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The Journal of Immunology, 2005, 175: 2843-2850.
Copyright © 2005 by The American Association of Immunologists

Intracellular Bacteria Differentially Regulated Endothelial Cytokine Release by MAPK-Dependent Histone Modification1

Bernd Schmeck*, Wiebke Beermann*, Vincent van Laak*, Janine Zahlten*,{dagger}, Bastian Opitz*, Martin Witzenrath*, Andreas C. Hocke*, Trinad Chakraborty{ddagger}, Michael Kracht§, Simone Rosseau*, Norbert Suttorp* and Stefan Hippenstiel2,*

* Department of Internal Medicine and Infectious Diseases, {dagger} Department of Periodontology, Charité-University Medicine Berlin, Berlin, Germany; {ddagger} Institute for Medical Microbiology, Justus-Liebig University of Giessen, Giessen, Germany; and § Institute of Pharmacology, Medizinische Hochschule Hannover, Hannover, Germany


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Epigenetic histone modifications contribute to the regulation of eukaryotic gene transcription. The role of epigenetic regulation in immunity to intracellular pathogens is poorly understood. We tested the hypothesis that epigenetic histone modifications influence cytokine expression by intracellular bacteria. Intracellular Listeria monocytogenes, but not noninvasive Listeria innocua, induced release of distinct CC and CXC chemokines, as well as Th1 and Th2 cytokines and growth factors by endothelial cells. Cytokine expression was in part dependent on p38 MAPK and MEK1. We analyzed global histone modification and modifications in detail at the gene promoter of IL-8, which depended on both kinase pathways, and of IFN-{gamma}, which was not blocked by kinase inhibition. Intracellular Listeria induced time-dependent acetylation (lysine 8) of histone H4 and phosphorylation/acetylation (serine 10/lysine 14) of histone H3 globally and at the il8 promoter in HUVEC, as well as recruitment of the histone acetylase CREB-binding protein. Inhibitors of p38 MAPK and MEK1 reduced lysine 8 acetylation of histone H4 and serine 10/lysine 14 phosphorylation/acetylation of histone H3 in Listeria-infected endothelial cells and disappearance of histone deacetylase 1 at the il8 promoter in HUVEC. In contrast, IFN-{gamma} gene transcription was activated by Listeria monocytogenes independent of p38 MAPK and MEK1, and histone phosphorylation/acetylation remained unchanged in infected cells at the IFN-{gamma} promoter. Specific inhibition of histone deacetylases by trichostatin A increased Listeria-induced expression of IL-8, but not of IFN-{gamma}, underlining the specific physiological impact of histone acetylation. In conclusion, MAPK-dependent epigenetic modifications differentially contributed to L. monocytogenes-induced cytokine expression by human endothelial cells.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The foodborne Gram-positive facultative intracellular bacterium Listeria monocytogenes (1) is a well-established model organism to study cellular microbiology of and immunity to intracellular bacteria (2). L. monocytogenes causes sepsis and meningitis, especially in newborns and immunocompromised patients (2). During disseminated infection, invasion of endothelial cells by L. monocytogenes is part of listeriosis (3). In recent studies, L. monocytogenes was found to activate the transcription factor NF-{kappa}B and induce endothelial expression of cytokines like IL-8, IL-6, GM-CSF, and MCP-1 (4, 5), as well as adhesion molecules P-selectin, E-selectin, ICAM-1, and VCAM-1 (3, 4). Finally, recruitment and activation of leukocytes by cytokines like IL-8, IFN-{gamma}, and TNF-{alpha} have been shown to be essential for clearance of L. monocytogenes infection (2, 6, 7, 8, 9). Inflammatory cell activation depends on listerial bacterial virulence factors like phospholipases and listeriolysin (4, 10) and requires evasion from the phagosome (11).

Increasing evidence indicates that histone modifications may serve as a combinatorial code for the transcriptional activity state of genes in many cellular processes by loosening the DNA-histone interaction and unmasking of transcription factor binding sites (12). In chromatin, 146 bp of DNA are wrapped 1.65 turns around a histone octamer (H2A, H2B, H3, H4) (13). A wide range of specific covalent modifications of accessible N-terminal histone tails is decisive for transcription repression or gene activation (14). To date, acetylation (mostly lysine), phosphorylation (serine/threonine), methylation (lysine), ADP ribosylation, and ubiquitination of histones have been described (15, 16). Phosphorylation at serine 10 (Ser10) on H3 and acetylation at lysine 14 (Lys14) of H4 seems to have a special impact on gene regulation (13). For example, it was found that LPS stimulation of dendritic cells induced p38 MAPK-dependent phosphorylation at Ser10 on H3 and acetylation at Lys14 on H4 specifically occurring at IL-8 and MCP-1, but not at TNF-{alpha} or MIP-1{alpha} genes (17). Both modifications have been correlated with the immediate early gene induction. H3 seems to be phosphorylated at Ser10 by p38 MAPK effectors mitogen- and stress-activated protein kinase-1 and kinase-2 (18). H4 is subsequently acetylated by histone acetyl transferases at Lys14. Moreover, LPS induced methylation of Lys4 on H3 of the IL-8 gene, which is considered to enhance transcriptional activity, but no silencing by methylation of Lys9 could be detected (19).

