Sepsis and septic shock are leading causes of disability and death among hospitalized patients, and their incidence continues to grow (1, 2). Despite advances in critical care and early aggressive initiation of antimicrobial therapy, sepsis remains difficult to treat due to its pathogenic complexity and dynamic, temporal disease course.
The early innate response and inflammatory cytokine cascade are associated with activation of the coagulation cascade (3). During sepsis, acute hypercoagulability may contribute to lethal sequelae of vascular thrombosis, tissue ischemia, and organ failure, and delayed hypocoagulability may contribute to bleeding complications due to depletion of coagulation factors (4, 5). As the balance between pro- and anti-inflammatory cytokines released affects the magnitude of physiologic and coagulation changes during sepsis, mitigating the initial inflammatory response may protect patients from sepsis-induced coagulopathy. Previously, the Recombinated Human Activated Protein C Worldwide Evaluation in Severe Sepsis (PROWESS) Trial demonstrated promise with delivery of drotrecogin alpha (activated), a serine protease with anti-inflammatory and antithrombotic properties, in adult patients with severe sepsis, with a 6.1% absolute reduction in risk of death (6). However, the sequel trial that enrolled septic patients with a low risk of death reported a lack of survival benefit, along with increased bleeding complications with administration of drotrecogin alpha (7). After a Cochrane review advising against use of drotrecogin alpha in patients with sepsis and septic shock, investigation of this agent has been discontinued from further trials (8).
The mechanism of action of drotrecogin alpha was not well understood and may have mediated procoagulant effects. Mitigating the initial inflammatory response may still prove to be a successful treatment strategy to protect patients from sepsis-induced coagulopathy. Macrophages are among the key early responders to infection (9). Tissue macrophages quickly respond to the septic insult by producing a number of cytokines, including tumor necrosis factor alpha (TNFα) and interleukin 10 (IL-10). As an early inflammatory cytokine released after bacteremia and endotoxemia, TNFα plays a central role in the manifestations of septic shock, including coagulation changes (10–12). In chimpanzees, blockade of TNFα with a monoclonal antibody or reduction of TNFα expression in macrophages have been demonstrated to decrease thrombin generation (13). In addition, a meta-analysis of sepsis trials on anti-TNF agents demonstrated survival benefit compared with control (14).
TNFα production can be triggered by ceramide (Cer) (15). Cer is the product of the hydrolysis of sphingomyelin by acid sphingomyelinase (Asm). Cer has been previously reported to mediate inflammation (16). Cer generation through Asm can be inhibited with the tricyclic antidepressant and functional Asm inhibitor Amitriptyline (AMIT) (17). Our laboratory has previously demonstrated that pretreated of mice with AMIT induces an anti-inflammatory response in scald-injured mice (18). Thus, we investigated whether modulating the initial inflammatory response with AMIT treatment would protect the host from dynamic sepsis-induced coagulopathy.
MATERIALS AND METHODS
Male CF-1 outbred mice aged 6 to 8 weeks (Charles River Laboratories, Wilmington, Mass) were used for experiments. Mice were acclimated for 1 week before experimentation, and were housed in standard environmental conditions with corn-cobb bedding in groups of four. A standard pellet diet and water ab libitum was provided. All murine experiments were performed between 8 AM and 1 PM using protocols approved by the Institutional Animal Care and Use Committee of the University of Cincinnati (protocol 10-05-10-01).
Polymicrobial sepsis model
Mice underwent cecal ligation and puncture (CLP) (33% ligation and a single 25-gauge puncture) to induce polymicrobial sepsis, as previously described (18, 19). Briefly, mice were anesthetized with 2% isoflurane in oxygen, shaved over the abdomen, topically disinfected with povidone-iodine, and placed supine on a heating pad. After a 2-cm midline laparotomy incision, the distal 33% of the cecum was ligated with a 4-0 silk tie (Syneture, Norwalk, Conn). A single puncture was made on the antimesenteric side with a 25-gauge needle, and a small amount of enteric contents was extruded through the puncture holes to ensure a full thickness perforation. The cecum was replaced in its original location, and the midline incision was closed in two layers with 4-0 silk (AD Surgical, Sunnyvale, Calif). After CLP, mice were resuscitated with 1 mL normal saline (NS) (Hospira, Lake Forest, Ill) injected subdermally, and were placed on a heating pad for 1 h. Sham mice underwent the above process save for ligation and puncture of the cecum.
