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Protection of Staphylococcus aureus-infected septic mice by suppression of early acute inflammation and enhanced antimicrobial activity by ginsan

Ji-Yeon Ahn, Jie-Young Song, Yeon-Sook Yun, Gajin Jeong, In-Soo Choi
DOI: http://dx.doi.org/10.1111/j.1574-695X.2005.00021.x 187-197 First published online: 1 March 2006

Abstract

Ginsan, an acidic polysaccharide prepared from Panax ginseng, demonstrated multiple immunomodulatory effects in previous studies. This study was conducted to elucidate the antiseptic mechanism induced by ginsan in mice infected with Staphylococcus aureus. When mice were treated with ginsan before the bacterial challenge with S. aureus, they were highly protected from sepsis-induced death. The numbers of S. aureus recovered from ginsan-treated mice were considerably lower than those recovered from nontreated mice. The in vivo depletion of monocytes/macrophages caused more S. aureus to be recovered from the bacteria-infected mice. Nevertheless, mice treated with both etoposide and ginsan were able to maintain an antibacterial activity. In addition, the phagocytic activity of ginsan-treated macrophage against S. aureus was considerably enhanced. The synthesis of inflammatory cytokines, such as tumor necrosis factor-α interleukin (IL)-1β, IL-6, IFN-γ, IL-12, IL-18 and interferon γ, was significantly downregulated at the early phase of sepsis in mice that were treated with ginsan before the bacterial challenge. Expression of Toll-like receptors (TLRs), including TLR2, TLR4, and TLR9, as well as the adaptor molecule MyD88, was considerably reduced in peritoneal macrophages that were treated with ginsan before a subsequent contact with S. aureus. These data indicated that ginsan protected mice from S. aureus-induced sepsis through the suppression of acute inflammatory responses at an early phase and the enhancement of antimicrobial activities at subsequent phases of infection.

Keywords
  • Panax ginseng
  • Staphylococcus aureus
  • sepsis
  • antibacterial activity
  • inflammation

Introduction

Sepsis is a severe and systemic illness caused by the excessive inflammatory response to microbial infections, often with fatal results. Although antibiotics are used to prevent and cure bacterial infections, the incidence of fatal cases of sepsis continues to increase because of the emergence of new antibiotic-resistant bacteria and inevitable nosocomial infections (Marshall et al., 1998; Wheeler & Bernard, 1999). According to a recent report, sepsis occurs in about 700 000 people and results in approximately 200 000 deaths in the United States each year (Angus et al., 2001).

Both Gram-negative and Gram-positive bacterial infections are capable of causing sepsis. Lipopolysaccharide (LPS) is known as a major sepsis-inducing component of the outer membrane of Gram-negative bacteria, such as Escherichia coli. In Gram-positive bacteria, peptidoglycan and lipoteichoic acid in the cell wall are suggested as possible candidates for sepsis-inducing materials (Wang et al., 2000). Staphylococcus aureus is the best-known cause of acute sepsis in Gram-positive bacteria. Due to its ability to produce exotoxins and to induce inflammatory cytokines from infected animals, S. aureus is considered an appropriate bacterium in the study of sepsis (Bone, 1994; Cohen, 2002). The bacterial peptidoglycan recognized by Toll-like receptor (TLR) 2 on monocytes/macrophages induces inflammatory responses by activating mitogen-activated protein kinases (MAPKs) and nuclear factor-κB (NFκB) (Underhill et al., 1999; Yoshimura et al., 1999; Ulloa et al., 2002; Chabaud-Riou & Firestein, 2004; Chen et al., 2004).

One of the important features of sepsis is the intense production of inflammatory cytokines, such as tumor necrosis factor-α (TNF-α), interleukin 1-β (IL-1β), and IL-6. These are major inflammatory mediators in sepsis induced by bacterial infections. TNF-α has been known as an important indicator of septic shock in animal models. In the presence of interferon (IFN)-γ, TNF-α synergistically induces lethality in animals by LPS-induced septic shock (Doherty et al., 1992; Smith et al., 1993). IL-12 and IL-18 ultimately induce the production of IFN-γ. This phenomenon was evident in a study of sepsis, in which the reduction of IL-12 or IL-18 synthesis diminished sepsis-induced death rates in animal models (Hultgren et al., 2001; Joshi et al., 2002). However, therapeutic application of inflammatory cytokine-specific antibodies, such as anti-TNF-α, for sepsis did not improve the survival of patients (Abraham et al., 1998).

