Secondary Logo

Journal Logo

Basic Science Aspects

Delayed but not Early Treatment with DNase Reduces Organ Damage and Improves Outcome in a Murine Model of Sepsis

Mai, Safiah H. C.*†; Khan, Momina*†; Dwivedi, Dhruva J.†‡; Ross, Catherine A.§; Zhou, Ji; Gould, Travis J.*†; Gross, Peter L.*†‡; Weitz, Jeffrey I.*†‡; Fox-Robichaud, Alison E.*†‡∥; Liaw, Patricia C.*†‡∥ for the Canadian Critical Care Translational Biology Group

Author Information
doi: 10.1097/SHK.0000000000000396

Abstract

INTRODUCTION

Sepsis is characterized by systemic activation of inflammatory and coagulation pathways in response to microbial infection (1). Severe sepsis, defined as sepsis associated with organ dysfunction, afflicts approximately 3 per 1,000 people in the United States annually (2) and is associated with mortality rates of 33% to 45% (1). The incidence of severe sepsis continues to increase by 1.5% per annum because of the aging population, increased prevalence of obesity and diabetes, and wider use of immunosuppressive agents and invasive procedures (3).

During the past several decades, many potential treatments for sepsis have shown early promise yet have failed to improve survival in more than 100 phase II and III clinical trials (4). These trials attempted to treat sepsis by dampening inflammation or coagulation. Current strategies for sepsis are largely supportive and include fluid resuscitation, mechanical ventilation, and early administration of broad-spectrum antibiotics (1). Despite these strategies, the mortality rate from sepsis remains high, suggesting that some fundamental knowledge is lacking in our understanding of sepsis pathophysiology (1, 2, 4).

In recent years, cell-free DNA (cfDNA) has emerged as an important link between innate immunity and coagulation. When activated by microbes, neutrophils release weblike structures termed neutrophil extracellular traps (NETs), which are composed of cfDNA, histones, proteases, and granular proteins (5, 6). By binding to microorganisms, NETs prevent dissemination of pathogens and ensure a high local concentration of antimicrobial agents. However, the presence of NETs in the microcirculation results in increased coagulation and platelet activation (7, 8), microvascular thrombosis (7, 9), and organ damage (10). In sepsis, high levels of nucleosomes (DNA bound to histones) are found in blood (11) and we have previously reported that elevated levels of circulating cfDNA predict poor clinical outcome in severe sepsis patients (12). We have recently reported that neutrophils are the major source of cfDNA released from whole blood on inflammatory stimulation (13).

The NETs are counterregulated by deoxyribonuclease I (DNase I) (14), a calcium- and magnesium-dependent endonuclease that hydrolyzes double-stranded DNA (15) and degrades chromatin released during necrosis to prevent anti-DNA autoimmunity (5). Deoxyribonuclease I has been used to treat patients with various conditions associated with increased cfDNA levels, including cystic fibrosis (16) and pleural infection (17).

In experimental sepsis, the protective effects of DNase remain unclear. Meng et al. (10) reported an exacerbated inflammatory response and increased bacterial load in septic mice given repeated injections of DNase. In contrast, Luo et al. (18) observed that repeated administration of DNase in septic mice reduced lung edema and tissue damage. We hypothesize that these divergent results may reflect the timing of DNase administration, a crucial element in managing sepsis. To examine this possibility, we compared the effect of early and late DNase administration on levels of cfDNA, markers of coagulation and inflammation, and organ histopathology in mice subjected to a cecal ligation and puncture (CLP) model of polymicrobial sepsis.

MATERIALS AND METHODS

Experimental sepsis: cecal ligation and puncture

Eight-week-old (20 – 25 g) male C57Bl/6 mice (Helicobacter hepaticus–free) were purchased from Charles River Laboratories (Sherbrooke, Quebec, Canada) and bred at the Thrombosis and Atherosclerosis Research Institute (Hamilton, Ontario, Canada). Details of housing can be found in Methods, Supplemental Digital Content 1, at https://links.lww.com/SHK/A298. The CLP model used in these studies was adapted from well-established protocols by other groups (19, 20). Under anesthesia, a midline laparotomy was performed and, after it was exteriorized, the cecum was ligated distal to the ileocecal valve, 1 cm from the cecal end and perforated through-and-through with an 18-gauge needle. In mice subjected to sham surgery, the cecum was exteriorized and returned to the peritoneal cavity (n = 4 – 12 per time point per group). Mice were randomized to the CLP or sham groups according to ARRIVE guidelines (21), and methods for randomization and experimenter blinding were in place to reduce allocation, selection, and experimenter biases (22). Mice were randomized to both groups on each surgery date. The CLP and sham mice were given analgesia (buprenorphine 0.1 mg/kg) for pain relief and resuscitation fluids (2 mL, Ringer’s lactate) subcutaneously every 4 h postoperatively to account for surgical losses. Deoxyribonuclease (Pulmozyme, Roche Genentech; 20 mg/kg) or saline was administered by intraperitoneal injection at 2, 4, or 6 h postoperatively (n = 5 – 18 per time point per group). Two hours after DNase or saline administration, blood was collected via the inferior vena cava into a one-tenth volume of 3.2% sodium citrate, spun at 2,000g, and plasma was stored at −80°C.

