290910
Glucan phosphate attenuates myocardial HMGB1 translocation in severe sepsis through inhibiting NF-κB activation.
Myocardial dysfunction is a major consequence of septic shock and contributes to the high mortality of sepsis. High-mobility group box 1 (HMGB1) serves as a late mediator of lethality in sepsis. We have reported that glucan phosphate (GP) attenuates cardiac dysfunction and increases survival in cecal ligation and puncture (CLP)-induced septic mice. In the present study, we examined the effect of GP on HMGB1 translocation from the nucleus to the cytoplasm in the myocardium of septic mice. GP was administered to mice 1 h before induction of CLP. Sham-operated mice served as control. The levels of HMGB1, Toll-like receptor 4 (TLR4), and NF-κB binding activity were examined. In an in vitro study, H9C2 cardiomyoblasts were treated with lipopolysaccharide (LPS) in the presence or absence of GP. H9C2 cells were also transfected with Ad5-IκBα mutant, a super repressor of NF-κB activity, before LPS stimulation. CLP significantly increased the levels of HMGB1, TLR4, and NF-κB binding activity in the myocardium. In contrast, GP administration attenuated CLP-induced HMGB1 translocation from the nucleus to the cytoplasm and reduced CLP-induced increases in TLR4 and NF-κB activity in the myocardium. In vitro studies showed that GP prevented LPS-induced HMGB1 translocation and NF-κB binding activity. Blocking NF-κB binding activity by Ad5-IκBα attenuated LPS-induced HMGB1 translocation. GP administration also reduced the LPS-stimulated interaction of HMGB1 with TLR4. These data suggest that attenuation of HMGB1 translocation by GP is mediated through inhibition of NF-κB activation in CLP-induced sepsis and that activation of NF-κB is required for HMGB1 translocation.
26170940
HMGB1: Endogenous Danger Signaling
While foreign pathogens and their products have long been known to activate the innate immune system, the recent recognition of a group of endogenous molecules that serve a similar function has provided a framework for understanding the overlap between the inflammatory responses activated by pathogens and injury. These endogenous molecules, termed alarmins, are normal cell constituents that can be released into the extracellular milieu during states of cellular stress or damage and subsequently activate the immune system. One nuclear protein, High mobility group box-1 (HMGB1), has received particular attention as fulfilling the functions of an alarmin by being involved in both infectious and non-infectious inflammatory conditions. Once released, HMGB1 signals through various receptors to activate immune cells involved in the immune process. Although initial studies demonstrated HMGB1 as a late mediator of sepsis, recent findings indicate HMGB1 to have an important role in models of non-infectious inflammation, such as autoimmunity, cancer, trauma, and ischemia reperfusion injury. Furthermore, in contrast to its pro-inflammatory functions, there is evidence that HMGB1 also has restorative effects leading to tissue repair and regeneration. The complex functions of HMGB1 as an archetypical alarmin are outlined here to review our current understanding of a molecule that holds the potential for treatment in many important human conditions.
4432093
Release of chromatin protein HMGB1 by necrotic cells triggers inflammation
This corrects the article DOI: 10.1038/nature00858
85601983
HMG-1 as a late mediator of endotoxin lethality in mice.
Endotoxin, a constituent of Gram-negative bacteria, stimulates macrophages to release large quantities of tumor necrosis factor (TNF) and interleukin-1 (IL-1), which can precipitate tissue injury and lethal shock (endotoxemia). Antagonists of TNF and IL-1 have shown limited efficacy in clinical trials, possibly because these cytokines are early mediators in pathogenesis. Here a potential late mediator of lethality is identified and characterized in a mouse model. High mobility group-1 (HMG-1) protein was found to be released by cultured macrophages more than 8 hours after stimulation with endotoxin, TNF, or IL-1. Mice showed increased serum levels of HMG-1 from 8 to 32 hours after endotoxin exposure. Delayed administration of antibodies to HMG-1 attenuated endotoxin lethality in mice, and administration of HMG-1 itself was lethal. Septic patients who succumbed to infection had increased serum HMG-1 levels, suggesting that this protein warrants investigation as a therapeutic target.
32196666
Cholinergic agonists inhibit HMGB1 release and improve survival in experimental sepsis
Physiological anti-inflammatory mechanisms can potentially be exploited for the treatment of inflammatory disorders. Here we report that the neurotransmitter acetylcholine inhibits HMGB1 release from human macrophages by signaling through a nicotinic acetylcholine receptor. Nicotine, a selective cholinergic agonist, is more efficient than acetylcholine and inhibits HMGB1 release induced by either endotoxin or tumor necrosis factor-alpha (TNF-α). Nicotinic stimulation prevents activation of the NF-κB pathway and inhibits HMGB1 secretion through a specific 'nicotinic anti-inflammatory pathway' that requires the α7 nicotinic acetylcholine receptor (α7nAChR). In vivo, treatment with nicotine attenuates serum HMGB1 levels and improves survival in experimental models of sepsis, even when treatment is started after the onset of the disease. These results reveal acetylcholine as the first known physiological inhibitor of HMGB1 release from human macrophages and suggest that selective nicotinic agonists for the α7nAChR might have therapeutic potential for the treatment of sepsis.
