcripts on BMM responding to LPS, with or without TSA treatment. Three distinct classes of LPS-responsive genes were identified: 1) LPS-inducible transcripts that were unaffected by TSA pretreatment; 2) LPS-inducible transcripts that were superinduced by TSA pretreatment; 3) LPS-induced transcripts that were repressed or blocked completely by TSA treatment (data not shown). Each class effectively acts as an internal control for the others, suggesting that TSA-sensitive HDACs have distinct roles on different classes of target genes. The arrays used were not comprehensive and contain many replicate targets. Hence, gene lists for the three classes do not provide global coverage and are therefore not reported in this study. Instead, we focused on a small number of genes and confirmed the selective effects of TSA on LPS-inducible gene expression in BMM by real-time PCR. Cyclooxygenase 2 (Cox-2), chemokine (C-X-C motif) ligand 2 (Cxcl2), and IFN-induced protein with tetratricopeptide repeats 2 (Ifit2) mRNAs were all strongly up-regulated at 10 h post-LPS treatment and were superinduced by TSA + LPS (Fig. 3 a). Chemokine (C-C motif) ligand 2 (Ccl2), Ccl7, and endothelin 1 (Edn1) mRNAs were also inducible by LPS (10 h), but the response was substantially impaired in the presence of TSA (Fig. 3a ). TSA also impaired basal mRNA expression of Ccl2 and Ccl7, but not Edn1. In isolation, these data suggest that HDACs act as negative regulators of Cox-2 and coregulated genes, but function as positive regulators of Ccl7 and coregulated genes. We confirmed that these divergent effects of TSA on gene expression in BMM were also apparent at the protein level. TSA treatment enhanced the level of LPS-induced Cox-2 protein in BMM (Fig. 3b ), but completely blocked production of LPS-induced endothelin-1 in BMM culture supernatants over a 24 h time course (Fig. 3c ). Subsequent analysis focused on Cox-2 and Ccl7 as representatives of either class of gene.


Figure 3
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  		Figure 3. The effect of the histone deacetylase inhibitor TSA on LPS-inducible gene expression in BMM. a) BMM were treated with medium, LPS (10 ng/ml), TSA (0.5 µM), or LPS + TSA for 10 h. RNA and cDNA was prepared, and levels of Cox-2, Cxcl2, Ifit2, Ccl2, Ccl7, and Edn1 mRNAs were estimated by real-time PCR. Data points represent the average of triplicate determinations ± SD. Similar results were obtained in at least 3 independent experiments. b) BMM were treated with medium or LPS (10 ng/ml). After another 8 h, BMM were stimulated with medium or TSA (0.5 µM). 24 h after the initial LPS stimulation, whole cell lysates were prepared from all samples (con, LPS, TSA, LPS+TSA). Levels of Cox-2 protein, relative to the STAT-1 loading control, were quantified by immunoblotting and densitometry. c) BMM were treated with medium, LPS (10 ng/ml), TSA (0.5 µM) or LPS + TSA over a 24 h time course. Levels of endothelin-1 secreted into culture supernatants were determined by ELISA. Data points represent the average of duplicates and the errors represent the range.

Since LPS transiently repressed, then induced many of the HDACs (Fig. 2) , we also assessed the kinetics of Cox-2 (LPS/TSA superinduced) and Ccl7 (TSA repressed) mRNA regulation by LPS in BMM (Fig. 4 a). Cox-2 and Ccl7 mRNAs peaked at 4–6 h in response to LPS (Fig. 4a ) and correlated with the transient repression of Hdac-4, 5, 6, and 7 expression (Fig. 2) . The subsequent decline in Cox-2 and Ccl7 mRNA levels at 8–24 h (Fig. 4a ) also correlated with the delayed induction of Hdac-1, 4, 5, and 7 mRNAs (Fig. 2) . Hence, the kinetics of Cox-2 and Ccl7 regulation by LPS was consistent with HDACs as negative regulators of Cox-2 gene expression, but was not consistent with HDACs as positive regulators of Ccl7 gene expression. Since we hypothesized that HDACs were responsible for the decline in expression of Cox-2 (and similarly regulated genes) that occurs 6–8 h after LPS stimulation (Fig. 4a ), we also assessed the effect of TSA when given at later time points after the initial stimulation with LPS. TSA was given to BMM after 7 h of LPS treatment, and the effect on subsequent declines in mRNA levels was assessed 11 and 20 h after the initial stimulation with LPS (Fig. 4b ). Consistent with the hypothesis, the decay in mRNA expression of the superinduced gene Cox-2 was prevented by TSA. Conversely, mRNA for the TSA-repressed gene Ccl7 decayed more rapidly in the presence of TSA.