Ginsan is a soluble acidic polysaccharide extracted from the roots of Panax Ginseng C. A. Meyer. It is mainly composed of β-(2→6)-fructofuranose and α-(1→6)-glucopyranose (Lee et al., 1997). Previous studies have shown that ginsan functioned as an effective biological response modifier (BRM). In particular, ginsan stimulated NK and T cells, produced multiple cytokines from activated immune cells, and induced tumoricidal and antimicrobial activities in macrophages (Song et al., 2002). The purpose of this study was to elucidate the ginsan-mediated antisepsis mechanism in S. aureus-infected mice. Ginsan induced anti-inflammatory responses and also enhanced the antibacterial activity that eventually prevented a fatal outcome in sepsis-induced animals.

Materials and methods

Experimental animals

All experiments made use of male BABL/c mice aged 5–7 weeks (18–22 g), purchased from the Charles River Breeding Laboratory (Charles River Japan, Inc., Atsugi Breeding Center, Yokohama, Japan). The mice were kept in the experimental facility at 60% humidity and 23±2°C, on a 12-h light–dark cycle. Five to 12 mice were randomly assigned to a specific experimental group depending on the purpose of each study. All animal experiments were performed in accordance with the guidelines set by the National Institute of Health, based on the protocols approved by the Institutional Animal Care and Use Committee of the Korea Institute of Radiological & Medical Sciences (KIRAMS).

Cell culture

BABL/c mouse-originated macrophage-like J774A.1 [American Type Culture Collection (ATCC)] TIP-67 cells were purchased from ATCC. The cells were cultured with Dulbecco's modified Eagle's medium (DMEM) (Gibco, Grand Island, NY) supplemented with 10% fetal bovine serum (JRH Biosciences, Lenexa, KA), 100 units mL−1 penicillin and 100 µg mL−1 streptomycin at 37°C in a humidified atmosphere with 5% CO2.

Bacteria

Staphylococcus aureus acquired from ATCC (ATCC 25923) were cultured on sheep's blood agar plate (Komed, Korea) for 24 h at 37°C to get a single colony of the bacteria. The selected colony was inoculated into tryptic soy broth (Difco, Sparks, MD) and incubated for 16 h in a shaker incubator at 37°C. The proliferated bacteria were collected by centrifugation at 4000 g for 10 min, and the bacterial pellet washed three times with phosphate-buffered saline (PBS). After determining the concentration of bacteria using a spectrophotometer, the number of bacteria was adjusted to 1.5 × 109 CFUs mL−1 in PBS. The mice were intraperitoneally injected with 1.5 × 108 CFUs of S. aureus.

Preparation of ginsan

Ginsan was purified from Panax ginseng C. A. Meyer (Araliaceae) in a soluble form, and the concentration of Ginsan was determined as described previously (Song et al., 2002). Routine quality controls of ginsan verified no contamination of endotoxin of Gram-negative bacteria or other microbial products in preparations of ginsan. Ginsan was stored as a dried powder at 4°C for future use. Ginsan was dissolved in PBS (pH 7.4) and filtered through 0.25 µm membranes (Millipore, Bedford, MA) just previously to being injected into the mice.

Etoposide and antibodies

Etoposide (Sigma, St Louis, MO) was used to selectively depopulate monocytes/macrophages in mice, as described in previous studies (Puliti et al., 2002). Before they were infected with S. aureus, the mice were subcutaneously injected for three consecutive days with 12.5 mg kg−1 etoposide that was resuspended in PBS containing 1% DMSO. The optimal dose of etoposide was administered at the optimal time based on the established practice set in previous studies (Calame et al., 1994; Verdrengh & Tarkowski, 2000; Puliti et al., 2002). Antibodies specific for c-Jun N-terminal kinase 1 (JNK1) and phosphorylated JNK1/2 were obtained from Santa Cruz Biotechnology (Santa Cruz, CA), while antibodies specific for p38 MAPK and phospho-p38 MAPK were acquired from Cell Signaling Technology (Cell Signaling, Beverly, MA).

