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Cardiovascular Research Advance Access originally published online on January 14, 2008
Cardiovascular Research 2008 78(1):26-35; doi:10.1093/cvr/cvn011
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Published on behalf of the European Society of Cardiology. All rights reserved. © The Author 2008. For permissions please email: journals.permissions@oxfordjournals.org

Bacterial DNA induces myocardial inflammation and reduces cardiomyocyte contractility: role of Toll-like receptor 9

Pascal Knuefermann1,*,{dagger}, Markus Schwederski1,{dagger}, Markus Velten1, Peter Krings1, Heidi Ehrentraut1, Myriam Rüdiger2, Olaf Boehm1, Klaus Fink3, Ulrike Dreiner2, Christian Grohé4, Andreas Hoeft1, Georg Baumgarten1, Alexander Koch5, Kai Zacharowski5 and Rainer Meyer2

1 Department for Anesthesiology and Intensive Care Medicine, University Hospital Bonn, Sigmund-Freud-Strasse 25, Bonn 53105, Germany
2 Institute of Physiology II, University Hospital Bonn, Bonn, Germany
3 Department of Pharmacology and Toxicology, University Hospital Bonn, Bonn, Germany
4 Department of Internal Medicine 2, University Hospital Bonn, Bonn, Germany
5 Molecular Cardioprotection and Inflammation Group, Department of Anaesthesia, Bristol Royal Infirmary, Bristol, UK

* Corresponding author. Tel: +49 228 2871 4110; fax: +49 228 2871 4115. E-mail address: pascal.knuefermann{at}ukb.uni-bonn.de

Received 26 April 2007; revised 30 December 2007; accepted 7 January 2008

Time for primary review: 24 days


   Abstract
Top
 Abstract
1. Introduction
 2. Methods
 3. Results
 4. DISCUSSION
 Funding
 References
 
Aims: Myocardial function is severely compromised during sepsis. Several underlying mechanisms have been proposed. The innate immune system, i.e. Toll-like receptor (TLR) 2 and 4, significantly contributes to cardiac dysfunction. Little is known regarding TLR9 and its pathogenic ligand bacterial DNA in the myocardium. We therefore studied the role of TLR9 in myocardial inflammation and cardiac contractility.

Methods and results: Wild-type (WT, C57BL/6) and TLR9-deficient (TLR9-D) mice and isolated cardiomyocytes were challenged with synthetic bacterial DNA (CpG-ODN). Myocardial contractility as well as markers of inflammation/signalling were determined. Isolated cardiomyocytes incorporated fluorescence-marked CpG-ODN. In WT mice, CpG-ODN caused a robust response in hearts demonstrated by increased levels of tumour necrosis factor (TNF-{alpha}), interleukin (IL)-1β, IL-6, inducible nitric oxide synthase (iNOS), and nuclear factor {kappa}B activity. This inflammatory response was absent in TLR9-D mice. Under similar conditions, contractility measurements of isolated ventricular cardiomyocytes demonstrated a TLR9-dependent loss of sarcomeric shortening after CpG-ODN exposure. This observation was iNOS dependent as the application of a specific iNOS inhibitor reversed sarcomeric shortening to normal levels.

Conclusion: Our data suggest that bacterial DNA contributes to myocardial cytokine production and loss of cardiomyocyte contractility via TLR9.

KEYWORDS Sepsis; Contractile function; Infection/inflammation; Cardiomyocytes


   1. Introduction
Top
 Abstract
 1. Introduction
2. Methods
 3. Results
 4. DISCUSSION
 Funding
 References
 
Sepsis represents a significant health problem in the western world. This disease is the leading cause of death in non-coronary intensive care units, with a mortality rate ranging from 20 to 80% of severely affected patients.1,2 In the United States, the incidence of sepsis has risen from ~160 000 cases in 1979 to 700 000 in 2000 (an increase of 13.7% per year).3 Recent data estimate that >150 000 patients suffer from sepsis each year in Germany.4 Cardiovascular dysfunction is a serious complication occurring in ~40% of patients with sepsis,5 increasing mortality from 20–70%.6 During experimental sepsis high levels of tumour necrosis factor (TNF-{alpha}) and interleukin (IL)-1β are detectable in the heart.7,8 There is consent that these cytokines severely compromise cardiac function during human sepsis.9,10 Altered cardiac nitric oxide (NO) production also plays an important role for cardiac dysfunction during sepsis.11 However, the proximal events that trigger and sustain the expression in the heart are not fully understood. The identification of toll-like receptors (TLRs) has been a major advance in the understanding of the pathogenesis of septic shock.12 To date, 13 TLRs (TLR1-13) have been described; TLR2 and TLR4 are the best-characterized receptors so far.13,14 We have recently reported that TLR4 and CD14 mediate the induction of cardiac TNF-{alpha}, IL-1β, and NO following lipopolysaccharide (LPS) challenge.15–17 More important, mice deficient for either TLR4 or CD14 are protected against LPS-induced left ventricular dysfunction.16,18 In addition, other virulence factors that act as TLR ligands have been discussed in the pathophysiology of myocardial dysfunction.19–21 These studies provided new insights into the pathogenesis of sepsis-induced cardiac depression; however, the full picture needs to be elucidated.

Bacterial DNA (CpG-ODN) can initiate an innate immune response via TLR9 potentially leading to septic shock,22,23 septic arthritis,24 or meningitis.25 Bacterial CpG motifs are un-methylated cytosine–phosphate–guanine (CpG) dinucleotides and are predominantly prevalent in bacterial DNA, but not in mammalian DNA.26 The effects of bacterial DNA can be mimicked by synthetic oligonucleotides via the same receptor system.27,28

Little is known about the role of TLR9 in the myocardium, but constitutive expression levels have been detected in mouse hearts.29,30 We therefore investigated whether TLR9 activation via CpG-ODN contributes to cardiac depression using in vivo and in vitro models.


   2. Methods
Top
 Abstract
 1. Introduction
 2. Methods
3. Results
 4. DISCUSSION
 Funding
 References
 
2.1 Animals
TLR9-deficient (TLR9-D) mice, kindly provided by Professor Shizuo Akira, Osaka University, Osaka, Japan,27 back-crossed to a C57BL/6 background, were handled according to the principles of laboratory animal care (NIH publication no: 85-23, revised 1996) and experimental procedures were approved by the German government ethical and research boards (50.203.2-BN 43, 28/01).

