Resistance of Mice of the 129 Background to Yersinia pestis Maps to Multiple Loci on Chromosome 1.

Yersinia pestis is a Gram-negative bacterium that is the causative agent of bubonic and pneumonic plague. It is commonly acquired by mammals such as rodents and humans via the bite of an infected flea. We previously reported that multiple substrains of the 129 mouse background are resistant to pigmentation locus-negative (pgm(-)) Yersinia pestis and that this phenotype maps to a 30-centimorgan (cM) region located on chromosome 1. In this study, we have further delineated this plague resistance locus to a region of less than 20 cM through the creation and phenotyping of recombinant offspring arising from novel crossovers in this region. Furthermore, our experiments have revealed that there are at least two alleles in this initial locus, both of which are required for resistance on a susceptible C57BL/6 background. These two alleles work in trans since resistance is restored in offspring possessing one allele contributed by each parent. Our studies also indicated that the Slc11a1 gene (formerly known as Nramp1) located within the chromosome1 locus is not responsible for conferring resistance to 129 mice.

Protease activated receptor-2 contributes to heart failure.

Heart failure is a major clinical problem worldwide. Previous studies have demonstrated an important role for G protein-coupled receptors, including protease-activated receptors (PARs), in the pathology of heart hypertrophy and failure. Activation of PAR-2 on cardiomyocytes has been shown to induce hypertrophic growth in vitro. PAR-2 also contributes to myocardial infarction and heart remodeling after ischemia/reperfusion injury. In this study, we found that PAR-2 induced hypertrophic growth of cultured rat neonatal cardiomyocytes in a MEK1/2 and p38 dependent manner. In addition, PAR-2 activation on mouse cardiomyocytes increased expression of the pro-fibrotic chemokine MCP-1. Furthermore, cardiomyocyte-specific overexpression of PAR-2 in mice induced heart hypertrophy, cardiac fibrosis, inflammation and heart failure. Finally, in a mouse model of myocardial infarction induced by permanent ligation of the left anterior descending coronary artery, PAR-2 deficiency attenuated heart remodeling and improved heart function independently of its contribution to the size of the initial infarct. Taken together, our data indicate that PAR-2 signaling contributes to the pathogenesis of hypertrophy and heart failure.

Hematopoietic and nonhematopoietic cell tissue factor activates the coagulation cascade in endotoxemic mice.

Tissue factor (TF) is the primary activator of the coagulation cascade. During endotoxemia, TF expression leads to disseminated intravascular coagulation. However, the relative contribution of TF expression by different cell types to the activation of coagulation has not been defined. In this study, we investigated the effect of either a selective inhibition of TF expression or cell type-specific deletion of the TF gene (F3) on activation of coagulation in a mouse model of endotoxemia. We found that inhibition of TF on either hematopoietic or nonhematopoietic cells reduced plasma thrombin-antithrombin (TAT) levels 8 hours after administration of bacterial lipopolysaccharide (LPS). In addition, plasma TAT levels were significantly reduced in endotoxemic mice lacking the TF gene in either myeloid cells (TF(flox/flox),LysM(Cre) mice) or in both endothelial cells (ECs) and hematopoietic cells (TF(flox/flox),Tie-2(Cre) mice). However, deletion of the TF gene in ECs alone had no effect on LPS-induced plasma TAT levels. Similar results were observed in mice lacking TF in vascular smooth muscle cells. Finally, we found that mouse platelets do not express TF pre-mRNA or mRNA. Our data demonstrate that in a mouse model of endotoxemia activation of the coagulation cascade is initiated by TF expressed by myeloid cells and an unidentified nonhematopoietic cell type(s).

Insulin activation of the phosphatidylinositol 3-kinase/protein kinase B (Akt) pathway reduces lipopolysaccharide-induced inflammation in mice.

