Histone deacetylase inhibitor LAQ824 augments inflammatory responses in macrophages through transcriptional regulation of IL-10.
ABSTRACT APCs are important in the initiation of productive Ag-specific T cell responses and the induction of T cell anergy. The inflammatory status of the APC at the time of encounter with Ag-specific T cells plays a central role in determining such divergent T cell outcomes. A better understanding of the regulation of proinflammatory and anti-inflammatory genes in its natural setting, the chromatin substrate, might provide novel insights to overcome anergic mechanisms mediated by APCs. In this study, we show for the first time, to our knowledge, that treatment of BALB/c murine macrophages with the histone deacetylase inhibitor LAQ824 induces chromatin changes at the level of the IL-10 gene promoter that lead to enhanced recruitment of the transcriptional repressors HDAC11 and PU.1. Such an effect is associated with diminished IL-10 production and induction of inflammatory cells able of priming naive Ag-specific T cells, but more importantly, capable of restoring the responsiveness of anergized Ag-specific CD4(+) T cells.
- SourceAvailable from: Gosse J Adema[Show abstract] [Hide abstract]
ABSTRACT: Epigenetic modifications, like histone acetylation, are essential for regulating gene expression within cells. Cancer cells acquire pathological epigenetic modifications resulting in gene expression patterns that facilitate and sustain tumorigenesis. Epigenetic manipulation therefore is emerging as a novel targeted therapy for cancer. Histone Acetylases (HATs) and Histone Deacetylases (HDACs) regulate histone acetylation and hence gene expression. Histone deacetylase (HDAC) inhibitors are well known to affect cancer cell viability and biology and are already in use for the treatment of cancer patients. Immunotherapy can lead to clinical benefit in selected cancer patients, especially in patients with limited disease after tumor debulking. HDAC inhibitors can potentially synergize with immunotherapy by elimination of tumor cells. The direct effects of HDAC inhibitors on immune cell function, however, remain largely unexplored. Initial data have suggested HDAC inhibitors to be predominantly immunosuppressive, but more recent reports have challenged this view. In this review we will discuss the effects of HDAC inhibitors on tumor cells and different immune cell subsets, synergistic interactions and possible mechanisms. Finally, we will address future challenges and potential application of HDAC inhibitors in immunocombination therapy of cancer.Oncotarget 07/2014; · 6.63 Impact Factor
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ABSTRACT: APCs are critical in T cell activation and in the induction of T cell tolerance. Epigenetic modifications of specific genes in the APC play a key role in this process, and among them histone deacetylases (HDACs) have emerged as key participants. HDAC6, one of the members of this family of enzymes, has been shown to be involved in regulation of inflammatory and immune responses. In this study, to our knowledge we show for the first time that genetic or pharmacologic disruption of HDAC6 in macrophages and dendritic cells results in diminished production of the immunosuppressive cytokine IL-10 and induction of inflammatory APCs that effectively activate Ag-specific naive T cells and restore the responsiveness of anergic CD4(+) T cells. Mechanistically, we have found that HDAC6 forms a previously unknown molecular complex with STAT3, association that was detected in both the cytoplasmic and nuclear compartments of the APC. By using HDAC6 recombinant mutants we identified the domain comprising amino acids 503-840 as being required for HDAC6 interaction with STAT3. Furthermore, by re-chromatin immunoprecipitation we confirmed that HDAC6 and STAT3 are both recruited to the same DNA sequence within the Il10 gene promoter. Of note, disruption of this complex by knocking down HDAC6 resulted in decreased STAT3 phosphorylation-but no changes in STAT3 acetylation-as well as diminished recruitment of STAT3 to the Il10 gene promoter region. The additional demonstration that a selective HDAC6 inhibitor disrupts this STAT3/IL-10 tolerogenic axis points to HDAC6 as a novel molecular target in APCs to overcome immune tolerance and tips the balance toward T cell immunity.The Journal of Immunology 08/2014; · 5.36 Impact Factor
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ABSTRACT: Microbial infections are hypothesized to play a key role in the mechanism leading to type 1 diabetes (T1D). We used the LEW1.WR1 rat model of Kilham rat virus (KRV)-induced islet destruction to better understand how virus infection triggers T1D. Inoculation of the LEW1.WR1 rat with KRV results in systemic inflammation followed by insulitis and islet destruction 2-4 weeks post-infection. In this study, we evaluated the effect of treatment with the anti-inflammatory histone deacetylase inhibitor (HDACi) ITF-2357 on KRV-induced immunity and disease progression. Administering LEW1.WR1 rats with KRV plus ITF-2357 on 14 consecutive days beginning on the day of infection protected animals from islet infiltration and T1D. ITF-2357 reversed KRV-induced T and B cell accumulation in the spleen or pancreatic lymph nodes on day 5 following infection. Moreover, ITF-2357 reduced the expression level of KRV-induced p40 subunit of IL-12/IL-23 in spleen cells in vitro and in the peripheral blood in vivo. ITF-2357 suppressed the KRV-induced expression of transcripts for IRF-7 in the rat INS-1 beta cell line. ITF-2357 increased the virus-induced IL-6 gene expression in the spleen, but did not alter the ability of LEW1.WR1 rats to develop normal KRV-specific humoral and cellular immune responses and clear the virus from the pancreatic lymph nodes, spleen, and serum. Finally, ITF-2357 reversed virus-induced modulation of bacterial communities in the intestine early following infection. The data suggest that targeting innate immune pathways with inhibitors of HDAC might represent an efficient therapeutic strategy for preventing T1D. Microbial infections have been implicated in triggering type 1 diabetes in humans and animal models. The LEW1.WR1 rat develops inflammation and T1D following infection with Kilham rat virus. The histone deacetylase inhibitor ITF-2357 suppresses virus-induced inflammation and prevents diabetes. ITF-2357 prevents T1D without altering virus-specific adaptive immunity or virus clearance. ITF-2357 therapy may be an efficient approach to prevent T1D in genetically susceptible individuals.Journal of Molecular Medicine 08/2013; · 4.77 Impact Factor
The Journal of Immunology
Histone Deacetylase Inhibitor LAQ824 Augments
Inflammatory Responses in Macrophages through
Transcriptional Regulation of IL-10
Hongwei Wang,* Fengdong Cheng,* Karrune Woan,* Eva Sahakian,* Oscar Merino,*
Jennifer Rock-Klotz,* Ildefonso Vicente-Suarez,* Javier Pinilla-Ibarz,* Kenneth L. Wright,*
Edward Seto,†Kapil Bhalla,‡Alejandro Villagra,* and Eduardo M. Sotomayor*
APCs are important in the initiation of productive Ag-specific T cell responses and the induction of T cell anergy. The inflammatory
statusof theAPC atthe timeofencounter withAg-specific T cells playsa centralroleindetermining such divergent T cell outcomes.
