Combination Therapy of 15-Epi-Lipoxin A4 With Antibiotics Protects Mice From Escherichia coli–Induced Sepsis* Tomomi Ueda, MD1; Koichi Fukunaga, MD, PhD2; Hiroyuki Seki, MD, PhD1; Jun Miyata, MD, PhD2; Makoto Arita, PhD3; Taku Miyasho, PhD4; Toru Obata, PhD5; Koichiro Asano, MD, PhD6; Tomoko Betsuyaku, MD, PhD2; Junzo Takeda, MD, PhD1
Objectives: Inflammation occurs along with infection during sepsis. 15-Epi-lipoxin A4 has protective and resolving effects in experimental models of infection. In this study, we examined the effects of 15-epi-lipoxin A4 combined with antibiotics on Escherichia coli–induced peritonitis. Design: Prospective experimental study. Setting: University research laboratory. Subjects: Male C57BL/6 mice. Interventions: Mice were injected with E. coli to induce peritonitis and were given either 15-epi-lipoxin A4 (1 μg/mouse) or placebo (saline) with antibiotics (ceftazidime). The effects of 15-epi-lipoxin A4 on peritoneal cell populations, bacterial burden, and cytokine production were assessed. Survival rates were observed for up to 7 days. In addition, we examined the effects of 15-epi-lipoxin A4 on peritoneal macrophages stimulated with lipopolysaccharide, CpG DNA, or live E. coli. Measurements and Main Results: Treatment with 15-epi-lipoxin A4 significantly reduced the number of neutrophils in the peritoneum, inhibited production of cytokines and chemokines, and decreased bacterial load in the serum. Combined treatment *See also p. 1012. 1 Department of Anesthesiology, Keio University School of Medicine, Tokyo, Japan. 2 Pulmonary Division, Department of Medicine, Keio University School of Medicine, Tokyo, Japan. 3 Department of Health Chemistry, Graduate School of Pharmaceutical Sciences, University of Tokyo, Tokyo, Japan. 4 School of Veterinary Medicine, Rakuno Gakuen University, Hokkaido, Japan. 5 Department of Surgery, Shiga University of Medical Science, Shiga, Japan. 6 Division of Pulmonary Medicine, Department of Medicine, Tokai University School of Medicine, Kanagawa, Japan. Dr. Asano received grant support from Novartis and Merck Sharp and Dohme (MSD) and lectured for GlaxoSmithKline, MSD, and Astellas Pharma. The remaining authors have disclosed that they do not have any potential conflicts of interest. For information regarding this article, E-mail: [email protected] Copyright © 2014 by the Society of Critical Care Medicine and Lippincott Williams & Wilkins DOI: 10.1097/CCM.0000000000000162
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of 15-epi-lipoxin A4 with antibiotics significantly improved survival in E. coli–infected mice. 15-Epi-lipoxin A4 also attenuated the production of interleukin-6 and tumor necrosis factor-α by lipopolysaccharide- or CpG DNA-stimulated peritoneal macrophages. Furthermore, 15-epi-lipoxin A4 combined with antibiotics synergistically reduced the production of interleukin-6 and tumor necrosis factor-α by peritoneal macrophages stimulated with live E. coli. Conclusions: 15-Epi-lipoxin A4 combined with antibiotics attenuated systemic inflammation, inhibited bacteria dissemination, and improved survival in E. coli–infected mice. The reduced production of interleukin-6 and tumor necrosis factor-α by peritoneal macrophages suggested that 15-epi-lipoxin A4 blocked the initial proinflammatory response. Taken together, these data suggested that 15-epi-lipoxin A4 combined with antibiotics was beneficial in regulating the proinflammatory response in sepsis without exacerbating infection. (Crit Care Med 2014; 42:e288–e295) Key Words: anti-inflammatory lipid mediators; combination drug therapy; lipoxin A4; mortality; peritonitis; sepsis
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epsis is a complex clinical syndrome characterized by systemic inflammatory responses caused by the host’s excessive reaction to severe infection (1). Despite the development of powerful antibiotics and advances in the management of intensive care patients, mortality and morbidity in sepsis remain high (2). Administration of antibiotics is one of the essential treatments for sepsis (3). However, in severe sepsis, bactericidal or bacteriostatic agents cannot adequately inhibit the release of microbial constituents, such as lipopolysaccharide (LPS) or unmethylated CpG dinucleotides of microbial DNA sequences (CpG), which contribute to the expansion of systemic inflammation (4, 5). Furthermore, overuse of antibiotics increases the occurrence of drug-resistant bacteria, which is a growing concern, especially in the ICU. The dampening of systemic inflammatory response syndrome (SIRS) is considered favorable in the treatment of April 2014 • Volume 42 • Number 4
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sepsis. However, inadequate inhibition of the inflammatory response could disrupt the ability of the immune system to eliminate invading pathogens (6, 7). Therefore, it is necessary to develop new strategies to control systemic inflammatory responses without impairing the host antimicrobial defense system. Recently, resolution of inflammation has been redefined as an active process, where anti-inflammatory lipid mediators, such as lipoxins, resolvins, and protectins, termed “specialized proresolving mediators” (SPMs), are biosynthesized (8, 9). During infection, specific SPMs are temporally and differentially regulated and enhance the capacity of bacterial clearance by accelerating resolution (10, 11). Lipoxin A4 (LxA4), a lipid mediator derived from arachidonic acid, is a well-characterized SPM, biosynthesized by interactions between platelets and neutrophils or the conversion of 15S-hydroxy-eicosatetraenoic acid (HETE) released from activated monocytes and epithelial cells by neutrophils (12, 13). The properties of LxA4 are mediated via the specific G-protein-coupled receptors, ALX and GPR32 (14). 15-Epi-LxA4 is an isomer generated from 15R-HETE, which is synthesized from arachidonate by aspirin-acetylated cyclooxygenase-2. In experimental murine models, LxA4 has beneficial effects in acute lung injury (15, 16) and bronchial asthma (17). The protective effects of LxA4 have been reported in a rat model of peritonitis induced by cecal ligation and puncture (18). In humans, LxA4 is also thought to play a significant role in inflammatory disease, such as severe asthma. A retrospective cohort study on the use of aspirin in patients with SIRS and sepsis showed a potential benefit of 15-epi-LxA4 in survival (19). In the present study, we assessed the role of 15-epi-LxA4 in a murine model of sepsis induced by Escherichia coli peritonitis. Using this model, we showed that 15-epi-LxA4 had a synergetic effect with antibiotics in controlling sepsis by decreasing the production of inflammatory cytokines, decreasing the recruitment of neutrophils to the inflammatory site, enhancing microbial clearance, and improving survival. We also showed in in vitro studies that 15-epi-LxA4 decreased the production of interleukin (IL)-6 and tumor necrosis factor (TNF)-α
B
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Animals Six- to 8-week-old male C57BL/6J mice weighing 18–22 g were obtained from Charles River Laboratories Japan (Yokohama, Japan). Mice were given free access to water and standard rodent chow and were housed in pathogen-free cages. All animal experiments were approved by the Animal Care and Use Committees of the Keio University School of Medicine. E. coli–Induced Peritonitis Mice were infected with 5 × 107 colony forming units (CFUs) of E. coli (ATCC 19138 American Type Culture Collection, Manassas, VA) to induce peritonitis as previously described (20). Fifteen minutes later, 15-epi-LxA4 (50 μg/kg; Cayman, Ann Arbor, MI) or saline (0.9%) as a vehicle control was administered intraperitoneally, and 20 mg/kg of ceftazidime (Glaxo Smith Kline, Middlesex, United Kingdom) was administered IV 1 hour after bacterial challenge. Four or eight hours (n = 9–18 mice per group) after bacterial challenge, mice were euthanized using 50 mg/kg pentobarbital sodium. The peritoneal cavity was washed with ice-cold phosphate-buffered saline (PBS). Peritoneal lavage fluid was pooled and centrifuged at 400 × g for 5 minutes at 4°C, and the supernatant was stored at –80°C until use. The cell pellet was resuspended in cold saline, and cell numbers were counted using a hematocytometer. Differential cell counts were performed using cytocentrifuged smears stained with Diff-Quik (Sysmez, Kobe, Japan). Blood was collected by cardiac puncture and centrifuged at 500 × g for 15 minutes to obtain plasma. For observation of survival, mice infected with E. coli (5 × 107 CFU/mouse) were treated with ceftazidime (20 mg/kg) and 15-epi-LxA4 (50 μg/kg) at 1 and 9 hours after the infection (n = 26 mice per group). Survival was observed over 7 days.
