This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowReprints and Permissions
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Li, W.
Right arrow Articles by Yoshikai, Y.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Li, W.
Right arrow Articles by Yoshikai, Y.

 Previous Article  |  Next Article 

Infection and Immunity, December 2004, p. 7005-7011, Vol. 72, No. 12
0019-9567/04/$08.00+0     DOI: 10.1128/IAI.72.12.7005-7011.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Immunostimulating Properties of Intragastrically Administered Acetobacter-Derived Soluble Branched (1,4)-ß-D-Glucans Decrease Murine Susceptibility to Listeria monocytogenes

Wei Li,1 Toshiki Yajima,1* Kimika Saito,1 Hitoshi Nishimura,1 Takashi Fushimi,2 Yoshifumi Ohshima,2 Yoshinori Tsukamoto,2 and Yasunobu Yoshikai1

Division of Host Defense, Research Center of Prevention of Infectious Disease, Medical Institute of Bioregulation, Kyushu University, Fukuoka,1 Central Research Institute, Mitsukan Group Co., Ltd., Handa, Japan2

Received 27 April 2004/ Returned for modification 3 June 2004/ Accepted 27 August 2004


arrow
ABSTRACT
 
We previously found that AC-1, an extracellular polysaccharide, produced by Acetobacter xylinum and composed of (1,4)-ß-D-glucan with branches of glucosyl residues, showed a strong activity to induce production of interleukin-12 (IL-12) p40 and tumor necrosis factor alpha by macrophages in vitro via Toll-like receptor 4 (TLR-4) signaling. In the present study, we examined the effect of oral administration of AC-1 on protective immunity against Listeria monocytogenes. Mice were given AC-1 or phosphate-buffered saline (PBS) intragastrically 2 days before, on the day of, and 2 days after an intraperitoneal inoculation of L. monocytogenes. The survival rate of AC-1-treated mice was significantly improved and bacterial growth in AC-1-treated mice was severely retarded compared to those of PBS-treated mice after infection with L. monocytogenes. IL-12 p40 levels in serum and magnitudes of CD4+ Th1 and CD8+ Tc1 responses against Listeria antigen were significantly higher in AC-1-treated mice than in PBS-treated mice. The effect of AC-1 on antilisterial activity was diminished in C3H/HeJ mice carrying mutated TLR-4. Thus, AC-1, a potent IL-12 inducer through TLR-4, enhanced protective immunity against L. monocytogenes via augmentation of Th1 responses. These results suggest that infectious processes driven by intracellular microorganisms could be prevented to develop by the (1,4)-ß-D-glucan.


arrow
INTRODUCTION
 
There have been many reports on antibacterial activities of (1,3)-ß-D-glucans, major structural components of fungal cell walls such as lentinan, schizophyllan, and krestin, all of which contain branched (1,3)-ß-D-glucan (4, 24, 26, 29). It has been suggested that signals from (1,3)-ß-D-glucan are transmitted through heterodimers of Toll-like receptor 2 (TLR-2) and TLR-6 (2, 27), although these are thought to contain multiple stimulators for macrophages in addition to (1,3)-ß-D-glucan (46). Cellulose (1,4)-ß-D-glucan is a polysaccharide produced by plant cells, fungi, and bacteria (21, 23, 25, 34, 35). We previously found that AC-1, an extracellular polysaccharide produced by Acetobacter xylinum and composed of (1,4)-ß-D-glucan with branches of glucosyl residues, showed a strong activity to induce production of interleukin-12 (IL-12) p40 and tumor necrosis factor alpha (TNF-{alpha}) by macrophage cell lines in vitro via Toll-like receptor 4 (TLR-4) signaling (23, 32, 34, 35). When oral administration of AC-1 was begun immediately after ovalbumin (OVA) immunization, the serum levels of OVA-specific immunoglobulin E (IgE) and IgG1 were significantly decreased and this was accompanied by augmentation of gamma interferon (IFN-{gamma}) production. These results suggest that AC-1, a potent IL-12 inducer, suppresses allergic inflammation with IgE production, thus offering an approach for the treatment of allergic disorders.