11059269
Convergence and amplification of toll-like receptor (TLR) and receptor for advanced glycation end products (RAGE) signaling pathways via high mobility group B1 (HMGB1)
Sustained proinflammatory responses in rheumatoid arthritis, atherosclerosis, and diabetic retinopathy, as well as in cancer, are often associated with increased angiogenesis that contributes to tissue disruption and disease progression. High mobility group B1 (HMGB1) has been recognized as a proinflammatory cytokine and more recently, as a proangiogenic factor. HMGB1 can either be passively released from necrotic cells or actively secreted in response to angiogenic and inflammatory signals. HMGB1 itself may signal through the receptor for advanced glycation end products (RAGE), and via toll-like receptors, TLR2 and TLR4. Activation of these receptors results in the activation of NFκB, which induces the upregulation of leukocyte adhesion molecules and the production of proinflammatory cytokines and angiogenic factors in both hematopoietic and endothelial cells, thereby promoting inflammation. Interestingly, HMGB1 seems to be involved in a positive feedback mechanism, that may help to sustain inflammation and angiogenesis in several pathological conditions, thereby contributing to disease progression. Endothelial cells express HMGB1, as well as the receptors RAGE, TLR2, and TLR4, and in diverse pathologies HMGB1 and its receptors are overexpressed. Furthermore, HMGB1-induced signaling can activate NFκB, which can subsequently induce the expression of HMGB1 receptors. Thus, HMGB1 can mediate amplification of inflammation and angiogenesis through increased secretion of HMGB1 and increased expression of the receptors it can interact with. In this review, we discuss signaling cascades that HMGB1 can induce via TLRs and RAGE, as well as its contribution to pathologies involving endothelial cells.
21350171
High mobility group box 1 protein interacts with multiple Toll-like receptors.
High mobility group box 1 (HMGB1), originally described as a DNA-binding protein, can also be released extracellularly and functions as a late mediator of inflammatory responses. Although recent reports have indicated that the receptor for advanced glycation end products (RAGE) as well as Toll-like receptor (TLR)2 and TLR4 are involved in cellular activation by HMGB1, there has been little evidence of direct association between HMGB1 and these receptors. To examine this issue, we used fluorescence resonance energy transfer (FRET) and immunoprecipitation to directly investigate cell surface interactions of HMGB1 with TLR2, TLR4, and RAGE. FRET images in RAW264.7 macrophages demonstrated association of HMGB1 with TLR2 and TLR4 but not RAGE. Transient transfections into human embryonic kidney-293 cells showed that HMGB1 induced cellular activation and NF-kappaB-dependent transcription through TLR2 or TLR4 but not RAGE. Coimmunoprecipitation also found interaction between HMGB1 and TLR2 as well as TLR4, but not with RAGE. These studies provide the first direct evidence that HMGB1 can interact with both TLR2 and TLR4 and also supply an explanation for the ability of HMGB1 to induce cellular activation and generate inflammatory responses that are similar to those initiated by LPS.
25677564
Involvement of Toll-like Receptors 2 and 4 in Cellular Activation by High Mobility Group Box 1 Protein*
High mobility group box 1 (HMGB1) protein, originally described as a DNA-binding protein that stabilizes nucleosomes and facilitates transcription, can also be released extracellularly during acute inflammatory responses. Exposure of neutrophils, monocytes, or macrophages to HMGB1 results in increased nuclear translocation of NF-κB and enhanced expression of proinflammatory cytokines. Although the receptor for advanced glycation end products (RAGE) has been shown to interact with HMGB1, other putative HMGB1 receptors are known to exist but have not been characterized. In the present experiments, we explored the role of RAGE, Toll-like receptor (TLR) 2, and TLR 4, as well as associated kinases, in HMGB1-induced cellular activation. Culture of neutrophils or macrophages with HMGB1 produced activation of NF-κB through TLR 4-independent mechanisms. Unlike lipopolysaccharide (LPS), which primarily increased the activity of IKKβ, HMGB1 exposure resulted in activation of both IKKα and IKKβ. Kinases and scaffolding proteins downstream of TLR 2 and TLR 4, but not TLR/interleukin-1 receptor (IL-1R)-independent kinases such as tumor necrosis factor receptor-associated factor 2, were involved in the enhancement of NF-κB-dependent transcription by HMGB1. Transfections with dominant negative constructs demonstrated that TLR 2 and TLR 4 were both involved in HMGB1-induced activation of NF-κB. In contrast, RAGE played only a minor role in macrophage activation by HMGB1. Interactions of HMGB1 with TLR 2 and TLR 4 may provide an explanation for the ability of HMGB1 to generate inflammatory responses that are similar to those initiated by LPS.
