LPS-induced IL-8 activation in human intestinal epithelial cells is accompanied by specific histone H3 acetylation and methylation changes
© Angrisano et al; licensee BioMed Central Ltd. 2010
Received: 18 February 2010
Accepted: 14 June 2010
Published: 14 June 2010
The release of LPS by bacteria stimulates both immune and specific epithelial cell types to release inflammatory mediators. It is known that LPS induces the release of IL-8 by intestinal mucosal cells. Because it is now emerging that bacteria may induce alteration of epigenetic patterns in host cells, we have investigated whether LPS-induced IL-8 activation in human intestinal epithelial cells involves changes of histone modifications and/or DNA methylation at IL-8 gene regulatory region.
RT-PCR analysis showed that IL-8 mRNA levels rapidly increase after exposure of HT-29 cells to LPS. DNA demethylating agents had no effects on IL-8 expression, suggesting that DNA methylation was not involved in IL-8 gene regulation. Consistently we found that 5 CpG sites located around IL-8 transcription start site (-83, -7, +73, +119, +191) were unmethylated on both lower and upper strand either in LPS treated or in untreated HT-29 cells, as well as in normal intestinal mucosa.
Conversely, pretreatment of HT-29 cells with deacetylase inhibitors strengthened the LPS-mediated IL-8 activation. Inhibitors of histone deacetylases could induce IL-8 mRNA expression also in the absence of LPS, suggesting that chromatin modifications could be involved in IL-8 gene regulation. Chromatin immunoprecipitation analyses showed that, concurrently with IL-8 activation, transient specific changes in H3 acetylation and H3K4, H3K9 and H3K27 methylation occurred at IL-8 gene promoter during LPS stimulation. Changes of H3-acetyl, H3K4me2 and H3K9me2 levels occurred early, transiently and corresponded to transcriptional activity, while changes of H3K27me3 levels at IL-8 gene occurred later and were long lasting.
The results showed that specific chromatin modifications occurring at IL-8 gene, including histone H3 acetylation and methylation, mark LPS-mediated IL-8 activation in intestinal epithelial cells while it is unlikely that DNA methylation of IL-8 promoter is directly involved in IL-8 gene regulation in these cells.
A possible novel additional strategy used by bacterial pathogens during infection is to interfere with host cellular processes by inducing epigenetic modifications and, consequently, determining a new specific cell transcriptional profile. Bacteria or their components could be a stimulus to change the genetic program of the target cells through epigenetic mechanisms [1, 2]. These mechanisms may operate at gene-specific level and include both chromatin modifications, orchestrated by chromatin-remodeling complexes and histone-modifying enzymes, and DNA methylation, directed by DNA-methyltransferases. Histone acetylation is in general associated to an active state of the chromatin while the effects of histone methylation may be associated with either transcriptional activation or repression, depending on which lysyl residue is modified [3, 4] and whether this residue is mono, di or trimethylated. Among the best studied H3 lysine modifications are di- and trimethylation of H3 on lysine 9 and lysine 27 (H3K9me2 and H3K27me3), associated with closed chromatin, and dimethylation of H3 on lysine 4 (H3K4me2) that marks active chromatin state. DNA methylation of CpG sites at gene regulatory regions is in general related to transcriptional repression and it is believed to be a more stable epigenetic mark compared to histone modifications [5, 6]. However, chromatin modifications and DNA methylation are strictly linked and can associate or interfere with each other [5, 7].