Although the endothelium lining the inner vessel wall is a primary target of pathogens entering the blood stream, including L. monocytogenes, the role of epigenetic modifications in a bacterial infection of endothelial cells is unknown. We hypothesized that a bacterial infection induced epigenetic modifications leading to increased proinflammatory cytokine expression in human endothelial cells.

In this study we show that L. monocytogenes activated p38 MAPK and ERK1/2 signaling pathways and induced cytokine expression in human endothelial cells. Expression of IL-8, but not IFN-{gamma} depended on histone modifications. L. monocytogenes induced p38 MAPK- and ERK1/2-dependent acetylation of histone H4 and phosphorylation/acetylation of histone H3 both globally and at the il8 gene promoter, but not at the ifn{gamma} promoter. Thus, bacteria-induced histone modifications contributed to cytokine expression of human endothelial cells.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Materials

U0126, SP600125, trichostatin A (TSA),3 SB202190, SB203580, and SB202474 were purchased from Calbiochem. TNF-{alpha} was purchased from R&D Systems. All other chemicals used were of analytical grade and obtained from commercial sources.

Cell culture

HUVEC were isolated from umbilical cord veins and identified as previously described (20, 21, 22). Briefly, cells obtained from collagenase digestions were washed, resuspended in MCDB-131/10% FCS and seeded into T75 flasks, or 6-well or 24-well plates. Studies performed were done using confluent endothelial cell monolayers in their second passage (3, 10).

Bacterial strains and infection

The wild-type L. monocytogenes serotype 1/2a strain, EGD, and the Listeria innocua serotype 6b strain (33090; American Type Culture Collection) INN were grown in brain heart infusion broth (Difco) at 37°C to the logarithmic growth phase and then given to HUVEC grown in medium without antibiotics with indicated concentrations and for indicated times as described (3, 10). To verify intracellular infection, HUVEC were incubated for 2 h with the bacteria, and monolayers were washed three times with plain medium to remove unbound bacteria, incubated for 1 h with gentamicin, and treated with 10% (w/v) saponin (Sigma-Aldrich) to lyse the host cells. Serial dilutions were plated on agar, and colonies were counted after 24 h.

IL-8 ELISA

Confluent HUVEC were stimulated for 15 h as indicated in a humidified atmosphere. After incubation supernatants were collected and processed for IL-8 quantification by sandwich ELISA as previously described (23).

Bioplex protein array system

Confluent HUVEC were stimulated for 15 h as indicated in a humidified atmosphere. After incubation supernatants were collected, and cytokines were analyzed with the Bioplex protein array system (Bio-Rad) using specific beads, according to manufacturer’s instructions.

Confocal microscopy

HUVEC monolayers were infected as described and fixed with 3% PFA (w/v; Sigma-Aldrich) for 20 min. Subsequently, DAPI (4',6'-diamidino-2-phenylindole; Sigma-Aldrich) staining for cellular and bacterial DNA was performed. For visualization of intracellular Listeria, additional F-actin staining was conducted with phalloidin Alexa Fluor 488 (Molecular Probes). Fluorescent images were acquired using an Axioskop 2 mot (objective, PlanNeoFluar x100; NA 1.4) equipped with an AxioCam MRm cooled greyscale camera (Zeiss). Deconvolution and digital image processing was executed by ImageProPlus 5.0 software (Media Cybernetics). To reach higher contrast for bacterial DNA and F-actin cladding in overlay images, red color was given for the DAPI channel.

