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Am J Physiol Endocrinol Metab 290: E490-E493, 2006. First published October 18, 2005; doi:10.1152/ajpendo.00406.2005
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Molecular inhibition of histone deacetylation results in major enhancement of the production of IL-1beta in response to LPS

Timothy A. Sato1 and Murray D. Mitchell1,2

1Liggins Institute, University of Auckland; and 2National Research Centre for Growth and Development, Auckland, New Zealand

Submitted 26 August 2005 ; accepted in final form 12 October 2005


   ABSTRACT
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 ABSTRACT
MATERIALS AND METHODS
 RESULTS AND DISCUSSION
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It has been postulated that the progression of human pregnancy to term is, in part, the result of a relative maternal Th2 immunological state. This can be activated in some cell types by modifying DNA methylation and histone acetylation status. We demonstrate that the molecular inhibition of histone deacetylation, using trichostatin A (TSA), in human choriodecidual explants leads to a massive increase in lipopolysaccharide (LPS)-stimulated IL-1beta. The inhibition of histone deacetylation had no effect on LPS-stimulated TNF-{alpha} production or production of the other cytokines studied (IL-10, IL-1 receptor antagonist). The molecular inhibition of DNA methylation and histone deacetylation, using 5-aza-2'-deoxycytidine and TSA, respectively, in human choriodecidual explants also results in an increase in the basal production of TNF-{alpha} but not that of IL-1beta. The differential response is unique, and the relative uncoupling of IL-1beta and TNF-{alpha} responsiveness may have importance in other biological systems and provide new therapeutic targets for pathologies where upregulation of IL-1beta is known to be a causative factor.

interleukin-1beta; lipopolysaccharide; histane deacetylation; human pregnancy

CYTOKINES ARE KEY INFLAMMATORY MEDIATORS that regulate numerous cellular responses (20). They can be categorized as proinflammatory (e.g., IL-1beta and TNF-{alpha}) or anti-inflammatory [e.g., IL-10 and IL-1 receptor antagonist (IL-1Ra)] depending on the cellular response initiated. Cytokines can further be categorized as Th1 or Th2 on the basis of whether they initiate a cellular or antibody-generating immune response, respectively (18). Maintenance of a Th2 type phenotype is critical to the maintenance of pregnancy (11, 17, 34). Recent studies have demonstrated that alterations of DNA methylation in T cells can lead to an adaption to a Th2 type phenotype (14).

Prostaglandin H synthase (PGHS)-2 is a key biosynthetic enzyme of arachidonic acid metabolism. Recent reports have demonstrated that PGHS-2 activity is suppressed in cancer cell lines by aberrantly enhanced DNA methylation and reduced histone acetylation; activity can be restored by treatment with inhibitors of DNA methylation and histone deacetylation (12, 32). Enhanced intrauterine prostaglandin production is requisite for the onset and progression of labor at term and preterm, and the most extremely preterm deliveries are mainly a result of an intrauterine infection associated with increased cytokine production (24). Recently, we (16) have demonstrated that prostaglandin production by intrauterine tissues of women at term of pregnancy can be regulated by inhibition of DNA methylation and histone deacetylation. Hence, we hypothesize that the regulation of DNA methylation and histone acetylation in key genes in human gestational tissues may be critical for the initiation of labor and delivery both at term and preterm.

To evaluate this proposition, we have now determined the effects of inhibiting DNA methylation and histone deacetylation on cytokine production by human choriodecidua, which is the major site of intrauterine cytokine production.


   MATERIALS AND METHODS
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 MATERIALS AND METHODS
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Reagents. Lipopolysaccharide (LPS), 5-aza-2'-deoxycytidine (ADC), and trichostatin A (TSA) were purchased from Sigma Chemical (St. Louis, MO). Media were from Irvine Scientific (Santa Ana, CA) and the fetal calf serum (FCS) from GIBCO (Grand Island, NY). Recombinant IL-1beta and anti-IL-1beta antisera were purchased from R&D Systems (Minneapolis, MN). Antibodies to IL-10 and TNF-{alpha} were purchased from PharMingen (San Diego, CA). Recombinant IL-10 and TNF-{alpha} used to calibrate the ELISAs were purchased from R&D Systems. A dual set of antibodies for IL-1Ra detection was also purchased from R&D Systems.

Explant cultures. The Auckland Ethics Committee approved all procedures involving human placentas. All patients gave informed consent for the use of their placentas. Placentas were obtained from women undergoing elective caesarean section at term. The amnion was separated from the choriodecidua membrane and rinsed in PBS to remove residual red blood cells. By use of a cork borer, 6-mm disks from the choriodecidual membrane were made (31). Three disks were placed in each well of a 12-well plate in triplicate. The tissues were cultured in DMEM-199 containing 10% heat-inactivated FCS plus antibiotics. Choriodecidua explants were established and treated with LPS (5 µg/ml) in the presence or absence of ADC, an inhibitor of DNA methylation, or a lower concentration of ADC with TSA, a treatment known to inhibit histone deacetylation. Concentrations and treatment protocols of the ADC and TSA were chosen on the basis of previously reported effective concentrations and times (12, 16, 32) so that our results could be put into the context of the current literature. The specificity of ADC has previously been confirmed (8). The specificity for the actions of TSA has also been established in previous studies conducted by other research groups (35).