Sterile inflammatory models
A 1 mg lipopolysaccharide (LPS) challenge, derived from Escherichia coli 0111:B4 strain (Sigma-Aldrich, St. Louis, Mo) and solubilized in NS (Hospira), was administered by intraperitoneal injection to induce endotoxemia. To evaluate the role of TNFα during the early phase of sepsis, naive mice were randomized to penile vein injection of NS or 400 ng recombinant murine TNFα (BioLegend, San Diego, Calif) solubilized in NS.
At the time of CLP or LPS challenge, mice were randomized to intraperitoneal delivery of NS (Hospira) or 16 mg/kg AMIT (Sigma-Aldrich) solubilized in NS, as previously described (20). For in vitro experiments, cells were treated with 100 μM AMIT solubilized in NS.
Platelet count determination
Whole blood samples were anticoagulated with heparin, and platelet counts were obtained using the Coulter AcT 10 Hematology Analyzer (Beckman Coulter, Brea, Calif).
Ferric chloride-induced carotid artery thrombosis
Mice were anesthetized with 100 mg/kg sodium pentobarbital (Virbac AH, Fort Worth, Tex), administered by intraperitoneal injection. They were then placed supine on a heating pad, underneath an operating microscope. Mice were subjected to ferric chloride-induced carotid artery injury, and time to complete carotid artery thrombosis was measured, as previously described (21, 22). Briefly, a 5-mm midline neck incision was performed. The anterior fascia and neck musculature were dissected to expose the right common carotid artery. After isolating the artery, a 1 × 2 mm piece of filter paper soaked in 4% w/v ferric chloride (Sigma-Aldrich) solution was applied to the ventral side of the vessel for 3 min. Carotid flow was measured using a flowmeter (Transonic, Ithaca, NY) and displayed as a waveform using LabChart (ADInstruments, Dunedin, New Zealand). Time to carotid occlusion was measured as the interval from removal of the filter paper to cessation of flow.
Whole blood samples were anticoagulated with 10% citrate. Changes in coagulation parameters were determined using rotational thromboelastometry (ROTEM, TEM Systems Inc, Durham, NC) analyses, performed within 10 min of sample collection. Overall coagulation (NATEM), fibrin contribution to clot (FIBTEM), and the extrinsic pathway coagulation (EXTEM) were determined. Viscoelastic coagulation testing included analysis of clot formation time (CFT), maximum clot firmness (MCF), and rate of clot formation (α-angle). In the EXTEM test, 20 μL of thromboplastin was added to 300 μL of citrated whole blood to initiate clot formation. In addition to thromboplastin, cytochalasin D was added to FIBTEM samples to negate the contribution of platelets during clot formation. Percent of platelet contribution (%MCF-Platelet) was calculated by the equation (EXTEMMCF−FIBTEMMCF)/EXTEMM, as previously described (23).
Whole blood was harvested by cardiac puncture, collected in serum separator tubes (BD Biosciences, San Jose, Calif), and centrifuged at 10,000 × g for 10 min at 20°C. Peritoneal fluid was harvested by peritoneal lavage and centrifuged at 450 × g for 10 min at 9°C. The serum and supernatant peritoneal fluid were stored in sterile tubes and frozen at −80°C until cytokine analysis. The TNFα and IL-6 concentrations were analyzed by cytometric bead array (BD Biosciences), as previously described (24).
