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.

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-κB. 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 p85α 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 IκBα or NF-κB nuclear translocation. LPS induction of TNF-α, 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 p85α subunit in all cells. Taken together, our results indicate that the PI3K-Akt pathway negatively regulates LPS signaling and gene expression in monocytes/macrophages.

Macrophages represent the first line of defence in innate immune responses and additionally serve important functions for the regulation of host inflammation and tissue homeostasis. The M1/M2 model describes the two extremes of macrophage polarization states, which can be induced by multiple stimuli, most notably by LPS/IFN‐γ and IL‐4/IL‐13. Historically, the expression of two genes encoding for enzymes, which use the same amino acid as their substrate, iNOS and ARG1, has been used to define classically activated M1 (iNOS) and alternatively activated M2 (ARG1) macrophages. This ‘arginine dichotomy’ has recently become a matter of debate; however, in parallel with the emerging field of immunometabolism there is accumulating evidence that these two enzymes and their related metabolites are fundamentally involved in the intrinsic regulation of macrophage polarization and function. The aim of this review is to highlight recent advances in macrophage biology and immunometabolism with a specific focus on amino acid metabolism and their related metabolic pathways: iNOS/ARG1 (arginine), TCA cycle and OXPHOS (glutamine) as well as the one‐carbon metabolism (serine, glycine).

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-α (TNF-α), 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.

Tissue factor (TF) has been shown to be up‐regulated in endothelial cells by the inflammatory cytokine tumor necrosis factor a (TNF‐α) as well as by the main angiogenic factor VEGF. Since both stimuli induce the transcription factor EGR‐1, which is critically involved in TF gene regulation, we used EGR‐1‐dependent TF induction as a model to identify potential cross‐talks between the various signal transduction cascades initiated by VEGF and TNF‐α. The data show that at the MAP kinase level, VEGF mainly activates ERK1/2 and p38 MAP kinases in human endothelial cells. TNF‐α is able to activate all three MAP kinase cascades as well as the classical inflammatory IκB/NFκB pathway. Furthermore, the MEK/ERK module of MAP kinases appears to act as the convergence point of VEGF‐ and TNF‐α‐initiated signaling cascades, which lead to the activation of EGR‐1 and subsequent TF expression, whereas the upstream signals are distinct. We found that induction of TF by VEGF via EGR‐1 is strongly PKC dependent. The TNF‐ α‐ initiated MEK/ERK cascade connected to EGR‐1 and TF expression is clearly less sensitive to PKC inhibition. TNF‐ α ‐mediated activation of MEK/ERK and EGR‐1 can be blocked by adenoviral expression of a dominant negative mutant of IKK2, whereas the VEGF signaling pathway is unaffected. Thus, our data demonstrate a new link between the classical inflammatory IKK/IκB and the MEK/ERK cascades triggered by TNF‐ α . The additional finding that EGF induces ERK and EGR‐1 in a PKC independent manner and that this signal is not sufficient to up‐regulate TF emphasizes the importance of a VEGF‐specific signaling pattern for the induction of TF.— Mechtcheriakova, D., Schabbauer, G., Lucerna, M., Clauss, M., de Martin, R., Binder, B. R., Hofer, E. Specificity, diversity, and convergence in VEGF and TNF‐α signaling events leading to tissue factor up‐regulation via EGR‐1 in endothelial cells. FASEB J. 15, 230–242 (2001)