EPCR-dependent PAR2 activation by the blood coagulation initiation complex regulates LPS-triggered interferon responses in mice.

Infection and inflammation are invariably associated with activation of the blood coagulation mechanism, secondary to the inflammation-induced expression of the coagulation initiator tissue factor (TF) on innate immune cells. By investigating the role of cell-surface receptors for coagulation factors in mouse endotoxemia, we found that the protein C receptor (ProcR; EPCR) was required for the normal in vivo and in vitro induction of lipopolysaccharide (LPS)-regulated gene expression. In cultured bone marrow-derived myeloid cells and in monocytic RAW264.7 cells, the LPS-induced expression of functionally active TF, assembly of the ternary TF-VIIa-Xa initiation complex of blood coagulation, and the EPCR-dependent activation of protease-activated receptor 2 (PAR2) by the ternary TF-VIIa-Xa complex were required for the normal LPS induction of messenger RNAs encoding the TLR3/4 signaling adaptor protein Pellino-1 and the transcription factor interferon regulatory factor 8. In response to in vivo challenge with LPS, mice lacking EPCR or PAR2 failed to fully initiate an interferon-regulated gene expression program that included the Irf8 target genes Lif, Iigp1, Gbp2, Gbp3, and Gbp6. The inflammation-induced expression of TF and crosstalk with EPCR, PAR2, and TLR4 therefore appear necessary for the normal evolution of interferon-regulated host responses.

Coagulation factor V mediates inhibition of tissue factor signaling by activated protein C in mice.

The key effector molecule of the natural protein C pathway, activated protein C (aPC), exerts pleiotropic effects on coagulation, fibrinolysis, and inflammation. Coagulation-independent cell signaling by aPC appears to be the predominant mechanism underlying its highly reproducible therapeutic efficacy in most animal models of injury and infection. In this study, using a mouse model of Staphylococcus aureus sepsis, we demonstrate marked disease stage-specific effects of the anticoagulant and cell signaling functions of aPC. aPC resistance of factor (f)V due to the R506Q Leiden mutation protected against detrimental anticoagulant effects of aPC therapy but also abrogated the anti-inflammatory and mortality-reducing effects of the signaling-selective 5A-aPC variant that has minimal anticoagulant function. We found that procofactor V (cleaved by aPC at R506) and protein S were necessary cofactors for the aPC-mediated inhibition of inflammatory tissue-factor signaling. The anti-inflammatory cofactor function of fV involved the same structural features that govern its cofactor function for the anticoagulant effects of aPC, yet its anti-inflammatory activities did not involve proteolysis of activated coagulation factors Va and VIIIa. These findings reveal a novel biological function and mechanism of the protein C pathway in which protein S and the aPC-cleaved form of fV are cofactors for anti-inflammatory cell signaling by aPC in the context of endotoxemia and infection.

Fetal gene defects precipitate platelet-mediated pregnancy failure in factor V Leiden mothers.

We describe a mouse model of fetal loss in factor V Leiden (FvL) mothers in which fetal loss is triggered when the maternal prothrombotic state coincides with fetal gene defects that reduce activation of the protein C anticoagulant pathway within the placenta. Fetal loss is caused by disruption of placental morphogenesis at the stage of labyrinth layer formation and occurs in the absence of overt placental thrombosis, infarction, or perfusion defects. Platelet depletion or elimination of protease-activated receptor 4 (Par4) from the mother allows normal placentation and prevents fetal loss. These findings establish a cause-effect relationship for the observed epidemiologic association between maternal FvL status and fetal loss and identify fetal gene defects as risk modifiers of pregnancy failure in prothrombotic mothers. Pregnancy failure is mediated by Par4-dependent activation of maternal platelets at the fetomaternal interface and likely involves a pathogenic pathway independent of occlusive thrombosis. Our results further demonstrate that the interaction of two given thrombosis risk factors produces markedly disparate consequences on disease manifestation (i.e., thrombosis or pregnancy loss), depending on the vascular bed in which this interaction occurs.

Endotoxemia and sepsis mortality reduction by non-anticoagulant activated protein C.