40793675
Toll-like receptor 9–dependent activation by DNA-containing immune complexes is mediated by HMGB1 and RAGE
Increased concentrations of DNA-containing immune complexes in the serum are associated with systemic autoimmune diseases such as lupus. Stimulation of Toll-like receptor 9 (TLR9) by DNA is important in the activation of plasmacytoid dendritic cells and B cells. Here we show that HMGB1, a nuclear DNA-binding protein released from necrotic cells, was an essential component of DNA-containing immune complexes that stimulated cytokine production through a TLR9–MyD88 pathway involving the multivalent receptor RAGE. Moreover, binding of HMGB1 to class A CpG oligodeoxynucleotides considerably augmented cytokine production by means of TLR9 and RAGE. Our data demonstrate a mechanism by which HMGB1 and RAGE activate plasmacytoid dendritic cells and B cells in response to DNA and contribute to autoimmune pathogenesis.
4311321
A human homologue of the Drosophila Toll protein signals activation of adaptive immunity
Induction of the adaptive immune response depends on the expression of co-stimulatory molecules and cytokines by antigen-presenting cells. The mechanisms that control the initial induction of these signals upon infection are poorly understood. It has been proposed that their expression is controlled by the non-clonal, or innate, component of immunity that preceded in evolution the development of an adaptive immune system in vertebrates. We report here the cloning and characterization of a human homologue of the Drosophila toll protein (Toll) which has been shown to induce the innate immune response in adult Drosophila. Like Drosophila Toll, human Toll is a type I transmembrane protein with an extracellular domain consisting of a leucine-rich repeat (LRR) domain, and a cytoplasmic domain homologous to the cytoplasmic domain of the human interleukin (IL)-1 receptor. Both Drosophila Toll and the IL-1 receptor are known to signal through the NF-κB pathway. We show that a constitutively active mutant of human Toll transfected into human cell lines can induce the activation of NF-κB and the expression of NF-κB-controlled genes for the inflammatory cytokines IL-1, IL-6 and IL-8, as well as the expression of the co-stimulatory molecule B7.1, which is required for the activation of naive T cells.
71447916
Inhibiting early activation of tissue nuclear factor-κB and nuclear factor interleukin 6 with (1→3)-β-D -glucan increases long-term survival in polymicrobial sepsis
Abstract Background: Recent data implicate the activation of nuclear factor-κB (NF-κB) and nuclear factor interleukin 6 (NF-IL6) as important steps in the pathophysiologic mechanisms of adult respiratory distress syndrome and systemic inflammatory response syndrome. Methods: This study evaluated the effect of immunomodulating polysaccharides on transcription factor activation, cytokine expression, and mortality in a murine cecal ligation and puncture (CLP) model. ICR/HSD mice were treated with glucan (50 mg/kg) 1 hour before or 15 minutes after CLP. Liver and lung tissue were harvested at 3 hours and mortality trends were observed for 20 days. Results: CLP increased liver and lung NF-κB and NF-IL6 nuclear binding activity as well as tumor necrosis factor-α and interleukin 6 messenger RNA levels at 3 hours. Pretreatment or posttreatment with glucans inhibited tissue NF-κB and NF-IL6 nuclear binding activity and tissue cytokine messenger RNA levels. Prophylaxis with glucan phosphate or scleroglucan increased ( P P Conclusions: Pretreatment or posttreatment with biologic response modifiers decreased tissue transcription factor nuclear binding activity and cytokine message in liver and lung of septic mice. Inhibiting early transcription factor activation and cytokine message expression correlates with improved outcome in polymicrobial sepsis as denoted by increased long-term survival. (Surgery 1999;126:54-65.)
7567487
Modulation of tissue Toll-like receptor 2 and 4 during the early phases of polymicrobial sepsis correlates with mortality.
OBJECTIVE
To determine whether there was a correlation between induction of polymicrobial sepsis, modulation of tissue Toll-like receptor (TLR) gene, and protein expression and survival outcome.


DESIGN
Prospective, randomized animal study.


SETTING
University medical school research laboratory.


SUBJECTS
Age- and weight-matched ICR/HSD mice.


INTERVENTIONS
Sepsis was induced by cecal ligation and puncture (CLP). No-surgery and sham (laparotomy)-operated mice were controls. We also examined tissue TLR2 and TLR4 messenger RNA and TLR4 protein levels in mice treated with an immunomodulator that increases survival in polymicrobial sepsis. In the immunomodulator study, mice were treated with glucan phosphate (50 mg/kg, intraperitoneally) 1 hr before CLP. No-surgery, sham surgery, glucan + no-surgery, sham surgery + glucan, and CLP groups were employed as controls.