A better understanding of the regulation of proinflammatory and anti-inflammatory genes in its natural setting, the chromatin
substrate, might provide novel insights to overcome anergic mechanisms mediated by APCs. In this study, we show for the first
time, to our knowledge, that treatment of BALB/c murine macrophages with the histone deacetylase inhibitor LAQ824 induces
chromatin changes at the level of the IL-10 gene promoter that lead to enhanced recruitment of the transcriptional repressors
HDAC11 and PU.1. Such an effect is associated with diminished IL-10 production and induction of inflammatory cells able of
priming naive Ag-specific T cells, but more importantly, capable of restoring the responsiveness of anergized Ag-specific CD4+
T cells. The Journal of Immunology, 2011, 186: 3986–3996.
such factors as the particular APC type as well as the context,
inflammatory versus non-inflammatory, in which the APC acquires
the Ag for processing and presentation to Ag-specific T cells (1, 2).
Not surprisingly, APCs isolated from a noninflammatory tumor
microenvironment are relatively inefficient at priming protective
responses, inducing instead T cell anergy (3–5).
During the past several years, numerous studies in experimental
models as well as in humans have provided sufficient evidence
supporting the conclusion that the induction of T cell anergy to tu-
mor Ags represents a significant barrier to harness antitumor immu-
nity (5–9). Importantlessonslearnedfromthesestudies point toma-
nipulation of the inflammatory status of the APC as an enticing
strategy to overcome anergic mechanisms in cancer (10–13). A bet-
ter understanding of the molecular/signaling mechanism(s) regulat-
and might unveil novel targets to overcome anergy to tumor Ags.
Recently, a significant effort has been devoted to better un-
derstand the regulation of pro- and anti-inflammatory genes in their
he potency of an immune response is dictated in large part
by the potency of the APC and its ability to optimally
prime the T cell response. This, in turn, is influenced by
natural setting, the chromatin substrate (14). Chromatin modifi-
cation by acetylation/deacetylation of histone tails is an important
mechanism of regulation of gene transcription, including genes
involved in the inflammatory response (15). In general, histone
acetylation mediated by histone acetyl transferases results in
transcriptionally active chromatin. In contrast, histone deacetyla-
tion mediated by histone deacetylases (HDACs) leads to an in-
active chromatin and gene repression (16).
HDACs exist as large multimeric complexes and are recruited to
gene promoters by corepressors or by multiprotein transcriptional
complexes. Eighteen HDACs have been identified, and they have
been grouped into four principal classes (17, 18). HDACs are the
molecular target of several structurally diverse compounds known
as histone deacetylase inhibitors (HDIs). Existing HDIs inhibit
proliferation of malignant cells in vitro by inducing cell cycle
arrest and apoptosis, and some of them have already demonstrated
significant antitumor activity in cancer patients (19, 20). In con-
trast to their well-known effects upon cancer cells, little is still
known about the immunological effects of HDIs. Although some
studies have shown that HDIs have anti-inflammatory properties
(21, 22), promote the expression of the suppressive factor IDO in
dendritic cells (DCs) (23), and diminish the morbidity and mor-
tality of graft-versus-host disease (24), others have highlighted
the proinflammatory effects of these compounds. For instance,
Tomasi’s group (25, 26) has shown that treatment of melanoma
cells with HDIs augments their Ag-presenting capabilities, leading
to activation of IFN-g–secreting T cells via the class I pathway. Vo
et al. (27) have recently demonstrated that in vivo treatment of
tumor-bearing mice with the hydroxamic acid analog pan-HDI
LAQ824 significantly enhances the antitumor activity of adop-
tively transferred Ag-specific T cells. Needless to say, the un-
derlying molecular mechanism(s) by which HDIs influence in-
flammatory responses remain to be fully elucidated.
In this study, we show that the pan-HDI LAQ824 induces several
chromatin changes in macrophages that resulted in enhanced
recruitment of the transcriptional repressors HDAC11 and PU.1
*Department of Immunology and Malignant Hematology, H. Lee Moffitt Cancer
Center, Tampa, FL 33613;
Cancer Center and Research Institute, Tampa, FL 33613; and‡University of Kansas
Medical Center, Kansas City, KS 66160
†Department of Molecular Oncology, H. Lee Moffitt
Received for publication April 15, 2010. Accepted for publication January 29, 2011.
This work was supported by Public Health Service Grants CA87583 and CA134807
(to E.M.S.) and by a grant from the Donald A. Adam Comprehensive Melanoma
Address correspondence and reprint requests to Dr. Eduardo M. Sotomayor, H. Lee
Moffit Cancer Center and Research Institute, 12902 Magnolia Drive, FOB-3 Room
5.3125, Tampa, FL 33613. E-mail address: Eduardo.Sotomayor@moffitt.org
Abbreviations used in this article: ChIP, chromatin immunoprecipitation; DC, den-
dritic cell; HA, hemagglutinin; HDAC, histone deacetylase; HDI, histone deacetylase
inhibitor; PEM, peritoneal elicited macrophage.
to the IL-10 gene promoter. Such an effect is associated with in-
hibition of IL-10 production and induction of cells able of priming
naive Ag-specific T cells and capable of restoring the responsive-
ness of anergized CD4+T cells.
Materials and Methods
Male BALB/c mice (6–8 wk old) were obtained from the National Insti-
tutes of Health (Frederick, MD). TCR-transgenic mice expressing an ab
TCR specific for aa 110–120 from influenza hemagglutinin (HA) presented
by I-Edwere a generous gift of H. von Boehmer (28). All experiments
involving the use of mice were performed in accordance with protocols
approved by the Animal Care and Use Committee of the University of
South Florida College of Medicine.
the B-cell lymphoma cell line A20 was obtained from the American Type
Culture Collection. A20HA was generated by electroporation-mediated
plasmid transfection, and transfected cells were selected as previously
reported (3, 6). Cells were cultured in vitro in RPMI 1640 media, supple-
mented with 10% FBS, penicillin/streptomycin (50 U/ml), L-glutamine (2
mM), and 2-ME (50 mM) (complete media), and grown at 37˚C and 5%
CO2. A20 mediawas also supplemented with additionalsodiumpyruvate (1
mM) and nonessential amino acids (13; Mediatech, Manassas, VA).