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MATERIALS AND METHODS
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from E. coli–infected macrophages treated with antibiotics. We report for the first time that 15-epi-LxA4 may potentiate the effects of antibiotics in sepsis, in part, through its direct modulatory effect on macrophages.
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Figure 1. Effects of 15-epi-lipoxin A4 (15-epi-LxA4) on leukocyte populations after Escherichia coli infection. Fifteen minutes after E. coli infection, 15-epi-LxA4 (50 μg/kg) or vehicle was administered intraperitoneally, and after 1 hr, ceftadizime (20 mg/kg) was administered. Peritoneal fluids were collected 4 hr after infection. The numbers of neutrophils (A), macrophages (B), and total leukocytes (C) were analyzed. Data are expressed as the mean ± sem (n = 3–9 mice/group). *p < 0.05 compared with uninfected mice; **p < 0.05 compared with vehicle-treated mice.
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Bacterial Load A 1:10 dilution of blood and peritoneal fluid was made and plated on blood agar. After incubation for 24 hours at 37°C, the number of colonies was counted. Endotoxin Assay Plasma endotoxin levels were measured using the endotoxin scattering photometry (ESP) method as previously described (21). Plasma samples were diluted 100 times with ESP sample dilution buffer www.ccmjournal.org
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Effect of 15-Epi-Lipoxin A4 Treatment on Systemic Inflammatory Response After Escherichia coli Injection
Table 1.
Escherichia coli
–
+
+
15-epi-LxA4
–
–
+
Serum (pg/mL) IL-1β
150.9 ± 49.0
1,105.7 ± 201.1a
439.5 ± 119.8b
IL-6
ND
1,239.4 ± 185.0a
13.2 ± 8.7b
256.6 ± 88.3
665.3 ± 93.8a
339.9 ± 68.0b
13.9 ± 1.2
434.6 ± 108.6a
127.8 ± 37.2b
173.9 ± 53.7
87,858.9 ± 38,359.4a
11,807.9 ± 4,536.9b
36.9 ± 12.8
92.4 ± 16.1a
59.9 ± 8.0b
TNF-α Macrophage inflammatory protein-1α Monocyte chemotactic protein-1 Interferon-γ IL-10
148.6 ± 44.4
1,488.0 ± 229.4a
628.0 ± 155.0b
29.6 ± 7.6a
1.7 ± 1.0b
17.4 ± 2.2a
1.7 ± 1.5b
Peritoneal lavage fluid (pg/mL) IL-6
0.1 ± 0.0 ND
TNF-α
IL = interleukin, ND = not detected, TNF = tumor necrosis factor. a p < 0.05 versus uninfected mice. b p < 0.05 versus vehicle-treated mice. Values are in pg/mL and represent the mean ± sem (n = 15–18 in each group).
and heated for 10 minutes at 70°C. An aliquot of the plasma was mixed with Limulus amebocyte lysate reagent (Single Test Wako ES, Wako Pure Chemical, Tokyo, Japan). The ESP reaction was observed using an aggregometer (PA-200, Kowa, Tokyo, Japan).
(1 × 105 CFU/mL) and ceftadizime (100 μg/mL) or saline (0.9%) were administered. After the specified time, the supernatant was collected to measure the production of IL-6 and TNF-α. No bacterial growth was seen in ceftadizime-treated cells after 3 hours of incubation.
Cell Culture and Treatment Peritoneal exudate macrophages were obtained from mice 48 hours after the intraperitoneal injection of 1.0 mL of 4% sterile thioglycollate broth (Sigma Life Science, St. Louis, MO). Peritoneal exudate cells were harvested and resuspended in RPMI 1640 culture medium supplemented with 10% heat-inactivated fetal bovine serum. Additionally, 100 U/mL penicillin and 100 μg/mL streptomycin were added to the culture medium. In experiments using viable bacteria, antibiotics were not added to the culture medium during the isolation, washing, or subsequent culturing period. We did not observe bacterial contamination in macrophages cultured in the absence of antibiotics. Cells were counted and plated in 24-well cell culture plates (Costar, Cambridge, MA) at an approximate density of 0.5 × 106 cells/well. Cells were incubated in a 5% Co2 humidified culture incubator at 37°C for 2 hours to allow macrophages to adhere to the plates. Nonadherent cells were removed by washing twice with PBS. Peritoneal macrophages were treated with 15-epi-LxA4 (1 μM) or ethanol as a vehicle control for 30 minutes, and LPS (100 ng/mL) from E. coli O55:B5 (Sigma-Aldrich, St. Louis, MO) or CpG (0.1 μM; HyCult Biotechnology, Plymouth, MA) was administered. In experiments using live bacteria, peritoneal macrophages were treated with 15-epi-LxA4 (1 μM) or saline (0.9%) as a vehicle control for 30 minutes, and then E. coli
Cytokine Analysis The concentrations of IL-6 and TNF-α were determined by enzyme-linked immunosorbent assay (R&D Systems, Minneapolis, MN), according to the manufacturer’s instructions. For the measurement of IL-1β, IL-10, monocyte chemotactic protein (MCP)-1, and macrophage inflammatory protein (MIP)-1α, a multiplex cytokine bead array system (Bio-Plex; Bio-Rad, Hercules, CA) was used.
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Statistical Analysis All data were expressed as means ± sems. Comparisons between groups were conducted using analysis of variance, Student t tests, or Wilcoxon rank-sum tests. Survival curves after E. coli infection were estimated using the Kaplan-Meier method and compared using the log-rank test. The resulting p values of less than 0.05 (95% CI) were considered significant. Statistics were analyzed using Graphpad Prism 4.0 for Windows (San Diego, CA).
RESULTS 15-Epi-LxA4 Inhibited Neutrophil Infiltration into the Peritoneum in E. coli–Induced Peritonitis To examine the effects of 15-epi-LxA4 on leukocyte infiltration during the early phases of E. coli–induced peritonitis, April 2014 • Volume 42 • Number 4
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A
B
Blood
Peritoneum
*
* 100 Bacterial count (10 CFU/ml)
7.5
75
2
Bacterial count (10 2 CFU/ml)
10.0
5.0
2.5
0.0
vehicle
15-epi-LxA4
50
25
0
vehicle
15-epi-LxA4
Endotoxin
C 1500
**
pg/ml
1000
500
*
0 E.coli
-
+
+
15-epi-LxA4
-
-
+
Figure 2. Effects of 15-epi-lipoxin A4 on bacterial growth in the blood and peritoneum, and serum endotoxin concentration 8 hr after Escherichia coli infection. 15-Epi-LxA4 (50 μg/ kg) or vehicle was administered intraperitoneally 15 min after, and 1 hr later, ceftadizime (20 mg/ kg) was administered. Peritoneal fluid and serum were obtained 8 hr after E. coli infection. Bacterial growth in the blood (A) and the peritoneum (B) were analyzed. (c) Endotoxin concentration in the serum 8 hr after E. coli infection. n = 10–18 mice/group. *p < 0.05 compared to vehicle-treated mice; **p < 0.05 compared to uninfected mice.