Figure 4
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  		Figure 4. Kinetics of regulation of TSA-sensitive genes by LPS. a) BMM were stimulated with LPS (10 ng/ml) for 0, 2, 4, 6, 8, or 24 h. RNA and cDNA was prepared, and Cox-2 (filled squares) and Ccl7 (open circles) mRNA levels were quantitated by real-time PCR. Data points represent the average of duplicate determinations ± SD. Similar results were obtained in 2 independent experiments. b) BMM were stimulated with LPS (10 ng/ml) for 0, 4, 7, 11, and 20 h. At 7 h post-LPS treatment, the 11 and 20 h treatment groups were treated with either medium or TSA (0.5 µM). RNA and cDNA was prepared and Cox-2 and Ccl7 mRNA levels were quantitated by real-time PCR. Data points represent the average of triplicate determinations ± SD; Similar results were obtained in 2 independent experiments.

Hdac-8 and transcriptional repression of LPS-responsive genes
One caveat about the studies here is that TSA might be acting through mechanisms other than HDACs. The selective actions on a subset of LPS-responsive genes argue against a distal effect of TSA on some aspect of LPS signaling. However, to complement the evidence that HDACs negatively regulate LPS-inducible expression of Cox-2 and similarly regulated genes, we examined the effect of HDAC overexpression in macrophages. In a recent study, overexpression of the class I HDAC, Hdac-1, suppressed activity of the Cox-2 promoter (26) . We examined the effect of Hdac-8, the only other class 1 HDAC expressed in BMM, on Cox-2expression. Ectopic overexpression of Hdac-8 in stably transfected RAW264 cells blocked the ability of LPS to induce Cox-2, but not Ccl7 mRNA (Fig. 5 ). This finding supports the view that blockade of HDAC action by TSA mediated the superinduction of Cox-2 mRNA on LPS stimulation of BMM. However, Hdac-8 overexpression in RAW264 cells did not superinduce Ccl7 mRNA expression, thus suggesting that TSA may act independent of HDACs to mediate repression of this class of LPS-inducible gene in macrophages.


Figure 5
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  		Figure 5. Effect of ectopic Hdac-8 expression on Cox-2 mRNA regulation in RAW264 cells. Pooled RAW264 cells stably expressing Hdac-8 (filled bars) or the empty vector, pEF6 (unfilled bars) were generated. Each population was stimulated with LPS (10 ng/ml) for 0, 2, or 7 h. RNA and cDNA was prepared and levels of Cox-2 and Ccl7 mRNA were quantitated by real-time PCR. Data points indicate average of triplicates ± SD; similar results were obtained in 2 independent experiments.

LPS and TSA act at the level of the Cox-2 promoter to regulate expression
To determine whether the effects of TSA are mediated at the level of promoter activation, 1.4 kb of the Cox-2 proximal promoter region was chosen to include regions conserved across mammalian species (www.dcode.org) and linked to a luciferase reporter gene. In transient transfections in RAW264 cells, regulation of the Cox-2 promoter mirrored that of the endogenous gene; the promoter was induced by LPS (5.5-fold) and was superinduced by the combination of LPS + TSA (18-fold) (Fig. 6 a). In RAW264 cells stably transfected with pGL2b1.4KbCox2, a similar phenomenon was also apparent (Fig. 6b ). Hence, TSA enhanced LPS-induced Cox-2 expression via its actions on the promoter.