Isolation of peritoneal elicited macrophages
BALB/c mice were injected i.p. with 1 ml thioglycollate (DIFCO Labo-
ratories, Detroit, MI). Four days later, peritoneal elicited macrophages
(PEM) were isolated by peritoneal lavage as previously described (30).
Isolation of splenic macrophages
BALB/c mice were injected i.v. with 1 3 106A20 lymphoma cells. Three
weeks later, animals were sacrificed, and their spleens were removed.
Splenocytes were isolated, washed with PBS, and then suspended in
complete RPMI 1640 medium. Cells were then cultured at 37˚C and 5%
CO2for 2 h to allow macrophages to adhere to the plate. Nonadherent cells
were then removed with two washes of cold PBS. Splenic macrophages
were then incubated for 1 additional h, and the cells were treated as in-
LPS (Escherichia coli 055:B5, catalog number L-2880) was purchased
from Sigma-Aldrich (St. Louis, MO). The HDAC inhibitors LAQ824 and
LBH589 were provided by Dr. K. Bhalla (University of Kansas Medical
Center). TSA was obtained from Sigma-Aldrich, and MS-275 is from
Calbiochem (San Diego, CA). HDIs were first reconstituted in DMSO for
stock preparation (10 mM). Solutions were then diluted in RPMI 1640 for
in vitro use in cell cultures.
Abs and immunoblottings
Histone H3 (D1H2) mAb (4499), histone H4 (Lys8) polyclonal Ab (2592),
acetyl-histone H3 (Lys9) polyclonal Ab (9671), and acetyl-histone H4
(Lys8) polyclonal Ab (2594) were purchased from Cell Signaling Tech-
nology (Danvers, MA) for Western blotting. The GAPDH (FL-335, sc-
25778), PolII (H-224, sc-9001), and PU.1 (Spi-1/T-21, sc-352) Abs were
purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Hyper-
acetylated H3 (06-599), hyperacetylated H4 (06-598), Sp1 (07-645), and
STAT3 (06-596) polyclonal Abs were purchased from Millipore (Billerica,
MA). Two different polyclonal Abs against HDAC11 were used: ab47036
from Abcam (Cambridge, MA) for immunoblots and Ab H4539 from
Sigma-Aldrich was used for chromatin immunoprecipitation (ChIP) anal-
R&D Systems (Minneapolis, MN). Recombinant mouse IL-10 (550070)
was purchased from BD Biosciences (San Diego, CA).
Phenotypic and functional analysis of macrophages
The expression of B7.2 in PEMs was determined by staining with biotin-
conjugated anti-CD86 (GL1 Ab; BD Pharmingen, San Jose, CA) and
followed by strepavidin-PE (Caltag Laboratories, Carlsbad, CA) Abs. Ten
thousand gated events were collected on an FACScan (BD Biosciences) and
analyzed using FlowJo software (Tree Star). In a parallel plate, PEMs were
treated with HDI alone, LPS alone, or a combination thereof. Then super-
natants were collected, and production of IL-10, IL-12p70, IL-1a, IL-1b,
IL-6, RANTES, TNF-a and GM-CSF was determined by ELISA (R&D
PEMs or splenic macrophages (1 3 105/well) were treated with LPS (2 mg/
ml) or with LPS plus HDI for 24 h. Cells were then washed, and 5 3 104
purified naive Ag-specific CD4+T cells (isolated from the spleen of HA
TCR-transgenic mice) or a similar number of anergized Ag-specific CD4+
T cells (isolated from the spleen of A20HA lymphoma-bearing mice) were
(HApeptide110–120SFERFEIFPKE). After48h, supernatants were collected
and stored at 220˚C until assayed for IL-2 and IFN-g production by ELISA.
Valuesfor Tcellsculturedinmediaalone areusually.10%ofthevaluesfor
Ag-stimulated T cells. The amount of cytokine production is expressed as
picograms per milliliter per 100 clonotype-positive CD4+T cells.
Real-time PCR analysis
Macrophage cell line or primary murine macrophages were plated at 2 3 106
cells per 35-mm well and cultured under conditions detailed for each exper-
and cDNA obtained with the iScript cDNA synthesis kit (Bio-Rad, Hercules,
CA). Target mRNA was quantified using MyIQ single-color real-time PCR
detection system(Bio-Rad) and iQSYBRgreen Supermix (Bio-Rad). Mouse
IL-12p40 primers (left, 59-GCAACGTTGGAAAGGAAAGA-39; right, 59-
AAAGCCAACCAAGCAGAAGA-39) and mouse IL-10 primers (left, 59-
CAGGGATCTTAGCTAACGGAAA-39; right, 59-GCTCAGTGAATAAAT-
AGAATGGGAAC-39) were used for PCR amplification (cycling parameters:
∼100% in all of the experiments performed. Quantification is expressed in
arbitrary units, and target mRNA levels were normalized to GAPDH ex-
pression using the method of Pfaffl (31).
ChIP studies were performed as previously described (32). The primers
used were: IL-10 (proximal region) sense, 59-GGAGGAGGAGCCT-
GAATAAC-39 and antisense, 59-CTGTTCTTGGTCCCCCTTTT-39; IL-10
(distal region) sense, 59-AACTCAGCCTGGAACTGACC-39 and antisense,
59-GCCTCTCCTCCTGACACTCTT-39; and IL-12 sense, 59-GTGGAGC-
CAAACAGGGAGGTA-39 and antisense, 59-GACGTCGAAATCCCAGTT-
TA-39. All samples and inputs were quantified using MyIQ single-color
real-time PCR detection system (Bio-Rad) and iQ SYBR green Supermix
(Bio-Rad). Single-product amplification was confirmed by melt curve
analysis, and primer efficiency was ∼100% in all experiments performed.
