Activated protein C ameliorates Bacillus anthracis lethal toxin-induced lethal pathogenesis in rats
- Jyh-Hwa Kau†1,
- Yung-Luen Shih†2, 3,
- Te-Sheng Lien†4,
- Chin-Cheng Lee2,
- Hsin-Hsien Huang1,
- Hung-Chi Lin1,
- Der-Shan Sun4Email author and
- Hsin-Hou Chang4Email author
© Kau et al.; licensee BioMed Central Ltd. 2012
Received: 14 May 2012
Accepted: 18 October 2012
Published: 21 November 2012
Lethal toxin (LT) is a major virulence factor of Bacillus anthracis. Sprague Dawley rats manifest pronounced lung edema and shock after LT treatments, resulting in high mortality. The heart failure that is induced by LT has been suggested to be a principal mechanism of lung edema and mortality in rodents. Since LT-induced death occurs more rapidly in rats than in mice, suggesting that other mechanisms in addition to the heart dysfunction may be contributed to the fast progression of LT-induced pathogenesis in rats. Coagulopathy may contribute to circulatory failure and lung injury. However, the effect of LT on coagulation-induced lung dysfunction is unclear.
To investigate the involvement of coagulopathy in LT-mediated pathogenesis, the mortality, lung histology and coagulant levels of LT-treated rats were examined. The effects of activated protein C (aPC) on LT-mediated pathogenesis were also evaluated.
Fibrin depositions were detected in the lungs of LT-treated rats, indicating that coagulation was activated. Increased levels of plasma D-dimer and thrombomodulin, and the ameliorative effect of aPC further suggested that the activation of coagulation-fibrinolysis pathways plays a role in LT-mediated pathogenesis in rats. Reduced mortality was associated with decreased plasma levels of D-dimer and thrombomodulin following aPC treatments in rats with LT-mediated pathogenesis.
These findings suggest that the activation of coagulation in lung tissue contributes to mortality in LT-mediated pathogenesis in rats. In addition, anticoagulant aPC may help to develop a feasible therapeutic strategy.
Anthrax is a disease caused by infection with the Gram-positive bacterium Bacillus anthracis. Anthrax is characterized by massive bacteremia in the absence of an effective immune response. Lethal toxin (LT) is a major virulence factor for B. anthracis that plays vital role in pathogenesis and the suppression of the host immune response [1, 2]. LT is a binary protein complex that is composed of protective antigen (PA), a host-cell receptor binding subunit that mediates the cellular entry of its LT-counterpart, the lethal factor (LF), which is a metalloprotease that inactivates the mitogen-activated protein kinase kinases (MEKs) signaling pathways . Although LT treatments might not reflect all features of B. anthracis infections, reductionistic approaches investigating the effects of LT in vitro and in vivo have aided in the elucidation the underlying pathogenic mechanisms in anthrax [3, 4]. LT treatments in rodents resulted in hemorrhage, shock, and hypoxic tissue damages with high mortality [5–10].
In contrast to sepsis, treatments of LT do not induce strong proinflammatory cytokine and nitric oxide releases in rodents [4, 8, 11]. LT-treated rats displayed acute lung edema and accelerated progression of pathogenesis, compared with mice . Recent findings suggest that LT can suppress cardiac function [3, 12–14]. Because acute heart dysfunction can cause the accumulation of body fluid in the lungs [15, 16], the lung edema associated with LT treatments in rodents may be a secondary manifestation of the failing heart [3, 4, 7, 14]. Since LT-induced death is much faster in rats than in mice, this suggests that other mechanisms in addition to the heart dysfunction may be contributed to the fast progression of LT-induced pathogenesis in rats. Coagulopathy causes hemodynamic changes [17, 18], and the conversion of extravascular fibrinogen into fibrin in the airway lumen can exacerbate lung injury [19, 20]. LT–induced coagulopathy has been observed in mice [5, 6]. However, the role of coagulopathy in LT-treated rats remains largely unclear.
