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Circulation Research. 2008;102:888-895
Published online before print February 28, 2008, doi: 10.1161/CIRCRESAHA.107.161307
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(Circulation Research. 2008;102:888.)
© 2008 American Heart Association, Inc.

Molecular Medicine

Simvastatin Reduces Chlamydophila pneumoniae–Mediated Histone Modifications and Gene Expression in Cultured Human Endothelial Cells

Bernd Schmeck*, Wiebke Beermann*, Philippe Dje N’Guessan, Andreas C. Hocke, Bastian Opitz, Julia Eitel, Quoc Thai Dinh, Martin Witzenrath, Matthias Krüll, Norbert Suttorp, Stefan Hippenstiel

From the FORSYS Junior Research Group "Systems Biology of Lung Inflammation" (B.S.), Department of Internal Medicine/Infectious Diseases and Pulmonary Medicine (B.S., W.B., P.D.N, A.C.H., B.O., J.E., M.W., M.K., N.S., S.H.), and Department of Internal Medicine/Psychosomatic Medicine (Q.T.D.), Charité-Universitätsmedizin Berlin, Germany.

Correspondence to Bernd Schmeck, MD, Department of Internal Medicine/Infectious Diseases and Pulmonary Medicine, FORSYS Junior Research Group "Systems Biology of Lung Inflammation", Charité-Universitätsmedizin Berlin, Augustenburger Platz 1, 13353 Berlin, Germany. E-mail bernd.schmeck{at}charite.de


*    Abstract
up arrowTop
 *Abstract
down arrowIntroduction
 down arrowMaterials and Methods
 down arrowResults
 down arrowDiscussion
 down arrowReferences
 
Inflammatory activation of the endothelium by Chlamydophila pneumoniae infection has been implicated in the development of chronic vascular lesions and coronary heart disease by seroepidemiological and animal studies. We tested the hypothesis that C pneumoniae induced inflammatory gene expression is regulated by Rho-GTPase–related histone modifications. C pneumoniae infection induced the liberation of proinflammatory interleukin-6, interleukin-8, granulocyte colony-stimulating factor, macrophage inflammatory protein-1β, granulocyte/macrophage colony-stimulating factor, and interferon-{gamma} by human endothelial cells. Cytokine secretion was reduced by simvastatin and the specific Rac1 inhibitor NSC23766 but was synergistically enhanced by inhibitors of histone deacetylases trichostatin A and suberoylanilide hydroxamic acid. Infection of endothelial cells with viable C pneumoniae, but not exposure to heat-inactivated C pneumoniae or infection with C trachomatis, induced acetylation of histone H4 and phosphorylation and acetylation of histone H3. Pretreatment of C pneumoniae–infected cells with simvastatin or NSC23766 reduced global histone modifications as well as specific modifications at the il8 gene promoter, as shown by chromatin immunoprecipitation. Reduced recruitment of nuclear factor {kappa}B p65/RelA as well as of RNA polymerase II was observed in statin-treated cells. Taken together, Rac1-mediated histone modifications seem to play an important role in C pneumoniae–induced cytokine production by human endothelial cells.


Key Words: endothelial cells • cytokines • statins • Chlamydophila pneumoniae • histones


*    Introduction
up arrowTop
 up arrowAbstract
 *Introduction
down arrowMaterials and Methods
 down arrowResults
 down arrowDiscussion
 down arrowReferences
 
Chlamydia are Gram-negative bacteria that are obligate intracellular parasites of eukaryotic cells, including endothelial cells. Although chronic or recurrent infections with Chlamydophila pneumoniae have been associated with the development of vascular lesions and atherosclerosis, little is known about the molecular pathogenesis.1–4 Infection of endothelial cells with C pneumoniae may initiate and perpetuate local inflammation by inducing cytokine release (eg, interleukin [IL]-6, IL-8) and adhesion molecule expression (P-/E-selectin, intercellular adhesion molecule-1, vascular cell adhesion molecule-1), which subsequently results in recruitment of inflammatory cells to the endothelium.5,6