Activated protein C (APC) reduces mortality of severe sepsis patients but increases the risk of serious bleeding. APC exerts anticoagulant activity by proteolysis of factors Va/VIIIa. APC also exerts antiinflammatory and antiapoptotic effects and stabilizes endothelial barrier function by APC-initiated cell signaling that requires two receptors, endothelial cell protein C receptor (EPCR) and protease-activated receptor 1 (PAR1). The relative importance of APC's various activities for efficacy in sepsis is unknown. We used protein engineering of mouse APC and genetically altered mice to clarify mechanisms for the efficacy of APC in mouse sepsis models. Mortality reduction in LPS-induced endotoxemia required the enzymatic active site of APC, EPCR, and PAR-1, highlighting a key role for APC's cytoprotective actions. A recombinant APC variant with normal signaling but <10% anticoagulant activity (5A-APC) was as effective as wild-type APC in reducing mortality after LPS challenge, and enhanced the survival of mice subjected to peritonitis induced by gram-positive or -negative bacteria or to polymicrobial peritoneal sepsis triggered by colon ascendens stent implantation. Thus, APC's efficacy in severe sepsis is predominantly based on EPCR- and PAR1-dependent cell signaling, and APC variants with normal cell signaling but reduced anticoagulant activities retain efficacy while reducing the risk of bleeding.

Hyperantithrombotic, noncytoprotective Glu149Ala-activated protein C mutant.

Activated protein C (APC) reduces mortality in severe sepsis patients. APC exerts anticoagulant activities via inactivation of factors Va and VIIIa and cytoprotective activities via endothelial protein C receptor and protease-activated receptor-1. APC mutants with selectively altered and opposite activity profiles, that is, greatly reduced anticoagulant activity or greatly reduced cytoprotective activities, are compared here. Glu149Ala-APC exhibited enhanced in vitro anticoagulant and in vivo antithrombotic activity, but greatly diminished in vitro cytoprotective effects and in vivo reduction of endotoxin-induced murine mortality. Thus, residue Glu149 and the C-terminal region of APC's light chain are identified as functionally important for expression of multiple APC activities. In contrast to Glu149Ala-APC, 5A-APC (Lys191-193Ala + Arg229/230Ala) with protease domain mutations lacked in vivo antithrombotic activity, although it was potent in reducing endotoxin-induced mortality, as previously shown. These data imply that APC molecular species with potent antithrombotic activity, but without robust cytoprotective activity, are not sufficient to reduce mortality in endotoxemia, emphasizing the need for APC's cytoprotective actions, but not anticoagulant actions, to reduce endotoxin-induced mortality. Protein engineering can provide APC mutants that permit definitive mechanism of action studies for APC's multiple activities, and may also provide safer and more effective second-generation APC mutants with reduced bleeding risk.

Caveolin-1-dependent apoptosis induced by fibrin degradation products.

In mice lacking the blood coagulation regulator thrombomodulin, fibrinolytic degradation products (FDP) of fibrin induce apoptotic cell death of a specialized cell type in the placenta, polyploid trophoblast giant cells. Here, we document that this bioactivity of FDP is conserved in human FDP, is not limited to trophoblast cells, and is associated with an Aalpha-chain segment of fibrin fragment E (FnE). The majority of proapoptotic activity is arginine-glycine-aspartic acid (RGD)-independent and requires caveolin-1-dependent cellular internalization of FnE. Internalization through caveoli is mediated by an epitope contained within Aalpha52-81 that is necessary and sufficient for cellular uptake of FnE. Aalpha52-81 does not cause apoptosis itself, and competitively inhibits FnE internalization and apoptosis induction. Apoptotic activity per se resides within Aalpha17-37 and requires the N-terminal neoepitope generated by release of fibrinopeptide A. Cellular internalization of FnE elicits depression of mitochondrial function and consequent apoptosis that is strictly dependent on the activity of caspases 9 and 3. These findings describe the molecular details of a novel mechanism linking fibrin degradation to cell death in the placenta, which may also contribute to pathologic alterations in nonplacental vascular beds that are associated with fibrinolysis.

Impaired survival of peripheral T cells, disrupted NK/NKT cell development, and liver failure in mice lacking Gimap5.