Figure 6
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  		Figure 6. Regulation of the Cox-2 promoter by LPS and TSA. a) RAW264 cells were transiently transfected with pGL2b1.4KbCox2, or pGL-2B. Transfections were split and, 24 h later, treated with medium (control), LPS (10 ng/ml), TSA (0.5 µM), or LPS+TSA for 7 h. Luciferase activity relative to total protein was calculated, and results are presented as fold induction relative to control values. Data points represent the average of triplicates ± SD, and similar results were obtained in 3 independent experiments. b) Pools of RAW264 cells stably transfected with pGL2b1.4KbCox2 were generated. Cells were treated for 6 h with medium, LPS (10 ng/ml), TSA (0.5 µM) or LPS+TSA. Luciferase activity relative to the untreated control is displayed. Data are representative of 2 experiments.

Changes in the extent of H4K12 acetylation are correlated with LPS-inducible gene expression
We predicted that during the time course of LPS action there would be detectable alterations in the level of histone acetylation associated with the promoters of LPS target genes. We therefore carried out chromatin immunoprecipitation (ChIP) analysis to examine the extent of histone acetylation on the promoters of TSA-sensitive genes across an extended LPS time course of 0 (unstimulated), 0.5, 2, 4, 7, 24, and 48 h (Fig. 7 ). Figure 7a demonstrates the promoter architecture of the genes examined and the PCR primers used in this analysis. Within the same experiment, we confirmed the alterations in mRNA levels for the genes of interest (Fig. 7b ). When Ab for histone H4 acetylated at lysine 12 was used for the ChIP analysis, there was an increase and subsequent decrease in the extent of histone acetylation at H4K12, which paralleled that of the mRNA level detected by real-time PCR for each the five genes tested here; the three genes enhanced by TSA (Cox-2, Cxcl2, and Ifit2) as well as the two genes repressed by TSA (Ccl2 and Ccl7) (Fig. 7b ). To confirm that the regions tested were indeed representative of inducible promoters, ChIP analysis was also performed with an RNA polymerase II Ab. For the three genes superinduced by TSA, the level of RNApol II association with their promoters increased substantially before the peak of mRNA production, suggesting that the induction is indeed primarily transcriptional. By contrast, the TSA-repressed genes showed a relatively small increase in RNApol II association upon LPS stimulation (Fig. 7b ). We suggest that, like the TNF-{alpha} and MacMarcks genes studied in our laboratory (33 , 34) , these genes may have constitutively active promoters, and a significant proportion of the regulation is post-transcriptional. These data indicate that LPS acutely regulated histone acetylation. However, there was no correlation between H4K12 acetylation and differential effects of TSA on subsets of LPS-inducible genes.


Figure 7
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  		Figure 7. ChIP analysis of H4 lysine 12 (H4K12) acetylation at TSA-regulated promoters during LPS activation of BMM. a) The maps for the promoter regions of LPS-inducible genes in which gene expression was enhanced (Cox-2, Cxcl2, and Ifit2) or repressed (Ccl2 and Ccl7) by TSA are shown. The PCR fragments amplified in ChIP analysis are also shown. b) Duplicate sets of BMM were stimulated with LPS for 0, 0.5, 2, 4, 7, 24, or 48 h; one time course set was used to prepare RNA to quantitate endogenous gene expression while the second time course set was used for immunoprecipitation experiments (either acetylated histone H4 lysine 12 or RNA polII). Levels of acetylated histone H4 lysine 12 and RNA polII at each of the 5 promoters, as well as endogenous gene expression, were determined by real-time PCR. All data are expressed as fold induction with control values set to 1 except for mRNA levels of Cox-2 and Ifit2; here control levels were set at 0.1. Similar results were obtained in 2 independent experiments.

