Low-tidal-volume prevent ventilation induced inflammation in a mouse model of sepsis.

BACKGROUND AND GOAL OF THE STUDY: Pulmonary inflammation, increased vascular permeability, and pulmonary edema, occur in response to primary pulmonary infections like pneumonia but are also evident in endotoxemia or sepsis. Mechanical ventilation augments pre-existing lung injury and inflammation resulting from exposure to microbial products. The objective of this study was to test the hypothesis that low-tidal-volume prevent ventilation induced lung injury in sepsis. MATERIALS AND METHODS: 10-12-week-old male C57BL/6N-mice received an intraperitoneal (i.p.) injection with equipotent dosages of LPS, 1668-thioate, 1612-thioate, or PBS. 120 min after injection, mice were randomized to low- (LV, 7 ± 1 ml/kg) or high-tidal-volume (HV, 25 ± 1 ml/kg) ventilation. Hemodynamic and ventilatory parameters were recorded and inflammatory markers were analyzed form BAL that was generated after 90 minute ventilation. RESULTS AND DISCUSSION: Arterial blood pressures declined during mechanical ventilation in all groups. pO2 decreased in LPS injected and CO2 increased in sham, LPS, and 1612-thioate administered mice at 45 min and in 1668-thioate injected mice after 90 minute LV ventilation compared to respective HV groups. BAL protein concentrations increased in HV ventilated and 1668- or 1612-thioat pre-treated mice. BAL TNF-α protein concentrations increased in both LPS- and 1668-thioate-injected and IL-1ß protein concentrations only in LPS-injected and HV ventilated mice. Most notably, no increased protein concentrations were observed in any of the LV ventilated groups. CONCLUSION: We conclude that low-tidal-volume ventilation may be a potential strategy for the prevention of ventilator induced lung injury in a murine model of systemic TLR agonist induced lung injury.

Soluble adenylyl cyclase: A novel player in cardiac hypertrophy induced by isoprenaline or pressure overload.

AIMS: In contrast to the membrane bound adenylyl cyclases, the soluble adenylyl cyclase (sAC) is activated by bicarbonate and divalent ions including calcium. sAC is located in the cytosol, nuclei and mitochondria of several tissues including cardiac muscle. However, its role in cardiac pathology is poorly understood. Here we investigate whether sAC is involved in hypertrophic growth using two different model systems. METHODS AND RESULTS: In isolated adult rat cardiomyocytes hypertrophy was induced by 24 h ß1-adrenoceptor stimulation using isoprenaline (ISO) and a ß2-adrenoceptor antagonist (ICI118,551). To monitor hypertrophy cell size along with RNA/DNA- and protein/DNA ratios as well as the expression level of α-skeletal actin were analyzed. sAC activity was suppressed either by treatment with its specific inhibitor KH7 or by knockdown. Both pharmacological inhibition and knockdown blunted hypertrophic growth and reduced expression levels of α-skeletal actin in ISO/ICI treated rat cardiomyocytes. To analyze the underlying cellular mechanism expression levels of phosphorylated CREB, B-Raf and Erk1/2 were examined by western blot. The results suggest the involvement of B-Raf, but not of Erk or CREB in the pro-hypertrophic action of sAC. In wild type and sAC knockout mice pressure overload was induced by transverse aortic constriction. Hemodynamics, heart weight and the expression level of the atrial natriuretic peptide were analyzed. In accordance, transverse aortic constriction failed to induce hypertrophy in sAC knockout mice. Mechanistic analysis revealed a potential role of Erk1/2 in TAC-induced hypertrophy. CONCLUSION: Soluble adenylyl cyclase might be a new pivotal player in the cardiac hypertrophic response either to long-term ß1-adrenoceptor stimulation or to pressure overload.

Antiepileptic Drugs Impair Shortening of Isolated Cardiomyocytes.