2.1.1 CpG-ODN challenge
Mice were intraperitoneally (i.p.) sensitized with 1 mg/kg D-galactosamine (D-GalN; Roth, Karlsruhe, Germany; in control experiments D-GalN alone did not induce an inflammatory response; data not shown). Thirty minutes later, mice received i.p. 1 nmol/g CpG-ODN (1668-Thioat; containing a ‘CG-motif’: 5'-TCC-ATG-ACG-TTC-CTG-ATG-CT; TibMolBiol, Berlin, Germany). According to the manufacturer, the oligonucleotides are endotoxin-free because they were synthetically synthesized like primers for polymerase chain reactions (PCR) with the difference of a thioat backbone. Oligonucleotides were dissolved in endotoxin-free water (Aqua ad Injectabili). Other groups have used these oligonucleotides finding no indication of LPS contamination.31–33 The CG sequence in a concentration of 1 nmol/g was chosen according to earlier publications.22,23,27 The selected dose was sufficient to induce clinical symptoms of sepsis. Both groups used 20 nmol per mouse, because the average body weight of their experimental animals was around 20 g. We have administered 1 nmol/g CpG-ODN in order to guarantee a weight-independent stimulatory dose. Preliminary data examining different concentrations of CpG-ODN (0–50 nmol) on cytokine expression in pulmonary tissue demonstrate a concentration-dependent increase up to 20 nmol. At 50 nmol no further increase was observed indicating that 20 nmol is a saturating concentration with respect to cytokine induction (data not shown). Two other non-CG containing oligonucleotides ODN 1612 (5'-GCT-AGA-TGT-TAG-CGT5) and ODN H154 (5'-CCT-CAA-GCT-TGA-GGG-G-3)34 or 1 mL/kg saline were used as negative controls. Hearts were prepared immediately after sacrificing the animals. Beating hearts were transferred into cold sterile potassium buffer solution (PBS) and allowed to beat spontaneously until no more blood was pumped out of the aortic root, snap-frozen in liquid nitrogen, and kept at –80°C until assayed.

2.1.2 Real-time polymerase chain reaction for TRL9
Total RNA from whole murine hearts was isolated with the guanidinum thiocyanate method.35 RNA concentration was determined by absorbance at 260 nm. Until further processing, RNA was dissolved in 100 µl of RNase-free water and stored at –80°C. Reverse transcription was performed using QIAGEN Omniscript Reverse Transcription kit (Qiagen, Hilden, Germany) according to the manufacturer's protocol. One microgram RNA was used in 20 µl reaction mixtures containing 2 µl 10x reverse transcription buffer, 2 µl dNTP mixture (5 mM of each dNTP), 1 µl Omniscript Reverse Transcriptase, and 2 µl oligo-dT primers. The specific pre-made TaqMan®Gene Expression Assays (Applied Biosystems, Foster City, CA, USA) for murine TLR9 (Mm00446193_m1, amplicon length: 60 bp) and murine GAPDH (Mm99999915_g1) as housekeeping gene were used in this study. Real-time PCR (RT–PCR) was performed according to the manufacturer's protocol. Hundred nanogram of single-stranded cDNA was mixed with supplied 2x TaqMan Universal Master Mix (PN 4304437, Applied Biosystems) and 0.5 µl of TaqMan®Gene Expression Assay to a final volume of 10 µl in a 384-well optical reaction plate. Each sample underwent 40 cycles of amplification in a 384-well optical reaction plate on an ABI PRISM® Sequence Detection Systems. Relative quotients (RQ) of TLR9 gene expression comparing control mice with stimulated mice at different time points were calculated with SDS Software 2.2 (Applied Systems, Foster City, CA, USA). RQ results were analysed with GraphPad Prism 4.05 (GraphPad Software, San Diego, USA).

2.1.3 Western blot analysis for TLR9
Frozen tissue was lysed in ice-cold buffer (150 mM NaCl, 50 mM Tris–HCl, pH 7.4, 1 mM EDTA, 5 µg/mL Leupeptin, 5 µg/mL aprotinin, 1 mM PMSF, 0.1% SDS, 1% sodium deoxycholate, 1% Triton X-100) as previously published.36 After brief centrifugation (16 800g), supernatants were removed, total protein was determined (bicinchoninic acid method), separated by SDS–PAGE and blotted onto nitrocellulose membranes. The blots were incubated with anti-TLR9-antibody (1:1000, IMG-431, Imgenex, San Diego, CA, USA) at 4°C overnight. Horseradish peroxidase-conjugated anti-rabbit secondary antibody (1:3000, GE Healthcare, Braunschweig, Germany) was used. Signals were visualized by enhanced chemiluminescence.

2.1.4 Polymerase chain reaction for iNOS
Whole hearts from wild-type (WT) and TLR9-D mice were collected 4 h after stimulation. RNA was isolated with the Perfect RNA Eukaryotic Mini kit (Eppendorf, Hamburg, Germany). First-strand cDNA was synthesized with the Omniscript Reverse Transcriptase kit (Qiagen). The single-stranded cDNA was amplified by PCR. Inducible nitric oxid synthase (iNOS) primer sequences, PCR conditions, and amplification length were defined as follows: 5'-ACA ACG TGA AGA AAA CCC CTT GTG-3' (sense) and 5'-ACA GTT CCG AGC GTC AAA GAC C-3' (anti-sense); 30 s at 94°C, 30 s at 57°C, and 60 s at 72°C; 35 cycles, and 557 bp. The same cDNA was used for β-actin amplification [5'-ATG GAT GAC GAT ATC GCT-3' (sense) and 5'-ATG AGG TAG TCT GTC AGG T-3' (anti-sense); 45 s at 94°C, 30 s at 56°C, and 60 s at 72°C; 30 cycles; and 570 bp] to confirm that equal amounts of RNA were transcribed. For semi-quantitative analysis, PCR cycles were chosen within the exponential phase of the cDNA amplification. Equal amounts of RT–PCR products were separated on 2% agarose gel. Optical densities of ethidium bromide-stained DNA bands were quantified. The results were expressed as iNOS/β-actin OD ratios.

2.1.5 Electrophoretic mobility shift assay
Myocardial protein extracts were prepared with NE-PERTM Nuclear and Cytoplasmic Extraction Reagents (Perbio, Bonn, Germany) according to the manufacturer's protocol.37 Nuclear factor {kappa}B (NF{kappa}B) oligonucleotides were end-labelled with [{gamma}-32P] ATP. Binding reactions (25 µL total) were performed with nuclear extracts and the specificity of the DNA-protein binding was determined by cold chase analysis as well as with supershift assays. Nuclear extracts were incubated with 2 mg of polyclonal anti-p50 (sc-114x) or anti-p65 (sc-109x) antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA). DNA-protein complexes were electrophoresed, gels were dried, exposed overnight, and scanned with a phosphoimager (FLA3000, Fujifilm Europe, Düsseldorf, Germany).

