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.
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-1β, 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.
Previously, we have found that changes in the location of intracellular heat shock protein (HSP)60 are associated with apoptosis. HSP60 has been reported to be a ligand of toll-like receptor (TLR)-4.
...We hypothesized that extracellular HSP60 (exHSP60) would mediate apoptosis via TLR4.
Adult rat cardiac myocytes were treated with HSP60, either recombinant human or with HSP60 purified from the media of injured rat cardiac myocytes. ExHSP60 induced apoptosis in cardiac myocytes, as detected by increased caspase 3 activity and increased DNA fragmentation. Apoptosis could be reduced by blocking antibodies to TLR4 and by nuclear factor kappaB binding decoys, but not completely inhibited, even though similar treatment blocked lipopolysaccharide-induced apoptosis. Three distinct controls showed no evidence for involvement of a ligand other than exHSP60 in the mediation of apoptosis.
This is the first report of HSP60-induced apoptosis via the TLRs. HSP60-mediated activation of TLR4 may be a mechanism of myocyte loss in heart failure, where HSP60 has been detected in the plasma.
Sepsis is a leading cause of death worldwide, despite the use of multimodal therapies. Common antibiotic regimens are being affected by a rising number of multidrug-resistant pathogens, and new ...therapeutic approaches are therefore needed. Antibiotics have immunomodulatory properties which appear to be beneficial in the treatment of sepsis. We hypothesized that the last-resort antibiotics vancomycin (VAN) and daptomycin (DMC) modulate cell migration, phagocytosis, and protein cytokine levels in a murine model of lipopolysaccharide (LPS)-induced sepsis. Ten to twelve-week-old C57BL/6 mice (n = 4–6 animals per group) were stimulated with LPS for 20 h, followed by the administration of VAN or DMC. The outcome parameters were leukocyte accumulation and effector function. Quantification of the immune cells in the peritoneal lavage was performed using flow cytometry analysis. Phagocytosis was measured using pHrodo E. coli BioParticles. The response of the cytokines TNFα, IL-6, and IL-10 was measured in vitro using murine peritoneal macrophages stimulated with LPS and VAN or DMC. VAN decreased both the peritoneal macrophage and the dendritic cell populations following LPS stimulation. DMC reduced the dendritic cell population in the peritoneal cavity in LPS-infected mice. Both antibiotics increased the phagocytic activity in peritoneal macrophages, but this effect was diminished in response to LPS. Phagocytosis of dendritic cells was increased in LPS-infected animals treated with VAN. VAN and DMC differently modulated the levels of pro-and anti-inflammatory cytokines. In a murine model of LPS-induced sepsis, VAN and DMC exhibit immunomodulatory effects on cells involved in innate immunity. The question of whether these antibiotics exhibit synergistic effects in the treatment of septic patients, beyond their bactericidal properties, should be further evaluated in future studies.
Beyond lipid lowering, statins are supposed to exert pleiotropic effects positively influencing the progression of atherosclerotic lesions. The development of such lesions is associated with ...increased release of angiopoietin-2 (Ang-2), an endothelial cell-specific protein growth factor stored in Weibel-Palade bodies (WPBs). The aim of our study was to examine whether statin pretreatment influences the release of Ang-2 from endothelial cells. Stimulation of HUVECs and HMVECs with PMA, thrombin or histamine resulted in significant release of Ang-2, as evidenced by ELISA. Pretreatment with simvastatin and mevastatin suppressed this release to basal level, while pravastatin had no effect. Simvastatin itself increased nitric oxide (NO, EC number 1.14.13.39) synthesis, measured by Griess reaction. Combining the statin pretreatment with the eNOS inhibitor L-NNA as well as bypassing the HMG-CoA reductase (EC number: 1.1.1.34) by adding mevalonic acid or geranyl pyrophosphate restored the exocytotic effect of PMA. Immunofluorescence microscopy showed that depletion of WPBs upon PMA stimulation ceased after pretreatment with simvastatin. This study demonstrates a potent suppressive effect of statins on the release of Ang-2 from endothelial cells. Regarding its harmful effects in the development of atherosclerotic lesions, our data provide further insight into the mechanisms of the anti-atherogenic potential of statins.
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.