Isolation and quantification of cfDNA

Cell-free DNA was isolated from 200 μL of mouse plasma using the Qiagen QIAamp DNA Blood Mini Kit (QIAgen, Valencia, Calif) and quantified by measuring absorbance at 260 nm using an Eppendorf BioPhotometer Plus (Hamburg, Germany) as previously described (12).

Quantification of interleukin-6, interleukin-10, and thrombin-antithrombin complexes

Interleukin-6 (IL-6) and IL-10 were quantified using enzyme-linked immunosorbent assay–based Quantikine Mouse IL-6 and IL-10 Immunoassay kits (R&D Systems, Minneapolis, Minn), whereas thrombin-antithrombin (TAT) complex levels were quantified using the Enzygnost TAT Micro immunoassay (Siemens Healthcare Diagnostics Products, Marburg, Germany) according to the manufacturer’s protocol.

Determination of myeloperoxidase activity, alanine transaminase activity, and creatinine levels

Enzymatic myeloperoxidase (MPO) is found in azurophilic granules of polymorphonuclear cells and is indicative of neutrophil accumulation in inflamed tissues. Mice were subjected to sham or CLP surgery and administered either DNase or saline 6 h after surgery. Lung tissues were frozen in liquid nitrogen and stored at −80°C, and MPO enzymatic activity in the lungs was quantified. Lung tissue (0.050 g) was homogenized in ice-cold PBS and centrifuged at 10,000g for 10 min in 4°C. The supernatant was discarded, and the pellet was resuspended and homogenized in potassium phosphate buffer (K2HPO4 50 mM, pH 6.0) containing hexadecyltrimethylammonium bromide (0.5% wt/vol). After centrifugation at 3,000g for 15 min, the supernatant was collected and MPO enzymatic activity was quantified by measuring the H2O2-dependent oxidation of o-dianisidine. One unit (U) of MPO activity is defined as MPO levels per gram of lung tissue that caused a change in absorbance of 1.0/min at 450 nm. Plasma levels of creatinine and alanine transaminase (ALT) activity were quantified using commercially available assays (Abcam, Cambridge, Mass). Creatinine levels were expressed in millimoles per liter, and ALT activity was expressed in units per liter of plasma. One unit of ALT activity was defined as the amount of ALT that generates 1.0 μmol of pyruvate per minute at 37°C.

Bacterial load, organ histology, and staining procedures

In bacterial culture experiments, lungs were homogenized and peritoneal cavity fluid was collected. Fresh blood, lung lysates, and peritoneal cavity fluid diluted in phosphate-buffered saline (Sigma-Aldrich, St. Louis, Mo) were plated on 5% sheep blood agar plates (Teknova, Hollister, Calif) and incubated for 48 h (see Methods, Supplemental Digital Content 1, at https://links.lww.com/SHK/A298). In all other experiments, mice were perfused with saline and 10% neutral buffered formalin and organs were collected in formalin, embedded in paraffin wax, processed, and sectioned at 5 μm thick. Organ sections were stained with hematoxylin and eosin to visualize overall morphology (nuclei and extracellular DNA appear bluish-purple and cytoplasm, connective tissue, and collagen appear pink). Other sections were stained with phosphotungstic acid and hematoxylin (American MasterTech Scientific, Lodi, Calif) to identify fibrin and collagen (red blood cells [RBCs], fibrin, nuclei, and striated fibers appear blue and collagen appears brownish-red). Photomicrographs of stained lung and kidney sections were visualized under 400× magnification. The length of the scale bar represents 25 μm. Organ damage was assessed by clinical pathologists blinded to the surgery and treatment allocation. Organ sections were scored by evaluating the level of inflammatory cell infiltration, interstitial edema, and vascular congestion (from 0 representing healthy or absence of abnormal organ pathology up to 3 for severe organ damage indicated by significant inflammatory infiltration, distinct regions of vascular congestion, and cell necrosis). Additional details are provided in Methods, Supplemental Digital Content 1, at https://links.lww.com/SHK/A298.

Mortality studies

Mice were subjected to either sham or CLP surgery and monitored continuously until end point or 20 h after surgery. A separate cohort of mice was subjected to CLP surgery and given saline control, early DNase treatment (2 h after CLP, repeated injections every 2 h), or late DNase treatment (6 h after CLP, repeated injections every 6 h) and monitored continuously until end point of 20 h after surgery. All mice were given Ringer’s lactate and buprenorphine (0.1 mg/kg) subcutaneously every 4 h postoperatively.

