TR-05493; No of Pages 5 Thrombosis Research xxx (2014) xxx–xxx
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Original article
Combination of antithrombin and recombinant thrombomodulin modulates neutrophil cell-death and decreases circulating DAMPs levels in endotoxemic rats Toshiaki Iba a,⁎, Takahiro Miki a, Naoyuki Hashiguchi a, Yoko Tabe b, Isao Nagaoka c a b c
Department of Emergency Disaster Medicine, Juntendo University Graduate School of Medicine Department of Clinical Laboratory Medicine, Juntendo University Graduate School of Medicine Department of Host Defense and Biochemical Research, Juntendo University Graduate School of Medicine
a r t i c l e
i n f o
Article history: Received 27 January 2014 Received in revised form 15 March 2014 Accepted 17 April 2014 Available online xxxx Keywords: Antithrombin Recombinant thrombomodulin Histone Cell-free DNA Damage-associated molecular patterns (DAMPs) Sepsis
a b s t r a c t Introduction: The activation of coagulation is recognized as a universal event in severe sepsis. Both antithrombin and thrombomodulin play pivotal roles as suppressors of coagulation. Since the levels of both anticoagulants decrease significantly, we hypothesized that a combination therapy would be beneficial. Methods: A sepsis model was established using the intravenous infusion of lipopolysaccharide (LPS). Either 125 IU/kg of antithrombin, 0.25 mg/kg of recombinant thrombomodulin, or a combination of both agents was injected immediately after LPS infusion (n = 7 each), while only a physiological saline was injected in the control group (n = 7). Blood samples were obtained at eight hours after LPS infusion, and organ damage markers and the plasma levels of damage-associated molecular patterns (DAMPs), such as histone H3 and cell-free DNA (cf-DNA), were measured. In another series, the leukocytes harvested from normal rats were cultured in plasma obtained from each group (n = 7). Eight hours later, the leukocytes were stained with green fluorescent protein, Annexin V and 7-AAD, and the proportion of alive + apoptic/necrotic cells was calculated. Results: Organ damage markers such as ALT and BUN were maintained best in the combination group (P b 0.05). The circulating levels of histone H3 and cf-DNA were both significantly lower in the combination therapy group (P b 0.01, 0.05, respectively). The proportion of alive + apoptic/necrotic cells was significantly higher in the combination therapy group (P b 0.05). Conclusion: The coadministration of antithrombin and recombinant thrombomodulin can modulate cell death and decrease the circulating levels of histone H3 and cf-DNA, leading to protection against organ damage in a rat model of sepsis. © 2014 Elsevier Ltd. All rights reserved.
Introduction Excess inflammatory responses play important roles in the worsening of the clinical condition during severe sepsis [1]. Research in this area has been focused on microorganisms and their related pathogenic substances, or pathogen-associated molecular patterns (PAMPs) and the host immune responses [2]. PAMPs are recognized by particular receptors and then initiate the production of immune-related proteins that exacerbate the damage, which is known as ‘alarmins’ [3,4]. Other than alarmins, there is increasing interest in sepsis associated cell death-related nucleic substances, such as DNA and histones. Together ⁎ Corresponding author at: Emergency and Disaster Medicine, Juntendo University Graduate School of Medicine, 2-1-1 Hongo Bunkyo-ku, Tokyo 113–8421, Japan. Tel.: +81 3 3813 3111x5818; fax: +81 3 3813 5431. E-mail addresses: [email protected] (T. Iba), [email protected] (T. Miki), [email protected] (N. Hashiguchi), [email protected] (Y. Tabe), [email protected] (I. Nagaoka).
with alarmins and PAMPs, these substances are known as damageassociated molecular patterns (DAMPs) [5,6]. The term ‘DAMPs’ is still controversial as it was formerly used in a manner similar to alarmins [7], or the representative of alarmins and PAMPs [4], but DAMPs often be used as the generic name of the intrinsic inflammatory initiators currently. In this report, we refer to ‘DAMPs’ as intrinsic substances released from dying cells that propagate further inflammation. There is a general agreement that the excess activation of coagulation during sepsis leads to inappropriate intravascular thrombus formation and malcirculation [8]. However, the results of clinical trials evaluating the effects of anticoagulants have been controversial [9–11]. We hypothesized that the maintenance of both major physiological anticoagulant systems, i.e. antithrombin (AT) and the thrombomodulin/ activated protein C (APC) system are essential and have reported that the combination of AT and recombinant thrombomodulin (rTM) improved survival in an experimental rat model of sepsis [12]. In addition, we have also reported that combination therapy with AT and rTM
http://dx.doi.org/10.1016/j.thromres.2014.04.015 0049-3848/© 2014 Elsevier Ltd. All rights reserved.