Effect of HDAC inhibition on LPS tolerance
Pretreatment of macrophages with LPS leads to a state of LPS insensitivity that may be partly related to the phenomenon of LPS tolerance in vivo (35 , 36) . To determine whether chromatin modification by HDACs contributes to LPS insensitivity, macrophages were initially activated with LPS for 7 h. LPS was then removed and cells were washed, fresh media added, and cells grown overnight. On the following day cells were restimulated with either medium or LPS for 7 h in the presence or absence of TSA. Levels of Cox-2 and Ccl7 mRNA were then assessed (Fig. 8 ). As expected, induction of Cox-2 mRNA by a secondary LPS challenge was reduced compared to the primary LPS response, while Ccl7 mRNA was not induced at all by a secondary LPS challenge (Fig. 8) . Addition of TSA alone for 7 h on the day after the initial LPS stimulation reactivated Cox-2 expression, suggesting that HDACs, induced by the primary LPS stimulation actively repressed Cox-2 expression. Hence, for Cox-2 and similarly regulated genes, LPS-induced HDACs are likely to contribute to the phenomenon of endotoxin tolerance. However, because of the dramatic impact of TSA alone on gene expression in this system, the effects of TSA on a secondary LPS challenge are difficult to interpret; Cox-2 mRNA was actually 5-fold repressed by a secondary LPS challenge whereas induction of Ccl7 mRNA (in terms of fold induction) was restored (Fig. 8) . While this finding is difficult to interpret, it nonetheless highlights the differential regulation of these two classes of gene.


Figure 8
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  		Figure 8. Effect of TSA on in vitro endotoxin tolerance. BMM were stimulated with LPS (10 ng/ml) for 0, 7, or 10 h, and RNA and cDNA were prepared. In addition, BMM were stimulated for 7 h with LPS, then washed and grown overnight in medium free of LPS. On the following day, these cells were restimulated for 7 h with medium or LPS (Booster LPS) in the presence (unfilled boxes) or absence (filled boxes) of TSA (0.5 µM); RNA and cDNA were prepared. For all samples, levels of Cox-2 and Ccl7 mRNA were determined by real-time PCR. Data points represent the average of triplicates ± SD; the experiment is representative of 2 independent experiments.

The role of PU.1 in regulating LPS-inducible gene expression regulation
The data described herein suggest that TSA enhanced LPS-induced Cox-2 expression by blocking the inhibitory actions of HDACs. However, despite the effects of TSA on gene expression, much of our data were inconsistent with HDACs as positive regulators of Ccl7 and coregulated genes. For example, there was no positive correlation between LPS-regulated HDAC expression and LPS-regulated Ccl7 expression (Fig. 2 , Fig. 4a ), and Hdac-8 overexpression in RAW264 did not induce or enhance LPS-induced Ccl7 expression (Fig. 5) . Suzuki and colleagues claimed that PU.1, an Ets family transcription factor that regulates macrophage-specific gene expression (37) , was degraded in murine macrophages in response to TSA (38) . Thus, we determined whether the negative effect of TSA on Ccl2 and Ccl7 expression occurs via targeting of PU.1. We first confirmed that TSA does lead to loss of PU.1 protein in the BMM being studied here, which are distinct from peritoneal macrophages and cell lines used by others (38) . Western blot analysis confirmed that the level of PU.1 protein in BMM was indeed repressed by treatment with TSA in a dose-response manner (Fig. 9 a). Consensus PU.1 binding sites were present in the proximal 1 kb region of the Ccl2 and Ccl7 promoters, while an imperfect PU.1 binding site is also present in the TSA superinduced Cox-2 promoter (Fig. 7a , Fig. 9b ). Gel mobility shift assays showed that PU.1 (as confirmed by supershifting with an anti-PU.1 Ab) bound the predicted binding sites within the Ccl2, Ccl7, and Cox-2 promoters (Fig. 9c ). To confirm that PU.1 was recruited to these promoters, we performed ChIP assays on the promoters of interest across the LPS time course using an anti-PU.1 Ab. Both real-time and conventional PCR analyses were performed on chromatin immunoprecipitated with an anti-PU.1 Ab (Fig. 9d ). Chromatin before immunoprecipitation was used as input control for both types of PCR. The ChIP profile confirmed an earlier report that PU.1 is recruited to the Cox-2 promoter in response to LPS (19) . By contrast, PU.1 was constitutively associated with the Ccl2 and Ccl7 promoters, suggesting it contributes to basal transcription (Fig. 9d ). Hence, the inhibitory effect of TSA on Ccl2 and Ccl7 gene expression is most likely mediated by PU.1 degradation.