Statistical analyses

Data are represented as mean ± SEM, and results across multiple groups were compared using one-way analysis of variance and Newman-Keuls post hoc test. Data were considered significant at P < 0.05. Analyses were performed using GraphPad Prism 4.0 (La Jolla, Calif) and SigmaPlot 11.0 (San Jose, Calif).

RESULTS

Increases in cfDNA accompany the early proinflammatory and procoagulant response in sepsis

To examine the temporal changes in cfDNA in a murine model of sepsis, mice were subjected to CLP or sham surgery and blood was collected 2, 4, 6, or 8 h postoperatively. A significant time-dependent increase in cfDNA levels in septic mice from 4 to 8 h after CLP surgery was observed (Fig. 1A). In septic mice, the increases in cfDNA levels were accompanied by a significant (P < 0.001) time-dependent increase in IL-6 (Fig. 1B), a decrease in IL-10 (Fig. 1C), and increases in TAT (Fig. 1D). In contrast, levels of cfDNA, IL-6, IL-10, and TAT complexes remained low in sham-operated mice.

F1-11
Fig. 1:
Time course of plasma cell-free DNA, IL-6, IL-10, and TAT complexes in sham- and CLP-operated mice. Mice were subjected to CLP or sham surgery. Blood was collected at 2-h increments after surgery. Plasma levels of cell-free DNA (A), IL-6 (B), IL-10 (C), and TAT complexes (D) were quantified and compared with levels observed in healthy control mice (analysis of variance, P < 0.001, *P < 0.05, **P < 0.01, ***P < 0.005; n = 4 – 8 sham, n = 8 – 12 CLP).

Histological staining was performed in the organs of septic mice harvested 6 h after CLP surgery (see Figure, Supplemental Digital Content 2, at https://links.lww.com/SHK/A299). Photomicrographs of hematoxylin and eosin–stained lung sections revealed edema in the interstitium and alveolar border and neutrophil infiltration. Phosphotungstic acid and hematoxylin staining showed intra-alveolar RBC congestion and thickening of the alveolar septum in the lungs of septic mice. These pathological characteristics were absent in healthy and sham-operated mice. Hematoxylin and eosin–stained kidney sections exhibited intense staining of tubular brush borders in sham-operated and healthy mice, which was lost in the kidney sections from septic mice. Tubular dilation and cell sloughing were also observed in the kidneys of septic mice, indicative of tubular necrosis and kidney injury. Phosphotungstic acid and hematoxylin stains of kidneys of septic mice confirmed intraglomerular and intertubular RBC trapping as well as RBC congestion in the glomeruli (see Figure, Supplemental Digital Content 2, at https://links.lww.com/SHK/A299).

Early administration of DNase 2 h after CLP surgery results in increased inflammation and organ damage

In mice subjected to CLP, administration of DNase 2 h after surgery resulted in a decrease in plasma levels of cfDNA (Fig. 2A), an increase in IL-6 (Fig. 2B) and IL-10 (Fig. 2C), and no significant changes in TAT levels (Fig. 2D) compared with CLP-operated mice given saline control. Deoxyribonuclease increased infiltration of inflammatory cells trapped within thickened alveolar borders in the lungs (see Figure, Supplemental Digital Content 3, at https://links.lww.com/SHK/A300; Fig. 3A). Kidneys from septic mice treated with DNase 2 h postoperatively showed increased intertubular and intraglomerular RBC congestion, dismantling of tubular structure, vacuolization of tubule centers, and inflammatory infiltration at the periphery of kidney tubules compared with septic mice given saline (see Figure, Supplemental Digital Content 4, at https://links.lww.com/SHK/A301; Fig. 3B). Organ histology scores for lungs and kidneys of septic mice given DNase 2 h after CLP were higher compared with septic mice given saline (Fig. 3). There were no significant differences in bacterial load in the whole blood, lungs, or peritoneal cavities with DNase or saline administration (Fig. 4). These findings suggest that early administration of DNase is associated with increased inflammation, RBC congestion, and exacerbated organ pathology compared with septic mice given saline.