Explants were treated with ADC (5 µM or 200 nM) for 2 days and kept in 5% CO2 in air at 37°C, the medium was replaced every day. After day 2, the explants were treated with LPS (5 µg/ml). Additionally, the explants treated with 200 nM ADC had TSA (300 nM) added as well. After a further 24-h incubation, the medium was removed and the wet weight of the tissue in each well determined so that production rates could be normalized.

Assays. Measurements of pro- and anti-inflammatory cytokines were conducted on cultured media using ELISAs. IL-1beta, IL-6, IL-10, and TNF-{alpha} were measured by ELISA, as previously described (26, 31). The manufacturer's specifications for the detection of IL-1Ra were followed. LDH levels were determined using a commercially available kit (Sigma), following the manufacturer's specifications.

Presentation of data. Production rates of cytokines were calculated as picograms per milligram wet tissue weight per 24 h (means ± SE, n = 5). Statistical significance was determined by ANOVA, and P < 0.05 was considered to be significant.


   RESULTS AND DISCUSSION
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 ABSTRACT
 MATERIALS AND METHODS
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The basal production rates of the cytokines studied are presented in Table 1. In all experiments, LPS caused a significant (P < 0.05) increase in IL-1beta (~4-fold) and TNF-{alpha} (~25-fold) production by human choriodecidual explants (Fig. 1). Inhibition of histone deacetylation caused a massive increase in IL-1beta production upon stimulation with LPS. This ~60-fold increase was observed only in IL-1beta production. The apparent decrease in TNF-{alpha} production upon LPS stimulation can be attributed solely to the increase in basal production rate, as the absolute LPS-stimulated production rate was very similar. There were no changes in the basal production rate of IL-1beta. There was no effect on the LDH levels (data not shown), so none of the treatments had an adverse effect on the explants.


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  		Table 1. Basal production rates of IL-1beta, TNF-{alpha}, IL-10, and IL-1Ra by human choriodecidual explants

 

Figure 1
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  		Fig. 1. Effects of altering DNA methylation and histone acetylation status on IL-1beta and TNF-{alpha} production by human choriodecidual explants. Rates of production of IL-1beta and TNF-{alpha} in the presence (filled bars) and absence (open bars) of LPS (5 µg/ml) and treatment with inhibitors of DNA methylation [5-aza-2'-deoxycytidine (ADC)] and histone deacetylation [ADC + trichostatin A (TSA)]. Data are presented as production of cytokines (as %control values). *P < 0.05 vs. unstimulated controls; #P < 0.05 vs. LPS-stimulated controls, n = 3.

 
Upon stimulation with LPS, there was a significant (P < 0.05) increase in the rates of production of IL-10 (~4-fold) and IL-1Ra (~2.5-fold) (Fig. 2). Alteration of DNA methylation or histone deacetylation status had no effect on the basal rates of production of IL-10 or IL-1Ra by human choriodecidual explants (Fig. 2). Inhibition of DNA methylation or histone deacetylation had no significant effects on IL-10, TNF-{alpha}, or IL-1Ra production upon LPS stimulation.


Figure 2
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  		Fig. 2. Effects of altering DNA methylation and histone acetylation status on IL-10 and IL-1 receptor antagonist (IL-1Ra) production by human choriodecidual explants. Rates of production of IL-10 and IL-1Ra in the presence (filled bars) and absence (open bars) of LPS (5 µg/ml) and treatment with inhibitors of DNA methylation (ADC) and histone deacetylation (ADC + TSA). Data are presented as production of cytokines (as %control values); n = 3.

 
Conventionally, in response to a stimulus, any observed increase in either IL-1beta or TNF-{alpha} production is also associated with an increase in the production of the other. Given the massive increase in IL-1beta production in response to LPS during inhibition of histone deacetylation, it was unexpected that there was very little effect on TNF-{alpha} production. In fact, if plotted as a stimulation index (Fig. 3), when histone deacetylation was inhibited the production rates of TNF-{alpha}, IL-10, and IL-1Ra actually decreased, whereas that of IL-1beta was massively increased.


Figure 3
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  		Fig. 3. Stimulation index for cytokine production by human choriodecidual explants after histone acetylation status alteration. Stimulation index (ratio of %stimulation by LPS in explants treated with an inhibitor of histone deacetylation over the same %stimulation in the absence of inhibition of histone deacetylation) for IL-1beta, IL-10, TNF-{alpha}, and IL-1Ra production for the histone acetylation-inhibited/LPS-stimulated treatment group.