BACKGROUND: Most antiepileptic drugs (AEDs) inhibit seizure generation by acting on voltage-dependent ion channels. Voltage-dependent sodium and calcium channels are commonly expressed in brain and heart, suggesting that AEDs may have considerable cardiodepressive effects, thereby facilitating sudden cardiac death as a potential cause of sudden unexpected death in epilepsy. Here, we investigated the effects of carbamazepine (CBZ), lamotrigine (LTG), and levetiracetam (LEV) alone and in combination on the shortening properties of isolated ventricular cardiomyocytes of wild-type mice. METHODS: Properties of murine cardiomyocytes were determined by recording the sarcomere shortening with a video imaging system before, during, and after administration of AEDs in different concentrations and combinations. We assessed (i) the number of successful shortenings during continuous electrical stimulation (electromechanical coupling) and (ii) the shortening amplitude as well as other shortening-related properties upon repetitive electrical stimulation at 4 Hz. Data are given as mean ± SEM. RESULTS: At 100 µM, CBZ (10 cells), LTG (11 cells), and LEV (11 cells) alone had no effect on the electromechanical coupling but reversibly reduced shortening amplitudes by 15 ± 4, 24 ± 3, and 11 ± 3%, respectively. Increasing the LTG concentration to 250 (21 cells) and 500 µM (4 cells) reversibly inhibited the electromechanical coupling in 62 and 100% of the experiments. Importantly, simultaneous application of CBZ, LTG, and LEV at 100 µM also impaired the electromechanical coupling in 8 of 19 cardiomyocytes (42%) and reduced the shortening amplitude by 21 ± 4%. CONCLUSION: Our data show that AEDs reversibly impair cardiac excitation and contraction. Importantly, the blocking effect on electromechanical coupling appears to be additive when different AEDs are simultaneously applied. The translational value of these experimental findings into clinical practice is limited. Our results, however, suggest that rationale AED therapy may be important with respect to cardiac side effects and potential facilitation of serious cardiac dysfunction especially when AEDs are used in combination or at very high doses.

Engineered Context-Sensitive Agonism: Tissue-Selective Drug Signaling through a G Protein-Coupled Receptor.

Drug discovery strives for selective ligands to achieve targeted modulation of tissue function. Here we introduce engineered context-sensitive agonism as a postreceptor mechanism for tissue-selective drug action through a G protein-coupled receptor. Acetylcholine M2-receptor activation is known to mediate, among other actions, potentially dangerous slowing of the heart rate. This unwanted side effect is one of the main reasons that limit clinical application of muscarinic agonists. Herein we show that dualsteric (orthosteric/allosteric) agonists induce less cardiac depression ex vivo and in vivo than conventional full agonists. Exploration of the underlying mechanism in living cells employing cellular dynamic mass redistribution identified context-sensitive agonism of these dualsteric agonists. They translate elevation of intracellular cAMP into a switch from full to partial agonism. Designed context-sensitive agonism opens an avenue toward postreceptor pharmacologic selectivity, which even works in target tissues operated by the same subtype of pharmacologic receptor.

Influence of Apnea-induced Hypoxia on Catecholamine Release and Cardiovascular Dynamics.

Prolonged breath-hold causes complex compensatory mechanisms such as increase in blood pressure, redistribution of blood flow, and bradycardia. We tested whether apnea induces an elevation of catecholamine-concentrations in well-trained apneic divers.11 apneic divers performed maximal dry apnea in a horizontal position. Parameters measured during apnea included blood pressure, ECG, and central, in addition to peripheral hemoglobin oxygenation. Peripheral arterial hemoglobin oxygenation was detected by pulse oximetry, whereas peripheral (abdominal) and central (cerebral) tissue oxygenation was measured by Near Infrared Spectroscopy (NIRS). Exhaled O2 and CO2, plasma norepinephrine and epinephrine concentrations were measured before and after apnea.Averaged apnea time was 247±76 s. Systolic blood pressure increased from 135±13 to 185±25 mmHg. End-expiratory CO2 increased from 29±4 mmHg to 49±6 mmHg. Norepinephrine increased from 623±307 to 1 826±984 pg ml-1 and epinephrine from 78±22 to 143±65 pg ml-1 during apnea. Heart rate reduction was inversely correlated with increased norepinephrine (correlation coefficient -0.844, p=0.001). Central (cerebral) O2 desaturation was time-delayed compared to peripheral O2 desaturation as measured by NIRSabdominal and SpO2.Increased norepinephrine caused by apnea may contribute to blood shift from peripheral tissues to the CNS and thus help to preserve cerebral tissue O2 saturation longer than that of peripheral tissue.