The effects of lipopolysaccharide (LPS) on some aspects of host epigenetics have been recently reported in macrophages and T lymphocytes. In T lymphocytes, LPS stimulation of TLR4 induces histone acetylation and H3S10 phosphorylation allowing for NF-κB to gain access to the IL-12 promoter [11, 12]. Moreover LPS-tolerance, associated with immunosuppression and poor prognosis , has been shown to be controlled by epigenetic changes including methylation of H3K9 [14–16]. LPS is the major component of the outer membrane of gram negative bacteria. The release of LPS by bacteria stimulates both immune and specific epithelial cell types to release inflammatory mediators. Although the effects of LPS have been deeply studied on macrophages and T-cells, only few studies addressed the LPS effects on the intestinal epithelial cells [17, 18]. This is of particular importance because the intestinal epithelial cells represent a key component of the mucosal immune system and are able to express inflammatory genes in response to LPS [17, 18]. These studies addressed the signaling pathways leading to LPS responsiveness of HT-29 cells, a human intestinal epithelial cell line, and demonstrated that LPS response is mediated by gamma interferon (IFN-γ) that induces the expression of the Toll-like receptor 4-MD-2 complex . As a result of LPS stimulation, the proinflammatory cytokine IL-8 accumulates in the culture medium of HT-29 cells.
In this work we have investigated whether epigenetic mechanisms are involved in LPS induced IL-8 gene activation in human intestinal epithelial cells. We found that both histone acetylation and methylation changes at IL-8 promoter, but not DNA methylation, are involved in IL-8 gene activation upon LPS induction.
Results and Discussion
Kinetics of LPS-mediated IL-8 gene activation in HT-29 cells
Because NF-κB has a critical role in LPS-mediated gene activation [17, 19], we measured by western blot analysis the protein levels of the NF-κB inhibitor IκB-α at short intervals after LPS treatment. Results shown in Figure 1B demonstrate that IκB-α was rapidly degraded in HT-29 cells upon LPS stimulation. A significant decrease in IκB-α was already observed 5 minutes after stimulation and persisted up to 60 minutes. These data are consistent with the observed kinetics of IL-8 mRNA expression (Figure 1A).
Inhibitors of histone deacetylases but not of DNA methyltransferases reactivate IL-8 gene expression in HT29 cells
Then we examined the effects of TSA and 5-dAZA on LPS induced IL-8 expression (Figure 2B). HT-29 cells were primed with IFN-γ, pretreated for 24 hours with TSA or 5-dAZA, and then the cells were stimulated with 50 ng/ml LPS. We found that cells pretreated with 5-dAZA showed an IL-8 activation pattern very similar (p = n.s.) to that observed in cells treated with LPS alone (Figure 1A), while TSA pretreatment significantly enhanced the LPS-mediated IL-8 activation (p < 0.05). Taken together these data suggest that histone acetylation state but not DNA methylation may influence IL-8 expression in intestinal derived HT-29 cells.
DNA methylation analysis of IL-8 promoter region
LPS-mediated IL-8 gene activation is accompanied by both histone H3 acetylation and methylation changes
Very interestingly, H3K27me3 levels were initially very low but then increased substantially starting at 6 hours and remained high 24 hours after LPS stimulation (Figure 4E).
H3K27 trimethylation is catalyzed by Polycomb group (PcG) protein complexes [21, 22], which have been shown to be involved in cytokines genes reprogramming occurring in both epithelial and macrophage cells in response to bacterial products and inflammation-related stimuli [23, 24]. It is also worth to note that when all the above ChIP assays were performed in unprimed HT-29 cells (not pre-treated with interferon-γ) we did not detect significant changes in histone H3 methylation state during the same time course (data not shown) suggesting that the observed chromatin modifications are dependent on the MD2/TLR4 pathway. However, because it is well known that even "pure" LPS preparations may be contaminated with lipoproteins, we cannot definitively exclude that the observed chromatin modifications could be influenced by TLR2 signaling.
Taken together our data indicate that while changes of H3-acetyl, H3K4me2 and H3K9me2 state in the IL-8 promoter region occur rapidly, transiently and correspond to transcription activation, the changes of H3K27me3 levels occur at a later time and are long lasting.
Finally it should be considered that a strong mark of gene repression, such as H3K27me3, could predispose to a more repressed state of IL-8 gene and, thus, could render the gene less responsive to further LPS stimulation. Moreover, H3K27me3 is also related to DNA hypermethylation that has been shown to occur in intestinal cancer at PcG target genes. In particular, it has been recently demonstrated that hypermethylation of PcG target genes in intestinal cancer is mediated by inflammation . Thus, although our data indicate that DNA methylation is not directly involved in LPS response, such phenomenon may occur later, after prolonged exposure to LPS, as a consequence of PcG proteins recruitment at IL-8 gene.