Western blot

For determination of p38 MAPK or ERK1/2 phosphorylation, HUVEC were stimulated as indicated, washed twice, and harvested. Cells were lysed in buffer containing Triton X-100, subjected to SDS-PAGE and blotted on Hybond-ECL membrane (Amersham Biosciences). Immunodetection was conducted with phospho-specific p38 MAPK Ab, phospho-specific ERK1/2 Ab, or phosphor-specific JNK Ab (Cell Signaling Technology), respectively (20). For histone analysis, cells were lysed in lysis buffer (10 mM HEPES, pH 7.9, 1.5 mM MgCl2, 10 mM KCl, 1.5 mM PMSF, 0.5 mM DTT) and H2SO4 was added to a final concentration of 0.2 M. Protein was precipitated with 50% TCA in a final concentration of 20%, subjected to SDS-PAGE, and blotted on Hybond-ECL membrane (Amersham Biosciences). Immunodetection was conducted with Abs specifically detecting Ac-Lys8-H4 and P-Ser10/Ac-Lys14-H3 (Cell Signaling Technology). In all experiments, ERK2 or p38 MAPK (Santa Cruz Biotechnology) was detected simultaneously to confirm equal protein loading. Proteins were visualized by incubation with secondary IRDye 800- or Cy5.5-labeled Abs, respectively (Odyssey infrared imaging system; LI-COR) (24, 25).

Chromatin immunoprecipitation (ChIP)

HUVEC were stimulated, culture medium was removed, and 1% formaldehyde was added. After 1 min, cells were washed in ice-cold 0.125 M glycin in PBS and then rapidly collected in ice-cold PBS, centrifuged and washed twice with ice-cold PBS. Cells were lysed in RIPA buffer (10 mM Tris pH 7.5, 150 mM NaCl, 1% Nonidet P-40, 1% desoxycholic acid, 0.1% SDS, 1 mM EDTA, 1% aprotinin) and the chromatin was sheared by sonication. Lysates were cleared by centrifugation and supernatants were stored in aliquots at –80°C until further use. Abs used for immunoprecipitation were purchased from Cell Signaling Technology (Ac-Lys8-H4 and P-Ser10/Ac-Lys14-H3) and Santa Cruz Biotechnology (CREB-binding protein (CBP), histone deacetylase (HDAC)1, polymerase II (Pol II)).

Immunoprecipitation from soluble chromatin was conducted overnight at 4°C. Immune complexes were collected with protein A/G-agarose for 60 min and washed twice with RIPA buffer, once with high-salt buffer (2 M NaCl, 10 mM Tris, pH 7.5, 1% Nonidet P-40, 0.5% desoxycholic acid, 1 mM EDTA) followed by another wash in RIPA buffer and one wash with TE buffer (10 mM Tris, 1 mM EDTA pH 7.5). Immune complexes were extracted in elution buffer (1 TE buffer containing 1% SDS) by shaking the lysates for 15 min at 30°C. They were then digested with RNase (1 µg/20 µl) for 30 min at 37°C. After proteinase K digestion (1 µg/8 µl for 6 h at 37°C and 6 h at 65°) DNA was extracted using a PCR purification kit (Qiagen). IL-8 promoter DNA was amplified by PCR using HotstarTaq (Qiagen) polymerase. The PCR conditions were 95°C for 15 min, 34–36 cycles of 94°C for 20 s, 60°C for 20 s, 72°C for 20 s. PCR products were separated by agarose gel electrophoresis and detected by ethidium bromide staining of gels. Equal amounts of input DNA were controlled by gel electrophoresis (23).

The following promoter-specific primers were used: IL-8 sense 5'-AAG AAA ACT TTC GTC ATA CTC CG-3', antisense 5'-TGG CTT TTT ATA TCA TCA CCC TAC-3'; IFN-{gamma} sense 5'-TGC CTC AAA GAA TCC CAC C-3', antisense 5'-CAG TAA CAG CCA AGA GAA CC-3'.

Statistical methods

Data are shown as the mean ± SEM of at least three independent experiments. A one-way ANOVA was used for data (see Figs. 1, 2A, 2B, 3A, 3B, 5A, and 5B). Main effects were then compared by a Newman-Keuls’ post test. A value of p < 0.05 was considered to be significant as indicated.



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  		FIGURE 1. L. monocytogenes induced MAPK-dependent release of cytokines by human endothelial cells. HUVEC were preincubated for 60 min with p38 MAPK inhibitor SB202190 (SB, 10 µM) or the MEK1 inhibitor U0126 (U, 10 µM) and infected with 107 CFU/ml L. monocytogenes for 15 h. Cytokine release was measured in the supernatant by Bioplex assay. Data are shown as mean ± SEM of at least three independent experiments. *, p < 0.05 compared with infected cells without inhibitors.