2.1.6 Ribonuclease protection assay
Myocardial RNA was extracted with the guanidinium thiocyanate method.35 The mRNA-expression was determined with a RNase protection assay system as earlier described in detail.15

2.1.7 Enzyme-linked immunosorbent assay
Whole hearts were dispersed with an Ultra-Turrax TP18-10 (IKA, Staufen, Germany), homogenized and incubated on ice for 5 min in 1 mL of ELISA buffer containing PBS, Triton X-100 (1 µL/mL), PMSF (250 mM in isopropanol, 1 µL/mL), and protease inhibitors (Roche, Mannheim, Germany). Samples were incubated on ice for 20 min, homogenized and centrifuged for 15 min at 4°C. TNF-{alpha}, IL-1β, and IL-6 were determined in the supernatant using an ELISA technique (R&D Systems, Minneapolis, MN, USA).

2.1.8 Multiplex cytokine assay
Blood was obtained from arterial puncture of the abdominal aortic vessel. Two millilitre syringes were flushed once with 1 mL of heparin (5000 IU/mL). Thereafter, the heparin was removed and blood samples taken. Blood was centrifuged at 3400 g for 10 min and the supernatant (plasma) was stored at –20°C. After thawing, samples were analysed immediately at 37°C to facilitate the comparison of results. Levels of TNF-{alpha}, IL-1β, and IL-6 (Mouse Cytokine multi-Plex for LuminexTM laser, BioSource Europe) were determined using the microsphere array technique (Luminex 100 system, Luminex Corporatio, Austin, TX, USA) as previously described.36

2.2 Single-cell experiments
2.2.1 Primary murine cardiomyocytes
Isolation of murine cardiomyocytes was performed as previously described38. Cardiomyocytes of WT- and TLR9-D mice were investigated immediately after isolation to determine baseline contractility. Treatment groups were incubated in Dulbecco's modified Eagle medium, supplemented with 5% minimal essential medium, 10% fetal calf serum, 50 µg/mL gentamicin (culture media from Gibco, New York, USA) with and without CpG-ODN (1 µg/mL, equivalent to 0.3 µM according to27) for 1–6 h. In the literature, concentrations for CpG-ODN range from 0.1 to 4 µM.22,23,27,39–41 In our experimental setting, this rather low concentration of CpG was sufficient to depress cardiac contractility by ~50%. For monitoring the time course of the CpG-ODN effect samples of control cardiomyocytes were taken every hour and the sarcomere shortening was recorded. In our model the maximum effect of CpG-ODN appeared after 5–6 h, therefore WT and TLR9-D cells were investigated at this time point with and without CpG-ODN. To further determine whether depressed cardiac contractility was iNOS dependent, a specific inhibitor of iNOS, S-methylisothiourea (SMT) was applied. Cells were incubated in culture medium supplemented with CpG-ODN (1 µg/mL) for 5–6 h. After 4 h of incubation SMT (100 µM; Sigma-Aldrich, Taufkirchen, Germany) was added and shortenings were recorded as described below.

2.2.2 Single-cell contractility
Sarcomere shortening of ventricular myocytes was recorded with a video imaging system and SarcLen software as published previously (IonOptix, Milton, MA, USA).17 The regular striation patterns of the sarcomeres were analysed by fast Fourier transformation. The video system is mounted to an inverted microscope (Zeiss Axiovert 135TV, Jena, Germany, lens Fluar 40x 1.3) and equipped with an experimental chamber with permanent perfusion Tyrode's solution (~600 µL/min leading to an exchange rate of three times per minute in the 200 µL volume of the chamber) heated to 36°C. Independently of the pretreatment sarcomere shortening was always monitored in Tyrode's solution (in mM: NaCl, 135; KCl, 4; CaCl2, 1.8; MgCl2, 1; Hepes, 2; glucose 10) to avoid direct effects of the incubation media on the contractile response. Contractions were induced by bipolar external stimuli (0.4 ms, 30 V, SD9, Grass, Quincy, MA, USA). Stimuli were applied in pulse trains of 20 stimuli interrupted by a 30 s stimulation pause. The stimulation protocol was: 0.5, 10, 1, 8, 2, 6, 4 Hz.

To obtain representative shortening frequency relationships the five last shortening signals of each train were averaged. The resulting signal was evaluated for the following parameters: resting sarcomere length, amplitude of sarcomere shortening, maximal speed of sarcomere shortening, and re-lengthening. For the demonstration and evaluation of staircases, trains of 20 original recordings of sarcomere shortening of 5–10 cardiomyocytes were averaged. The peak values were fitted by a double exponential function as previously published.41

2.2.3 Histology
Histological analysis was performed to document the uptake of fluorescently-labelled oligonucleotides (1668-ODN-Cy5, 1612-ODN-Cy5; H154-ODN-Cy5) in freshly isolated cardiomyocytes of both genotypes. Coverslips were pretreated with laminin solution (Laminin 10 µg/mL in PBS, L2020 Sigma) for 30 min at room temperature (RT) for adhesion of cardiomyocytes. Cells were stimulated with 1 µg/mL (0.3 µM) 1668-ODN-Cy5 for 30 min at 37°C. Further steps were performed at RT. After removal of culture medium cells were fixed for 10 min in freshly prepared 4% paraformaldehyde and permeablized with 0.2% Triton X-100 in PBS for 5 min. After washing (twice with 0.2% Tween in PBS), nuclei were stained with 4'-6-Diamidino-2-phenylindole (DAPI) for 30 min. Cells were analysed on an Olympus IX71 inverted microscope (Olympus, Hamburg, Germany).

2.3 Statistical evaluation
All values are expressed as mean ± SEM. A one-way or two-way ANOVA followed by Bonferroni-corrected post hoc analysis was used when appropriate. For statistical comparison of iNOS-mRNA a t-test was applied. Significant differences were considered to exist at P ≤ 0.05.


   3. Results
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
4. DISCUSSION
 Funding
 References
 
3.1 Clinical manifestations of CpG-ODN challenge
Mice were i.p. sensitized with 1 mg/kg D-GalN. Thirty minutes later, mice received i.p. either 1 nmol/g CpG-ODN, 1 nmol/g non-CpG-ODN or 1 mL/kg saline. In control experiments, D-GalN alone did not induce an inflammatory response (data not shown). Mice were monitored for clinical symptoms. Following 2 h of CpG-ODN challenge, WT mice developed shock-like symptoms such as lethargy and diarrhoea, whereas TLR9-D mice remained healthy. Mice challenged with non-CpG containing oligonucleotides or saline did not exhibit inflammatory signs.

3.2 Uptake of CpG-ODN into cardiomyocytes and TLR9 expression
Using fluorescence microscopy, we could demonstrate an uptake of CpG-ODN into freshly isolated cardiomyocytes of both genotypes within 30 min after stimulation (Figure 1A–D). However, uptake of CpD-ODN was delayed in TLR9-D cells. CpG-ODN was found in the cytoplasm as well as in the nucleus. Non-CpG-ODN was also tested and its uptake was detected in WT cells (data not shown). The expression of TLR9 in whole myocardial tissue was confirmed by RT–PCR and Western blot analysis. After CpG-ODN challenge the expression of TLR9 demonstrated a time-dependent regulation. Figure 1E depicts that TLR9 mRNA was significantly up-regulated after 2 h in comparison to baseline and in comparison to the receptor expression in hearts from TLR9-D mice. The time-dependent TLR9 regulation was also detectable on protein level (Figure 1F).