MEASUREMENTS AND MAIN RESULTS
Total RNA was isolated from liver, lung, and spleen at 0, 1, 3, 6, 8, and 24 hrs after CLP. TLR gene expression was assessed by reverse transcription-polymerase chain reaction. Tissue TLR4 protein levels were evaluated at 24 hrs by Western blot and immunohistochemistry. CLP sepsis increased (p <.05) liver and lung TLR2 and TLR4 gene expression compared with controls. TLR4 protein concentrations also were increased. Increased TLR2/4 gene and TLR4 protein expression correlated with mortality. Immunoprophylaxis with glucan phosphate increased (p <.001) long-term survival (20% vs. 70%) but inhibited (p <.05) CLP-induced increases in tissue TLR2 and TLR4 messenger RNA expression as well as TLR4 protein expression.


CONCLUSIONS
Early increases in TLR2/4 gene and TLR4 protein expression correlated with mortality, whereas blunting TLR gene and protein expression correlated with improved long-term survival. This suggests that early up-regulation of tissue TLR2/4 may play a role in the proinflammatory response and pathophysiology of polymicrobial sepsis.
15204534
Cells migrating to sites of tissue damage in response to the danger signal HMGB1 require NF-κB activation
Tissue damage is usually followed by healing, as both differentiated and stem cells migrate to replace dead or damaged cells. Mesoangioblasts (vessel-associated stem cells that can repair muscles) and fibroblasts migrate toward soluble factors released by damaged tissue. Two such factors are high mobility group box 1 (HMGB1), a nuclear protein that is released by cells undergoing unscheduled death (necrosis) but not by apoptotic cells, and stromal derived factor (SDF)–1/CXCL12. We find that HMGB1 activates the canonical nuclear factor κB (NF-κB) pathway via extracellular signal-regulated kinase phosphorylation. NF-κB signaling is necessary for chemotaxis toward HMGB1 and SDF-1/CXCL12, but not toward growth factor platelet-derived growth factor, formyl-met-leu-phe (a peptide that mimics bacterial invasion), or the archetypal NF-κB–activating signal tumor necrosis factor α. In dystrophic mice, mesoangioblasts injected into the general circulation ingress inefficiently into muscles if their NF-κB signaling pathway is disabled. These findings suggest that NF-κB signaling controls tissue regeneration in addition to early events in inflammation.
43410271
Overexpression of cardiac I-κBα prevents endotoxin-induced myocardial dysfunction
Nuclear factor-κ B (NF-κB) is an inducible transcription factor that regulates expression of many genes, such as tumor necrosis factor-α (TNF-α), which may contribute to myocardial dysfunction. We ...
26011784
Modulation of the Phosphoinositide 3-Kinase Pathway Alters Innate Resistance to Polymicrobial Sepsis 1
We examined the effect of modulating phosphoinositide 3-kinase (PI3K) activity in a murine model of cecal ligation and puncture-induced polymicrobial sepsis. Inhibition of PI3K activity with wortmannin increased serum cytokine levels and decreased survival time in septic mice. We have reported that an immunomodulator, glucan phosphate, induces protection in murine polymicrobial sepsis. We observed that glucan stimulated tissue PI3K activity, which positively correlated with increased survival in septic mice. We investigated the effect of PI3K inhibition on survival in septic mice treated with glucan. Treatment of mice with the PI3K inhibitors, wortmannin and LY294002, completely eliminated the protective effect of glucan, indicating that protection against septic mortality was mediated through PI3K. Inhibition of PI3K resulted in increased serum levels of IL1-β, IL-2, IL-6, IL-10, IL-12, and TNF-α in septic mice. Apoptosis is thought to play a central role in the response to septic injury. We observed that inhibition of PI3K activity in septic mice resulted in increased splenocyte apoptosis and a change in the anatomic distribution of splenocyte apoptosis. We conclude that PI3K is a compensatory mechanism that suppresses proinflammatory and apoptotic processes in response to sepsis and/or inflammatory injury. Thus, PI3K may play a pivotal role in the maintenance of homeostasis and the integrity of the immune response during sepsis. We also observed that glucan phosphate decreased septic morbidity and mortality through a PI3K-dependent mechanism. This suggests that stimulation of the PI3K pathway may be an effective approach for preventing or treating sepsis and/or septic shock.
