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Deoxyribonuclease Reduces Tissue Injury and Improves Survival After Hemorrhagic Shock
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Deoxyribonuclease Reduces Tissue Injury and Improves Survival After Hemorrhagic Shock Joaquin Cagliani, MD, PhD,a,b,c,e Weng-Lang Yang, PhD,a,b,c,d Max Brenner, MD, PhD,b,e and Ping Wang, MDa,b,c,* a
Elmezzi Graduate School of Molecular Medicine, Manhasset, New York Center for Immunology and Inflammation, Feinstein Institutes for Medical Research, Manhasset, New York c Department of Surgery, Zucker School of Medicine at Hofstra/Northwell, Manhasset, New York d Department of Radiation Oncology, Albert Einstein College of Medicine, Bronx, New York e Department of Molecular Medicine, Zucker School of Medicine at Hofstra/Northwell, Manhasset, New York b
article info
abstract
Article history:
Background: Hemorrhagic shock (HS) caused by rapid loss of a large amount of blood is the
Received 18 June 2019
leading cause of early death after severe injury. When cells are damaged during HS, many
Received in revised form
intracellular components including DNA are released into the circulation and function as
16 September 2019
endogenous damage-associated molecular patterns (DAMPs) that can trigger excessive
Accepted 23 November 2019
inflammatory response and subsequently multiple organ dysfunction. We hypothesized
Available online xxx
that the administration of deoxyribonuclease I (DNase I) could reduce cell-free DNA and attenuate tissue damage in HS. Methods: Eight-week-old male C57BL/6 mice underwent HS by controlled bleeding from the femoral artery for 90 min, followed by resuscitation with Ringer’s lactate solution (vehicle) or DNase I (10 mg/kg BW). Results: At 20 h after HS, serum levels of cell-free DNA were increased by 7.6-fold in the vehicle-treated HS mice compared with sham, while DNase I reduced its levels by 47% compared with the vehicle group. Serum levels of tissue injury markers (lactate dehydrogenase, aspartate aminotransferase, and alanine aminotransferase) and proinflammatory cytokine interleukin 6 were significantly reduced in the DNase Ietreated mice. In the lungs, messenger RNA levels of proinflammatory cytokines (interleukin 6 and interleukin 1 b), chemoattractant macrophage inflammatory protein - 2, and myeloperoxidase activity were significantly decreased in HS mice after DNase I. Finally, DNase I significantly improved the 10-day survival rate in HS mice. Conclusions: Administration of DNase I attenuates tissue damage and systemic and lung inflammation, leading to improvement of survival in HS mice. Thus, DNase I may potentially serve as an adjunct therapy for managing patients with HS. ª 2019 Elsevier Inc. All rights reserved.
* Corresponding author. Department of Surgery, Zucker School of Medicine at Hofstra/Northwell The Feinstein Institutes for Medical Research, 350 Community Drive, Manhasset, NY 11030. Tel.: þ(516) 562 3411; fax: þ516 562-1022. E-mail address: [email protected] (P. Wang). 0022-4804/$ e see front matter ª 2019 Elsevier Inc. All rights reserved. https://doi.org/10.1016/j.jss.2019.11.036
cagliani et al cfdna in hemorrhagic shock
Introduction
Methods
Hemorrhagic shock (HS) induced by a rapid loss of a large amount of blood is the leading cause of early death after severe injury.1-3 In the United States, HS causes more than 60,000 deaths yearly, 80% of which are due to trauma.4 When hemorrhage occurs, the blood supply to the vital organs is compromised, causing ischemia. The subsequent restoration of blood supply or reperfusion after hemorrhage can acutely trigger complex inflammatory cascades which play a key role in generating cellular stress and postischemia injury.5 When cells are damaged during HS, intracellular components such as nuclear and mitochondrial DNA are released into the circulation. These cell-free components function as endogenous damage-associated molecular patterns (DAMPs) that can trigger primary innate immune response mediated by pattern-recognition receptors (PRRs) such as toll-like receptors (TLRs), nucleotide oligomerization domain e such as receptors (NLRs), or receptors for advanced glycation endproducts (RAGEs) after HS. 6 The interaction between DAMPs and PRRs activates numerous signal transduction pathways, leading to the release of proinflammatory cytokines to activate monocytes, neutrophils, and endothelial cells. While the inflammatory response in the setting of hemorrhage may be protective by promoting the activation of the clotting system, 7 the uncontrolled amplification of the proinflammatory pathways during reperfusion is particularly detrimental for the host.8,9 This exuberant immune response leads to cellular apoptosis, thereby propagating tissue damage and organ failure.10-12 In patients admitted to an intensive care unit with trauma-related injuries, cell-free DNA (cfDNA) has been proposed as a biomarker for diagnostic and prognostic implications.13 Several reports have indicated that the amount of cfDNA correlates with mortality, trauma severity, and post-traumatic complications.14-16 Although deoxyribonuclease (DNase)-based therapeutic strategies to decrease the levels of cfDNA have been shown to be beneficial in models of ischemia reperfusion injury and acute lung injury,17-19 the potential therapeutic value of DNase I in the physiopathology of HS remains unknown. Given that cfDNA can act as a DAMP to modulate the immune response and its levels are elevated in trauma patients, we then hypothesized that administering exogenous DNase I to reduce the levels of circulating cfDNA may attenuate inflammation and organ injury as well as have survival benefits within the scope of HS. In this study, we used a mouse model of HS to investigate the role of cfDNA. We first measured the change of circulating cfDNA levels in HS mice and after DNase I. We then compared the serum levels of organ injury markers, histological damage, and expression of proinflammatory cytokines and chemokines in the lungs between the HS mice with and without DNase I. Finally, we determined the effect of DNase I on the 10-day survival of HS mice.
Mouse model of HS
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Eight-week-old male C57BL/6 mice (20-25 g) purchased from The Jackson Laboratory (Bar Harbor, ME) were housed in a 12-h light and dark cycle and temperature-controlled environment. Mice were fed a standard laboratory mouse chow diet and water ad libitum. After acclimation for 7 d, mice were subjected to HS. All experiments were performed in accordance with the guidelines for the use of experimental animals by the National Institutes of Health (Bethesda, MD) and were approved by the Institutional Animal Care and Use Committee of the Feinstein Institutes for Medical Research. The animal model of HS was performed as previously described.20 Succinctly, mice were randomly assigned to undergo sham, HS adjuvantly treated with DNase I, or HS adjuvantly treated with vehicle (control) groups. Anesthesia was induced with inhalation of 2.5% isoflurane and then maintained with 1.5% isoflurane. The inguinal regions were shaved and antiseptically prepared with povidone iodine and chlorhexidine gluconate. Two 0.5-cm inguinal incisions were made to expose the right and left femoral arteries, which were both cannulated with pressure equalizer-10 tubing after careful dissection of the femoral vein and femoral nerves. One catheter was used for controlled hemorrhage, fluid resuscitation, and administration of DNase I or vehicle, while the second catheter was connected to a transducer to continuously measure the mean arterial pressure (MAP) and heart rate. The results were recorded with a blood pressure analyzer Digi-Med (Micro-Med, Louisville, KY). The MAP values were averaged over 5-s intervals to record the oscillations in the blood pressure. Blood was withdrawn from the femoral artery to reach and maintain an MAP of 27.5 2.5 mmHg over 90 min. Mice were then resuscitated with prewarmed Ringer’s lactate solution totaling two times the volume of shed blood over the 30-min resuscitation phase. Mice were not heparinized during the procedure. The animal’s body temperature was kept at 37 C using a water-recirculating heating pad during the HS and resuscitation phases. After the resuscitation, the femoral artery was ligated, and the incision was closed with nonabsorbable suture. Animals were allowed to recover from anesthesia before being returned to their cages. Sham mice underwent the same procedure, including bilateral femoral artery cannulation, without induction of hemorrhage or resuscitation. All efforts were made to minimize suffering.
Administration of DNase I During the 30-min resuscitation phase, the mice received Ringer’s lactate solution alone (vehicle) or containing bovineextracted pancreatic DNase I (10 mg/kg BW) from SigmaAldrich (St. Louis, MO). We chose the dose of DNase I based on previous studies in murine models.21,22
Blood and lung tissue collection At 20 h after the initiation of resuscitation, when hemorrhageassociated acute lung injury can be detected,23 all mice were
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euthanized by CO2 asphyxiation. Whole blood was drawn from the inferior vena cava and centrifuged twice at 12,000 rpm for 10 min at 4 C to separate the cellular fraction from the serum and thus exclude the contribution of DNA from the lysis of the white cells. After the blood draw, lung tissues were harvested and stored at 80 C until analysis.