 


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  		FIGURE 2. L. monocytogenes dose-dependently induced release of IL-8 and IFN-{gamma} by human endothelial cells. HUVEC were infected with L. monocytogenes or L. innocua at the indicated doses for 15 h and IL-8 (A) or IFN-{gamma} (B) release was measured in the supernatant by ELISA or Bioplex assay, respectively. Data are shown as mean ± SEM of at least three independent experiments. *, p < 0.05 compared with untreated cells. Intracellular bacteria were visualized 2 h after infection by staining of DNA (DAPI, red) and F-actin (green) and subsequent confocal fluorescence microscopy (C). Only wild-type L. monocytogenes (strain EGD) were observed in HUVEC cytosol and recruited host cell F-actin, but no L. inoccua (strain INN) were detected. Representative photographs of three independent experiments are shown.

 


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  		FIGURE 3. L. monocytogenes activated MAPK and inhibition of p38 MAPK and MEK1 but not JNK reduced L. monocytogenes-induced IL-8 release. HUVEC were preincubated (60 min) with the indicated concentrations of p38 MAPK inhibitors SB202190 and SB203580, and the control compound SB202474 (A), or MEK1 inhibitor U0126 or JNK inhibitor SP600125 (B), and then infected with L. monocytogenes (107 CFU/ml) for 15 h. IL-8 release was measured in the supernatant by ELISA. Data are shown as mean ± SEM of at least three independent experiments. *, p < 0.05 from L. monocytogenes-infected cells without inhibitor. C, HUVEC were stimulated with 10 ng/ml TNF-{alpha} or infected with L. monocytogenes (107 CFU/ml) for 30 min and phosphorylation of p38 MAPK, ERK1/2, and JNK were detected by Western blot. p38 MAPK and ERK2 expression were detected as loading control. Representative blots of three blots are shown.

 


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  		FIGURE 5. L. monocytogenes-induced histone modifications at the promoter of IL-8, but not IFN-{gamma}, were p38 MAPK- and MEK1-dependent. HUVEC were preincubated with p38 MAPK inhibitor SB202190 (10 µM) or MEK1 inhibitor U0126 (10 µM) and infected with L. monocytogenes (107 CFU/ml) for 120 min. A, Histone modifications were detected by Western blot using acetylation (Lys8) H4-specific or phosphorylation/acetylation (Ser10/Lys14) H3-specific Abs. HUVEC were preincubated with the indicated concentrations of HDAC inhibitor TSA (0.01 ng/ml) and then infected with L. monocytogenes (5 x 105 CFU/ml) for 15 h. Release of IL-8 (B) and IFN-{gamma} (C) were measured in the supernatant by ELISA or Bioplex assay, respectively. Data are shown as mean ± SEM of at least three independent experiments. *, p < 0.05 from L. monocytogenes- or TSA-exposed cells. Histone modifications (acetylation at Lys8 H4, phosphorylation/acetylation at Ser10/Lys14 H3) and RNA Pol II were detected at the promoter of il8 (D) and ifn{gamma} (E) by ChIP. Representative of three independent experiments is shown.

 

   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
L. monocytogenes induced MAPK-dependent cytokine release by human endothelial cells

HUVECs were infected by pathogenic invasive L. monocytogenes strain EGD and noninvasive L. innocua. Endothelial cells infected with L. monocytogenes (107 CFU/ml, 15 h) released G-CSF, IFN-{gamma}, IL-1{beta}, IL-4, IL-5, IL-6, IL-7, IL-8, IL-13, MCP-1, and TNF-{alpha} (Fig. 1). No significant increase in secretion of GM-CSF, IL-2, IL-10, IL-12 (p70), and IL-17 could be detected in EGD-infected endothelial cells. L. innocua (107 CFU/ml, 15 h) did not induce any significant cytokine release (data not shown).

MAPKs were considered as important regulators of cell activation (23) and contributed to the regulation of epigenetic gene regulation (17). Using p38 MAPK inhibitor SB202190 (10 µM) or the MEK1 inhibitor U0126 (10 µM) we therefore tested the role of MAPK pathways for L. monocytogenes-induced cytokine expression. Release of IL-4, IL-6, and IL-8 was reduced by p38 MAPK and MEK1 inhibition, whereas IL-5, IL-7, and MCP-1 release could be reduced only by inhibition of p38 MAPK (Fig. 1).