Please cite this article as: Iba T, et al, Combination of antithrombin and recombinant thrombomodulin modulates neutrophil cell-death and decreases circulating DAMPs levels ..., Thromb Res (2014), http://dx.doi.org/10.1016/j.thromres.2014.04.015
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T. Iba et al. / Thrombosis Research xxx (2014) xxx–xxx
suppressed leukocyte activation in microvasculature, leading to the maintenance of microcirculation and subsequent organ protection. Since AT and rTM suppressed the elevation of circulating levels of nucleosome during the early stage of sepsis in a former study [13], we speculated that the combination of these anticoagulants may express effects through the suppression of the expulsion of neutrophil extracellular traps (NETs) and leukocyte necrosis. In the current study, we focused on further details of the mechanisms of this combination therapy, especially leukocyte cell death. Materials And Methods Sepsis model and treatment groups Ten-week-old Wistar rats (purchased from Japan Clea Co., Tokyo Japan) were used. All the experimental procedures were conducted after obtaining the approval of the Ethical Committee for Animal Experiments of Juntendo University. All the rats were provided with standard rat chow and water ad libitum. Rats were anesthetized with intraperitoneal sodium thiopental (100 mg/kg; Pentothal; Sigma Chemical Co., St. Louis, USA). Then, 8.0 mg/kg of lipopolysaccharide (LPS, E. coli O55-B5; Difco Laboratories, Detroit, USA) diluted with 0.15 mL of sterile physiological saline was infused intravenously. The animals were divided into four groups. Either 125 IU/kg of antithrombin (CSL Behring Japan Co., Tokyo, Japan) (AT group), 0.25 mg/kg of recombinant thrombomodulin (ART-123, Asahi Kasei Pharma Co., Tokyo, Japan) (rTM group), or 125 IU/kg of antithrombin and 0.25 mg/kg of rTM (AT/rTM group) were administered intravenously immediately after LPS infusion (n = 7, each). In the control group (n = 7), animals were given LPS and saline. Blood sampling and measurement At eight hours after the LPS infusion, the rats were sacrificed under anesthesia in an ether chamber. Blood samples were obtained from the inferior vena cava and a blood cell count was performed. The white blood cell (WBC) count and the platelet count were determined using a multi-automatic blood cell counter for animals (MICROS abc LC-152; HORIBA, Ltd. Tokyo, Japan). The citrated plasma samples were obtained by whole blood centrifugation and were stored at -80 ºC until assay. An additional five rats without any treatment were used for blood sampling as a normal group. Fibrin/fibrinogen degradation products (FDP), and fibrinogen levels and the levels of the organ damage markers such as alanine aminotransferase (ALT), total bilirubin (T-Bil), creatinine, and blood urea nitrogen (BUN) were measured in the samples. The FDP and fibrinogen levels were determined using an enzyme-linked immunosorbent assay kit (Teikoku Laboratories, Tokyo, Japan). Quantification of cf-DNA and Histone H3 levels in plasma In the other series, the citrated plasma taken from each group (n = 7) was used to measure the histone H3 and cf-DNA levels. The histone H3 levels were measured using an originally established sandwich enzyme-linked immunosorbent assay (ELISA). Briefly, 20 μL of citrated plasma was diluted 1:4 in 1% bovine serum albumin, 0.5% Tween, and 1 mmol/L ethylenediamine tetraacetic acid in phosphate buffered saline and added to streptavidin-coated microtiter plates containing biotinylated mouse anti-histone antibody (clone H11-4; Roche Diagnostics, Basel Switzerland) and peroxidase-conjugated anti-histone antibodies (rabbit anti-histone H3 antibody (HRP)(ab53528, Abcam, Cambridge UK). After standard washing steps with cleaning buffer, the peroxidase activity of the retained immunocomplex was developed by incubation with ABTS (2,2'-azino-bis-(3-ethylbenzothiazoline-6-sulphonate) and read in a spectrophotometer at 405 nm. cf-DNA was isolated from 250 μL of plasma by using the FitAmp™ Circulating DNA Quantification Kit (Epigentek Inc, NY USA). The concentration of the DNA was measured for the UV
absorbance at 260 nm using a spectrophotometer (Beckman DU 7400; Beckman Coulter Inc., Brea, CA, USA). Immunostaining of the leukocyte Leukocytes extracted from healthy rats were utilized for the immunostaining. Rats were given intraperitoneal injections of glycogen. Eight hours later, their peritoneal cavities were washed with 10 mL of medium RPMI 1640. The sampled peritoneal lavage was centrifuged at 400 ×g for 5 min and then washed again with the collection buffer. Then, the leukocytes were resuspended and cultured in a chamber slide (#4722-040; Iwaki Inc, Tokyo Japan) with 50 μL of the plasma obtained from each group for 8 hours and then, stained with Apoptosis/Necrosis Detection Kit (ENZ-51002–25, Enzo Life Sciences, NY USA) according to the manufacturer's instructions with slight modifications. Briefly, recipitated leukocytes were suspended in 500 μL of detection solution (apoptosis detection reagent containing Annexin V-EnzoGold conjugate, necrosis detection reagent containing 7-AAD, and green fluorescent protein for the viable cell detection). Seven fields in each sample were examined under a fluorescent microscope by two individuals, and the mean ratio of the viable plus apoptotic cells relative to the necrotic cells was calculated. Statistical analysis All the data were expressed as the mean ± standard error. A statistical analysis was performed using the StatView II statistical software package for Macintosh. Statistical comparisons were performed using a non-parametric Kruskal-Wallis one-way ANOVA with Dunnett posthoc test for multiple comparisons and the Mann–Whitney test for single comparisons. Statistical differences were deemed significant at a level of P b 0.05. Results I) Laboratory data findings The normal WBC count was approximately 6000/mm3, and the number decreased to 2412.5 ± 248.2/mm3 in the control group at 8 hours after LPS injection. These reductions were suppressed in all of the AT, rTM and AT/rTM groups (P b 0.01 respectively) (Fig. 1, left). Similarly, the platelet count decreased to less than one third after LPS injection, and these reductions were again suppressed in all of the treatment groups (P b 0.01 respectively). The platelet count was maintained at over 500 x 103/mm3 in the AT/rTM group, and this count was significantly higher than those for the AT and rTM groups (P b 0.05, 0.01, respectively) (Fig. 1, right). For the hemostatic markers, both the AT and the AT/rTM groups showed significant reductions in the elevated FDP level, compared with that in the control group (P b 0.05 and 0.01, respectively). The fibrinogen level was significantly higher in the AT, rTM and AT/rTM groups than in the control group (P b 0.05, 0.05 and 0.01, respectively). Regarding the organ damage markers, elevated levels of ALT, T-Bil, creatinine, and BUN were observed at 8 hours after LPS infusion in the control group, and the significant suppression of the elevated ALT and BUN levels were recognized in the AT/rTM group (P b 0.05, respectively) (Table 1). The histone H3 level was 17.0 ± 6.0 pg/mL in the control group. Meanwhile, the levels were 6.0 ± 1.0 pg/mL in the AT group, 5.0 ± 1.0 pg/mL in the rTM group, and 3.0 ± 1.0 pg/mL in the AT/rTM group (Fig. 2, left), with a significantly lower level in the AT/rTM group (P b 0.01 compared with the control group, P b 0.05 compared with AT and rTM group). The cf-DNA level was 46.2 ± 6.2 ng/mL in the control group. Meanwhile, the levels were suppressed to about two thirds of that in the AT group and the rTM group. The cf-DNA level was most significantly suppressed in the AT/rTM group, and
Please cite this article as: Iba T, et al, Combination of antithrombin and recombinant thrombomodulin modulates neutrophil cell-death and decreases circulating DAMPs levels ..., Thromb Res (2014), http://dx.doi.org/10.1016/j.thromres.2014.04.015
T. Iba et al. / Thrombosis Research xxx (2014) xxx–xxx
7000
800
6000
700 600
Platelet count
5000
4000
3000
2000
500 400 300
AT/rTM
rTM
normal
AT
AT/rTM
0 rTM
0 control
100
normal
1000
AT
200
control
WBC count
3
Fig. 1. Changes in WBC and platelet count. The WBC count decreased significantly at 8 hours after LPS injection, and these reductions were suppressed in the AT, rTM and AT/rTM groups. The platelet count also decreased significantly after LPS injection, and these reductions were suppressed in all the treatment groups. The count in the AT/rTM group was significantly higher than those in the AT and rTM groups. WBC: white blood cells, LPS: lipopolysaccharide, AT: antithrombin, rTM: recombinant thrombomodulin *: P b 0.05, **: P b 0.01.