Figure 1
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  		Figure 1 (A–D) In vitro stimulation of isolated cardiomyocytes with 1 µg/mL 1668-ODN-Cy5. Uptake of fluorescently-labelled CpG-ODN was documented after 30 min in isolated cardiomyocytes in the cytoplasm as well as in the nucleus from (A and B) WT cells as well as (C and D) TLR9-D cells. A difference in the kinetics of uptake was documented as cardiomyocytes from WT mice show this fluorescence signal earlier than cells of TLR9-D mice. A DAPI staining indicates the location of the nucleus. (E and F) Regulation of TLR9 expression in whole myocardial tissue after stimulation with CpG-ODN. Expression of TLR9 mRNA was measured by real-time polymerase chain reaction (E) and Western blot analysis (F). Significant up-regulation of TLR9 mRNA was detected after 2 h in comparison to baseline expression and to hearts of TLR9-D mice (*P ≤ 0.05, n = 3). TLR9 protein was also up-regulated after 2 h of stimulation.

 
3.3 NF{kappa}B activation in the heart after CpG-ODN stimulation
Figure 2 illustrates the time course of myocardial NF{kappa}B-DNA binding activity following CpG-ODN stimulation. CpG-ODN treatment led to a substantial time-dependent activation (starting at 1 h and lasting up to 4 h) of myocardial NF{kappa}B in WT mice. This effect was not detectable in TLR9-D mice. Supershift assays revealed that the NF{kappa}B-complex mainly consists of p50 and p65.


Figure 2
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  		Figure 2 Myocardial NF{kappa}B activation measured by electrophoretic mobility shift assay is impaired in CpG-ODN-treated TLR9-D mice. Crude nuclear extracts were prepared from whole hearts of WT and TLR9-D mice at indicated time points after CpG-ODN administration. Results are representatives of three experiments. Supershift experiments for p50 and p65 as well as a cold chase (CC) experiment were performed on WT hearts 120 min after stimulation. Results are depicted in the last three lanes.

 
3.4 Myocardial cytokine mRNA and protein expression after CpG-ODN challenge
To determine the contribution of the TLR9 signalling pathway for CpG-induced myocardial cytokine gene expression RNase Protection Assays were performed. Figure 3A–D illustrates the time course of the cytokine mRNA expression of TNF-{alpha}, IL-1β, and IL-6 following CpG-ODN administration. CpG-ODN induced a rapid and robust increase in TNF-{alpha}, IL-1β, and IL-6 mRNA transcripts in hearts of WT mice. Peak cytokine expression was detected 1–2 h after injection of CpG-ODN. Densitometric analysis (Figure 3B–D) revealed a significant increase in the expression of the above mentioned cytokines in WT mice. This effect was not detectable in TLR9-D animals. In addition, the inflammatory response of various control oligonucleotide sequences (ODN 1612, ODN H154) was analysed indicating no significant up-regulation of cytokine gene mRNA in the heart (Table 1). To determine a direct relationship between increased mRNA expression and protein content in the heart, we carried out ELISAs. Figure 4 illustrates that CpG-ODN administration led to a significant increase in protein expression of TNF-{alpha}, IL-1β, and IL-6 in myocardial tissue from WT mice. A strong increase in cytokine production was observed after injection of CpG-ODN with a peak protein expression at 1 h for TNF-{alpha} (~5 x fold) and at 2 h for IL-1β (~5 xfold) and IL-6 (~7 x fold). This effect was not detectable in TLR9-D animals.


Figure 3
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  		Figure 3 Impaired CpG-ODN-induced myocardial TNF-{alpha}, IL-1β, and IL-6 mRNA in TLR9-D mice. (A) A representative autoradiograph of an RNase Protection Assay is shown. (B–D) Adjacent graphs illustrate relative TNF-{alpha}, IL-1β, and IL-6 mRNA levels. Data are expressed as TNF-{alpha}, IL-1β, or IL-6:L32 ratio; values represent mean ± SEM (*P ≤ 0.05, n = 5).

 


Figure 4
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  		Figure 4 Impaired CpG-ODN-induced myocardial TNF-{alpha}, IL-1β, and IL-6 protein expression in TLR9-D mice. Myocardial protein expression was measured by ELISA (*P ≤ 0.05, n = 5).

 


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  		Table 1 Influence of three different oligonucleotides (1668, H154, 1612) on cardiac mRNA of TNF-{alpha}, IL-6, and IL-1β detected 2, 4, and 6 h after injection into WT mice. The oligonucleotide 1668 caused an increase of all cytokine mRNA, whereas the other two oligonucleotides did not induce significant increases (*P < 0.05 compared with respective control values; n = 3–5/time point)

 
3.5 Plasma cytokine levels following systemic CpG-ODN challenge
CpG-ODN-treated WT animals showed a significant increase in the plasma levels of the cytokines TNF-{alpha} and IL-6 after 2 h. Plasma levels of IL-1β increased much less after 2 h without reaching statistical significance. These effects were not detectable in CpG-ODN-treated TLR9-D mice. After 6 h, cytokine levels in WT mice returned to baseline levels (Figure 5).


Figure 5
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  		Figure 5 Plasma cytokine levels of CpG-ODN-treated WT- and TLR9-D mice. Plasma levels of TNF-{alpha} and IL-6 were significantly increased after 2 h. Plasma levels of IL-1β were elevated after 2 h without reaching statistical significance (*P ≤ 0.05, n = 5).

 
3.6 Influence of CpG-ODN on sarcomere shortening
Sarcomere shortening of isolated ventricular cardiomyocytes from WT and TLR9-D mice was monitored in order to investigate whether CpG-ODN affects cardiomyocyte shortening and whether these changes are mediated via TLR9. Amplitude of sarcomere shortening from WT and TLR9-D cardiomyocytes depended on stimulation frequency. After a stimulation pause the first shortening is characterized by a high amplitude (post-rest shortening). Subsequently, the post-rest shortening is followed by a negative staircase at low frequencies (<6 Hz) and a positive staircase at high frequencies (>6 Hz) (Figure 6). This contractility pattern was similar in cardiomyocytes from WT and TLR9-D mice. Treatment of WT cells with CpG-ODN for >4 h depressed both post-rest and steady-state shortening. However, neither frequency-dependent shortening behaviour nor resting sarcomere length was influenced by CpG-ODN. A detailed analysis of the time course of CpG-ODN effects exhibits a tendency to decreased shortenings 3 h after the start of treatment and reaches the level of significance after 4 h (Figure 7A).