The loss of Gimap5 (GTPase of the immune-associated protein 5) gene function is the underlying cause of lymphopenia and autoimmune diabetes in the BioBreeding (BB) rat. The in vivo function of murine gimap5 is largely unknown. We show that selective gene ablation of the mouse gimap5 gene impairs the final intrathymic maturation of CD8 and CD4 T cells and compromises the survival of postthymic CD4 and CD8 cells, replicating findings in the BB rat model. In addition, gimap5 deficiency imposes a block of natural killer (NK)- and NKT-cell differentiation. Development of NK/NKT cells is restored on transfer of gimap5(-/-) bone marrow into a wild-type environment. Mice lacking gimap5 have a median survival of 15 weeks, exhibit chronic hepatic hematopoiesis, and in later stages show pronounced hepatocyte apoptosis, leading to liver failure. This pathology persists in a Rag2-deficient background in the absence of mature B, T, or NK cells and cannot be adoptively transferred by transplanting gimap5(-/-) bone marrow into wild-type recipients. We conclude that mouse gimap5 is necessary for the survival of peripheral T cells, NK/NKT-cell development, and the maintenance of normal liver function. These functions involve cell-intrinsic as well as cell-extrinsic mechanisms.

Gr1+ cells control growth of YopM-negative yersinia pestis during systemic plague.

YopM, a protein toxin of Yersinia pestis, is necessary for virulence in a mouse model of systemic plague. We previously reported YopM-dependent natural killer (NK) cell depletion from blood and spleen samples of infected mice. However, in this study we found that infection with Y. pestis KIM5 (YopM(+)) caused depletion of NK cells in the spleen, but not in the liver, and antibody-mediated ablation of NK cells had no effect on bacterial growth. There was no YopM-associated effect on the percentage of dendritic cells (DCs) or polymorphonuclear leukocytes (PMNs) in the early stage of infection; however, there was a YopM-associated effect on PMN integrity and on the influx of monocytes into the spleen. Ablation of Gr1(+) cells caused loss of the growth defect of YopM(-) Y. pestis in both the liver and spleen. In contrast, ablation of macrophages/DCs inhibited growth of both parent and mutant bacteria, accompanied by significantly fewer lesion sites in the liver. These results point toward PMNs and inflammatory monocytes as major cell types that control growth of YopM(-) Y. pestis. Infection with fully virulent Y. pestis CO92 and a YopM(-) derivative by intradermal and intranasal routes showed that the absence of YopM significantly increased the 50% lethal dose only in the intradermal model, suggesting a role for YopM in bubonic plague, in which acute inflammation occurs soon after infection.

Pharmacological targeting of the thrombomodulin-activated protein C pathway mitigates radiation toxicity.

Tissue damage induced by ionizing radiation in the hematopoietic and gastrointestinal systems is the major cause of lethality in radiological emergency scenarios and underlies some deleterious side effects in patients undergoing radiation therapy. The identification of target-specific interventions that confer radiomitigating activity is an unmet challenge. Here we identify the thrombomodulin (Thbd)-activated protein C (aPC) pathway as a new mechanism for the mitigation of total body irradiation (TBI)-induced mortality. Although the effects of the endogenous Thbd-aPC pathway were largely confined to the local microenvironment of Thbd-expressing cells, systemic administration of soluble Thbd or aPC could reproduce and augment the radioprotective effect of the endogenous Thbd-aPC pathway. Therapeutic administration of recombinant, soluble Thbd or aPC to lethally irradiated wild-type mice resulted in an accelerated recovery of hematopoietic progenitor activity in bone marrow and a mitigation of lethal TBI. Starting infusion of aPC as late as 24 h after exposure to radiation was sufficient to mitigate radiation-induced mortality in these mice. These findings suggest that pharmacologic augmentation of the activity of the Thbd-aPC pathway by recombinant Thbd or aPC might offer a rational approach to the mitigation of tissue injury and lethality caused by ionizing radiation.

Mice naturally resistant to Yersinia pestis Delta pgm strains commonly used in pathogenicity studies.