TM efficiently suppressed the coagulation, and which lead to the improvement of organ dysfunction. Interestingly, AT/rTM suppressed not only the activated coagulation but also inhibited leukocyte necrosis. When viewed according to leukocyte cell death, apoptosis was dominant under normal situations, and the population of necrotic leukocytes was rather high after the LPS treatment. The necrotic leukocytes expelled DAMPs and accelerated further inflammation during sepsis. In this study, AT and rTM were revealed to inhibit leukocyte necrosis, reduce the circulating DAMPs, suppress the coagulatory responses and thereby demonstrated the organ protection. Monocytes/macrophages are widely accepted as the major players in the procoagulant process; however, recent evidence has suggested that neutrophils also play important roles by expressing tissue factor [16,17], micro particles [18] and ejecting NETs [19,20]. Neutrophils accumulate and adhere tightly to the endothelium, and expel web-like structures named NETs that serve as a scaffold for thrombus formation [21]. As a matter of fact, the major components of NETs, histones [22] and DNA [23,24] are strong initiators of coagulation. In this study, circulating levels of histone H3 cf-DNA were recognized after LPS treatment. Regarding the source of these DAMPs, leukocyte necrosis is likely to contribute to the elevation in DAMPs as well as NETs. Histones are DNA-binding proteins that account for the majority of nucleic proteins. Once histones are released into the bloodstream, they express an innately damaging effect on tissues that is most
the difference was significant compared with that in the control group (P b 0.05) (Fig. 2, right). II) Alive and apoptotic leukocyte/necrotic leukocyte The surfaces of leukocytes incubated in medium with plasma obtained from the control group were mainly stained with 7-ADD (red), which represents necrosis (Fig. 3, left). In contrast, the percentage of leukocytes that were stained green (green fluorescent protein) or yellow (Annexin V) increased in the AT group and the rTM group, and this trend was most prominent in the AT/rTM group (Fig. 3, middle). The ratio of alive plus apoptotic/necrotic cells calculated in the AT/rTM group was almost twice as much as that in the control group, and the difference was statistically significant (P b 0.05) (Fig. 3, right). Discussion The activation of coagulation is an important part of host defense mechanisms [14] and is a universal event during severe sepsis. Activated coagulation limits microbial spreading and localizes infection [15]. If it occurs systemically, however, it can cause obstructions in the microvasculature and can induce tissue ischemia, thereby contributing to multiple organ failure and death. Therefore, the basic concept of anticoagulant therapy is to limit the excess activation of the coagulation. In the current study, we demonstrated that AT and Table 1 Changes in biochemical data.
Normal Control group AT group rTM group AT/rTM group
FDP (μg/dL)
fibrinogen (mg/dl)
ALT (IU/L)
T-Bil (mg/dL)
creatinine (mg/dL)
BUN (mg/dL)
10.4 33.1 19.2 27.5 12.3
242.2 91.4 128.0 125.1 189.3
25.1 85.4 60.9 57.3 29.6
0.74 1.59 1.19 1.49 1.34
0.18 0.98 0.77 0.77 0.72
14.1 61.8 57.9 56.4 44.6
± ± ± ± ±
0.9 6.8 3.6⁎ 5.7 1.9⁎⁎
± ± ± ± ±
16.1 9.4 12.3⁎ 12.6⁎ 17.2⁎⁎
± ± ± ± ±
2.6 14.6 12.4 13.2 9.9⁎
± ± ± ± ±
0.07 0.27 0.13 0.18 0.18
± ± ± ± ±
0.04 0.34 0.39 0.36 0.36
± ± ± ± ±
0.7 5.5 4.9 5.1 4.8⁎
FDP = fibrin/fibrinogen degradation products ALT = alanine aminotransferase; T-Bil = total bilirubin; BUN = blood urea nitrogen ⁎ P b 0.05 ⁎⁎ P b 0.01 compared to the control
Please cite this article as: Iba T, et al, Combination of antithrombin and recombinant thrombomodulin modulates neutrophil cell-death and decreases circulating DAMPs levels ..., Thromb Res (2014), http://dx.doi.org/10.1016/j.thromres.2014.04.015
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(pg/mL)
(ng/mL) 60
25.0
50 20.0
15.0
cf-DNA
Histone H3
40
30
10.0 20 5.0
10
AT/rTM
rTM
AT
control
AT/rTM
rTM
AT
control
normal
normal
0
0
Fig. 2. Changes in histone H3 and cf-DNA. The histone H3 level was elevated at 8 hours after LPS injection in the control group. The levels were significantly lower in the AT, rTM and AT/rTM groups. The cf-DNA level was also elevated in the control group. The cf-DNA level was significantly suppressed in the AT/rTM group compared with the control group. cf-DNA: cell-free DNA, LPS: lipopolysaccharide, AT: antithrombin, rTM: recombinant thrombomodulin *: P b 0.05, **: P b 0.01.
elevated after stimulation with LPS. The findings that the combination therapy with AT/rTM suppressed these DAMPs levels suggest that this anticoagulant therapy can control the dangerous cycle of increased DAMPs, activated coagulation and leukocyte necrosis. The above theory was further confirmed in the ex vivo experiment. The manner of leukocyte cell death was examined using the fluorescent staining. As a result, the leukocytes treated with plasma from the control group were stained mainly in red with 7-AAD, a dye that binds to DNA in membrane-permeabilized cells, suggesting that the majority of the leukocytes fell into necrotic cell death. In contrast, necrosis was efficiently suppressed in the AT/rTM group, in which green or yellow
Alive+apoptotic/necrotic cells
prominent for histone H3 and H4 [25]. Xu et al. [25] demonstrated that APC can degrade histones H3 and H4 and minimize tissue damage, thereby improving survival rates. Interestingly, Semeraro et al. [26] reported that histones activate coagulation in a dose dependent manner through the inhibition of the thrombomodulin-mediated activation of protein C. Other than APC, rTM was also reported to reduce the damage induced by histones [27]. As for cf-DNA, its level reportedly increases in sepsis, and a higher level indicates a poor outcome [28,29]. cf-DNA is also known to induce inflammation through the activation of Toll-like receptor in sepsis [30]. In our study, the concentrations of histone H3 and cf-DNA were low in the normal group, but they were significantly
*
1.5
1.0
0.5
AT/rTM
AT
rTM
AT/rTM
control
control
normal
0
Fig. 3. Immunostaining of leukocytes and ratio of viable and apoptotic leukocytes/necrotic leukocytes. The leukocytes incubated in the medium and the plasma obtained from the control group were stained predominantly by 7-AAD (red) (left). In contrast, the percentage of leukocytes that were stained green (green fluorescent protein) or yellow (Annexin V) increased in the AT/rTM group (middle). The populations of viable and apoptotic/necrotic leukocytes were calculated in the AT/rTM group. The ratio in the AT/rTM group was almost twice that in the control group (right). The photographs are overlays of the immunofluorescent and bright views. AT: antithrombin, rTM: recombinant thrombomodulin.