Figure 6
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  		Figure 6 Averaged original recordings of sarcomere shortening (peak values ± SD, n = 6). Shortenings of WT cardiomyocytes are shown at 1 and 10 Hz stimulation frequency, measured immediately after isolation (Tyrode solution) and after 6 h in culture medium substituted with 1 µg/mL CpG-ODN. After a stimulation pause of 30 s, a train of 20 pulses was applied and the resulting sarcomere shortenings are plotted as function of time.

 
In plots of steady-state shortening amplitude vs. stimulation frequency shortening amplitude exhibits a biphasic pattern with a negative shortening-frequency relationship <2 Hz and a positive relationship >2 Hz. At 2 Hz shortening was minimal (Figure 7B). This pattern was comparable in both WT and TLR9-D cells (Figure 7C). Six hours of cell culture did not change this pattern of contractility nor did it alter the shortening amplitudes (Figure 7B). Addition of CpG-ODN induced a significant decrease of shortening amplitude in WT cells at all frequencies (Figure 7B and D), whereas in TLR9-D cells shortening amplitude was insensitive to CpG-ODN treatment (Figure 7C). The speed of sarcomere shortening as well as re-lengthening showed the same frequency relationship as the sarcomere-shortening amplitude, and in WT cells CpG-ODN suppressed both parameters significantly (data not shown). In a second set of experiments the influence of iNOS inhibition on CpG-ODN-dependent suppression of myocyte shortening was tested (Figure 7D). In these experiments steady-state shortening amplitude was significantly reduced at all tested frequencies. Addition of SMT, a selective iNOS blocker, during the last hour of CpG-ODN treatment reversed the reduction of shortening completely.

3.7 Myocardial iNOS mRNA expression
We compared myocardial iNOS mRNA expression after CpG-ODN challenge in WT and TLR9-D mice. CpG-ODN application caused a pronounced induction of iNOS mRNA expression in WT compared with TLR9-D mice (Figure 7E and F).


Figure 7
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  		Figure 7 Influence of CpG-ODN on sarcomere shortening amplitude. (A) Time course of CpG-ODN effect on sarcomere shortening recorded at 2 Hz stimulation frequency under control conditions (0 h) and after 1, 2, 3, 4, 5, and 6 h of CpG-ODN application. Compared with control cells CpG-ODN decreased sarcomere shortening significantly after 4 h stimulation (*P ≤ 0.05, n = 15–21). (B–D) Averaged sarcomere shortening amplitudes of cardiomyocytes of WT (B and D) and TLR9-D (C) mice plotted vs. the stimulation frequency. WT cells were recorded in Tyrode (WT Tyrode) immediately after isolation and after 6 h of cell culture in the absence (WT CM) or presence of 1 µg/mL CpG-ODN (WT CM+CpG; n = 19–71). The shortening amplitudes of cardiomyocytes from WT mice were significantly reduced by CpG-ODN (B and D), whereas they did not change in cells from TLR9-D mice (TLR9-D CM vs. TLR9-D CM +CpG) (C). The presence of the iNOS inhibitor SMT (100 µM) prevented the 1 µg/mL CpG-ODN-mediated effects (D) (P ≤ 0.05; *WT Tyrode vs. WT CM+CPG, # WT CM+CpG vs. WT CM+CpG+SMT; n = 11–51). Myocardial iNOS mRNA expression 4 h after stimulation (E and F). (E) RT polymerase chain reaction of iNOS mRNA expression from WT and TLR9-D mice after CpG-ODN administration. (F) iNOS mRNA levels normalized to β-actin (*P ≤ 0.05, n = 4).

 

   4. DISCUSSION
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. DISCUSSION
Funding
 References
 
Cardiac depression is a serious and frequent complication of human sepsis. Several mechanisms have been postulated for the impaired myocardial function during sepsis including accumulation of (i) bacterial wall fragments like LPS,42 (ii) various cytokines such as IL-1β and TNF-{alpha}9, and (iii) reactive oxygen/nitrogen species.43,44 Altered NO production within the heart is believed to participate in the pathogenesis of heart failure originated from sepsis.11 There is good evidence that the endocrine, immune, and nervous system are in concert to depress cardiac contractility under inflammatory conditions. This has led to the new concept that innate immunity, i.e. the TLR system may play an important role in this scenario. We have previously shown that TLR4- as well as CD14-deficiency abolishes LPS-induced contractile dysfunction in vivo.15,16,18 In a similar experimental setting we could also demonstrate that Staphylococcus aureus mediates cardiac dysfunction via TLR2.19

Bacterial DNA (CpG-ODN) can initiate an innate immune response via TLR9 potentially leading to septic shock.22,23 TLR9 is localized in the endoplasmatic reticulum and following CpG stimulation recruited to endosomal vesicles.45,46 Then, TLR9 and CpG-ODN co-localize resulting in cell activation. Ligand-binding studies showed that bacterial DNA binds directly to TLR9. Following CpG-ODN binding, TLR9 associates with the adaptor molecule MyD88 resulting in activation of the IL-1 receptor-associated kinase family and mitogen-activated protein kinases. The latter events activate NF{kappa}B among other transcription factors (for detailed review please ref. 47).

Little is known about the role of TLR9 in the myocardium, but constitutive TLR9 expression levels have been detected in the heart.29,30,48 Our study demonstrates a TLR9-dependent mechanism of cardiac inflammation in vivo (i.p. application 1 nmol/g BW), which was absent in TLR9-D animals. After incubation with 1 µg/mL CpG-ODN, cardiomyocyte contractility in vitro was reduced in WT- and unaffected in TLR9-D cells. Despite the fact that various papers suggest similar in vivo and in vitro doses for CpG-ODN, it is unclear whether these concentrations correlate with the clinical setting in vivo. Preliminary data shown in the methods section demonstrate that 20 nmol in vivo is a saturating dose on the cytokine expression in murine pulmonary tissue. So far, levels of CpG-ODN in tissue have not been reported in the literature. In the clinical setting of sepsis this question is even harder to answer since the amplification of short fragments derived from bacterial DNA is technically difficult. At this point amplification of bacterial products is used to identify different bacteria, but we are not aware of an experimental approach quantifying bacteria or bacterial products. Our data are in accordance with others that TLR9 mRNA is expressed in homogenates of cardiac tissue.29,30,34,48,49 Furthermore, we demonstrated intracellular as well as nuclear incorporation of fluorescence-labelled 1668-ODN-Cy5 into isolated cardiomyocytes of both genotypes within 30 min of treatment indicating that CpG-ODN is internalized to interact with the receptor. InternaIization of ODNs seems to be independent of the presence of TLR9 as well as independent of the presence of the CG motif. This observation is consistent with previous reports indicating that CpG-ODNs appear to be sequence-independently endocytosed into glioblastoma cells.50 However, uptake of 1668-ODN-Cy5 in TLR9-D cells was slower. In addition, CpG-ODN led to a significant time dependent up-regulation of TLR9 mRNA and protein in WT mice (Figure 1E and F).