 
Any factors produced by the choriodecidua would have direct access to the myometrium, thus potentially initiating or inhibiting contractions via enhanced/decreased production of uterotonic agents, such as prostaglandins, that are regulated by cytokines (24). The events that lead to parturition and delivery are complex and involve a coordinated event involving not only the choriodecidua but the myometrium, amnion, and placenta as well. Any disruptions or alterations in one component are bound to have effects on the others. It has been reported that inhibition of histone deacetylation lengthened the gestational period in mice by approximately one day (2). Results presented here seemingly are not consistent with the implications from those studies, since IL-1beta has been demonstrated to initiate the mechanisms that instigate parturition (2). The increase that we observed in IL-1beta production in response to LPS during inhibition of histone deacetylation would most likely be sufficient to contribute to the processes of labor. It is tempting to speculate that the enhanced inhibitory influences exerted via progesterone actions (2) might outweigh the stimulatory actions of IL-1beta (30); however, the precise mechanism(s) are not known at this time.

There are increased concentrations of cytokines in the amniotic fluid of patients undergoing preterm labor as a result of an intrauterine infection (23). Intra-amniotic IL-1{alpha} has been shown to induce fetal lung maturity by inducing the production of surfactant (1, 13). The increase in IL-1beta production that we have observed could be a mechanism by which, given the impending preterm birth of the fetus, mechanisms are initiatedto attempt to facilitate the maturation of the fetal lungs. The elevation of only IL-1beta production that we have observed supports this theory, as TNF-{alpha} was demonstrated to have no effect on the maturation of the sheep fetal lung, whereas IL-1{alpha} was (5, 10). A recent study has demonstrated that surfactant from the fetal lung may mediate the initiation of parturition via stimulation of macrophage migration to the uterus (3) and subsequent release of IL-1beta. Taken together, these studies point to a potentially fascinating feedforward system by which the IL-1beta produced drives the production of surfactant which in turn initiates parturition.

Our results implicate a new regulatory pathway for the production of IL-1beta that is potentially critical for parturition and survival of the newborn. In addition to its role in term and preterm labor, it has been demonstrated that IL-1beta plays a critical part in early pregnancy. For instance, the precise timing of IL-1beta upregulation is vital for porcine conceptus elongation and placental implantation to occur (25). Moreover the shift to a Th2 state that seems to be requisite for successful maintenance of pregnancy may be IL-1beta regulated, since Th2 proliferation of T helper cells requires IL-1beta expression by antigen-presenting cells (33). The potentially central role of IL-1beta in early pregnancy is further emphasized by the actions of this cytokine in enhancing both estrogen and progesterone biosynthesis in placental cells and cell lines (6, 18). In our studies, the basal production of TNF-{alpha} was upregulated when methylation and histone acetylation status were altered. TNF-{alpha} has been demonstrated to be important in placental growth and differentiation (9). A recent report demonstrated that histone acetylation is required for TNF-{alpha} production by alveolar cells, but regulation of histone acetylation was not solely responsible for its production (15), whereas another report demonstrated that TNF-{alpha} could induce histone acetylation in monocytes and macrophages (21). Clearly there are complex and intertwined regulatory pathways in place that require further exploration.

The upregulation of IL-1beta has been implicated in Alzheimer's disease (AD) and Down’s syndrome, as there was up to 30 times more IL-1beta present in the glial cells of affected individuals (7). IL-1beta treatment of rat brains caused a six- to sevenfold increase in a marker that is overexpressed in neurons and dystrophic neuritis in AD patients (29). Additionally, treatment of peripheral blood mononuclear cells isolated from AD patients with an acetylcholinesterase inhibitor, a compound that has shown some degree of effectiveness in slowing down the progression of AD, resulted in a decrease in IL-1beta production (22). The regulation of methylation, specifically of promoters, has been implicated in the onset and progression of AD (27, 28); however, there is nothing in the literature examining the histone acetylation status in AD patients at present.

Findings reported here have far-ranging implications that not only include pregnancy and delivery but also extend to include conditions where IL-1beta has been shown to be a key regulatory mediator, such as many infectious diseases (4). Given the wide range of systemic effects that IL-1beta exerts, further investigation of this novel regulatory pathway will need to be undertaken to better understand the potential implications for better-targeted therapeutics for these conditions.


   GRANTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
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REFERENCES
 
This work was funded by the Health Research Council of New Zealand and the Auckland University Research Committee.


   ACKNOWLEDGMENTS
 
We acknowledge and thank the theater staff at National Women's hospital, with a special thanks to Olivia Tupusi for organizing patient consent and collection of the placentas.


   FOOTNOTES
 

Address for reprint requests and other correspondence: M. D. Mitchell, Liggins Institute, Faculty of Medical and Health Sciences, Univ. of Auckland, Private Bag 92019, Auckland, New Zealand (e-mail: m.mitchell{at}auckland.ac.nz)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
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