Target sequence levels were normalized to the input signal using the
method of Pfaffl (31). Quantification is expressed in arbitrary units as the
ratio between treated and untreated cells. All ChIP experiments were re-
peated three times, and final quantitative real-time PCR was done in trip-
T cell anergy model
We used a well-established experimental model of tumor-induced Ag-
specific CD4+T cell anergy (3, 6). Briefly, 2.5 3 106naive CD4+trans-
genic T cells specific for an MHC class II epitope of influenza HA were
injected i.v. into A20HA lymphoma-bearing mice. Twenty-one days after
T cell transfer, animals were sacrificed, and T cells were reisolated from
their spleens. Cytokine production by clonotypic CD4+T cells in response
to HA peptide110–120presented by untreated, LPS-treated, or PEM-treated
with LPS plus LAQ824 was then determined as described under Ag pre-
Unpaired t tests were performed using Microsoft Excel (Microsoft) with
significance at p , 0.05.
Phenotypic and functional changes in macrophages treated
with the HDI LAQ824
First, we asked whether treatment of macrophages with the hy-
droxamic acid analog pan-HDI LAQ824 would result in changes
in histone acetylation and the induction or not of inflammatory
cells. As shown in Fig. 1A, treatment of PEM with LAQ824 was
associated with increased acetylation of histones H3 and H4
The Journal of Immunology 3987
relative to their acetylated status in untreated PEM (None). No
changes in the expression of MHC molecules or costimulatory
molecules were observed in PEM treated with LAQ824 alone
(data not shown). However, higher expression of B7.2 was ob-
served in PEMs treated with LPS plus LAQ824 as compared with
cells treated with LPS alone (Fig. 1B). Furthermore, treatment of
PEMs with LPS in the presence of increasing concentrations of
LAQ824 resulted in a dose-dependent inhibition of the anti-
inflammatory cytokine IL-10 (Fig. 1C). Of note, this inhibitory
effect was accompanied by an increased production of the pro-
inflammatory cytokine IL-12 (Fig. 1D). It should be pointed out
that LAQ824 was not toxic to PEMs because these cells were
viable (as determined by trypan blue exclusion) and capable of
producing IL-12 even when they were exposed to the highest
concentration of HDI (100 nM) used in our experiments.
To further confirm the proinflammatory effects of LAQ824, we
determined next the production of cytokines/chemokines by PEMs
treated with HDI. As shown in Fig. 2, PEMs treated with LPS and
LAQ824 produced higher levels of IL-1a, IL-1b, IL-6, RANTES,
TNF-a, and GM-CSF as compared with PEMs treated with LPS
alone. Of note, LAQ824 also inhibits the production of IL-10 by
macrophages isolated from the spleen of tumor-bearing mice (Fig.
3A). No changes in IL-12 production were observed in tumor-
bearing macrophages treated with HDI plus LPS as compared
with macrophages treated with LPS alone (Fig. 3B).
Pan-HDIs LBH589 and TSA, but not the class I selective HDI
MS-275, inhibit IL-10 production by macrophages
HDIs represent a family of structurally diverse compounds that
inhibit the enzymatic function of HDACs. LAQ824 as well as
LBH589, TSA, and SAHA are pan-HDIs that belong to the
hydroxamic acid family of compounds. To determine whether the
other members of this family share the proinflammatory effects of
LAQ824, we treated PEMs with either LBH589 or TSA. Remi-
in a dose-dependent manner the production of IL-10 by PEMs in
response to LPS (Fig. 4A, 4B, left panel). Such an inhibition was
associated with increased production of IL-12 (Fig. 4A, 4B, right
panel). Similar changes, although of a lesser magnitude, were
observed in PEMs treated with SAHA (data not shown).
In contrast to the above results, treatment of PEMs with a more
selective HDI, compound MS-275, which inhibits the enzymatic
function of class I HDACs (HDAC 1, 2, 3, and 8) did not result in
inhibition of IL-10 or augmentation of IL-12 production by treated
cells (Fig. 4C). These findings argue against a role for class I
HDACs in regulating IL-10 production in PEMs and point to class
II and/or class IV HDACs as potential molecular targets involved
in the inhibitory effect on IL-10 mediated by pan-HDIs.
LAQ824-treated PEMs efficiently prime naive Ag-specific
T cells and restore the responsiveness of anergized T cells
Next, we determined whether the acquisition of inflammatory
properties by LAQ824-treated macrophages renders these cells
better activators of Ag-specific CD4+T cells. PEMs were therefore
treated with LAQ824, LPS, or a combination of LPS plus LAQ824
for 24 h. Following this treatment, naive CD4+T cells specific for
an MHC class II-restricted epitope of influenza HA were added to
the PEM monolayer and stimulated, or not, with cognate HA
peptide. First, clonotypic T cells encountering cognate peptide on
untreated PEM (None) or on PEM treated with LAQ824 (HDI
alone) produce similar levels of IL-2 (Fig. 5A, top panel). A
slightly increased production of IL-2 was observed in T cells
encountering Ag in LPS-treated PEMs (Fig. 5A, top panel, LPS
alone). IL-2 production was further enhanced in CD4+T cells that
encountered HA peptide in PEMs treated with LPS plus increas-
ing concentrations of LAQ824 (Fig. 5A, top panel, LAQ+LPS).
LAQ824-treated PEMs also triggered an enhanced effector func-
tion of clonotypic CD4+T cells, as determined by their capacity to
produce higher levels of IFN-g in response to cognate peptide
(Fig. 5A, bottom panel).
Using a TCR-transgenic model, we have previously demon-
strated that CD4+T cells specific for an MHC class II epitope of
influenza HA are rendered anergic during the growth of a B cell
lymphoma expressing HA as a model tumor Ag (A20HA). Iso-
tional changes in macrophages treated
with the HDI LAQ824. A, PEMs were
treated or not with LAQ824 (12.5 nM)
for 24 h. Then, cell lysates were ob-
tained, and acetylation of histones H3
and H4 was determined by immuno-
blotting using anti-acetyl H3 and H4
Abs, respectively. B, PEMs were treated
with LPS (2 mg/ml) or with LPS plus
LAQ824 (12.5 nM) for 24 h. Cells were
then stained with an anti-B7.2 Ab or an
isotype control. In a parallel experiment,
PEMs were treated with LPS alone (2
mg/ml) or LPS plus increasing concen-
trations of LAQ824 as indicated. After
24 h, supernatants were collected and the
levels of IL-10 (C) and IL-12 (D) were
determined by ELISA. Shown is a rep-
resentative experiment of five experi-
ments with similar results. *p , 0.05,
**p , 0.01.
Phenotypic and func-
3988 HDAC INHIBITORS AND IL-10 GENE REGULATION
lation of these clonotype-positive T cells from tumor-bearing mice
followed by their in vitro restimulation with HA peptide plus
APCs demonstrated that these cells were anergic given their
inability to produce IL-2 or IFN-g (3, 6). However, in vitro
incubation of these same anergic T cells with LAQ824-treated
PEMs resulted in restoration of T cell responsiveness (Fig. 5B).