Insulin is used to control pro-inflammatory hyperglycemia in critically ill patients. However, recent studies suggest that insulin-induced hypoglycemia may negate its beneficial effects in these patients. It is noteworthy that recent evidence indicates that insulin has anti-inflammatory effects that are independent of controlling hyperglycemia. To date, the mechanism by which insulin directly reduces inflammation has not been elucidated. It is well established that insulin activates phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt) signaling in many cell types. We and others have shown that this pathway negatively regulates LPS-induced signaling and pro-inflammatory cytokine production in monocytic cells. We hypothesized that insulin inhibits inflammation during endotoxemia by activation of the PI3K/Akt pathway. We used a nonhyperglycemic mouse model of endotoxemia to determine the effect of continuous administration of a low dose of human insulin on inflammation and survival. It is noteworthy that insulin treatment induced phosphorylation of Akt in muscle and adipose tissues but did not exacerbate lipopolysaccharide (LPS)-induced hypoglycemia. Insulin decreased plasma levels of interleukin-6, tumor necrosis factor-alpha, monocyte chemotactic protein 1 (MCP1)/JE, and keratinocyte chemoattractant, and decreased mortality. The PI3K inhibitor wortmannin abolished the insulin-mediated activation of Akt and the reduction of chemokine and interleukin-6 levels. We conclude that insulin reduces LPS-induced inflammation in mice in a PI3K/Akt-dependent manner without affecting blood glucose levels.

Genetic analysis of the role of the PI3K-Akt pathway in lipopolysaccharide-induced cytokine and tissue factor gene expression in monocytes/macrophages.

LPS stimulation of monocytes/macrophages induces the expression of genes encoding proinflammatory cytokines and the procoagulant protein, tissue factor. Induction of these genes is mediated by various signaling pathways, including mitogen-activated protein kinases, and several transcription factors, including Egr-1, AP-1, ATF-2, and NF-kappaB. We used a genetic approach to determine the role of the phosphatidylinositol-3-kinase (PI3K)-protein kinase B (Akt) pathway in the regulation of LPS signaling and gene expression in isolated macrophages and in mice. The PI3K-Akt pathway is negatively regulated by the phosphatase and tensin homologue (PTEN). We used peritoneal exudate cells from Pik3r1-deficient mice, which lack the p85alpha regulatory subunit of PI3K and have reduced PI3K activity, and peritoneal macrophages from PTEN(flox/flox)/LysMCre mice (PTEN(-/-)), which have increased Akt activity. Analysis of LPS signaling in Pik3r1(-/-) and PTEN(-/-) cells indicated that the PI3K-Akt pathway inhibited activation of the ERK1/2, JNK1/2, and p38 mitogen-activated protein kinases and reduced the levels of nuclear Egr-1 protein and phosphorylated ATF-2. Modulating the PI3K-Akt pathway did not affect LPS-induced degradation of IkappaBalpha or NF-kappaB nuclear translocation. LPS induction of TNF-alpha, IL-6, and tissue factor gene expression was increased in Pik3r1(-/-) peritoneal exudate cells and decreased in PTEN(-/-) peritoneal macrophages compared with wild-type (WT) cells. Furthermore, LPS-induced inflammation and coagulation were enhanced in WT mice containing Pik3r1(-/-) bone marrow compared with WT mice containing WT bone marrow and in mice lacking the p85alpha subunit in all cells. Taken together, our results indicate that the PI3K-Akt pathway negatively regulates LPS signaling and gene expression in monocytes/macrophages.

Protease-activated receptor-1 contributes to cardiac remodeling and hypertrophy.

BACKGROUND: Protease-activated receptor-1 (PAR-1) is the high-affinity receptor for the coagulation protease thrombin. It is expressed by a variety of cell types in the heart, including cardiomyocytes and cardiac fibroblasts. We have shown that tissue factor (TF) and thrombin contribute to infarct size after cardiac ischemia-reperfusion (I/R) injury. Moreover, in vitro studies have shown that PAR-1 signaling induces hypertrophy of cardiomyocytes and proliferation of cardiac fibroblasts. The purpose of the present study was to investigate the role of PAR-1 in infarction, cardiac remodeling, and hypertrophy after I/R injury. In addition, we analyzed the effect of overexpression of PAR-1 on cardiomyocytes. METHODS AND RESULTS: We found that PAR-1 deficiency reduced dilation of the left ventricle and reduced impairment of left ventricular function 2 weeks after I/R injury. Activation of ERK1/2 was increased in injured PAR-1(-/-) mice compared with wild-type mice; however, PAR-1 deficiency did not affect infarct size. Cardiomyocyte-specific overexpression of PAR-1 in mice induced eccentric hypertrophy (increased left ventricular dimension and normal left ventricular wall thickness) and dilated cardiomyopathy. Deletion of the TF gene in cardiomyocytes reduced the eccentric hypertrophy in mice overexpressing PAR-1. CONCLUSIONS: Our results demonstrate that PAR-1 contributes to cardiac remodeling and hypertrophy. Moreover, overexpression of PAR-1 on cardiomyocytes induced eccentric hypertrophy. Inhibition of PAR-1 after myocardial infarction may represent a novel therapy to reduce hypertrophy and heart failure in humans.