Determination of cytokine levels

The concentrations of murine cytokines in sera, such as IFN-γ, IL-1β, IL-2, IL-4, IL-6, IL-10, IL-12, and TNF-α, were determined by enzyme-linked immunosorbent assay (ELISA) kits according to the manufacturers' protocols (R&D Systems, Minneapolis, MN).

Determination of CFUs

Blood, spleens, and kidneys were obtained from S. aureus-infected and control mice under aseptic conditions 24 h after bacterial infection. The spleens and kidneys were minced in 2.7 mL of PBS before being spread on the plate. After samples were diluted 300-fold in PBS, 0.1 mL was plated on the blood agar plates. The plates were incubated at 37°C for 24 h, after which the number of CFUs was calculated by counting the number of colonies.

Determination of phagocytic activity of macrophages

Fluorescence-isothiocynate-(FITC)-labeled S. aureus were prepared, as described by Peiser (2000). Briefly, S. aureus were cultured for 20 h at 37°C, and were inactivated for 90 min at 70°C. The heat-killed bacteria were labeled with 0.25% FITC for 12 h at 4°C, before fixed with 4% paraformaldehyde for 1 h at 4°C. The fixed and FITC-labeled S. aureus were washed four times in PBS before use. J774A.1 macrophages were pretreated with 0.1 µg mL−1 of ginsan for 3 h at 37°C in a humidified atmosphere with 5% CO2 before they were used. 5 × 106 macrophages were incubated with the heat-killed FITC-labeled S. aureus for 30 min at 37°C in a humidified atmosphere with 5% CO2. The cells were then placed on ice to prevent any reaction. The nonspecific extracellular fluorescence was quenched with 0.2% Trypan blue in PBS, as previously described (Ramet et al., 2002). The cells were analyzed with flow cytometry using CellQuest software (Becton Dickinson, Franklin Lakes, NJ).

Determination of expression of Toll-like receptors and MyD88 by RT-PCR

Total RNA was isolated from peritoneal macrophages of mice by using the Trizol reagent (Gibco BRL, Rockville, MD). One µg of RNA was reversely transcribed into cDNA for 1 h at 37°C, using 15 units of MMLV RT (Promega, Madison, WI) in the presence of 1 µL pd(N)6 random hexamer (Amersham Biotechnology AB, San Francisco, CA). The RT activity was inactivated by incubating the reaction tube for 5 min at 94°C. The synthesized cDNA was used as a template for the amplification of Toll-like receptors 2, 4, and 9, and MyD88 by PCR (Applied Biosystem, Foster City, CA). The reaction mixture consisted of 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 1.5 mM MgCl2, 2.5 mM deoxynucleoside triphosphates (Takara, Japan), 1.25 units of Taq DNA polymerase (Takara, Japan), and 0.05 mM of each gene primer set. The primers for amplifying murine TLR2, TLR4, TLR9, MyD88, and glyseraldehyde-3-phosphate dehydrogenase (GAPDH) were prepared (Bioneer, Daejeon, Korea). The sequences for TLR2 sense and antisense primers were 5′-CAAACTGGAGACTCTGGAAG-3′ and 5′-CTGTAGGAAACAAAGGCATC-3′, respectively. The sequences for TLR4 sense and antisense primers were 5′-GACACCCTCCATAGACTTCA-3′ and 5′-TGTTCAACATTCACCAAGAA-3′, respectively. The sequences for TLR9 sense and antisense primers were 5′-CCTGTCTCAAAATAACCTGC-3′ and 5′-CTGAGGTTGACCTCTTTCAG-3′, respectively. The sequences for MyD88 sense and antisense primers were 5′-CGATGCCTTTATCTGCTACT-3′ and 5′-TTCTTCATCGCCTTGTATTT-3′, respectively. The sequences for GAPDH sense and antisense primers were 5′-GAGTCTACTGGCGTCTTCAC-3′ and 5′-CCATCCACAG TCTTCTGAGT-3′, respectively. The predicted DNA sizes of amplified TLR2, TLR4, TLR9, MyD88, and GAPDH were 375, 266, 297, 303, and 285 bp, respectively. The conditions of the thermal cycle were 94°C for 1 min, 52°C to 58°C for 30 s, and 72°C for 30 s for 32 cycles. The PCR products were analyzed through electrophoresis using 1.5% agarose gel in a Tris-acetate-EDTA buffer supplemented with 0.005% ethidium bromide, and were quantified using an image analyzer (Bio-Rad, Hercules, CA).