Indeed, anergized Ag-specific CD4+T cells encountering cognate
Ag on PEMs treated with LAQ824 and LPS regained their ability
to produce IL-2 (Fig. 5B, top panel) and IFN-g (Fig. 5B, bottom
panel). In contrast, anergic T cells encountering cognate Ag on
untreated (None), HDI alone, or LPS-treated PEM were unable to
produce IL-2 (Fig. 5B, top panel) and produced minimal amounts
of IFN-g (Fig. 5B, bottom panel). Therefore, LAQ824-treated
PEMs effectively prime naive Ag-specific CD4+T cells and re-
store the responsiveness of anergic CD4+T cells.
To better define the role of IL-10 inhibition induced by LAQ824
in the augmentation of the APC function of macrophages, we
cultured PEMs with naive Ag-specific CD4+T cells in the presence
of recombinant murine IL-10. As shown in Fig. 5C, T cells pro-
duced less IFN-g in response to cognate Ag presented by LPS-
treated macrophages when rIL-10 was added (Fig. 5C, LPS alone,
black bar versus gray bar). Similarly, the enhanced APC function
displayed by macrophages treated with 12.5 nM LAQ824+LPS
(Fig. 5C, LAQ+LPS, gray bar) was abrogated when rIL-10 was
added back to the cultures (Fig. 5C, LAQ+LPS, black bar). This
result was not surprising given the well-known ability of IL-10 to
inhibit Th1-type responses (33). Given that the inhibition of IL-10
induced by LAQ824 at the dose of 12.5 nM is incomplete (Fig.
1C), we asked next whether neutralization of the remaining se-
creted IL-10 with anti–IL-10 Abs could further augment the APC
duction by macrophages from tumor-bearing
mice. A, Splenic macrophages from A20
bearing mice were treated with LPS (2 mg/
ml), LAQ824 alone (12.5 nM), or LPS (2 mg/
ml) plus LAQ824 for 24 h. The supernatants
were collected and production of IL-10 (A)
and IL-12 (B) were determined by ELISA.
Shown is a representative experiment of two
experiments with similar results. **p , 0.01.
LAQ824 inhibits IL-10 pro-
(12.5 nM), or LPS (2 mg/ml) plus increasing concentrations of LAQ824 as indicated. After 24 h, supernatants were collected, and cytokine/chemokine
levels were determined by ELISA. Shown is a representative experiment of three experiments with similar results. *p , 0.05, **p , 0.01.
Production of proinflammatory mediators by macrophages treated with HDI LAQ824. PEMs were treated with LPS (2 mg/ml), LAQ824
The Journal of Immunology3989
function of LAQ824-treated macrophages. As shown in Fig. 5D,
IL-10 blockade was insufficient to enhance the APC function of
LPS-treated macrophages to augment IFN-g production by CD4+
T cells (Fig. 5D, LPS alone, black bar versus gray bar), and it was
not able to significantly enhance the effect of LAQ824 treatment
(Fig. 5D, LAQ+LPS, black bar versus gray bar). Taken together,
these data point to a contributory role of IL-10 inhibition in the
enhanced APC function displayed by LAQ824-treated macro-
phages because this effect was reversed when rIL-10 was added
back to the cultures. However, neutralizing the remaining IL-10
protein that could have still been produced by LAQ824-treated
macrophages did not result in further enhancement of their APC
IL-10 mRNA expression and histone acetylation of the IL-10
gene promoter in PEMs treated with LAQ824
IL-10 is an anti-inflammatory cytokine that plays an important role
intheestablishmentandmaintenance ofTcell anergy(34,35).One
of the most striking and consistent effects of pan-HDIs was their
ability to inhibit IL-10 production (Figs. 1C, 3, 4). To better un-
derstand the mechanism(s) underlying this inhibitory effect, we
evaluated next the kinetics of IL-10 mRNA expression in mac-
rophages treated with LPS, LAQ824, or LPS plus LAQ824. In
response to LPS stimulation, a rapid increase in IL-10 mRNAwas
observed at 1 h, followed by a rapid decline, and, after 6 h, the IL-
10 mRNA levels were back to baseline (Fig. 6A, open triangles).
In PEMs treated with LPS plus LAQ824, a decreased induction of
IL-10 mRNA was observed at 1 h (Fig. 6A, closed circles). This
initial response was followed by a progressive decline, and, by 6 h,
IL-10 mRNA levels were back to baseline levels. No changes in
IL-10 mRNA levels were observed in PEMs treated with LAQ824
alone (Fig. 6A, open squares). Therefore, treatment of PEMs with
LAQ824 resulted in decreased IL-10 gene transcriptional activity
in response to LPS stimulation.
Next, we determined the acetylation status of histones at the
level of the IL-10 promoter. First, by using a ChIP assay, we as-
sessed the kinetics of acetylation of histones H3 and H4 in PEMs
treated with LPS alone. As seen in Fig. 6B, acetylation changes
occurred within a particular time window, with a peak acetylation
occurring at 60 min. This was followed by a progressive decline,
and by 4 h, H3 and H4 acetylation levels were almost back to
baseline. Given that the peak acetylation was observed 1 h after
LPS stimulation, in the next set of experiments, PEMs were pre-
treated, or not, with LAQ824 for 24 h and then stimulated or not
with LPS and replenished LAQ824 as appropriate to maintain
pretreatment conditions for an additional 1 h. By ChIP assay, we
treated with LPS (2 mg/ml), HDI alone, or LPS (2 mg/ml) plus increasing concentrations of the HDI LBH589 (A), TSA (B), or MS-275 (C) as indicated.