Figure 9
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  		Figure 9. Involvement of PU.1 in regulating expression of TSA-repressed genes. a) BMM were treated either with LPS (10 ng/ml) for 0, 2, 7, or 24 h, TSA (0.75 µM or 1.5 µM) for 7 h, or with LPS + TSA for 7 h. Whole cell extracts or nuclear extracts were prepared and the total amount of PU.1 and ERK-1/2 was determined by immunoblotting. A 150 kDa nonspecific band (*) appeared when immunoblotting nuclear extracts with the anti-PU.1 Ab and is displayed as an internal loading control. b) Putative PU.1 binding sites within the Cox-2, Ccl2, and Ccl7 promoters are indicated (boldface underlined) relative to the transcription start sites. c) Gel mobility shift assays were performed using BMM nuclear extracts and ds radiolabeled probes (shown in Table 1 ). Nuclear extract and probe cocktail was treated with or without an anti-PU.1 Ab prior to separation by PAGE. Arrows mark PU.1 bound to probe and PU.1-containing complexes supershifted (ss) with the Ab. d) BMM, treated over a 0–48 h LPS time course, were fixed, nuclei isolated, and chromatin prepared. Immunoprecipitations were performed with an anti PU.1 Ab. Fragments (Fig. 7a ) were amplified by conventional and real-time PCR. Chromatin before immunoprecipitation was used as an input control.

Regulation of HDAC expression in macrophages by TSA
The low basal expression of the HDACs in BMM (Fig. 2) prompted us to determine whether HDACs act on their own promoters to maintain low basal expression in macrophages. Hence, BMM were treated for 10 or 24 h with TSA, and expression of Hdac-1 to 11 was assessed. Fig. 10 shows that, with the exception of Hdac-7 and 9, TSA strongly up-regulated expression of all of the HDACs examined at both time points. Hence, in macrophages HDACs are likely to negatively regulate their own expression. In contrast, Hdac-9 expression was repressed by TSA at both time points, suggesting that this Hdac may be a PU.1 target gene.


Figure 10
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  		Figure 10. Regulation of Hdac expression in BMM by TSA. BMM were treated with TSA (0.5 µM) for 10 or 24 h. RNA and cDNA were prepared and Hdac expression was estimated by real-time PCR. Data represent the average of triplicates ± SD. Similar results were obtained in 2 independent experiments.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In this study, we have shown that LPS-inducible genes in mouse BMM can be divided into distinct classes depending on their sensitivity to TSA. We have identified several genes in which the LPS response was enhanced by TSA (Cox-2, Cxcl2, and Ifit2). This extends a previous finding which showed that TSA enhanced LPS-induced expression of the dual specificity phosphatase, MKP-M, in macrophages (21) . Apart from the positive effects of TSA on LPS-induced gene expression, we provide several other lines of evidence that HDACs negatively regulate this class of gene. First, induction of Cox-2 mRNA by LPS coincided with down-regulation of Hdac-4, 5, 6, and 7 mRNAs, while the subsequent decline in LPS-induced Cox-2 mRNA levels coincided with the induction of Hdac-1 mRNA (Fig. 2 , Fig. 4a ). The LPS-induced Cox-2 expression profile also correlated with acetylation and deacetylation at histone H4K12 at the Cox-2 promoter (Fig. 7b ). Further, overexpression of Hdac-8 in RAW264 cells selectively blocked LPS-induced Cox-2 expression (Fig. 5) .