the number of total inflammatory cells was determined, and differential cell analysis in the peritoneal lavage fluid was performed 4 hours after E. coli administration. As shown in Figure 1A, intraperitoneal treatment with 15-epi-LxA4 reduced the infiltration of neutrophils, as compared to that observed in vehicle-treated mice. A reduction in the number of macrophages was observed in both vehicle- and 15-epi-LxA4-treated mice when compared with uninfected mice (Fig. 1B). 15-Epi-LxA4 Attenuated Systemic Inflammation in E. coli–Induced Peritonitis To further investigate the systemic effects of 15-epi-LxA4 in E. coli–induced peritonitis, levels of cytokines and chemokines were measured. The serum and peritoneal lavage fluid were obtained 8 hours after E. coli infection because the maximum production of inflammatory cytokines in serum and peritoneal lavage fluid was observed at 8 hours (data not shown). Treatment with 15-epi-LxA4 significantly reduced serum levels of IL-6, TNF-α, IL-1β, IL-10, MCP-1, and MIP-1α (Table 1). IL-6 and TNF-α were also decreased in the peritoneal lavage fluid of E. coli–infected mice when treated with 15-epiLXA4 (Table 1). Critical Care Medicine
15-Epi-LxA4 Treatment Reduced Bacterial Load and Endotoxin Release During E. coli Infection Because the reduction in neutrophils and cytokine production, as seen in the present study, often leads to exacerbation of infection (22), we examined the effects of 15-epi-LxA4 on bacterial growth in the peritoneal cavity and blood 8 hours after E. coli infection. In both the blood and peritoneal cavity, there were significant reductions in bacterial growth in the 15-epi-LxA4-treated group compared with the vehicletreated group (Fig. 2, A and B). To investigate the effects of 15-epi-LxA4 on endotoxin release, we measured the serum endotoxin concentration 8 hours after E. coli infection. An increase in endotoxin levels was observed 8 hours after E. coli injection, and treatment with 15-epi-LxA4 significantly reduced serum endotoxin levels compared to treatment with vehicle (Fig. 2C).
15-Epi-LxA4 Reduced the Production of IL-6 and TNF-α in Peritoneal Macrophages Stimulated With LPS or CpG Next, we sought to determine the role of peritoneal macrophages in E. coli–induced peritonitis because macrophages are the dominant population in the peritoneal cavity in an aseptic environment. We examined the effects of 15-epi-LxA4 on the production of IL-6 and TNF-α in peritoneal macrophages by ex vivo stimulation with LPS or CpG. In LPS-stimulated macrophages, pretreatment with 15-epi-LxA4 decreased the production of IL-6 and TNF-α (Fig. 3A). In CpG-stimulated macrophages, 15-epi-LxA4 also reduced the production of TNF-α (Fig. 3B). Combination Treatment With 15-epi-LxA4 and Antibiotics Reduced the Production of IL-6 and TNF-α in Peritoneal Macrophages Stimulated With Live E. coli To further investigate the therapeutic potential of this modality for the treatment of sepsis, antibiotics were administered simultaneously with E. coli to observe whether there were any beneficial effects of 15-epi-LxA4 in combination with antibiotics on E. coli–infected macrophages. E. coli–treated macrophages failed to survive for up to 6 www.ccmjournal.org
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A
of 15-epi-LxA4 and antibiotics further reduced IL-6 and TNF-α production (Fig. 4). These findings indicated that 15-epi-LxA4 treatment could synergistically reduce cytokine production in combination with antibiotics.
LPS 6000 TNF- α(pg/ml)
IL-6 (pg/ml)
12000 8000
*
4000
4000
*
2000
N.D.
N.D.
15-Epi-LxA4 and Antibiotic Treatment Improved + Survival in a Murine + 15-epi-LxA4 Peritonitis Model To investigate the contribuB CpG tion of 15-epi-LxA4 and anti10000 6000 biotics to survival during E. coli–induced peritonitis, either 7500 15-epi-LxA4 or vehicle was 4000 administered with ceftadizime 5000 * after injection of E. coli. When 2000 2500 mice were not treated with antibiotics, all of them died N.D. N.D. 0 0 within 24 hours after E. coli + CpG + + + infection. When 15-epi-LxA4 + + 15-epi-LxA4 and ceftadizime were given 1 and 9 hours after infection, Figure 3. Effects of 15-epi-lipoxin A4 on the production of interleukin (IL)-6 and tumor necrosis factor (TNF)-α after 7 days, 23% of the antibiby peritoneal macrophages. Harvested peritoneal macrophages were incubated with 15-epi-LxA4 (1 μM) or otic-treated mice (n = 26) were vehicle for 30 min and were stimulated with lipopolysaccharide (LPS) (100 ng/mL) (A) or CpG (0.1 μM) (B) for 3 hr, and supernatants were collected. Data are presented as the mean ± sem (n = 3 in each group). alive compared with 54% of *p < 0.05 compared to vehicle-treated cells. The results presented were obtained in three identical independent mice in the combination therexperiments. ND = not detected. apy group (n = 26) (Fig. 5), and a significant improvement in survival was observed (p < hours after infection without cotreatment with antibiotics; therefore, cytokines were measured at 3 hours after E. 0.05 vs the control group). These results demonstrated that 15-epi-LxA4 enhanced survival in E. coli–induced peritonitis coli administration. 1 5-Epi-LxA4 alone did not reduce the when combined with antibiotic treatment. production of IL-6 or TNF-α. However, treatment with antibiotics significantly reduced the production of IL-6 and TNF-α compared to the untreated cells. The combination DISCUSSION -
0
+
+
-
+
+
IL-6 (pg/ml)
TNFα (pg/ml)
0 LPS
Figure 4. Effects of 15-epi-lipoxin A4 (15-epi-LxA4) on the production of interleukin (IL)-6 and tumor necrosis factor (TNF)-α after 3 hr of incubation with Escherichia coli and ceftadizime. Harvested peritoneal macrophages were incubated with 15-epi-LxA4 (1 μM) or vehicle for 30 min, infected with E. coli, and treated with ceftadizime (100 μg/mL or saline). Supernatants were collected 3 hr later. Values are the mean ± sem (n = 3–4 in each group). *p < 0.05 versus E. coli (+), ceftadizime (–), and 15-epi-LxA4 (–); **p < 0.05 versus E. coli (+), ceftadizime (+), and 15-epi-LxA4 (–). The results presented were obtained in three identical independent experiments.