Assessment of cfDNA in serum cfDNA was extracted from 500 mL of serum of each mice using the QIAmp DNA Mini Kit (Qiagen, Valencia, CA) in accordance with the manufacturer’s instructions. The quality and quantification of the isolated DNA was determined at 260 and 280 nm, respectively, on a NanoDrop microvolume spectrophotometer (Thermo Fisher Scientific Inc, Waltham, MA).
Analysis of serum injury markers and interleukin 6 Serum activities of alanine aminotransferase (ALT), aspartate aminotransferase (AST), and lactate dehydrogenase (LDH) were determined using assay kits (Pointe Scientific, Canton, MI). Serum interleukin 6 (IL-6) levels were analyzed using a commercial mouse enzyme-linked immunosorbent assay (ELISA) kit (BD Biosciences, San Diego, CA). All assays were carried out in accordance with their manufacturer protocol instructions.
Quantitative real-time polymerase chain reaction Total RNA was extracted from tissues using Trizol (Invitrogen, Carlsbad, CA) and reverse transcribed into using murine leukemia virus reverse transcriptase (Applied Biosystems, Foster City, CA). A quantitative real-time polymerase chain reaction (PCR) assay was carried out in a final volume of 20 mL containing 0.25 mmol of each forward and reverse primer, complimentary DNA, and 10 mL SYBR Green PCR master mix (Applied Biosystems). Amplification was conducted in an Applied Biosystems Step One Plus real-time PCR machine under the thermal profile of 50 C for 2 min, 95 C for 10 min followed by 45 cycles of 95 C for 15 s and 60 C for 1 min. The levels of mouse bactin messenger RNA (mRNA) were used for normalization. Relative quantity of mRNA was expressed as the fold change in comparison with the sham tissues. The primers sequences used for quantitative real-time are the following: IL-6 (NM_031168): 50 -CCGGAGAGGAGACTTCACAG-30 and 50 -GGA AATTGGGGTAGGAAGGA-30 ; IL-1b (NM_008361): 50 -CAGGATG AGGACATGAGCACC-30 , and 50 -CTCTGCAGACTCAAACTCCAC30 ; macrophage inflammatory protein-2 (MIP-2) (NM_009140): 50 -CCCTGGTTCAGAAAATCATCCA-30 and 50 -GCTCCTCCTTTC CAGGTCAGT-30 ; b-actin (NM_007393): 50 -CGTGAAAAGATGA CCCAGATCA-30 and 50 -TGGTACGACCAGAGGCATACAG-30 .
Histology analysis Lung tissue segments collected at 20 h after reperfusion were fixed in 10% formalin before paraffin embedding. The tissue blocks were sectioned into 5-mm cuts, transferred to glass slides, and stained with hematoxylin and eosin (H&E). Ten fields per sample were then assessed in a blinded fashion using a modified American Thoracic Society lung injury
scoring system, consisting of semiquantitative scores for inflammatory cell infiltration into the alveolar and interstitial space, presence of hyaline membranes, proteinaceous debris inside airspaces, and alveolar septal thickening.23 Based on the presence of these parameters, scores per visual field were categorized as 0 (no injury), 1 (moderate injury), and 2 (severe injury). Using the weighted equation with a maximum score of 100 per field presented by Matute-Bello et al.,24 the parameter scores were calculated on a scale of 0-1 and then averaged as the final lung injury score in each group.
Myeloperoxidase activity assay Myeloperoxidase (MPO) activity in the lung was determined using the peroxidase-catalyzed reaction as previously described.25 Lung tissues were sonicated in 50 mM potassium phosphate buffer containing 0.5% hexadecyltrimethylammonium bromide (pH 6.0). After centrifugation, the supernatant was diluted in a reaction solution containing o-dianisidine hydrochloride and H2O2. The rate of change in optical density per 1 min was measured at 460 nm, and the MPO activity was expressed as change in absorbance per min per gram of protein.
Terminal deoxynucleotidyl transferaseemediated deoxyuridine triphosphate nick-end labeling assay Lung tissue sections were immersed in 20 mg/mL of proteinase K (Thermo Fisher Scientific) at room temperature for 20 min and then stained using an in situ labeling of DNA fragmentation with terminal deoxynucleotidyl transferaseemediated deoxyuridine triphosphate nick-end labeling assay kit (Roche Diagnostics, Florham Park, NJ) and counterstained with Vectashield mounting medium containing 40 ,6-diamidino-20 -phenylindole dihydrochloride (Vector Laboratories, Inc, Burlingame, CA). Apoptotic cells were visualized green at 200 magnification under a fluorescence microscope (Nikon Eclipse Ti-S, Melville, NY) and were then counted in five visual fields/sections. The average number of apoptotic cells/field was calculated.
Survival study The survival rate was assessed in a separate group of animals, which were randomly assigned to adjuvant treatment with vehicle (n ¼ 10) or DNase I (n ¼ 10). Sham animals were not included because they are expected to have a survival rate of 100%. Animals were subjected to HS and fluid resuscitation plus adjuvant treatment with Ringer’s lactate solution (vehicle) or DNase I (10 mg/kg BW) via the right femoral artery over 30 min as described previously. After resuscitation, the femoral arteries were ligated, and the incisions were closed in layers. The mice were returned to their cages for recovery, allowed food, and water ad libitum and monitored for 10 d.
Statistical analysis For nonsurvival experiments, we calculated that a sample size of nine mice per group for a 7-group analysis of variance (ANOVA) would have a power of 0.8 to detect a difference of 25% between groups with a standard deviation of 25% of the
cagliani et al cfdna in hemorrhagic shock
Fig. 1 e Monitoring of blood pressure during HS induction, maintenance, and resuscitation phases. Arterial blood pressure at the induction phase (0-20 min), the hemorrhage phase (20-110 min), and the first 30 min of resuscitation phase (110-140 min) in the vehicle- and DNase Ietreated mice was recorded from the beginning of the operation. Sham-operated mice with the same time frame were also recorded. Data presented are mean ± SEM (n [ 6 mice/ group). (Color version of figure is available online.)
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Fig. 2 e Effect of DNase I on the serum levels of cfDNA after HS. Mice were subjected to HS as described in Methods. Serum from vehicle- and DNase Ietreated mice at 20 h after HS and sham mice were collected for the analysis. The serum levels of cfDNA were quantified spectrophotometrically. Data are expressed as mean ± SEM (n [ 6 mice/group) and compared by ANOVA and SNK test. *P < 0.05 versus sham; and #P < 0.05 versus vehicle. (Color version of figure is available online.)
DNase I decreases serum cfDNA after HS mean and a P 0.05. For survival experiments, we calculated that a sample size of 20 mice per group would have a power of 0.8 to detect a difference in survival of 50% or more with a P 0.05. Data are expressed as mean standard error of the mean (SEM) and compared by one-way ANOVA using the Student-Newman-Keuls (SNK) test for multiple group comparisons. The survival rate was estimated by the Kaplan-Meier method and compared using the log-rank test. Differences between the experimental groups with P 0.05 were considered statistically significant.
Results Adjuvant treatment with DNase I does not affect the MAP during the resuscitation phase HS is a complicated, heterogeneous condition involving the systemic activation of inflammation and coagulation in response to significant blood loss. It may progress to severe HS if there is dysfunction in one or multiple organs. Thus, we sought to determine if mice adjuvantly treated with vehicle and DNase I had undergone differences in blood loss before receiving resuscitation with or without the adjuvant treatment or showed different hemodynamic responses during the 30min resuscitation phase. Indeed, both groups of mice had the same amount of blood withdrawn (0.8 0.19 mL per mouse) to maintain the target MAP during the hemorrhage phase before resuscitation plus adjuvant treatment with vehicle or DNase I. Similarly, the MAPs of the two groups of mice did not differ during the fluid resuscitation phase (Fig. 1). As expected, the sham group MAP remained stable throughout the study.
To explore the potential role cfDNA as an inflammatory mediator in HS, we examined the release of DNA in the serum of mice undergoing HS followed by reperfusion. At 20 h after HS, serum levels of cfDNA increased significantly from 11.5 ng/ mL in the sham to 117.1 ng/mL in the vehicle group, while adjuvant treatment with DNase I significantly decreased the levels of cfDNA by 47% compared with the vehicle group (Fig. 2).
DNase I attenuates tissue injury and systemic inflammation after HS The HS group showed marked elevation of serum levels of organ injury markers in comparison with the sham group. Remarkably, the levels of LDH, AST, and ALT were significantly higher in HS-vehicle mice by 14.0-, 19.6-, and 8.8-fold, respectively (Fig. 3). Mice adjuvantly treated with DNase I, however, had serum levels of LDH, AST, and ALT significantly decreased by 39.0%, 33.9%, and 63.8%, respectively, compared with the HS-vehicle group (Fig. 3A-C). Because excessive elevated levels of proinflammatory cytokines in circulation is a major contributor to remote organ injury after HS, we then measured the levels of IL-6 in the serum. The IL-6 protein levels increased significantly by 8.9-fold in the vehicle group as compared with sham, while its levels were significantly decreased in mice adjuvantly treated with DNase I by 28% compared with the HSvehicle group (Fig. 3D). These results suggest that DNase I can mitigate the systemic inflammation and organ injury after HS.