A novel class of antioxidants inhibit LPS induction of tissue factor by selective inhibition of the activation of ASK1 and MAP kinases.

OBJECTIVE: Oxidative stress contributes to the pathogenesis of many diseases, including atherosclerosis and sepsis. We have previously described a novel class of therapeutic compounds with antioxidant and antiinflammatory properties. However, at present, the intracellular targets of these compounds have not been identified. The purpose of this study was to elucidate the mechanism by which 2 structurally-related antioxidants (AGI-1067 and AGI-1095) inhibit LPS induction of tissue factor (TF) expression in human monocytic cells and endothelial cells. METHODS AND RESULTS: We found that succinobucol (AGI-1067) and AGI-1095 inhibited LPS induction of TF expression in both monocytic cells and endothelial cells. These compounds also reduced LPS induction of nuclear AP-1 and expression of Egr-1 without affecting nuclear translocation of NF-kappaB. Importantly, these antioxidants inhibited LPS activation of the redox-sensitive kinase, apoptosis signal-regulating kinase-1 (ASK1) and the mitogen-activated protein kinases (MAPKs) p38, ERK1/2, and JNK1/2. CONCLUSIONS: AGI-1067 and AGI-1095 inhibit TF gene expression in both monocytic cells and endothelial cells through a mechanism that involves the inhibition of the redox-sensitive MAP3K, ASK1. These compounds selectively reduce the activation/induction of MAPK, AP-1, and Egr-1 without affecting NF-kappaB nuclear translocation.

Tissue factor: a link between C5a and neutrophil activation in antiphospholipid antibody induced fetal injury.

Fetal loss in patients with antiphospholipid (aPL) antibodies has been ascribed to thrombosis of placental vessels. However, we have shown that inflammation, specifically activation of complement with generation of the anaphylotoxin C5a, is an essential trigger of fetal injury. In this study, we analyzed the role of the procoagulant molecule tissue factor (TF) in a mouse model of aPL antibody-induced pregnancy loss. We found that either blockade of TF with a monoclonal antibody in wild-type mice or a genetic reduction of TF prevented aPL antibody-induced inflammation and pregnancy loss. In response to aPL antibody-generated C5a, neutrophils express TF potentiating inflammation in the deciduas and leading to miscarriages. Importantly, we showed that TF in myeloid cells but not fetal-derived cells (trophoblasts) was associated with fetal injury, suggesting that the site for pathologic TF expression is neutrophils. We found that TF expression in neutrophils contributes to respiratory burst and subsequent trophoblast injury and pregnancy loss induced by aPL antibodies. The identification of TF as an important mediator of C5a-induced oxidative burst in neutrophils in aPL-induced fetal injury provides a new target for therapy to prevent pregnancy loss in the antiphospholipid syndrome.

A non-anticoagulant synthetic pentasaccharide reduces inflammation in a murine model of kidney ischemia-reperfusion injury.

Fondaparinux is a synthetic pentasaccharide that selectively inhibits factor Xa (FXa) in an antithrombin-dependent fashion. This newly developed anticoagulant is used in the prevention and treatment of venous thromboembolism. Recently, we showed that fondaparinux reduces inflammation and protects the kidney from ischemia-reperfusion (I/R) injury. However, the relative contributions of the anticoagulant and anti-inflammatory activities of fondaparinux to the observed protection is unknown. To address this, we chemically modified fondaparinux to abolish its affinity for antithrombin and analyzed the effect of this non-anticoagulant (NAC)-pentasaccharide on binding of U937 cells to P-selectin in vitro and on inflammation in a murine model of kidney I/R injury. NAC-pentasaccharide was as effective as fondaparinux at inhibiting the binding of U937 cells to P-selectin. In addition, NAC-pentasaccharide significantly reduced IL-6 and MIP-2 expression and injury in the kidney I/R model. These findings indicate that the anti-inflammatory activity of fondaparinux can be dissociated from its anticoagulant activity and that NAC-pentasaccharide is protective in kidney I/R injury.

