Insulin-like growth factor-1 improves survival in sepsis via enhanced hepatic bacterial clearance.
ABSTRACT: Both insulin-like growth factor (IGF)-1 and bacterial clearance by Kupffer cells are significantly reduced in severe sepsis. Kupffer cell apoptosis is triggered by tumor necrosis factor (TNF)-alpha and activation of the PI-3 kinase pathway prevents TNF-induced Kupffer cell death.We evaluated if the marked decline in IGF-1 is related to bacterial clearance in sepsis.Sepsis was induced in C57BL/6 mice by intratracheal inoculation with Pseudomonas aeruginosa (strain PA103). Some mice received IGF-1 24 mg/kg either before infection or 12 hours after infection. In vitro studies were performed using the clonal Kupffer cell line KC13-2.Sepsis resulted in decreased levels of IGF-1. In vitro studies with KC13-2 cells demonstrated that IGF-1 protected Kupffer cells against TNF-alpha-induced apoptosis by activating the PI-3 kinase pathway and stabilizing the inhibitor of apoptosis protein, XIAP. In the animal model, pretreatment with IGF-1 decreased hepatic TNF-alpha and IL-6, improved hepatic bacterial clearance as demonstrated by real-time polymerase chain reaction with primers specific for P. aeruginosa, and improved survival in severe sepsis. Moreover, we rescued mice from severe sepsis by IGF-1 treatment 12 hours after infection.These studies show that the decline in IGF-1 levels in sepsis is related to bacterial clearance and that replacement of IGF-1 in a murine model of sepsis improves overall survival.
Project description:Liver injury has a critical effect on the severity and outcome of sepsis. The impact of stored red blood cells (RBCs) on the pathogenesis of sepsis-associated hepatic injury is not well understood. Therefore, to investigate the effects of stored-RBC transfusion on sepsis-induced liver damage as well as the associated mechanism, we constructed a sepsis mouse model enabling noninvasive imaging of bacterial infection caused by Pseudomonas aeruginosa, a common gram-negative respiratory pathogen. We showed that transfusions with stored RBCs enhanced sepsis-induced liver injury in vivo, and liver injury exacerbated the severity of sepsis and decreased survival in P aeruginosa-infected mice. Stored-RBC transfusions enhanced the production of proinflammatory cytokines such as tumor necrosis factor (TNF)-?, interleukin 6 (IL-6), and IL-1?, which play important roles in sepsis-associated liver injury in P aeruginosa-infected mice. Further study showed that the enhanced inflammation observed was associated with increased activation of M1-polarized Kupffer cells, which produce many inflammatory cytokines, including TNF-? and IL-6. Moreover, the M1-polarized Kupffer cells and secreted proinflammatory cytokines exerted their effects on hepatocytes through enhanced Jun N-terminal kinase activation and inhibited nuclear factor-kappaB activation, demonstrating that transfusion with stored RBCs disrupted the balance between cell survival and cell death in the liver. Understanding the mechanisms whereby stored RBCs might contribute to these complications will likely be helpful in providing guidance toward making transfusions safer.
Project description:In a previous publication, we were able to show that irradiation of Kupffer cells, the liver resident macrophages, leads to an increased TNF-alpha concentration in the culture medium. The pathomechanisms underlying this phenomenon, however, remained to be elucidated. Here, we show that following irradiation of Kupffer cells, the apoptosis rate increased drastically within 48 h. At the same time, the total TNF-alpha concentration in cell lysates of Kupffer cells attached to the culture plate decreased. However, normalization of the TNF-alpha concentration with respect to cell number revealed that TNF-alpha concentration per attached cell remained constant during the observation period. Western blot analysis showed that heat shock protein 27 (Hsp27) is strongly downregulated and bax is upregulated in irradiated Kupffer cells as compared to sham-irradiated cells. Overexpression of Hsp27 in Kupffer cells was shown to prevent the effect of irradiation on bax expression, apoptosis and, at the same time, on increase of TNF-alpha concentration in the Kupffer cell medium. We conclude that irradiation of Kupffer cells leads to apoptosis because of downregulation of Hsp27 and consecutive upregulation of bax expression. Furthermore, we suggest that apoptosis of Kupffer cells leads to an increase of TNF-alpha concentration in the culture medium which may be due to cell death rather than active release or synthesis.