24590409
Protection against Myocardial Ischemia/Reperfusion Injury in TLR4-Deficient Mice Is Mediated through a Phosphoinositide 3-Kinase-Dependent Mechanism1
TLRs play a critical role in the induction of innate and adaptive immunity. However, TLRs have also been reported to mediate the pathophysiology of organ damage following ischemia/reperfusion (I/R) injury. We have reported that TLR4−/− mice show decreased myocardial injury following I/R; however, the protective mechanisms have not been elucidated. We examined the role of the PI3K/Akt signaling pathway in TLR4−/− cardioprotection following I/R injury. TLR4−/− and age-matched wild-type (WT) mice were subjected to myocardial ischemia for 45 min, followed by reperfusion for 4 h. Pharmacologic inhibitors of PI3K (wortmannin or LY294002) were administered 1 h before myocardial I/R. Myocardial infarct size/area at risk was reduced by 51.2% in TLR4−/− vs WT mice. Cardiac myocyte apoptosis was also increased in WT vs TLR4−/− mice following I/R. Pharmacologic blockade of PI3K abrogated myocardial protection in TLR4−/− mice following I/R. Specifically, heart infarct size/area at risk was increased by 98% in wortmannin and 101% in LY294002-treated TLR4−/− mice, when compared with control TLR4−/− mice. These data indicate that protection against myocardial I/R injury in TLR4−/− mice is mediated through a PI3K/Akt-dependent mechanism. The mechanisms by which PI3K/Akt are increased in the TLR4−/− myocardium may involve increased phosphorylation/inactivation of myocardial phosphatase and tensin homolog deleted on chromosome 10 as well as increased phosphorylation/inactivation of myocardial glycogen synthase kinase-3β. These data implicate innate immune signaling pathways in the pathology of acute myocardial I/R injury. These data also suggest that modulation of TLR4/PI3K/Akt-dependent signaling pathways may be a viable strategy for reducing myocardial I/R injury.
7839677
Development of a water-soluble, sulfated (1-->3)-beta-D-glucan biological response modifier derived from Saccharomyces cerevisiae.
This report describes a method for the solubilization of micro-particulate (1-->3)-beta-D-glucan. Insoluble glucan is dissolved in methyl sulfoxide and urea (8 M) and partially sulfated at 100 degrees. The resulting water-soluble product is called glucan sulfate. The conversion rate is 98%, and the preparation is endotoxin free as determined by the Limulus lysate procedure. Glucan sulfate is composed of 34.06% C, 6.15% H, 50.30% O, 5.69% S and 3.23% N, and has a repeating unit empirical formula of (C6H10O5)8.3 SO3NH4+.4 H2O, suggesting that, on the average, a sulfate group is substituted on every third glucose subunit along the polymer. Molecular weight averages, polydispersity, and intrinsic viscosity were determined by aqueous high-performance size-exclusion chromatography (HPSEC). Two polymer peaks were resolved. Peak 1 (Mw = 1.25 x 10(6) g/mol) represents < 1% of the total polymer mass. Peak 2 (Mw = 1.45 x 10(4) g/mol) comprises > 99% of polymers. 13C NMR spectroscopy confirmed the beta-(1-->3) interchain linkage. In solution, glucan sulfate polymers self-associate in a triple helix. Glucan sulfate stimulates murine bone marrow proliferation following intravenous administration. The ability to prepare a immunologically active, water-soluble (1-->3)-beta-D-glucan preparation will greatly enhance the clinical utility of this class of compounds.
22428190
Modulating Toll-like receptor mediated signaling by (1-->3)-beta-D-glucan rapidly induces cardioprotection.
OBJECTIVE
Immune and inflammatory signaling pathways, initiated by the innate response, are involved in myocardial ischemia/reperfusion (I/R) injury. Toll-like receptor (TLR) mediated MyD88-dependent NFkappaB pathways play a role in the induction of innate immunity. We have reported that glucan phosphate (GP) improved survival in experimental sepsis, which correlated with decreased tissue NFkappaB activation. In the present study, we report that GP rapidly induced cardioprotection against I/R injury in vivo.


METHODS
Sprague-Dawley rats were pretreated with GP (40 mg/kg, i.p) 1 h before 45 min of ligation of the left anterior descending coronary followed by reperfusion for 4 and 24 h. Infarction size was examined by triphenyltetrazolium chloride (TTC) staining. NFkappaB activation was analyzed by electrophoretic mobility shift assay (EMSA). IkappaB kinase-beta (IKKbeta), IL-1 receptor-associated kinase (IRAK) and Phosphoinositide 3-kinase (PI3K) activities were determined by kinase assay with appropriate substrates. Association of TLR4 with MyD88 or with PI3K p85 was assessed by immunoprecipitation with anti-TLR4 followed by immunoblotting with anti-MyD88 or anti-p85.


RESULTS
GP treatment reduced infarct size by 47% in rat hearts subjected to reperfusion for 4 h and by 50% following reperfusion for 24 h. The same protective effect was observed when GP was administrated 5 min after initiation of ischemia. The mechanisms of GP induced cardioprotection involve decreased association of TLR4 with MyD88, inhibition of I/R induced IRAK and IKKbeta activity and decreased NFkappaB activity. In addition, GP increased TLR4 phosphotyrosine, resulting in increasing PI3K/Akt activity in the myocardium, which correlated with decreased cardiac myocyte apoptosis following I/R.