In the near future it will be very important to investigate whether the observed increase in H3K27me3 levels at IL-8 gene in response to LPS may occur in intestinal mucosa and may represent a stable epigenetic mark; in this case the observed modifications could be potentially involved in two important phenomena such as the LPS tolerance, and the hypermethylation of PcG target genes in intestinal cancer.
Our data demonstrate an important role of histone modifications, including histone H3 acetylation and H3K4, H3K9 and H3K27 methylation state, in LPS-mediated IL-8 gene activation in intestinal epithelial cells. In particular we demonstrate that H3-acetyl, H3K4me2 and H3K9me2 changes are early, transient and correlate with the modulation of IL-8 transcriptional activity. Conversely, increase of H3K27me3 levels at IL-8 gene occurs later and is long lasting. Our data provide novel insights into the epigenetic mechanisms that control transcription and gene expression in LPS response.
The human colon cell lines HT-29 were grown in Dulbecco's Modified Eagle's Medium supplemented with 10% fetal bovine serum (Life Technologies), 2 mM glutamine, penicillin (25 U/mL) and streptomycin (25 μg/mL) in a 5% CO2 atmosphere at 37°C. Cells were pretreated with Human interferon-γ (INF-γ) (Roche Applied Science, Germany) 10 ng/ml for 12 hours or control medium, washed, and then stimulated with LPS 50 ng/ml. LPS (Escherichia coli, O55:B5) were purchased from Sigma-Aldrich (St. Louis, MO) and reconstituted in endotoxin-free water. 5-aza-2-deoxyazacytidine (ICN Biomedical Inc.) treatments were performed at 5 μM and 50 μM final concentration while trichostatin (TSA) (Sigma Aldrich) was used at 25 and 100 nM.
Western Blot Analysis
Cell extracts were prepared in Nonidet P40 lysis buffer with 1 mM PMSF and Complete™ protease inhibitors mix (Roche, Indianapolis, IN, USA). 50 μg of proteins were resolved by electrophoresis using 10% SDS-PAGE gels and transferred to BA 85 0.45 μm PROTAN nitrocellulose filters (Schleicher & Schnell, Inc., Dassel, Germany). The blots were incubated with rabbit anti-IκB-α antibodies (Santa Cruz Biotechnology, Santa Cruz, CA, USA) and mouse anti-γ-tubulin antibodies (Sigma-Aldrich Corp. St. Louis, MO, USA) as a control for protein loading. Immunoblots were stained with correspondent secondary antibodies IgG (Amersham Pharmacia Biotech, Buckinghamshire, UK), and revealed with the enhanced chemiluminescence detection system IgG (ECL, Amersham Pharmacia Biotech, Buckinghamshire, UK). Western blot analyses of each sample were performed more than three times. Protein levels were quantified using the software Quantity One (Bio-Rad).
