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Am J Physiol Lung Cell Mol Physiol 290: L818-L826, 2006. First published January 6, 2006; doi:10.1152/ajplung.00428.2005
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TRANSLATIONAL PHYSIOLOGY

Moraxella catarrhalis induces inflammatory response of bronchial epithelial cells via MAPK and NF-{kappa}B activation and histone deacetylase activity reduction

Hortense Slevogt,1 Bernd Schmeck,1 Carola Jonatat,1 Janine Zahlten,1,2 Wiebke Beermann,1 Vincent van Laak,1 Bastian Opitz,1 Solveig Dietel,1 Philippe Dje N'Guessan,1 Stefan Hippenstiel,1 Norbert Suttorp,1 and Joachim Seybold1

Departments of 1Internal Medicine/Infectious Diseases and 2Periodontology and Synoptic Dentistry, Charité-Universitätsmedizin Berlin, Berlin, Germany

Submitted 6 October 2005 ; accepted in final form 23 December 2005


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Moraxella catarrhalis is a major cause of infectious exacerbations of chronic obstructive lung disease (COPD) and may also contribute to the pathogenesis of COPD. Little is known about M. catarrhalis-bronchial epithelium interaction. We investigated activation of M. catarrhalis infected bronchial epithelial cells and characterized the signal transduction pathways. Moreover, we tested the hypothesis that the M. catarrhalis-induced cytokine expression is regulated by acetylation of histone residues and controlled by histone deacetylase activity (HDAC). We demonstrated that M. catarrhalis induced a strong time- and dose-dependent inflammatory response in the bronchial epithelial cell line (BEAS-2B), characterized by the release of IL-8 and GM-CSF. For this cytokine liberation activation of the ERK and p38 mitogen-activated protein (MAP) kinases and transcription factor NF-{kappa}B was required. Furthermore, M. catarrhalis-infected bronchial epithelial cells showed an enhanced acetylation of histone H3 and H4 globally and at the promoter of the il8 gene. Preventing histone deacetylation by the histone deacetylase inhibitor trichostatin A augmented the M. catarrhalis-induced IL-8 response. After exposure to M. catarrhalis, we found a decrease in global histone deacetylase expression and activity. Our findings suggest that M. catarrhalis-induced activation of il8 gene transcription was caused by interference with epigenetic mechanisms regulating il8 gene accessibility. Our findings provide insight into important molecular and cellular mechanisms of M. catarrhalis-induced activation of human bronchial epithelium.

chronic obstructive pulmonary disease; bronchial epithelium; immune response; mitogen-activated protein kinase; nuclear factor-{kappa}B

CHRONIC OBSTRUCTIVE PULMONARY DISEASE (COPD) is a leading cause of morbidity and mortality worldwide. Disease progression is characterized by frequent acute exacerbations caused by viral or bacterial infection (8, 26, 37). Although many studies support the causative role of Haemophilus influenzae and Streptococcus pneumoniae in the pathogenesis of COPD (26, 38), Moraxella catarrhalis was widely ignored for decades because this pathogen was considered an irrelevant saprophyte of the upper respiratory tract (27). Increasing recent evidence underlined the importance of M. catarrhalis for acute exacerbations and disease progression of COPD. For example, Murphy et al. (27) demonstrated in a prospective study that M. catarrhalis colonized the lower respiratory tract of 5–32% of adults with COPD at any time and caused ~10% of all COPD-exacerbations, accounting for 2–4 million episodes annually in the United States. However, little is known about M. catarrhalis-bronchial epithelium interaction. Inflammation in COPD is characterized by increased infiltration of neutrophils, lymphocytes, and macrophages into the airways (4). Neutrophils play an important role in the pathogenesis of airway inflammation in COPD because of their ability to release a number of mediators including elastase, metalloproteases, and oxygen radicals, which promote tissue inflammation and damage (4, 8). Neutrophil accumulation in the airways of patients with COPD is driven by increased release of cytokines. Among them, important mediators involved in the recruitment and activation of leucocytes are interleukin 8 (IL-8) and granulocyte-macrophage colony stimulating factor (GM-CSF), suggesting that these mediators may play an important role in the pathogenesis of the disease (2, 4). Bronchial epithelial cells contribute significantly to the orchestration of the immune response through release of a variety of inflammatory chemokines such as IL-8 and GM-CSF (4, 37), thereby inducing COPD-characteristic alteration such as chronic bronchial inflammation and air flow limitation (7, 36). Transcriptional regulation of inflammatory genes is achieved by a variety of complex signal transduction pathways involving the mitogen-activated protein (MAP) kinases, extracellular signal-regulated kinase (ERK), c-Jun kinase (JNK), and p38 MAP kinase and the transcription factor NF-{kappa}B, which is particularly strongly activated in epithelial cells of COPD (4, 7, 8). There is increasing evidence for the importance of covalent, posttranslational modifications of histone tails in proinflammatory gene regulation resulting in altered chromatin structure and therefore DNA accessibility (25, 31). Barnes et al. (3) recently emphasized the importance of such epigenetic mechanisms in the context of COPD. Investigation revealed that the effects of histone modifications depend on specific amino acids that are modified, e.g., by acetylation of lysine and phosphorylation of serine residues (25). Enzymes that partly determine histone acetylation pattern are the histone deacetylases (HDACs) (3). Ito et al. (16) recently demonstrated a pronounced reduction of HDAC2 activity and expression in peripheral lung tissue of patients with COPD that correlated with disease severity.