We report that females of some substrains of inbred mouse strain 129 are resistant to systemic plague due to conditionally virulent Deltapgm strains of Yersinia pestis; however, fully virulent Y. pestis is not attenuated in these mice. Therefore, these mice offer a powerful system in which to map in parallel host resistance traits and opposing bacterial virulence properties for plague.

Antibody against V antigen prevents Yop-dependent growth of Yersinia pestis.

The V antigen (LcrV) of the plague bacterium Yersinia pestis is a potent protective antigen that is under development as a vaccine component for humans. LcrV is multifunctional. On the bacterial surface it mediates delivery of a set of toxins called Yops into host cells, and as a released protein it can cause production of the immunosuppressive cytokine interleukin-10 (IL-10) and can inhibit chemotaxis of polymorphonuclear neutrophils. It is not known how these mechanisms of LcrV operate, what their relative importance is, when they function during plague, and which are critical to protection by antibody. This study investigated several of these issues. C57BL/6 mice, mice unable to express IL-10, or mice with the macrophage lineage eliminated were treated with a protective anti-LcrV antibody or a nonprotective antibody against YopM and infected intravenously by Y. pestis KIM5 or a strain that lacked the genes encoding all six effector Yops. Viable bacterial numbers were determined at various times. The data indicated that Yops were necessary for Yersinia growth after the bacteria had seeded liver and spleen. Anti-LcrV antibody prevented this growth, even in IL-10-/- mice, demonstrating that one protective mechanism for anti-LcrV antibody is independent of IL-10. Anti-LcrV antibody had no effect on persistence in organs of Y. pestis lacking effector Yops, even though the yersiniae could strongly express LcrV, suggesting that Yops are necessary for building sufficient bacterial numbers to produce enough LcrV for its immunosuppressive effects. In vitro assays showed that anti-LcrV antibody could partially block delivery of Yops and downstream effects of Yops in infected macrophage-like J774A.1 cells. However, cells of the macrophage lineage were found to be dispensable for protection by anti-LcrV antibody in spleen, although they contributed to protection in liver. Taken together, the data support the hypothesis that one protective effect of the antibody is to block delivery of Yops to host cells and prevent early bacterial growth. The findings also identified the macrophage lineage as one host cell type that mediates protection.

The plague virulence protein YopM targets the innate immune response by causing a global depletion of NK cells.

Yersinia pestis, the etiologic agent of plague, delivers six Yersinia outer proteins (Yops) into host cells upon direct bacterial contact. One of these, YopM, is necessary for virulence in a mouse model of septicemic plague, but its pathogenic function is unknown. We report here the immune processes affected by YopM during infection. To test whether the innate or adaptive immune system is targeted by YopM, C57BL/6 (B6) and B6 SCID mice were infected with either the conditionally virulent Y. pestis KIM5 or a yopM deletion mutant and evaluated for bacterial growth in spleen and liver. Both B6 and SCID mice succumbed to infection with Y. pestis KIM5, whereas both mouse strains survived infection by the YopM(-) mutant. These data showed that YopM counteracts innate defenses present in SCID mice. The YopM(-) strain grew more slowly than the parent Y. pestis during the first 4 days of infection in both mouse strains, indicating an early pathogenic role for YopM. In B6 mice, populations of cells of the immune system were not differentially affected by the two Y. pestis strains, with one major exception: the parent Y. pestis KIM5 but not the YopM(-) mutant caused a significant global decrease in NK cell numbers (blood, spleen, and liver), beginning early in infection. NK cells and macrophages isolated early (day 2) from livers and spleens of mice infected with either Y. pestis strain contained comparable levels of cytokine mRNA: interleukin (IL)-1 beta, IL-12, IL-15, IL-18, and tumor necrosis factor alpha in macrophages and gamma interferon in NK cells. However, by day 4 postinfection, cells from mice infected with the parent Y. pestis expressed lower levels of these messages, while those from mice infected with the mutant retained strong expression. Significantly, mRNA for the IL-15 receptor alpha chain was not expressed in NK cells from Y. pestis KIM5-infected mice as early as day 2 postinfection. These findings suggest that YopM interferes with innate immunity by causing depletion of NK cells, possibly by affecting the expression of IL-15 receptor alpha and IL-15.