DNase I reduces lung inflammatory cytokines after HS To determine the role of DNase I in the lung inflammation induced by HS, we measured proinflammatory cytokines in
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A
B
C
D
Fig. 3 e Effect of DNase I on organ injury after HS. Mice were subjected to HS as described in Methods. Serum from vehicleand DNase Ietreated mice at 20 h after HS and sham mice were collected for the analysis. The serum levels of (A) LDH, (B) AST, and (C) ALT were determined by enzymatic method. (D) The serum levels of IL-6 were measured by ELISA. Data are expressed as mean ± SEM (n [ 6 mice/group) and compared by ANOVA and SNK test. *P < 0.05 versus sham; #P < 0.05 versus vehicle. (Color version of figure is available online.)
the lungs at 20 h after HS. The mRNA expression in the lungs of both IL-6 and IL-1b in the HS-vehicle mice were significantly higher than that in the sham, by 3.9- and 2.4-fold, respectively (Fig. 4A and B). Meanwhile, the mice adjuvantly treated with DNase I exhibited in a significant reduction of IL-6 and IL-1b levels by 59.0% and 83.3%, respectively, as compared with the
A
B
group treated with vehicle (Fig. 4A and B). To analyze the effect of DNase I on neutrophil infiltration which is another mechanism of injury involving immune cells, we examined the chemokine expression of MIP-2 in the lungs. MIP-2 functions as chemoattractant to neutrophils at inflamed sites.26-28 The HS-vehicle group showed significantly elevated mRNA
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Fig. 4 e Effect of DNase I on the proinflammatory cytokine expression in the lungs after HS. Mice were subjected to HS as described in Methods. Lung tissues from vehicle- and DNase Ietreated mice at 20 h after HS and sham mice were collected for the analysis. The mRNA levels of (A) IL-6, (B) IL-1b, and (C) MIP-2 in the lungs were assessed by quantitative real-time polymerase chain reaction. Each gene expression level was normalized to b-actin. The value in the sham group is designated as 1 for comparison. Data are expressed as mean ± SEM (n [ 6 mice/group) and compared by ANOVA and SNK test. *P < 0.05 versus sham; #P < 0.05 versus vehicle. (Color version of figure is available online.)
cagliani et al cfdna in hemorrhagic shock
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Fig. 5 e Effect of DNase I on lung morphology and neutrophil infiltration after HS. Mice were subjected to HS as described in Methods. Lung tissues from vehicle- and DNase Ietreated mice at 20 h after HS and sham mice were harvested, sectioned, and subjected to histologic analysis. (A-C) Representative images of tissue sections with H&E staining at an original magnification of 200 3 . (D) Histologic injury score in each group was blindly graded as described in Methods. (E) Lung myeloperoxidase (MPO) activity was determined spectrophotometrically. Data are expressed as mean ± SEM (n [ 6 mice/ group) and compared by ANOVA and SNK test. *P < 0.05 versus sham; #P < 0.05 versus vehicle. (Color version of figure is available online.)
expression levels of MIP-2 by 34.1-fold, compared with sham (Fig. 4C). Conversely, adjuvant treatment with DNase significantly decreased the levels of MIP-2 by 53.9% compared with the HS-vehicle group (Fig. 4C).
DNase I improves the integrity of the lung histology after HS The effects of adjuvant treatment with DNase I on hemorrhageinduced acute lung injury were further assessed by histological analyses (Fig. 5A-C). The HS-vehicle group showed alterations in the alveolar capillary barrier, septal thickening, and hyaline deposits as compared with the sham (Fig. 5A). The lung architecture and morphology of the HS mice adjuvantly treated with DNase I, however, resembled the sham group more than the HS-vehicle group (Fig. 5A-C). Accordingly, the lung injury score in the HSvehicle group was 5.2-fold greater than that in the sham (P < 0.05), while the DNase Ietreated mice showed a significant 42.5% decrease in the lung injury score when compared with the mice treated with vehicle (Fig. 5D). To further quantify hemorrhage-induced lung inflammation, we measured the MPO activity as an indicator of infiltration by activated neutrophils.26 We found a significant 3.7-fold increase in the MPO activity of the lungs of mice in the HS-vehicle group as compared with those in the sham group, while mice adjuvantly treated with DNase I showed a significant 59.8% decrease in the MPO activity as compared with mice in the HS-vehicle group (Fig. 5E).
DNase I attenuates apoptosis in the lungs after HS The TUNEL assay was used to study the effect of DNase I on apoptosis in lung tissues. The sham group showed almost no detection of TUNEL-positive cells, while a significant increase of TUNEL-positive cells was observed in the HS-vehicle group (Fig. 6A-C). In mice adjuvantly treated with DNase I, the number of apoptotic cells was significantly decreased by 75.0% compared with the animals in the HS-vehicle (Fig. 6D).
DNase I improves survival after HS Then, we examined the role of DNase I on 10-day survival after HS. We found that mice adjuvantly treated with DNase I had a significant improvement in the 10-day survival when compared with those in the HS-vehicle group (80% versus 30%, P < 0.05; Fig. 7).
Discussion The physiologic changes in response to HS are directed at maintaining blood pressure and cardiac output to support the function of vital organs.29 However, the extent of compensation is limited, and in the absence of acute intervention, severe hypoxia and inflammation can cause irreversible damage
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Fig. 6 e Effect of DNase I on apoptosis in the lungs after HS. Mice were subjected to HS as described in Methods. Lung tissues from vehicle- and DNase Ietreated mice at 20 h after HS and sham mice were harvested, sectioned, and subjected to terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assay. (A-C) Representative images of tissue sections stained with TUNEL (green fluorescence) and nuclear counterstaining with 40 ,6-diamidino-2-phenylindole (DAPI) (blue fluorescence). Bar scale indicates 100 mm at an original magnification of 200 3 . (D) The number of TUNEL-positive cells was counted and averaged over five high-power fields (HPFs)/section. Data are expressed as mean ± SEM (n [ 6 mice/group) and compared by ANOVA and SNK test. *P < 0.05 versus sham; #P < 0.05 versus vehicle. dUTP [ deoxyuridine triphosphate. (Color version of figure is available online.)
at the cellular and organ levels and may result in eventual death.4 In the present study, we proposed that better outcomes for HS could be achieved by the control of DAMP levels in the circulation through the degradation of cfDNA by DNase I. Our results show that adjuvant treatment with DNase I along with fluid resuscitation provides a strong protective effect by attenuating organ injury and systemic and pulmonary inflammation and reducing neutrophil infiltration and TUNEL-positive cells in the lungs of mice subjected to HS. Importantly, we also demonstrate that DNase I improves the 10-day survival rate in a lethal mouse model of HS. Therefore, our results confirm the detrimental role of cfDNA contributing to the severity and worse outcomes in HS and clearly indicate the beneficial effects of adjuvant treatment of severe hemorrhage with DNase I. DAMPs activate the innate immune response and have been shown to play a key role in the pathogenesis of ischemiareperfusion.30 Examples of DAMPs include extracellular coldinducible RNA-binding protein, high-mobility group box 1 protein, and genomic or mitochondrial DNA.6,31-33 We demonstrate that cfDNA is released and markedly elevated after the initial traumatic injury after HS. The cfDNA can
regulate the innate immune response through a wide variety of pathways mediated by various PRRs in macrophages.34-36 For example, when binding with TLR9, cfDNA triggers the activation of mitogen-activated protein kinases (MAPKs) and IkB kinase complex through the adapter protein MyD88 to promote inflammation. Along these lines, it has been demonstrated that cfDNA signaling through IkB activates interferon regulatory factor-7 and nuclear factor kappa B, leading to the transcription of type 1 interferons and enhanced expression of proinflammatory cytokines such as IL-6 and IL-1b.34,37 Indeed, we found that degrading cfDNA with DNase I diminished the production of proinflammatory cytokines and resulted in decreased lung injury after HS. Exogenous administration of DNase I has also been shown to be beneficial in treating atelectasis38,39 and pleural infections40 and improving the lung function in patient with cystic fibrosis41 as well as is under a randomized phase Ib trial for the treatment of lupus nephritis.42 In addition, a decrease in DNase activity has recently been shown to correlate with the development of systemic inflammatory response syndrome in patients with trauma,43 indicating the therapeutic benefit of targeting cfDNA in these patients.
cagliani et al cfdna in hemorrhagic shock
Fig. 7 e Effect of DNase I on the survival of hemorrhaged mice. Mice were subjected to HS, as described in Methods, and treated with Ringer’s lactate solution (vehicle) or containing DNase I (10 mg/kg) during the reperfusion phase. Mice were monitored for survival for 10 d. Survival rates were analyzed by the Kaplan-Meier estimator using a log-rank test. (n [ 10 mice/group; P < 0.05). (Color version of figure is available online.)