Project description:Proinflammatory cytokines released by hepatic macrophages (Kupffer cells) have a central role in the pathogenesis of liver injury and the cardiovascular abnormalities of sepsis. Because cytokine release is controlled primarily at the level of gene expression, intracellular signalling mechanisms that control the transcription of cytokine genes are critical links to organ injury. Oxidant stress up-regulates and antioxidants down-regulate the pleiotropic transcription factor NF-kappa B, a DNA-binding protein that induces the expression of cytokines and vascular adhesion molecules. Thiol-bearing molecules are also important inhibitors of NF-kappa B activation, but whether this inhibition represents an antioxidant effect is unknown. This study was undertaken to determine whether important endogenous and pharmacological thiols modulate the activation of NF-kappa B and the release of tumour necrosis factor alpha (TNF-alpha) from Kupffer cells and to ascertain whether these effects are mediated through glutathione. Exposure of rat Kupffer cells to a physiologically relevant concentration of lipopolysaccharide (10 ng/ml) activated NF-kappa B within 1 h and induced the release of TNF-alpha over 5 h. Cellular glutathione content remained unchanged after lipopolysaccharide exposure, but both glutathione monoethyl ester and N-acetyl-L-cysteine increased cellular glutathione levels, blocked NF-kappa B activation and inhibited the release of TNF-alpha. Inhibition of glutathione synthesis prevented the NAC-induced increase in Kupffer cell glutathione, yet it did not prevent the inhibition of TNF-alpha release by NAC. Thus the inhibition of NF-kappa B activation by pharmacological thiols such as NAC might reflect a more general role of the intracellular thiol redox status in NF-kappa B regulation rather than the antioxidant properties of these agents.
Project description:<h4>Background</h4>An imbalance between overwhelming inflammation and lymphocyte apoptosis is the main cause of high mortality in patients with sepsis. Baicalin, the main active ingredient of the Scutellaria root, exerts anti-inflammatory, anti-apoptotic, and even antibacterial properties in inflammatory and infectious diseases. However, the therapeutic effect of baicalin on polymicrobial sepsis remains unknown.<h4>Methodology/principal findings</h4>Polymicrobial sepsis was induced by cecal ligation and puncture (CLP) in C57BL/6 mice. Mice were infused with baicalin intraperitoneally at 1 h, 6 h and 12 h after CLP. Survival rates were assessed over the subsequent 8 days. Bacterial burdens in blood and peritoneal cavity were calculated to assess the bacterial clearance. Neutrophil count in peritoneal lavage fluid was also calculated. Injuries to the lung and liver were detected by hematoxylin and eosin staining. Levels of cytokines, including tumor necrosis factor (TNF)-alpha, interleukin (IL)-6, IL-10 and IL-17, in blood and peritoneum were measured by enzyme-linked immunosorbent assay. Adaptive immune function was assessed by apoptosis of lymphocytes in the thymus and counts of different cell types in the spleen. Baicalin significantly enhanced bacterial clearance and improved survival of septic mice. The number of neutrophils in peritoneal lavage fluid was reduced by baicalin. Less neutrophil infiltration of the lung and liver in baicalin-treated mice was associated with attenuated injuries to these organs. Baicalin significantly reduced the levels of proinflammatory cytokines but increased the level of anti-inflammatory cytokine in blood and peritoneum. Apoptosis of CD3(+) T cell was inhibited in the thymus. The numbers of CD4(+), CD8(+) T lymphocytes and dendritic cells (DCs) were higher, while the number of CD4(+)CD25(+) regulatory T cells was lower in the baicalin group compared with the CLP group.<h4>Conclusions/significance</h4>Baicalin improves survival of mice with polymicrobial sepsis, and this may be attributed to its antibacterial property as well as its anti-inflammatory and anti-apoptotic effects.