Statistics analysis

The GraphPad Prism v3.02 software was used to conduct all statistical analyses (San Diego, CA). Each experimental group consisted of at least 10 animals. The significance of the differences between the groups was determined using one- or two-tailed Student's t-test. A value of P<0.05 was regarded as a statistical significance. The results were presented as mean±standard error of mean (SEM).

Results

Protection of mice by ginsan from acute septic death

To determine the optimal time for the administration of ginsan, the mice were intravenously injected, either once with a single dose of ginsan at 3, 24, or 48 h, or twice with the same dose at 3 and 24 h before they were challenged with Staphylococcus aureus. On day 6 post-infection (PI), the highest survival rate (83%) was observed in mice that were treated with a single dose of ginsan (0.025 mg kg−1) at 24 h prior to the bacterial infection with S. aureus (Fig. 1a). The mice that were treated with ginsan at 3 h before the bacterial challenge showed the lowest survival rate (58%) among the ginsan-treated mice, although this survival rate was still considerably higher than that of control mice (17%). To determine the optimal administration dose of ginsan, the mice were injected with 0.012, 0.025, 0.5, 25, and 250 mg kg−1 of ginsan 24 h before they were challenged with S. aureus. On day 6 PI, the mice injected with 0.025, 25, and 250 mg kg−1 of ginsan demonstrated the highest survival rates of 88% (Fig. 1b). In contrast, the control group of mice that were not treated with ginsan showed only a 10% survival rate. These results indicated that ginsan had a strong antiseptic activity at a broad range of concentrations with no or very low toxicity at a high dose. These results collectively suggested that ginsan played a significant role in protection of mice from S. aureus-induced septic death. The optimal dose (0.025 mg kg−1) and treatment time (24 h prior to bacterial infection) determined in this study were applied to the following experiments with some exceptions.

1

Protection of mice from Staphylococcus aureus-induced septic death by pre-treatment with ginsan. (a) The survival rates of S. aureus-infected mice were measured to determine the optimal time for the administration of ginsan before bacterial infection. The four groups of mice were administered with a single dose of ginsan (0.025 mg kg−1, intravenously) at 0, 3, 24, and 48 h before S. aureus infection (1.5 × 108 CFUs, intraperitoneally). The mice in the last group were given the same dose of ginsan twice at 3 and 24 h before S. aureus infection. *, P<0.003. (b) The survival rates of S. aureus-infected mice were measured to determine the optimal dose of ginsan. The mice were administered intravenously with various doses of ginsan at 24 h before bacterial infection; n=10–12 mice in each group. These data represent one of three repeated experiments that yielded similar results. *, P<0.003.

Enhanced in vivo bactericidal activity by ginsan

Prior to examining in vivo bactericidal activity induced by ginsan, we determined in vitro a direct bactericidal effect of ginsan. When the S. aureus was cultured in tryptic soy broth containing 250 mg mL−1 ginsan for 16 h at 37°C, no bactericidal activity was observed (data not shown). The mice treated with ginsan prior to the bacterial infection were sacrificed on day 4 PI to verify the enhanced bactericidal activity in vivo. The numbers of bacteria present in their spleens, kidneys, and blood were determined (Fig. 2a). The numbers of bacteria recovered from the ginsan-treated mice were significantly lower than those recovered from the control mice (Fig. 2b). The average CFUs of S. aureus detected in the spleens, the kidneys, and blood of ginsan-treated mice were 52-, 30-, and 9-fold less, respectively, than those of S. aureus determined in the nontreated control mice. These results demonstrated that ginsan enhanced the bactericidal activity in the ginsan-treated mice.