F2-11
Fig. 2:
Plasma cell-free DNA, IL-6, IL-10, and TAT complexes in sham- and CLP-operated mice administered DNase 2, 4, and 6 h postoperatively. Mice were subjected to sham or CLP surgery and administered either DNase or saline 2, 4, or 6 h postoperatively. Blood was collected 2 h after DNase or saline administration, and levels of plasma cfDNA (A), IL-6 (B), IL-10 (C), and TAT complexes (D) were quantified (*P < 0.05, **P < 0.01, ***P < 0.005; n = 8 – 12 per sham group, n = 10 – 14 per CLP group).
F3-11
Fig. 3:
Histology scores, MPO activity, creatinine levels, and ALT activity of sham- and CLP-operated mice administered DNase or saline. Organs harvested from DNase- or saline-treated mice subjected to sham or CLP surgery were scored for organ damage by a blinded clinical pathologist. Lungs (A) and kidneys (B) were evaluated for inflammatory cell infiltration, interstitial edema, cell necrosis, and vascular congestion and given a score ranging from 0 to 3 inclusively (*P < 0.05, **P < 0.01, ***P < 0.005, n = 4 – 6 per sham group, n = 6 – 9 per CLP group). Organs from healthy control mice were given histology scores of 0. Mice were subjected to either CLP or sham surgery and given saline or DNase 2, 4, or 6 h after surgery. Lungs were harvested and snap frozen in liquid nitrogen, and enzyme activity levels of MPO were quantified (C). Plasma levels of creatinine (D) and enzyme activity of ALT (E) were determined (*P < 0.05, **P < 0.01, ***P < 0.005; n = 4 – 6 per sham group, n = 7 – 8 per CLP group).
F4-11
Fig. 4:
Bacterial culture counts from the lungs, blood, and peritoneal cavity fluid of CLP-operated mice administered DNase or saline. Mice were subjected to sham or CLP surgery and given DNase at 2, 4, or 6 h postoperatively. Two hours after DNase or saline administration, homogenized lung lysate (A), blood (B), and peritoneal cavity fluid (C) were collected and cultured on blood agar plates to determine the bacterial load in nonseptic and septic mice after administration of DNase or saline (*P < 0.05, **P < 0.01, ***P < 0.005; n = 5 – 8 per CLP + saline group, n = 8 – 12 per CLP + DNase group). No colony-forming units were observed in nonseptic mice administered DNase or saline (data not shown).

Delayed administration of DNase 4 or 6 h after CLP surgery reduces inflammation, organ damage, and bacterial dissemination

Administration of DNase 4 or 6 h after CLP surgery resulted in decreases in cfDNA (Fig. 2A) and IL-6 (Fig. 2B), increases in IL-10 (Fig. 2C), and modest decreases in TAT (Fig. 2D). Changes in plasma biomarkers were accompanied by attenuated organ damage in the lungs and kidneys (see Figure, Supplemental Digital Content 3, at https://links.lww.com/SHK/A300; Fig. 4). Lungs from septic mice treated with DNase showed decreased neutrophil infiltration, interstitial edema, interalveolar RBC congestion, fibrin deposition, and obstruction of pulmonary microvessels compared with septic mice given saline (see Figure, Supplemental Digital Content 3, at https://links.lww.com/SHK/A300; Fig. 3A). Kidneys of DNase-treated septic mice showed modest improvements in organ damage with a general resolution of tubular structure demarcated by the staining of tubular brush borders and decreases in intraglomerular and intertubular vascular congestion, nuclear dropoff, and tubular necrosis (see Figure, Supplemental Digital Content 4, at https://links.lww.com/SHK/A301; Fig. 3B). Lung MPO activity in septic mice given delayed DNase treatment was reduced by 38% compared with MPO activity in the lungs of septic mice administered saline (Fig. 3C). Creatinine levels were elevated in all septic mice versus nonseptic mice; however, no significant changes in creatinine levels between septic mice given saline versus DNase were observed (Fig. 3D). Alanine transaminase activity, a marker of liver injury, was increased in septic mice given saline and significantly reduced in septic mice given DNase when DNase was administered at 4 or 6 h after CLP surgery (Fig. 3E). Organ histology scores of lungs and kidneys were lower in septic mice given DNase versus saline when DNase was administered 4 or 6 h after CLP. A decreased bacterial load was also observed in the lungs (Fig. 4A), blood (Fig. 4B), and peritoneal cavity fluid (Fig. 4C) of septic mice given delayed administration of DNase. Taken together, these data suggest that an increased window between sepsis induction (via CLP surgery) and DNase treatment is associated with decreased inflammation, improved organ health, and decreased bacterial dissemination.

Administration of DNase to sham-operated mice exacerbates inflammation without inducing neutrophil accumulation

To determine the effects of DNase administration in the nonseptic condition, DNase was administered to mice 2, 4, or 6 h after sham surgery and blood was collected 2 h after DNase injection. Deoxyribonuclease administration in mice after sham surgery resulted in increased levels of cfDNA, IL-6, and IL-10 and no changes in TAT complexes (Fig. 2), MPO activity (Fig. 3C), creatinine levels (Fig. 3D), and ALT activity (Fig. 3E). Changes in organ pathology in these mice include pulmonary edema and vascular congestion in the lungs (see Figure, Supplemental Digital Content 3, at https://links.lww.com/SHK/A300) and microvascular congestion in the kidneys (see Figure, Supplemental Digital Content 4, at https://links.lww.com/SHK/A301) accompanied by increased organ damage scores when DNase was administered 4 or 6 h after sham surgery (Fig. 3A). These results suggest that administration of DNase to mice subjected to sham surgery results in increased inflammation in the lungs but no increase in neutrophil accumulation or organ injury.