Plasmacytoid dendritic cells (pDCs) are a major source of type I interferon (IFN) and are important for host defense by sensing microbial DNA via TLR9. pDCs also play a critical role in the ...pathogenesis of IFN‐driven autoimmune diseases. Yet, this autoimmune reaction is caused by the recognition of self‐DNA and has been linked to TLR9‐independent pathways. Increasing evidence suggests that the cytosolic DNA receptor cyclic GMP‐AMP (cGAMP) synthase (cGAS) is a critical component in the detection of pathogens and contributes to autoimmune diseases. It has been shown that binding of DNA to cGAS results in the synthesis of cGAMP and the subsequent activation of the stimulator of interferon genes (STING) adaptor to induce IFNs. Our results show that the cGAS‐STING pathway is expressed and activated in human pDCs by cytosolic DNA leading to a robust type I IFN response. Direct activation of STING by cyclic dinucleotides including cGAMP also activated pDCs and knockdown of STING abolished this IFN response. These results suggest that pDCs sense cytosolic DNA and cyclic dinucleotides via the cGAS‐STING pathway and that targeting this pathway could be of therapeutic interest.
Transfection of DNA or cyclic dinucleotides into the cytosol of human pDCs elicit a type I IFN response independent of TLR9. cGAS and STING are expressed in pDCs and IFN induction in response to DNA or cyclic dinucleotides is dependent on the activation of STING and TBK1 suggesting that human pDC express a functional active cGAS‐STING pathway.
Objective. TLR7 ligation in plasmacytoid dendritic cells is promising for the treatment of cancer, allergy, and infectious diseases; however, high doses of ligands are required. We hypothesized that ...the combination of structurally different TLR7 ligands exponentiates the resulting immune response. Methods. CAL-1 (human pDC line) cells were incubated with the TLR7-specific adenine analog CL264 and single-stranded 9.2s RNA. Protein secretion was measured by ELISA. Microarray technique was used to detect modified gene expression patterns upon synergistic stimulation, revealing underlying functional groups and networks. Cell surface binding properties were studied using FACS analysis. Results. CL264 in combination with 9.2s RNA significantly enhanced cytokine and interferon secretion to supra-additive levels. This effect was due to a stronger stimulation of already regulated genes (by monostimulation) as well as to recruitment of thus far unregulated genes. Top scoring canonical pathways referred to immune-related processes. Network analysis revealed IL-1β, IL-6, TNF, and IFN-β as major regulatory nodes, while several minor regulatory nodes were also identified. Binding of CL264 to the cell surface was enhanced by 9.2s RNA. Conclusion. Structurally different TLR7 ligands act synergistically on gene expression patterns and on the resulting inflammatory response. These data could impact future strategies optimizing TLR7-targeted drug design.
IFNA1 (interferon alpha) is a key cytokine regulating the activity of numerous immune cells. Plasmacytoid dendritic cells (pDCs) as natural interferon-producing cells play critical roles as sensors ...of pathogens and link innate to adaptive immunity. CpG motifs within DNA sequences activating toll-like receptor 9 (TLR9) are the main stimuli eliciting IFNA1 secretion from pDCs. Adrenergic substances are capable of differentially modulating the response from various immune cells. Hence, the aim of this study was to examine how adrenoceptor stimulation influences TLR9-induced IFNA1 secretion from human pDCs.
PBMCs generated from human whole blood and pDCs enriched from buffy coats were stimulated with LPS and CpG-ODN 2336 in the presence or absence of epinephrine and different adrenoceptor antagonists. Secretion of TNF and IFNA1 was measured by ELISA. Flow cytometry was used to determine efficacy of pDC enrichment and adrenoceptor expression of PBMC subsets. The influence of modified IFNA1 secretion on NK cell activity was evaluated using a colorimetric tumor cell lysis assay.
TLR9-induced IFNA1 secretion as well as TLR4-induced TNF secretion from PBMCs was dose-dependently attenuated by coincubation with epinephrine. Combination with different specific adrenoceptor antagonists revealed that this effect was mediated by the adrenoceptor β2 (ADRB2). Since flow cytometric analysis could exclude the presence of ADRB2 on pDCs, highly enriched pDCs lacked any visible impact of adrenoceptor stimulation on TLR9-induced IFNA1 release. Combination of pDCs with PBMCs restored the effect, even when they were separated by a permeable membrane. Suppression of TLR9-mediated IFNA1 secretion from PBMCs by adrenoceptor stimulation reduced the lytic activity of NK cells on K562 tumor cells.
We provide insights into the underlying mechanisms of the interrelation between immune responses and pharmacological agents widely used in clinical practice. Our results have implications for the future treatment of human patients, in which the endogenous immune response plays a pivotal role, such as during viral infections, inflammatory diseases and cancers.
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.
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