Protective mechanisms against infection with Listeria monocytogenes, an intracellular bacterium, are mediated by two major waves of host responses (18, 19, 31, 41, 42). The first one is "innate immunity" depending mainly on phagocytes, natural killer (NK) cells and {gamma}{delta} T cells (11, 14). The activity of phagocytes is modified by IFN-{gamma} produced by NK cells and {gamma}{delta} T cells at the early stages during L. monocytogenes infection. The second is "acquired immunity" that is mediated by CD4+ Th1 and CD8+ Tc1 cells (12, 18, 43). T cells initially stimulated in the presence of IL-12 tend to develop into Th1 cells capable of producing IFN-{gamma} (5, 16, 37-40). IL-12 produced by macrophages/dendritic cells in the early stage of infection plays an important role in determining whether naive T cells will differentiate into Th1 cells. Microbial adjuvants such as nonmethylated palindromic DNA containing CpG-ODN are potent IL-12 inducers that act via TLRs and enhance protective immunity against L. monocytogenes infection via augmentation of Th1 responses not only by parenteral administration but also by oral administration (20, 30). Thus, oral administration of potent inducers of IL-12 can be used to prevent and control infection with intracellular bacteria in which Th1 responses are important for protection.

At very early time points after the delivery of L. monocytogenes, in tissues such as the liver, the immunostimulatory properties of microbial molecules are assessed by a rapid reduction of the initial bacterial load. Several factors, particularly IL-12 produced by macrophages and dendritic cells and IFN-{gamma} produced by NK cells, have been reported to be intensively involved in the early clearance of L. monocytogenes (5, 13, 16, 37-40), suggesting that accumulation of IL-12 p40 is critical for elimination of Listeria at the innate immunity phase responsible for a symbiosis between innate immunity and the T-cell system of resistance of AC-1-treated mice against Listeria infection. Both Th1 and Tc1 response plays a critical role in protective immunity against L. monocytogenes infection. Th1 cells bearing the surface receptor CD4 are generated after infection by various intracellular pathogens for production of IFN-{gamma}, and Tc1 cells of cytotoxic CD8+ T cells also produce IFN-{gamma} (12, 18).

In the present study, we examined the effects of AC-1, a potent inducer of IL-12 production by macrophages/dendritic cells, on protection against L. monocytogenes. The protection was significantly augmented in mice orally given AC-1. The AC-1-treated mice showed increased levels of IL-12 p40 in serum and augmented CD4+ Th1 and CD8+ Tc1 responses to Listeria antigen in the spleen. The protective effect of AC-1 was diminished in C3H/HeJ mice that carry mutated TLR-4. Thus, AC-1 enhanced antilisterial activity via augmentation of Th1 responses by stimulating macrophages/dendritic cells to produce IL-12 at least partly via TLR-4 signaling. These results suggest possible prophylactic and therapeutic applications of a (1,4)-ß-D-glucan for preventing infection with intracellular bacteria.


arrow
MATERIALS AND METHODS
 
Animals. C57BL/6, C3H/HeN, and C3H/HeJ mice were obtained from Japan SLC (Hamamatsu, Japan). These mice were bred at our institute under specific-pathogen-free conditions. All experiments were carried out in accordance with the Guide for the Care and Use of Laboratory Animals prepared by the National Academy of Sciences.

Purification of AC. The polysaccharide AC series of Acetobacter species used in the present study were prepared and purified by a method reported previously (23, 32, 34, 35). Briefly, five strains of polysaccharide-producing Acetobacter species were cultivated in a shaking flask at 30°C for 5 days, and the cells were removed by both centrifugation (10,000 x g) of diluted broth and filtration with celite. The cell-free polysaccharides were precipitated by the addition of isopropyl alcohol, and the precipitate was dissolved in water. Then, 5% aqueous CTAB (cetyltrimethylammonium bromide) solution was added until no more precipitate was formed. The insoluble acidic polysaccharide-CTAB complex was collected by centrifugation and redissolved in 20% sodium chloride solution. After dialysis against running water, the polysaccharide was precipitated with ethanol and dissolved in water. The acidic polysaccharide thus obtained was dialyzed against distilled water and lyophilized. The endotoxin content of AC-1 (100 µg/ml) was estimated by using an endotoxin-specific chromogenic Limulus test (Wako Pure Chemicals, Osaka, Japan) to be 32 pg/ml.

Bacteria. L. monocytogenes, strain EGD, was used in experiments. In some experiments, recombinant L. monocytogenes expressing OVA (rLM-OVA), kindly provided by Subash Sad (Institute for Biological Sciences, Ottawa, Ontario, Canada) was used (10, 28). Bacterial virulence was maintained by serial passages in C57BL/6 mice. Fresh isolates were obtained from infected spleens, grown in tryptic soy broth (Nissui Pharmaceutical Co., Ltd., Tokyo, Japan), washed repeatedly, resuspended in phosphate-buffered saline (PBS), and stored at –70°C in small aliquots. Heat-killed L. monocytogenes (HKL) was prepared by incubating viable L. monocytogenes at 74°C for 120 min.