The aim of the study presented was to investigate the role of M. catarrhalis infection for proinflammatory cytokine expression in bronchial epithelium and to characterize the signal transduction pathways contributing to cell activation. By using IL-8 and GM-CSF as model cytokines, we analyzed the role of relevant signaling pathways in M. catarrhalis-induced activation of airway epithelial cells. Furthermore, we demonstrated the influence of M. catarrhalis on the accessibility of the Il8 gene, which, in part, was shown to be regulated by the histone acetylation-deacetylation balance. Overall, our data provide evidence that M. catarrhalis induced a complex proinflammatory signaling and interfered with epigenetic mechanisms, thus representing a potent stimulus to initiate or maintain an inflammatory bronchial epithelial response.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cell lines. Human bronchial epithelial cell line BEAS-2B was a kind gift of C. Harris (National Institutes of Health, Bethesda, MD) (30). BEAS-2B cells were grown in Keratinocyte-SFM (Gibco-BRL, Life Technologies, Paisley, UK) supplemented with 2 mM L-glutamine, 100 U/ml penicillin, and 100 µg/ml streptomycin. Cells were grown to confluence in 75-cm2 flasks and subsequently cultured in different well plates (both Falcon; Corning Star, Wiesbaden, Germany). Twelve hours before the experiment cells were grown in medium without antibiotic supplements.

Materials. The MAP kinase inhibitors SP-600125, U0126, and SB-202190, SB-203580, as well as the nonfunctional control compound SB-202474, were purchased from Calbiochem (Merck, Bad Soden, Germany), TNF-{alpha} and IL-lbeta from R & D Systems (Wiesbaden, Germany), and IKK-NEMO binding domain (NBD) and trichostatin A (TSA) from Biomol (Plymouth). All other chemicals used were of analytical grade and obtained from commercial sources.

Bacterial strains. M. catarrhalis wild-type strain O35E (serotype A) was kindly provided by Eric Hansen (University of Texas Southwestern Medical Center, Dallas, TX). M. catarrhalis strain was grown overnight at 37°C on brain-heart infusion (BHI) agar (Dibco Laboratories, Heidelberg, Germany) supplemented with 5% heated sheep blood. For infection experiments, single colonies of bacterial overnight cultures were expanded by resuspension in BHI broth and incubation at 37°C for 2–3 h to midlog phase (A405 0.4–0.6), harvested by centrifugation, resuspended in cell culture medium without antibiotics, and adjusted to an optical density (OD) at 405 nm of 0.3 [{approx}1 x 106 colony-forming units (cfu)/ml] and used for infecting the BEAS-2B cells at the indicated multiplicity of infection (MOI). To confirm the viability of M. catarrhalis in cell culture medium, bacteria were resuspended and OD was measured over time. To exclude an influence of the inhibitors used in this study on the viability of M. catarrhalis, bacteria were resuspended in cell culture medium including the inhibitors and incubated for 12 h. Subsequently the bacterial suspensions were serially diluted in PBS, and 100-µl aliquots were plated on BHI agar containing 5% sheep blood to determine the number of viable M. catarrhalis. After a 12-h incubation, the number of cfu were counted and compared with a bacterial control suspension without inhibitors.

IL-8 and GM-CSF ELISA. Confluent BEAS-2B cells were stimulated for 15 h as indicated. After incubation, supernatants were collected and processed for IL-8 or GM-CSF quantification by sandwich ELISA as described previously (29, 35).