Infection of the endothelium by Chlamydia is recognized by different pattern-recognition receptors. Membrane-bound Toll-like receptors TLR2 and TLR4 have been demonstrated to mediate host defense against C pneumoniae or chlamydial components.7–10 In addition, the nucleotide-binding oligomerization domain protein NOD1 was identified as part of a cytosolic surveillance system detecting intracellular Chlamydia in human endothelial cells.11 The detection of pathogens by these pattern-recognition receptors resulted in the activation of complex signaling pathways, including the stimulation of nuclear factor (NF)-{kappa}B–dependent gene transcription.12

Increasing evidence indicates that histone modifications may serve as 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.13 In chromatin, 146 base pairs of DNA are wrapped 1.65 turn around a histone octamer (H2A, H2B, H3, H4)2.14 Transcription repression or gene activation is regulated by specific covalent modifications of accessible N-terminal histone tails,15,16 including acetylation (mostly lysine), phosphorylation (serine/threonine), and methylation (lysine).17,18 Phosphorylation at Ser10 on H3 and acetylation at Lys14 of H4 seems to have a special impact on gene regulation.14 These modifications were implicated in lipopolysaccharide (LPS)-stimulated activation of dendritic cells,19 as well as in Listeria monocytogenes–induced activation of human endothelial cells.20

In this study, we tested the hypothesis that C pneumoniae regulated inflammatory gene expression in endothelial cells by histone modifications. C pneumoniae induced expression of IL-6, IL-8, granulocyte colony-stimulating factor (G-CSF), macrophage inflammatory protein (MIP)-1β, granulocyte/macrophage colony-stimulating factor (GM-CSF), and interferon (IFN)-{gamma} was reduced by simvastatin and the Rac1 inhibitor NSC23766 but was synergistically enhanced by inhibitors of histone deacetylases (HDACs). Pretreatment of cells infected with C pneumoniae with simvastatin or NSC23766 reduced global histone modifications, as well as specific modifications at the il8 gene promoter. Moreover, reduced recruitment of NF-{kappa}B p65/RelA as well as of RNA polymerase II (Pol II) was observed in statin-treated cells. Taken together, Rac1-mediated histone modifications seem to play an important role in C pneumoniae–induced cytokine production by human endothelial cells.


*    Materials and Methods
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 *Materials and Methods
down arrowResults
 down arrowDiscussion
 down arrowReferences
 
Human umbilical vein endothelial cells (HUVECs) and human aortic endothelial cells (HAECs) were infected by C pneumoniae strain CWL 029 or Chlamydia trachomatis serovar K. Cytokine release was measured by ELISA and Bioplex Protein Array system. Histone modifications were analyzed globally by Western blot and on the promoter level by chromatin immunoprecipitation (ChIP). Details regarding reagents and methodology are provided in the online data supplement, available at http://circres.ahajournals.org.


*    Results
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowMaterials and Methods
 *Results
down arrowDiscussion
 down arrowReferences
 
C pneumoniae–Induced Cytokine Expression Was Inhibited by Simvastatin and Rac1 Inhibitor NSC23766
C pneumoniae infection of the endothelium is suspected to contribute to the initiation or progression of atherosclerosis,3,21–25 and cytokine release of infected endothelial cells may promote inflammation of the vessel wall.23,26,27 As shown in Figure 1A, infection of HUVECs with C pneumoniae CWL (5 multiplicities of infection [mois]) for 24 hours induced the expression of IL-6, IL-8, G-CSF, GM-CSF, MIP-1β, and IFN-{gamma}. Infection with C trachomatis did not induce IL-8 or GM-CSF (Figure 1B; 5 mois). However, infection with C pneumoniae did not induce significant release of IL-2, IL-4, IL-7, IL-10, IL-12(p70), and IL-13 by endothelial cells in our experimental setup (data not shown).


Figure 1
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  		Figure 1. C pneumoniae–induced cytokine release was blocked by simvastatin or specific Rac1 inhibition. A, HUVECs were preincubated for 24 hours with simvastatin (15 µg/mL) or the specific Rac1 inhibitor NSC23766 (200 µmol/L) and infected with 5 mois (MOI) of C pneumoniae CWL for 15 hours. Cytokine release was measured in the supernatant by Bioplex assay. Data are shown as means±SEM of at least 3 independent experiments. *P<0.05 compared with uninfected control cells; #P<0.05 compared with infected cells without inhibitors. B, HUVECs were infected with 5 mois of viable or heat-inactivated C pneumoniae CWL or viable C trachomatis serovar K (CTK) for 15 hours. Cytokine release was measured in the supernatant by ELISA. Data are shown as means±SEM of at least 3 independent experiments. *P<0.05, viable CWL compared with uninfected control cells; #P<0.05, heat-inactivated CWL or C trachomatis serovar K compared with viable CWL.

