Olaf Boehm

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

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)-1beta, IL-6, inducible nitric oxide synthase (iNOS), and nuclear factor kappaB 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.

Clinical chemistry reference database for Wistar rats and C57/BL6 mice.

Abstract Clinical chemistry data are decisive for evaluating altered organ function or damage in experimental animals. Few publications provide reliable clinical chemistry reference intervals, and analytical methods are often not described. Here, we investigated common clinical chemistry values in adult male and female Wistar rats and C57/BL6 mice (n=30/group). Blood samples were taken and analysed for electrolytes, substrates, metabolites and enzymes. In addition, we investigated cystatin C, an important marker of glomerular dysfunction. All data were obtained using commercially available kits frequently employed in most clinical chemistry laboratories and compared with data from other studies, as well as with human data. Significant gender-specific differences were observed in rats (electrolytes, retention parameters and transaminases) and in mice (cholesterol, glucose). High variability was noted for sodium, potassium, glucose, creatine kinase, lactate dehydrogenase and transaminase levels. Both rodent species showed markedly higher α-amylase activity than humans. This report demonstrates significant differences between genders for many analytes in rats and for fewer parameters in mice. Some reference values displayed major discrepancies between rodents and humans.

The fibrin-derived peptide Bβ15- 42 is cardioprotective in a pig model of myocardial ischemia-reperfusion injury

Objective:The fibrin-derived peptide B&bgr;15–42 has been shown to reduce infarct size in rodent models of ischemia-reperfusion injury. To increase its potential for translation into the clinic, we studied the effects of B&bgr;15–42 in pigs, whose coronary anatomy is similar to that of humans. In addition, we evaluated the pharmacokinetics and safety of B&bgr;15–42 in several species, including humans. Design:Animal study and phase I trial. Setting:University hospital and contract research laboratories. Subjects:Pigs/healthy volunteers. Interventions:Male farm-bred Landrace pigs were subjected to 1 hr of left anterior descending coronary artery occlusion followed by 3 hrs of reperfusion. At the time of reperfusion, B&bgr;15–42 (2.4 mg/kg, n = 6) or random peptide (control; 2.4 mg/kg, n = 6) was administered as an intravenous bolus. As a positive control, pigs were subjected to ischemic preconditioning (n = 6). Cardiac damage and hemodynamics were recorded. Biodistribution and pharmacokinetics of B&bgr;15–42 were determined in rats and dogs. In a phase I trial involving 30 male healthy volunteers, pharmacokinetics and safety were tested in a randomized, double-blinded, placebo-controlled, parallel-group, single ascending dose study. Measurements and Main Results:B&bgr;15–42 and ischemic preconditioning significantly reduced myocardial infarct size and troponin I levels. B&bgr;15–42 also reduces interleukin-6 levels, underlining its anti-inflammatory properties. Furthermore, in humans, the pharmacokinetics of the peptide B&bgr;15–42 were comparable to those of animals, and no serious adverse effects were observed. Conclusions:B&bgr;15–42 elicits cardioprotection in pigs and is clinically safe in phase I testing of humans. This study confirms the new concept of a pathogenic role of fibrin derivatives in myocardial reperfusion injury, which can be inhibited by peptide B&bgr;15–42.

Preconditioning by toll-like receptor 2 agonist Pam3CSK4 reduces CXCL1-dependent leukocyte recruitment in murine myocardial ischemia/reperfusion injury

Objective:To test whether preconditioning with a toll-like receptor (TLR) 2 agonist protects against myocardial ischemia and reperfusion by interfering with chemokine CXCL1 release from cardiomyocytes. Design:C3H mice were challenged with vehicle or synthetic TLR2 agonist Pam3Cys-Ser-Lys4 (Pam3CSK4; 1 mg/kg) 24 hrs before myocardial ischemia (20 mins) and reperfusion (2 hrs or 24 hrs). Infarct size, troponin T release, and leukocyte recruitment were quantified. In murine cardiomyocytes (HL-1), we studied the expression/activation profile of TLR2 in response to stimulation with Pam3CSK4 (0.01–1 mg/mL). Furthermore, we studied the chemokine ligand 1 (CXCL1) response to Pam3CSK4 and ischemia/reperfusion in vivo and in vitro. Setting:University hospital research laboratory. Subjects:Anesthetized male mice and murine cardiomyocytes. Measurements and Main Results:Preconditioning by Pam3CSK4 reduced infarct size and troponin T release. This was accompanied by a decreased recruitment of leukocytes into the ischemic area and an improved cardiac function. In HL-1 cells, TLR2 activation amplified the expression of the receptor in a time-dependent manner and led to CXCL1 release in a concentration-dependent manner. Preconditioning by Pam3CSK4 impaired CXCL1 release in response to a second inflammatory stimulus in vivo and in vitro. Conclusions:Preconditioning by TLR2 agonist Pam3CSK4 reduces myocardial infarct size after myocardial ischemia/reperfusion. One of the mechanisms involved is a diminished chemokine release from cardiomyocytes, which subsequently limits leukocyte infiltration.