RT-PCR analysis. RNA was extracted using RNeasy Mini kit (Qiagen, Hilden, Germany), according to the manufacturer's instructions.. All primers were purchased from TIB MOLBIOL (Berlin, Germany). PCR products were analyzed on 1.5% agarose gels, stained with ethidium bromide, and subsequently visualized. To confirm equal loading, PCR for glyceraldehyde-3-phosphate dehydrogenase was performed in parallel.

Immunoblot analysis. The activation status of ERK1/2 as well as p38 and JNK MAPK was assessed by immunoblot analysis with antibodies that recognize the phosphorylated (activated) form of the respective kinases (35). BEAS-2B cells were stimulated as indicated, washed twice, and harvested as described previously. Immunodetection of phosphorylated kinases was carried out as described previously (14). Degradation of I{kappa}B{alpha} was analyzed in BEAS-2B cell lysates using a rabbit polyclonal antibody (Santa Cruz Biotechnologies, Heidelberg, Germany) as described previously. The HDAC1 (H54) and HDAC2 (H51) antibodies were purchased from Santa Cruz Biotechnology. In all experiments, the respective unphosphorylated MAP kinases, p38, ERK2, or JNK-1 (Santa Cruz Biotechnology), were detected simultaneously to confirm equal protein load. The proteins were visualized by incubation with secondary IRDye 800- or Cy5.5-labeled antibodies, respectively (Odyssey infrared imaging system; LI-COR Biosciences, Lincoln, NE). For each blot, relative phosphoprotein or HDAC1 and -2 levels were calculated from the ratio of fluorescence intensities of the phosphoprotein-panprotein or the HDAC/ERK2 protein to correct for differences in protein loading. Differences between experimental and control conditions were obtained by comparison to the normalized control ratio (arbitrarily set at 1 or 100%, respectively).

Electrophoretic mobility shift assay. After stimulation of BEAS-2B cells, nuclear protein was isolated and analyzed by electrophoretic mobility shift assay (EMSA) as described previously (35). IRDye800-labeled consensus NF-{kappa}B oligonucleotides were purchased from Metabion (Planegg-Martinsried, Germany). Briefly, EMSA binding reactions were performed by incubating 1.25 µg of nuclear extract with the labeled oligonucleotides according to the manufacturer's instructions. The reaction mixture was subjected to electrophoresis on SDS-PAGE (4%) and analyzed by the Odyssey infrared imaging system (LI-COR).

Histone isolation and immunodetection. Histone was isolated from BEAS-2B cells by acid extraction as follows. After the indicated stimulation, BEAS-2B cells were washed twice with ice-cold PBS supplemented with 100 mM NaF, 2 mM Na3VO4, and 15 mM Na4P2O7, scraped into 800 µl of ice-cold lysis buffer containing 10 mM HEPES (pH 7.9), 1.5 mM MgCl2, 10 mM KCL, 1.5 mM PMSF, and 0.5 mM DTT, transferred into Eppendorf tube at 4°C, and mixed with 0.2 M H2SO4. Subsequently the lysate was incubated on ice for 60 min and centrifuged at 24,980 g for 10 min. After centrifugation supernatants were collected and were mixed with trichloroacetic acid (TCA) making 20% TCA solution, incubated for 45 min at 4°C, and centrifuged at 24,980 g for 10 min. The pellet was washed twice with 1 ml of acetone and resuspended in PBS containing 100 mM NaF, 2 mM Na3VO4, and 15 mM Na4P2O7. Immunodetection of acetylated-phosphorylated and nonacetylated-phosphorylated histones was carried out with primary antibodies targeting acetylated-phosphorylated (P-Ser-10/Ac-Lys-14) and the nonacetylated-phosphorylated histone H3, respectively, as well as the acetylated (Ac-Lys-8)-panacetylated histone H4 and the nonacetylated histone H4. The proteins were visualized by incubation with secondary IRDye 800- or Cy5.5-labeled antibodies, respectively (Odyssey infrared imaging system, LI-COR Biosciences) as described previously (32, 33).

HDAC activity. HDAC activities of BEAS-2B nuclear extracts were measured by fluorometric detection with an HDAC activity assay (BIOMOL, Hamburg, Germany) according to the manufacturer's protocol.