To analyze whether only viable bacteria induce cytokines, we exposed cells to viable or heat-inactivated C pneumoniae and measured release of cytokine IL-8 and growth factor GM-CSF. Induction of both factors by heat-inactivated bacteria was significantly lower (Figure 1B).

Because both heat-inactivated C pneumoniae and viable C trachomatis did not induce significant expression of LPS-inducible IL-8 and GM-CSF in endothelial cells (Figure 1B), cell activation by viable C pneumoniae seems to be independent of LPS, which could be present in the given experimental setting.

The beneficial effects of clinically used statins, which blocked HMG-CoA (3-hydroxy-3-methylglutaryl coenzyme A; EC 1.1.1.34) depended, at least in part, on the inhibition of small GTP-binding Rho protein family members.28,29 Pretreatment of endothelial cells with simvastatin for 24 hours (15 µg/mL) or the Rac1-specific inhibitor NSC2376630 for 24 hours (200 µmol/L) abolished C pneumoniae–related release of the analyzed cytokines (Figure 1). By using the important chemotactic cytokine IL-8 as a model, we demonstrated the dose dependency of the simvastatin (Figure 2A) and NSC23766 (Figure 2B) effect with respect to C pneumoniae–induced IL-8 release in HUVECs. Thus, inhibition of small GTP-binding Rho proteins reduced C pneumoniae-related cytokine expression in HUVECs.


Figure 2
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  		Figure 2. C pneumoniae–induced IL-8 release was dose-dependently blocked by simvastatin or specific Rac1 inhibition. HUVECs were preincubated for 24 hours with the indicated doses of simvastatin (A) or the specific Rac1 inhibitor NSC23766 (B) and infected with 5 mois of C pneumoniae CWL for 15 hours. Cytokine release was measured in the supernatant by ELISA. Data are shown as means±SEM of at least 3 independent experiments. *P<0.05 compared with uninfected control cells; #P<0.05 compared with infected cells without inhibitors.

C pneumoniae, but Not C trachomatis, Induced Histone Modifications in Endothelial Cells
Increasing evidence indicates that the transcription of inflammatory genes is regulated by specific covalent modifications of N-terminal histone tails at Ser10 on H3 (phosphorylation) and at Lys14 of H4 (acetylation).14,19,20 Analysis of HUVECs infected with C pneumoniae for 60 minutes demonstrated rapid induction of global acetylation of H4 and phosphorylation/acetylation of H3 at Ser10/Lys14 starting after 30 minutes of infection, as demonstrated by Western blot (Figure 3A). In addition, the HDAC inhibitor trichostatin A (TSA) (0.01 ng/mL, 60 minutes) induced acetylation of H4 and, to a lesser extend than C pneumoniae, of H3 (Figure 3A).


Figure 3
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  		Figure 3. Viable C pneumoniae, but not heat-inactivated bacteria or C trachomatis, time-dependently induced phosphorylation/acetylation of H3 and acetylation of H4. A, HUVECs were infected with C pneumoniae (5 mois) for the indicated time periods or exposed to TSA (0.01 ng/mL, 60 minutes). Histone modifications were detected by Western blot using antibodies specifically detecting Ac-H4 or phosphorylated/acetylated (Ser10/Lys14) H3. B, HUVECs were exposed to viable C pneumoniae (CWL), heat-inactivated C pneumoniae (hiCWL), or C trachomatis serovar K (CTK), each with 5 mois, for 60 minutes. Histone modifications were detected by Western blot using antibodies specifically detecting Ac-H4 or phosphorylated/acetylated (Ser10/Lys14) H3. Representatives of 3 independent experiments are shown. C, HUVECs were exposed to viable or heat-inactivated C pneumoniae or C trachomatis (both, 5 mois). After maturation (72 hours), cells were scraped, briefly sonicated, passaged (1:3) onto HEp-2 cells seeded on glass coverslips, and visualized by confocal microscopy (red, F-actin; green, bacteria).