It is known that TLR9 stimulation leads to the activation of NF{kappa}B in various tissues.47,51 However, we show for the first time that also cardiac NF{kappa}B activity is up-regulated following CpG-ODN application. Constitutive expression of TLR9 seems to be responsible for NF{kappa}B activation and cytokine induction as well as the increase in TLR9 expression (at 2 h). TLR9 up-regulation may not lead to further NF{kappa}B activation/cytokine induction because the injected CpG-ODN may already have been degraded after 2 h.

In WT mice, CpG-ODN led to a significant increase of TNF-{alpha}, IL-1β, and IL-6 mRNA and protein expression in the heart. TNF-{alpha} mRNA did not clearly precede the respective protein expression. This observation may be related to the low time resolution of 1 h. However, in case of IL-1β and IL-6 mRNA expression rises earlier than protein expression. Furthermore, increased plasma levels of TNF-{alpha}, IL-1β, and IL-6 indicate systemic inflammation in WT animals. In contrast, the level of these cytokines did not change in TLR9-D mice after CpG challenge.

In our study cardiac contractility measured as sarcomere shortening of isolated cardiomyocytes from WT- and TLR9-D mice was recorded at stimulation frequencies between 0.5 and 10 Hz after incubation with 1 µg/mL CpG-ODN for up to 6 h. Treatment for >4 h depressed both post-rest and steady-state shortening. Detailed analysis of the time course of CpG-ODN effects exhibited tendency to decreased shortenings 3 h after the start of treatment reaching statistical significance after 4 h. As iNOS levels and cardiomyocyte contractility are linked, we investigated whether iNOS was regulated via TLR9 in vivo. CpG-ODN application caused a pronounced induction of iNOS mRNA in WT compared with TLR9-D mice (Figure 7E and i). Inhibition of iNOS with SMT prevented CpG-ODN-mediated effects on shortening in the current study, and this suggests that iNOS and NO are relevant members of the TLR9 signalling cascade within cardiomyocytes.

A study by Paladugu et al.52 reported that bacterial DNA (from S. aureus and Escherichia coli) depressed myocardial contraction in rat neonatal cardiomyocytes in a concentration-dependent fashion, ranging from 43–61%. Since pretreatment with DNase led to abrogation of depression of myocyte contraction, the observed myocyte contraction depression was attributed to the ligand itself and not potential contaminating proteins. On one hand, this study supports our data indicating that bacterial DNA has the potential to depress cardiac contractility. On the other hand, these authors have not examined whether this effect was mediated by a specific receptor, particularly by TLR9. Furthermore, it is unclear whether the TLR9 expression pattern might change from neonatal to adult cells. Therefore, our data demonstrate for the first time that bacterial DNA depresses cardiac contractility via myocardial TLR9.

Whether other TLR ligands (e.g. LPS) directly act on cardiomyocytes or function via the presence of leukocytes remains controversial.17,53 Since contractility studies were performed in pure cardiomyocyte cultures, our results indicate that this effect can be attributed to cardiomyocytes alone and that cardiomyocytes might have the potential to recognize pathogenic ligands via TLRs. In addition, cardiomyocytes have the capability to produce inflammatory and cardiac depressive mediators, including TNF-{alpha} and NO supporting the fact that ligand-mediated effects on sarcomere shortening occur in absence of immune cells.8,54

Recently, Boyd et al.30 reported that cognate ligands of TLR2, 4, and 5 induced mediator production in the conditioned media of a murine cardiomyocyte cell line (HL-1 cells) 24 h after stimulation. The fact that TLR2, 4, and 5 activation can lead to cytokine induction in the heart has been reported before, generally documenting cytokine production within 2–6 h after stimulation using the respective pathogenic ligand.15,17,19,20 In contrast to our study, Boyd et al.30 reported that stimulation with CpG-ODN for 24 h did not lead to cytokine (IL-6) and chemokine (KC, MIP-2) production. These differences might be due to the HL-1 cell line itself or to the long duration of stimulation (24 h). In addition, the most important mediators for cardiac depression during sepsis (TNF-{alpha}, IL-1β) were not determined. Figures 3 and 4 indicate that CpG-ODN induces maximal cytokine production within 1–2 h of stimulation. This is in context with our observation that WT mice showed clinical symptoms of inflammation 2 h following CpG-ODN stimulation.

We propose that bacterial DNA causes myocardial dysfunction. The fact that spontaneous or drug-induced bacterial degradation eventually leads to the liberation of bacterial DNA has not been taken into consideration yet in respect to ligand-induced organ dysfunction. Furthermore, our results demonstrate that the presence of immune cells is not essential for TLR9-mediated myocardial depression.


   Funding
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. DISCUSSION
 Funding
References
 
This work was supported in part by BonFor (to P.K.), the Deutsche Forschungsgemeinschaft (KN 521/2-1 to P.K.; BN 1726/2-1 to G.B; ZA 243/9-1 to K.Z.), and the Deutsche Stiftung für Herzforschung (F/16/03 to P.K. and R.M.).


   Acknowledgements
 
The authors thank Shizuo Akira, Department of Host Defense, Research Institute for Microbial Diseases, Osaka University, Japan for kindly providing the TLR9-deficient mice. The authors thank Patrik Efferz and Dirk Böker for expert technical assistance.

Conflict of interest: none declared.


   Notes
 
{dagger}These two authors contributed equally to this work. Back


   References
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. DISCUSSION
 Funding
 References
 