Chromatin immunoprecipitation. BEAS-2B cells were stimulated, culture medium was removed, and 1% formaldehyde was added as described previously (35). After 1 min, the cells were washed in ice-cold 0.125 M glycine in PBS, then rapidly collected in ice-cold PBS, centrifuged, and washed twice with ice-cold PBS. The cells were lysed in chromatin immunoprecipitation (ChIP) radioimmunoprecipitation assay (RIPA) buffer (10 mM Tris, pH 7.5, 150 mM NaCl, 1% Nonidet P-40, 1% desoxycholic acid, 0.1% SDS, 1 mM EDTA, and 1% aprotinin), and chromatin was sheared by sonication. The samples were cleared by centrifugation, and the supernatants were stored in aliquots at –80°C until further use. HDAC1 antibodies (H-51) and HDAC2 antibodies (H54) were purchased from Santa Cruz Biotechnology. The anti-phospho-acetyl-histone H3 (anti-phospho-Ser-10/Ac-Lys-14-histone H3) and anti-acetyl-histone H4 (anti-acetyl-histone H4) antibodies were purchased from Upstate Biotechnology (Lake Placid, NY). Immunoprecipitations from soluble chromatin were carried out overnight at 4°C. Immune complexes were collected with protein A/G agarose (Santa Cruz Biotechnology) for 60 min and washed thoroughly with RIPA buffer and high-salt buffer (2 M NaCl, 10 mM Tris, pH 7.5, 1% Nonidet P-40, 0.5% desoxycholic acid, 1 mM EDTA). We extracted immune complexes in elution buffer [1 TE buffer: 0.01 M Tris, pH 7.5, 0.001 EDTA (TE) buffer containing 1% SDS] by shaking the samples for 15 min at 1,200 rpm, 30°C. They were then digested with RNase for 30 min at 37°C. After proteinase K digestion for 6 h at 37°C and 6 h at 65°C, DNA was extracted with a PCR purification kit (Qiagen, Hilden, Germany). Il-8 promotor DNA was amplified by PCR using Hot Star Taq (Qiagen) DNA polymerase. The PCR conditions were 95°C for 15 min and 33–35 cycles of 94°C for 20 s, 60°C for 20 s, 72°C for 20 s and 72°C for 7 min. PCR products were separated by agarose gel electrophoresis and detected by ethidium bromide staining. Equal amounts of input DNA was controlled by gel electrophoresis. The following promotor-specific primers for IL8 were used: sense, 5'-AAG AAA ACT TTC GTC ATA CTC CG-3'; antisense, 5'-TGG CTT TTT ATA TCA TCA CCC TAC-3'.

Statistical analysis. Data are shown as means ± SE of at least three independent experiences. A one-way ANOVA was used for data of Figs. 2, B, D, and F; 3, A–E; 4, C and D; 6; and 7, B, D, and E. Main effects were then compared by a Newman-Keuls posttest. Statistical significance was accepted at a P < 0.01 indicated by asterisks.


Figure 2
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  		Fig. 2. M. catarrhalis (M. cat.) activates p38, ERK, and JNK MAPK. Confluent BEAS-2B cells were treated with M. catarrhalis at an MOI of 0.1 for up to 240 min followed by immunoblotting for phosphorylated p38 kinase, phosphorylated ERK kinase, and phosphorylated Jun kinase. MAPK phosphorylation was normalized to the respective unphosphorylated MAP kinases p38, ERK2, and JNK1, which were simultaneously detected by an infrared imaging system. Data are demonstrated as fold of the unstimulated normalized control and are presented as means ± SE of 3 separate experiments (B, D, and F); *P < 0.05. Positive controls for MAPK activation were performed with 50 ng/ml TNF-{alpha} and 10 ng/ml IL-1beta. One representative immunoblot out of 3 experiments is shown for detection of each MAPK (A, C, and E). *P < 0.01; T/I, TNF-{alpha} and IL-1beta; p-, phosphorylated.

 

Figure 3
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  		Fig. 3. ERK1/2 and p38 inhibition blocks M. catarrhalis-induced IL-8 and GM-CSF secretion. IL-8 (A and B) and GM-CSF (D and E) secretion by BEAS-2B cells infected with M. catarrhalis for 15 h were assessed by ELISA. Cells were pretreated with the indicated doses of specific p38 inhibitor SB-202190 or nonactive control compound SB-202474, ERK1/2-specific inhibitor U0126, or specific JNK inhibitor SP-600125 for 45 min or were left untreated. TNF-{alpha} (50 ng/ml) and IL-1beta (10 ng/ml) were used as positive control. C demonstrates the effect of pretreatment with SP-600125 on TNF-{alpha}- and IL-1beta-induced IL-8 expression. *P < 0.01 by Student's t-test.