Challenge with bacteria. Mice were injected intraperitoneally (i.p.) with various doses of L. monocytogenes.

Mice were intragastrically (i.g.) given a suspension of 100 µl of AC-1 (10 mg/ml) or PBS 2 days before and on days 0 and 2 after i.p. inoculation. From preliminary experiments, we determined the most effective schedule of AC-1 administration against listeria infection.

Bacterial growth in PEC, spleen cells, or liver cells. Mice were inoculated i.p. with viable L. monocytogenes in 0.2 ml of PBS on day 0 at the sublethal dose of 105 CFU, which corresponds to one-tenth of the 50% lethal dose for C57BL/6 mice. On day 6 after inoculation, the mice were killed by cutting the cervical artery. Peritoneal exudate cells (PEC) were lavaged with 5 ml of ice-cold Hanks balanced salt solution (HBSS) and harvested after gentle massage. The spleens and livers were removed and separately placed in homogenizers containing 2 ml of HBSS. The organs were completely homogenized, and the homogenates were serially diluted with cold HBSS. These samples were spread onto Trypto-Soya agar plates (Nissui Pharmaceutical Co.), and the colonies were counted after incubation for 24 h at 37°C.

Cell preparation. Spleen cells and PEC were obtained on day 6 after inoculation. Single spleen cell suspensions were prepared by using a glass slide. To enrich T cells, spleen cells were depleted of red blood cells by ammonium chloride-Tris buffer and passed through a nylon wool column (Wako Pure Chemicals, Osaka, Japan).

In vitro stimulation of T-cell-enriched spleen cells. T-cell-enriched spleen cells from mice infected with Listeria 6 days previously were incubated without any stimulation, with HKL, on immobilized anti-CD3 monoclonal antibody (MAb) in the presence of the mitomycin C-treated syngeneic naive splenocytes for 48 h at 37°C. Culture supernatants were then collected, and the amounts of IFN-{gamma} in the culture supernatants were determined by enzyme-linked immunosorbent assay (ELISA).

Analysis of intracellular cytokine synthesis. The spleen cells from mice infected with Listeria or rLM-OVA 6 days previously were harvested, washed, and suspended at 107 cells/ml in complete culture medium, and they were then incubated with HKL or 5 µg of OVA257-264 peptide/ml for 4 h at 37°C in the presence of 10 µg of brefeldin A (Sigma Chemical Co.)/ml. These cells were harvested, washed, and incubated for 30 min at 4°C with phycoerythrin-conjugated anti-CD44 MAb and Cy-Chrome-conjugated CD4 MAb or CD8 MAb. After surface staining, cells were subjected to intracellular cytokine staining by using the Fast-Immune cytokine system according to the institutions of the manufacturer (Becton Dickinson). The cells were washed and fixed in 1,000 µl of fluorescence-activated cell sorting (FACS) lysis solution (Becton Dickinson) for 10 min at room temperature and then washed again, resuspended in 500 µl of FACS permeabilizing solution (Becton Dickinson), and incubated for 10 min at room temperature. After being washed, the cells were stained with fluorescein isothiocyanate-conjugated IFN-{gamma} MAb or fluorescein isothiocyanate-conjugated isotype control rat IgG (Pharmingen) for 30 min at room temperature, and the fluorescence of the cells was analyzed by using a flow cytometer. In some experiments, spleen cells or PEC were stained with anti-CD44 MAb, anti-CD8 MAb, and OVA257-264 H-2Kb tetramer purchased from MBL (Nagoya).

ELISA of cytokines. IFN-{gamma}, IL-12 p40, and TNF-{alpha} in the culture supernatants and serum were measured by ELISA (Genzyme, Cambridge, Mass.). ELISAs were performed according to the manufacturer's instructions.

Statistical analysis. The statistical significance of the survival rate was determined by the generalized Wilcoxon test. Other data were analyzed by using the Student t test. A P of <0.05 was taken as significant. Analysis was done by using StatView 4.5 software (Abacus Concept, Berkeley, Calif.).


arrow
RESULTS
 
Resistance of AC-1-treated mice against Listeria infection. Mice were i.g. administered AC-1 or PBS 2 days before, on the day of, and 2 days after i.p. inoculation of L. monocyogenes. Mice were challenged i.p. with 0.2 ml of a single cell suspension containing a high dose (106 CFU) of L. monocytogenes, and survival rates were determined. As shown in Fig. 1, 70% of PBS-treated mice had died 7 days after L. monocytogenes infection. On the other hand, 80% of AC-1-treated mice survived beyond day 7 after inoculation with L. monocytogenes (P < 0.01). Thus, oral administration of AC-1-protected mice from lethal challenge with L. monocytogenes.