To test whether this is a C pneumoniae–specific effect, we compared the effect with C trachomatis–induced histone alterations. However, only C pneumoniae induced histone H3/H4 modifications (Figure 3B). Furthermore, heat-inactivated C pneumoniae did not induce the modification of histones in endothelial cells (Figure 3B).

To ensure intracellular infection of endothelial cells, HUVECs were exposed to viable or heat-inactivated C pneumoniae or C trachomatis (both, 5 mois). After maturation (72 hours), cells were scraped, briefly sonicated, and passaged (1:3) onto HEp-2 cells seeded on glass coverslips. Only in HEp-2 cells exposed to HUVEC extracts with viable C pneumoniae, but not to cells incubated with heat-inactivated C pneumoniae, an intracellular LPS signal could be detected (Figure 3C). Because HEp-2 cells do not efficiently endocytose LPS, C pneumoniae infection was demonstrated in concordance with the literature.11,31

Inhibition of HDACs Increased C pneumoniae–Related Cytokine Expression
To test whether C pneumoniae–related histone modifications are of functional importance for the observed cytokine induction, cells were infected with a low moi (0.5) of C pneumoniae and exposed to low doses of HDAC inhibitors, which did not induce cytokine expression per se (Figure 4). Exposure of endothelial cells to HDAC inhibitor TSA (0.1 ng/mL) and to C pneumoniae (0.5 moi) enhanced the expression of IL-6, IL-8, G-CSF, GM-CSF, MIP-1β, and IFN-{gamma} (Figure 4). Moreover, a chemically unrelated HDAC inhibitor, suberoylanilide hydroxamic acid (SAHA),32 also induced C pneumoniae–related IL-8 expression in HUVECs (Figure 5A). By analyzing IL-8 as an example, we extended our observation to HAECs (Figure 5B and 5C). In HAECs, preexposure of cells to both HDAC inhibitors significantly increased the C pneumoniae–induced IL-8 release. Thus, histone acetylation seems to contribute to C pneumoniae–related cytokine release in human endothelial cells in vitro.


Figure 4
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  		Figure 4. HDAC inhibition synergistically enhanced C pneumoniae–induced cytokine release. HUVECs were preincubated with the HDAC inhibitor TSA (0.01 ng/mL) and then infected with C pneumoniae (0.5 moi) for 15 hours. Release of cytokines was measured in the supernatant by Bioplex assay. Data are shown as means±SEM of at least 3 independent experiments. *P<0.05 from C pneumoniae– and TSA-exposed cells.


Figure 5
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  		Figure 5. HDAC inhibitors TSA and SAHA synergistically enhanced C pneumoniae–induced IL-8 release by human umbilical vein or aortic endothelial cells. HUVECs (A) or HAECs (B/C) were preincubated with HDAC inhibitors SAHA (1 nmol/L) (A and C) or TSA (0.01 ng/mL) (B) and then infected with C pneumoniae (0.5 moi) for 15 hours. Release of IL-8 was measured in the supernatant by ELISA. Data are shown as means±SEM of at least 3 independent experiments. *P<0.05 from C pneumoniae– and inhibitor-exposed cells. D, HUVECs were preincubated with the HDAC inhibitor TSA (0.01 ng/mL) (B) and then infected with viable or heat-inactivated C pneumoniae (hiCWL) (both, 5 mois) for 15 hours. Release of IL-8 was measured in the supernatant by ELISA. Data are shown as means±SEM of at least 3 independent experiments. *P<0.05 comparing viable and heat-inactivated C pneumoniae; #P<0.05 from heat-inactivated C pneumoniae with or without preincubation with TSA.

To analyze whether histone acetylation is only involved in cytokine induction by viable bacteria, we exposed cells to viable or heat-inactivated C pneumoniae. IL-8 induction by heat-inactivated bacteria was significantly lower but could also be enhanced by TSA (Figure 5D). The same results were obtained with respect to GM-CSF induction (data not shown).