After 24 h, supernatants were collected, and production of IL-10 (left panel) and IL-12 (right panel) was determined by ELISA. Shown is a representative
experiment of two experiments with similar results. *p , 0.05, **p , 0.01.
Production of IL-10 and IL-12 by PEMs treated with the pan-HDIs LBH589 or TSA or with the class I selective HDI MS-275. PEMs were
3990 HDAC INHIBITORS AND IL-10 GENE REGULATION
found that in response to LPS, histones H3 and H4 display in-
creased acetylation (Fig. 6C, Control LPS) relative to untreated
PEM (Fig. 6C, Control w/o LPS). When PEMs were treated with
HDI alone, no significant changes in acetylation of H3 and H4
were observed (Fig. 6C, LAQ w/o LPS). However, treatment of
PEMs with LPS in the presence of HDI resulted in decreased H3
and H4 acetylation (Fig. 6C, LAQ LPS). This effect was specific
for IL-10 because a similar analysis of H3 and H4 acetylation at
the level of the IL-12 promoter revealed no differences between
PEMs treated with LPS alone and PEMs treated with LPS plus
HDI (Fig. 6D, Control LPS versus LAQ/LPS). Taken together,
treatment of PEMs with LAQ824 resulted in decreased IL-10 gene
transcriptional activity and diminished H3 and H4 acetylation at
the level of the IL-10 gene promoter.
Chromatin changes in the IL-10 gene promoter induced by the
Previously, we have reported the sequence of chromatin mod-
ifications that occur at the level of the IL-10 gene promoter in
macrophages stimulated with LPS (32). Briefly, phosphorylation
of Ser10on H3 is an early event that occurs 30 min after LPS
stimulation. This is followed by increased acetylation of H3 and
H4 at 60 min, subsequent recruitment to the IL-10 gene promoter
of the transcriptional activators Sp1 and STAT3, and, ultimately,
IL-10 gene expression. This sequence of events was affected in
RAW264.7 macrophage cells pretreated with LAQ824 for 24 h
and then stimulated with LPS with replenished LAQ824 (Fig. 7).
First, phosphorylation of Ser10on H3 at the IL-10 promoter has
been shown necessary for transcriptional activation (36). No dif-
ferences in the kinetics of phosphorylation of H3 at the Ser10
position were observed among macrophages treated with LPS
alone (Fig. 7A, solid lines) or cells treated with LAQ824 and LPS
(Fig. 7A, dashed lines). Second, in macrophages treated with LPS,
we observed a peak acetylation of H3 (Fig. 7B) and H4 (Fig. 7C)
at 1 h, which was followed by a progressive decline, and, by 3 h,
H3 and H4 acetylation levels were back to baseline. In sharp
contrast, a diminished H3 and H4 acetylation was observed at all
evaluated time points in cells treated with LAQ824 plus LPS (Fig.
7B, 7C, dashed lines). The effect of LAQ824 upon the global
transcriptional activity of the IL-10 promoter was evaluated next
were treated with LPS alone (2 mg/ml), LAQ824 alone (12.5 nM), or LPS plus LAQ824 as indicated for 24 h. Then, cells were washed with RPMI 1640,
and 5 3 104naive anti-HA transgenic CD4+T cells (A) or 5 3 104anergized CD4+T cells isolated from A20HA-bearing mice (B) were added to the PEMs
in the presence of cognate HA peptide (12.5 mg/ml). After 48 h, supernatants were collected, and IL-2 and IFN-g production was measured by ELISA. The
amount of cytokine production is expressed as picograms per milliliter per 100 clonotype-positive CD4+T cells. Shown is a representative experiment of
three experiments with similar results. PEMs (1 3 105/well) were treated with LPS alone (2 mg/ml) or LPS plus LAQ824 (12.5 nM) in the presence or not
of recombinant murine IL-10 (C) or neutralizing anti-mouse IL-10 Ab (D) for 24 h. Then, cells were washed, with warmed RPMI 1640 and 5 3 104naive
anti-HA transgenic CD4+T cells were added to the PEMs in the presence of cognate HA peptide (12.5 mg/ml). Recombinant murine IL-10 (C) or
neutralizing anti-mouse IL-10 Ab (D) were replenished, or not, in the macrophages/T cell cultures to maintain the respective pretreated conditions, but
without LPS. After 48 h, supernatants were collected, and IFN-g production was measured by ELISA. *p , 0.05, **p , 0.01.
PEMs treated with LAQ824 effectively activate Ag-specific CD4+T cells and restore the function of anergized T cells. PEMs (1 3 105/well)
The Journal of Immunology3991
by determining the binding of PolII to the IL-10 gene promoter.
Unlike macrophages treated with LPS alone, only a minimal and
transient increase in PolII binding was observed in macrophages
treated with LAQ824 plus LPS (Fig. 7D).
Sp1 and STAT3 are known IL-10 transcriptional activators that
are recruited to the proximal region of the IL-10 gene promoter in
macrophages stimulated with LPS (37–39). As shown in Fig. 7E,
recruitment of Sp1 peaks at 1 h after LPS stimulation and is then
followed by a rapid decline at 2 h. Similarly, STAT3 binding to the
IL-10 promoter is evident within 60 min of LPS stimulation. This
is followed by a progressive decline to basal levels by 3 h (Fig.
7F). No such kinetics of recruitment of either Sp1 or STAT3 was
observed in cells treated with LAQ824 and LPS (Fig. 7E, 7F,
PU.1 is a transcriptional repressor that interacts with the distal
IL-10 gene promoter region (40, 41). Recently, we have demon-
treated with 2 mg/ml LPS (open triangles), 12.5 nM LAQ824 (open squares), or the combination thereof (black circles). Cell aliquots were obtained at 1, 6,
and 12 h, and total RNAwas isolated. IL-10 mRNA relative to GAPHD mRNAwas determined by quantitative real-time PCR. Data are depicted as the ratio
of treated cells versus untreated cells. Shown is a representative experiment of three experiments with similar results. B, Kinetics of H3 and H4 acetylation
of the IL-10 gene promoter in response to LPS stimulation. PEMs were treated with LPS (1 mg/ml) for different periods of time (minutes) as indicated.