2

Enhanced bactericidal activity in Staphylococcus aureus-infected mice by pretreatment with ginsan. (a) Mice were treated with ginsan (0.025 mg kg−1, intravenously) 24 h before S. aureus infection (1.5 × 108 CFUs, intraperitoneally). The spleens, the kidneys, and blood were obtained from mice on day 4 after infection, and the bacteria in the organs were cultured for 20 h at 37°C on blood agar plates. (b) The number of bacteria in each sample was presented as CFUs mL−1. Significant antibacterial effect of ginsan on bacterial propagation was demonstrated in the three organs represented by P values of P<0.0001, 0.0004, and 0.0003 in the spleen, the kidney, and blood, respectively; n=5 mice in each group. These data represent one of three repeated experiments that yielded similar results. The data were expressed as mean±standard error of mean.

Monocyte/macrophage-mediated antibacterial activity in ginsan-treated mice

The mice were treated with etoposide, a well-known monocyte/macrophage-depopulating agent, to deplete the monocytes/macrophages in vivo. The etoposide-treated or ginsan-treated mice were infected with a sublethal dose of S. aureus. Compared to the 100% survival rate of ginsan-treated mice, the survival rate of etoposide-treated mice was 50% on day 7 PI (Fig. 3a). Furthermore, the survival rate (90%) of mice treated with both etoposide and ginsan was still much higher than that (50%) of mice treated only with etoposide before the bacterial challenge. These results suggested that monocytes/macrophages played an important role in controlling S. aureus in vivo, and that ginsan enhanced the resistance of etoposide-treated mice against lethal sepsis. The numbers of S. aureus found in the spleens and the kidneys of mice that were treated with ginsan prior to the bacterial challenge were considerably lower than those determined in the control mice that were only infected with the bacteria (Fig. 3b). The numbers of bacteria isolated from the two organs of mice that were pretreated with both etoposide and ginsan were significantly lower than those determined in mice that were treated with only etoposide before the bacterial infections (Fig. 3b). These data suggested that the pretreatment of mice with ginsan enhanced monocytes/macrophage-mediated antibacterial activity. In addition, these results indicated that ginsan might induce other immune cell-mediated antibacterial activity in the etoposide-treated mice.

3

Monocytes/macrophages-mediated antibacterial activity in ginsan-treated septic mice. (a) The survival rates of mice that were treated with ginsan alone, etoposide alone, or etoposide plus ginsan before Staphylococcus aureus infection were determined. The mice were subcutaneously injected with etoposide (12.5 mg kg−1) once a day for three consecutive days to deplete monocytes/macrophages. One group of the etoposide-treated mice was infected with a sublethal dose of S. aureus (1.0 × 108 CFUs mL−1), while the other etoposide-treated group was treated with ginsan (0.025 mg kg−1, intravenously) 24 h before the bacterial infection. The survival rates of the mice were observed for 7 days. Mice treated with etoposide plus ginsan demonstrated a significantly enhanced bacterial clearance, compared to mice treated with etoposide alone; n=10 in each group. (b) The numbers of S. aureus were determined in ginsan-treated mice after pretreatment of etoposide. The spleens and the kidneys were obtained from S. aureus-infected mice on day 2 after bacterial infection. The results from one of three repeated experiments were presented. The data were expressed as mean±standard error of mean. (*, P<0.005 when compared with the mice that were infected with S. aureus, and **, P<0.003, when compared with the mice that were infected with S. aureus after etoposide-treatment); n=10–12 mice in each group.