View larger version (22K):
[in this window]
[in a new window]
  		FIG. 1. Effect of oral administration of AC-1 on survival rates after i.p. inoculation with a lethal dose of L. monocytogenes. C57BL/6 mice were inoculated i.p. with 106 CFU L. monocytogenes in 0.2 ml of PBS on day 0. Mice were challenged i.g. with a suspension of 200 µl of AC-1 (10 mg/ml) or PBS 2 days before, on the day of, and 2 days after inoculation with L. monocytogenes. *, Significantly different from the value for PBS-treated mice (P < 0.01 by the generalized Wilcoxon's test).

Mice were challenged i.p. with 0.2 ml of a single cell suspension containing a sublethal dose (105 CFU) of L. monocytogenes, and bacterial growth was measured in the spleen and liver on day 6 after infection. As shown in Fig. 2, the bacterial growth was significantly retarded in AC-1-treated mice compared to that in PBS-treated mice (P < 0.01). These results indicated that oral administration of AC-1 suppressed the in vivo growth of L. monocytogenes.



View larger version (23K):
[in this window]
[in a new window]
  		FIG. 2. Effect of oral administration of AC-1 on bacterial growth in the spleen and liver after L. monocytogenes infection. C57BL/6 mice were challenged i.p. with 0.2 ml of a single cell suspension containing a sublethal dose of L. monocytogenes, and bacterial growth was measured in the spleen and liver on day 6 after infection. The data were obtained from three separate experiments and are expressed as means + the standard deviation (SD) for five mice at each point. Statistically significant differences between AC-1-treated and PBS-treated mice are indicated (**, P < 0.01).

IL-12 p40 and TNF-{alpha} levels in the sera of AC-1-treated mice after Listeria infection. We previously reported that AC-1 is a potent inducer of IL-12 p40 and TNF-{alpha} by macrophages in vitro (32). We therefore measured the IL-12 p40 and TNF-{alpha} levels in serum in AC-treated mice on days 1, 2, and 3 after infection. As shown in Fig. 3, the IL-12 p40 level was significantly higher in the sera of AC-1-treated mice than in the sera of PBS-treated mice at 24, 48, and 72 h after infection (P < 0.01). TNF-{alpha} was not detected in serum at these stages after infection in either AC-1- or PBS-treated mice (data not shown).



View larger version (23K):
[in this window]
[in a new window]
  		FIG. 3. IL-12 production in the sera of AC-1-treated mice after Listeria infection. Sera were collected from AC-1-treated and PBS-treated mice at 24, 48, and 72 h after Listeria infection, and IL-12 protein levels were determined by ELISA. The data were obtained from three independent experiments and are expressed as the means of triplicate determinations ± the SD (**, P < 0.01).

Generation of Listeria antigen-specific Th1/Tc1 cells in AC-1-treated mice. Accumulation of IL-12 p40 is critical for a symbiosis between innate immunity and adaptive immunity mediated by Th1/Tc1 responses that are important for Listeria eradication. To determine antigen-specific Th1/Tc1 responses in AC-1-treated mice after infection with L. monocytogenes, we first examined IFN-{gamma} production by spleen T cells of infected mice in response to Listeria antigen. T cells from mice infected with L. monocytogenes 6 days previously were cultured with HKL in the presence of APC, and IFN-{gamma} levels in the culture supernatant were measured 48 h later by ELISA. As shown in Fig. 4, T cells from AC-1-treated mice produced a significantly higher level of IFN-{gamma} in response to HKL than did T cells from PBS-treated mice (P < 0.01).



View larger version (22K):
[in this window]
[in a new window]
  		FIG. 4. Generation of Listeria antigen-specific T cells in AC-1-treated mice. Nylon wool-enriched T cells from AC-1-treated mice or PBS-treated mice on day 6 after Listeria infection were cultured without any stimulation or with HKL and immobilized CD3 MAb in the presence of APC for 2 days. Cytokine activity in the culture supernatants was determined by ELISA. The data are representative of three independent experiments with pooled cells from three AC-1-treated or PBS-treated mice and are shown as the means of triplicate determinations + the SD. Statistically significant differences between AC-1-treated mice and PBS-treated mice are shown (*, P < 0.05). ND, not detectable.