Suppressing the initial proinflammatory response in sepsis leads to a better prognosis, but exacerbation of infection due to excessive anti-inflammatory and immunosuppressive treatment is also well recognized (23, 24). These facts emphasize the need for modulation of the proinflammatory response with adequate antiphlogistic agents. In the present study, we demonstrated the effects of treatment with 15-epi-LxA4 in combination with antibiotics in an E. coli–induced peritonitis model. We showed that treatment with 15-epiLxA4 plus antibiotics inhibited
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in sepsis and also shorten the duration of the immunosuppressive state vehicle + ceftadizime by reducing the production of IL-10, 15-epi-LxA 4 + ceftadizime 75 which may have contributed to the improvement in survival. * In infections caused by gram-neg50 ative bacteria, major constituents such as LPS or CpG DNA activate Toll25 like receptor (TLR)-4 or TLR-9 and initiate the inflammatory response (37–39). Furthermore, LxA4 has been 0 shown to decrease TNF-α expression 0 2 4 6 by inhibiting nuclear factor-κB sigdays naling in RAW264.7 cells and human macrophages (28, 40). Consistent Figure 5. Effects of 15-epi-lipoxinA4 (15-epi-LxA4) on survival in Escherichia coli–induced peritonitis. Mice were treated IV with ceftadizime and vehicle (black square) or 15-epi-LxA4 (white pyramid) at 1 with this, in our study, 15-epi-LxA4 and 9 hr after E. coli infection. *p < 0.05 versus the vehicle-treated group. suppressed the production of IL-6 neutrophil infiltration, promoted the clearance of bacteria, and TNF-α from murine peritoneal macrophages stimulated with LPS or CpG, suggesting that 15-epi-LxA4 may counterregdecreased proinflammatory mediators, and improved survival ulate both TLR-4- and TLR-9-mediated signaling. When macin E. coli–infected mice. rophages were cocultured with live E. coli, 15-epi-LxA4 failed In general, inhibition of neutrophil infiltration to the infecto attenuate IL-6 and TNF-α production. However, when maction foci impedes bacterial clearance and aggravates infection (25, 26). However, in our study, treatment with 15-epi-LxA4 rophages were cocultured with live E. coli in the presence of decreased the number of neutrophils in the peritoneum, as antibiotics, 15-epi-LxA4 synergistically reduced IL-6 and TNFreported previously (27), but simultaneously reduced the α production. In our preliminary studies, 15-epi-LxA4 itself did not show antibacterial properties and, as stated previously, bacterial load. The decrease in the number of neutrophils can be explained by both inhibition of recruitment and enhance- did not enhance bacterial phagocytosis by peritoneal macroment of apoptosis by 15-epi-LxA4, as previously reported (28). phages. Proinflammatory cytokines can be secreted not only by 15-Epi-LxA4 itself does not possess bactericidal effects, and we LPS or CpG stimuli but also by direct phagocytosis of E. coli by were not able to clarify the precise mechanisms mediating the macrophages, which activates the NFκB pathway via a different pathway from TLRs (41, 42). Therefore, it is plausible that in antimicrobial effect of 15-epi-LxA4. Phagocytosis of E. coli was not enhanced by 15-epi-LxA4 in primary murine macrophages cells not treated with antibiotics, 15-epi-LxA4 was not able to inhibit bacterial growth and cytokine production triggered by in our study (data not shown), although it was enhanced in human monocytes in previous studies (29, 30). In humans, direct phagocytosis of E. coli. These observations are the first indication that 15-epi-LxA4 and antibiotics synergistically reg15-epi-LxA4 stimulates the antimicrobial activity of the epithelia (31), but this phenomenon is not observed in mice. ulate proinflammatory cytokine production by macrophages. Importantly, in the current study, we observed a signifiFurther studies are needed to reveal the antibacterial effects of cant survival benefit in mice injected with the combination 15-epi-LxA4 in mice. of 15-epi-LxA4 and antibiotics compared to antibiotics alone. During gram-negative bacterial infections, release of endotoxin initiates the production of proinflammatory cytokines. Our findings implied that 15-epi-LxA4 contributed to improvIncreases in the expression of proinflammatory cytokines, ing survival by decreasing the bacterial load and attenuating the primary proinflammatory response, which prevented the including IL-6, IL-1β, and TNF-α, contribute to the disease severity (32–35). The production of MCP-1, a CC chemo- further development of sepsis. Our in vitro studies showed that repeated administration of 15-epi-LxA4 abrogated the detrikine that is also produced during sepsis, is associated with mental effects induced by bacterial components such as LPS increased mortality (36). In the current study, administraand CpG and synergistically attenuated inflammation during tion of 15-epi-LxA4 dramatically inhibited the production of inflammatory mediators, such as IL-1β, IL-6, TNF-α, MCP-1, the later stages of sepsis. Our data suggested that 15-epi-LxA4 and MIP-1α. Our results suggested that both the reduction in could enhance the effects of antibiotics in sepsis and possibly the release of endotoxin and the decrease in the production shorten the antibiotic treatment period, which would prevent the occurrence of new drug-resistant pathogens. of IL-6 and TNF-α by 15-epi-LxA4 in pathogen-stimulated In the present study, we were able to show that 15-epiperitoneal macrophages resulted in diminished systemic cytoLxA4 improved survival, attenuated the inflammatory kine production. In the current study, we also observed a mild response, and enhanced bacterial clearance when adminisdecrease in the production of IL-10 following treatment with tered with antibiotics. However, we were not able to explain 15-epi-LxA4, and we suspect that this was the result of suppression of the proinflammatory response. Our results implied that the mechanism through which 15-epi-LxA4 enhanced bac15-epi-LxA4 can attenuate the initial inflammatory response terial clearance, and further studies are needed. Also in survival rate (%)
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the clinical setting, immunosuppression is subsequently observed in sepsis, and the magnitude of suppression affects the clinical outcome. Our current model of E. coli–induced peritonitis failed to reproduce this phenomenon, and further studies are needed to elucidate the possible role of 15-epiLxA4 in this phase of sepsis.
CONCLUSIONS In conclusion, 15-epi-LxA4 exhibited potent anti-inflammatory effects without concomitant immunosuppressive effects, and administration of 15-epi-LxA4 with antibiotics counterregulated the inflammatory response both in vivo and in vitro. Antibacterial effects occurring with anti-inflammatory effects distinguish 15-epi-LxA4 from other adjuvant therapies, such as corticosteroids and antibody-targeted therapies, in the treatment of sepsis. In sepsis, combination therapy of 15-epiLxA4 and antibiotics may represent a new and effective treatment to control infection-initiated systemic inflammation.
ACKNOWLEDGMENTS We thank Shizuko Kagawa and Miyuki Yamamoto for outstanding technical assistance.