C pneumoniae Induced Histone Alterations at the il8 Gene Promoter
To gain more insight into the impact of histone modification on C pneumoniae–related cytokine regulation, we analyzed IL-8 expression as a model at promoter level by applying ChIP. Infection of endothelial cells with C pneumoniae induced rapid acetylation of H4 and phosphorylation/acetylation of H3 at Ser10/Lys14 at the il8 promoter (Figure 6A). Moreover, we observed recruitment of NF-{kappa}B p65/Rela subunit to the il8 promoter in infected cells, which is known to be essential for IL-8 expression.33 In addition, binding of Pol II at the il8 promoter further indicates the start of gene transcription. As expected from results in Figures 3B and 5UpD, heat-inactivated C pneumoniae induced no modification of analyzed histone residues and only weak recruitment of p65 and Pol II to the il8 promoter in ChIP analysis (Figure 6B).


Figure 6
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  		Figure 6. C pneumoniae induced phosphorylation/acetylation of H3 and acetylation of H4 at the il8 promoter. HUVECs were exposed to viable (A and B) or heat-inactivated (hiCWL) (B) C pneumoniae (5 mois) for the indicated time periods. Binding of Pol II and NF-{kappa}B/RelA, as well as histone modifications (acetylated H4, phosphorylated/acetylated Ser10/Lys14 on H3), was detected at the il8 gene promoter by ChIP. Representatives of 3 independent experiments are shown.

Simvastatin Reduced C pneumoniae–Induced Global and Gene-Specific Histone Modifications
Because simvastatin and Rac1 inhibitor NSC23766 both inhibited C pneumoniae–related cytokine production, we assessed their effect on global histone modification and on gene-specific (IL-8) regulation. As can be seen in Figure 7A, preincubation with both simvastatin and NSC23766 reduced global acetylation of H4 and phosphorylation/acetylation of H3 at Ser10/Lys14. ChIP experiments (Figure 7B) showed that simvastatin reduced the C pneumoniae–related acetylation of H4 and phosphorylation/acetylation of H3 at Ser10/Lys14 at the il8 promoter. In parallel, recruitment of p65, as well as of Pol II, was abolished back to control levels in simvastatin-exposed endothelial cells, further suggesting that C pneumoniae–related histone modifications are a prerequisite for the initiation of cytokine expression.


Figure 7
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  		Figure 7. C pneumoniae–induced histone modifications were blocked by simvastatin or specific Rac1 inhibition. HUVECs were preincubated for 24 hours with simvastatin (15 mg/mL) (A and B) or the specific Rac1 inhibitor NSC23766 (200 µmol/L) (A) and infected with 5 mois C pneumoniae CWL for 60 minutes. A, Histone modifications were detected by Western blot using acetylation (H4)- or phosphorylation/acetylation (Ser10/Lys14) H3-specific antibodies. B, Histone modifications (acetylation of H4, phosphorylation/acetylation at Ser10/Lys14 H3) and Pol II binding were detected at the promoter of il8 (D) by ChIP. Representatives of 3 independent experiments are shown.


*    Discussion
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowMaterials and Methods
 up arrowResults
 *Discussion
down arrowReferences
 
The study presents evidence that statins modulate C pneumoniae–induced inflammatory activation of human endothelial cells by reducing infection-related histone modifications. Viable C pneumoniae–induced expression of proatherosclerotic cytokines IL-6, IL-8, G-CSF, MIP-1β, GM-CSF, and IFN-{gamma} was reduced by simvastatin and the Rac1 inhibitor NSC23766 but was synergistically enhanced by inhibitors of HDACs. Pretreatment with simvastatin or NSC23766 reduced global C pneumoniae–induced histone modifications, as well as specific modifications at the il8 gene promoter. Moreover, reduced recruitment of NF-{kappa}B p65/RelA and Pol II was observed in statin-treated cells. Although C trachomatis infected endothelial cells, they did neither induce cytokine expression nor histone modifications.

Impaired endothelial function is observed in a variety of pathological conditions, such as hypertension and atherosclerosis,34,35 and the endothelium is a primary target in several infectious diseases.26 In particular, C pneumoniae infection has been suggested as a trigger of endothelial inflammation, promoting vascular diseases.36 In our model, C pneumoniae infection induced the release of chemoattractants, proinflammatory cytokines, as well as myeloid growth factors, by primary human endothelial cells, whereas C trachomatis infected endothelial cells but did not induce cytokine release (this study and Krüll et al31).