 

Figure 4
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  		Fig. 4. M. catarrhalis-induced I{kappa}B{alpha} degradation and translocation of NF-{kappa}B in bronchial epithelial cells. A: M. catarrhalis-induced I{kappa}B{alpha} degradation in BEAS-2B cells. Confluent BEAS-2B cells were treated with indicated doses (MOI: 1) of M. catarrhalis for 30 and 60 min, and immunoblotting was performed for I{kappa}B{alpha} and the loading control ERK2. Representative gels out of 3 are shown. B: EMSA demonstrates nuclear translocation of NF-{kappa}B in BEAS-2B cells infected with M. catarrhalis for 30, 60, and 120 min. C and D: BEAS-2B cells were preincubated with IKK-NBD at the indicated concentration for 30 min and then infected with M. catarrhalis (MOI 0.1) or stimulated with 50 ng/ml TNF-{alpha} and 10 ng/ml IL-1beta (T/I) for 15 h. IL-8 (C) and GM-CSF levels (D) were measured by ELISA. The values represent means ± SE of 3 values. *P < 0.01 by Student's t-test.

 

Figure 6
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  		Fig. 6. Prevention of histone deacetylation with TSA increased M. catarrhalis-induced IL-8 release. BEAS-2B cells were left untreated or were pretreated with TSA (10 ng/ml) for 45 min following infection with M. catarrhalis (MOI: 0.1) for 15 h. IL-8 concentrations in the supernatants were assessed by ELISA. *P < 0.01 by Student’s t-test.

 

Figure 7
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  		Fig. 7. M. catarrhalis reduced global histone deacetylase (HDAC)1 and 2 expression and HDAC activity. A and B: BEAS-2B cells were infected with M. catarrhalis (MOI: 0.1) for the indicated times. Reduction of the M. catarrhalis induced HDAC1 and HDAC2 expression in BEAS-2B cells was verified by immunoblotting. As positive control cells were stimulated with LPS (1 µg/ml) for 4 h. HDAC1 and -2 protein expression was normalized to the expression of ERK2, which was simultaneously detected. Data are demonstrated as percentages of the unstimulated normalized control and are presented as means ± SE of 3 separate experiments (B and D). One representative immunoblot out of 3 experiments is shown A and C. E: HDAC activity was measured in BEAS-2B cells after exposure with M. catarrhalis for the indicated times and expressed as percentage of activity in unstimulated cells. As positive control cells were stimulated with LPS (1 µg/ml) for 16 h. The bars represent means of 3 values ± SD. *P < 0.05, **P < 0.01.

 

   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
M. catarrhalis induces IL-8 and GM-CSF expression in epithelial cells. We first investigated the M. catarrhalis-induced IL-8 (Fig. 1A) and GM-CSF (Fig. 1B) protein secretion in BEAS-2B cells. We found an increase 6 h postinfection (p.i.) with further enhancement at 12 and 24 h in a dose-dependent manner. Moreover, we confirmed the induction of IL-8 and GM-CSF mRNA transcription in M. catarrhalis-infected BEAS-2B cells by RT-PCR. M. catarrhalis infection (MOI: 0.1) induced an increase in transcription of IL-8 and GM-CSF mRNA 2 h p.i. (Fig. 1C).


Figure 1
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  		Fig. 1. Moraxella catarrhalis induced a time- and dose-dependent IL-8 and granulocyte-macrophage colony-stimulating factor (GM-CSF) expression and transcription in BEAS-2B cells. Confluent BEAS-2B cells were infected for 6, 12, or 24 h with M. catarrhalis strain O35E [multiplicity of infection (MOI): 0.1] as indicated. IL-8 (A) or GM-CSF (B) concentrations were assessed in the supernatants by ELISA. C: semiquantitative RT-PCR for IL-8 and GM-CSF after infection of confluent BEAS-2B cells with M. catarrhalis at an MOI of 0.1 for the indicated time periods. PCR for GAPDH served as an internal control. One representative gel out of 3 is shown. M.c., M. catarrhalis.