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inflammation: Stimulation of macrophage phagocytosis of apoptotic neutrophils in vivo. J Am Soc Nephrol 2002; 13:2497–2507 14. Chiang N, Arita M, Serhan CN: Anti-inflammatory circuitry: Lipoxin, aspirin-triggered lipoxins and their receptor ALX. Prostaglandins Leukot Essent Fatty Acids 2005; 73:163–177 15. Jin SW, Zhang L, Lian QQ, et al: Posttreatment with aspirin-triggered lipoxin A4 analog attenuates lipopolysaccharide-induced acute lung injury in mice: The role of heme oxygenase-1. Anesth Analg 2007; 104:369–377 16. Fukunaga K, Kohli P, Bonnans C, et al: Cyclooxygenase 2 plays a pivotal role in the resolution of acute lung injury. J Immunol 2005; 174:5033–5039 17. Levy BD, Lukacs NW, Berlin AA, et al: Lipoxin A4 stable analogs reduce allergic airway responses via mechanisms distinct from CysLT1 receptor antagonism. FASEB J 2007; 21:3877–3884 18. Walker J, Dichter E, Lacorte G, et al: Lipoxin a4 increases survival by decreasing systemic inflammation and bacterial load in sepsis. Shock 2011; 36:410–416 19. Eisen DP, Reid D, McBryde ES: Acetyl salicylic acid usage and mortality in critically ill patients with the systemic inflammatory response syndrome and sepsis. Crit Care Med 2012; 40:1761–1767 20. Christ WJ, Asano O, Robidoux AL, et al: E5531, a pure endotoxin antagonist of high potency. Science 1995; 268:80–83 21. Obata T, Nomura M, Kase Y, et al: Early detection of the Limulus amebocyte lysate reaction evoked by endotoxins. Anal Biochem 2008; 373:281–286 22. Rios-Santos F, Benjamim CF, Zavery D, et al: A critical role of leukotriene B4 in neutrophil migration to infectious focus in cecal ligation and puncture sepsis. Shock 2003; 19:61–65 23. Adib-Conquy M, Cavaillon JM: Compensatory anti-inflammatory response syndrome. Thromb Haemost 2009; 101:36–47 24. Muenzer JT, Davis CG, Chang K, et al: Characterization and modulation of the immunosuppressive phase of sepsis. Infect Immun 2010; 78:1582–1592 25. Kasten KR, Prakash PS, Unsinger J, et al: Interleukin-7 (IL-7) treatment accelerates neutrophil recruitment through gamma delta T-cell IL-17 production in a murine model of sepsis. Infect Immun 2010; 78:4714–4722 26. Craciun FL, Schuller ER, Remick DG: Early enhanced local neutrophil recruitment in peritonitis-induced sepsis improves bacterial clearance and survival. J Immunol 2010; 185:6930–6938 27. Clish CB, O’Brien JA, Gronert K, et al: Local and systemic delivery of a stable aspirin-triggered lipoxin prevents neutrophil recruitment in vivo. Proc Natl Acad Sci U S A 1999; 96:8247–8252 28. Godson C, Mitchell S, Harvey K, et al: Cutting edge: Lipoxins rapidly stimulate nonphlogistic phagocytosis of apoptotic neutrophils by monocyte-derived macrophages. J Immunol 2000; 164:1663–1667 29. Chiang N, Fredman G, Bäckhed F, et al: Infection regulates pro-resolving mediators that lower antibiotic requirements. Nature 2012; 484:524–528 30. Prescott D, McKay DM: Aspirin-triggered lipoxin enhances macrophage phagocytosis of bacteria while inhibiting inflammatory cytokine production. Am J Physiol Gastrointest Liver Physiol 2011; 301:G487–G497 31. Campbell EL, Serhan CN, Colgan SP: Antimicrobial aspects of inflammatory resolution in the mucosa: A role for proresolving mediators. J Immunol 2011; 187:3475–3481 32. Fisher CJ Jr, Dhainaut JF, Opal SM, et al: Recombinant human interleukin 1 receptor antagonist in the treatment of patients with sepsis syndrome. Results from a randomized, double-blind, placebo-controlled trial. Phase III rhIL-1ra Sepsis Syndrome Study Group. JAMA 1994; 271:1836–1843 33. Abraham E, Wunderink R, Silverman H, et al: Efficacy and safety of monoclonal antibody to human tumor necrosis factor alpha in patients with sepsis syndrome. A randomized, controlled, double-blind, multicenter clinical trial. TNF-alpha MAb Sepsis Study Group. JAMA 1995; 273:934–941 34. Blackwell TS, Christman JW: Sepsis and cytokines: Current status. Br J Anaesth 1996; 77:110–117 April 2014 • Volume 42 • Number 4
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39. Sparwasser T, Miethke T, Lipford G, et al: Bacterial DNA causes septic shock. Nature 1997; 386:336–337 40. Kure I, Nishiumi S, Nishitani Y, et al: Lipoxin A(4) reduces lipopolysaccharide-induced inflammation in macrophages and intestinal epithelial cells through inhibition of nuclear factor-kappaB activation. J Pharmacol Exp Ther 2010; 332:541–548 41. Maiti G, Naskar D, Sen M: The Wingless homolog Wnt5a stimulates phagocytosis but not bacterial killing. Proc Natl Acad Sci U S A 2012; 109:16600–16605 42. Kim J, Kim J, Kim DW, et al: Wnt5a induces endothelial inflammation via beta-catenin-independent signaling. J Immunol 2010; 185:1274–1282
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Objectives: Inflammation occurs along with infection during sepsis. 15-Epi-lipoxin A4 has protective and resolving effects in experimental models of infection. In this study, we examined the effects of 15-epi-lipoxin A4 combined with antibiotics on Escherichia coli–induced peritonitis. Design: Prospective experimental study. Setting: University research laboratory. Subjects: Male C57BL/6 mice. Interventions: Mice were injected with E. coli to induce peritonitis and were given either 15-epi-lipoxin A4 (1 μg/mouse) or placebo (saline) with antibiotics (ceftazidime). The effects of 15-epi-lipoxin A4 on peritoneal cell populations, bacterial burden, and cytokine production were assessed. Survival rates were observed for up to 7 days. In addition, we examined the effects of 15-epi-lipoxin A4 on peritoneal macrophages stimulated with lipopolysaccharide, CpG DNA, or live E. coli. Measurements and Main Results: Treatment with 15-epi-lipoxin A4 significantly reduced the number of neutrophils in the peritoneum, inhibited production of cytokines and chemokines, and decreased bacterial load in the serum. Combined treatment *See also p. 1012. 1 Department of Anesthesiology, Keio University School of Medicine, Tokyo, Japan. 2 Pulmonary Division, Department of Medicine, Keio University School of Medicine, Tokyo, Japan. 3 Department of Health Chemistry, Graduate School of Pharmaceutical Sciences, University of Tokyo, Tokyo, Japan. 4 School of Veterinary Medicine, Rakuno Gakuen University, Hokkaido, Japan. 5 Department of Surgery, Shiga University of Medical Science, Shiga, Japan. 6 Division of Pulmonary Medicine, Department of Medicine, Tokai University School of Medicine, Kanagawa, Japan. Dr. Asano received grant support from Novartis and Merck Sharp and Dohme (MSD) and lectured for GlaxoSmithKline, MSD, and Astellas Pharma. The remaining authors have disclosed that they do not have any potential conflicts of interest. For information regarding this article, E-mail: [email protected] Copyright © 2014 by the Society of Critical Care Medicine and Lippincott Williams & Wilkins DOI: 10.1097/CCM.0000000000000162
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of 15-epi-lipoxin A4 with antibiotics significantly improved survival in E. coli–infected mice. 15-Epi-lipoxin A4 also attenuated the production of interleukin-6 and tumor necrosis factor-α by lipopolysaccharide- or CpG DNA-stimulated peritoneal macrophages. Furthermore, 15-epi-lipoxin A4 combined with antibiotics synergistically reduced the production of interleukin-6 and tumor necrosis factor-α by peritoneal macrophages stimulated with live E. coli. Conclusions: 15-Epi-lipoxin A4 combined with antibiotics attenuated systemic inflammation, inhibited bacteria dissemination, and improved survival in E. coli–infected mice. The reduced production of interleukin-6 and tumor necrosis factor-α by peritoneal macrophages suggested that 15-epi-lipoxin A4 blocked the initial proinflammatory response. Taken together, these data suggested that 15-epi-lipoxin A4 combined with antibiotics was beneficial in regulating the proinflammatory response in sepsis without exacerbating infection. (Crit Care Med 2014; 42:e288–e295) Key Words: anti-inflammatory lipid mediators; combination drug therapy; lipoxin A4; mortality; peritonitis; sepsis