L. monocytogenes induced p38 MAPK- and ERK1/2-dependent IL-8 release by human endothelial cells

Focusing on the important chemoattractant IL-8 and IFN-{gamma} as model cytokines for Listeria-induced endothelial activation we analyzed the role of p38 MAPK and MEK1 in more detail. Endothelial cells infected with L. monocytogenes (15 h) released IL-8 and IFN-{gamma} in a dose-dependent manner (Fig. 2, A and B), whereas L. innocua (105–107 CFU/ml, 15 h) had no effect on cytokine secretion within the dose range and time frame tested. Intracellular localization could be verified for L. monocytogenes, but not for L. innocua, by DAPI and F-actin staining after 2 h of infection (Fig. 2C) or by plating and colony count after 2 h (data not shown). Inhibition of p38 MAPK by specific chemical inhibitors SB202190 and SB203580 reduced L. monocytogenes-induced IL-8 expression in a concentration-dependent manner, whereas inactive control compound SB202474 had no effect (Fig. 3A). Moreover, blocking of ERK1/2 pathway with the MEK1 inhibitor U0126 reduced also IL-8 release, whereas inhibition of JNK by SP600125 did not influence cytokine expression (Fig. 3B). In addition, L. monocytogenes induced phosphorylation of p38 MAPK as well as ERK1/2, but not JNK (Fig. 3C).

L. monocytogenes-induced histone acetylation at the il8 gene promoter

Next we tested the hypothesis that L. monocytogenes induced epigenetic modifications in endothelial cells. First, global histone modification was analyzed by extraction of acid-stable proteins and Western blot with modification-specific histone Abs. L. monocytogenes (107 CFU/ml) induced phosphorylation at Ser10 and acetylation at Lys14 of histone H3 as well as acetylation at Lys8 of histone H4 after 60–180 min (Fig. 4A). Second, we addressed histone modifications occurring specifically at the il8 gene promoter of HUVEC by ChIP (Fig. 4B). L. monocytogenes-induced phosphorylation/acetylation of histone H3 was detected 60 min after stimulation at the il8 gene promoter, as early as was found in global H3 analysis by Western blot. However, it was not detectable after 180 min of cell infection, when in global analysis H3 was still modified (Fig. 4B). Acetylation of histone H4 at Lys8 was seen 120 min after bacterial infection and persisted up to 180 min at the il8 promoter. Moreover, after 120 min, binding of RNA Pol II to the il8 promoter could be observed and was still present after 180 min, indicating gene transcription (Fig. 4B). HDAC inhibition by TSA increased promoter binding of Lys8-acetylated histone H4 but not of phosphorylated/acetylated H3 or Pol II (Fig. 4B). Thus, L. monocytogenes induced epigenetic modifications in endothelial cells contributing to IL-8 expression.



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  		FIGURE 4. L. monocytogenes induced phosphorylation/acetylation of H3 and acetylation of H4. A, HUVEC were infected with L. monocytogenes (107 CFU/ml) for the indicated time periods. Histone modifications were detected by Western blot using Abs specifically detecting (Ac-Lys8) H4 or phosphorylated/acetylated (Ser10/Lys14) H3. B, HUVEC were stimulated with TSA (0.01 ng/ml) for 60 min or infected with L. monocytogenes (5 x 106 CFU/ml) for the indicated time periods. Binding of RNA Pol II and histone modifications (acetylated Lys8 at H4, phosphorylated/acetylated Ser10/Lys14 on H3) were detected at the il8 gene promoter by ChIP. Representatives of three independent experiments are shown.

 
Listeria-induced histone modifications were p38 MAPK- and ERK1/2-dependent at the IL-8, but not at the IFN-{gamma} promoter

We next analyzed the impact of p38 MAPK and ERK1 pathways on the histone modifications and transactivation observed. HUVEC were preincubated with p38 MAPK inhibitor SB202190 or MEK1 inhibitor U0126, infected with L. monocytogenes for 2 h and acid-stable proteins were isolated to assess global histone modification. Inhibition of both p38 MAPK and ERK pathway blocked L. monocytogenes-induced phosphorylation/acetylation of histone H3 (Ser10/Lys14) and acetylation of histone H4 (Lys8) (Fig. 5A). In accordance with the IL-8 protein data obtained by ELISA, SB202190 and U0126 reduced L. monocytogenes-stimulated Pol II binding at the il8 gene promoter and hence subsequent gene transcription (Fig. 5D). In addition, specific modifications of H3 (Ser10/Lys14) and H4 (Lys8) at the il8 gene promoter were also reduced by inhibition of p38 MAPK and ERK pathway (Fig. 5D).

IFN-{gamma} plays a central role in host immune reaction to L. monocytogenes (7), but its Listeria-induced release was not blocked by inhibition of p38 MAPK or MEK1 (Fig. 1). Therefore, we performed ChIP analysis addressing the ifn{gamma} gene promoter and accordingly found increased binding of Pol II in infected cells but no reduction after SB202190 or U0126 preincubation (Fig. 5E). In contrast to the IL-8 data, modifications of H3 (Ser10/Lys14) and H4 (Lys8) at the ifn{gamma} gene promoter were not increased by L. monocytogenes infection nor were they changed by p38 MAPK or MEK1 inhibition.