CONCLUSION
The results suggest that activation of the TLR mediated MyD88-dependent NFkappaB signaling pathway may play an important role in myocardial I/R injury, while stimulation of the PI3K/Akt signaling could serve a protective role. The data indicates that GP treatment shifts the TLR mediated activation signal in I/R from a predominantly NFkappaB pathway to a predominant PI3K/Akt signaling pathway.
4518545
Early activation of transcription factor NF-κB during ischemia in perfused rat heart.
The transcription factor nuclear factor κB (NF-κB) regulates multiple immediate-early gene expressions involved in immune and inflammatory responses and cellular defenses. Ischemia-reperfusion induces many immediate-early gene expressions, but little is known about the NF-κB activation in myocardium during ischemia and reperfusion. This study demonstrated that ischemia alone rapidly induced NF-κB activation in the myocardium of isolated working rat hearts. Electrophoretic mobility shift assay showed that NF-κB binding activity significantly increased in the nucleus after 5 min of ischemia and remained elevated for up to 30 min. Western blot analysis suggested that the levels of inhibitory IκBα protein in the cytoplasm became markedly decreased at 4, 5, 7.5, and 10 min of ischemia but were gradually restored following 10 min of ischemia. Reduction of IκBα protein in the cytoplasm by ischemia resulted in NF-κB translocation to the nucleus. Northern blot hybridization showed that IκBα mRNA levels were not significantly elevated during myocardial ischemia. Pyrrolidine dithiocarbamate, an antioxidant, significantly inhibited the loss of IκBα protein from the cytoplasm and prevented NF-κB binding activity in the nucleus. Reperfusion following short periods of ischemia augmented NF-κB binding activity in the nucleus induced by ischemia. The results suggest that early activation of NF-κB induced by ischemia in the myocardium could be a signal mechanism for controlling and regulating immediate-early gene expression during ischemia-reperfusion.
6763641
Blockade of MyD88 attenuates cardiac hypertrophy and decreases cardiac myocyte apoptosis in pressure overload-induced cardiac hypertrophy in vivo.
In this study, we evaluated whether blocking myeloid differentiation factor-88 (MyD88) could decrease cardiac myocyte apoptosis following pressure overload. Adenovirus expressing dominant negative MyD88 (Ad5-dnMyD88) or Ad5-green fluorescent protein (GFP) (Ad5-GFP) was transfected into rat hearts (n = 8/group) immediately followed by aortic banding for 3 wk. One group of rats (n = 8) was subjected to aortic banding for 3 wk without transfection. Sham surgical operation (n = 8) served as control. The ratios of heart weight to body weight (HW/BW) and heart weight to tibia length (HW/TL) were calculated. Cardiomyocyte size was examined by FITC-labeled wheat germ agglutinin staining of membranes. Cardiac myocyte apoptosis was determined by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling assay, and myocardial interstitial fibrosis was examined by Masson's Trichrome staining. Aortic banding significantly increased the HW/BW by 41.0% (0.44 +/- 0.013 vs. 0.31 +/- 0.008), HW/TL by 47.2% (42.7 +/- 1.30 vs. 29.0 +/- 0.69), cardiac myocyte size by 49.6%, and cardiac myocyte apoptosis by 11.5%, and myocardial fibrosis and decreased cardiac function compared with sham controls. Transfection of Ad5-dnMyD88 significantly reduced the HW/BW by 18.2% (0.36 +/- 0.006 vs. 0.44 +/- 0.013) and HW/TL by 22.3% (33.2 +/- 0.95 vs. 42.7 +/- 1.30) and decreased cardiomyocyte size by 56.8%, cardiac myocyte apoptosis by 76.2%, as well as fibrosis, and improved cardiac function compared with aortic-banded group. Our results suggest that MyD88 is an important component in the Toll-like receptor-4-mediated nuclear factor-kappaB activation pathway that contributes to the development of cardiac hypertrophy. Blockade of MyD88 significantly reduced cardiac hypertrophy, cardiac myocyte apoptosis, and improved cardiac function in vivo.
84683660
NF-κB activation is required for the development of cardiac hypertrophy in vivo
In the present study, we examined whether NF-κB activation is required for cardiac hypertrophy in vivo. Cardiac hypertrophy in rats was induced by aortic banding for 1, 3, and 5 days and 1–6 wk, an...
795399
TLR2 ligands attenuate cardiac dysfunction in polymicrobial sepsis via a phosphoinositide 3-kinase-dependent mechanism.