S
epsis is a complex clinical syndrome characterized by systemic inflammatory responses caused by the host’s excessive reaction to severe infection (1). Despite the development of powerful antibiotics and advances in the management of intensive care patients, mortality and morbidity in sepsis remain high (2). Administration of antibiotics is one of the essential treatments for sepsis (3). However, in severe sepsis, bactericidal or bacteriostatic agents cannot adequately inhibit the release of microbial constituents, such as lipopolysaccharide (LPS) or unmethylated CpG dinucleotides of microbial DNA sequences (CpG), which contribute to the expansion of systemic inflammation (4, 5). Furthermore, overuse of antibiotics increases the occurrence of drug-resistant bacteria, which is a growing concern, especially in the ICU. The dampening of systemic inflammatory response syndrome (SIRS) is considered favorable in the treatment of April 2014 • Volume 42 • Number 4
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sepsis. However, inadequate inhibition of the inflammatory response could disrupt the ability of the immune system to eliminate invading pathogens (6, 7). Therefore, it is necessary to develop new strategies to control systemic inflammatory responses without impairing the host antimicrobial defense system. Recently, resolution of inflammation has been redefined as an active process, where anti-inflammatory lipid mediators, such as lipoxins, resolvins, and protectins, termed “specialized proresolving mediators” (SPMs), are biosynthesized (8, 9). During infection, specific SPMs are temporally and differentially regulated and enhance the capacity of bacterial clearance by accelerating resolution (10, 11). Lipoxin A4 (LxA4), a lipid mediator derived from arachidonic acid, is a well-characterized SPM, biosynthesized by interactions between platelets and neutrophils or the conversion of 15S-hydroxy-eicosatetraenoic acid (HETE) released from activated monocytes and epithelial cells by neutrophils (12, 13). The properties of LxA4 are mediated via the specific G-protein-coupled receptors, ALX and GPR32 (14). 15-Epi-LxA4 is an isomer generated from 15R-HETE, which is synthesized from arachidonate by aspirin-acetylated cyclooxygenase-2. In experimental murine models, LxA4 has beneficial effects in acute lung injury (15, 16) and bronchial asthma (17). The protective effects of LxA4 have been reported in a rat model of peritonitis induced by cecal ligation and puncture (18). In humans, LxA4 is also thought to play a significant role in inflammatory disease, such as severe asthma. A retrospective cohort study on the use of aspirin in patients with SIRS and sepsis showed a potential benefit of 15-epi-LxA4 in survival (19). In the present study, we assessed the role of 15-epi-LxA4 in a murine model of sepsis induced by Escherichia coli peritonitis. Using this model, we showed that 15-epi-LxA4 had a synergetic effect with antibiotics in controlling sepsis by decreasing the production of inflammatory cytokines, decreasing the recruitment of neutrophils to the inflammatory site, enhancing microbial clearance, and improving survival. We also showed in in vitro studies that 15-epi-LxA4 decreased the production of interleukin (IL)-6 and tumor necrosis factor (TNF)-α
B
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Animals Six- to 8-week-old male C57BL/6J mice weighing 18–22 g were obtained from Charles River Laboratories Japan (Yokohama, Japan). Mice were given free access to water and standard rodent chow and were housed in pathogen-free cages. All animal experiments were approved by the Animal Care and Use Committees of the Keio University School of Medicine. E. coli–Induced Peritonitis Mice were infected with 5 × 107 colony forming units (CFUs) of E. coli (ATCC 19138 American Type Culture Collection, Manassas, VA) to induce peritonitis as previously described (20). Fifteen minutes later, 15-epi-LxA4 (50 μg/kg; Cayman, Ann Arbor, MI) or saline (0.9%) as a vehicle control was administered intraperitoneally, and 20 mg/kg of ceftazidime (Glaxo Smith Kline, Middlesex, United Kingdom) was administered IV 1 hour after bacterial challenge. Four or eight hours (n = 9–18 mice per group) after bacterial challenge, mice were euthanized using 50 mg/kg pentobarbital sodium. The peritoneal cavity was washed with ice-cold phosphate-buffered saline (PBS). Peritoneal lavage fluid was pooled and centrifuged at 400 × g for 5 minutes at 4°C, and the supernatant was stored at –80°C until use. The cell pellet was resuspended in cold saline, and cell numbers were counted using a hematocytometer. Differential cell counts were performed using cytocentrifuged smears stained with Diff-Quik (Sysmez, Kobe, Japan). Blood was collected by cardiac puncture and centrifuged at 500 × g for 15 minutes to obtain plasma. For observation of survival, mice infected with E. coli (5 × 107 CFU/mouse) were treated with ceftazidime (20 mg/kg) and 15-epi-LxA4 (50 μg/kg) at 1 and 9 hours after the infection (n = 26 mice per group). Survival was observed over 7 days.
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Figure 1. Effects of 15-epi-lipoxin A4 (15-epi-LxA4) on leukocyte populations after Escherichia coli infection. Fifteen minutes after E. coli infection, 15-epi-LxA4 (50 μg/kg) or vehicle was administered intraperitoneally, and after 1 hr, ceftadizime (20 mg/kg) was administered. Peritoneal fluids were collected 4 hr after infection. The numbers of neutrophils (A), macrophages (B), and total leukocytes (C) were analyzed. Data are expressed as the mean ± sem (n = 3–9 mice/group). *p < 0.05 compared with uninfected mice; **p < 0.05 compared with vehicle-treated mice.
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Bacterial Load A 1:10 dilution of blood and peritoneal fluid was made and plated on blood agar. After incubation for 24 hours at 37°C, the number of colonies was counted. Endotoxin Assay Plasma endotoxin levels were measured using the endotoxin scattering photometry (ESP) method as previously described (21). Plasma samples were diluted 100 times with ESP sample dilution buffer www.ccmjournal.org
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Effect of 15-Epi-Lipoxin A4 Treatment on Systemic Inflammatory Response After Escherichia coli Injection
Table 1.
Escherichia coli
–
+
+
15-epi-LxA4
–
–
+
Serum (pg/mL) IL-1β
150.9 ± 49.0
1,105.7 ± 201.1a
439.5 ± 119.8b
IL-6
ND
1,239.4 ± 185.0a
13.2 ± 8.7b
256.6 ± 88.3
665.3 ± 93.8a
339.9 ± 68.0b
13.9 ± 1.2
434.6 ± 108.6a
127.8 ± 37.2b
173.9 ± 53.7
87,858.9 ± 38,359.4a
11,807.9 ± 4,536.9b
36.9 ± 12.8
92.4 ± 16.1a
59.9 ± 8.0b
TNF-α Macrophage inflammatory protein-1α Monocyte chemotactic protein-1 Interferon-γ IL-10
148.6 ± 44.4
1,488.0 ± 229.4a
628.0 ± 155.0b
29.6 ± 7.6a
1.7 ± 1.0b
17.4 ± 2.2a
1.7 ± 1.5b
Peritoneal lavage fluid (pg/mL) IL-6
0.1 ± 0.0 ND
TNF-α
IL = interleukin, ND = not detected, TNF = tumor necrosis factor. a p < 0.05 versus uninfected mice. b p < 0.05 versus vehicle-treated mice. Values are in pg/mL and represent the mean ± sem (n = 15–18 in each group).
and heated for 10 minutes at 70°C. An aliquot of the plasma was mixed with Limulus amebocyte lysate reagent (Single Test Wako ES, Wako Pure Chemical, Tokyo, Japan). The ESP reaction was observed using an aggregometer (PA-200, Kowa, Tokyo, Japan).
(1 × 105 CFU/mL) and ceftadizime (100 μg/mL) or saline (0.9%) were administered. After the specified time, the supernatant was collected to measure the production of IL-6 and TNF-α. No bacterial growth was seen in ceftadizime-treated cells after 3 hours of incubation.