Cell enumeration and characterization
Peritoneal fluid was harvested by peritoneal lavage and centrifuged at 450 × g for 10 min. The resuspended cells were enumerated with a cell counter (Beckman Coulter), and analyses of cell surface antigen and intracellular cytokine expression were performed by flow cytometry. The following antibodies were used: Ly6G (Clone: 1A8, BD Biosciences), Ly6C (Clone: AL-21, BD Biosciences), CD11b (Clone: M1/70, BD Biosciences), F4/80 (Clone: T45-2342, BD Biosciences), TNFα (Clone: MP6-XT22, BioLegend), CD80 (Clone: 16-10A1, BD Biosciences), and CD86 (Clone GL-1, BioLegend). Flow cytometry acquisition and analysis were performed on an Attune Flow Cytometer (Life Technologies, Foster City, Calif).
To evaluate leukocyte intracellular TNFα expression in vitro, we isolated leukocytes from peritoneal lavages of untreated mice. Cells were then stimulated with 100 ng LPS in the presence of protein transport inhibitor moensin (2 μM, Sigma) for 3 h. Intracellular staining was subsequently performed as previously described (25).
To analyze intracellular TNFα expression in vivo, mice were pretreated with protein transport inhibitor brefeldin A (BFA; BioLegend), as previously described (26). BFA (250 μg) was administered i.v. 30 min before challenge with LPS. Peritoneal macrophages were isolated 90 min after LPS injection and intracellular staining was performed as previously described (25).
GraphPad Prism 6.0 (Graphpad Software, La Jolla, Calif) was used to perform statistical comparisons. At least two independent iterations were conducted for each panel in each figure and the data combined. For inclusion into the manuscript, the iterations demonstrated a similar trend or significance. One- or two-tailed Student t test was performed for two group comparisons as indicated, or one-way ANOVA with Tukey post hoc analysis was used to compare more than two groups. A P value of less than 0.05 was considered statistically significant.
AMIT treatment mitigates acute sepsis-associated coagulopathy
We first observed, using ferric chloride-induced carotid artery thrombosis, that septic control mice were hypercoagulable compared with sham mice (Fig. 1A: sham 407.7 ± 29 s vs. CLP+NS 171.3 ± 10 s, P < 0.05). We next measured the time to carotid occlusion in septic AMIT-treated mice (CLP+AMIT 326.7 ± 28 s, P < 0.01 vs. sham and CLP+NS), and found that not only were they significantly different than septic control mice, but they were also similar to the results of sham mice. These findings were reinforced by differences observed in NATEM CFT between septic control and AMIT-treated mice (Fig. 1B: sham 78.5 ± 6 s, CLP+NS 3.3 ± 3 s, and CLP+AMIT 75.3 ± 9 s, P = 0.01). There were no differences in platelet contribution to clot between the septic control and AMIT-treated mice (data not shown).
AMIT-treated mice are protected from late sepsis-associated hypocoagulability
Next, we examined the coagulation profile of sham and septic control mice 16 h after CLP. In contrast to the 2-h findings, septic control mice were hypocoagulable compared with sham mice, as measured by time to carotid artery occlusion (Fig. 2A: sham 273.1 ± 27 s, CLP+NS 419.2 ± 48 s, and CLP+AMIT 289.4 ± 18 s, P < 0.01) and CFT (Fig. 2B: sham 70.1 ± 10 s and CLP+NS 189.6 ± 21 s; P < 0.05). However, septic AMIT-treated mice were protected from delayed hypocoagulability, and had a similar coagulation profile as sham mice (Fig. 2A: CLP+AMIT 289.4 ± 18 s, P < 0.01 vs. sham and CLP+NS). Similar to the early 2-h time point, there were no differences in platelet contribution to clot between the septic control and AMIT-treated mice (data not shown).