Neutrophils play a pivotal role in the inflammatory component after injury. In the lungs, the innate immune response can recruit neutrophils which may cause injury by releasing reactive oxygen species and proteolytic enzymes such as elastase and MPO. It has been reported that disproportionate production of these enzymes is associated with poor clinical outcomes.12,28,44 Here, we report that adjuvant treatment of HS with DNase I led to a significant reduction in MIP-2 e a key neutrophil chemoattractant e and in the amount of activated neutrophils infiltrating the lungs. These results were further supported by the reduction in lung proinflammatory cytokines and the improved lung histology in hemorrhaged mice adjuvantly treated with DNase I. Neutrophil degranulation in the lungs releases toxic reactive oxygen species and proteolytic enzymes to the extracellular space, resulting in increased rates of epithelial cell death. Indeed, we noticed a marked increase in the number of apoptotic cells, a type of cellular death, in the lungs after HS. Interestingly, a reduction in the number of apoptotic cells and consequent improvement in lung injury was attained by using DNase I, which indicates that lowering cfDNA levels ultimately limits cell death secondary to lung inflammation in our model of HS. In addition to degranulation, during inflammation, activated neutrophils can also form extracellular traps (NETs).45 NETs are a major source of cfDNA,46 and aside from their antimicrobial defense by preventing the dissemination of pathogens, they can be detrimental to trauma by causing endothelial barrier dysfunction47 and microvascular thrombosis.19,45 DNase is able to dismantle the cytotoxic NETs scaffold and digest the extracellular DNA derived from NETs in experimental models of ischemia and reperfusion injury and acute lung injury.17,18 Although DNase is naturally
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present in human blood and produced as a defense mechanism associated with NETs, our data show that administration of exogenous DNase I is clearly beneficial in the setting of pure HS. Given the complex sequence of the trauma, the consecutive circulatory shock, and the pathophysiology underlying the response to the injury, developing a consistent and reproducible model of HS is paramount. However, we acknowledge that our experimental model presents some limitations as the selfcompensatory mechanisms that occur during the isobaric hypotension are disrupted. In addition, we have not explored the effects of clinically important variables such as resuscitation volume, administration of blood and blood products, and presence of trauma. Nonetheless, the increased release of cfDNA associated with severe trauma and trauma surgery14,15 suggests that these cases will likely benefit even more from adjuvant treatment with DNase I. While our study emphasizes the effects of DNase I on the immune and lung injury responses after pure HS, it will be important to verify the beneficial effects of DNase I in preclinical models of uncontrolled bleeding and combined trauma hemorrhage. In conclusion, our data show further evidence of cfDNA’s detrimental proinflammatory effects in HS and indicate that cfDNA’s targeting with DNase I during reperfusion represents a new and innovative therapeutic strategy to modulate the systemic inflammatory response and subsequent organ dysfunction caused by HS.
Acknowledgment This work was supported by National Institutes of Health grant R01HL076179 (to PW) and the Elmezzi Graduate School of Molecular Medicine (to J.C.). J.C., W.-L.Y., M.B., and P.W. conceived and designed the experiment. J.C. performed the experiments. J.C. and W.-L.Y. analyzed the data. J.C. wrote the manuscript and prepared the figures. W.-L.Y. and M.B. revised the manuscript. P.W. reviewed and supervised the research. All authors read and approved the final manuscript. All experiments involving live animals were carried out in accordance with the National Institutes of Health guidelines for the use of experimental animals and were reviewed and approved by the Institutional Animal Care and Use Committee at the Feinstein Institutes for Medical Research. The data sets used and analyzed during the present study are available from the corresponding author on reasonable request.
Disclosure The authors declare that the research was conducted in the absence of any commercial or financial relationship that could be construed as a potential conflict of interest.
references
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2. Pidcoke HF, Aden JK, Mora AG, et al. Ten-year analysis of transfusion in Operation Iraqi Freedom and Operation Enduring Freedom: increased plasma and platelet use correlates with improved survival. J Trauma Acute Care Surg. 2012;73:S445eS452. 3. Bardes JM, Inaba K, Schellenberg M, et al. The contemporary timing of trauma deaths. J Trauma Acute Care Surg. 2018;84:893e899. 4. Cannon JW. Hemorrhagic shock. N Engl J Med. 2018;378:370e379. 5. Wang CH, Hsieh WH, Chou HC, et al. Liberal versus restricted fluid resuscitation strategies in trauma patients: a systematic review and meta-analysis of randomized controlled trials and observational studies. Crit Care Med. 2014;42:954e961. 6. Zhang Q, Raoof M, Chen Y, et al. Circulating mitochondrial DAMPs cause inflammatory responses to injury. Nature. 2010;464:104e107. 7. Mitchell TA, Herzig MC, Fedyk CG, et al. Traumatic hemothorax blood contains elevated levels of microparticles that are prothrombotic but inhibit platelet aggregation. Shock. 2017;47:680e687. 8. Rushing GD, Britt LD. Reperfusion injury after hemorrhage: a collective review. Ann Surg. 2008;247:929e937. 9. Bickell WH, Wall Jr MJ, Pepe PE, et al. Immediate versus delayed fluid resuscitation for hypotensive patients with penetrating torso injuries. N Engl J Med. 1994;331:1105e1109. 10. Lomas-Neira J, Perl M, Venet F, Chung CS, Ayala A. The role and source of tumor necrosis factor-a in hemorrhage-induced priming for septic lung injury. Shock. 2012;37:611e620. 11. Timmermans K, Kox M, Vaneker M, et al. Plasma levels of danger-associated molecular patterns are associated with immune suppression in trauma patients. Intensive Care Med. 2016;42:551e561. 12. Itagaki K, Kaczmarek E, Lee YT, et al. Mitochondrial DNA released by trauma induces neutrophil extracellular traps. PLoS One. 2015;10:e0120549. 13. Lo YM, Rainer TH, Chan LY, Hjelm NM, Cocks RA. Plasma DNA as a prognostic marker in trauma patients. Clin Chem. 2000;46:319e323. 14. McIlroy DJ, Bigland M, White AE, et al. Cell necrosisindependent sustained mitochondrial and nuclear DNA release following trauma surgery. J Trauma Acute Care Surg. 2015;78:282e288. 15. Simmon JD, Lee Y-L, Mulekar S, et al. Elevated levels of plasma mitochondrial DNA DAMPs are linked to clinical outcome in severely injured human subjects. Ann Surg. 2013;258:591. 16. Margraf S, Lo¨gters T, Reipen J, et al. Neutrophil-derived circulating free DNA (cf-DNA/NETs): a potential prognostic marker for posttraumatic development of inflammatory second hit and sepsis. Shock. 2008;30:352e358. 17. Caudrillier A, Kessenbrock K, Gilliss BM, et al. Platelets induce neutrophil extracellular traps in transfusion-related acute lung injury. J Clin Invest. 2012;122:2661e2671. 18. Savchenko AS, Borissoff JI, Martinod K, et al. VWF-mediated leukocyte recruitment with chromatin decondensation by PAD4 increases myocardial ischemia/reperfusion injury in mice. Blood. 2014;123:141e148. 19. Jansen MP, Emal D, Teske GJ, et al. Release of extracellular DNA influences renal ischemia reperfusion injury by platelet activation and formation of neutrophil extracellular traps. Kidney Int. 2017;91:352e364. 20. Zhang F, Yang WL, Brenner M, Wang P. Attenuation of hemorrhage-associated lung injury by adjuvant treatment with C23, an oligopeptide derived from cold-inducible RNA-binding protein. J Trauma Acute Care Surg. 2017;83:690e697.