PI3K-Akt pathway suppresses coagulation and inflammation in endotoxemic mice.

OBJECTIVE: In endotoxemia, lipopolysaccharide (LPS) induces a systemic inflammatory response and intravascular coagulation. Monocytes orchestrate the innate immune response to LPS by expressing a variety of pro-inflammatory cytokines, such as tumor necrosis factor-alpha (TNF-alpha), and the procoagulant molecule, tissue factor (TF). In this study, we analyzed the role of the phosphoinositide 3-kinase (PI3K)-Akt pathway in the activation of coagulation and the innate immune response in a mouse model of endotoxemia. METHODS AND RESULTS: Wortmannin and LY294002 were used to inhibit the PI3K-Akt pathway. We found that wortmannin inhibited LPS-induced Akt phosphorylation in blood cells. Inhibition of the PI3K-Akt pathway significantly increased TF mRNA expression in blood cells, TF antigen, and thrombin-antithrombin III levels in the plasma, and fibrin deposition in the liver of endotoxemic mice. Inhibition of the PI3K-Akt pathway also strongly enhanced LPS-induced cytokine expression and the levels of soluble E-selectin in the plasma, suggesting enhanced activation of both monocytes and endothelial cells. Wortmannin treatment also increased the number of macrophages in the liver and kidney of endotoxemic mice. Finally, wortmannin and LY294002 dramatically reduced the survival time of endotoxemic mice. CONCLUSIONS: These data suggest that the PI3K-Akt pathway suppresses LPS-induced inflammation and coagulation in endotoxemic mice.

Dexamethasone enhances LPS induction of tissue factor expression in human monocytic cells by increasing tissue factor mRNA stability.

Glucocorticoids, such as dexamethasone (Dex), are used clinically in the treatment of various inflammatory diseases. Dex acts by inhibiting the expression of inflammatory mediators, such as tumor necrosis factor alpha (TNF-alpha) and monocyte chemoattractant protein-1 (MCP-1). It is surprising that Dex enhances bacterial lipopolysaccharide (LPS) induction of tissue factor (TF) expression in human monocytic cells. TF is a transmembrane glycoprotein that activates the coagulation protease cascade. In this study, we analyze the mechanism by which Dex enhances LPS-induced TF expression in human monocytic cells. We found that Dex reduced LPS-induced TF gene transcription but increased the stability of TF mRNA. Dex decreased the stability of MCP-1 mRNA and did not affect TNF-alpha mRNA stability. Finally, we showed that Dex increased the stability of a transcript consisting of the final 297 nucleotides of the TF mRNA in in vitro decay assays. This region contains AU-rich elements that regulate mRNA stability and may mediate the Dex response. Therefore, despite an inhibition of TF gene transcription, Dex enhances TF expression in human monocytic cells by increasing the stability of TF mRNA.

Role of tissue factor and protease-activated receptors in a mouse model of endotoxemia.

Sepsis is associated with a systemic activation of coagulation and an excessive inflammatory response. Anticoagulants have been shown to inhibit both coagulation and inflammation in sepsis. In this study, we used both genetic and pharmacologic approaches to analyze the role of tissue factor and protease-activated receptors in coagulation and inflammation in a mouse endotoxemia model. We used mice expressing low levels of the procoagulant molecule, tissue factor (TF), to analyze the effects of TF deficiency either in all tissues or selectively in hematopoietic cells. Low TF mice had reduced coagulation, inflammation, and mortality compared with control mice. Similarly, a deficiency of TF expression by hematopoietic cells reduced lipopolysaccharide (LPS)-induced coagulation, inflammation, and mortality. Inhibition of the down-stream coagulation protease, thrombin, reduced fibrin deposition and prolonged survival without affecting inflammation. Deficiency of either protease activated receptor-1 (PAR-1) or protease activated receptor-2 (PAR-2) alone did not affect inflammation or survival. However, a combination of thrombin inhibition and PAR-2 deficiency reduced inflammation and mortality. These data demonstrate that hematopoietic cells are the major pathologic site of TF expression during endotoxemia and suggest that multiple protease-activated receptors mediate crosstalk between coagulation and inflammation.