 
M. catarrhalis-induced phosphorylation of ERK1/2, p38, and JNK MAP kinase in BEAS-2B cells. Because MAP kinases are considered as important regulators of proinflammatory cytokine expression we analyzed MAP kinase activation and its impact in M. catarrhalis-related cytokine release. Infection of BEAS-2B cells with M. catarrhalis was associated with the activation of the MAPK p38, ERK1/2, and JNK as demonstrated by immunoblot analysis of phosphorylated ERK1/2, p38, and JNK (Fig. 2, A–F). The degree of MAP kinase phosphorylation observed was comparable to that seen following TNF-{alpha}/IL-1beta exposure. The p38 MAPK inhibitor, but not its inactive control compound, significantly inhibited IL-8 (Fig. 3A) and GM-CSF (Fig. 3D) release induced by M. catarrhalis in BEAS-2B cells. Similar results were obtained with SB-203580, another specific p38 inhibitor (data not shown). Preexposure of BEAS-2B cells to the ERK pathway inhibitor U0126 also abrogated M. catarrhalis-induced IL-8 (Fig. 3B) and GM-CSF secretion (Fig. 3E). In contrast, no reduction of cytokine release could be detected with the JNK inhibitor SP-600125 (Fig. 3, B and E). JNK is known to be essential for TNF-{alpha}- and IL-1beta-induced IL-8 expression in human airway epithelial cells (18, 20, 22, 28). Therefore, we compared the inhibitory effect of SP-600125 on the M. catarrhalis-induced IL-8 secretion with the TNF-{alpha}/ IL-1beta-induced IL-8 secretion. In Fig. 3C, our results show a significant reduction of IL-8 release after SP-600125 pretreatment of the cells. In contrast to the JNK-dependent IL-8 release induced by TNF-{alpha}/IL-1beta, the M. catarrhalis-induced IL-8 production was again not inhibited by the JNK inhibitor. Moreover, control experiments clearly demonstrated that the inhibitors used did not affect the viability of M. catarrhalis (data not shown).

Involvement of the transcription factor NF-{kappa}B in M. catarrhalis-induced IL-8 and GM-CSF expression. Next we tested the role of NF-{kappa}B in M. catarrhalis-related gene expression. As shown in Fig. 4A, I{kappa}B{alpha}, the cytosolic inhibitor of NF-{kappa}B, was degraded 30–60 min after infection of BEAS-2B cells with M. catarrhalis. Parallel to this, EMSA analysis revealed a nuclear translocation of NF-{kappa}B 1 h after M. catarrhalis infection (Fig. 4B). Next, BEAS-2B cells were pretreated with the cell-permeable NF-{kappa}B inhibitor IKK-NBD (23) before they were exposed to M. catarrhalis. Indeed, IL-8 and GM-CSF secretion induced by M. catarrhalis was significantly reduced by IKK-NBD (Fig. 4, C and D).

Role of histone phosphorylation-acetylation induced by M. catarrhalis. To test the hypothesis that M. catarrhalis induced epigenetic modifications we investigated M. catarrhalis-related global histone modifications in BEAS-2B cells (Fig. 5A). BEAS-2B cells were incubated with M. catarrhalis for 1 and 2 h, respectively. We then subjected cell lysates to immunoblot analysis, using an antibody targeting phosphorylated histone-H3 at the serine 10 and acetylated histone H3 at the lysine 14 residue (P-Ser-10/Ac-Lys-14-H3) or to the acetylated histone-H4 at the lysine 8 residue (anti-acetyl-histone H4). Phosphorylation-acetylation on serine 10/lysine 14-H3 was significantly increased 1 and 2 h p.i. Compared with the noninfected control cell lysates (Fig. 5A). Notably, M. catarrhalis also caused a time-dependent increase in acetylated histone H4 compared with the control (Fig. 5A). Because acetylation of core histones is critically involved in the induction of the il8 gene transcription (25, 32) ChIP analysis was applied. It revealed an increased binding of modified histone H3 and H4 to the IL8 promotor in M. catarrhalis-infected BEAS-2B cells (Fig. 5B). Concomitantly, we demonstrated enhanced recruitment of RNA polymerase II to the IL8 promoter, which is known to be part of the transcription machinery facilitating IL-8 expression (25, 32). Inhibition of HDAC by TSA treatment is known to cause an increase in acetylated histone H3 and H4 at the IL8 promotor (12); therefore, we used TSA-treated BEAS-2B cells as positive control (Fig. 5B). Our data provide evidence that M. catarrhalis induced histone H3 and H4 modifications at the IL8 promotor of BEAS-2B cells, increasing the accessibility of the il8 gene.