Nuclear extracts were then obtained, and ChIP using anti-hyperacetylated H3 or anti-hyperacetylated H4 Abs was performed. Shown is a representative
experiment of two experiments performed with similar results. C and D, PEMs were treated or not with LAQ824 (20 nM) for 24 h. Then LAQ824 was
replenished as appropriate to maintain pretreatment conditions, and LPS (1 mg/ml) was added or not to PEM cultures for an additional 60 min. Cells were
then harvested, and nuclear extracts were obtained. ChIP, using anti-hyperacetylated H3 or anti-hyperacetylated H4 Abs, was then performed, and samples
were subject to PCR analysis using primers specific for the IL-10 (C) or IL-12 (D) promoters. IgG and Input (10%) controls are also shown. Signal in-
tensities obtained from ethidium bromide-stained gels were quantified using the ImageQuant 5.2 software (Molecular Dynamics). Shown is a representative
experiment of three performed with similar results.
Changes in IL-10 mRNA expression and histone acetylation of the IL-10 gene promoter in PEMs treated with LAQ824. A, PEMs were
3992HDAC INHIBITORS AND IL-10 GENE REGULATION
strated that HDAC11, by interacting with the distal segment of
the IL-10 gene promoter, negatively regulates the expression of
this cytokine in murine and human APCs (32). As shown in Fig.
7G and 7H, ChIP analysis of the IL-10 gene distal promoter in
macrophages treated with LAQ824 revealed some interesting
findings. Recruitment of PU.1 to the IL-10 gene promoter in cells
treated with LPS reached a peak within 2 h and remained elevated
for the duration of the analysis (Fig. 7G, solid line). In macro-
phages treated with LAQ824, the binding of PU.1 was already
elevated at time zero and increased further in response to LPS
stimulation (Fig. 7G, dashed line). A similar pattern was observed
when we evaluated the kinetics of HDAC11 recruitment to the IL-
10 gene promoter. First, in macrophages treated with LPS alone,
HDAC11 binding was detectable after 2 h, the change was modest
in magnitude, and it remained slightly elevated at 3 h (Fig. 7H,
solid line). In macrophages treated with LAQ824, recruitment of
HDAC11 reached a higher peak (relative to macrophages treated
with LPS alone) within 2 h, and it was followed by a rapid return
to baseline levels at 3 h after LPS stimulation (Fig. 7H, dashed
Therefore, treatment of macrophages with LAQ824 is associated
with several chromatin changes at the level of the IL-10 gene
promoter, among them an enhanced recruitment of the transcrip-
tional repressors PU.1 and HDAC11.
In this study, we have demonstrated that the pan-HDI LAQ824, by
inhibiting IL-10 and increasing the expression of B7.2 and the
production of several proinflammatory mediators, induced inflam-
matory macrophages that effectively activate Ag-specific CD4+
T cells and restore the responsiveness of anergic T cells.
cells were treated with LAQ824 (20 nM) for
24 h. Cells were then washed and treated
or not with LPS (1 mg/ml) and replenished
LAQ824 or not to maintain pretreatment
conditions. Cells were collected at base-
line (time 0) or at 30, 60, 120, or 180 min
after LPS treatment and then subjected to
ChIP analysis using: an Ab to phosphory-
lated Ser10of H3 (A), anti-hyperacety-
lated H3 (B), anti-hyperacetylated H4 (C),
anti-polymerase II (D), anti-Sp1 (E), anti-
STAT3 (F), anti-PU.1 (G), or anti-HDAC11
(H). Quantitative real-time PCR analysis of
the IL-10 gene promoter was performed.
Values were obtained with the Pfaffl method
and are presented relative to input before
immunoprecipitation. Arbitrary units are
presented as percentage relative to no-treat-
ment condition. Error bars represent SD
Chromatin modifications in
The Journal of Immunology 3993
Among the above changes, the most striking effect of LAQ824
was its ability to inhibit the production of the immunosuppressive
cytokine IL-10. Such an effect was also displayed by other mem-
bers of the hydroxamic acid family like LBH589, TSA, and
SAHA, but not by the more specific HDI MS-275, which mainly
targetclassI HDACs.The central role ofIL-10 inthe establishment
and maintenance of T cell anergy (34, 35, 42–44) prompted us to
further investigate the underlying mechanism(s) by which these
particular HDIs inhibit IL-10 production in macrophages.
The dynamic production of pro- and anti-inflammatory medi-
ators at the site of Ag encounter has been shown to shape the
10 plays a key role in negatively regulating these dynamic changes
to prevent self-tissue damage that might otherwise occur if an
ongoing immune response is not kept in check (39). This pro-
tective property of IL-10 imposes, however, a significant barrier to
our efforts to effectively harness antitumor immune responses.
Indeed, production of this cytokine at the tumor site by tumor cells
themselves or infiltrating cells, such as APCs or regulatory T cells,
creates a microenvironment that is conducive to T cell anergy
rather than T cell activation (5). A better understanding of the
genetic and/or epigenetic mechanisms regulating IL-10 production
therefore has important implications to manipulate the inflam-
matory status of APCs and their intrinsic ability to prime, or not,
Ag-specific T cell responses.
Recent studies have shown that IL-10 production is regulated at
the chromatin level by changes in the acetylation status of the gene
promoter (46, 47). For instance, changes in the chromatin struc-
ture of the IL-10 promoter in T cells differentiated into the Th1 or
Th2 phenotype closely regulate IL-10 expression (48). In mac-
rophages, increased acetylation of the IL-10 promoter has been
associated with enhanced transcriptional activity (46). Conversely,
we have recently demonstrated that decreased acetylation of the
IL-10 promoter is associated with decreased IL-10 transcriptional
activity in murine and human APCs (32). Given the above, we
expected that treatment of macrophages with HDIs would result in
increased histone acetylation and increased IL-10 production.