Late DNase administration rescues septic mice from death

To determine whether DNase administration would improve outcome in our sepsis model, mice were subjected to CLP and given saline, early DNase treatment (every 2 h after CLP), or late DNase treatment (every 6 h after CLP). In our model, there is a 100% mortality rate at 20 h after CLP (vs. 0% in sham-operated mice; Fig. 5A). A total of 80% of mice given late DNase treatment survived compared with 0% in mice given saline control. In mice administered DNase every 2 h after CLP surgery, only 25% of mice survived (Fig. 5B). There was a significantly prolonged time to death in septic mice given late DNase treatment versus septic mice given saline control (21.10 ± 1.18 h DNase-treated vs. 14.39 ± 1.17 h saline control; Fig. 5C). Taken together, these findings indicate that repeated DNase administration after a period of sepsis progression significantly reduces mortality of septic mice.

F5-11
Fig. 5:
Mortality curves of sham-operated mice and CLP-operated mice given saline, early DNase, or late DNase treatment. Mice were subjected to sham or CLP surgery and monitored until end point. A separate group of mice was subjected to CLP surgery and given saline, early DNase treatment (2 h after CLP), or late DNase treatment (6 h after CLP) and monitored until end point. The number of hours from CLP surgery to death in each treatment group was documented (***P < 0.005; n = 5 – 8 per group).

DISCUSSION

Our studies suggest that delayed DNase treatment to septic mice exerts a protective effect. We have documented elevations in levels of cfDNA and markers of inflammation and coagulation from sepsis induction to 8 h after surgery (Fig. 1). In clinical and experimental sepsis, increases in IL-6 levels and decreases in IL-10 are indicative of the early inflammatory response and sustained increases of IL-6 correlate with poor outcome (23, 24). The IL-6 and IL-10 profiles in mice subjected to CLP are consistent with previous reports of an early and gradual rise in IL-6 levels and a transient increase in IL-10 levels during the 8- to 10-h period after the onset of sepsis (25). Elevations in cfDNA were associated with elevations in TAT, indicating activation of coagulation. Thrombin-antithrombin levels increased by 2 h after sepsis induction and remained elevated for 8 h (Fig. 1). The time course of these changes is consistent with previous studies in murine sepsis models (26) and with the results of clinical studies, which document increases in TAT levels in early sepsis and severe sepsis patients (27). Using plasma samples from severe sepsis patients, we recently showed that elevations in cfDNA correlate with increases in thrombin generation (13), consistent with the procoagulant effects of cfDNA. Using IL-6, IL-10, and TATs as markers of proinflammation, anti-inflammation, and coagulation, respectively, the current data suggest that increases in cfDNA accompany the early proinflammatory and procoagulant response in sepsis.

Increases in cfDNA and the early proinflammatory and procoagulant responses are associated with organ damage (see Figure, Supplemental Digital Content 2, at https://links.lww.com/SHK/A299). Septic mice with high cfDNA levels have significant accumulations of inflammatory cells and RBCs as well as intravascular fibrin deposition in the lungs and kidneys. In contrast, these pathological changes are absent in mice subjected to sham surgery with low levels of cfDNA. Clinical observations support a potential association between cfDNA and organ dysfunction in sepsis (11, 12), and we have shown that elevated levels of circulating cfDNA are strongly predictive of mortality in severe sepsis patients (12).

We sought to further investigate the relationship between cfDNA and sepsis pathophysiology by modifying levels of cfDNA with DNase. Deoxyribonuclease treatment was administered at doses based on pharmacokinetic and toxicology studies investigating the use of DNase in cystic fibrosis (28). The lowest dose of DNase at which a decrease in cfDNA levels was observed in mice was chosen for our studies (unpublished data). The half-life of rhDNaseI is short (7 – 25 min), and the rapid clearance of DNase from the circulation into highly perfused tissues (lung, liver, and kidney) occurs within 2 to 30 min (28). Deoxyribonuclease treatment after a prolonged period of sepsis progression (i.e., 4 or 6 h) resulted in reduced levels of circulating cfDNA, reduced proinflammatory IL-6, and increased levels of anti-inflammatory IL-10 (Fig. 2), suggesting that modulating cfDNA may alter the immune response in experimental sepsis. Deoxyribonuclease administration at this delayed time point also resulted in decreased pulmonary edema, fibrin deposition, microvessel obstruction, and vascular congestion in the end organs of septic mice (see Figures, Supplemental Digital Content 3 and 4, at https://links.lww.com/SHK/A300 and https://links.lww.com/SHK/A301; Fig. 3), reduced neutrophil accumulation in the lungs indicated by decreased MPO activity, reduced ALT (Fig. 3), decreased bacterial dissemination in the lungs, blood, and peritoneal cavity (Fig. 4), and improved outcome (Fig. 5), suggesting that late DNase treatment may attenuate inflammation and reverse lung and liver damage in sepsis.