Figure 5
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  		Fig. 5. M. catarrhalis enhanced phosphoacetylation of histone H3 and acetylation of H4 at the IL8 promotor. A: BEAS-2B cells were cultured with M. catarrhalis (MOI: 0.1) for the times indicated. Whole cell extracts were prepared, and 20 µl of the extract protein were loaded in SDS 12.5% PAGE gel and immunoblotted with antibodies targeting phosphoacetylated histone H3 (P-Ser-10/Ac-Lys-14-H3) or acetylated histone H4 (anti-acetyl-histone-H4). A duplicate gel and membrane were blotted with antibodies targeting the nonphospho-acetylated histone H3 (H3) and the nonacetylated histone H4 (H4), respectively. B: BEAS-2B cells were left untreated, infected with M. catarrhalis (MOI 0.1), or treated with trichostatin A (TSA, 10 ng/ml) for the indicated times. Chromatin immunoprecipitation (ChIP) assays were performed to demonstrate the binding of phosphoacetylated histone H3 (P-Ser-10/Ac-Lys-14-H3) or acetylated H4 (anti-acetyl-histone-H4) to the IL8 promotor. To indicate gene transcription recruitment of polymerase II (Pol II) to the IL-8 promotor was analyzed by ChIP. PCR products were separated by agarose gel electrophoresis and detected by ethidium bromide staining. PCR amplifications of the IL-8 promotor of the total DNA (without prior immune precipitation) was used as control (INPUT DNA). One representative gel out of 3 is shown. {alpha}-P/AC-H3, P-Ser-10/Ac-Lys-14-H3; {alpha}-Ac-H4, Ac-Lys-8-H4.

 
Effects of M. catarrhalis on HDAC expression and activity. To test the role of HDAC activity on the M. catarrhalis-induced IL-8 release, we pretreated BEAS-2B cells with the HDAC inhibitor TSA (Fig. 6). Inhibition of HDAC activity by TSA markedly increased M. catarrhalis-induced IL-8 production in BEAS-2B cells, suggesting an important role of HDAC activity for IL-8 expression. Interestingly, an M. catarrhalis-dependent reduction in HDAC1 protein expression of ~25% and a reduction of HDAC2 protein expression of ~50% was observed in BEAS-2B cells 16 h p.i. (Fig. 7, B and D). These findings correlate with our results that after BEAS-2B cells were directly exposed to M. catarrhalis (MOI: 0.1) their nuclear extracts showed a marked time-dependent decrease of HDAC activity of 67% of the activity of unexposed cells. Nuclear extracts of BEAS-2B cells stimulated with 1 µg/ml LPS for 16 h showed a reduction of HDAC activity of about 50% (Fig. 7C). Together, these data suggest that the M. catarrhalis-induced reduction of HDAC1 and -2 expression and activity may lead to an increase in IL-8 expression in bronchial epithelial cells.


   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
An increasing number of epidemiologic studies demonstrating an association between M. catarrhalis and respiratory morbidity in the course of COPD (26, 27, 38) led us to a detailed analysis of the M. catarrhalis-bronchial epithelium interaction. The study presented demonstrates that infection of human bronchial epithelium with M. catarrhalis resulted in secretion of IL-8 and GM-CSF. Our results show that M. catarrhalis-induced cytokine secretion was dependent on activation of the MAP kinases p38 and ERK1/2. Pathogen-related stimulation of p38 and ERK MAPK pathway seems to be essential for proinflammatory activation of airway epithelial cells and has previously also been demonstrated for the important pulmonary pathogens H. influenzae and S. pneumoniae (6, 33, 40). Thus pathogen (including M. catarrhalis)-triggered and p38 and ERK MAPK pathway-regulated signaling may significantly contribute to an innate immune response of the epithelial lung surface. Interestingly our data show that M. catarrhalis-induced activation of JNK MAP kinases was not important for IL-8 and GM-CSF liberation. JNK activation is known to lead to the phosphorylation of a number of transcription factors, most notably the c-Jun component of activator protein-1 (22). Induction of JNK activation by the potentially pulmonary pathogens H. influenzae and Pseudomonas aeruginosa has been reported previously (11, 17), but the role of JNK activation for cytokine release of pulmonary epithelial cells to different stimuli such as bacteria is incompletely understood. However, Krull et al. (19) recently reported that the C. pneumoniae-induced GM-CSF secretion in bronchial epithelial cells was mediated by the activation of the ERK1/2 and p38 but not by the activation of the JNK kinases. Moreover, macrolide antibiotics-induced IL-8 and GM-CSF secretion in human bronchial epithelial cells has been shown to be independent of JNK phosphorylation (39). Emerging evidence indicates that specificity in JNK kinases activation is controlled by distinct intracellular complexes of multiple signaling proteins upstream to the phosphorylation of JNK as well as by scaffolding proteins downstream to the phosphorylation of JNK (5, 22). Together, our results suggest that M. catarrhalis-induced IL-8 transcriptional regulation might be a complex cell- and stimulus-specific signaling network between different MAPK pathways. Thus its precise mechanism of action in pulmonary epithelial cells deserves further attention.