Enhanced in vitro phagocytic activity of monocyte/macrophage given by ginsan

The phagocytic activity of macrophages was investigated to verify whether it increased with ginsan treatment. The S. aureus killed by heat and labeled with FITC were added to the culture of ginsan-treated or nontreated J774A.1 cells. The ginsan-treated J774A.1 cells demonstrated an enhanced phagocytic activity against S. aureus compared to the nontreated control cells (Fig. 4). In particular, the ginsan-treated macrophages demonstrated 43% and 42% increases in phagocytic activity before and after trypan blue quenching, respectively. These data implied that the ginsan-mediated stimulation of monocytes/macrophages accelerated the antibacterial and phagocytic activities of macrophages.

4

Enhanced phagocytic activity of macrophages for Staphylococcus aureus by pretreatment with ginsan. Ginsan-treated (0.1 µg mL−1) or nontreated macrophage cell line J774A.1 cells were incubated with heat-inactivated and fluorescein isothiocyanate-labeled S. aureus for 10 min at 4°C, and then for 30 min at 37°C. The nonspecific binding of bacteria was quenched by the treatment of cells with 0.2% trypan blue. The phagocytic activities of the cells were determined through fluorescence activated cell sorter analysis, and were measured by mean fluorescent intensity. The results from one of three repeated experiments were presented.

Reduction of inflammatory cytokines at the early phase of sepsis by ginsan

Inflammatory cytokines are known as important mediators in development of lethal sepsis. To confirm this, the production profiles of inflammatory cytokines in ginsan-treated and subsequently sepsis-induced mice were evaluated for 24 h. The highest production of almost all cytokines examined in this study was observed at 8–12 h PI in mice (Fig. 5af). Mice infected with S. aureus abundantly produced typical inflammatory cytokines, such as TNF-α, IL-1β and IL-6. In contrast, the mice that were treated with ginsan 24 h before the bacterial infection produced considerably lower levels of the cytokines (Fig. 5ac). It is also well known that IFN-γ and TNF-α synergistically promote severe sepsis in animals and that IL-18 and IL-12 stimulate production of IFN-γ. Therefore, the high concentrations of IFN-γ, IL-12 and IL-18 in septic animals may indicate a poor prognosis of the disease. Given this basic fact, the effect of ginsan on the production of IFN-γ, IL-12, and IL-18 in septic mice was evaluated. The production of all three inflammatory cytokines dramatically decreased in mice that were treated with ginsan before S. aureus infection (Fig. 5df). In contrast to the downregulated synthesis of inflammatory cytokines in ginsan-treated septic mice, the production of IL-2, a typical Th 1 type cytokine, and IL-4, a typical Th2 type cytokine, neither increased nor decreased in all mice examined in this study (data not shown). These data indicated that ginsan significantly downregulated the production of inflammatory cytokines at the early phase of sepsis, which effectively diminished septic symptoms and led to higher survivals of mice.

5

Reduction of inflammatory cytokines in ginsan-treated septic mice. The mice were divided into four groups: (i) the nontreated control group, (ii) the ginsan-treated group (0.025 mg kg−1, intravenously), (iii) the Staphylococcus aureus-infected group, and (iv) the ginsan-treated group (0.025 mg kg−1, intravenously), before infection with S. aureus. Sera were collected from mice at 0, 2, 4, 8, 11, and 24 h post-infection in each group. The concentrations of inflammatory cytokines, such as (a) tumor nercosis factor-α, (b) interleukin (IL)-1β, (c) IL-6, (d) interferon-γ, (e) IL-12 and (f) IL-18, were determined using enzyme-linked immunosorbent assay kits. The data were expressed as mean±standard error of mean (*, P<0.05). The results from one of three repeated experiments were presented; n=5–6 mice in each group.