Myocardial dysfunction is a major consequence of septic shock and contributes to the high mortality of sepsis. In the present study, we examined the effect of Toll-like receptor 2 (TLR2) ligands, peptidoglycan (PGN), and Pam3CSK4 (Pam3) on cardiac function in cecal ligation and puncture (CLP)-induced sepsis in mice. We also investigated whether the phosphoinositide 3-kinase (PI3K)/Akt signaling pathway is involved in the effect of TLR2 ligands on cardiac function in CLP mice. PGN was administered to C57B6/L mice 1 h before the induction of CLP. Sham surgically operated mice served as a control. Cardiac function indexes (rate of change in left ventricular pressure, stroke work, cardiac output, and ejection fraction) were examined by a microconductance pressure catheter. Cardiac function was significantly decreased 6 h after CLP-induced sepsis compared with sham-operated control. In contrast, PGN administration attenuated CLP-induced cardiac dysfunction. Importantly, the therapeutic treatment with Pam3 1 h after CLP also significantly attenuated cardiac dysfunction in CLP mice. However, the beneficial effect of TLR2 ligands on cardiac dysfunction in CLP-mice was abolished in TLR2-deficient mice. PGN administration significantly increased the levels of phospho-Akt and phospho-GSK-3beta in the myocardium compared with the levels in untreated CLP mice. PI3K inhibition abolished the PGN-induced attenuation of cardiac dysfunction in CLP mice. In conclusion, these data demonstrate that the administration of TLR2 ligands, PGN, or Pam3 attenuates cardiac dysfunction in septic mice via a TLR2/PI3K-dependent mechanism. More significantly, Pam3 therapeutic treatment will have a potential clinical relevance.
9335248
TLR2 ligands induce cardioprotection against ischaemia/reperfusion injury through a PI3K/Akt-dependent mechanism.
AIMS
Toll-like receptor (TLR)-mediated signalling pathways have been implicated in myocardial ischaemia/reperfusion (I/R) injury. Activation of the phosphoinositide 3-kinase (PI3K)/Akt pathway protects the myocardium from ischaemic injury. We hypothesized that the modulation of TLR2 would induce cardioprotection against I/R injury via activation of the PI3K/Akt signalling.


METHODS AND RESULTS
Mice were treated with TLR2 ligands, peptidoglycan (PGN) or Pam3CSK4, respectively, 1 h before the hearts were subjected to ischaemia (1 h), followed by reperfusion (4 h). Infarct size was determined by triphenyltetrazolium chloride staining. Cardiac function and haemodynamic performance were evaluated. Infarct size was significantly reduced in PGN- or Pam3CSK4-treated mice compared with untreated I/R mice. Administration of TLR2 ligands improved cardiac function following I/R. PGN treatment increased the levels of phospho-Akt and phospho-GSK-3beta (glycogen synthase kinase-3beta), compared with untreated I/R hearts. PGN stimulation increased TLR2 tyrosine phosphorylation and association of the p85 subunit of PI3K with TLR2. To investigate the role of PI3K/Akt signalling in PGN-induced cardioprotection, we administered the PI3K inhibitor, Wortmannin, to the mice 15 min before PGN treatment. We also administered PGN to kinase-deficient Akt (kdAkt) transgenic mice 1 h before myocardial I/R. Both PI3K inhibition and kdAkt mice abolished the cardioprotection induced by PGN. To examine the role of TLR2 in PGN-induced cardioprotection, we administrated PGN to TLR2 knockout mice 1 h before the hearts were subjected to I/R. PGN-induced cardioprotection was lost in TLR2-deficient mice.


CONCLUSION
These results demonstrate that TLR2 ligands induced cardioprotection, which is mediated through a TLR2/PI3K/Akt-dependent mechanism.
73111012
IMPLICATION OF TOLL-LIKE RECEPTOR AND TUMOR NECROSIS FACTOR α SIGNALING IN SEPTIC SHOCK
Septic shock is initiated by a systemic inflammatory response to microbial infection that frequently leads to impaired perfusion and multiple organ failure. Because of its high risk of death, septic shock is a major problem particularly for patients in the intensive care unit. In general, bacterial lipopolysaccharide (LPS) is a strong activator of various immune responses and stimulates monocytes/macrophages to release a variety of inflammatory cytokines. However, overproduction of inflammatory factors in response to bacterial infections is known to cause septic shock, similar to that induced by LPS. Studies of LPS-signaling pathways and downstream inflammatory cytokines may have critical implications in the treatment of sepsis. In recent years, there has been significant progress in understanding the signaling pathways activated by LPS and its receptor Toll-like receptor 4 (TLR4), as well as by tumor necrosis factor α (TNFα), a potent inflammatory cytokine induced by LPS stimulation. This review briefly summarizes our current knowledge of these signaling pathways and critical signal transducers. Characterization of key signal transducers may allow us to identify tractable, novel targets for the therapeutic interventions of sepsis.