Signaling via NF-{kappa}B mediates multiple aspects of host response to bacterial infection (13). In our study we provide evidence for the central role of NF-{kappa}B for M. catarrhalis related cell activation and cytokine liberation. Interestingly, Di Stefano et al. (7) observed a marked increase in the expression of p65 protein, the major subunit of NF-{kappa}B, in bronchial biopsies of COPD patients. This finding was significantly correlated with the degree of airflow limitation and with increasing severity of the disease. Similar results were found in COPD patients regarding an upregulation of bronchial epithelial IL-8 expression (7, 36). In the light of these results, our data suggest a similar role for M. catarrhalis with respect to airway inflammation and overall to the pathophysiology of COPD.

Recent studies imply that inflammatory gene transcription is controlled, at least in part, by the degree of local unwinding of nucleosomal DNA, which is regulated by histone modification such as acetylation (10, 15). In histone H3 from most species, the main sides for phosphorylation include serine 10 and for acetylation the lysine 14 residue (9, 10, 25). Acetylation of histone H4 is known to often occur at the lysine 8 residue (25). Increased acetylation is known to result in a more loosely wound structure allowing access of transcription factors and RNA polymerase II (25). There is increasing evidence that in patients with COPD, expression of inflammatory genes, such as Il8, is regulated by such modification of core histones(3). However, little is known about the role of these mechanisms in bacterial infections. Schmeck et al. (32) were the first to demonstrate bacteria-induced histone modifications at the Il8 promotor in human endothelial cells after infection with Listeria monocytogenes. The authors hypothesized that bacteria might play an important role for epigenetic modifications altering inflammatory gene expression (32). Concordantly, an important finding of our study is that M. catarrhalis induced an enhancement of the Ser-10/Lys-14-histone H3 phospho-acetylation and histone H4 acetylation globally and at the IL8 promotor of bronchial epithelial cells. In addition, we could demonstrate M. catarrhalis-related enhancement of RNA polymerase II recruitment to the IL8 promotor. Thus our findings suggest that M. catarrhalis may facilitate the bronchial epithelial cell activation via histone H3 and H4 modifications.

Hypoacetylation and deacetylation of histones, respectively, lead to tighter winding of DNA and reduced gene transcription (15). HDACs are known to decrease acetylation of individual lysines of histones H3 or H4 that are important for IL-8 transcription (1, 15). The proposed link between M. catarrhalis-infection and HDACs was supported by studies that made use of TSA, a specific inhibitor of HDAC activity, for IL-8 expression (3, 12, 21). Preventing histone deacetylation clearly increased the M. catarrhalis-induced cellular IL-8 production. Moreover, we demonstrated that M. catarrhalis reduced the global expression and activity of HDAC1 and -2 in airway epithelial cells. In this context it is of interest that HDAC activity was also decreased in lung biopsy specimens from patients with COPD and that a progressive reduction in total HDAC activity in this patients reflected the severity of the disease (16). Oxidative stress and cigarette smoke could already be identified as inducing a significant decrease in HDAC activity in respiratory epithelium (24, 41). Our data suggest also an effect of M. catarrhalis on the histone acetylation-deacetylation balance in bronchial epithelium. It is tempting to speculate that other bacteria such as H. influenzae or S. pneumoniae might also be capable of reducing HDAC expression and activity in bronchial epithelium.

In conclusion, our study provides novel data on important molecular mechanisms that were used by M. catarrhalis to activate bronchial epithelial cells to affect gene transcription resulting in a strong inflammatory response. After M. catarrhalis infection, we found activation of p38 and ERK MAP kinase pathways, activation of NF-{kappa}B, reduction of global HDAC expression and activity, as well as alterations in the nuclear histone acetylation-deacetylation balance in bronchial epithelial cells. Our results suggest that M. catarrhalis is an important pathogen that can induce a strong proinflammatory response in human bronchial epithelium and may thus contribute to the pathogenesis of COPD.


   FOOTNOTES
 

Address for reprint requests and other correspondence: H. Slevogt, Dept. of Internal Medicine/Infectious Diseases, Charité-Universitätsmedizin Berlin, Augustenburger Platz 1, 13353 Berlin, Germany (e-mail: hortense.slevogt{at}charite.de)

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
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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