Cell Culture and Treatment Peritoneal exudate macrophages were obtained from mice 48 hours after the intraperitoneal injection of 1.0 mL of 4% sterile thioglycollate broth (Sigma Life Science, St. Louis, MO). Peritoneal exudate cells were harvested and resuspended in RPMI 1640 culture medium supplemented with 10% heat-inactivated fetal bovine serum. Additionally, 100 U/mL penicillin and 100 μg/mL streptomycin were added to the culture medium. In experiments using viable bacteria, antibiotics were not added to the culture medium during the isolation, washing, or subsequent culturing period. We did not observe bacterial contamination in macrophages cultured in the absence of antibiotics. Cells were counted and plated in 24-well cell culture plates (Costar, Cambridge, MA) at an approximate density of 0.5 × 106 cells/well. Cells were incubated in a 5% Co2 humidified culture incubator at 37°C for 2 hours to allow macrophages to adhere to the plates. Nonadherent cells were removed by washing twice with PBS. Peritoneal macrophages were treated with 15-epi-LxA4 (1 μM) or ethanol as a vehicle control for 30 minutes, and LPS (100 ng/mL) from E. coli O55:B5 (Sigma-Aldrich, St. Louis, MO) or CpG (0.1 μM; HyCult Biotechnology, Plymouth, MA) was administered. In experiments using live bacteria, peritoneal macrophages were treated with 15-epi-LxA4 (1 μM) or saline (0.9%) as a vehicle control for 30 minutes, and then E. coli
Cytokine Analysis The concentrations of IL-6 and TNF-α were determined by enzyme-linked immunosorbent assay (R&D Systems, Minneapolis, MN), according to the manufacturer’s instructions. For the measurement of IL-1β, IL-10, monocyte chemotactic protein (MCP)-1, and macrophage inflammatory protein (MIP)-1α, a multiplex cytokine bead array system (Bio-Plex; Bio-Rad, Hercules, CA) was used.
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Statistical Analysis All data were expressed as means ± sems. Comparisons between groups were conducted using analysis of variance, Student t tests, or Wilcoxon rank-sum tests. Survival curves after E. coli infection were estimated using the Kaplan-Meier method and compared using the log-rank test. The resulting p values of less than 0.05 (95% CI) were considered significant. Statistics were analyzed using Graphpad Prism 4.0 for Windows (San Diego, CA).
RESULTS 15-Epi-LxA4 Inhibited Neutrophil Infiltration into the Peritoneum in E. coli–Induced Peritonitis To examine the effects of 15-epi-LxA4 on leukocyte infiltration during the early phases of E. coli–induced peritonitis, April 2014 • Volume 42 • Number 4
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A
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0 E.coli
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Figure 2. Effects of 15-epi-lipoxin A4 on bacterial growth in the blood and peritoneum, and serum endotoxin concentration 8 hr after Escherichia coli infection. 15-Epi-LxA4 (50 μg/ kg) or vehicle was administered intraperitoneally 15 min after, and 1 hr later, ceftadizime (20 mg/ kg) was administered. Peritoneal fluid and serum were obtained 8 hr after E. coli infection. Bacterial growth in the blood (A) and the peritoneum (B) were analyzed. (c) Endotoxin concentration in the serum 8 hr after E. coli infection. n = 10–18 mice/group. *p < 0.05 compared to vehicle-treated mice; **p < 0.05 compared to uninfected mice.
the number of total inflammatory cells was determined, and differential cell analysis in the peritoneal lavage fluid was performed 4 hours after E. coli administration. As shown in Figure 1A, intraperitoneal treatment with 15-epi-LxA4 reduced the infiltration of neutrophils, as compared to that observed in vehicle-treated mice. A reduction in the number of macrophages was observed in both vehicle- and 15-epi-LxA4-treated mice when compared with uninfected mice (Fig. 1B). 15-Epi-LxA4 Attenuated Systemic Inflammation in E. coli–Induced Peritonitis To further investigate the systemic effects of 15-epi-LxA4 in E. coli–induced peritonitis, levels of cytokines and chemokines were measured. The serum and peritoneal lavage fluid were obtained 8 hours after E. coli infection because the maximum production of inflammatory cytokines in serum and peritoneal lavage fluid was observed at 8 hours (data not shown). Treatment with 15-epi-LxA4 significantly reduced serum levels of IL-6, TNF-α, IL-1β, IL-10, MCP-1, and MIP-1α (Table 1). IL-6 and TNF-α were also decreased in the peritoneal lavage fluid of E. coli–infected mice when treated with 15-epiLXA4 (Table 1). Critical Care Medicine
15-Epi-LxA4 Treatment Reduced Bacterial Load and Endotoxin Release During E. coli Infection Because the reduction in neutrophils and cytokine production, as seen in the present study, often leads to exacerbation of infection (22), we examined the effects of 15-epi-LxA4 on bacterial growth in the peritoneal cavity and blood 8 hours after E. coli infection. In both the blood and peritoneal cavity, there were significant reductions in bacterial growth in the 15-epi-LxA4-treated group compared with the vehicletreated group (Fig. 2, A and B). To investigate the effects of 15-epi-LxA4 on endotoxin release, we measured the serum endotoxin concentration 8 hours after E. coli infection. An increase in endotoxin levels was observed 8 hours after E. coli injection, and treatment with 15-epi-LxA4 significantly reduced serum endotoxin levels compared to treatment with vehicle (Fig. 2C).
15-Epi-LxA4 Reduced the Production of IL-6 and TNF-α in Peritoneal Macrophages Stimulated With LPS or CpG Next, we sought to determine the role of peritoneal macrophages in E. coli–induced peritonitis because macrophages are the dominant population in the peritoneal cavity in an aseptic environment. We examined the effects of 15-epi-LxA4 on the production of IL-6 and TNF-α in peritoneal macrophages by ex vivo stimulation with LPS or CpG. In LPS-stimulated macrophages, pretreatment with 15-epi-LxA4 decreased the production of IL-6 and TNF-α (Fig. 3A). In CpG-stimulated macrophages, 15-epi-LxA4 also reduced the production of TNF-α (Fig. 3B). Combination Treatment With 15-epi-LxA4 and Antibiotics Reduced the Production of IL-6 and TNF-α in Peritoneal Macrophages Stimulated With Live E. coli To further investigate the therapeutic potential of this modality for the treatment of sepsis, antibiotics were administered simultaneously with E. coli to observe whether there were any beneficial effects of 15-epi-LxA4 in combination with antibiotics on E. coli–infected macrophages. E. coli–treated macrophages failed to survive for up to 6 www.ccmjournal.org
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of 15-epi-LxA4 and antibiotics further reduced IL-6 and TNF-α production (Fig. 4). These findings indicated that 15-epi-LxA4 treatment could synergistically reduce cytokine production in combination with antibiotics.
LPS 6000 TNF- α(pg/ml)
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12000 8000
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15-Epi-LxA4 and Antibiotic Treatment Improved + Survival in a Murine + 15-epi-LxA4 Peritonitis Model To investigate the contribuB CpG tion of 15-epi-LxA4 and anti10000 6000 biotics to survival during E. coli–induced peritonitis, either 7500 15-epi-LxA4 or vehicle was 4000 administered with ceftadizime 5000 * after injection of E. coli. When 2000 2500 mice were not treated with antibiotics, all of them died N.D. N.D. 0 0 within 24 hours after E. coli + CpG + + + infection. When 15-epi-LxA4 + + 15-epi-LxA4 and ceftadizime were given 1 and 9 hours after infection, Figure 3. Effects of 15-epi-lipoxin A4 on the production of interleukin (IL)-6 and tumor necrosis factor (TNF)-α after 7 days, 23% of the antibiby peritoneal macrophages. Harvested peritoneal macrophages were incubated with 15-epi-LxA4 (1 μM) or otic-treated mice (n = 26) were vehicle for 30 min and were stimulated with lipopolysaccharide (LPS) (100 ng/mL) (A) or CpG (0.1 μM) (B) for 3 hr, and supernatants were collected. Data are presented as the mean ± sem (n = 3 in each group). alive compared with 54% of *p < 0.05 compared to vehicle-treated cells. The results presented were obtained in three identical independent mice in the combination therexperiments. ND = not detected. apy group (n = 26) (Fig. 5), and a significant improvement in survival was observed (p < hours after infection without cotreatment with antibiotics; therefore, cytokines were measured at 3 hours after E. 0.05 vs the control group). These results demonstrated that 15-epi-LxA4 enhanced survival in E. coli–induced peritonitis coli administration. 1 5-Epi-LxA4 alone did not reduce the when combined with antibiotic treatment. production of IL-6 or TNF-α. However, treatment with antibiotics significantly reduced the production of IL-6 and TNF-α compared to the untreated cells. The combination DISCUSSION -
0
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Figure 4. Effects of 15-epi-lipoxin A4 (15-epi-LxA4) on the production of interleukin (IL)-6 and tumor necrosis factor (TNF)-α after 3 hr of incubation with Escherichia coli and ceftadizime. Harvested peritoneal macrophages were incubated with 15-epi-LxA4 (1 μM) or vehicle for 30 min, infected with E. coli, and treated with ceftadizime (100 μg/mL or saline). Supernatants were collected 3 hr later. Values are the mean ± sem (n = 3–4 in each group). *p < 0.05 versus E. coli (+), ceftadizime (–), and 15-epi-LxA4 (–); **p < 0.05 versus E. coli (+), ceftadizime (+), and 15-epi-LxA4 (–). The results presented were obtained in three identical independent experiments.