Please cite this article as: Iba T, et al, Combination of antithrombin and recombinant thrombomodulin modulates neutrophil cell-death and decreases circulating DAMPs levels ..., Thromb Res (2014), http://dx.doi.org/10.1016/j.thromres.2014.04.015
T. Iba et al. / Thrombosis Research xxx (2014) xxx–xxx
staining was predominant. Green fluorescent protein is widely used to stain viable cells, and Annexin V is a popular fluorescence that is used to detect apoptosis because it has a high affinity to phosphatidylserine, which is translocated from the inner to the outer plasma membrane of apoptotic cells. In the current study, both of global coagulation tests such as PT and APTT and bleeding tendency have not been examined. Since the bleeding adverse event causes critical damage to the patients with septic DIC, these issues should be addressed before the clinical application. In summary, the combination therapy successfully suppressed activated coagulation and leukocyte necrosis and decreased the levels of circulating DAMPs such as histone H3 and cf-DNA. Since activated coagulation and leukocyte necrosis plays a significant role in the amplification of inflammation and organ damage, the combination therapy with AT and rTM may be useful for the treatment of severe sepsis. Conclusion The combination of antithrombin and rTM efficiently suppressed leukocyte necrosis and decreased the circulating levels of DAMPs including histone H3 and cf-DNA. These effects lead to the suppression of activated coagulation and subsequent organ protection. Conflict of interest statement The authors state that Iba T, Miki T, Hashiguchi Y, Tabe Y and Nagaoka I have no conflicts of interest. Authors’ contributions Iba T and Hashiguchi N designed the study. Miki T and Tabe Y performed the experiment. Iba T and Nagaoka I wrote the manuscript. All authors read and approved the final manuscript. Acknowledgements This work was supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology (#25462831). A part of this study was presented at the 40th Annual Meeting of the Japanese Society of Intensive Care Medicine. References [1] Angus DC, van der Poll T. Severe sepsis and septic shock. N Engl J Med 2013; 369:2063. [2] Kumar H, Kawai T, Akira S. Pathogen recognition by the innate immune system. Int Rev Immunol 2011;30:16–34. [3] Harris HE, Raucci A. Alarmin(g) news about danger: workshop on innate danger signals and HMGB1. EMBO Rep 2006;7:774–8. [4] Rittirsch D, Flierl MA, Ward PA. Harmful molecular mechanisms in sepsis. Nat Rev Immunol 2008;8:776–87. [5] Seong SY, Matzinger P. Hydrophobicity: an ancient damage-associated molecular pattern that initiates innate immune responses. Nat Rev Immunol 2004;4:469–78. [6] Cinel I, Opal SM. Molecular biology of inflammation and sepsis: a primer. Crit Care Med 2009;37:291–304.
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[7] Bianchi ME. DAMPs, PAMPs and alarmins: all we need to know about danger. J Leukoc Biol 2007;81:1–5. [8] Iba T, Nagaoka I, Boulat M. The anticoagulant therapy for sepsis-associated disseminated intravascular coagulation. Thromb Res 2013;31:383–9. [9] Bernard GR, Vincent JL, Laterre PF, LaRosa SP, Dhainaut JF, Lopez-Rodriguez A, et al. Recombinant human protein C Worldwide Evaluation in Severe Sepsis (PROWESS) study group: Efficacy and safety of recombinant human activated protein C for severe sepsis. N Engl J Med 2001;344:699–709. [10] Abraham E, Reinhart K, Opal S, Demeyer I, Doig C, Rodriguez AL, et al. OPTIMIST Trial Study Group: Efficacy and safety of tifacogin (recombinant tissue factor pathway inhibitor) in severe sepsis: a randomized controlled trial. J Am Med Assoc 2003;290:238–47. [11] Warren BL, Eid A, Singer P, Pillay SS, Carl P, Novak I, et al. Caring for the critically ill patient. High-dose antithrombin III in severe sepsis: a randomized controlled trial. J Am Med Assoc 2001;286:1869–78. [12] Iba T, Nakarai E, Takayama T, Nakajima K, Sasaoka T, Ohno Y. Combination effect of antithrombin and recombinant human soluble thrombomodulin in an LPS induced rat sepsis model. Crit Care 2009;13:R203. [13] Iba T, Miki T, Hashiguchi N, Yamada A, Nagaoka I. Combination of antithrombin and recombinant thrombomodulin attenuates leukocyte-endothelial interaction and suppresses the increase of intrinsic damage-associated molecular patterns in endotoxemic rats. J Surg Res 2014;187:581–6. [14] Engelmann B, Massberg S. Thrombosis as an intravascular effector of innate immunity. Nat Rev Immunol 2013;13:34–45. [15] Esmon CT, Xu J, Lupu F. Innate immunity and coagulation. J Thromb Haemost 2011;9:182–8. [16] Maugeri N, Brambilla M, Camera M, Carbone A, Tremoli E, Donati MB, et al. Human polymorphonuclear leukocytes produce and express functional tissue factor upon stimulation. J Thromb Haemost 2006;4:1323–30. [17] Darbousset R, Thomas GM, Mezouar S, Frère C, Bonier R, Mackman N, et al. Tissue factor-positive neutrophils bind to injured endothelial wall and initiate thrombus formation. Blood 2012;120:2133–43. [18] Aras O, Shet A, Bach RR, Hysjulien JL, Slungaard A, Hebbel RP, et al. Induction of microparticle- and cell-associated intravascular tissue factor in human endotoxemia. Blood 2004;103:4545–53. [19] Fuchs TA, Abed U, Goosmann C, Hurwitz R, Schulze I, Wahn V, et al. Novel cell death program leads to neutrophil extracellular traps. J Cell Biol 2007;176:231–41. [20] Clark SR, Ma AC, Tavener SA, McDonald B, Goodarzi Z, Kelly MM, et al. Platelet TLR4 activates neutrophil extracellular traps to ensnare bacteria in septic blood. Nat Med 2007;13:463–9. [21] Levi M. The coagulant response in sepsis. Clin Chest Med 2008;29:627–42. [22] Xu J, Zhang