The formation and release of NETs and NET-associated proteins in the vasculature have recently been documented in murine models of endotoxemia (29) and experimental sepsis (10, 18). It is possible that the degradation of cfDNA by DNase dismantles DNA traps, thereby attenuating NET-mediated inflammation and coagulation in the microvasculature and reducing the hyperinflammatory state and associated organ pathology in sepsis. It is also possible that the protective benefit of DNase is a result of alleviating inflammatory responses such as NET formation and inflammatory infiltration, which in turn decreases cfDNA levels, although it is not known whether cfDNA promotes inflammation.

Although delayed DNase treatment (4 or 6 h after CLP) reduced organ damage, DNase treatment at an earlier time point (2 h after CLP) resulted in increased inflammation (Fig. 2), organ damage (Fig. 3), and decreased survival (Fig. 5B). These findings support the hypothesis that NETs have a protective role in the early immune response to a septic insult but an injurious one when NETs are not cleared in later stages of severe sepsis. Removing NETs during the immediate immune response (within 2 h postoperatively) resulted in exacerbated organ pathology and earlier death (starting 5 h after CLP; Fig. 5B) possibly because NETs are required to ensnare and kill pathogens. This concept is supported by findings from groups that demonstrate early DNase administration at just 1 h after CLP surgery (with subsequent DNase treatments at 4, 7, 10, 21, 24, and 27 h postoperatively) resulted in increased bacterial dissemination and bacterial colony-forming units in the lungs, livers, and peritoneal cavities, increased serum IL-6, increased neutrophil recruitment, and decreased survival (14). Other groups have also reported similar findings of increased bacterial dissemination and organ damage resulting from early DNase administration (1 – 2 h after CLP surgery [30]). In contrast, Luo et al. (18) reported moderate improvements in lung pathology and modest decreases in bacterial load in septic mice given repeated doses of DNase after 24 h but not at 6 h after CLP. In contrast to our model, Luo et al. used a low-grade less severe model of CLP (punctures with 21 gauge) previously reported to result in decreased mortality (19) and no resuscitation procedure was described in this study (18). In our model, mice are resuscitated early after sepsis induction and at high fluid volumes, the benefits of which have been shown in clinical sepsis (31–33). Studies from both Meng et al. (10) and Luo et al. (18) report the effects of repeated DNase administration with injections beginning at 1 h after CLP, a time point that precedes any observable pathological changes in our experiments and may not be relevant for investigating the therapeutic potential of DNase in clinical sepsis. However, our studies show that DNase exerts protective effects when administered in mice after a prolonged period of sepsis progression (4 or 6 h after CLP), at a time point that is relevant to the experimental and clinical presentation of severe sepsis, as evidenced by inflammatory biomarkers, changes in organ pathology, increased time to death, and increased survival. Thus, the effects of removing NETs via DNase administration are time sensitive and seem effective when administered after a prolonged period of sepsis progression.

Interestingly, administration of DNase in sham-operated mice resulted in an increase in cfDNA, IL-6, and IL-10 levels (Fig. 2) and inflammation in the end organs (see Figures, Supplemental Digital Content 3 and 4, at https://links.lww.com/SHK/A300 and https://links.lww.com/SHK/A301; Fig. 3). These findings are consistent with previous pharmacological reports investigating the toxicology of rhDNase. Rats administered DNase intravenously exhibited a mild short-term inflammatory response, with pulmonary changes consistent with injection of a foreign protein (28). Deoxyribonuclease has also been shown to induce an initial proinflammatory response when administered in cystic fibrosis (34, 35). Deoxyribonuclease administration via the intravenous route induces a mild inflammatory response in highly perfused end organs.

Our studies show that DNase given 2 h postoperatively (before detectable changes in markers of inflammation and coagulation) increases inflammation and organ injury, and that a therapeutic effect is observed when DNase is administered 4 or 6 h postoperatively. This narrow therapeutic window suggests that, whereas DNase administration may have a therapeutic potential, there are limitations to DNase as an antisepsis therapy. The timing and use of DNase administration should be further investigated. In addition, we recognize that the use of antibiotics is not accounted for in this study and its use in future studies may alter mortality rates of septic mice administered DNase.

In summary, our studies are the first to demonstrate that delayed administration of DNase may be protective in experimental sepsis. The timing of DNase administration may be a crucial element in future investigations of the therapeutic potential of DNase in sepsis.