Suppression of TLR-mediated inflammatory signals in ginsan-treated macrophages

Toll-like receptor (TLR) 2 in macrophages is well known for its ability to recognize components of Gram-positive bacteria. In addition, TLR2-mediated signaling induces the activation of inflammatory cytokine genes. Therefore, this study examined whether ginsan could modulate the expression of TLR2 and an adaptor molecule MyD88 in macrophages that were co-cultured with the heat-inactivated S. aureus. The macrophages (PMs) co-cultured only with S. aureus expressed considerably high levels of TLR2 and MyD88, and moderately high levels of TLR4 and TLR9 (Fig. 6). In contrast, peritoneal macrophages treated with ginsan before co-culturing with S. aureus almost completely downregulated expressions of TLR2 and MyD88 (Fig. 6). Moreover, expressions of TLR4 and TLR9 were also blocked in the same cells. Therefore, these results indicated that ginsan downregulated the expression of TLR2, TLR4, TLR9, and MyD88 in the macrophages that recognized the bacterial cell walls. The production of inflammatory cytokines, including TNF-α IL-1 β and IFN-γ was induced by the translocation of NFκB and the phosphorylation of p38 MAPK and JNK (O'Neill, 2003; Bachar et al., 2004). The reduction of free NFκB and the downregulated synthesis of phospho-JNK1/2 and p38 MAPK were demonstrated in PMs that were treated with ginsan prior to contact with heat-killed S. aureus (data not shown). These data, therefore, collectively implied that ginsan mediated the suppression of TLRs and downstream-signal-transduction molecules in septic mice. These events ultimately led to the reduced production of inflammatory cytokines and the enhanced survival of the septic mice.

6

Ginsan-mediated suppression of Toll-like receptor (TLR)s and MyD88 in peritoneal macrophages stimulated with Staphylococcus aureus. Peritoneal macrophages (4 × 106 cells per well) of mice were pretreated with ginsan (0.1 µg mL−1) for 10 h, before being stimulated with heat-killed S. aureus (8 × 107 CFUs well−1) for 6 h at 37°C. The same number of peritoneal macrophages was cultured for 6 h at 37°C, with or without S. aureus. Reverse transcriptase polymerase chain reaction was conducted to evaluate the expression levels of TLR2, TLR4, and TLR9, as well as MyD88. Glyseraldehyde-3-phosphate dehydrogenase was used as a control gene. These results represent one of three repeated experiments.

Discussion

This study elucidated the antiseptic action of ginsan, which modulated inflammatory responses in Staphylococcus aureus-infected mice and prevented them dying from infection. In this study, mice that were treated with 25 µg mL−1 of ginsan 24 h before the bacterial challenge exhibited the best protection against sepsis. One of the other immunomodulators, poly-[1,6]-BD-glucopyranosyl-[1,3]-BD-glucopyranose (PGG) glucan, also demonstrated a protective effect when it was administered to mice 4–6 h before bacterial infection (Onderdonk et al., 1992). No toxic or side effect was observed even though high concentrations of ginsan (0.5–250 mg mL−1) were administrated to the mice. The main problem described in other studies conducted with different kinds of the BRM was the very narrow range of dose in which the biological responses were reproduced (Onderdonk et al., 1992). In the case of ginsan, however, antiseptic activity was maintained even though the optimal dose was exceeded 10 000 times. Ginsan could therefore be considered as a safe immunomodulator.

Mice that were treated with ginsan before the challenge with S. aureus demonstrated a potent bactericidal activity. When compared to the control mice, 98.1% and 96.7% bacterial clearance were observed in the spleens and the kidneys, respectively, of ginsan-treated septic mice. The bacterial load in rats that were treated with an aqueous extract of ginseng was also reduced (Song et al., 1997). It is therefore evident that components of Panax ginseng naturally induce antibacterial activity in the animals. This study indicated that the antibacterial activity induced by ginsan was mainly performed by monocytes/macrophages. The survival rate of etoposide-treated septic mice was much lower than that of septic mice that were treated with both etoposide and ginsan, and that of nontreated mice. The numbers of bacteria determined in the spleens, kidneys, and blood of etoposide-treated mice were considerably higher than those found in the same organs of mice treated with both etoposide and ginsan, and in nontreated mice. These data implied that macrophages played an important role in the clearance of S. aureus in the infected mice. Monocyte/macrophage-mediated protection of animals was also demonstrated in S. aureus-induced endocarditis and sepsis models in different systems (Verdrengh & Tarkowski, 2000; Veltrop et al., 2000). How did the mice treated with both etoposide and ginsan retain their antibacterial activity? We assumed that the remaining few monocytes/macrophages or the unaffected neutrophils and other immune cells in mice contributed the clearance of bacteria. This hypothesis, however, needs to be further evaluated in future studies. The immunostimulatory effects of ginsan, including the activation of macrophages and the proliferation of T and B lymphocytes, were already demonstrated in previous studies (Lee et al., 1997; Lim et al., 2002; Song et al., 2002). In the present study, the highly enhanced phagocytic activity of macrophages was further verified after in vitro treatment with ginsan. Taken together, these findings highlight the importance of macrophage-mediated innate immune responses enhanced by ginsan in S. aureus-infected mice.