We next determined CD4+ Th1 and CD8+ Tc1 responses in AC-1-treated mice against infection by cytokine FACS analysis of the expression of CD4, CD44, and intracellular IFN-{gamma} or of CD8, CD44 and intracellular IFN-{gamma}. As shown in Fig. 5, a significant fraction of CD44, CD4+ T cells from both groups of mice infected with L. monocytogenes 6 days previously produced IFN-{gamma} in response to HKL. The number of CD4+ Th1 cells producing IFN-{gamma} was significantly larger in AC-1-treated mice than in PBS-treated mice at this stage. IFN-{gamma}-producing CD8+ cells in response to HKL were not detected in either group (data not shown).



View larger version (33K):
[in this window]
[in a new window]
  		FIG. 5. Intracellular expression of IFN-{gamma} by CD4+ T cells from AC-1-treated mice infected with L. monocytogenes. Splenocytes or PEC were pooled from three mice of each group on day 6 after infection and cultured with or without HKL in the presence of brefeldin A for 4 h at 37°C. After culture, the cells were surface stained with anti-CD4 and anti-CD44 MAb. Intracellular cytokine-producing cells were examined by using a flow cytometer and analyzed by gating on CD4+ T cells. The data are representative of two independent experiments with pooled cells from three mice and are shown as typical two-color profiles.

Because there is no immunodominant epitope in Listeria antigen in C57BL/6 mice, we decided to use rLM-OVA for detection of CD8+ Tc1 response in AC-1-treated mice. The relative numbers of OVA-specific CD8+ T cells in the total splenocytes, as assessed by staining with an H-2Kb tetramer coupled with an OVA-derived SIINFEKL peptide (OVA257-264Kb tetramer), were higher in AC-1-treated mice than those in PBS-treated mice on day 6 after infection with rLM-OVA (Fig. 6A). To further compare the frequency of CD8+ Tc1 cells, we examined intracellular IFN-{gamma} production in response to OVA257-264 peptide. As shown in Fig. 6B, a higher levels of CD8+ T cells with a high level of CD44 in AC-1-treated mice were stained with intracellular IFN-{gamma} than those in PBS-treated mice on day 6 after rLM-OVA infection. Thus, the numbers of Th1 and Tc1 cells in the spleen and PEC of AC-1-treated mice were higher than those in the spleen of PBS-treated mice after Listeria infection.



View larger version (50K):
[in this window]
[in a new window]
  		FIG. 6. Generation of OVA257-264-specific CD8+ T cells in AC-1-treated mice infected with rLM-OVA. (A) AC-1-treated or PBS-treated mice were inoculated i.p. with 5 x 105 CFU rLM-OVA. On day 6 after infection, spleen cells or PEC from these mice were stained with OVA257-264 Kb tetramer, anti-CD44 MAb and anti-CD8 MAb. The results of flow cytometry are presented as typical profiles after an analysis gate had been set on CD8+ cells. (B) Spleen cells or PEC from AC-1-treated or PBS-treated mice were prepared on day 6 after rLM-OVA infection and cultured with or without OVA257-264 peptide in the presence of brefeldin A for 4 h at 37°C. Intracellular cytokine-producing cells were examined by using a flow cytometer and analyzed by gating on CD8+ T cells.

Effects of AC-1 on protection against Listeria infection in C3H/HeJ mice. We previously reported that TLR-4 is mainly involved in the activity of AC-1 for IL-12 production by macrophages (32). To determine the involvement of TLR-4 in the protective effect of AC-1 on Listeria infection, we compared the effects of oral administration of AC-1 in protection against Listeria infection in C3H/HeN and C3H/HeJ mice. Consistent with our data obtained from C57BL/6 mice (Fig. 1), AC-1-treated C3H/HeN mice showed marked resistance to Listeria infection, as assessed by bacterial growth in the spleen and liver (Fig. 7). On the other hand, AC-1-treated C3H/HeJ mice showed only a slight resistance to Listeria infection, although the number of bacteria was significantly decreased by AC-1 treatment (P < 0.05). These results indicate that TLR-4 is involved in the protective effect of AC-1 on Listeria infection.