12444354
Bench-to-bedside review: Toll-like receptors and their role in septic shock
The Toll-like receptors (TLRs) are essential transmembrane signaling receptors of the innate immune system that alert the host to the presence of a microbial invader. The recent discovery of the TLRs has rapidly expanded our knowledge of molecular events that initiate host–pathogen interactions. These functional attributes of the cellular receptors provide insights into the nature of pattern recognition receptors that activate the human antimicrobial defense systems. The fundamental significance of the TLRs in the generation of systemic inflammation and the pathogenesis of septic shock is reviewed. The potential clinical implications of therapeutic modulation of these recently characterized receptors of innate immunity are also discussed.
639653
Toll-like receptors: critical proteins linking innate and acquired immunity
Recognition of pathogens is mediated by a set of germline-encoded receptors that are referred to as pattern-recognition receptors (PRRs). These receptors recognize conserved molecular patterns (pathogen-associated molecular patterns), which are shared by large groups of microorganisms. Toll-like receptors (TLRs) function as the PRRs in mammals and play an essential role in the recognition of microbial components. The TLRs may also recognize endogenous ligands induced during the inflammatory response. Similar cytoplasmic domains allow TLRs to use the same signaling molecules used by the interleukin 1 receptors (IL-1Rs): these include MyD88, IL-1R–associated protein kinase and tumor necrosis factor receptor–activated factor 6. However, evidence is accumulating that the signaling pathways associated with each TLR are not identical and may, therefore, result in different biological responses.
1145018
High Mobility Group 1 Protein (Hmg-1) Stimulates Proinflammatory Cytokine Synthesis in Human Monocytes
Lipopolysaccharide (LPS) is lethal to animals because it activates cytokine release, causing septic shock and tissue injury. Early proinflammatory cytokines (e.g., tumor necrosis factor [TNF] and interleukin [IL]-1) released within the first few hours of endotoxemia stimulate mediator cascades that persist for days and can lead to death. High mobility group 1 protein (HMG-1), a ubiquitous DNA-binding protein, was recently identified as a “late” mediator of endotoxin lethality. Anti–HMG-1 antibodies neutralized the delayed increase in serum HMG-1, and protected against endotoxin lethality, even when passive immunization was delayed until after the early cytokine response. Here we examined whether HMG-1 might stimulate cytokine synthesis in human peripheral blood mononuclear cell cultures. Addition of purified recombinant HMG-1 to human monocyte cultures significantly stimulated the release of TNF, IL-1α, IL-1β, IL-1RA, IL-6, IL-8, macrophage inflammatory protein (MIP)-1α, and MIP-1β; but not IL-10 or IL-12. HMG-1 concentrations that activated monocytes were within the pathological range previously observed in endotoxemic animals, and in serum obtained from septic patients. HMG-1 failed to stimulate cytokine release in lymphocytes, indicating that cellular stimulation was specific. Cytokine release after HMG-1 stimulation was delayed and biphasic compared with LPS stimulation. Computer-assisted image analysis demonstrated that peak intensity of HMG-1–induced cellular TNF staining was comparable to that observed after maximal stimulation with LPS. Administration of HMG-1 to Balb/c mice significantly increased serum TNF levels in vivo. Together, these results indicate that, like other cytokine mediators of endotoxin lethality (e.g., TNF and IL-1), extracellular HMG-1 is a regulator of monocyte proinflammatory cytokine synthesis.
22073796
The Phosphatidylinositol 3-Kinase-Akt Pathway Limits Lipopolysaccharide Activation of Signaling Pathways and Expression of Inflammatory Mediators in Human Monocytic Cells*
Monocytes and macrophages express cytokines and procoagulant molecules in various inflammatory diseases. In sepsis, lipopolysaccharide (LPS) from Gram-negative bacteria induces tumor necrosis factor-alpha (TNF-α) and tissue factor (TF) in monocytic cells via the activation of the transcription factors Egr-1, AP-1, and nuclear factor-κB. However, the signaling pathways that negatively regulate LPS-induced TNF-α and TF expression in monocytic cells are currently unknown. We report that inhibition of the phosphatidylinositol 3-kinase (PI3K)-Akt pathway enhances LPS-induced activation of the mitogen-activated protein kinase pathways (ERK1/2, p38, and JNK) and the downstream targets AP-1 and Egr-1. In addition, inhibition of PI3K-Akt enhanced LPS-induced nuclear translocation of nuclear factor-κB and prevented Akt-dependent inactivation of glycogen synthase kinase-β, which increased the transactivational activity of p65. We propose that the activation of the PI3K-Akt pathway in human monocytes limits the LPS induction of TNF-α and TF expression. Our study provides new insight into the inhibitory mechanism by which the PI3K-Akt pathway ensures transient expression of these potent inflammatory mediators.