21. Chen G, Zhang D, Fuchs TA, et al. Heme-induced neutrophil extracellular traps contribute to the pathogenesis of sickle cell disease. Blood. 2014;123:3818e3827. 22. Czaikoski PG, Mota JM, Nascimento DC, et al. Neutrophil extracellular traps induce organ damage during experimental and clinical sepsis. PLoS One. 2016;11:e0148142. 23. Cagliani J, Yang W-L, McGinn JT, Wang Z, Wang P. Antiinterferon-a receptor 1 antibodies attenuate inflammation and organ injury following hemorrhagic shock. J Trauma Acute Care Surg. 2019;86:881e890. 24. Matute-Bello G, Downey G, Moore BB, et al. An official American Thoracic Society workshop report: features and measurements of experimental acute lung injury in animals. Am J Respir Cell Mol Biol. 2011;44:725e738. 25. Kuncewitch M, Yang W-L, Jacob A, et al. Stimulation of Wnt/ beta-catenin signaling pathway with Wnt agonist reduces organ injury after hemorrhagic shock. J Trauma Acute Care Surg. 2015;78:793. 26. Schmekel B, Karlsson SE, Linden M, et al. Myeloperoxidase in human lung lavage. Inflammation. 1990;14:447e454. 27. Dorman RB, Gujral JS, Bajt ML, Farhood A, Jaeschke H. Generation and functional significance of CXC chemokines for neutrophil-induced liver injury during endotoxemia. Am J Physiol Gastrointest Liver Physiol. 2005;288:G880eG886. 28. Frevert CW, Huang S, Danaee H, Paulauskis JD, Kobzik L. Functional characterization of the rat chemokine KC and its importance in neutrophil recruitment in a rat model of pulmonary inflammation. J Immunol. 1995;154:335e344. 29. Gutierrez G, Reines HD, Wulf-Gutierrez ME. Clinical review: hemorrhagic shock. Crit Care. 2004;8:373e381. 30. Eltzschig HK, Eckle T. Ischemia and reperfusion–from mechanism to translation. Nat Med. 2011;17:1391e1401. 31. Qiang X, Yang W-L, Wu R, et al. Cold-inducible RNAbinding protein (CIRP) triggers inflammatory responses in hemorrhagic shock and sepsis. Nat Med. 2013;19:1489e1495. 32. Piccinini AM, Midwood KS. DAMPening inflammation by modulating TLR signalling. Mediators Inflamm. 2010;2010:672395. 33. Zhang JZ, Liu Z, Liu J, Ren JX, Sun TS. Mitochondrial DNA induces inflammation and increases TLR9/NFkappaB expression in lung tissue. Int J Mol Med. 2014;33:817e824. 34. Zhang Q, Itagaki K, Hauser CJ. Mitochondrial DNA is released by shock and activates neutrophils via p38 map kinase. Shock. 2010;34:55e59. 35. Wei X, Shao B, He Z, et al. Cationic nanocarriers induce cell necrosis through impairment of Na(þ)/K(þ)-ATPase and cause subsequent inflammatory response. Cell Res. 2015;25:237e253. 36. Julian MW, Shao G, Vangundy ZC, Papenfuss TL, Crouser ED. Mitochondrial transcription factor A, an endogenous danger signal, promotes TNFalpha release via RAGE- and TLR9responsive plasmacytoid dendritic cells. PLoS One. 2013;8:e72354. 37. Hendriks T, de Hoog M, Lequin MH, Devos AS, Merkus PJ. DNase and atelectasis in non-cystic fibrosis pediatric patients. Crit Care. 2005;9:R351eR356. 38. El Hassan NO, Chess PR, Huysman MW, Merkus PJ, de Jongste JC. Rescue use of DNase in critical lung atelectasis and mucus retention in premature neonates. Pediatrics. 2001;108:468e470. 39. Rahman NM, Maskell NA, West A, et al. Intrapleural use of tissue plasminogen activator and DNase in pleural infection. N Engl J Med. 2011;365:518e526. 40. Quan JM, Tiddens HA, Sy JP, et al. A two-year randomized, placebo-controlled trial of dornase alfa in young patients with
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cystic fibrosis with mild lung function abnormalities. J Pediatr. 2001;139:813e820. 41. Davis Jr JC, Manzi S, Yarboro C, et al. Recombinant human Dnase I (rhDNase) in patients with lupus nephritis. Lupus. 1999;8:68e76. 42. McIlroy DJ, Minahan K, Keely S, et al. Reduced deoxyribonuclease enzyme activity in response to high postinjury mitochondrial DNA concentration provides a therapeutic target for systemic inflammatory response syndrome. J Trauma Acute Care Surg. 2018;85:354e358. 43. Fan J, Marshall JC, Jimenez M, et al. Hemorrhagic shock primes for increased expression of cytokine-induced neutrophil chemoattractant in the lung: role in pulmonary
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inflammation following lipopolysaccharide. J Immunol. 1998;161:440e447. Liu FC, Chuang YH, Tsai YF, Yu HP. Role of neutrophil extracellular traps following injury. Shock. 2014;41:491e498. Fuchs TA, Brill A, Duerschmied D, et al. Extracellular DNA traps promote thrombosis. Proc Natl Acad Sci U S A. 2010;107:15880e15885. Meegan JE, Yang X, Coleman DC, Jannaway M, Yuan SY. Neutrophil-mediated vascular barrier injury: role of neutrophil extracellular traps. Microcirculation. 2017;24. Meng W, Paunel-Gorgulu A, Flohe S, et al. Deoxyribonuclease is a potential counter regulator of aberrant neutrophil extracellular traps formation after major trauma. Mediators Inflamm. 2012;2012:149560.
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Deoxyribonuclease Reduces Tissue Injury and Improves Survival After Hemorrhagic Shock Joaquin Cagliani, MD, PhD,a,b,c,e Weng-Lang Yang, PhD,a,b,c,d Max Brenner, MD, PhD,b,e and Ping Wang, MDa,b,c,* a
Elmezzi Graduate School of Molecular Medicine, Manhasset, New York Center for Immunology and Inflammation, Feinstein Institutes for Medical Research, Manhasset, New York c Department of Surgery, Zucker School of Medicine at Hofstra/Northwell, Manhasset, New York d Department of Radiation Oncology, Albert Einstein College of Medicine, Bronx, New York e Department of Molecular Medicine, Zucker School of Medicine at Hofstra/Northwell, Manhasset, New York b
article info
abstract
Article history:
Background: Hemorrhagic shock (HS) caused by rapid loss of a large amount of blood is the
Received 18 June 2019
leading cause of early death after severe injury. When cells are damaged during HS, many
Received in revised form
intracellular components including DNA are released into the circulation and function as
16 September 2019
endogenous damage-associated molecular patterns (DAMPs) that can trigger excessive
Accepted 23 November 2019
inflammatory response and subsequently multiple organ dysfunction. We hypothesized
Available online xxx
that the administration of deoxyribonuclease I (DNase I) could reduce cell-free DNA and attenuate tissue damage in HS. Methods: Eight-week-old male C57BL/6 mice underwent HS by controlled bleeding from the femoral artery for 90 min, followed by resuscitation with Ringer’s lactate solution (vehicle) or DNase I (10 mg/kg BW). Results: At 20 h after HS, serum levels of cell-free DNA were increased by 7.6-fold in the vehicle-treated HS mice compared with sham, while DNase I reduced its levels by 47% compared with the vehicle group. Serum levels of tissue injury markers (lactate dehydrogenase, aspartate aminotransferase, and alanine aminotransferase) and proinflammatory cytokine interleukin 6 were significantly reduced in the DNase Ietreated mice. In the lungs, messenger RNA levels of proinflammatory cytokines (interleukin 6 and interleukin 1 b), chemoattractant macrophage inflammatory protein - 2, and myeloperoxidase activity were significantly decreased in HS mice after DNase I. Finally, DNase I significantly improved the 10-day survival rate in HS mice. Conclusions: Administration of DNase I attenuates tissue damage and systemic and lung inflammation, leading to improvement of survival in HS mice. Thus, DNase I may potentially serve as an adjunct therapy for managing patients with HS. ª 2019 Elsevier Inc. All rights reserved.