Featured by abnormal activation of coagulation system, coagulopathy is frequently observed in a severe pathological condition like sepsis, which manifests with prolonged plasma clotting time and decreased circulating anticoagulant protein C [21, 22]. Protein C is an inactive zymogen of a soluble, vitamin K–dependent, plasma serine protease that plays a central role in endogenous anticoagulation . The activation of protein C requires binding to the endothelial protein C receptor and the thrombomodulin-thrombin complexes on the endothelium . Decreased circulating protein C and increased circulating thrombomodulin are indicators of dysfunctional anticoagulation that lead to the prothrombotic, hypercoagulable states which are associated with sepsis and acute lung injury [21, 24–28]. B. anthracis infections in humans and animals may lead to coagulopathy with elevated levels of plasma D-dimer [29–31]. Released into the circulation during the degradation of fibrin during fibrinolysis, D-dimer is widely used as a marker for the detection of coagulopathy . Anthrax LT is a virulence factor of B. anthracis. However, such coagulant pathological changes were primarily observed in B. anthracis infections but not clearly demonstrated in LT-treated rats.
We investigated the role of coagulopathy in LT-induced pathogenesis in rats. Analyses of tissue sections, plasma clotting time, key coagulant factors, and hemodynamic parameters were performed to evaluate the coagulant status of LT-treated rats. Our analysis of plasma D-dimer and thrombomodulin levels suggest that coagulopathy is involved in LT-induced pathogenesis, and our observations of the ameliorative effects of activated protein C (aPC) on LT-induced coagulopathy support our findings. The regulation of coagulation and the pathological responses in rats are compared with those of mouse models.
Male Sprague Dawley rats aged 8 to 10 weeks that were free from specific pathogens, were purchased from the National Laboratory Animal Center, Taiwan. Rats were intravenously treated with either 0.25 mg/kg of PA or 0.25 mg/kg of LT (LF : PA = 15 : 85; a lethal dose), respectively. Rats will die from LT-induced pathogenesis at 4 to 7 hours following the LT injections. Lipopolysaccharide (LPS, Sigma-Aldrich, St. Louis, MO, USA) was dissolved in phosphate-buffered saline (PBS, pH 7.4) and used as a control to induce coagulopathy. The LPS treatments (20 mg/kg) were performed over the same time course as PA and LF injections. Xigris® (drotrecogin alfa, Eli Lilly & Co., Giessen, Germany), a recombinant form of human aPC, was administrated intravenously every 4 hour (96 μg/kg/injection) for the total of three injections as a modification of the manufacturer’s instructions. Because the Xigris was diluted in normal saline, the control rats were injected with an equivalent amount of saline for comparison. The experiments were performed in accordance with institutional guidelines. All animal research methods were approved by the Animal Care and Use Committee of Tzu Chi University (approval ID: 97005).
Toxin preparation and usage
Components of Bacillus anthracis lethal toxins, LF and PA, were prepared and purified as previously described [33–35]. Doses of LT contained LF and PA at a ratio of 15:85, respectively, such that 100 μg LT contained 15 μg LF plus 85 μg PA, which is roughly equivalent to the composition of native toxin. Purified proteins were analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Using Image J software (National Institutes of Health, Bethesda, MD, USA), 85-90% purity was estimated for the total protein fractions. LPS was monitored using a Limulus Amoebocyte Lysate QCL-1000 kit (Lonza, Walkersville, MD, USA), and the level of LPS contamination was less than 1 EU/mg LT, a level of LPS that we observed to be incapable of eliciting significant inflammatory cytokine IL-1 and TNF-α secretion in vitro and in vivo (data not shown).