View larger version (29K):
[in this window]
[in a new window]
  		FIG. 7. Effect of oral administration of AC-1 on bacterial growth in the spleen and liver from C3H/HeJ mice after L. monocytogenes infection. C3H/HeN and C3H/HeJ mice were challenged i.p. with a sublethal dose of L. monocytogenes (2 x 105 CFU), and bacterial growth was measured in the spleens and livers on day 6 after infection. The data were obtained from three separate experiments and are expressed as means + the SD for five mice at each point. Statistically significant differences between AC-1-treated and PBS-treated mice are shown (*, P < 0.05; **, P < 0.01).


arrow
DISCUSSION
 
Parenteral administration of botanical and yeast extracts containing (1,3)(1,6)-ß-D-glucan has shown antimicrobial effects in animal studies and patients with high risks of development of infections (8, 9, 26, 45). Although (1,3)(1,6)-ß-D-glucan was the primary focus of most previous investigations, its in vivo immunomodulatory effects, especially when it is administered orally, have not been tested. Furthermore, ß-glucan, despite its wide distribution in daily diets, has not been rigorously tested in purified forms. In the present study, we demonstrated that oral administration of AC-1, a soluble branched (1,4)-ß-D-glucan from Acetobacter species, protected mice from infection the intracellular bacterium L. monocytogenes and enhanced responses of Th1 cells producing IFN-{gamma} in response to Listeria antigen. The most important finding in the present study is that an antibacterial effect could be exerted by the oral administration of soluble (1,4)-ß-D-glucan alone. Administration three times over a period of 1 week resulted in prolongation of survival and retardation of bacterial growth in the infected mice. We previously found that oral administration of AC-1 with almost the same schedule inhibited the induction of Th2 response with IgE production in mice (32). (1,4)-ß-D-glucan may have prophylactic and therapeutic applications for preventing not only allergic disorders but also infectious diseases.

(1,4)-ß-D-glucan with branches of glucosyl residues showed a strong activity to induce production of IL-12 p40 and TNF-{alpha} by macrophages in vitro via TLR-4 signaling. The molecular size of (1,4)-ß-D-glucan seems to be important for exerting this biological activity because (1,4)-ß-D-endoglucanase treatment abolished the immunomodulatory effects of AC-1, and low-molecular weight (1,4)-ß-D-glucan was less effective (32). Therefore, (1,4)-ß-D-glucan should be absorbed in its original form for in vivo immunomodulatory effects of (1,4)-ß-D-glucan when administered by the oral route in infection development. In the case of (1,3)-ß-D-glucan, the (1,3) backbone and the (1,6)-linked branches with high molecular weight were thought to be important for its immunomodulatory effect (3). (1,3)-ß-D-glucan with high molecular weight appeared in blood after oral administration. This suggests that it entered the blood and behaved pharmacokinetically in a manner similar to that in the case of parenteral administration of ß-glucan. These studies also suggest that processing by the gastrointestinal tract produced ß-glucan with strong activity. Although (1,4)(1,6)-ß-D-glucan may not be processed in the gastrointestinal tract of humans and mice because of a lack of bacteria producing (1,4)-ß-D-endoglucanase in the gastrointestinal tract, it is possible that (1,4)-ß-D-glucan in its original form is absorbed from the digestive tract, stimulating macrophage/dendritic cells in secondary lymphoid tissues. Alternatively, (1,4)-ß-D-glucan may directly stimulate macrophage and dendritic cells to produce cytokines in gut-associated lymphoid tissues. Further investigation is needed to confirm these possibilities.

Naive T cells initially stimulated in the presence of IL-12 tend to develop into Th1/Tc1 cells that produce IFN-{gamma} and display cytotoxic activity, which are for induction of cell-mediated immunity against intracellular parasites and tumors (5, 16, 37, 38). Induction of IL-12 production by macrophages/dendritic cells via TLR-4 signaling may be a major mechanism by which AC-1 promotes a Th1/Tc1-biased response to Listeria antigen. AC-1 may exert its antibacterial effect in mice mainly through the production of IL-12, which activates CD4+ Th1 and CD8+ Tc1 cells. IL-12 also exerts its antibacterial effect through enhancement of IFN-{gamma} production and cytotoxicity of NK cells (13, 39, 40). In our preliminary study, AC-1-treated mice showed increased NK activity and resistance against major histocompatibility complex class I-deficient malignant melanoma. Therefore, NK cells may contribute to the enhancement of protection against Listeria infection in AC-1 treatment. Furthermore, it has been shown that host defense responses related to ß-glucan include activation of an alternative complement pathway, release of lysosomal enzymes by monocytes, and generation of antimicrobial products such as superoxide and defensins (6, 7, 15, 22, 33). Thus, (1,4)-ß-D-glucan may activate both innate immunity and adaptive immunity, resulting in augmentative protective immunity against L. monocytogenes.