* Corresponding author. Department of Surgery, Zucker School of Medicine at Hofstra/Northwell The Feinstein Institutes for Medical Research, 350 Community Drive, Manhasset, NY 11030. Tel.: þ(516) 562 3411; fax: þ516 562-1022. E-mail address: [email protected] (P. Wang). 0022-4804/$ e see front matter ª 2019 Elsevier Inc. All rights reserved. https://doi.org/10.1016/j.jss.2019.11.036
cagliani et al cfdna in hemorrhagic shock
Introduction
Methods
Hemorrhagic shock (HS) induced by a rapid loss of a large amount of blood is the leading cause of early death after severe injury.1-3 In the United States, HS causes more than 60,000 deaths yearly, 80% of which are due to trauma.4 When hemorrhage occurs, the blood supply to the vital organs is compromised, causing ischemia. The subsequent restoration of blood supply or reperfusion after hemorrhage can acutely trigger complex inflammatory cascades which play a key role in generating cellular stress and postischemia injury.5 When cells are damaged during HS, intracellular components such as nuclear and mitochondrial DNA are released into the circulation. These cell-free components function as endogenous damage-associated molecular patterns (DAMPs) that can trigger primary innate immune response mediated by pattern-recognition receptors (PRRs) such as toll-like receptors (TLRs), nucleotide oligomerization domain e such as receptors (NLRs), or receptors for advanced glycation endproducts (RAGEs) after HS. 6 The interaction between DAMPs and PRRs activates numerous signal transduction pathways, leading to the release of proinflammatory cytokines to activate monocytes, neutrophils, and endothelial cells. While the inflammatory response in the setting of hemorrhage may be protective by promoting the activation of the clotting system, 7 the uncontrolled amplification of the proinflammatory pathways during reperfusion is particularly detrimental for the host.8,9 This exuberant immune response leads to cellular apoptosis, thereby propagating tissue damage and organ failure.10-12 In patients admitted to an intensive care unit with trauma-related injuries, cell-free DNA (cfDNA) has been proposed as a biomarker for diagnostic and prognostic implications.13 Several reports have indicated that the amount of cfDNA correlates with mortality, trauma severity, and post-traumatic complications.14-16 Although deoxyribonuclease (DNase)-based therapeutic strategies to decrease the levels of cfDNA have been shown to be beneficial in models of ischemia reperfusion injury and acute lung injury,17-19 the potential therapeutic value of DNase I in the physiopathology of HS remains unknown. Given that cfDNA can act as a DAMP to modulate the immune response and its levels are elevated in trauma patients, we then hypothesized that administering exogenous DNase I to reduce the levels of circulating cfDNA may attenuate inflammation and organ injury as well as have survival benefits within the scope of HS. In this study, we used a mouse model of HS to investigate the role of cfDNA. We first measured the change of circulating cfDNA levels in HS mice and after DNase I. We then compared the serum levels of organ injury markers, histological damage, and expression of proinflammatory cytokines and chemokines in the lungs between the HS mice with and without DNase I. Finally, we determined the effect of DNase I on the 10-day survival of HS mice.
Mouse model of HS
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Eight-week-old male C57BL/6 mice (20-25 g) purchased from The Jackson Laboratory (Bar Harbor, ME) were housed in a 12-h light and dark cycle and temperature-controlled environment. Mice were fed a standard laboratory mouse chow diet and water ad libitum. After acclimation for 7 d, mice were subjected to HS. All experiments were performed in accordance with the guidelines for the use of experimental animals by the National Institutes of Health (Bethesda, MD) and were approved by the Institutional Animal Care and Use Committee of the Feinstein Institutes for Medical Research. The animal model of HS was performed as previously described.20 Succinctly, mice were randomly assigned to undergo sham, HS adjuvantly treated with DNase I, or HS adjuvantly treated with vehicle (control) groups. Anesthesia was induced with inhalation of 2.5% isoflurane and then maintained with 1.5% isoflurane. The inguinal regions were shaved and antiseptically prepared with povidone iodine and chlorhexidine gluconate. Two 0.5-cm inguinal incisions were made to expose the right and left femoral arteries, which were both cannulated with pressure equalizer-10 tubing after careful dissection of the femoral vein and femoral nerves. One catheter was used for controlled hemorrhage, fluid resuscitation, and administration of DNase I or vehicle, while the second catheter was connected to a transducer to continuously measure the mean arterial pressure (MAP) and heart rate. The results were recorded with a blood pressure analyzer Digi-Med (Micro-Med, Louisville, KY). The MAP values were averaged over 5-s intervals to record the oscillations in the blood pressure. Blood was withdrawn from the femoral artery to reach and maintain an MAP of 27.5 2.5 mmHg over 90 min. Mice were then resuscitated with prewarmed Ringer’s lactate solution totaling two times the volume of shed blood over the 30-min resuscitation phase. Mice were not heparinized during the procedure. The animal’s body temperature was kept at 37 C using a water-recirculating heating pad during the HS and resuscitation phases. After the resuscitation, the femoral artery was ligated, and the incision was closed with nonabsorbable suture. Animals were allowed to recover from anesthesia before being returned to their cages. Sham mice underwent the same procedure, including bilateral femoral artery cannulation, without induction of hemorrhage or resuscitation. All efforts were made to minimize suffering.
Administration of DNase I During the 30-min resuscitation phase, the mice received Ringer’s lactate solution alone (vehicle) or containing bovineextracted pancreatic DNase I (10 mg/kg BW) from SigmaAldrich (St. Louis, MO). We chose the dose of DNase I based on previous studies in murine models.21,22
Blood and lung tissue collection At 20 h after the initiation of resuscitation, when hemorrhageassociated acute lung injury can be detected,23 all mice were
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euthanized by CO2 asphyxiation. Whole blood was drawn from the inferior vena cava and centrifuged twice at 12,000 rpm for 10 min at 4 C to separate the cellular fraction from the serum and thus exclude the contribution of DNA from the lysis of the white cells. After the blood draw, lung tissues were harvested and stored at 80 C until analysis.
Assessment of cfDNA in serum cfDNA was extracted from 500 mL of serum of each mice using the QIAmp DNA Mini Kit (Qiagen, Valencia, CA) in accordance with the manufacturer’s instructions. The quality and quantification of the isolated DNA was determined at 260 and 280 nm, respectively, on a NanoDrop microvolume spectrophotometer (Thermo Fisher Scientific Inc, Waltham, MA).
Analysis of serum injury markers and interleukin 6 Serum activities of alanine aminotransferase (ALT), aspartate aminotransferase (AST), and lactate dehydrogenase (LDH) were determined using assay kits (Pointe Scientific, Canton, MI). Serum interleukin 6 (IL-6) levels were analyzed using a commercial mouse enzyme-linked immunosorbent assay (ELISA) kit (BD Biosciences, San Diego, CA). All assays were carried out in accordance with their manufacturer protocol instructions.
Quantitative real-time polymerase chain reaction Total RNA was extracted from tissues using Trizol (Invitrogen, Carlsbad, CA) and reverse transcribed into using murine leukemia virus reverse transcriptase (Applied Biosystems, Foster City, CA). A quantitative real-time polymerase chain reaction (PCR) assay was carried out in a final volume of 20 mL containing 0.25 mmol of each forward and reverse primer, complimentary DNA, and 10 mL SYBR Green PCR master mix (Applied Biosystems). Amplification was conducted in an Applied Biosystems Step One Plus real-time PCR machine under the thermal profile of 50 C for 2 min, 95 C for 10 min followed by 45 cycles of 95 C for 15 s and 60 C for 1 min. The levels of mouse bactin messenger RNA (mRNA) were used for normalization. Relative quantity of mRNA was expressed as the fold change in comparison with the sham tissues. The primers sequences used for quantitative real-time are the following: IL-6 (NM_031168): 50 -CCGGAGAGGAGACTTCACAG-30 and 50 -GGA AATTGGGGTAGGAAGGA-30 ; IL-1b (NM_008361): 50 -CAGGATG AGGACATGAGCACC-30 , and 50 -CTCTGCAGACTCAAACTCCAC30 ; macrophage inflammatory protein-2 (MIP-2) (NM_009140): 50 -CCCTGGTTCAGAAAATCATCCA-30 and 50 -GCTCCTCCTTTC CAGGTCAGT-30 ; b-actin (NM_007393): 50 -CGTGAAAAGATGA CCCAGATCA-30 and 50 -TGGTACGACCAGAGGCATACAG-30 .
Histology analysis Lung tissue segments collected at 20 h after reperfusion were fixed in 10% formalin before paraffin embedding. The tissue blocks were sectioned into 5-mm cuts, transferred to glass slides, and stained with hematoxylin and eosin (H&E). Ten fields per sample were then assessed in a blinded fashion using a modified American Thoracic Society lung injury
scoring system, consisting of semiquantitative scores for inflammatory cell infiltration into the alveolar and interstitial space, presence of hyaline membranes, proteinaceous debris inside airspaces, and alveolar septal thickening.23 Based on the presence of these parameters, scores per visual field were categorized as 0 (no injury), 1 (moderate injury), and 2 (severe injury). Using the weighted equation with a maximum score of 100 per field presented by Matute-Bello et al.,24 the parameter scores were calculated on a scale of 0-1 and then averaged as the final lung injury score in each group.
Myeloperoxidase activity assay Myeloperoxidase (MPO) activity in the lung was determined using the peroxidase-catalyzed reaction as previously described.25 Lung tissues were sonicated in 50 mM potassium phosphate buffer containing 0.5% hexadecyltrimethylammonium bromide (pH 6.0). After centrifugation, the supernatant was diluted in a reaction solution containing o-dianisidine hydrochloride and H2O2. The rate of change in optical density per 1 min was measured at 460 nm, and the MPO activity was expressed as change in absorbance per min per gram of protein.