X, Monestier M, Esmon NL, Esmon CT. Extracellular histones are mediators of death through TLR2 and TLR4 in mouse fatal liver injury. J Immunol 2011; 187:2626–31. [23] Fuchs TA, Brill A, Duerschmied D, Schatzberg D, Monestier M, Myers Jr DD, et al. Extracellular DNA traps promote thrombosis. Proc Natl Acad Sci U S A 2010; 107:15880–5. [24] Brinkmann V, Zychlinsky A. Neutrophil extracellular traps: is immunity the second function of chromatin? J Cell Biol 2012;198:773–83. [25] Xu J, Zhang X, Pelayo R, Monestier M, Ammollo CT, Semeraro F, et al. Extracellular histones are major mediators of death in sepsis. Nat Med 2009;15:1318–21. [26] Ammollo CT, Semeraro F, Xu J, Esmon NL, Esmon CT. Extracellular histones increase plasma thrombin generation by impairing thrombomodulin-dependent protein C activation. J Thromb Haemost 2011;9:1795–803. [27] Nakahara M, Ito T, Kawahara K, Yamamoto M, Nagasato T, Shrestha B, et al. Recombinant thrombomodulin protects mice against histone-induced lethal thromboembolism. PLoS One 2013;8:e75961. [28] Saukkonen K, Lakkisto P, Pettila V, Varpula M, Karlsson S, Ruokonen E, Pulkki K. Cellfree. Plasma DNA as a predictor of outcome in severe sepsis and septic shock. Clin Chem 2008;54:1000–7. [29] Zeerleder S, Zwart B, Wuillemin WA, Aarden LA, Groeneveld AB, Caliezi C, van Nieuwenhuijze AE, et al. Elevated nucleosome levels in systemic inflammation and sepsis. Crit Care Med 2003;31:1947–51. [30] Duramad O, Fearon KL, Chang B, Chan JH, Gregorio J, Coffman RL, Barrat FJ. Inhibitors of TLR-9 act on multiple cell subsets in mouse and man in vitro and prevent death in vivo from systemic inflammation. J Immunol 2005;174:5193–200.
Please cite this article as: Iba T, et al, Combination of antithrombin and recombinant thrombomodulin modulates neutrophil cell-death and decreases circulating DAMPs levels ..., Thromb Res (2014), http://dx.doi.org/10.1016/j.thromres.2014.04.015
Contents lists available at ScienceDirect
Thrombosis Research journal homepage: www.elsevier.com/locate/thromres
Original article
Combination of antithrombin and recombinant thrombomodulin modulates neutrophil cell-death and decreases circulating DAMPs levels in endotoxemic rats Toshiaki Iba a,⁎, Takahiro Miki a, Naoyuki Hashiguchi a, Yoko Tabe b, Isao Nagaoka c a b c
Department of Emergency Disaster Medicine, Juntendo University Graduate School of Medicine Department of Clinical Laboratory Medicine, Juntendo University Graduate School of Medicine Department of Host Defense and Biochemical Research, Juntendo University Graduate School of Medicine
a r t i c l e
i n f o
Article history: Received 27 January 2014 Received in revised form 15 March 2014 Accepted 17 April 2014 Available online xxxx Keywords: Antithrombin Recombinant thrombomodulin Histone Cell-free DNA Damage-associated molecular patterns (DAMPs) Sepsis
a b s t r a c t Introduction: The activation of coagulation is recognized as a universal event in severe sepsis. Both antithrombin and thrombomodulin play pivotal roles as suppressors of coagulation. Since the levels of both anticoagulants decrease significantly, we hypothesized that a combination therapy would be beneficial. Methods: A sepsis model was established using the intravenous infusion of lipopolysaccharide (LPS). Either 125 IU/kg of antithrombin, 0.25 mg/kg of recombinant thrombomodulin, or a combination of both agents was injected immediately after LPS infusion (n = 7 each), while only a physiological saline was injected in the control group (n = 7). Blood samples were obtained at eight hours after LPS infusion, and organ damage markers and the plasma levels of damage-associated molecular patterns (DAMPs), such as histone H3 and cell-free DNA (cf-DNA), were measured. In another series, the leukocytes harvested from normal rats were cultured in plasma obtained from each group (n = 7). Eight hours later, the leukocytes were stained with green fluorescent protein, Annexin V and 7-AAD, and the proportion of alive + apoptic/necrotic cells was calculated. Results: Organ damage markers such as ALT and BUN were maintained best in the combination group (P b 0.05). The circulating levels of histone H3 and cf-DNA were both significantly lower in the combination therapy group (P b 0.01, 0.05, respectively). The proportion of alive + apoptic/necrotic cells was significantly higher in the combination therapy group (P b 0.05). Conclusion: The coadministration of antithrombin and recombinant thrombomodulin can modulate cell death and decrease the circulating levels of histone H3 and cf-DNA, leading to protection against organ damage in a rat model of sepsis. © 2014 Elsevier Ltd. All rights reserved.
Introduction Excess inflammatory responses play important roles in the worsening of the clinical condition during severe sepsis [1]. Research in this area has been focused on microorganisms and their related pathogenic substances, or pathogen-associated molecular patterns (PAMPs) and the host immune responses [2]. PAMPs are recognized by particular receptors and then initiate the production of immune-related proteins that exacerbate the damage, which is known as ‘alarmins’ [3,4]. Other than alarmins, there is increasing interest in sepsis associated cell death-related nucleic substances, such as DNA and histones. Together ⁎ Corresponding author at: Emergency and Disaster Medicine, Juntendo University Graduate School of Medicine, 2-1-1 Hongo Bunkyo-ku, Tokyo 113–8421, Japan. Tel.: +81 3 3813 3111x5818; fax: +81 3 3813 5431. E-mail addresses: [email protected] (T. Iba), [email protected] (T. Miki), [email protected] (N. Hashiguchi), [email protected] (Y. Tabe), [email protected] (I. Nagaoka).