Despite the effect of Hdac-8 on Cox-2 expression, the complex patterns of expression of different HDACs across the time course of the LPS response preclude a definitive identification of a particular corepressor that is responsible for the actions of TSA in each of the circumstances studied. Hdac-1 induction by LPS peaked at 8 h (Fig. 2) and correlated most closely with the onset of repression of gene expression (Fig. 4a ) and histone deacetylation of target promoters (Fig. 7b ). Others have also reported that Hdac-1 cotransfection blocked activity of the Cox-2 promoter in macrophages (26) . However, the addition of TSA to BMM 24 h after an initial stimulation with LPS still reactivated Cox-2 expression (Fig. 8) even though Hdac-1 mRNA was only transiently induced by LPS (Fig. 2) . Hence, it is probable that sequential regulation of HDACs controls LPS-inducible Cox-2 expression. A proposed mechanism for the role of histone acetylation and deacetylation in regulation of Cox-2 and similarly regulated genes is outlined in Fig. 11 . LPS transiently represses expression of Hdac-4, 5, 6, and 7 (Fig. 2) and up-regulates components of the histone acetyltransferase complex (Fig. 1) . The combined effect is histone H4 acetylation at target promoters (Fig. 7b ) and transcriptional activation. LPS also triggers histone H3 phosphorylation and phosphoacetylation at target promoters via a MAPK p38-dependent pathway (39) . Hdac-1 is then induced (Fig. 2) and acts to shut down gene expression. As Hdac-1 expression declines, Hdac-4, 5, and 7 are induced and act to maintain gene expression in a basal state. We have not assessed cellular localization of HDACs in this study, but many of the class II HDACs can shuttle between cytoplasm and nucleus (40) . It is therefore likely that regulation of HDAC localization also contributes to LPS-inducible gene expression. The observation that TSA regulated the activity of the Cox-2 promoter in transient transfection analysis (Fig. 6a ) might be considered surprising, since it implies that transiently transfected DNA is subjected to epigenetic gene regulation. Nonetheless, a range of studies has demonstrated that HATs and HDACs do regulate promoter activity in transient transfection experiments, thus indicating that histone modification can indeed regulate promoter activity in such analyses.


Figure 11
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  		Figure 11. Proposed model for regulation of Cox-2 (and coregulated genes) by histone modification. In unstimulated macrophages there are low levels of histone H3/H4 acetylation at the promoters of LPS-inducible genes that contain CpG islands (due to the action of histone deacetylases). Activation by LPS transiently down-regulates expression of Hdac-4, 5, 6, and 7 (HDACs) and up-regulates expression of p300, P/CAF, Cited-2, and CBP (HATs). Consequently, histone H3/H4 acetylation increases, transcription factors and RNApol II are recruited, and transcriptional activation occurs. Expression of Hdac-1 is also increased, and Hdac-1 acts to deacetylate histone H3/H4 and shut down gene expression. Subsequently, as Hdac-1 mRNA levels decline and expression of Hdac-4, 5, and 7 increases, this acts to maintain low levels of histone acetylation, RNApol II recruitment, and transcriptional activation.

Although TSA negatively regulated Ccl2, Ccl7, and Edn1 gene expression, we did not find supporting evidence that HDACs positively regulate expression of this gene class. For example, acetylated histone H4K12 levels at the Ccl2 and Ccl7 promoters were maximal when Ccl2 and Ccl7 mRNA levels were maximal (Fig. 7b ). Further, overexpression of Hdac-8 in RAW264 cells did not induce Ccl7 expression (Fig. 5) . Instead, the most likely explanation for the effect of TSA on these genes is that they are PU.1 target genes and that TSA inhibits basal and inducible expression by down-regulating PU.1 expression (Fig. 9a ). This is supported by the observation that PU.1 was associated with the Ccl2 and Ccl7 promoters in unstimulated BMM (Fig. 9d ). A comprehensive analysis of the effects of TSA on gene expression in macrophages is likely to identify other PU.1 target genes. It should be noted that there were differences between the effects of TSA on Edn1 and Ccl2/Ccl7 gene expression. Whereas TSA inhibited both basal and LPS-induced Ccl2/Ccl7 expression, TSA only affected LPS-inducible expression of Edn1. The inference is that PU.1 regulates LPS-inducible, but not basal activity, of the Edn1 promoter. Another possibility is that HDACs do indeed act as positive regulators of Edn1 expression. Regardless of the mechanism, given the involvement of Edn1 in vasoconstriction and cardiovascular disease (41) , TSA might have therapeutic applications in cardiovascular disease associated with inflammation.