The toll-like receptor 4-antagonist eritoran reduces murine cardiac hypertrophy.

Toll‐like receptor 4 (TLR4) recognizes lipopolysaccharides and endogenous ligands released after organ injury. Deficiency of TLR4 attenuates the development of left ventricular hypertrophy after transverse aortic constriction (TAC) in mice. We hypothesized that application of the TLR4 antagonist eritoran may also reduce cardiac hypertrophy after TAC surgery.

Priming with Synthetic Oligonucleotides Attenuates Pressure Overload-Induced Inflammation and Cardiac Hypertrophy in Mice

AIMS Inflammation and Toll-like receptor (TLR) signalling have been linked to the development of cardiac hypertrophy following transverse aortic constriction (TAC). In the present study, we investigated whether pre-treatment with the synthetic TLR9 ligands 1668-thioate or 1612-thioate modulates the progression of TAC-induced cardiac inflammation and hypertrophy. METHODS AND RESULTS C57BL/6N-mice were pre-treated with 1668-thioate, 1612-thioate (0.25 nmol/g, i.p.), or phosphate-buffered saline 16 h prior to TAC or sham surgery. Heart-weight/body-weight ratio (HW/BW), cardiomyocyte cell size, cellular macrophage accumulation, myofibroblast differentiation, and collagen deposition were investigated for up to 28 days. Cardiac function was monitored using a pressure-volume catheter and M-mode echocardiography. Inflammatory gene expression in the heart was analysed via gene array, while the time course of mRNA expression of key inflammatory mediators was assessed via RT-qPCR. TAC increased the HW/BW ratio and cardiomyocyte cell size and induced macrophage accumulation, myofibroblast differentiation, and collagen deposition. These changes were accompanied by cardiac inflammation and a significant loss of left ventricular function. Pre-treatment with cytosine-phosphate-guanine (CpG)-containing 1668-thioate attenuated the inflammatory response, the progression of cardiac hypertrophy, and cardiac remodelling, which resulted in a prolonged preservation of left ventricular function. These changes were induced to a smaller extent by the use of the non-CG-containing oligodeoxynucleotide 1612-thioate. CONCLUSION Pre-treatment with 1668-thioate attenuated cardiac hypertrophy following pressure overload, possibly by modifying the hypertrophy-induced inflammatory response, thereby reducing cardiac growth and fibrosis as well as delaying loss of cardiac function.

The effects of the fibrin-derived peptide Bβ15-42 in acute and chronic rodent models of myocardial ischemia-reperfusion

ABSTRACT Many compounds have been shown to prevent reperfusion injury in various animal models, although to date, translation into clinic has revealed several obstacles. Therefore, the National Heart, Lung, and Blood Institute convened a working group to discuss reasons for such failure. As a result, the concept of adequately powered, blinded, randomized studies for preclinical development of a compound has been urged. We investigated the effects of a fibrin-derived peptide B&bgr;15-42 in acute and chronic rodent models of ischemia-reperfusion at three different study centers (Universities of Dusseldorf and Vienna, TNO Biomedical Research). A total of 187 animals were used, and the peptide was compared with the free radical scavenger Tempol, CD18 antibody, &agr;-C5 antibody, and the golden standard, ischemic preconditioning. We show that B&bgr;15-42 robustly and reproducibly reduced infarct size in all models of ischemia-reperfusion. Moreover, the peptide significantly reduced plasma levels of the cytokines interleukin 1&bgr;, tumor necrosis factor &agr;, and interleukin 6. In rodents, B&bgr;15-42 inhibits proinflammatory cytokine release and is cardioprotective during ischemia-reperfusion injury.

Ly6C(low) and not Ly6C(high) macrophages accumulate first in the heart in a model of murine pressure-overload.