with alarmins and PAMPs, these substances are known as damageassociated molecular patterns (DAMPs) [5,6]. The term ‘DAMPs’ is still controversial as it was formerly used in a manner similar to alarmins [7], or the representative of alarmins and PAMPs [4], but DAMPs often be used as the generic name of the intrinsic inflammatory initiators currently. In this report, we refer to ‘DAMPs’ as intrinsic substances released from dying cells that propagate further inflammation. There is a general agreement that the excess activation of coagulation during sepsis leads to inappropriate intravascular thrombus formation and malcirculation [8]. However, the results of clinical trials evaluating the effects of anticoagulants have been controversial [9–11]. We hypothesized that the maintenance of both major physiological anticoagulant systems, i.e. antithrombin (AT) and the thrombomodulin/ activated protein C (APC) system are essential and have reported that the combination of AT and recombinant thrombomodulin (rTM) improved survival in an experimental rat model of sepsis [12]. In addition, we have also reported that combination therapy with AT and rTM
http://dx.doi.org/10.1016/j.thromres.2014.04.015 0049-3848/© 2014 Elsevier Ltd. All rights reserved.
Please cite this article as: Iba T, et al, Combination of antithrombin and recombinant thrombomodulin modulates neutrophil cell-death and decreases circulating DAMPs levels ..., Thromb Res (2014), http://dx.doi.org/10.1016/j.thromres.2014.04.015
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suppressed leukocyte activation in microvasculature, leading to the maintenance of microcirculation and subsequent organ protection. Since AT and rTM suppressed the elevation of circulating levels of nucleosome during the early stage of sepsis in a former study [13], we speculated that the combination of these anticoagulants may express effects through the suppression of the expulsion of neutrophil extracellular traps (NETs) and leukocyte necrosis. In the current study, we focused on further details of the mechanisms of this combination therapy, especially leukocyte cell death. Materials And Methods Sepsis model and treatment groups Ten-week-old Wistar rats (purchased from Japan Clea Co., Tokyo Japan) were used. All the experimental procedures were conducted after obtaining the approval of the Ethical Committee for Animal Experiments of Juntendo University. All the rats were provided with standard rat chow and water ad libitum. Rats were anesthetized with intraperitoneal sodium thiopental (100 mg/kg; Pentothal; Sigma Chemical Co., St. Louis, USA). Then, 8.0 mg/kg of lipopolysaccharide (LPS, E. coli O55-B5; Difco Laboratories, Detroit, USA) diluted with 0.15 mL of sterile physiological saline was infused intravenously. The animals were divided into four groups. Either 125 IU/kg of antithrombin (CSL Behring Japan Co., Tokyo, Japan) (AT group), 0.25 mg/kg of recombinant thrombomodulin (ART-123, Asahi Kasei Pharma Co., Tokyo, Japan) (rTM group), or 125 IU/kg of antithrombin and 0.25 mg/kg of rTM (AT/rTM group) were administered intravenously immediately after LPS infusion (n = 7, each). In the control group (n = 7), animals were given LPS and saline. Blood sampling and measurement At eight hours after the LPS infusion, the rats were sacrificed under anesthesia in an ether chamber. Blood samples were obtained from the inferior vena cava and a blood cell count was performed. The white blood cell (WBC) count and the platelet count were determined using a multi-automatic blood cell counter for animals (MICROS abc LC-152; HORIBA, Ltd. Tokyo, Japan). The citrated plasma samples were obtained by whole blood centrifugation and were stored at -80 ºC until assay. An additional five rats without any treatment were used for blood sampling as a normal group. Fibrin/fibrinogen degradation products (FDP), and fibrinogen levels and the levels of the organ damage markers such as alanine aminotransferase (ALT), total bilirubin (T-Bil), creatinine, and blood urea nitrogen (BUN) were measured in the samples. The FDP and fibrinogen levels were determined using an enzyme-linked immunosorbent assay kit (Teikoku Laboratories, Tokyo, Japan). Quantification of cf-DNA and Histone H3 levels in plasma In the other series, the citrated plasma taken from each group (n = 7) was used to measure the histone H3 and cf-DNA levels. The histone H3 levels were measured using an originally established sandwich enzyme-linked immunosorbent assay (ELISA). Briefly, 20 μL of citrated plasma was diluted 1:4 in 1% bovine serum albumin, 0.5% Tween, and 1 mmol/L ethylenediamine tetraacetic acid in phosphate buffered saline and added to streptavidin-coated microtiter plates containing biotinylated mouse anti-histone antibody (clone H11-4; Roche Diagnostics, Basel Switzerland) and peroxidase-conjugated anti-histone antibodies (rabbit anti-histone H3 antibody (HRP)(ab53528, Abcam, Cambridge UK). After standard washing steps with cleaning buffer, the peroxidase activity of the retained immunocomplex was developed by incubation with ABTS (2,2'-azino-bis-(3-ethylbenzothiazoline-6-sulphonate) and read in a spectrophotometer at 405 nm. cf-DNA was isolated from 250 μL of plasma by using the FitAmp™ Circulating DNA Quantification Kit (Epigentek Inc, NY USA). The concentration of the DNA was measured for the UV
absorbance at 260 nm using a spectrophotometer (Beckman DU 7400; Beckman Coulter Inc., Brea, CA, USA). Immunostaining of the leukocyte Leukocytes extracted from healthy rats were utilized for the immunostaining. Rats were given intraperitoneal injections of glycogen. Eight hours later, their peritoneal cavities were washed with 10 mL of medium RPMI 1640. The sampled peritoneal lavage was centrifuged at 400 ×g for 5 min and then washed again with the collection buffer. Then, the leukocytes were resuspended and cultured in a chamber slide (#4722-040; Iwaki Inc, Tokyo Japan) with 50 μL of the plasma obtained from each group for 8 hours and then, stained with Apoptosis/Necrosis Detection Kit (ENZ-51002–25, Enzo Life Sciences, NY USA) according to the manufacturer's instructions with slight modifications. Briefly, recipitated leukocytes were suspended in 500 μL of detection solution (apoptosis detection reagent containing Annexin V-EnzoGold conjugate, necrosis detection reagent containing 7-AAD, and green fluorescent protein for the viable cell detection). Seven fields in each sample