ACKNOWLEDGMENTS

The authors thank Drs Vinai Bhagirath and Mark Inman at McMaster University for their suggestions, guidance, and critical review of this work.

REFERENCES

1. Dellinger RP, Levy MM, Rhodes A, Annane D, Gerlach H, Opal SM, Sevransky JE, Sprung CL, Douglas IS, Jaeschke R, et al.: Surviving sepsis campaign: international guidelines for management of severe sepsis and septic shock: 2012. Crit Care Med 41: 580–637, 2013.
2. Angus DC, Linde-Zwirble WT, Lidicker J, Clermont G, Carcillo J, Pinsky MR: Epidemiology of severe sepsis in the United States: analysis of incidence, outcome, and associated costs of care. Crit Care Med 29: 1303–1310, 2001.
3. Wang HE, Shapiro NI, Griffin R, Safford MM, Judd S, Howard G: Chronic medical conditions and risk of sepsis. PLoS One 7: e48307, 2012.
4. Marshall JC: Why have clinical trials in sepsis failed? Trends Mol Med 20: 195–203, 2014.
5. Brinkmann V, Reichard U, Goosmann C, Fauler B, Uhlemann Y, Weiss DS, Weinrauch Y, Zychlinsky A: Neutrophil extracellular traps kill bacteria. Science 303: 1532–1535, 2004.
6. Papayannopoulos V, Metzler KD, Hakkim A, Zychlinsky A: Neutrophil elastase and myeloperoxidase regulate the formation of neutrophil extracellular traps. J Cell Biol 191: 677–691, 2010.
7. 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 9: 1795–1803, 2011.
8. Fuchs TA, Brill A, Duerschmied D, Schatzberg D, Monestier M, Myers DD Jr, Wrobleski SK, Wakefield TW, Hartwig JH, Wagner DD: Extracellular DNA traps promote thrombosis. Proc Natl Acad Sci U S A 107: 15880–15885, 2010.
9. Semeraro F, Ammollo CT, Morrissey JH, Dale GL, Friese P, Esmon NL, Esmon CT: Extracellular histones promote thrombin generation through platelet-dependent mechanisms: involvement of platelet TLR2 and TLR4. Blood 118: 1952–1961, 2011.
10. Meng W, Paunel-Görgülü A, Flohé S, Hoffmann A, Witte I, MacKenzie C, Baldus SE, Windolf J, Lögters TT: Depletion of neutrophil extracellular traps in vivo results in hypersusceptibility to polymicrobial sepsis in mice. Crit Care 16: R137, 2012.
11. Zeerleder S, Zwart B, Wuillemin WA, Aarden LA, Groeneveld AB, Caliezi C, van Nieuwenhuijze AE, van Mierlo GJ, Eerenberg AJ, Lämmle B, et al.: Elevated nucleosome levels in systemic inflammation and sepsis. Crit Care Med 31: 1947–1951, 2003.
12. Dwivedi DJ, Toltl LJ, Swystun LL, Pogue J, Liaw KL, Weitz JI, Cook DJ, Fox-Robichaud AE, Liaw PC, Canadian Critical Care Translational Biology Group: Prognostic utility and characterization of cell-free DNA in patients with severe sepsis. Crit Care 16: R151, 2012.
13. Gould TJ, Vu TT, Swystun LL, Dwivedi DJ, Mai SH, Weitz JI, Liaw PC: Neutrophil extracellular traps promote thrombin generation through platelet-dependent and platelet-independent mechanisms. Arterioscler Thromb Vasc Biol 34: 1977–1984, 2014.
14. Meng W, Paunel-Görgülü A, Flohé S, Witte I, Schädel-Höpfner M, Windolf J, Lögters TT: Deoxyribonuclease is a potential counter regulator of aberrant neutrophil extracellular traps formation after major trauma. Mediators Inflamm 2012: 149560, 2012.
15. Prince WS, Baker DL, Dodge AH, Ahmed AE, Chestnut RW, Sinicropi DV: Pharmacodynamics of recombinant human DNase I in serum. Clin Exp Immunol 113: 289–296, 1998.
16. Shak S: Aerosolized recombinant human DNase I for the treatment of cystic fibrosis. Chest 107(2 Suppl): 65S–70S, 1995.
17. Rahman NM, Maskell NA, West A, Teoh R, Arnold A, Mackinlay C, Peckham D, Davies CW, Ali N, Kinnear W, et al.: Intrapleural use of tissue plasminogen activator and DNase in pleural infection. N Engl J Med 365: 518–526, 2011.
18. Luo L, Zhang S, Wang Y, Rahman M, Syk I, Zhang E, Thorlacius H: Proinflammatory role of neutrophil extracellular traps in abdominal sepsis. Am J Physiol Lung Cell Mol Physiol 307(7): L586–L596, 2014. Available at: http://ajplung.physiology.org/content/307/7/L586. Accessed February 27, 2015.