Sarcomeric lesions and remodeling proximal to intercalated disks in overload-induced cardiac hypertrophy.

Pressure overload induces cardiac remodeling involving both the contractile machinery and intercalated disks (IDs). Filamin C (FlnC) and Xin actin-binding repeat-containing proteins (XIRPs) are multi-adapters localizing in IDs of higher vertebrates. Knockout of the gene encoding Xin (Xirp1) in mice leads to a mild cardiac phenotype with ID mislocalization. In order to amplify this phenotype, we performed transverse aortic constriction (TAC) on control and Xirp1-deficient mice. TAC induced similar left ventricular hypertrophy in both genotypes, suggesting that the lack of Xin does not lead to higher susceptibility to cardiac overload. However, in both genotypes, FlnC appeared in "streaming" localizations across multiple sarcomeres proximal to the IDs, suggesting a remodeling response. Furthermore, FlnC-positive areas of remodeling, reminiscent of sarcomeric lesions previously described for skeletal muscles (but so far unreported in the heart), were also observed. These adaptations reflect a similarly strong effect of the pressure induced by TAC in both genotypes. However, 2 weeks post-operation TAC-treated knockout hearts had reduced levels of connexin43 and slightly increased incidents of ventricular tachycardia compared to their wild-type (WT) counterparts. Our findings highlight the FlnC-positive sarcomeric lesions and ID-proximal streaming as general remodeling responses in cardiac overload-induced hypertrophy.

Tlr2 deficiency does not limit the development of left ventricular hypertrophy in a model of transverse aortic constriction induced pressure overload.

BACKGROUND: Toll-like receptors (TLRs) are involved in a variety of cardiovascular disorders, including septic cardiomyopathy, ischemia/reperfusion, heart failure, and cardiac hypertrophy. Previous research revealed that TLR4 promotes cardiac hypertrophy in vivo. Therefore, we investigated whether TLR2 is also involved in the development of cardiac hypertrophy. METHODS: Tlr2 deficient and wild type mice were subjected to transverse aortic constriction (TAC) or sham operation procedure. Left ventricular, heart and lung weights as well as hemodynamic parameters were determined after 3, 14 or 28 days. Real-time RT PCR was used to evaluate left ventricular gene expression. Protein content was determined via ELISA. RESULTS: TAC increased systolic left ventricular pressure, contraction and relaxations velocities as well as the heart weight in both genotypes. Tlr2 deficiency significantly enhanced cardiac hypertrophy after 14 and 28 days of TAC. Left ventricular end-diastolic pressure and heart rate increased in Tlr2(-/-) TAC mice only. Fourteen days of TAC led to a significant elevation of ANP, BNP, TGFß and TLR4 mRNA levels in Tlr2(-/-) left ventricular tissue. CONCLUSION: These data suggest that Tlr2 deficiency may promote the development of cardiac hypertrophy and ventricular remodeling after transverse aortic constriction.

Loss of the LIM-only protein Fhl2 impairs inflammatory reaction and scar formation after cardiac ischemia leading to better hemodynamic performance.

AIMS: The pathogenesis of myocardial ischemia-reperfusion injury (MI/R) involves an inflammatory response. Since the four-and-a-half LIM domain-containing protein 2 (Fhl2) has been observed to modulate immune cell migration, we aimed to study the consequences of Fhl2(-/-) under MI/R with respect to immune reaction, scar formation, and hemodynamic performance. MATERIAL AND METHODS: In a closed chest model of 1h MI/R, immune cell invasion of phagocytic monocytes was characterized by flow cytometry and immunohistochemistry. In addition, infarct size was assessed by triphenyltetrazolium chloride/Masson trichrome staining 24h/21days after reperfusion and a set of hemodynamic parameters was recorded by catheterisation in Fhl2(-/-) mice and controls. KEY FINDINGS: While flow cytometry did not reveal differences in myocardial CD45(high) immune cell infiltrate, histological analysis showed that infiltrating immune cells in Fhl2(-/-) animals were preferentially located in the perivascular area, whereas in wild type, immune cells were well dispersed within the area at risk. After 24h and 21days of reperfusion, infarct size was significantly reduced in Fhl2(-/-) compared to WT animals. In addition, hemodynamic performance was better in Fhl2(-/-) mice, compared to WT mice up to day 21 of reperfusion. The loss of Fhl2 leads to an altered immune response to myocardial ischemia, which results in smaller infarcts and better hemodynamic performance up to 21days after myocardial ischemia reperfusion. SIGNIFICANCE: Immune cell invasion plays a pivotal role in the context of MI/R. Fhl2 significantly influences immune cell function and immune cell interaction with injured cardiac tissue leading to altered scar composition.