Although an increased global acetylation of histones H3 and H4
was observed in cells treated with LAQ824 (Fig. 1A), the opposite
outcome was observed at the level of the IL-10 gene promoter of
HDI-treated cells. To our surprise, diminished H3 and H4 acety-
lation at the IL-10 promoter was observed at all evaluated time
points in treated macrophages (Fig. 7B, 7C). This decrease in
histone acetylation, which occurs early, might explain the de-
creased recruitment of the transcriptional activators STAT3 and
Sp1 to the IL-10 gene promoter (Fig. 7E, 7F). It is plausible that
a more compacted chromatin due to diminished histone acet-
ylation in the IL-10 promoter region might block the access of
these transcriptional activators to the promoter region resulting in
the decreased IL-10 gene transcriptional activity observed in
However, we are left to explain how HDIs induce decreased
histone acetylation at the level of the IL-10 gene promoter in the
first place. Kinetic analysis of H3 and H4 acetylation provided
some hints. In macrophages pretreated with LAQ824 and then
stimulated with LPS and replenished LAQ824, we observed an
initial acetylation of H3 and H4 that reaches its peak at 1 h
poststimulation. However, the magnitude of these changes was
significantly lower than in macrophages treated with LPS alone.
Following this peak acetylation, we observed a rapid abrogation of
such a response in cells treated with HDI, suggesting either a lack
of stimuli to support H3 and H4 acetylation and/or the induction of
counterregulatory mechanism(s) that attenuated the degree of H3
and H4 acetylation observed in cells treated with LPS alone.
Of particular interest is the finding that following stimulation,
enhanced recruitment of two transcriptional repressors to the distal
promoter of IL-10 was observed, a process that occurs faster for
PU.1 than for HDAC11 (Fig. 7G, 7H). It is plausible therefore that
recruitment of PU.1 and HDAC11 to the IL-10 gene promoter
might represent a counterregulatory mechanism triggered by HDIs
to diminish H3 and H4 acetylation and block the sequence of
events that lead to IL-10 gene transcriptional activation. Sup-
porting the above, we have recently demonstrated that over-
expression of HDAC11 in the macrophage cell line RAW264.7
resulted in decreased H3 and H4 acetylation of the IL-10 gene
promoter and inhibition of IL-10 gene transcriptional activity (32).
Interestingly, Bradbury et al. (49) have found that treatment of
myeloid leukemic blasts with TSA, a member of the hydroxamic
acid family of HDIs, resulted in a 60–200-fold induction of
HDAC11 mRNA expression. Similarly, we have also found that
RAW264.7 cells treated with either TSA or LAQ824 display in-
creased HDAC11 mRNA and protein expression (data not shown).
Such an effect of HDIs upon HDAC11 expression might explain,
at least in part, the increased recruitment of this particular HDAC
to the IL-10 gene promoter in cells treated with LAQ824. The
mechanism(s) by which HDI increases the expression of HDAC11
in macrophages, however, remains to be elucidated.
An additional observation in macrophages treated with LAQ824
is their enhanced expression of B7.2 and increased production
of several proinflammatory mediators, in particular IL-12. It is
plausible that these effects are secondary to the inhibition of IL-10,
a cytokine that has been shown to downregulate the expression
of costimulatory molecules and the production of IL-12 in APCs
(50, 51). This switch from an IL-10– to an IL-12–producing and
B7.2-expression state is critical in polarizing T cells toward Th1
cell-mediated immunity. In our system, the demonstration that H3
and H4 acetylation in the IL-12 promoter was neither inhibited
nor enhanced in cells treated with LAQ824 (Fig. 6D) suggests
that the enhanced production of this proinflammatory mediator in
treated macrophages might be secondary to the abrogation of IL-10
production by LAQ824. Our experiments using rIL-10 also point
to a contributory role of IL-10 inhibition (induced by LAQ824) in
the enhanced APC function displayed by treated macrophages
because this effect was reversed when rIL-10 was added back to
the cultures. It remains to be demonstrated whether the upregu-
lation of B7.2 is a direct or indirect effect of LAQ824. It is
noteworthy to mention that B7.2 was preferentially upregulated in
treated macrophages, whereas no changes were observed in vari-
ous other costimulatory molecules (data not shown). Although the
exact mechanism for this selectivity has yet to be elucidated, our
results are consistent with observations in human DCs whereby
IL-10 mediated downregulation of B7.2 but not B7.1 (50).
Our findings are at odds with the studies by Reddy et al. (23),
who recently found that the HDI SAHA actually attenuated in-
flammatory responses in DCs through IDO-dependent mecha-
nisms. Injection of DCs treated ex vivo with SAHA also decreased
the severity of graft-versus-host disease in their murine allogeneic
bone marrow transplantation model. Several differences between
Reddy’s study (23) and ours may explain these seemingly con-
flicting data. First, in our in vitro system measuring cytokine se-
cretion, we used macrophages that were treated with HDI and LPS
given at the same time. In their study, DCs primarily, but in one
experiment macrophages, were pretreated with SAHA prior to
stimulation with TLR agonist. Second, they found that SAHA
treatment did not induce significant changes in the production of
IL-10 by DCs. Supporting their observation, we have also found
that among all of the members of the hydroxamic family of HDI,
SAHA is the weakest inhibitor of IL-10 production in macro-
3994 HDAC INHIBITORS AND IL-10 GENE REGULATION
phages (data not shown). It is plausible therefore that differences
among members of the same family of HDI, with LAQ824 and
LBH589 being more potent inhibitors of IL-10 production than
SAHA, might explain at least in part the divergent effects of these
HDI upon the inflammatory status of DCs and/or macrophages. Of
note, the overall potencies of HDIs must also be reconciled with
the relative potencies, as pan-HDIs do not inhibit all members of
the HDAC family at equimolar concentrations, which may explain
the differing effects on a single target such as IL-10 (52). Our
findings are, however, consistent with reports by others indicating
that HDIs can potentiate inflammatory and antitumor responses
in vitro and in vivo (25–27). Among the latter, Vo et al. (27) has
demonstrated an enhanced T cell-mediated immunity in tumor-
bearing mice treated with LAQ824. Whether the preferential ex-
pansion of adoptively transferred Ag-specific T cells observed in
this model is the result of T cell interaction with inflammatory
APCs induced by in vivo treatment with LAQ824 treatment re-
mains to be explored.
Taken together, LAQ824-treated macrophages cannot only more
potently activate naive T cells, but also restore the reactivity of
anergized T cells from tumor-bearing mice. LAQ824 by inducing
inflammatory cells can potentially tip the balance toward T cell
activation rather than T cell anergy, an effect that holds promise for
the use of HDIs in the adjuvant setting to ultimately improve the
efficacy of cancer immunotherapy.
The authors have no financial conflicts of interest.
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