Whereas the three superinducible genes studied here (Cox-2, Cxcl2, Ifit2) have classical CpG islands, the Ccl7 and Ccl2 promoters are not CpG-rich and have classical TATA boxes. In the case of the CpG islands, it is possible that partial or complete methylation is involved in maintaining the basal transcription state of the genes, through the recruitment of histone deacetylases by methyl CpG-binding proteins (42 43 44) . This possibility is currently under investigation. The PU.1 sites in this class of promoter are distal to the transcription start site, and the recruitment of PU.1 to the promoter region occurs late in the activation response (Fig. 9d ). The decay in PU.1 nuclear protein induced by TSA might serve to prevent PU.1 association, but this could either be inconsequential or function in repression. By contrast, in the Ccl7 gene a strong conserved PU.1 site is located close to the transcription start site, a functional location shared with many constitutively expressed macrophage genes (45) . In this distinct context, the loss of nuclear PU.1 could prevent transcriptional initiation and repress constitutive promoter activity. The Ccl7 promoter has not been analyzed in detail, but in humans, at least, the purine-rich elements may be functional (46) . The Ccl2 promoter has been more widely studied, and there is evidence that a distal NF-{kappa}B site controls regulated histone acetylation of a Sp1 site at the proximal promoter (47) . Ccl7 and Ccl2 share one other distinct feature in that they form part of a limited set of macrophage genes that are targets of the repressor BCL-6 in mice (48) , although the relevance of this to our findings has not been investigated.

The ChIP studies of the pattern of histone acetylation across the promoter regions of both TSA-superinducible and TSA-repressible genes revealed a common pattern of rapidly inducible acetylation of histone H4, followed by deacetylation as the level of mRNA declined (Fig. 7b ). By contrast, acetylation of histone H3 was not well correlated with transcription and increased across the time course (data not shown). This observation is consistent with the study by Laribee and Klemsz (49) , who reported that TSA treatment increased histone H4, but not H3, acetylation in macrophages. However, post-translational modification of histone H3 is still an important regulator of proinflammatory gene expression (39) . The decline in mRNA levels of inducible genes in macrophages responding to LPS is due, at least in part, to inducible degradation of the mRNA. Our studies show there is also a dynamic sequence of acetylation and deacetylation of histone H4 with time that correlates with the recruitment of RNApol II to the LPS-inducible promoters, so that transient induction does in fact represent transient transcriptional activation for each of the genes we have studied. The importance of histone deacetylation in switching off transcription is evident from the ability of TSA to prevent the decline in Cox-2 mRNA levels at later time points in the temporal cascade.

There is considerable interest in the use of TSA inhibitors in cancer chemotherapy, where they can induce differentiation and growth arrest. The complex effects on inducible gene expression in macrophages, as well as lineage-specific differentiation reported previously (49) , suggest that such agents could have unpredictable effects on innate immune responses and inflammation. Indeed, peroxynitrite-induced damage of Hdac-2 has been implicated in the steroid insensitivity of patients with chronic obstructive pulmonary disease (50) , implying that clinical use of HDAC inhibitors might have unwanted effects in exacerbating inflammatory disease. Such an effect of TSA was reported in a neural inflammatory model (51) . Nonetheless, there may also be occasions when inhibition of HDACs is desirable for treatment of macrophage-mediated inflammatory disease, since TSA potently down-regulated inducible expression of the proinflammatory genes, Ccl2, Ccl7, and Edn1. Separation of LPS-inducible genes into classes based on responsiveness to TSA opens up the possibility that HDAC inhibition, perhaps directed against specific macrophage-expressed isoforms, could have a subtle therapeutic benefit. In overview, we have shown a direct link between epigenetic gene regulation and environmental factors (LPS in this case). In particular, we have shown that regulated expression of multiple histone deacetylases contributes to feedback control of macrophage activation and that TSA can be used to distinguish different classes of LPS-responsive genes with distinct promoter architectures. HDAC inhibitors that selectively target Ccl2, Ccl7, Edn1, and coregulated genes may have anti-inflammatory properties that could be utilized for therapeutic applications.


   ACKNOWLEDGMENTS
 
We thank the SRC microarray facility at the University of Queensland for providing us with the microarray Chips used for these analyses. This work was funded by grants from the National Health and Medical Research Council of Australia (NHMRC) to D.A.H. Research Grant for Advanced and Innovational Research Program in Life Science to Y.H. Research Grant for Advanced and Innovational Research Program in Life Science to J.K. CREST (Core Research for Evolutional Science and Technology) of Japan Science and Technology Corporation (JST) to Y.H. Research Grant for the Genome Network Project from the Ministry of Education, Culture, Sports, Science and Technology (MEXT) of the Japanese Government.


   FOOTNOTES
 
1 These authors contributed equally to this work.

Received for publication October 24, 2005. Accepted for publication March 3, 2006.


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