were examined under a fluorescent microscope by two individuals, and the mean ratio of the viable plus apoptotic cells relative to the necrotic cells was calculated. Statistical analysis All the data were expressed as the mean ± standard error. A statistical analysis was performed using the StatView II statistical software package for Macintosh. Statistical comparisons were performed using a non-parametric Kruskal-Wallis one-way ANOVA with Dunnett posthoc test for multiple comparisons and the Mann–Whitney test for single comparisons. Statistical differences were deemed significant at a level of P b 0.05. Results I) Laboratory data findings The normal WBC count was approximately 6000/mm3, and the number decreased to 2412.5 ± 248.2/mm3 in the control group at 8 hours after LPS injection. These reductions were suppressed in all of the AT, rTM and AT/rTM groups (P b 0.01 respectively) (Fig. 1, left). Similarly, the platelet count decreased to less than one third after LPS injection, and these reductions were again suppressed in all of the treatment groups (P b 0.01 respectively). The platelet count was maintained at over 500 x 103/mm3 in the AT/rTM group, and this count was significantly higher than those for the AT and rTM groups (P b 0.05, 0.01, respectively) (Fig. 1, right). For the hemostatic markers, both the AT and the AT/rTM groups showed significant reductions in the elevated FDP level, compared with that in the control group (P b 0.05 and 0.01, respectively). The fibrinogen level was significantly higher in the AT, rTM and AT/rTM groups than in the control group (P b 0.05, 0.05 and 0.01, respectively). Regarding the organ damage markers, elevated levels of ALT, T-Bil, creatinine, and BUN were observed at 8 hours after LPS infusion in the control group, and the significant suppression of the elevated ALT and BUN levels were recognized in the AT/rTM group (P b 0.05, respectively) (Table 1). The histone H3 level was 17.0 ± 6.0 pg/mL in the control group. Meanwhile, the levels were 6.0 ± 1.0 pg/mL in the AT group, 5.0 ± 1.0 pg/mL in the rTM group, and 3.0 ± 1.0 pg/mL in the AT/rTM group (Fig. 2, left), with a significantly lower level in the AT/rTM group (P b 0.01 compared with the control group, P b 0.05 compared with AT and rTM group). The cf-DNA level was 46.2 ± 6.2 ng/mL in the control group. Meanwhile, the levels were suppressed to about two thirds of that in the AT group and the rTM group. The cf-DNA level was most significantly suppressed in the AT/rTM group, and
Please cite this article as: Iba T, et al, Combination of antithrombin and recombinant thrombomodulin modulates neutrophil cell-death and decreases circulating DAMPs levels ..., Thromb Res (2014), http://dx.doi.org/10.1016/j.thromres.2014.04.015
T. Iba et al. / Thrombosis Research xxx (2014) xxx–xxx
7000
800
6000
700 600
Platelet count
5000
4000
3000
2000
500 400 300
AT/rTM
rTM
normal
AT
AT/rTM
0 rTM
0 control
100
normal
1000
AT
200
control
WBC count
3
Fig. 1. Changes in WBC and platelet count. The WBC count decreased significantly at 8 hours after LPS injection, and these reductions were suppressed in the AT, rTM and AT/rTM groups. The platelet count also decreased significantly after LPS injection, and these reductions were suppressed in all the treatment groups. The count in the AT/rTM group was significantly higher than those in the AT and rTM groups. WBC: white blood cells, LPS: lipopolysaccharide, AT: antithrombin, rTM: recombinant thrombomodulin *: P b 0.05, **: P b 0.01.
TM efficiently suppressed the coagulation, and which lead to the improvement of organ dysfunction. Interestingly, AT/rTM suppressed not only the activated coagulation but also inhibited leukocyte necrosis. When viewed according to leukocyte cell death, apoptosis was dominant under normal situations, and the population of necrotic leukocytes was rather high after the LPS treatment. The necrotic leukocytes expelled DAMPs and accelerated further inflammation during sepsis. In this study, AT and rTM were revealed to inhibit leukocyte necrosis, reduce the circulating DAMPs, suppress the coagulatory responses and thereby demonstrated the organ protection. Monocytes/macrophages are widely accepted as the major players in the procoagulant process; however, recent evidence has suggested that neutrophils also play important roles by expressing tissue factor [16,17], micro particles [18] and ejecting NETs [19,20]. Neutrophils accumulate and adhere tightly to the endothelium, and expel web-like structures named NETs that serve as a scaffold for thrombus formation [21]. As a matter of fact, the major components of NETs, histones [22] and DNA [23,24] are strong initiators of coagulation. In this study, circulating levels of histone H3 cf-DNA were recognized after LPS treatment. Regarding the source of these DAMPs, leukocyte necrosis is likely to contribute to the elevation in DAMPs as well as NETs. Histones are DNA-binding proteins that account for the majority of nucleic proteins. Once histones are released into the bloodstream, they express an innately damaging effect on tissues that is most
the difference was significant compared with that in the control group (P b 0.05) (Fig. 2, right). II) Alive and apoptotic leukocyte/necrotic leukocyte The surfaces of leukocytes incubated in medium with plasma obtained from the control group were mainly stained with 7-ADD (red), which represents necrosis (Fig. 3, left). In contrast, the percentage of leukocytes that were stained green (green fluorescent protein) or yellow (Annexin V) increased in the AT group and the rTM group, and this trend was most prominent in the AT/rTM group (Fig. 3, middle). The ratio of alive plus apoptotic/necrotic cells calculated in the AT/rTM group was almost twice as much as that in the control group, and the difference was statistically significant (P b 0.05) (Fig. 3, right). Discussion The activation of coagulation is an important part of host defense mechanisms [14] and is a universal event during severe sepsis. Activated coagulation limits microbial spreading and localizes infection [15]. If it occurs systemically, however, it can cause obstructions in the microvasculature and can induce tissue ischemia, thereby contributing to multiple organ failure and death. Therefore, the basic concept of anticoagulant therapy is to limit the excess activation of the coagulation. In the current study, we demonstrated that AT and Table 1 Changes in biochemical data.