A Model to Simulate Clinically Relevant Hypoxia in Humans.

In case of apnea, arterial partial pressure of oxygen (pO2) decreases, while partial pressure of carbon dioxide (pCO2) increases. To avoid damage to hypoxia sensitive organs such as the brain, compensatory circulatory mechanisms help to maintain an adequate oxygen supply. This is mainly achieved by increased cerebral blood flow. Intermittent hypoxia is a commonly seen phenomenon in patients with obstructive sleep apnea. Acute airway obstruction can also result in hypoxia and hypercapnia. Until now, no adequate model has been established to simulate these dynamics in humans. Previous investigations focusing on human hypoxia used inhaled hypoxic gas mixtures. However, the resulting hypoxia was combined with hyperventilation and is therefore more representative of high altitude environments than of apnea. Furthermore, the transferability of previously performed animal experiments to humans is limited and the pathophysiological background of apnea induced physiological changes is poorly understood. In this study, healthy human apneic divers were utilized to mimic clinically relevant hypoxia and hypercapnia during apnea. Additionally, pulse-oximetry and Near Infrared Spectroscopy (NIRS) were used to evaluate changes in cerebral and peripheral oxygen saturation before, during, and after apnea.

Ghrelin promotes oral tumor cell proliferation by modifying GLUT1 expression.

In our study, ghrelin was investigated with respect to its capacity on proliferative effects and molecular correlations on oral tumor cells. The presence of all molecular components of the ghrelin system, i.e., ghrelin and its receptors, was analyzed and could be detected using real-time PCR and immunohistochemistry. To examine cellular effects caused by ghrelin and to clarify downstream-regulatory mechanisms, two different oral tumor cell lines (BHY and HN) were used in cell culture experiments. Stimulation of either cell line with ghrelin led to a significantly increased proliferation. Signal transduction occurred through phosphorylation of GSK-3ß and nuclear translocation of ß-catenin. This effect could be inhibited by blocking protein kinase A. Glucose transporter1 (GLUT1), as an important factor for delivering sufficient amounts of glucose to tumor cells having high requirements for this carbohydrate (Warburg effect) was up-regulated by exogenous and endogenous ghrelin. Silencing intracellular ghrelin concentrations using siRNA led to a significant decreased expression of GLUT1 and proliferation. In conclusion, our study describes the role for the appetite-stimulating peptide hormone ghrelin in oral cancer proliferation under the particular aspect of glucose uptake: (1) tumor cells are a source of ghrelin. (2) Ghrelin affects tumor cell proliferation through autocrine and/or paracrine activity. (3) Ghrelin modulates GLUT1 expression and thus indirectly enhances tumor cell proliferation. These findings are of major relevance, because glucose uptake is assumed to be a promising target for cancer treatment.

Tlr4 Deficiency Protects against Cardiac Pressure Overload Induced Hyperinflammation.