The finding that the protective effect of AC-1 was significantly reduced but not completely abolished in C3H/HeJ mice that carry mutated TLR-4 is notable. We previously showed that TLR-4 is a pattern recognition receptor for AC-1 (32). Consistently, our present results indicate that AC-1 augmented antilisterial activity via augmentation of Th1 responses by stimulating macrophages/dendritic cells to produce IL-12 at least partly via TLR-4. However, receptors other than TLR-4 may also be involved in in vivo immunomodulatory effects of AC-1 (1,3)-ß-D-glucans such as zymosan have been reported to bind to membrane components such as {alpha}Mß2 integrin (complement receptor 3) (44, 45), a scavenger receptor, lactosylceramide, and dectin-1 (1, 36). It has been suggested that signals from zymosan are transmitted through heterodimers of TLR-2 and TLR-6 (2, 27), although zymosan is thought to contain multiple stimulators for macrophages in addition to (1,3)-ß-D-glucan. It has recently been reported that curdlan, a linear (1,3)-ß-D-glucan, stimulates the binding of macrophages to pattern-recognition receptors with MyD88 for its signal transduction, although the responsible receptors, including TLRs, have not been identified (17). Although we have excluded TLR-2/TLR-6 heterodimer as a possible pattern recognition receptor for AC-1, other receptors, including other TLR complexes, may be involved in the possible effect of AC-1 on bacteria infection.

In conclusion, oral administration of a (1,4)-ß-D-glucan enhances antibacterial activity against L. monocytogenes via increased IL-12 production and augmented Th1/Tc1 responses. Thus, a (1,4)-ß-D-glucan orally administrated may be applicable to prophylactic and therapeutic use for preventing infection with intracellular parasites.


arrow
ACKNOWLEDGMENTS
 
We thank Kazue Kaneda and Yoko Kobayashi for excellent technical assistance. We also thank Subash Sad for kindly providing recombinant L. monocytogenes expressing OVA.

This study was supported in part by a Grant-in-Aid for Scientific Research on Priority Areas and Young Scientists (B), Japan Society for Promotion of Science, and grants from the Japanese Ministry of Education, Science, and Culture (Y.Y.); Yakult Bioscience Foundation (Y.Y.); Uehara Memorial Foundation (Y.Y.); and Nakamura Jishirou Foundation (T.Y.).


arrow
FOOTNOTES
 
* Corresponding author. Mailing address: Division of Host Defense, Research Center for Prevention of Infectious Diseases, Medical Institute of Bioregulation, Kyushu University, Fukuoka 812-8582, Japan. Phone: 81-92-642-6962. Fax: 81-92-642-6973. E-mail: yajimato{at}bioreg.kyushu-u.ac.jp. Back