  1. Wheeler AP, Bernard GR. Treating Patients with Severe Sepsis. N Engl J Med (1999) 340:207–215.[Free Full Text]
  2. Rangel-Frausto MS, Pittet D, Costigan M, Hwang T, Davis CS, Wenzel RP. The natural history of the systemic inflammatory response syndrome (SIRS). A prospective study. Jama (1995) 273:117–123.[Abstract/Free Full Text]
  3. Martin GS, Mannino DM, Eaton S, Moss M. The epidemiology of sepsis in the United States from 1979 through 2000. N Engl J Med (2003) 348:1546–1554.[Abstract/Free Full Text]
  4. Brunkhorst FM. Epidemiology, economy and practice—results of the German Study on Prevalence by the Competence Network Sepsis (SepNet). Anasthesiol Intensivmed Notfallmed Schmerzther (2006) 41:43–44.[CrossRef][Medline]
  5. Parker MM, Shelhamer JH, Bacharach SL, Green MV, Natanson C, Frederick TM, et al. Profound but reversible myocardial depression in patients with septic shock. Ann Intern Med (1984) 100:483–490.[Abstract/Free Full Text]
  6. Parrillo JE, Parker MM, Natanson C, Suffredini AF, Danner RL, Cunnion RE, et al. Septic shock in humans. Advances in the understanding of pathogenesis, cardiovascular dysfunction, and therapy. Ann Intern Med (1990) 113:227–242.[Abstract/Free Full Text]
  7. Giroir BP, Johnson JH, Brown T, Allen GL, Beutler B. The tissue distribution of tumor necrosis factor biosynthesis during endotoxemia. J Clin Invest (1992) 90:693–698.[Web of Science][Medline]
  8. Kapadia S, Lee J, Torre-Amione G, Birdsall HH, Ma TS, Mann DL. Tumor necrosis factor-alpha gene and protein expression in adult feline myocardium after endotoxin administration. J Clin Invest (1995) 96:1042–1052.[Web of Science][Medline]
  9. Kumar A, Thota V, Dee L, Olson J, Uretz E, Parrillo JE. Tumor necrosis factor alpha and interleukin 1beta are responsible for in vitro myocardial cell depression induced by human septic shock serum. J Exp Med (1996) 183:949–958.[Abstract/Free Full Text]
  10. Cain BS, Meldrum DR, Dinarello CA, Meng X, Joo KS, Banerjee A, et al. Tumor necrosis factor-alpha and interleukin-1beta synergistically depress human myocardial function. Crit Care Med (1999) 27:1309–1318.[CrossRef][Web of Science][Medline]
  11. Ullrich R, Scherrer-Crosbie M, Bloch KD, Ichinose F, Nakajima H, Picard MH, et al. Congenital deficiency of nitric oxide synthase 2 protects against endotoxin-induced myocardial dysfunction in mice. Circulation (2000) 102:1440–1446.[Abstract/Free Full Text]
  12. Kopp EB, Medzhitov R. The toll-receptor family and control of innate immunity. Curr Opin Immunol (1999) 11:13–18.[CrossRef][Web of Science][Medline]
  13. Kawai T, Akira S. TLR signaling. Cell Death Differ (2006) 13:816–825.[CrossRef][Web of Science][Medline]
  14. Akira S, Uematsu S, Takeuchi O. Pathogen recognition and innate immunity. Cell (2006) 124:783–801.[CrossRef][Web of Science][Medline]
  15. Baumgarten G, Knuefermann P, Nozaki N, Sivasubramanian N, Mann DL, Vallejo JG. In vivo expression of proinflammatory mediators in the adult heart after endotoxin administration: the role of toll-like receptor-4. J Infect Dis (2001) 183:1617–1624.[CrossRef][Web of Science][Medline]
  16. Knuefermann P, Nemoto S, Misra A, Nozaki N, Defreitas G, Goyert SM, et al. CD14-deficient mice are protected against lipopolysaccharide-nduced cardiac inflammation and left ventricular dysfunction. Circulation (2002) 106:2608–2615.[Abstract/Free Full Text]
  17. Baumgarten G, Knuefermann P, Schuhmacher G, Vervolgyi V, von Rappard J, Dreiner U, et al. Toll-like receptor 4, nitric oxide, and myocardial depression in endotoxemia. Shock (2006) 25:43–49.[Medline]
  18. Nemoto S, Vallejo JG, Knuefermann P, Misra A, Defreitas G, Carabello BA, et al. Escherichia coli LPS-induced LV dysfunction: role of toll-like receptor-4 in the adult heart. Am J Physiol (2002) 282:H2316–H2323.[Web of Science]
  19. Knuefermann P, Sakata Y, Baker JS, Huang CH, Sekiguchi K, Hardarson HS, et al. Toll-like receptor 2 mediates Staphylococcus aureus-induced myocardial dysfunction and cytokine production in the heart. Circulation (2004) 110:3693–3698.[Abstract/Free Full Text]
  20. Eaves-Pyles T, Murthy K, Liaudet L, Virag L, Ross G, Soriano FG, et al. Flagellin, a novel mediator of Salmonella-induced epithelial activation and systemic inflammation: I kappa B alpha degradation, induction of nitric oxide synthase, induction of proinflammatory mediators, and cardiovascular dysfunction. J Immunol (2001) 166:1248–1260.[Abstract/Free Full Text]
  21. Linde A, Mosier D, Blecha F, Melgarejo T. Innate immunity and inflammation–new frontiers in comparative cardiovascular pathology. Cardiovasc Res (2007) 73:26–36.[Abstract/Free Full Text]
  22. Sparwasser T, Miethke T, Lipford G, Borschert K, Hacker H, Heeg K, et al. Bacterial DNA causes septic shock. Nature (1997) 386:336–337.[CrossRef][Medline]
  23. Sparwasser T, Miethke T, Lipford G, Erdmann A, Hacker H, Heeg K, et al. Macrophages sense pathogens via DNA motifs: induction of tumor necrosis factor-alpha-mediated shock. Eur J Immunol (1997) 27:1671–1679.[Web of Science][Medline]
  24. Deng GM, Nilsson IM, Verdrengh M, Collins LV, Tarkowski A. Intra-articularly localized bacterial DNA containing CpG motifs induces arthritis. Nat Med (1999) 5:702–705.[CrossRef][Web of Science][Medline]
  25. Deng GM, Liu ZQ, Tarkowski A. Intracisternally localized bacterial DNA containing CpG motifs induces meningitis. J Immunol (2001) 167:4616–4626.[Abstract/Free Full Text]
  26. Krieg AM, Yi AK, Matson S, Waldschmidt TJ, Bishop GA, Teasdale R, et al. CpG motifs in bacterial DNA trigger direct B-cell activation. Nature (1995) 374:546–549.[CrossRef][Medline]
  27. Hemmi H, Takeuchi O, Kawai T, Kaisho T, Sato S, Sanjo H, et al. A toll-like receptor recognizes bacterial DNA. Nature (2000) 408:740–745.[CrossRef][Medline]
  28. Bauer S, Kirschning CJ, Hacker H, Redecke V, Hausmann S, Akira S, et al. Human TLR9 confers responsiveness to bacterial DNA via species-specific CpG motif recognition. Proc Natl Acad Sci USA (2001) 98:9237–9242.[Abstract/Free Full Text]