Project description:G2A is a GPCR abundantly expressed in immune cells. G2A-/- mice showed higher lethality, higher plasma cytokines, and an impaired bacterial clearance in response to a murine model of sepsis (cecal ligation and puncture), which were blocked by GdCl3, an inhibitor of Kupffer cells. Anti-IL-10 Ab reversed the impaired bacterial clearance in G2A-/- mice. Indomethacin effectively blocked both the increased i.p. IL-10 levels and the impaired bacterial clearance, indicating that disturbed PG system is the proximal cause of these phenomena. Stimulation with LPS/C5a induced an increase in Escherichia coli phagocytosis and intracellular cAMP levels in G2A+/+ peritoneal macrophages but not G2A-/- cells, which showed more PGE2/nitrite release and intracellular reactive oxygen species levels. Heterologous coexpression of G2A and adenosine receptor type 2b (A2bAR) induced a synergistic increase in cAMP signaling in a ligand-independent manner, with the evidence of physical interaction of G2A with A2bAR. BAY 60-6583, a specific agonist for A2bAR, increased intracellular cAMP levels in Kupffer cells from G2A+/+ but not from G2A-/- mice. Both G2A and A2bAR were required for antiseptic action of lysophosphatidylcholine. These results show inappropriate activation of G2A-/- Kupffer cells to septic insults due to an impaired cAMP signaling possibly by lack of interaction with A2bAR.
Project description:INTRODUCTION: Ischemia/reperfusion occurs in myocardial infarction, cardiac dysfunction during sepsis, cardiac transplantation and coronary artery bypass grafting, and results in injury to the myocardium. Although reperfusion injury is related to the nature and duration of ischemia, it is also a separate entity that may jeopardize viable cells and ultimately may impair cardiac performance once ischemia is resolved and the organ heals. METHOD: The present study was conducted in an ex vivo murine model of myocardial ischemia/reperfusion injury. After 20 min of ischemia, isolated hearts were perfused for up to 2 hours with solution (modified Kreb's) only, solution plus insulin-like growth factor (IGF)-1, or solution plus tumor necrosis factor (TNF)-alpha. Cardiac contractility was monitored continuously during this period of reperfusion. RESULTS: On the basis of histologic evidence, IGF-1 prevented reperfusion injury as compared with TNF-alpha; TNF-alpha increased perivascular interstitial edema and disrupted tissue lattice integrity, whereas IGF-1 maintained myocardial cellular integrity and did not increase edema. Also, there was a significant reduction in detectable creatine phosphokinase in the perfusate from IGF-1 treated hearts. By recording transduced pressures generated during the cardiac cycle, reperfusion with IGF-1 was accompanied by markedly improved cardiac performance as compared with reperfusion with TNF-alpha or modified Kreb's solution only. The histologic and functional improvement generated by IGF-1 was characterized by maintenance of the ratio of mitochondrial to nuclear DNA within heart tissue. CONCLUSION: We conclude that IGF-1 protects ischemic myocardium from further reperfusion injury, and that this may involve mitochondria-dependent mechanisms.
Project description:Basophils are evolutionarily conserved in vertebrates, despite their small numbers and short life span, suggesting that they have beneficial roles in maintaining health. However, these roles are not fully defined. Here we demonstrate that basophil-deficient mice exhibit reduced bacterial clearance and increased morbidity and mortality in the cecal ligation and puncture (CLP) model of sepsis. Among the several proinflammatory mediators that we measured, tumor necrosis factor (TNF) was the only cytokine that was significantly reduced in basophil-deficient mice after CLP. In accordance with that observation, we found that mice with genetic ablation of Tnf in basophils exhibited reduced systemic concentrations of TNF during endotoxemia. Moreover, after CLP, mice whose basophils could not produce TNF, exhibited reduced neutrophil and macrophage TNF production and effector functions, reduced bacterial clearance, and increased mortality. Taken together, our results show that basophils can enhance the innate immune response to bacterial infection and help prevent sepsis.