Analysis of plasma proteins in plasma and lung tissue fluid
We measured the relative levels of albumin, immunoglobulin G (IgG) and fibrinogen in the plasma/lung homogenate fluid of surviving rats at four hours following the LT treatments (0.25 mg/kg) using enzyme-linked immunosorbent assay (ELISA). This time point demonstrated 30% mortality and therefore was expected to reveal pathophysiological changes of the lethal pathogenesis. The lung tissue of rats (0.5 g) was homogenized in 1 mL extraction buffer (10 mM Tris, 150 mM NaCl, pH 7.4) at 4°C using a tissue homogenizer (PRO Scientific, Oxford, CT, USA). The homogenates were transferred to 1.5 mL Eppendorf tubes, and centrifuged at 13,000 × g for 10 minutes at 4°C. The supernatant (lung-tissue fluid) was stored at −80°C until analyzed. The total protein in the plasma and lung-tissue fluid was determined by the method of Bradford. Plasma or lung tissue fluid (50 μg/well) was coated on 96-well dishes with at 37°C for 2 hours. After blocking with 5% BSA for additional 1 hour and 3 washes, the wells were then separately probed using anti-albumin (Abcam, Cambridge, MA, USA), anti-rat IgG (Jackson Immunoresearch Laboratory, West Grove, PA, USA) and anti-fibrinogen (Abcam) antibodies. Plasma of phosphate-buffered saline (PBS, pH 7.4)-treated rats served as a vehicle control. Plasma samples were used neat or diluted four-fold with PBS to keep the ELISA signal in a linear range. The plasma protein levels of treated rats were normalized to those of the vehicle controls.
Histology and immunohistochemistry
Lungs of animals treated with LT (0.25 mg/kg) for 4 hours were fixed with 10% buffered formalin (Sigma-Aldrich) and embedded in paraffin. Tissue sections were then deparaffinized and stained with hematoxylin and eosin (H&E), and immunoglobulin (Ig). Tissue section images were obtained using a Leica DMIRE2 microscope (Wetzlar, Germany). Staining of fibrin was performed as previously described  using deparaffinization; 3% H2O2, 10 minutes; Tris-buffered saline (TBS) with 0.05% Tween (TBST); 0.1% proteinase K in TBS, 10 minutes; TBST; 1:1000 chicken anti-human fibrinogen (Abcam, Cambridge, MA), which cross reacts with rat fibrinogen and fibrin (data not shown), in an overnight incubation at 4°C. Bound primary antibody was visualized using an anti-chicken antibody-peroxidase conjugate and the DAB (3,3’-diaminobenzidine tetrahydrochloride) substrate, and sections were counterstained with hematoxylin. Purified total IgGs from non-immunized rabbits were used for the control staining.
Hemoglobin analyses of lung fluid
Whole blood samples were collected from the tail veins of surviving rats at 4 hours following LT treatment and mixed 9:1 with ACD anticoagulant solution (38 mM citric acid, 75mM sodium citrate, 100mM dextrose) [37, 38]. These blood samples were used as controls for comparisons to lung tissue and interstitial fluid. The hemoglobin levels were determined using a Drabkin’s reagent (Sigma-Aldrich) (ACD and blood : Drabkin’s reagent = 1:500) according to previously described methods [39, 40]. Serially dilutions were used to plot a standard curve. Hemoglobin levels in the samples from LT-treated rats were normalized to the levels in the whole blood of vehicle controls. The 1:4 dilutions of control blood were used as reference points to maintain a linear data range within the detection limits of the method.
Blood oxygen measurement
At 4 hours following the LT treatments, surviving rats were anaesthetized by intraperitoneal injection of pentobarbital (30 mg/kg). Cardiac puncture of the (right ventricle) was used to collect blood in 50 IU/ml lithium heparinized syringes, and the blood oxygen content (PO2) was measured directly by the method of amperometry in a blood gas analysis machine (Synthesis series, Instrumentation Laboratory, Lexington, MA, USA).
Cell death analysis
The mouse macrophage-like cell line J774A.1 (ATCC TIB67) was used for cytotoxicity analysis [34, 35, 41]. The cells were maintained in Dulbeco’s modified Eagle’s medium (DMEM) containing 10% fetal bovine serum (FBS). The cells (1 × 105/well) were treated with a cytotoxic dose of LT (15 ng/mL), with or without additional treatments of aPC (Xigris; 1.5 μg/mL), in 96-well dishes for 3 hours. The level of viable cells was determined using the WST-1 kit (Roche Diagnostics, Indianapolis, IN, USA) according to the manufacturer’s instructions.
Plasma levels of D-dimer, thrombomodulin, protein C and antithrombin III levels in mice were determined before (0 hour), and after (2 and 4 hours) PA, LT and LPS treatments using a D-dimer ELISA kit (American Diagnostica, Stamford, CT, USA), a thrombomodulin ELISA kit (Abcam) and chromogenic protein C and antithrombin III kits (American Diagnostica), respectively . Activated partial thromboplastin time (aPTT) and prothrombin time (PT) analyses were performed to distinguish between the effects of LT on the intrinsic and extrinsic coagulation pathways, using an ACL Futura Plus coagulometer (Instrumentation Laboratory) as described .