Cardiac tissue remodeling in the course of chronic left ventricular hypertrophy requires phagocytes which degrade cellular debris, initiate and maintain tissue inflammation and reorganization. The dynamics of phagocytes in left ventricular hypertrophy have not been systematically studied. Here, we characterized the temporal accumulation of leukocytes in the cardiac immune response by flow cytometry and fluorescence microscopy at day 3, 6 and 21 following transverse aortic constriction (TAC). Cardiac hypertrophy due to chronic pressure overload causes cardiac immune response and inflammation represented by an increase of immune cells at all three time points among which neutrophils reached their maximum at day 3 and macrophages at day 6. The cardiac macrophage population consisted of both Ly6Clow and Ly6Chigh macrophages. Ly6Clow macrophages were more abundant peaking at day 6 in response to pressure overload. During the development of cardiac hypertrophy the expression pattern of adhesion molecules was investigated by qRT-PCR and flow cytometry. CD11b, CX3CR1 and ICAM-1 determined by qRT-PCR in whole cardiac tissue were up-regulated in response to pressure overload at day 3 and 6. CD11b and CX3CR1 were significantly increased by TAC on the surface of Ly6Clow but not on Ly6Chigh macrophages. Furthermore, ICAM-1 was up-regulated on cardiac endothelial cells. In fluorescence microscopy Ly6Clow macrophages could be observed attached to the intra- and extra-vascular vessel-wall. Taken together, TAC induced the expression of adhesion molecules, which may explain the accumulation of Ly6Clow macrophages in the cardiac tissue, where these cells might contribute to cardiac inflammation and remodeling in response to pressure overload.

IN VIVO TOLL-LIKE RECEPTOR 4 ANTAGONISM RESTORES CARDIAC FUNCTION DURING ENDOTOXEMIA

ABSTRACT Severe sepsis and septic shock are often accompanied by acute cardiovascular depression. Lipopolysaccharide (LPS) signaling via Toll-like receptor 4 (TLR4) can induce septic organ dysfunction. The aim of this study was to elucidate the in vivo impact of pharmacological TLR4 antagonism on LPS-induced cardiovascular depression using eritoran tetrasodium (E5564). To simulate sepsis, C3H/HeN mice were challenged i.p. with 2 mg/kg body weight LPS. With the intent to antagonize the LPS effects, eritoran was administered i.v. (4 mg/kg body weight). Physical activity, peripheral blood pressure, and heart frequency were recorded before and after LPS and eritoran injection. In addition, intracardiac hemodynamic parameters were analyzed with a pressure conductance catheter. After 2 and 6 h of LPS stimulation ± eritoran treatment, the hearts and aortae were harvested, and TLR as well as inflammatory mediator expression was measured using reverse transcription–quantitative polymerase chain reaction and enzyme-linked immunosorbent assay. Lipopolysaccharide significantly decreased arterial blood pressure over time. Administration of eritoran partially prevented the LPS-dependent reduction in blood pressure and preserved cardiac function. In addition, LPS increased the expression of CD14 and TLR2 in cardiac and aortic tissue. In aortic tissue, eritoran attenuated this increase, whereas no significant reduction was observed in the heart. Furthermore, cardiac and aortic inducible nitric oxide synthetase mRNA levels were significantly increased 6 h after LPS application. This effect was reduced in the presence of eritoran. In summary, the beneficial influence of eritoran on cardiovascular function in vivo seems to rely mainly on reduction of LPS-induced inducible nitric oxide synthetase expression as well as on attenuated cytokine expression in the vascular wall.

Antibiotics regulate the immune response in both presence and absence of lipopolysaccharide through modulation of Toll-like receptors, cytokine production and phagocytosis in vitro

The inflammatory response to pathogen-associated molecular patterns such as lipopolysaccharide (LPS) in sepsis is mediated via Toll-like receptors (TLRs). Since TLRs also trigger various immune functions, including phagocytosis, their modulation is a promising strategy in the treatment of sepsis. As antibiotics have immunomodulatory properties, this study examined the effect of commonly used classes of antibiotics on i) the expression of TLRs and cytokines and ii) the phagocytic activity under sepsis-like conditions in vitro. This was achieved by incubating THP-1 monocytes and peripheral blood mononuclear cells (PBMCs) obtained from patients after open-heart surgery with the addition of LPS and six key antibiotics (piperacillin, doxycycline, erythromycin, moxifloxacin or gentamicin). After 24h, mRNA levels of both cytokines (IL-1β, IL-6) and TLRs (1, 2, 4, and 6) were monitored and phagocytosis was determined following coincubation with Escherichia coli. Each antibiotic differentially regulated the gene expression of the investigated TLRs and cytokines in monocytes. Erythromycin, moxifloxacin and doxycyclin displayed the strongest effects and changed mRNA-levels of the investigated genes up to 5.6-fold. Consistent with this, antibiotics and, in particular, moxifloxacin, regulated the TLR-and cytokine expression in activated PBMCs obtained from patients after open-heart surgery. Furthermore, piperacillin, doxycyclin and moxifloxacin inhibited the phagocytic activity of monocytes. Our results suggest that antibiotics regulate the immune response by modulating TLR- and cytokine expression as well as phagocytosis under septic conditions. Moxifloxacin, doxycycline and erythromycin were shown to possess the strongest immunomodulatory effects and these antibiotic classes should be considered for future immunomodulatory studies in sepsis.