Terminal deoxynucleotidyl transferaseemediated deoxyuridine triphosphate nick-end labeling assay Lung tissue sections were immersed in 20 mg/mL of proteinase K (Thermo Fisher Scientific) at room temperature for 20 min and then stained using an in situ labeling of DNA fragmentation with terminal deoxynucleotidyl transferaseemediated deoxyuridine triphosphate nick-end labeling assay kit (Roche Diagnostics, Florham Park, NJ) and counterstained with Vectashield mounting medium containing 40 ,6-diamidino-20 -phenylindole dihydrochloride (Vector Laboratories, Inc, Burlingame, CA). Apoptotic cells were visualized green at 200 magnification under a fluorescence microscope (Nikon Eclipse Ti-S, Melville, NY) and were then counted in five visual fields/sections. The average number of apoptotic cells/field was calculated.
Survival study The survival rate was assessed in a separate group of animals, which were randomly assigned to adjuvant treatment with vehicle (n ¼ 10) or DNase I (n ¼ 10). Sham animals were not included because they are expected to have a survival rate of 100%. Animals were subjected to HS and fluid resuscitation plus adjuvant treatment with Ringer’s lactate solution (vehicle) or DNase I (10 mg/kg BW) via the right femoral artery over 30 min as described previously. After resuscitation, the femoral arteries were ligated, and the incisions were closed in layers. The mice were returned to their cages for recovery, allowed food, and water ad libitum and monitored for 10 d.
Statistical analysis For nonsurvival experiments, we calculated that a sample size of nine mice per group for a 7-group analysis of variance (ANOVA) would have a power of 0.8 to detect a difference of 25% between groups with a standard deviation of 25% of the
cagliani et al cfdna in hemorrhagic shock
Fig. 1 e Monitoring of blood pressure during HS induction, maintenance, and resuscitation phases. Arterial blood pressure at the induction phase (0-20 min), the hemorrhage phase (20-110 min), and the first 30 min of resuscitation phase (110-140 min) in the vehicle- and DNase Ietreated mice was recorded from the beginning of the operation. Sham-operated mice with the same time frame were also recorded. Data presented are mean ± SEM (n [ 6 mice/ group). (Color version of figure is available online.)
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Fig. 2 e Effect of DNase I on the serum levels of cfDNA after HS. Mice were subjected to HS as described in Methods. Serum from vehicle- and DNase Ietreated mice at 20 h after HS and sham mice were collected for the analysis. The serum levels of cfDNA were quantified spectrophotometrically. Data are expressed as mean ± SEM (n [ 6 mice/group) and compared by ANOVA and SNK test. *P < 0.05 versus sham; and #P < 0.05 versus vehicle. (Color version of figure is available online.)
DNase I decreases serum cfDNA after HS mean and a P 0.05. For survival experiments, we calculated that a sample size of 20 mice per group would have a power of 0.8 to detect a difference in survival of 50% or more with a P 0.05. Data are expressed as mean standard error of the mean (SEM) and compared by one-way ANOVA using the Student-Newman-Keuls (SNK) test for multiple group comparisons. The survival rate was estimated by the Kaplan-Meier method and compared using the log-rank test. Differences between the experimental groups with P 0.05 were considered statistically significant.
Results Adjuvant treatment with DNase I does not affect the MAP during the resuscitation phase HS is a complicated, heterogeneous condition involving the systemic activation of inflammation and coagulation in response to significant blood loss. It may progress to severe HS if there is dysfunction in one or multiple organs. Thus, we sought to determine if mice adjuvantly treated with vehicle and DNase I had undergone differences in blood loss before receiving resuscitation with or without the adjuvant treatment or showed different hemodynamic responses during the 30min resuscitation phase. Indeed, both groups of mice had the same amount of blood withdrawn (0.8 0.19 mL per mouse) to maintain the target MAP during the hemorrhage phase before resuscitation plus adjuvant treatment with vehicle or DNase I. Similarly, the MAPs of the two groups of mice did not differ during the fluid resuscitation phase (Fig. 1). As expected, the sham group MAP remained stable throughout the study.
To explore the potential role cfDNA as an inflammatory mediator in HS, we examined the release of DNA in the serum of mice undergoing HS followed by reperfusion. At 20 h after HS, serum levels of cfDNA increased significantly from 11.5 ng/ mL in the sham to 117.1 ng/mL in the vehicle group, while adjuvant treatment with DNase I significantly decreased the levels of cfDNA by 47% compared with the vehicle group (Fig. 2).
DNase I attenuates tissue injury and systemic inflammation after HS The HS group showed marked elevation of serum levels of organ injury markers in comparison with the sham group. Remarkably, the levels of LDH, AST, and ALT were significantly higher in HS-vehicle mice by 14.0-, 19.6-, and 8.8-fold, respectively (Fig. 3). Mice adjuvantly treated with DNase I, however, had serum levels of LDH, AST, and ALT significantly decreased by 39.0%, 33.9%, and 63.8%, respectively, compared with the HS-vehicle group (Fig. 3A-C). Because excessive elevated levels of proinflammatory cytokines in circulation is a major contributor to remote organ injury after HS, we then measured the levels of IL-6 in the serum. The IL-6 protein levels increased significantly by 8.9-fold in the vehicle group as compared with sham, while its levels were significantly decreased in mice adjuvantly treated with DNase I by 28% compared with the HSvehicle group (Fig. 3D). These results suggest that DNase I can mitigate the systemic inflammation and organ injury after HS.
DNase I reduces lung inflammatory cytokines after HS To determine the role of DNase I in the lung inflammation induced by HS, we measured proinflammatory cytokines in
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A
B
C
D
Fig. 3 e Effect of DNase I on organ injury after HS. Mice were subjected to HS as described in Methods. Serum from vehicleand DNase Ietreated mice at 20 h after HS and sham mice were collected for the analysis. The serum levels of (A) LDH, (B) AST, and (C) ALT were determined by enzymatic method. (D) The serum levels of IL-6 were measured by ELISA. Data are expressed as mean ± SEM (n [ 6 mice/group) and compared by ANOVA and SNK test. *P < 0.05 versus sham; #P < 0.05 versus vehicle. (Color version of figure is available online.)
the lungs at 20 h after HS. The mRNA expression in the lungs of both IL-6 and IL-1b in the HS-vehicle mice were significantly higher than that in the sham, by 3.9- and 2.4-fold, respectively (Fig. 4A and B). Meanwhile, the mice adjuvantly treated with DNase I exhibited in a significant reduction of IL-6 and IL-1b levels by 59.0% and 83.3%, respectively, as compared with the
A
B
group treated with vehicle (Fig. 4A and B). To analyze the effect of DNase I on neutrophil infiltration which is another mechanism of injury involving immune cells, we examined the chemokine expression of MIP-2 in the lungs. MIP-2 functions as chemoattractant to neutrophils at inflamed sites.26-28 The HS-vehicle group showed significantly elevated mRNA
C
Fig. 4 e Effect of DNase I on the proinflammatory cytokine expression in the lungs after HS. Mice were subjected to HS as described in Methods. Lung tissues from vehicle- and DNase Ietreated mice at 20 h after HS and sham mice were collected for the analysis. The mRNA levels of (A) IL-6, (B) IL-1b, and (C) MIP-2 in the lungs were assessed by quantitative real-time polymerase chain reaction. Each gene expression level was normalized to b-actin. The value in the sham group is designated as 1 for comparison. Data are expressed as mean ± SEM (n [ 6 mice/group) and compared by ANOVA and SNK test. *P < 0.05 versus sham; #P < 0.05 versus vehicle. (Color version of figure is available online.)
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Fig. 5 e Effect of DNase I on lung morphology and neutrophil infiltration after HS. Mice were subjected to HS as described in Methods. Lung tissues from vehicle- and DNase Ietreated mice at 20 h after HS and sham mice were harvested, sectioned, and subjected to histologic analysis. (A-C) Representative images of tissue sections with H&E staining at an original magnification of 200 3 . (D) Histologic injury score in each group was blindly graded as described in Methods. (E) Lung myeloperoxidase (MPO) activity was determined spectrophotometrically. Data are expressed as mean ± SEM (n [ 6 mice/ group) and compared by ANOVA and SNK test. *P < 0.05 versus sham; #P < 0.05 versus vehicle. (Color version of figure is available online.)
expression levels of MIP-2 by 34.1-fold, compared with sham (Fig. 4C). Conversely, adjuvant treatment with DNase significantly decreased the levels of MIP-2 by 53.9% compared with the HS-vehicle group (Fig. 4C).