Means and standard deviations for quantifiable data were calculated using Microsoft Office Excel 2003 for Windows. Comparisons between groups of nonparametric data were made using an ANOVA. For survival studies, the log-rank test was performed to determine significance using the Online Application Survival Analysis Lifespan Assays (http://sbi.postech.ac.kr/oasis). The unpaired Student’s t test was used to analyze the parametric data for other results. Results with p values less than 0.05 were considered statistically significant.
Ameliorative effect of activated protein C in LT-mediated pathogenesis
Pulmonary edema and lesions are frequently observed in inhalation anthrax [29, 47, 48]. Elevated plasma D-dimer indicates coagulopathy in anthrax patients [29, 30]. These manifestations are reflected in the results of in the rat experiments described in this study. Compared to the rat model, several pathological features of coagulant regulation are distinct in mice. LT induces thrombocytopenia in mice [5, 8], but not in rats . The results of our D-dimer analyses suggest that coagulopathy is involved in LT-mediated pathogenesis in rats (Figure 5B), which is consistent with anthrax in human [29, 30], but not in mice . The protective role of aPC in LT-mediated mortality in rats (Figure 4B), further suggests a role of coagulopathy. In contrast, the lack aPC-mediated amelioration of LT-induced pathogenesis in the C57Bl/6J mice in our lethal dose experiments indicates that coagulopathy does not contribute significantly to mortality in mice (data not shown). Despite these distinct differences in coagulopathy, LT-induced cardiopathy is similar in the rat and mouse models [3, 4, 7, 12–14]. The significant differences in lung pathology indicate a mechanism other than heart dysfunction. Thus, coagulopathy was a consideration. Although no obvious systemic changes in clotting time or plasma anticoagulants were observed, fibrin deposition was observed in the lung sections, suggesting that the lung is the primarily organ affected by LT-induced coagulopathy in rats.
In the absence of systemic coagulopathy, localized activation of coagulation in the lung may cause significant pathology. LT-induced pathogenesis in rats shares common features with acute lung injury and acute respiratory distress syndrome in humans, including multiple organ dysfunction, intra-alveolar coagulation with fibrin deposition on the hyaline membrane [49, 50], and decreased mortality with aPC treatment . Coagulant activation and the presence of fibrin is known to be associated with the pathophysiology of lung injury [19, 20, 51, 52], and LT has been shown to increase the paracellular permeability of endothelial cells . Our data indicate that this increased permeability results in the extravasation of fibrinogen and fibrin into the alveolar lumen (Figure 1B, 2L).
Fibrinogen, fibrin and related degradation products have been shown to suppress surfactant functions in the alveoli [51, 52]. The activation of coagulation pathways can induce local leukocyte traffic and endothelial cell permeability  that may exacerbate lung injury [20, 54]. The elevation of D-dimer following LT treatment indicates the initiation of the coagulation-fibrinolysis cascade (Figure 1C; Figure 5B). Unlike endothelial cells, the cells comprising the alveolar surface do not express thrombomodulin. The LT-induced release of endothelial thrombomodulin into the plasma that supposedly will suppress vascular anticoagulant protein C pathway. Because the LT-induced coagulopathy is primarily restricted to the lung, the circulating plasma level of aPC may not increase in the same manner as occurs in LPS-treated animals (Figure 5F, LT vs. LPS). Since aPC can inactivate the coagulation cascade and block fibrin formation , this suggests the ameliorative role of aPC in LT-treated animals (Figure 4f, 5). The anticoagulant and anti-inflammatory activities of aPC play protective roles in lung injury and asthma . Because LT-mediated pathogenesis does not induce significant secretion of proinflammatory cytokines [8, 11], anticoagulant activity of aPC is likely the major effect that contributes to the amelioration of LT-induced pathogenesis.