Transverse aortic constriction provokes a pro-inflammatory reaction and results in cardiac hypertrophy. Endogenous ligands contribute to cardiac hypertrophy via toll-like receptor (TLR)-4 binding. A lack of TLR4 signaling diminishes hypertrophy and inflammation. Wild type mice undergoing aortic constriction respond to a lipopolysaccharide second-hit stimulus with hyperinflammation. The objective of this study was to assess whether other second-hit challenges utilizing TLR ligands provoke a comparable inflammatory reaction, and to find out whether this response is absent in TLR4 deficient mice. Assuming that cardiac stress alters the expression of pattern recognition receptors we analyzed the effects of transverse aortic constriction and second-hit virulence factor treatment on TLR expression, as well as cytokine regulation. Wild type and Tlr4-/- mice were subjected to three days of TAC and subsequently confronted with gram-positive TLR2 ligand lipoteichoic acid (LTA, 15 mg/g bodyweight) or synthetic CpG-oligodesoxynucleotide 1668 thioate (20 nmol/kg bodyweight, 30 min after D-galactosamin desensitization) signaling via TLR9. Hemodynamic measurements and organ preservation were performed 6 h after stimulation. Indeed, the study revealed a robust enhancement of LTA induced pattern recognition receptor and cytokine mRNA expression and a LTA-dependent reduction of hemodynamic pressure in TAC wild type mice. Second-Hit treatment with CpG-ODNs led to similar results. However, second-hit effects were abolished in Tlr4-/- mice. In total, these data indicate for the first time that cardiac stress increases the inflammatory response towards both, gram-negative and gram-positive, TLR ligands as well as bacterial DNA. The decrease of the inflammatory response upon TLR2 and -9 ligand challenge in TAC Tlr4-/- mice demonstrates that a lack of TLR4 signaling does not only prevent left ventricular hypertrophy but also protects the mice from a cardiac stress induced hyperinflammatory reaction.

Antifungal antibiotics modulate the pro-inflammatory cytokine production and phagocytic activity of human monocytes in an in vitro sepsis model.

AIMS: The incidence of secondary systemic fungal infections has sharply increased in bacterial septic patients. Antimycotics exhibit immunomodulatory properties, yet these effects are incompletely understood in secondary systemic fungal infections following bacterial sepsis. We investigated a model of systemic inflammation to determine whether antimycotics (liposomal amphotericin B (L-AMB), itraconazol (ITC), and anidulafungin (ANI)) modulate the gene and protein expression as well as the phagocytic activity of lipopolysaccharide (LPS)-stimulated human monocytes. MAIN METHODS: THP-1 monocytes were incubated with L-AMB, ITC or ANI and LPS. Gene expression levels of cytokines (TNF-, IL-1, IL-6, and IL-10) were measured after 2h, 6h, and 24h. Cytokine protein levels were evaluated after 24h and phagocytic activity was determined following co-incubation with Escherichia coli. KEY FINDINGS: All antimycotics differentially modulated the gene and protein expression of cytokines in sepsis-like conditions. In the presence of LPS, we identified L-AMB as immunosuppressive, whereas ITC demonstrated pro-inflammatory properties. Both compounds induced remarkably less phagocytosis. SIGNIFICANCE: Our study suggests that antimycotics routinely used in septic patients alter the immune response in sepsis-like conditions by modulating cytokine gene and protein expression levels and phagocytic activity. Future treatment strategies should consider the immune status of the host and apply antimycotics accordingly in bacterial septic patients with secondary fungal infections.

Linezolid, vancomycin and daptomycin modulate cytokine production, Toll-like receptors and phagocytosis in a human in vitro model of sepsis.

Conventional antibiotics exhibit immunomodulatory properties beneficial in the treatment of sepsis. Antibiotic-resistant Gram-positive bacteria have become a problem in sepsis therapy, giving rise to increased use of last-resort antibiotics; for example, linezolid (LIN), vancomycin (VAN) and daptomycin (DAP). As the immunomodulatory properties of these antibiotics in treating sepsis are unknown, this study examined the effect of VAN, LIN and DAP on the immune response under sepsis-like conditions in vitro. Lipopolysaccharide (LPS)-activated THP-1 monocytes were incubated with LIN, VAN or DAP. Gene expression of cytokines (TNFα, IL-1ß, IL-6, IL-10) and Toll-like receptors (TLR1, 2, 4, 6, 7 and 9) was monitored and phagocytosis was determined following coincubation with E. coli. The antibiotics differentially modulated the gene expression of the investigated cytokines. While LIN and VAN upregulated the expression of all TLRs, DAP downregulated mRNA levels of TLR1, TLR2 and TLR6, which recognize pathogen-associated molecular patterns from Gram-positive bacteria. In addition, LIN inhibited, whereas VAN promoted the phagocytic activity of monocytes. Our results suggest that LIN and VAN possess pro-inflammatory properties, whereas DAP might reduce the immune response to Gram-positive bacteria in sepsis. Furthermore, VAN might be beneficial in the prevention of Gram-negative infections by increasing the phagocytosis of E. coli.

Evaluation of near-infrared spectroscopy under apnea-dependent hypoxia in humans.