These cytokines are also thought to play important roles in atherosclerosis: in aortic arches of atherosclerotic mice, expression of monocyte chemoattractant protein-1 and MIP-1β was plaque progression dependently increased.27 Monocyte chemoattractant protein-1 and also IL-6 were elevated in patients with myocardial infarction and unstable angina.37 In the Edinburgh Artery study, IL-6 showed a stronger independent predictive value for atherosclerosis and its progression than C-reactive protein or soluble adhesion molecules.38 Accordingly, IL-6 was associated with significantly increased risk for cardiovascular disease39 and was found to be the strongest independent biomarker for coronary artery disease–related death in a study with more than 1000 patients.40 IL-6 and IL-8 were significantly increased in infarct-related coronary artery thrombi and atherosclerotic plaque specimens obtained with a transluminal extraction catheter from cases of acute myocardial infarction.41 Moreover, GM-CSF was demonstrated to be an important factor in oxidized LDL–induced macrophage proliferation,42 and Gupta et al found that IFN-{gamma} potentiated atherosclerosis in ApoE knockout mice.43

Because release of IL-6, IL-8, and IFN-{gamma} was also observed in C pneumoniae–infected macrophages, these molecules may play an important role in the host defense against C pneumoniae.44 For example, IFN-{gamma} was demonstrated to impair C pneumoniae infection in macrophages,45,46 and mouse models deficient for the IFN-{gamma} receptor showed the higher sensitivity to C pneumoniae.47

Recent studies imply that histone modifications control eukaryotic gene transcription by effecting transcription factor binding and promoter transactivation.15,18,20,48,49 Tight wrapping of DNA around histone octamers appears to obstruct binding of the transcription machinery.50 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.15,51 Viable C pneumophila, but not heat-inactivated bacteria or C trachomatis, induced acetylation of histone H4 and phosphorylation and acetylation of H3. Stimulus-induced phosphorylation of H3 at Ser10 has been reported to be associated with the activation of promoters of mammalian immediate-early genes.19,52 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 Lys14.48,53,54 Moreover, Agalioti et al 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.51 In this line, we provided evidence that histone acetylation is necessary for L monocytogenes–related release of IL-8, but not IFN-{gamma}, by human endothelial cells.20 Here, we show that C pneumoniae–induced release of both cytokines was enhanced by inhibition of histone deacetylation in human venous and aortic endothelial cells pointing to pathogen-specific gene regulation. Heat-inactivated C pneumoniae were found to induce lower IL-8 and GM-CSF expression, as well as histone modification and p65-/Pol II-recruitment to the il8 promoter, suggesting that surface structures of C pneumoniae can activate HUVECs in high concentrations, but for full histone modification, additional effects of viable C pneumoniae seem to be necessary.

Autocrine effects on the histone modification analyzed seem to be unlikely, because the addressed modifications were observed as early as 30 minutes after stimulation. Furthermore, stimulation of HUVECs with high concentrations of recombinant human IL-8 (50 ng/mL) or tumor necrosis factor-{alpha} (50 ng/mL) did not result in comparable histone modifications (data not shown).

The proinflammatory and chemotactic cytokine IL-8 potently recruits leukocytes to sites of infection, thereby contributing to local tissue inflammation.55 The il8 gene promoter has been described previously as being regulated by histone phosphorylation and acetylation.20,56,57 Modifications of both histones H3 and H4 were observed in overall chromatin analysis and were also induced by C pneumoniae infection specifically at the il8 gene promoter, together with recruitment of NF-{kappa}B/p65 and Pol II. As Agalioti et al have shown, phosphorylation of H3 at Ser10 and acetylation of H3-Lys14 and H4-Lys8 were followed by recruitment of bromodomain-containing factors TFIID and SWI/SNF and subsequent transcription.51 Therefore, our observations, together with previous studies, implicate a sequence of C pneumoniae–related histone phosphorylation, histone acetylation, chromatin remodeling, and successful gene transcription.