Normal Control group AT group rTM group AT/rTM group
FDP (μg/dL)
fibrinogen (mg/dl)
ALT (IU/L)
T-Bil (mg/dL)
creatinine (mg/dL)
BUN (mg/dL)
10.4 33.1 19.2 27.5 12.3
242.2 91.4 128.0 125.1 189.3
25.1 85.4 60.9 57.3 29.6
0.74 1.59 1.19 1.49 1.34
0.18 0.98 0.77 0.77 0.72
14.1 61.8 57.9 56.4 44.6
± ± ± ± ±
0.9 6.8 3.6⁎ 5.7 1.9⁎⁎
± ± ± ± ±
16.1 9.4 12.3⁎ 12.6⁎ 17.2⁎⁎
± ± ± ± ±
2.6 14.6 12.4 13.2 9.9⁎
± ± ± ± ±
0.07 0.27 0.13 0.18 0.18
± ± ± ± ±
0.04 0.34 0.39 0.36 0.36
± ± ± ± ±
0.7 5.5 4.9 5.1 4.8⁎
FDP = fibrin/fibrinogen degradation products ALT = alanine aminotransferase; T-Bil = total bilirubin; BUN = blood urea nitrogen ⁎ P b 0.05 ⁎⁎ P b 0.01 compared to the control
Please cite this article as: Iba T, et al, Combination of antithrombin and recombinant thrombomodulin modulates neutrophil cell-death and decreases circulating DAMPs levels ..., Thromb Res (2014), http://dx.doi.org/10.1016/j.thromres.2014.04.015
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(pg/mL)
(ng/mL) 60
25.0
50 20.0
15.0
cf-DNA
Histone H3
40
30
10.0 20 5.0
10
AT/rTM
rTM
AT
control
AT/rTM
rTM
AT
control
normal
normal
0
0
Fig. 2. Changes in histone H3 and cf-DNA. The histone H3 level was elevated at 8 hours after LPS injection in the control group. The levels were significantly lower in the AT, rTM and AT/rTM groups. The cf-DNA level was also elevated in the control group. The cf-DNA level was significantly suppressed in the AT/rTM group compared with the control group. cf-DNA: cell-free DNA, LPS: lipopolysaccharide, AT: antithrombin, rTM: recombinant thrombomodulin *: P b 0.05, **: P b 0.01.
elevated after stimulation with LPS. The findings that the combination therapy with AT/rTM suppressed these DAMPs levels suggest that this anticoagulant therapy can control the dangerous cycle of increased DAMPs, activated coagulation and leukocyte necrosis. The above theory was further confirmed in the ex vivo experiment. The manner of leukocyte cell death was examined using the fluorescent staining. As a result, the leukocytes treated with plasma from the control group were stained mainly in red with 7-AAD, a dye that binds to DNA in membrane-permeabilized cells, suggesting that the majority of the leukocytes fell into necrotic cell death. In contrast, necrosis was efficiently suppressed in the AT/rTM group, in which green or yellow
Alive+apoptotic/necrotic cells
prominent for histone H3 and H4 [25]. Xu et al. [25] demonstrated that APC can degrade histones H3 and H4 and minimize tissue damage, thereby improving survival rates. Interestingly, Semeraro et al. [26] reported that histones activate coagulation in a dose dependent manner through the inhibition of the thrombomodulin-mediated activation of protein C. Other than APC, rTM was also reported to reduce the damage induced by histones [27]. As for cf-DNA, its level reportedly increases in sepsis, and a higher level indicates a poor outcome [28,29]. cf-DNA is also known to induce inflammation through the activation of Toll-like receptor in sepsis [30]. In our study, the concentrations of histone H3 and cf-DNA were low in the normal group, but they were significantly
*
1.5
1.0
0.5
AT/rTM
AT
rTM
AT/rTM
control
control
normal
0
Fig. 3. Immunostaining of leukocytes and ratio of viable and apoptotic leukocytes/necrotic leukocytes. The leukocytes incubated in the medium and the plasma obtained from the control group were stained predominantly by 7-AAD (red) (left). In contrast, the percentage of leukocytes that were stained green (green fluorescent protein) or yellow (Annexin V) increased in the AT/rTM group (middle). The populations of viable and apoptotic/necrotic leukocytes were calculated in the AT/rTM group. The ratio in the AT/rTM group was almost twice that in the control group (right). The photographs are overlays of the immunofluorescent and bright views. AT: antithrombin, rTM: recombinant thrombomodulin.
Please cite this article as: Iba T, et al, Combination of antithrombin and recombinant thrombomodulin modulates neutrophil cell-death and decreases circulating DAMPs levels ..., Thromb Res (2014), http://dx.doi.org/10.1016/j.thromres.2014.04.015
T. Iba et al. / Thrombosis Research xxx (2014) xxx–xxx
staining was predominant. Green fluorescent protein is widely used to stain viable cells, and Annexin V is a popular fluorescence that is used to detect apoptosis because it has a high affinity to phosphatidylserine, which is translocated from the inner to the outer plasma membrane of apoptotic cells. In the current study, both of global coagulation tests such as PT and APTT and bleeding tendency have not been examined. Since the bleeding adverse event causes critical damage to the patients with septic DIC, these issues should be addressed before the clinical application. In summary, the combination therapy successfully suppressed activated coagulation and leukocyte necrosis and decreased the levels of circulating DAMPs such as histone H3 and cf-DNA. Since activated coagulation and leukocyte necrosis plays a significant role in the amplification of inflammation and organ damage, the combination therapy with AT and rTM may be useful for the treatment of severe sepsis. Conclusion The combination of antithrombin and rTM efficiently suppressed leukocyte necrosis and decreased the circulating levels of DAMPs including histone H3 and cf-DNA. These effects lead to the suppression of activated coagulation and subsequent organ protection. Conflict of interest statement The authors state that Iba T, Miki T, Hashiguchi Y, Tabe Y and Nagaoka I have no conflicts of interest. Authors’ contributions Iba T and Hashiguchi N designed the study. Miki T and Tabe Y performed the experiment. Iba T and Nagaoka I wrote the manuscript. All authors read and approved the final manuscript. Acknowledgements This work was supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology (#25462831). A part of this study was presented at the 40th Annual Meeting of the Japanese Society of Intensive Care Medicine. References [1] Angus DC, van der Poll T. Severe sepsis and septic shock. N Engl J Med 2013; 369:2063. [2] Kumar H, Kawai T, Akira S. Pathogen recognition by the innate immune system. Int Rev Immunol 2011;30:16–34. [3] Harris HE, Raucci A. Alarmin(g) news about danger: workshop on innate danger signals and HMGB1. EMBO Rep 2006;7:774–8. [4] Rittirsch D, Flierl MA, Ward PA. Harmful molecular mechanisms in sepsis. Nat Rev Immunol 2008;8:776–87. [5] Seong SY, Matzinger P. Hydrophobicity: an ancient damage-associated molecular pattern that initiates innate immune responses. Nat Rev Immunol 2004;4:469–78. [6] Cinel I, Opal SM. Molecular biology of inflammation and sepsis: a primer. Crit Care Med 2009;37:291–304.
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