In sepsis induced by Gram-positive or Gram-negative bacteria, inflammatory cytokines such as TNF-α, IL-1β and IL-6 functioned as the primary mediators for the induction of shock and death (Verhoef & Mattsson, 1995; Kox et al., 2000). The cytokines mentioned above were also known to enhance the growth of S. aureus (Meduri et al., 1999; Gogos et al., 2000). This means that the severe fatal consequence of sepsis can be attributed to the profuse synthesis of inflammatory cytokines. In this respect, ginsan protected mice from sepsis-induced fatality by inhibiting the synthesis of inflammatory cytokines in S. aureus-infected mice. Ginsan significantly reduced the production of inflammatory cytokines such as TNF-α, IL-1β, IL-6, IFN-γ, IL-12, and IL-18, as well as the synthesis of the anti-inflammatory cytokine IL-10 (data not shown). Virtually insignificant amounts of IL-2 and IL-4, however, were found in both S. aureus-infected and non-infected mice. The elevated level of IL-10 in patients with sepsis was correlated with death (Kanangat et al., 2001). The capacity of ginsan to downregulate sepsis-induced inflammation might therefore lead to the protection of mice from S. aureus-induced fatality.

To investigate the signaling pathways that regulate the production of inflammatory cytokines, the expression of TLR2, TLR4, and TLR9, as well as an adaptor molecule MyD88 in peritoneal macrophages, was examined. When the cells were treated with heat-killed S. aureus, such cells highly expressed TLR2, TLR4 and MyD88. When the cells were treated with ginsan prior to the contact with the heat-killed bacteria, however, the cells dramatically suppressed the expression of TLR2, TLR4 and MyD88. As the increased expression of TLR2 and TLR4 was reported in both septic humans and animals (Williams et al., 2003; Armstrong et al., 2004; Harter et al., 2004), TLRs have been speculated to play a key role in triggering the production of inflammatory cytokines. The ginsan-mediated alleviation of sepsis therefore seemed to be largely mediated by the downregulation of TLRs and MyD88 expression. In the downstream of the TLR-regulated signal transduction pathway, the phosphorylation of JNK1/2 and p38 MAPK, and the translocation of NFκB, induced expression of inflammatory cytokines in sepsis (Ulloa et al., 2002; O'Neill, 2003; Bachar et al., 2004). Peritoneal macrophages pretreated with ginsan and subsequently exposed to heat-killed S. aureus reduced localization of NFκB and expression of phospho-JNK1/2 and p38 MAPK (data not shown). These facts indicated that ginsan played important roles in the modulation of TLR-mediated signal transduction pathways and the following suppression of inflammatory cytokine expression.

In conclusion, this study demonstrates the protective mechanisms induced by ginsan in S. aureus-infected septic animals. The first protective effect given by ginsan was the upregulated phagocytic activity of monocytes/macrophages. The second protective effect was the reduced synthesis of sepsis-inducing inflammatory cytokines at the early period of bacterial infections. The diminished inflammatory responses were the consequence of down-regulated signals that were transmitted through TLRs. These harmonious modulations ultimately protected the animals from the lethal sepsis.

Acknowledgements

We thank In-Sung Jung and Sin-Keun Kang for their expert technical assistance in accomplishing this study. In-Soo Choi and Jie-Young Song contributed equally as authors.

Footnotes

  • Editor: Willem van Eden

References

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