Inhibition of HDACs increased L. monocytogenes-induced release of IL-8, but not of IFN-{gamma}

To further analyze the influence of histone acetylation on L. monocytogenes-related cytokine release, we increased global histone acetylation by incubation of HUVEC with TSA. Human endothelial cells were infected with L. monocytogenes at a low concentration (5 x 105 CFU/ml), which only slightly increased IL-8 release after 15 h of infection (Fig. 5B). Inhibition of HDAC by TSA alone did not stimulate cytokine secretion significantly. Preincubation with TSA and infection with L. monocytogenes increased IL-8 release (Fig. 6A) synergistically but not secretion of IFN-{gamma} (Fig. 5C).



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  		FIGURE 6. L. monocytogenes p38 MAPK- and ERK1/2-dependently regulated localization of histone modifying enzymes. HUVEC were infected with L. monocytogenes (107 CFU/ml) for 60 or 120 min or preincubated with p38 MAPK inhibitor SB202190 (10 µM) or MEK1 inhibitor U0126 (10 µM) and infected with L. monocytogenes (107 CFU/ml) for 120 min. Binding of RNA Pol II, CBP, or HDAC1 to the il8 promoter were detected by ChIP. Representatives of three independent experiments are shown.

 
L. monocytogenes p38 MAPK- and ERK1/2-dependent regulated localization of histone-modifying enzymes

We addressed promoter binding of histone acetylase CBP and HDAC1 to gain insight in the mechanisms underlying our observations. Infection with L. monocytogenes enhanced binding of CBP to the il8 promoter after 60 min (Fig. 6). CBP level in infected cells with inhibition of p38 MAPK and MEK1 was lower than in uninfected cells. HDAC1 was bound to il8 promoter in resting cells but disappeared after L. monocytogenes infection. Inhibition of p38 MAPK or MEK1 induced recruitment of HDAC1 to il8 promoter in infected cells.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
The data presented indicate that epigenetic mechanisms differentially contribute to the activation of proinflammatory cytokine expression by endothelial cells infected with intracellular bacteria. Intracellular Listeria induced time-dependent acetylation (Lys8) of histone H4 and phosphorylation/acetylation (Ser10/Lys14) of histone H3 globally and at the il8 promoter in HUVEC, as well as recruitment of histone acetylase CBP. Inhibitors of p38 MAPK and MEK1 reduced Lys8 acetylation of histone H4 and Ser10/Lys14 phosphorylation/acetylation of histone H3 in Listeria-infected endothelial cells and disappearance of HDAC1 at the il8 promoter in HUVEC. In contrast, Pol II recruitment to the ifn{gamma} gene promoter during L. monocytogenes infection remained unaffected by p38 MAPK or MEK1 inhibition. Furthermore, acetylation (Lys8) of histone H4 and phosphorylation/acetylation (Ser10/Lys14) of histone H3 at the ifn{gamma} gene promoter were not increased by L. monocytogenes infection, but appeared to have high basal levels in resting HUVEC.

Clearance of L. monocytogenes in murine infection models depended on cytokine-induced chemotaxis and activation of neutrophils and monocytes/macrophages (6, 9, 26, 27). In human endothelial cells, L. monocytogenes infection induced release of chemoattractants IL-8 (polymorphonuclear neutrophils) and MCP-1 (monocytes), Th1 cytokines TNF-{alpha} and IFN-{gamma}, and Th2 cytokines IL-4, IL-5, IL-6, and IL-13. In addition, the proinflammatory cytokine IL-1{beta} and the myeloid growth factors G-CSF and IL-7 were secreted. Of these, release of IL-4, IL-6, and IL-8 depended on p38 MAPK and MEK1 pathway, and IL-5, IL-7, and MCP-1 could be reduced only by inhibition of p38 MAPK.

Recent studies imply that epigenetic phenomena control eukaryotic gene transcription by effecting transcription factor binding and promoter transactivation (16, 28, 29, 30). Tight wrapping of DNA around histone octamers appears to obstruct binding of the transcription machinery (31). Acetylation and phosphorylation of histones change the polarity of histone tails, thereby facilitating uncoiling of DNA and binding of transcription factors and the basal transcription machinery (32, 33). However, the role of these mechanisms in pathogen-host cell interaction, including endothelial cell activation, has not been studied.