DNase I improves the integrity of the lung histology after HS The effects of adjuvant treatment with DNase I on hemorrhageinduced acute lung injury were further assessed by histological analyses (Fig. 5A-C). The HS-vehicle group showed alterations in the alveolar capillary barrier, septal thickening, and hyaline deposits as compared with the sham (Fig. 5A). The lung architecture and morphology of the HS mice adjuvantly treated with DNase I, however, resembled the sham group more than the HS-vehicle group (Fig. 5A-C). Accordingly, the lung injury score in the HSvehicle group was 5.2-fold greater than that in the sham (P < 0.05), while the DNase Ietreated mice showed a significant 42.5% decrease in the lung injury score when compared with the mice treated with vehicle (Fig. 5D). To further quantify hemorrhage-induced lung inflammation, we measured the MPO activity as an indicator of infiltration by activated neutrophils.26 We found a significant 3.7-fold increase in the MPO activity of the lungs of mice in the HS-vehicle group as compared with those in the sham group, while mice adjuvantly treated with DNase I showed a significant 59.8% decrease in the MPO activity as compared with mice in the HS-vehicle group (Fig. 5E).
DNase I attenuates apoptosis in the lungs after HS The TUNEL assay was used to study the effect of DNase I on apoptosis in lung tissues. The sham group showed almost no detection of TUNEL-positive cells, while a significant increase of TUNEL-positive cells was observed in the HS-vehicle group (Fig. 6A-C). In mice adjuvantly treated with DNase I, the number of apoptotic cells was significantly decreased by 75.0% compared with the animals in the HS-vehicle (Fig. 6D).
DNase I improves survival after HS Then, we examined the role of DNase I on 10-day survival after HS. We found that mice adjuvantly treated with DNase I had a significant improvement in the 10-day survival when compared with those in the HS-vehicle group (80% versus 30%, P < 0.05; Fig. 7).
Discussion The physiologic changes in response to HS are directed at maintaining blood pressure and cardiac output to support the function of vital organs.29 However, the extent of compensation is limited, and in the absence of acute intervention, severe hypoxia and inflammation can cause irreversible damage
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Fig. 6 e Effect of DNase I on apoptosis in the lungs after HS. Mice were subjected to HS as described in Methods. Lung tissues from vehicle- and DNase Ietreated mice at 20 h after HS and sham mice were harvested, sectioned, and subjected to terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assay. (A-C) Representative images of tissue sections stained with TUNEL (green fluorescence) and nuclear counterstaining with 40 ,6-diamidino-2-phenylindole (DAPI) (blue fluorescence). Bar scale indicates 100 mm at an original magnification of 200 3 . (D) The number of TUNEL-positive cells was counted and averaged over five high-power fields (HPFs)/section. Data are expressed as mean ± SEM (n [ 6 mice/group) and compared by ANOVA and SNK test. *P < 0.05 versus sham; #P < 0.05 versus vehicle. dUTP [ deoxyuridine triphosphate. (Color version of figure is available online.)
at the cellular and organ levels and may result in eventual death.4 In the present study, we proposed that better outcomes for HS could be achieved by the control of DAMP levels in the circulation through the degradation of cfDNA by DNase I. Our results show that adjuvant treatment with DNase I along with fluid resuscitation provides a strong protective effect by attenuating organ injury and systemic and pulmonary inflammation and reducing neutrophil infiltration and TUNEL-positive cells in the lungs of mice subjected to HS. Importantly, we also demonstrate that DNase I improves the 10-day survival rate in a lethal mouse model of HS. Therefore, our results confirm the detrimental role of cfDNA contributing to the severity and worse outcomes in HS and clearly indicate the beneficial effects of adjuvant treatment of severe hemorrhage with DNase I. DAMPs activate the innate immune response and have been shown to play a key role in the pathogenesis of ischemiareperfusion.30 Examples of DAMPs include extracellular coldinducible RNA-binding protein, high-mobility group box 1 protein, and genomic or mitochondrial DNA.6,31-33 We demonstrate that cfDNA is released and markedly elevated after the initial traumatic injury after HS. The cfDNA can
regulate the innate immune response through a wide variety of pathways mediated by various PRRs in macrophages.34-36 For example, when binding with TLR9, cfDNA triggers the activation of mitogen-activated protein kinases (MAPKs) and IkB kinase complex through the adapter protein MyD88 to promote inflammation. Along these lines, it has been demonstrated that cfDNA signaling through IkB activates interferon regulatory factor-7 and nuclear factor kappa B, leading to the transcription of type 1 interferons and enhanced expression of proinflammatory cytokines such as IL-6 and IL-1b.34,37 Indeed, we found that degrading cfDNA with DNase I diminished the production of proinflammatory cytokines and resulted in decreased lung injury after HS. Exogenous administration of DNase I has also been shown to be beneficial in treating atelectasis38,39 and pleural infections40 and improving the lung function in patient with cystic fibrosis41 as well as is under a randomized phase Ib trial for the treatment of lupus nephritis.42 In addition, a decrease in DNase activity has recently been shown to correlate with the development of systemic inflammatory response syndrome in patients with trauma,43 indicating the therapeutic benefit of targeting cfDNA in these patients.
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Fig. 7 e Effect of DNase I on the survival of hemorrhaged mice. Mice were subjected to HS, as described in Methods, and treated with Ringer’s lactate solution (vehicle) or containing DNase I (10 mg/kg) during the reperfusion phase. Mice were monitored for survival for 10 d. Survival rates were analyzed by the Kaplan-Meier estimator using a log-rank test. (n [ 10 mice/group; P < 0.05). (Color version of figure is available online.)
Neutrophils play a pivotal role in the inflammatory component after injury. In the lungs, the innate immune response can recruit neutrophils which may cause injury by releasing reactive oxygen species and proteolytic enzymes such as elastase and MPO. It has been reported that disproportionate production of these enzymes is associated with poor clinical outcomes.12,28,44 Here, we report that adjuvant treatment of HS with DNase I led to a significant reduction in MIP-2 e a key neutrophil chemoattractant e and in the amount of activated neutrophils infiltrating the lungs. These results were further supported by the reduction in lung proinflammatory cytokines and the improved lung histology in hemorrhaged mice adjuvantly treated with DNase I. Neutrophil degranulation in the lungs releases toxic reactive oxygen species and proteolytic enzymes to the extracellular space, resulting in increased rates of epithelial cell death. Indeed, we noticed a marked increase in the number of apoptotic cells, a type of cellular death, in the lungs after HS. Interestingly, a reduction in the number of apoptotic cells and consequent improvement in lung injury was attained by using DNase I, which indicates that lowering cfDNA levels ultimately limits cell death secondary to lung inflammation in our model of HS. In addition to degranulation, during inflammation, activated neutrophils can also form extracellular traps (NETs).45 NETs are a major source of cfDNA,46 and aside from their antimicrobial defense by preventing the dissemination of pathogens, they can be detrimental to trauma by causing endothelial barrier dysfunction47 and microvascular thrombosis.19,45 DNase is able to dismantle the cytotoxic NETs scaffold and digest the extracellular DNA derived from NETs in experimental models of ischemia and reperfusion injury and acute lung injury.17,18 Although DNase is naturally
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present in human blood and produced as a defense mechanism associated with NETs, our data show that administration of exogenous DNase I is clearly beneficial in the setting of pure HS. Given the complex sequence of the trauma, the consecutive circulatory shock, and the pathophysiology underlying the response to the injury, developing a consistent and reproducible model of HS is paramount. However, we acknowledge that our experimental model presents some limitations as the selfcompensatory mechanisms that occur during the isobaric hypotension are disrupted. In addition, we have not explored the effects of clinically important variables such as resuscitation volume, administration of blood and blood products, and presence of trauma. Nonetheless, the increased release of cfDNA associated with severe trauma and trauma surgery14,15 suggests that these cases will likely benefit even more from adjuvant treatment with DNase I. While our study emphasizes the effects of DNase I on the immune and lung injury responses after pure HS, it will be important to verify the beneficial effects of DNase I in preclinical models of uncontrolled bleeding and combined trauma hemorrhage. In conclusion, our data show further evidence of cfDNA’s detrimental proinflammatory effects in HS and indicate that cfDNA’s targeting with DNase I during reperfusion represents a new and innovative therapeutic strategy to modulate the systemic inflammatory response and subsequent organ dysfunction caused by HS.
Acknowledgment This work was supported by National Institutes of Health grant R01HL076179 (to PW) and the Elmezzi Graduate School of Molecular Medicine (to J.C.). J.C., W.-L.Y., M.B., and P.W. conceived and designed the experiment. J.C. performed the experiments. J.C. and W.-L.Y. analyzed the data. J.C. wrote the manuscript and prepared the figures. W.-L.Y. and M.B. revised the manuscript. P.W. reviewed and supervised the research. All authors read and approved the final manuscript. All experiments involving live animals were carried out in accordance with the National Institutes of Health guidelines for the use of experimental animals and were reviewed and approved by the Institutional Animal Care and Use Committee at the Feinstein Institutes for Medical Research. The data sets used and analyzed during the present study are available from the corresponding author on reasonable request.
Disclosure The authors declare that the research was conducted in the absence of any commercial or financial relationship that could be construed as a potential conflict of interest.
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