Project description:OBJECTIVES:In vivo lipopolysaccharide (LPS) tolerance on bacterial infection was investigated, focusing on liver macrophages. METHODS:LPS tolerance was induced by intraperitoneal injections with 5 μg/kg of LPS for 3 consecutive days, and then mice were intravenously infected with Escherichia coli. RESULTS:All LPS-primed mice survived lethal bacterial infection. Drastic enhancement of bactericidal activity of liver macrophages strongly contributed to bacterial clearance. Although LPS-primed mice produced substantial amounts of tumor necrosis factor (TNF) inside the liver, TNF efflux into the systemic circulation was markedly suppressed. These mice showed a dramatic increase in CD11b+ monocyte- derived macrophages in the liver. The CD11b+ macrophages that increased in LPS-primed mice were those with strong phagocytic/bactericidal activity and an upregulated expression of Fcγ receptor I, but the subfraction with a potent TNF-producing capacity and poor phagocytic activity diminished. The adoptive transfer of CD11b+ macrophages from LPS-primed mice to control mice increased survival after bacterial infection and reduced the elevation of plasma TNF. LPS priming did not affect the CD68+ resident Kupffer cells, and CD68+ Kupffer cell-depleted mice still exhibited LPS tolerance with strong resistance to bacteremia. CONCLUSIONS:LPS tolerance recruits CD11b+ macrophages to the liver with enhanced bactericidal activity, which plays a central role in resistance to lethal bacteremia.
Project description:Sepsis, a highly lethal systemic inflammatory syndrome, is associated with increases of proinflammatory cytokines (e.g., TNF-alpha, HMGB1) and the accumulation of apoptotic cells that have the potential to be detrimental. Depending on the timing and tissue, prevention of apoptosis in sepsis is beneficial; however, thwarting the development of secondary necrosis through the active removal of apoptotic cells by phagocytosis may offer a novel anti-sepsis therapy. Immature dendritic cells (IDCs) release exosomes that contain milk fat globule EGF factor VIII (MFGE8), a protein required to opsonize apoptotic cells for phagocytosis. In an experimental sepsis model using cecal ligation and puncture, we found that MFGE8 levels decreased in the spleen and blood, which was associated with impaired apoptotic cell clearance. Administration of IDC-derived exosomes promoted phagocytosis of apoptotic cells and significantly reduced mortality. Treatment with recombinant MFGE8 was equally protective, whereas MFGE8-deficient mice suffered from increased mortality. IDC exosomes also attenuated the release of proinflammatory cytokines in septic rats. Liberation of HMGB1, a nuclear protein that contributes to inflammation upon release from unengulfed apoptotic cells, was prevented by MFGE8-mediated phagocytosis in vitro. We conclude that IDC-derived exosomes attenuate the acute systemic inflammatory response in sepsis by enhancing apoptotic cell clearance via MFGE8.
Project description:Altered expression and activity of immunomodulatory cytokines plays a major role in the pathogenesis of alcoholic liver disease. Chronic ethanol feeding increases the sensitivity of Kupffer cells, the resident hepatic macrophage, to lipopolysaccharide (LPS), leading to increased tumor necrosis factor alpha (TNF-alpha) expression. This sensitization is normalized by treatment of primary cultures of Kupffer cells with adiponectin, an anti-inflammatory adipokine. Here we tested the hypothesis that adiponectin-mediated suppression of LPS signaling in Kupffer cells is mediated via an interleukin-10 (IL-10)/heme oxygenase-1 (HO-1) pathway after chronic ethanol feeding. Knockdown of IL-10 expression in primary cultures of Kupffer cells with small interfering RNA (siRNA) prevented the inhibitory effect of globular adiponectin (gAcrp) on LPS-stimulated TNF-alpha expression. gAcrp increased IL-10 mRNA and protein expression, as well as expression of the IL-10 inducible gene, HO-1; expression was higher in Kupffer cells from ethanol-fed rats compared with pair-fed controls. Although IL-10 receptor surface expression on Kupffer cells was not affected by ethanol feeding, IL-10-mediated phosphorylation of STAT3 and expression of HO-1 was higher in Kupffer cells after ethanol feeding. Inhibition of HO-1 activity, either by treatment with the HO-1 inhibitor zinc protoporphyrin or by siRNA knockdown of HO-1, prevented the inhibitory effect of gAcrp on LPS-stimulated TNF-alpha expression in Kupffer cells. LPS-stimulated TNF-alpha expression in liver was increased in mice after chronic ethanol exposure. When mice were treated with cobalt protoporphyrin to induce HO-1 expression, ethanol-induced sensitivity to LPS was ameliorated.gAcrp prevents LPS-stimulated TNF-alpha expression in Kupffer cells through the activation of the IL-10/STAT3/HO-1 pathway. Kupffer cells from ethanol-fed rats are highly sensitive to the anti-inflammatory effects of gAcrp; this sensitivity is associated with both increased expression and sensitivity to IL-10.