Systemic TNFα levels are reduced after AMIT treatment during sepsis and endotoxemia
Due to its role as an early inflammatory cytokine, we measured serum TNFα levels 6 h after CLP-induced sepsis and 90 min after LPS-induced endotoxemia. Compared with the control cohorts, there was a twofold decrease in TNFα levels in AMIT-treated septic (Fig. 3A: CLP+NS 79.9 ± 18 pg/mL vs. CLP+AMIT 36.8 ± 4 pg/mL, P = 0.03) and endotoxemic mice (Fig. 3B: LPS+NS 8,420 ± 457 ng/mL vs. LPS+AMIT 4,024 ± 285 ng/mL, P < 0.01). Serum TNFα levels in sham mice were below the detection limit (data not shown). No differences in systemic interleukin 6 (IL-6) levels were observed between septic control and AMIT-treated mice (data not shown).
Administration of recombinant TNFα recapitulates the acute coagulation profile of septic mice
To verify the role that TNFα plays in sepsis-associated coagulopathy, NS or 400 ng of recombinant murine TNFα was systemically administered to healthy mice with coagulation measured after 2 h. We observed that exogenous TNFα recapitulated the coagulation profile observed in septic mice, with hypercoagulability seen in time to carotid occlusion (Fig. 4A: NS 306.1 ± 36 s vs. TNFα 194 ± 11 s, P = 0.02) and NATEM CFT (Fig. 4B: NS 109.4 ± 2.63 s vs. TNFα 77.38 ± 8.65 s, P < 0.01). We did not observe any differences in the time to carotid occlusion and CFT at 16 h after TNFα or NS administration (data not shown).
Peritoneal macrophage TNFα production is suppressed in AMIT-treated septic mice
To determine cellular sources of AMIT-influenced TNFα suppression, peritoneal macrophages were treated with the protein transport inhibitor moensin and stimulated with PBS in vitro. Moensin inhibits cytokine release and allows sufficient cytokine accumulation to be detectable by flow cytometry, as previously described (26). Flow cytometric analysis demonstrated that peritoneal macrophage TNFα expression was influenced by AMIT treatment (Fig. 5, A–C). Specifically, there was a 2.7-fold decrease in intracellular TNFα expression in LPS-stimulated macrophages treated with AMIT (Fig. 5D: LPS+NS: 60.74 ± 3.93 vs. LPS+AMIT 22.79 ± 3.27, P < 0.0001). To confirm that AMIT influences TNFα in vivo as well, mice were pretreated with 250 μg of brefeldin A, a protein transport inhibitor similar to moensin. This was followed by LPS and either NS or AMIT administration. Flow cytometric analysis confirmed the reduction of TNFα in resident peritoneal macrophages upon AMIT treatment in endotoxemic mice (Fig. 5E: LPS+NS: 16.65 ± 7.32 vs. LPS+AMIT 7.08 ± 4.41, P = 0.0331).
Ceramide regulates peritoneal macrophage TNFα production through M1 polarization
We next investigated whether AMIT or increased ceramide could mediate changes to the macrophage phenotype. We already established that the in vitro treatment of peritoneal macrophages with AMIT significantly decreased TNFα production (Fig. 5D). Similarly, a reduction in overall macrophage activation, as measured by CD11b median fluorescence intensity (MFI), was observed upon AMIT treatment, but increased by exogenous ceramide addition, after 5 and 30 min (Fig. 6A: 5 min LPS 1.51 ± 0.02 × 106 MFI, LPS + AMIT 0.90 ± 0.01 × 106 MFI, and LPS + Cer 2.04 ± 0.04 × 106, P < 0.01; 30 min LPS 1.76 ± 0.04 × 106 MFI, LPS + AMIT 1.52 ± 0.03 × 106 MFI, and LPS + Cer 2.26 ± 0.04 × 106 MFI, P < 0.01). Macrophage activation was also blunted in monocytes of AMIT-treated CLP mice (Fig. 6B: CLP + NS 0.35 ± 0.06 × 106 MFI, CLP + AMIT 0.25 ± 0.09 × 106 MFI, P < 0.05).