Recombinant aPC (Xigris) was previously approved by United States Food and Drug Administration (FDA) for the reduction of mortality in adult patients with severe sepsis . Xigris was, however, withdrawn from the market by the manufacturer after the failure of its world wide trail to treat severe sepsis [57, 58]. LT-mediated pathogenesis does not induce a prominent inflammatory response [8, 11], and the changes in aPTT, PT, protein C, and antithrombin III are distinct from those of endotoxin-induced sepsis (Figure 5D and 5G). Thus, LT-induced pathogenesis is significantly different from sepsis. As a result, Xigris may, nonetheless, ameliorate LT-mediated pathogenesis by re-establishing the regulation of coagulation. Further investigations of the ameliorative effects of Xigris on LT-mediated pathogenesis are warranted.
Consistent with the results of previous studies [12, 13], the mean blood pressure of the LT-treated rats significantly decreased at approximately 4 h following LT treatment (Additional file 1: Figure S2A vs. Figure S2B). To ascertain whether the elicitation of lung edema occurred prior to the circulatory collapse, we examined the extent of lung injury by measuring the blood pressure and the lung wet-to-dry weight ratio of LT-treated rats. We found that both pulmonary and circulatory abnormalities were induced over a similar time course (Additional file 1: Figure S2A-B; 4-hour, LT + vehicle vs. vehicle groups). The changes in levels of plasma D-dimer and thrombomodulin were also associated with a similar time course (Figure 5B-C, 4-hour groups). Thus, the LT treatments elicited both coagulation-mediated lung injury (Figure 5 and Additional file 1: Figure S2C) and heart dysfunction (Additional file 1: Figure S2B) in rats. Therefore, we propose a hypothetical model for LT-mediated pathogenesis that is based on the hemodynamic changes and the altered coagulation occurring simultaneously (Additional file 1: Figure S3).
Severe heart failure leads to the redistribution and accumulation of body fluid in the lung [15, 16], which would likely exacerbate the increased vascular permeability and the coagulation-activation in the lungs of LT-treated rats. Coagulopathy may also negatively influence cardiac function, resulting in further hemodynamic changes [17, 18]. Dysregulation of protein C activation and thrombosis are also caused by secondary pulmonary hypertension, a known effect of severe heart failure . As a result, these two pathogenic events may have potential for mutual exacerbation (Additional file 1: Figure S3). Thus, the interruption of this exacerbating feedback by aPC treatment may contribute to the improved survival rate (Additional file 1: Figure S3). The cross-talks between these two types of pathogenic regulations are interesting issues and worthy to be further investigated.
In addition to LT, B. anthracis releases other toxins that perturb coagulation and vascular processes during infection. Bacteria-derived metalloproteases have been shown to degrade von Willebrand factor and ADAMTS13, which contribute to the recruitment of platelets to the injured vessel wall . In addition, bacterial protease InhA has been shown to inhibit fibrinolysis by activating plasminogen activator inhibitor-1 [61–63], and anthrolysin O, a cholesterol-dependent cytolysin and a Toll-like receptor 4 agonist, has been shown to disrupt endothelial and epithelial barriers [64–66]. Combined treatments of anthrolysin O and edema toxin significantly induced thrombin activity in human umbilical vein endothelial cells . These studies indicate that B. anthracis-derived secretory factors play important roles in anthrax consumptive coagulopathy. Further investigations are needed to reveal the combined effects of these virulence factors on circulatory homeostasis during infection.
In summary, our results suggest that the activation of the coagulation-fibrinolysis cascade is involved in LT-mediated pathogenesis in rats. The anticoagulant aPC significantly reduced mortality in LT-treated rats. Specific treatments to overcome LT-mediated pathogenesis are lacking. Our findings may aid in the development of new therapeutic strategies for treating anthrax infections.
This work was supported by National Science Council of Taiwan, Republic of China, under grant no. NSC 95-2311-B-320-006, 95-2811-B-320-001, 96-2311-B-320-005-MY3, 98-2320-B-320-004 -MY3, and 99-2311-B-320-003-MY3; and Tzu-Chi University under grant no. TCIRP 95002–02, TCIRP 96004–01, TCIRP 98001–01, TCIRP 101001, TCRPP 99020–01, TCRPP100003 and TCRPP101005.
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