  29. Rodriguez-Martinez S, Cancino-Diaz ME, Jimenez-Zamudio L, Garcia-Latorre E, Cancino-Diaz JC. TLRs and NODs mRNA expression pattern in healthy mouse eye. Br J Ophthalmol (2005) 89:904–910.[Abstract/Free Full Text]
  30. Boyd JH, Mathur S, Wang Y, Bateman RM, Walley KR. Toll-like receptor stimulation in cardiomyoctes decreases contractility and initiates an NF-kappaB dependent inflammatory response. Cardiovasc Res (2006) 72:384–393.[Abstract/Free Full Text]
  31. Hacker H, Vabulas RM, Takeuchi O, Hoshino K, Akira S, Wagner H. Immune cell activation by bacterial CpG-DNA through myeloid differentiation marker 88 and tumor necrosis factor receptor-associated factor (TRAF)6. J Exp Med (2000) 192:595–600.[Abstract/Free Full Text]
  32. Kranzer K, Bauer M, Lipford GB, Heeg K, Wagner H, Lang R. CpG-oligodeoxynucleotides enhance T-cell receptor-triggered interferon-gamma production and up-regulation of CD69 via induction of antigen-presenting cell-derived interferon type I and interleukin-12. Immunology (2000) 99:170–178.[CrossRef][Web of Science][Medline]
  33. Bartz H, Avalos NM, Baetz A, Heeg K, Dalpke AH. Involvement of suppressors of cytokine signaling in toll-like receptor-mediated block of dendritic cell differentiation. Blood (2006) 108:4102–4108.[Abstract/Free Full Text]
  34. Yamada H, Ishii KJ, Klinman DM. Suppressive oligodeoxynucleotides inhibit CpG-induced inflammation of the mouse lung. Crit Care Med (2004) 32:2045–2049.[CrossRef][Medline]
  35. Chomczynski P, Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate- phenol-chloroform extraction. Anal Biochem (1987) 162:156–159.[Web of Science][Medline]
  36. Zacharowski K, Zacharowski PA, Koch A, Baban A, Tran N, Berkels R, et al. Toll-like receptor 4 plays a crucial role in the immune-adrenal response to systemic inflammatory response syndrome. Proc Natl Acad Sci USA (2006) 103:6392–6397.[Abstract/Free Full Text]
  37. Knuefermann P, Chen P, Misra A, Shi SP, Abdellatif M, Sivasubramanian N. Myotrophin/V-1, a protein up-regulated in the failing human heart and in postnatal cerebellum, converts NF{kappa}B p50-p65 heterodimers to p50-p50 and p65-p65 homodimers. J Biol Chem (2002) 277:23888–23897.[Abstract/Free Full Text]
  38. Tiemann K, Weyer D, Djoufack PC, Ghanem A, Lewalter T, Dreiner U, et al. Increasing myocardial contraction and blood pressure in C57BL/6 mice during early postnatal development. Am J Physiol (2003) 284:H464–H474.[Web of Science]
  39. Jahrsdorfer B, Muhlenhoff L, Blackwell SE, Wagner M, Poeck H, Hartmann E, et al. B-cell lymphomas differ in their responsiveness to CpG oligodeoxynucleotides. Clin Cancer Res (2005) 11:1490–1499.[Abstract/Free Full Text]
  40. Kerkmann M, Costa LT, Richter C, Rothenfusser S, Battiany J, Hornung V, et al. Spontaneous formation of nucleic acid-based nanoparticles is responsible for high interferon-alpha induction by CpG-A in plasmacytoid dendritic cells. J Biol Chem (2005) 280:8086–8093.[Abstract/Free Full Text]
  41. Wagner M, Poeck H, Jahrsdoerfer B, Rothenfusser S, Prell D, Bohle B, et al. IL-12p70-dependent Th1 induction by human B cells requires combined activation with CD40 ligand and CpG DNA. J Immunol (2004) 172:954–963.[Abstract/Free Full Text]
  42. Natanson C, Eichenholz PW, Danner RL, Eichacker PQ, Hoffman WD, Kuo GC, et al. Endotoxin and tumor necrosis factor challenges in dogs simulate the cardiovascular profile of human septic shock. J Exp Med (1989) 169:823–832.[Abstract/Free Full Text]
  43. Pattanaik U, Prasad K. Reactive oxygen species and endotoxic shock: effect of dimethylthiourea. J Cardiovasc Pharmacol Ther (2001) 6:273–285.[Abstract/Free Full Text]
  44. Khadour FH, Panas D, Ferdinandy P, Schulze C, Csont T, Lalu MM, et al. Enhanced NO and superoxide generation in dysfunctional hearts from endotoxemic rats. Am J Physiol (2002) H1108–H1115.
  45. Latz E, Schoenemeyer A, Visintin A, Fitzgerald KA, Monks BG, Knetter CF, et al. TLR9 signals after translocating from the ER to CpG DNA in the lysosome. Nat Immunol (2004) 5:190–198.[CrossRef][Web of Science][Medline]
  46. Leifer CA, Kennedy MN, Mazzoni A, Lee C, Kruhlak MJ, Segal DM. TLR9 is localized in the endoplasmic reticulum prior to stimulation. J Immunol (2004) 173:1179–1183.[Abstract/Free Full Text]
  47. Vollmer J. TLR9 in health and disease. Int Rev Immunol (2006) 25:155–181.[CrossRef][Medline]
  48. Zarember KA, Godowski PJ. Tissue expression of human toll-like receptors and differential regulation of toll-like receptor mRNAs in leukocytes in response to microbes, their products, and cytokines. J Immunol (2002) 168:554–561.[Abstract/Free Full Text]
  49. Droemann D, Albrecht D, Gerdes J, Ulmer AJ, Branscheid D, Vollmer E, et al. Human lung cancer cells express functionally active toll-like receptor 9. Respir Res (2005) 6:1–6.[CrossRef][Medline]
  50. Zhang Z, Weinschenk T, Schluesener HJ. Uptake, cellular distribution and novel cellular binding proteins of immunostimulatory CpG oligodeoxynucleotides in glioblastoma cells. Mol Cell Biochem (2005) 272:35–46.[CrossRef][Medline]
  51. Yeo SJ, Yoon JG, Hong SC, Yi AK. CpG DNA induces self and cross-hyporesponsiveness of RAW264.7 cells in response to CpG DNA and lipopolysaccharide: alterations in IL-1 receptor-associated kinase expression. J Immunol (2003) 170:1052–1061.[Abstract/Free Full Text]
  52. Paladugu B, Kumar A, Parrillo JE, Der S, Osman J, Mensing J, et al. Bacterial DNA and RNA induce rat cardiac myocyte contraction depression in vitro. Shock (2004) 21:364–369.[CrossRef][Medline]
  53. Tavener SA, Long EM, Robbins SM, McRae KM, Van Remmen H, Kubes P. Immune cell toll-like receptor 4 is required for cardiac myocyte impairment during endotoxemia. Circ Res (2004) 95:700–707.[Abstract/Free Full Text]
  54. Feldman AM, Combes A, Wagner D, Kadakomi T, Kubota T, Li YY, et al. The role of tumor necrosis factor in the pathophysiology of heart failure. J Am Coll Cardiol (2000) 35:537–544.[Abstract/Free Full Text]

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