The M1, or cytotoxic macrophage phenotype, is known to promote inflammation and TNFα production (27). We evaluated macrophage surface markers for M1 polarization in vitro. There were significant decreases in CD80 (Fig. 6C: NS 8.3 ± 0.5 × 103 MFI, LPS 9.8 ± 0.5 × 103 MFI, LPS+ AMIT 4.5 ± 0.05 × 103, and LPS + Cer 9.2 ± 0.6 × 103 MFI, p < 0.01), and CD86 (Fig. 6D: NS 0.22 ± 0.003 × 106 MFI, LPS 0.28 ± 0.002 × 106 MFI, LPS + AMIT 0.07 ± 0.001 × 106 MFI, and LPS + Cer vs. 0.12 ± 0.01 × 106 MFI, P < 0.01) macrophage surface membrane expression after treatment with AMIT compared with addition of Cer after LPS stimulation.
Patients with septic shock often exhibit clinical manifestations of hypothermia, hypotension, multiorgan failure, and coagulopathy. The development of thromboembolic events contributes to tissue injury and physiologic insults that contribute to ongoing morbidity and early death. We demonstrated in septic mice a dynamic early hypercoagulability, followed by delayed hypocoagulability. However, septic mice treated with AMIT were resistant to these coagulation changes and exhibited a coagulation profile similar to naive sham mice. The inflammatory cytokine TNFα was markedly elevated in septic mice, but decreased in AMIT-treated mice. Exogenous administration of recombinant TNFα in naive mice recapitulated the acute sepsis-induced hypercoagulable profile. After endotoxemia, peritoneal macrophages were the predominant source of TNFα expression. AMIT treatment significantly decreased macrophage TNFα expression and activation. Finally, accumulation of Cer was observed to favor M1 phenotype, whereas administration of AMIT and reduction of Cer suppressed M1 phenotype. These findings are consistent with previous findings on cystic fibrosis lungs (16). In these experiments, we detected an increase of proinflammatory mediators in cystic fibrosis mice, which was normalized by application of AMIT. At present, it is unknown how ceramide mediates the increase of proinflammatory mediators or, vice versa, how AMIT reduces inflammation. It might be possible that ceramide activates NF-kappaB as previously described (28). In addition, inflammatory mediators might be regulated via JNK and p38, which are also targets of ceramide (29). However, future studies have to delineate whether these or other signaling molecules are involved in the present phenotype.
We used NATEM to measure coagulation during sepsis. Thrombelastometry is a viscoelastic assay that is increasingly being used in the clinical setting to predict need for blood product transfusion among trauma, solid organ transplant, and cardiac surgery patients (30). In addition, prospective comparisons of adult intensive care unit and trauma patients with hypercoagulability on admission NATEM demonstrated an association with an increased incidence of thromboembolic events (31, 32). Furthermore, we used ferric chloride-induced carotid artery thrombosis as a complimentary in vivo method of measuring coagulation. Free radicals generated from ferric chloride denude the vessel endothelial layer, triggering platelet activation and aggregation (33). Due to initiation of the coagulation cascade, this model enhances the data obtained by NATEM, as it replicates the downstream effects of vessel injury from trauma-induced sepsis or sepsis after surgical intervention (22).
It has been demonstrated after CLP-induced sepsis, a hypercoagulable phase is prevalent and followed by a hypocoagulable phase. These dynamic changes were attributed to early thrombin generation and clotting factor consumption (34). Similarly, we demonstrated these temporal changes using both NATEM and ferric chloride-induced carotid artery thrombosis at 2 and 16 ho after CLP. However, Wang et al. reported that antibiotic treatment ameliorated initial thrombin generation and preserved its formation at 24 h, we demonstrate a coagulation profile of septic AMIT-treated mice comparable to that of sham mice at 2 and 16 h.