In this study we investigated the responsiveness of near-infrared spectroscopy (NIRS) recordings measuring regional cerebral tissue oxygenation (rSO2) during hypoxia in apneic divers. The goal was to mimic dynamic hypoxia as present during cardiopulmonary resuscitation, laryngospasm, airway obstruction, or the "cannot ventilate cannot intubate" situation. Ten experienced apneic divers performed maximal breath hold maneuvers under dry conditions. SpO2 was measured by Masimo™ pulse oximetry on the forefinger of the left hand. NIRS was measured by NONIN Medical's EQUANOX™ on the forehead or above the musculus quadriceps femoris. Following apnea median cerebral rSO2 and SpO2 values decreased significantly from 71 to 54 and from 100 to 65%, respectively. As soon as cerebral rSO2 and SpO2 values decreased monotonically the correlation between normalized cerebral rSO2 and SpO2 values was highly significant (Pearson correlation coefficient = 0.893). Prior to correlation analyses, the values were normalized by dividing them by the individual means of stable pre-apneic measurements. Cerebral rSO2 measured re-saturation after termination of apnea significantly earlier (10 s, SD = 3.6 s) compared to SpO2 monitoring (21 s, SD = 4.4 s) [t(9) = 7.703, p < 0.001, r(2) = 0.868]. Our data demonstrate that NIRS monitoring reliably measures dynamic changes in cerebral tissue oxygen saturation, and identifies successful re-saturation faster than SpO2. Measuring cerebral rSO2 may prove beneficial in case of respiratory emergencies and during pulseless situations where SpO2 monitoring is impossible.

The toxic effect of R350P mutant desmin in striated muscle of man and mouse.

Mutations of the human desmin gene on chromosome 2q35 cause autosomal dominant, autosomal recessive and sporadic forms of protein aggregation myopathies and cardiomyopathies. We generated R349P desmin knock-in mice, which harbor the ortholog of the most frequently occurring human desmin missense mutation R350P. These mice develop age-dependent desmin-positive protein aggregation pathology, skeletal muscle weakness, dilated cardiomyopathy, as well as cardiac arrhythmias and conduction defects. For the first time, we report the expression level and subcellular distribution of mutant versus wild-type desmin in our mouse model as well as in skeletal muscle specimens derived from human R350P desminopathies. Furthermore, we demonstrate that the missense-mutant desmin inflicts changes of the subcellular localization and turnover of desmin itself and of direct desmin-binding partners. Our findings unveil a novel principle of pathogenesis, in which not the presence of protein aggregates, but disruption of the extrasarcomeric intermediate filament network leads to increased mechanical vulnerability of muscle fibers. These structural defects elicited at the myofiber level finally impact the entire organ and subsequently cause myopathy and cardiomyopathy.

Postconditioning with a CpG containing oligodeoxynucleotide ameliorates myocardial infarction in a murine closed-chest model.

AIMS: Toll-like receptor (TLR)9 ligand CpG-oligodeoxynucleotide (CpG-ODN) exerts preconditioning in myocardial ischemia/reperfusion. We hypothesized a postconditioning effect of CpG-ODN in a murine closed-chest model of myocardial infarction. MATERIALS AND METHODS: C57BL/6 (12 weeks, male, WT) mice were instrumented at the left anterior descending artery, then allowed 5d of recovery before 30 min ischemia. Treatments comprised: 1) PBS: 250 µl phosphate buffer solution intraperitoneally 5 min before reperfusion and 2) IPC (ischemic postconditioning): 3 twenty-second reperfusion and occlusion episodes at the end of ischemia 3) CpG-ODN: 1668 thioate 0.2 µmol/kg BW intraperitoneally 5 min before reperfusion. Infarct size was assessed via triphenyltetrazolium chloride (TTC) staining after 2 and 24h reperfusion. Myocardial mRNA-expression of cytokines was measured using real-time PCR after 2h reperfusion. Phosphatidylinositol-3 kinase (PI3K)-inhibitor wortmannin was injected intraperitoneally in WT 15 min before postconditioning and PBS in each group. Cardiac function in WT was assessed with a left-ventricular pressure-volume catheter at 24h reperfusion. KEY FINDINGS: Following 30 min ischemia and 2h reperfusion, infarct size was diminished by 90% in WT postconditioned with CpG-ODN (2.4 ± 1.55 IS/AAR%) and IPC (1.98 ± 1.03 IS/AAR%) compared to PBS mice (23.2 ± 3.97 IS/AAR%). Infarct size increased following 24h reperfusion but the differences remained robust. Expression of TNF-α and IL-10 was increased in CpG-ODN. Wortmannin abolished the postconditioning effect of CpG-ODN and IPC. Ejection fraction and preload-recruitable stroke work were significantly greater in CpG-ODN mice. SIGNIFICANCE: CpG-ODN confers postconditioning via activation of TLR9. Cardiac function is preserved following CpG-ODN postconditioning. The PI3K -inhibitor wortmannin attenuates CpG-ODN postconditioning.