Stimulus-induced phosphorylation of H3 at Ser10 has been reported to be associated with the activation of promoters of many mammalian immediate-early genes (17, 34). Moreover, several reports found coincidence of MAPK-dependent phosphorylation of histone H3 at Ser10 and acetylation of histone H3 at Lys14 at the gene promoters of fos and others and the expression of these genes (35, 36). Some evidence has been provided that H3 phosphorylation at Ser10 may have a role in the regulation of transcription by acting as a signal for subsequent acetylation of lysines and, in particular, histone H3 Lys14 (37, 38, 39). Moreover, Agalioti et al. (33) demonstrated that both phosphorylation/acetylation (Ser10/Lys14) of histone H3 and acetylation of Lys8 at histone H4 were necessary for the recruitment of general transcription factors and hence for gene transcription.

In this study we show that infection of human endothelial cells with L. monocytogenes resulted in increased acetylation (Lys8) of histone H4 and phosphorylation/acetylation (Ser10/Lys14) of histone H3. Moreover, inhibition of HDACs with TSA (0.01 ng/ml) had no effect on IL-8 secretion at this concentration, but acted synergistically with respect to IL-8 release when added in combination with L. monocytogenes (5 x 105 CFU/ml). To our knowledge, the only other report of bacteria-induced histone modification described phosphorylation and acetylation of H3 in intestinal epithelial cells exposed to Bacteroides vulgates (40). In that study, TSA blocked the inhibitory TGF-{beta} effect on B. vulgatus/LPS-induced histone phosphorylation/acetylation in intestinal epithelial cells.

The proinflammatory and chemotactic cytokine IL-8 potently recruits leukocytes to sites of infection thereby contributing to local tissue inflammation (41). The il8 gene promoter has been previously described as being regulated by histone phosphorylation and acetylation (42, 43). Therefore, we addressed IL-8-specific epigenetic phenomena by ChIP. Modifications of both histones H3 and H4 were observed in overall chromatin analysis and were also induced by L. monocytogenes infection specifically at the il8 gene promoter.

Activation of p38 MAPK and ERK1/2 was considered to be involved in proinflammatory activation of cells (44, 45, 46), and to contribute to epigenetic regulation of gene expression (47). Moreover, these kinases participated in IL-8 expression induced either by endotoxin (17, 20) or bacteria (48) in endothelial cells. By analyzing the pathways responsible for histone modifications, we found that inhibition of p38 MAPK and ERK1/2 blocked phosphorylation/acetylation (Ser10/Lys14) of histone H3 and acetylation (Lys8) of histone H4 in L. monocytogenes-infected endothelial cells. This finding is in accordance with other reports showing that both kinase pathways were involved in phosphorylation of histone H3 (17, 38) and activity of histone acetyltransferases (49). Both, p38 MAPK and ERK1/2 have been shown to phosphorylate histone H3 in vitro, and inhibitors of both kinase pathways could block UV-induced H3 phosphorylation at Ser10 (50). Therefore, Listeria-activated MAPKs might phosphorylate H3 directly at the il8 gene promoter. However, several other kinases including ribosomal S6 kinase-2 (51) and mitogen- and stress-activated protein kinase-1 (35), which are nuclear effectors of p38 MAPK and ERK1/2, have been implicated in H3 phosphorylation at Ser10, although results are somewhat controversial (52).

Addressing histone modifications at the il8 gene promoter, we found that inhibition of p38 MAPK and MEK1-ERK1/2 pathway blocked phosphorylation and acetylation at the histones. This finding is supported by the observation of Saccani et al. (17) that histone H3 was p38 MAPK-dependently phosphorylated at the il8 gene promoter in dendritic cells exposed to LPS. In contrast, there was no effect of JNK on IL-8 expression in L. monocytogenes-stimulated endothelium.

Apparently, histone acetyl transferases, e.g., the CBP or Gcn5, which acetylate H3 at Lys14, seem to prefer H3 phosphorylated at Ser10 as a substrate (38, 39, 53, 54). Agalioti et al. (33) have shown that, following phosphorylation of H3 at Ser10, acetylation of H3-Lys14 and H4-Lys8 were prerequisites for recruitment of bromodomain-containing factors TFIID and SWI-SNF, respectively. Subsequently, SWI-SNF complex was found to remodel the nucleosome masking the TATA box such that general transcription factor TFIID could bind to the TATA box and RNA Pol II was recruited (55). Similar results have been found in yeast (56). In agreement, we found a coincidence of phosphorylation and acetylation of H3 and recruitment of CBP to the il8 gene promoter in Listeria-infected HUVEC. Both events were reduced by inhibition of p38 MAPK or MEK1. Moreover, inhibition of p38 MAPK not only blocked Listeria-induced disappearance of HDAC1 from the il8 promoter, but even enhanced it. Recruitment to the site of action is considered as a mechanism of HDAC regulation and specificity (57). Therefore, our observations together with previous studies implicate a sequence of Listeria-induced MAPK activation, histone phosphorylation, histone acetylation, chromatin remodeling, and successful gene transcription.