Quantitative and semiquantitative RT-PCR analysis
Total RNA was isolated with RNeasy extraction kit QIAGEN (Qiagen,GmBh) according to the manufacturer instructions. The integrity of the RNA was assessed by denaturing agarose gel electrophoresis (presence of sharp 28S, 18S and 5S bands) and spectrophotometry. To ensure that RNA samples were not contaminated by DNA, negative controls were obtained by performing the PCR on samples that were not reversed-transcribed but otherwise identically processed. 1 μg of total RNA of each sample was reverse-transcribed with QuantiTect® Reverse Transcription (Qiagen) using an optimized blend of oligo-dT and random primers according to the manufacturer's instructions. Quantitative PCR amplifications were performed using QuantiTect SYBR Green (Qiagen) in a Chromo4 Real Time thermocycler (BIORAD). Following primers were used for IL-8 cDNA amplification: cIL-8F (forward) 5'-ggcacaaactttcagagacag-3' and cIL-8R (reverse) 5'-acacagagctgcagaaatcagg-3'; G6PD gene was used as housekeeping gene for PCR reaction: G6F (forward) 5'-acagagtgagcccttcttcaa-3' and G6R (reverse) 5'-ggaggctgcatcatcgtact-3'. The quantitative PCR conditions were: 95°C for 15 minutes followed by 40 cycles of 95°C for 15 seconds, 60°C for 30 seconds, and 72°C for 30 seconds. Calculations of relative expression levels were performed using the 2-ΔΔCt method  and take into account the values of at least three independent experiments. Semiquantitative PCR reactions were performed for the assessment of IL-8 expression, using cIL-8F and cIL-8R primers, and MD-2 expression using the following primers: MDF (forward) 5'-ggctcccagaaatagcttcaac-3' and MDR (reverse), 5'-ttccaccctgttttcttccata-3'; GAPDH was used as a housekeeping gene for normalization using the following primers: GAPF (forward) 5'-ggtcgtattgggcgcctggtcacc-3' and GAPR (reverse) 5'- cacacccatgacgaacatgggggc-3'. Each reaction was performed in triplicate. The conditions used for semiquantitative PCR were 1 minute at 94°C, 1 minute at 60°C and then 2 minutes at 68°C for 30 cycles. The PCR products were separated on a 1.5% agarose gel and stained with ethidium bromide.
DNA methylation analysis
Genomic DNA was isolated from cultured cells and from tissue samples using DNeasy Blood and Tissue extraction kit (Qiagen) according to the manufacturer's instructions. Colon samples were obtained from the tissue bank of the Naples Oncogenomic Center (NOGEC). Normal mucosa samples were taken from macroscopically and microscopically unaffected areas of a colon cancer specimen. Sodium bisulfite conversion of 1 μg of genomic DNA was performed using EZ DNA Methylation Kit (Zymo Research). DNA methylation analysis was performed using the SEQUENOM MassARRAY platform. This system utilizes MALDI-TOF mass spectrometry in combination with RNA base specific cleavage (MassCLEAVE). A detectable pattern is then analyzed for methylation status. PCR primers to analyze IL-8 promoter region, designed by using Epidesigner http://www.epidesigner.com, were: for upper strand region (-137 to +246) IL-8UF 5'-aggaagagagGGAAGTGTGATGATTTAGGTTTGTT-3' and IL-8UR 5' cagtaatacgactcactatagggagaaggctCCAAAACATCAAAAATAACTTTACTATCT-3'; for lower strand (region -113 to +264) IL-8LF 5'- aggaagagagAAAAAGGATGTTTGTTATTAAAGTATTAAG-3' and IL-8LR 5'- cagtaatacgactcactatagggagaaggctCCCTAAAAAAATAAACCATCAATTAC-3'. For reverse primer, an additional T7 promoter tag for in vivo transcription was added, as well as a 10-mer tag on the forward primer to adjust for melting-temperature differences. The sensitivity of methylation assay was evaluated using Universal methylated and unmethylated Human DNA Standards (Zymo Research Corporation, Orange USA) and the standard error was found to be ± 3%. The MassCLEAVE biochemistry was performed as previously described . Mass spectra were acquired by using a MassARRAY Compact MALDI-TOF (Sequenom) and spectra's methylation ratios were generated by the Epityper software v1.0 (Sequenom). The whole procedure was performed at Sequenom GmbH Laboratories (Hamburg, Germany).