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 Ly6C(low) and Ly6C(high) macrophages. Ly6C(low) 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 Ly6C(low) but not on Ly6C(high) macrophages. Furthermore, ICAM-1 was up-regulated on cardiac endothelial cells. In fluorescence microscopy Ly6C(low) 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 Ly6C(low) macrophages in the cardiac tissue, where these cells might contribute to cardiac inflammation and remodeling in response to pressure overload.

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-1ß, 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.

TLR2 stimulation induces cardiac inflammation but not cardiac depression in vivo.

BACKGROUND: Bacteria such as Staphylococcus aureus induce myocardial dysfunction in vivo. To rectify conflicting evidence about the role of TLR2 signaling and cardiac dysfunction, we hypothesized that the specific TLR2 agonist purified lipoteichoic acid (LTA) from S. aureus contributes to cardiac dysfunction in vitro and in vivo. METHODS: Wildtype (WT-) and TLR2-deficient (TLR2-D) mice were challenged with LTA and in comparison with equivalent doses of lipopolysaccharide (LPS) and CpG-oligodeoxynucleotide (CpG-ODN). TLR2-expression, NFκB as well as cytokine response were determined. Sarcomere shortening of isolated cardiomyocytes was analyzed in vitro and cardiac function in vivo after stimulation with LTA. RESULTS: LTA induced up-regulation of TLR2 mRNA, activation of NFκB and cytokine expression within 2-6 h in WT-, but not in TLR2-D hearts. Cytokines were also elevated in the serum. LPS and CpG-ODN induced a more severe cardiac inflammation. In vitro incubation of cardiomyocytes with LTA reduced sarcomere shortening via NO at stimulation frequencies ≤ 8 Hz only in WT cells. However, hemodynamic parameters in vivo were not affected by LTA challenge. CONCLUSIONS: LTA induced cardiac inflammation was relatively weak and sarcomere shortening was reduced only below physiological heart rates. This may explain the apparent contradiction between the in vivo and in vitro LTA effects.

Toll-like receptor 9 promotes cardiac inflammation and heart failure during polymicrobial sepsis.

BACKGROUND: Aim was to elucidate the role of toll-like receptor 9 (TLR9) in cardiac inflammation and septic heart failure in a murine model of polymicrobial sepsis. METHODS: Sepsis was induced via colon ascendens stent peritonitis (CASP) in C57BL/6 wild-type (WT) and TLR9-deficient (TLR9-D) mice. Bacterial load in the peritoneal cavity and cardiac expression of inflammatory mediators were determined at 6, 12, 18, 24, and 36 h. Eighteen hours after CASP cardiac function was monitored in vivo. Sarcomere length of isolated cardiomyocytes was measured at 0.5 to 10 Hz after incubation with heat-inactivated bacteria. RESULTS: CASP led to continuous release of bacteria into the peritoneal cavity, an increase of cytokines, and differential regulation of receptors of innate immunity in the heart. Eighteen hours after CASP WT mice developed septic heart failure characterised by reduction of end-systolic pressure, stroke volume, cardiac output, and parameters of contractility. This coincided with reduced cardiomyocyte sarcomere shortening. TLR9 deficiency resulted in significant reduction of cardiac inflammation and a sustained heart function. This was consistent with reduced mortality in TLR9-D compared to WT mice. CONCLUSIONS: In polymicrobial sepsis TLR9 signalling is pivotal to cardiac inflammation and septic heart failure.