We observed significant decreases in systemic TNFα levels with AMIT treatment. Numerous studies have established the contributory role of this inflammatory cytokine in promoting coagulation (10–13). To verify this relationship, we administered exogenous TNFα to naive mice, which resulted in acute hypercoagulability similar to septic control mice. Waage et al. previously demonstrated a dose–response curve for recombinant TNFα, and reported that 500 ng was the lowest dose that resulted in death (35). We administered 400 ng of TNFα to achieve toxicity without mortality. Differences in coagulation between naïve mice given NS and recombinant TNFα did not persist at 16 h after administration, likely due to the absence of bacteremia or endotoxemia, which has been reported to potentiate the effects of TNFα (34, 35). While an early rise in serum TNFα levels is appreciable and peaks at 90 min, gradual decline afterward suggests that other mediators during sepsis or endotoxemia are also responsible for the dynamic coagulation profile observed.
Tissue macrophages at the site of septic insult are a primary source of TNFα production (36). We observed decreased CD11b, CD80, and CD86 MFI among peritoneal macrophages in vitro when stimulated with LPS and treated with AMIT, suggestive of suppressed M1 polarization. This supports our finding of improved homeostasis of the coagulation cascade in the septic AMIT-treated mice, comparable to that of sham mice, as opposed to increased CD11b, CD80, and CD86 MFI seen in macrophages stimulated with LPS alone or LPS and Cer, suggestive of M1 phenotype. Nolan et al. demonstrated improved survival and attenuation of inflammatory cytokine production in murine sepsis with genetic and pharmacologic inhibition of macrophage CD80 expression (37). Of note, a key component to any potential therapeutic to alter the host immune response to sepsis should include immune staging. Prior to treatment, such staging should at least examine evidence of pro- or anti-inflammatory mediators, such as TNFα. Potentially, the use of an anti-TNFα antibody could be used as a therapy to reduce availability of this cytokine. However, the antibody is relatively stable and would promote a decrease of TNFα for a prolonged period. In contrast, one benefit for using AMIT as a potential treatment is its labileness in serum. Thus, one could potentially treat with AMIT and subsequent re-analyze the inflammatory mediators to determine if subsequent dose(s) are needed.
Treatment of sepsis sepsis-induced coagulopathy with AMIT may have clinical potential, as AMIT is a popular antidepressant with an established safety and dosing profile. In addition, we were able to demonstrate significant immunomodulatory changes and systemic effects with a single loading dose, which is more clinically relevant than prior studies, in which mice were pretreated for several days before inducing sepsis (20). AMIT can regulate a number of targets, including acid sphingomyelinase (Asm) (38). Asm inhibition with AMIT has been shown to reduce ceramide levels (17), thus mediating anti-inflammatory effects (16). We did not include Asm heterozygous or knockout mice in our coagulopathy experiments to test the specificity of the observed AMIT-related effects for Asm inhibition, as we felt that the Asm deficient mice would introduce potentially confounding compensatory mechanisms. To test the specificity of AMIT for reducing ceramide in our model, we treated macrophages with AMIT in vitro and observed a decrease of extracellular and total ceramide (Suppl. Fig. 1, see http://links.lww.com/SHK/A731). AMIT has been shown to block the acid sphingomyelinase, but it also blocks the acid ceramidase and may, therefore, induce also a decrease of sphingosine and sphingosine 1-phosphate, in addition to a decrease of ceramide (39). In addition to its known ability to prevent uptake of monoaminergic transmitters, AMIT has been demonstrated to have anticholinergic, antiadrenergic, and mild antihistaminergic effects. A recent paper also shows the specificity of AMIT for Asm in terms of cytokine production (15). Thus, the effects reported here could be due to Asm inhibition, although further studies are necessary to support this in the LPS and CLP models used for this study.
Altogether, during polymicrobial sepsis, AMIT treatment suppressed macrophage TNFα expression and macrophage M1 function, ameliorating an initial hypercoagulable state, and protecting septic mice from delayed coagulopathy. We propose that AMIT treatment is a promising therapeutic approach in the treatment of sepsis-associated coagulopathy that may prevent acute thromboembolic events and delayed bleeding complications.
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