19. Baker CC, Chaudry IH, Gaines HO, Baue AE: Evaluation of factors affecting mortality rate after sepsis in a murine cecal ligation and puncture model. Surgery 94: 331–335, 1983.
20. Hubbard WJ, Choudhry M, Schwacha MG, Kerby JD, Rue LW 3rd, Bland KI, Chaudry IH: Cecal ligation and puncture. Shock 24(Suppl 1): 52–57, 2005.
21. Kilkenny C, Browne WJ, Cuthill IC, Emerson M, Altman DG: Improving bioscience research reporting: the ARRIVE guidelines for reporting animal research. Osteoarthritis Cartilage 20: 256–260, 2012.
22. Mai S, Khan M, Liaw P, Fox-Robichaud A: Experimental sepsis models. In: Sepsis—An Ongoing and Significant Challenge. Azevedo L (ed.). 2012. Available at: http://www.intechopen.com/books/sepsis-an-ongoing-and-significant-challenge/experimental-sepsis-modelspdf. Accessed November 13, 2014.
23. Hack CE, De Groot ER, Felt-Bersma RJ, Nuijens JH, Strack Van Schijndel RJ, Eerenberg-Belmer AJ, Thijs LG, Aarden LA: Increased plasma levels of interleukin-6 in sepsis. Blood 74: 1704–1710, 1989.
24. Remick DG, Bolgos G, Copeland S, Siddiqui J: Role of interleukin-6 in mortality from and physiologic response to sepsis. Infect Immun 73: 2751–2757, 2005.
25. Remick DG, Newcomb DE, Bolgos GL, Call DR: Comparison of the mortality and inflammatory response of two models of sepsis: lipopolysaccharide vs. cecal ligation and puncture. Shock 13: 110–116, 2000.
26. Tucker EI, Verbout NG, Leung PY, Hurst S, McCarty OJ, Gailani D, Gruber A: Inhibition of factor XI activation attenuates inflammation and coagulopathy while improving the survival of mouse polymicrobial sepsis. Blood 119: 4762–4768, 2012.
27. Kinasewitz GT, Yan SB, Basson B, Comp P, Russell JA, Cariou A, Um SL, Utterback B, Laterre PF, Dhainaut JF, et al.: Universal changes in biomarkers of coagulation and inflammation occur in patients with severe sepsis, regardless of causative micro-organism [ISRCTN74215569]. Crit Care R82–R90, 2004.
28. Dayan AD: Pharmacological-toxicological expert report on recombinant human deoxyribonuclease I (rhDNase; Pulmozyme). Hum Exp Toxicol 13: S2–S42, 1994.
29. Clark SR, Ma AC, Tavener SA, McDonald B, Goodarzi Z, Kelly MM, Patel KD, Chakrabarti S, McAvoy E, Sinclair GD, et al.: Platelet TLR4 activates neutrophil extracellular traps to ensnare bacteria in septic blood. Nat Med 13: 463–469, 2007.
30. McDonald B, Urrutia R, Yipp BG, Jenne CN, Kubes P: Intravascular neutrophil extracellular traps capture bacteria from the bloodstream during sepsis. Cell Host Microbe 12: 324–333, 2012.
31. Rivers E, Nguyen B, Havstad S, Ressler J, Muzzin A, Knoblich B, Peterson E, Tomlanovich M, Early Goal-Directed Therapy Collaborative Group: Early goal-directed therapy in the treatment of severe sepsis and septic shock. N Engl J Med 345: 1368–1377, 2001.
32. ProCESS Investigators, Yealy DM, Kellum JA, Huang DT, Barnato AE, Weissfeld LA, Pike F, Terndrup T, Wang HE, Hou PC, et al.: A randomized trial of protocol-based care for early septic shock. N Engl J Med 370: 1683–1693, 2014.
33. Vincent JL, Gerlach H: Fluid resuscitation in severe sepsis and septic shock: an evidence-based review. Crit Care Med 32(11 Suppl): S451–S454, 2004.
34. Suri R, Marshall LJ, Wallis C, Metcalfe C, Bush A, Shute JK: Effects of recombinant human DNase and hypertonic saline on airway inflammation in children with cystic fibrosis. Am J Respir Crit Care Med 166: 352–355, 2002.
35. Shah PL, Scott SF, Knight RA, Marriott C, Ranasinha C, Hodson ME: In vivo effects of recombinant human DNase I on sputum in patients with cystic fibrosis. Thorax 51: 119–125, 1996.
Keywords:

Sepsis; multiple organ failure; inflammation; deoxyribonucleases; coagulation; therapy

Supplemental Digital Content

© 2015 by the Shock Society