Suppressing the initial proinflammatory response in sepsis leads to a better prognosis, but exacerbation of infection due to excessive anti-inflammatory and immunosuppressive treatment is also well recognized (23, 24). These facts emphasize the need for modulation of the proinflammatory response with adequate antiphlogistic agents. In the present study, we demonstrated the effects of treatment with 15-epi-LxA4 in combination with antibiotics in an E. coli–induced peritonitis model. We showed that treatment with 15-epiLxA4 plus antibiotics inhibited
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in sepsis and also shorten the duration of the immunosuppressive state vehicle + ceftadizime by reducing the production of IL-10, 15-epi-LxA 4 + ceftadizime 75 which may have contributed to the improvement in survival. * In infections caused by gram-neg50 ative bacteria, major constituents such as LPS or CpG DNA activate Toll25 like receptor (TLR)-4 or TLR-9 and initiate the inflammatory response (37–39). Furthermore, LxA4 has been 0 shown to decrease TNF-α expression 0 2 4 6 by inhibiting nuclear factor-κB sigdays naling in RAW264.7 cells and human macrophages (28, 40). Consistent Figure 5. Effects of 15-epi-lipoxinA4 (15-epi-LxA4) on survival in Escherichia coli–induced peritonitis. Mice were treated IV with ceftadizime and vehicle (black square) or 15-epi-LxA4 (white pyramid) at 1 with this, in our study, 15-epi-LxA4 and 9 hr after E. coli infection. *p < 0.05 versus the vehicle-treated group. suppressed the production of IL-6 neutrophil infiltration, promoted the clearance of bacteria, and TNF-α from murine peritoneal macrophages stimulated with LPS or CpG, suggesting that 15-epi-LxA4 may counterregdecreased proinflammatory mediators, and improved survival ulate both TLR-4- and TLR-9-mediated signaling. When macin E. coli–infected mice. rophages were cocultured with live E. coli, 15-epi-LxA4 failed In general, inhibition of neutrophil infiltration to the infecto attenuate IL-6 and TNF-α production. However, when maction foci impedes bacterial clearance and aggravates infection (25, 26). However, in our study, treatment with 15-epi-LxA4 rophages were cocultured with live E. coli in the presence of decreased the number of neutrophils in the peritoneum, as antibiotics, 15-epi-LxA4 synergistically reduced IL-6 and TNFreported previously (27), but simultaneously reduced the α production. In our preliminary studies, 15-epi-LxA4 itself did not show antibacterial properties and, as stated previously, bacterial load. The decrease in the number of neutrophils can be explained by both inhibition of recruitment and enhance- did not enhance bacterial phagocytosis by peritoneal macroment of apoptosis by 15-epi-LxA4, as previously reported (28). phages. Proinflammatory cytokines can be secreted not only by 15-Epi-LxA4 itself does not possess bactericidal effects, and we LPS or CpG stimuli but also by direct phagocytosis of E. coli by were not able to clarify the precise mechanisms mediating the macrophages, which activates the NFκB pathway via a different pathway from TLRs (41, 42). Therefore, it is plausible that in antimicrobial effect of 15-epi-LxA4. Phagocytosis of E. coli was not enhanced by 15-epi-LxA4 in primary murine macrophages cells not treated with antibiotics, 15-epi-LxA4 was not able to inhibit bacterial growth and cytokine production triggered by in our study (data not shown), although it was enhanced in human monocytes in previous studies (29, 30). In humans, direct phagocytosis of E. coli. These observations are the first indication that 15-epi-LxA4 and antibiotics synergistically reg15-epi-LxA4 stimulates the antimicrobial activity of the epithelia (31), but this phenomenon is not observed in mice. ulate proinflammatory cytokine production by macrophages. Importantly, in the current study, we observed a signifiFurther studies are needed to reveal the antibacterial effects of cant survival benefit in mice injected with the combination 15-epi-LxA4 in mice. of 15-epi-LxA4 and antibiotics compared to antibiotics alone. During gram-negative bacterial infections, release of endotoxin initiates the production of proinflammatory cytokines. Our findings implied that 15-epi-LxA4 contributed to improvIncreases in the expression of proinflammatory cytokines, ing survival by decreasing the bacterial load and attenuating the primary proinflammatory response, which prevented the including IL-6, IL-1β, and TNF-α, contribute to the disease severity (32–35). The production of MCP-1, a CC chemo- further development of sepsis. Our in vitro studies showed that repeated administration of 15-epi-LxA4 abrogated the detrikine that is also produced during sepsis, is associated with mental effects induced by bacterial components such as LPS increased mortality (36). In the current study, administraand CpG and synergistically attenuated inflammation during tion of 15-epi-LxA4 dramatically inhibited the production of inflammatory mediators, such as IL-1β, IL-6, TNF-α, MCP-1, the later stages of sepsis. Our data suggested that 15-epi-LxA4 and MIP-1α. Our results suggested that both the reduction in could enhance the effects of antibiotics in sepsis and possibly the release of endotoxin and the decrease in the production shorten the antibiotic treatment period, which would prevent the occurrence of new drug-resistant pathogens. of IL-6 and TNF-α by 15-epi-LxA4 in pathogen-stimulated In the present study, we were able to show that 15-epiperitoneal macrophages resulted in diminished systemic cytoLxA4 improved survival, attenuated the inflammatory kine production. In the current study, we also observed a mild response, and enhanced bacterial clearance when adminisdecrease in the production of IL-10 following treatment with tered with antibiotics. However, we were not able to explain 15-epi-LxA4, and we suspect that this was the result of suppression of the proinflammatory response. Our results implied that the mechanism through which 15-epi-LxA4 enhanced bac15-epi-LxA4 can attenuate the initial inflammatory response terial clearance, and further studies are needed. Also in survival rate (%)
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the clinical setting, immunosuppression is subsequently observed in sepsis, and the magnitude of suppression affects the clinical outcome. Our current model of E. coli–induced peritonitis failed to reproduce this phenomenon, and further studies are needed to elucidate the possible role of 15-epiLxA4 in this phase of sepsis.
CONCLUSIONS In conclusion, 15-epi-LxA4 exhibited potent anti-inflammatory effects without concomitant immunosuppressive effects, and administration of 15-epi-LxA4 with antibiotics counterregulated the inflammatory response both in vivo and in vitro. Antibacterial effects occurring with anti-inflammatory effects distinguish 15-epi-LxA4 from other adjuvant therapies, such as corticosteroids and antibody-targeted therapies, in the treatment of sepsis. In sepsis, combination therapy of 15-epiLxA4 and antibiotics may represent a new and effective treatment to control infection-initiated systemic inflammation.
ACKNOWLEDGMENTS We thank Shizuko Kagawa and Miyuki Yamamoto for outstanding technical assistance.
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