IFN-{gamma} seems to be critical for early defense against L. monocytogenes in murine models (2, 7) and was shown to prevent bacterial escape from the phagosomes of macrophages (11). We found that Pol II was recruited to the ifn{gamma} gene promoter and that IFN-{gamma} was released in L. monocytogenes-infected endothelial cells, but unlike the data analyzing IL-8 expression, inhibition of p38 MAPK or MEK1 did not block these events. It has been shown in Th1 cells that IFN-{gamma} expression depended on p38 MAPK but not ERK1/2 (58, 59). However, there are fundamental differences in the regulation of ifn{gamma} gene promoter activity in different cell types (60). In resting endothelial cells, the ifn{gamma} gene promoter appeared to have high basal levels of histone phosphorylation and acetylation, which remained unchanged either following L. monocytogenes infection or p38 MAPK or MEK1 inhibition. IFN-{gamma} secretion could not be stimulated by inhibition of HDACs. In resting NK cells, which rapidly release IFN-{gamma} after stimulation, histones H3 and H4 were also acetylated (60, 61) and DNA was unmethylated (62) at the ifn{gamma} gene promoter. In contrast, in CD4-postive T cells histone modification occurred only after cell stimulation. Therefore, it is possible that cells, which express IFN-{gamma} shortly after infection, may not need chromatin remodeling at this locus, and therefore require different signaling pathways (e.g., p38 MAPK).

When considering that clearance of L. monocytogenes infection in murine models required granulocyte and monocyte action (6, 9, 26, 27) and expression of CXC chemokines and IFN-{gamma} (7, 11, 63, 64), epigenetic regulation of proinflammatory genes may contribute significantly in preparing the host immune system against infection. In the intestine, altered histone acetylation may maintain normal intestine homeostasis to commensal luminal enteric bacteria by reducing inflammation as suggested by Haller et al. (40) underlining the specificity of epigenetic regulation in host-pathogen interaction.

As differences between cell types in chromatin modification exist, epigenetic responses to Listeria infection in other cell types as macrophages or intestinal epithelium should be addressed in the future. Moreover, epigenetic modifications could be compared with infections with other endothelial-activating bacteria like e.g., Chlamydia pneumoniae.

In conclusion, we report for the first time epigenetic phenomena in the innate immune reaction to intracellular bacteria. L. monocytogenes activated il8 transcription in human endothelial cells by p38 MAPK- and ERK1/2-dependent modification of histones H3 and H4. In contrast, ifn{gamma} gene transcription was activated by L. monocytogenes independently of p38 MAPK and MEK1-ERK1/2, and histone phosphorylation/acetylation remained unchanged in infected cells at this gene. Specific epigenetic mechanisms influencing inflammatory gene expression are indicated as novel strategies in the pathogenesis of intracellular bacteria and may pave the way for the rational development of new therapeutic interventions during infection.


   Acknowledgments
 
We greatly appreciate the excellent technical assistance of Kerstin Möhr, Sylvia Schapke, and Jenny Thiele (Department of Internal Medicine and Infectious Diseases, Charité-University Medicine Berlin). We thank Dr. Joseph R. Miller for proofreading the manuscript.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


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

1 This work was supported in part by the Bundesministerium für Bildung und Forschung (BMBF) Grants CAPNETZ C15 (to B.S.), CAPNETZ C15, BMBF-NBL3 (to S.H.), CAPNETZ C4 (to. N.S.), by the Nationales Genomforschungsnetz Deutschland (to T.C.), and by the Deutsche Forschungsgemeinschaft DFG Grants Kr1143/4-1 (to N.S.) and GRK325 (to J.Z.). Part of this work will be included in the doctoral thesis of W.B. Back

2 Address correspondence and reprint requests to Dr. Stefan Hippenstiel, Department of Internal Medicine and Infectious Diseases, Charité-University Medicine Berlin, Augustenburger Platz 1, 13353 Berlin, Germany. E-mail address: stefan.hippenstiel{at}charite.de Back

3 Abbreviations used in this paper: TSA, trichostatin A; ChIP, chromatin immunoprecipitation; HDAC, histone deacetylase; CBP, CREB-binding protein; Pol II, polymerase II. Back

Received for publication January 26, 2005. Accepted for publication June 14, 2005.


   References
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 Abstract
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 Materials and Methods
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 References
 

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