It has been suggested that the beneficial effects of HMG-CoA reductase–inhibiting statins on endothelial and vascular function depended not only on cholesterol reduction but also on blocking of geranylgeranylation, thereby inhibiting small GTP-binding Rho protein–dependent pathways.28,29 Rho-GTPases act as molecular switches in important signaling pathways, including IL-1β–,58 tumor necrosis factor-{alpha}–,59 TLR2-,60 and TLR4-related61 cell activation. By using simvastatin and the Rac1-specific inhibitor NSC23766,30,62 we found an inhibition of C pneumoniae–related cytokine release by both agents, thereby blocking major inflammatory and chemotactic endocrine pathways.55

However, further studies are needed to explore the effect of C pneumoniae on the overall gene expression response in C pneumoniae infection in the presence or absence of statins. In addition, release of possible anti- and proinflammatory molecules not detected by, eg, global gene expression analysis (such as nitric oxide, prostacyclin) should be investigated as well.

Accordingly, we demonstrated that simvastatin and the specific Rac1 inhibitor blocked C pneumoniae–induced acetylation of H4 and phosphorylation and acetylation of H3. At the il8 promoter, simvastatin inhibited C pneumoniae–related histone modifications, as well as NF-{kappa}B/p65 and Pol II recruitment. In accordance, RhoA and Rac1 activation was shown recently in C pneumoniae–infected type II lung epithelial cells,63 as well as in vascular smooth muscle cells,64 suggesting an important role of Rho protein–related cell activation in C pneumoniae pathogenesis. Taking our observations into account, (simva)statin-related Rac1 inhibition seems to reduce C pneumoniae–related inflammatory activation of (endothelial) cells.

The expression of important proinflammatory mediators is controlled by activation of the transcription factor NF-{kappa}B.33 Because Rac1 was found to be involved in NF-{kappa}B activation by C pneumoniae (this study and Dechend et al64), lipopolysaccharide,61 heat-inactivated Staphylococcus aureus,60 and viable S pneumoniae65 Rac1 may act as a central molecular switch in regulating NF-{kappa}B–related gene transcription in bacterial infections.

C trachomatis infected endothelial cells without initiating any detectable cytokine response with respect to the used colony-forming unit and the time frame analyzed. In addition, in contrast to C pneumoniae, C trachomatis did not induce the phosphorylation of mitogen-activated protein kinases or upregulation of adhesion molecule intercellular adhesion molecule-1 in endothelial cells.31 Although it could not be ruled out that replication of C trachomatis was less sufficient in endothelial cells (which are not their primary target cells), these data suggest that the infection process per se and the formation of a Chlamydia-containing organelle is not sufficient to activate endothelial cytokine release.

Initiation of the endothelial cytokine response against C pneumoniae depends, at least in part, on their recognition by transmembranous and cytosolic pattern-recognition receptors,7–11 and Rho proteins are considered to act as molecular switches in TLR2 and TLR4 signaling.60,61,65 Therefore, it seems likely that the effects of simvastatin and NSC23766 observed depended on the inhibition of Rho-GTPase–related host cell signaling pathways initiated after recognition of C pneumoniae by pattern-recognition receptors.

In conclusion, we provide evidence that Rac1-related histone modifications contribute to the control of the release of important chemokines in C pneumoniae–infected human endothelial cells.


*    Acknowledgments
 
The excellent technical support of Doris Stoll and Jacqueline Hellwig is greatly appreciated. Parts of this work will be included in the doctoral thesis of Wiebke Beermann.

Sources of Funding

This work was supported by Deutsche Forschungsgemeinschaft grants SPP-Kr 2197/1-2 (to M.K. and N.S.) and HI-789/6-1 (to S.H.) and by the Bundesministerium für Bildung und Forschung (BMBF) (to B.S., N.S., and S.H [BMBF-Competence network CAPNETZ, and FORSYS-Partner]).

Disclosures

None.


*    Footnotes
 
*Both authors contributed equally to this work. Back

Original received August 18, 2007; revision received February 16, 2008; accepted February 20, 2008.


*    References
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 up arrowAbstract
 up arrowIntroduction
 up arrowMaterials and Methods
 up arrowResults
 up arrowDiscussion
 *References
 

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