Quantitative ChIP analysis
Cells were plated at a density of ~ 3-5 106 in 100 mm Petri dish 24 h before the treatments. Cells were cross-linked by adding 1% formaldehyde for 15 minutes at room temperature in shaking. Glycine was added to a final concentration of 125 mM for 5 minutes at room temperature in shaking. Cells were rinsed twice with cold PBS supplemented with 500 μM PMSF and harvested in five pellet-volumes of Cell Lysis Buffer (5 mM PIPES pH 8.0, 85 mM KCl, 0.5% NP40) supplemented with 1 mM PMSF and Complete™ protease inhibitors mix. Lysates were incubated for 30 minutes at 4°C and then passed through ten dounce cycles. They were subsequently centrifuged and nuclei were collected. Nuclei were then resuspended in 250 μL Sonication Buffer (0.3% SDS, 10 mM EDTA, 50 mM Tris-HCl ph 8.0) supplemented with 1 mM PMSF and Complete™ protease inhibitors mix and incubated for 60 minutes at 4°C. Chromatin was sonicated to an average DNA length of 300-800 bp using a 3 mm (small size) tip equipped Bandelin Sonoplus UW-2070 sonicator with 5 × 10 seconds cycles of pulses (specific cycle 0.3, Power 30%) alternated by 60 seconds of rest. Sonicated samples were centrifuged and the supernatant was collected. 80 μg of chromatin were diluted with Dilution Buffer (0.01% SDS, 1.2 mM EDTA, 16.7 mM Tris-HCl pH 8.0, 1,1% TRITON X-100, 167 mM NaCl), precleared (2 hours) by incubation with 20 μL Salmon Sperm DNA/Protein A Agarose-50% Slurry (Upstate Biotechonology, Dundee; UK) and subjected to immunoprecipitation with specific antibodies with rotation over-night at 4°C. Antibodies used for ChIP assays were: anti-H3Ac, anti di-methyl-H3K9, anti tri-methyl-H3K27 (Upstate Biotechnology) and anti-di-methyl-H3K4 (Abcam Inc.). Immunocomplexes were collected by adsorption onto 30 μL Salmon Sperm DNA/Protein A Agarose-50% Slurry and the beads were washed (four times) sequentially with Low Salt Washing Buffer (0.1% SDS, 2 mM EDTA, 20 mM Tris-HCl pH 8.0, 1% Triton X-100, 150 mM NaCl), High Salt Washing Buffer (0.1% SDS, 2 mM EDTA, 20 mM Tris-HCl pH 8.0, 1% Triton X-100, 500 mM NaCl) and LiCl Washing Buffer (Upstate). Precipitates were washed with TE Buffer (10 mM Tris-HCl pH 8.0 and 1 mM EDTA), and antibody-chromatin fragments were eluted from the beads with 1% sodium dodecyl sulphate in 0.1 M NaCO3. Cross-links were reverted by adding 200 mM NaCl and heating at 65°C overnight. 40 mg/mL RNase A and 20 mg/mL proteinase K, 10 mM EDTA and 40 mM Tris-HCl pH 6.5 were added and samples were then incubated 2 hours at 45°C. Samples were then extracted in phenol-chloroform-isoamylic acid (25:24:1), ethanol-precipitated and finally centrifuged at 13000 rpm for 45 minutes at 4°C. Pellets were washed with 70% ethanol, centrifuged at 8000 rpm for 5 minutes at 4°C and finally resuspended in 60 μL of H2O. 2 μL of each sample were used as template for subsequent PCR analysis and 32 amplification cycles were used. Amplification of the IL-8 promoter fragment, using SYBR®Green Taq, was performed using the primers: pIL-8F (forward) 5'- CAGAGACAGCAGAGCACAC-3' and pIL-8R (reverse) 5'-ACGGCCAGCTTGGAAGTC-3' amplifying a 101 bp fragment. All PCR signals from immunoprecipitated DNA were normalized to PCR signals from non-immunoprecipitated input DNA. The signals obtained by precipitation with the control IgG were subtracted from the signals obtained with the specific antibodies. Results are expressed as percentage of the input: signals obtained from the ChIPs were divided by signals obtained from an input sample; this input sample represents the amount of chromatin used in the ChIP. Calculations take into account the values of at least three independent experiments.
Statistical significance between groups was assessed by Student's t test. Data are expressed as means ± standard deviation (SD). All experiments were repeated at least three times. A p value < 0.05 was considered to be statistically significant.
reverse-transcription-polymerase chain reaction
Toll-like receptor 4
myeloid differentiation protein 2
Polycomb protein complexes
inhibitors of NF-κB.
This work was supported by grant from MIUR (PRIN07) to LC.
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