Editor: J. N. Weiser


arrow
REFERENCES
 
    1
  1. Brown, G. D., P. R. Taylor, D. M. Reid, J. A. Willment, D. L. Williams, P. L. Martinez, S. Y. Wong, and S. Gordon. 2002. Dectin-1 is A major ß-glucan receptor on macrophages. J. Exp. Med. 196:407-412.[Abstract/Free Full Text]
    2. 2
  2. Bulut, Y., E. Faure, L. Thomas, O. Equils, and M. Arditi. 2001. Cooperation of Toll-like receptor 2 and 6 for cellular activation by soluble tuberculosis factor and Borrelia burgdorferi outer surface protein A lipoprotein: role of Toll-interacting protein and IL-1 receptor signaling molecules in Toll-like receptor 2 signaling. J. Immunol. 167:987-994.[Abstract/Free Full Text]
    3. 3
  3. Cheung, N., K., S. Modak, A. Vickers, and B. Knuckles. 2002. Orally administered beta-glucans enhance anti-tumor effects of monoclonal antibodies. Cancer Immunol. Immunother. 51:557-564.[Medline]
    4. 4
  4. Chihara, G., Y. Maeda, J. Hamuro, T. Sasaki, and F. Fukuoka. 1969. Inhibition of mouse sarcoma 180 by polysaccharides from Lentinus edodes (Berk.) sing. Nature 222:687-688.
    5. 5
  5. Coffman, R. L., K. Varkila, P. Scott, and R. Chatelain. 1991. Role of cytokines in the differentiation of CD4+ T-cell subsets in vivo. Immunol. Rev. 123:189-207.[Medline]
    6. 6
  6. Czop, J. K., and K. F. Austen. 1985. A beta-glucan inhibitable receptor on human monocytes: its identity with the phagocytic receptor for particulate activators of the alternative complement pathway. J. Immunol. 134:2588-2593.[Abstract]
    7. 7
  7. Czop, J. K., and K. F. Austen. 1985. Properties of glycans that activate the human alternative complement pathway and interact with the human monocyte beta-glucan receptor. J. Immunol. 135:3388-3393.[Abstract]
    8. 8
  8. de Felippe, J., Jr., M. da Rocha e Silva, Jr., F. M. Maciel, M. Soares Ade, and N. F. Mendes. 1993. Infection prevention in patients with severe multiple trauma with the immunomodulator ß1-3 polyglucose (glucan). Surg. Gynecol. Obstet. 177:383-388.[Medline]
    9. 9
  9. Di Luzio, N. R., D. L. Williams, R. B. McNamee, B. F. Edwards, and A. Kitahama. 1979. Comparative tumor-inhibitory and antibacterial activity of soluble and particulate glucan. Int. J. Cancer 24:773-779.[Medline]
    10. 10
  10. Dudani, R., Y. Chapdelaine, H. van Faassen, D. K. Smith, H. Shen, L. Krishnan, and S. Sad. 2002. Multiple mechanisms compensate to enhance tumor-protective CD8+ T cell response in the long-term despite poor CD8+ T cell priming initially: comparison between an acute versus a chronic intracellular bacterium expressing a model antigen. J. Immunol. 168:5737-5745.[Abstract/Free Full Text]
    11. 11
  11. Dunn, P. L., and R. J. North. 1991. Early gamma interferon production by natural killer cells is important in defense against murine listeriosis. Infect. Immun. 59:2892-2900.[Abstract/Free Full Text]
    12. 12
  12. Harty, J. T., L. L. Lenz, and M. J. Bevan. 1996. Primary and secondary immune response to Listeria monocytogenes. Curr. Opin. Immunol. 8:526-530.[CrossRef][Medline]
    13. 13
  13. Herberman, R. B., C. W. Reynolds, and J. R. Ortaldo. 1986. Mechanism of cytotoxicity by natural killer (NK) cells. Annu. Rev. Immunol. 4:651-680.[CrossRef][Medline]
    14. 14
  14. Hiromatsu, K., Y. Yoshikai, G. Matsuzaki, S. Ohga, K. Muramori, K. Matsumoto, J. A. Bluestone, and K. Nomoto. 1992. A protective role of {gamma}/{delta} T cells in primary infection with Listeria monocytogenes in mice. J. Exp. Med. 175:49-56.[Abstract/Free Full Text]
    15. 15
  15. Honda, S., H. Sugino, T. Asano, and A. Kakinuma. 1986. Activation of the alternative pathway of complement by an antitumor (1-3)-ß-D-glucan from Alcaligenes faecalis var. myxogenes IFO 13140, and its lower molecular weight and carboxymethylated derivatives. Immunopharmacology 1:29-37.
    16. 16
  16. Hsieh, C. S., S. E. Macatonia, C. S. Tripp, S. F. Wolf, A. O'Garra, and K. M. Murphy. 1993. Development of TH1 CD4+ T cells through IL-12 produced by Listeria-induced macrophages. Science 260:547-549.[Abstract/Free Full Text]
    17. 17
  17. Kataoka, K., T. Muta, S. Yamazaki, and K. Takeshige. 2002. Activation of macrophages by linear (1-3)-ß-D-glucans: implications for the recognition of fungi by innate immunity. J. Biol. Chem. 277:36825-36831.[Abstract/Free Full Text]
    18. 18
  18. Kaufmann, S. H. E. 1993. Immunity to intracellular bacteria. Annu. Rev. Immunol. 11:129-163.[CrossRef][Medline]
    19. 19
  19. Kaufmann, S. H. E., M. Emoto, G. Szalay, J. Barsig, and I. E. Flesch. 1997. Interleukin-4 and listeriosis. Immunol. Rev. 158:95-105.[CrossRef][Medline]
    20. 20
  20. Krieg, A. M., L. Love-Homan, A. K. Yi, and J. T. Harty. 1998. CpG DNA induces sustained IL-12 expression in vivo and resistance to Listeria monocytogenes challenge. J. Immunol. 161:2428-2434.[Abstract/Free Full Text]
    21. 21
  21. Lee, J. N., D. Y. Lee, I. H. Ji, G. E. Kim, H. N. Kim, J. Sohn, S. Kim, and C. W. Kim. 2001. Purification of soluble beta-glucan with immune-enhancing activity from the cell wall of yeast. Biosci. Biotechnol. Biochem. 65:837-841.[CrossRef][Medline]
    22. 22
  22. Matsushita, M. 1990. Curdlan, a (1-3)-ß-D-glucan from Alcaligenes faecalis var. myxogenes IFO13140, activates the alternative complement pathway by heat treatment. Immunol. Lett. 26:95-97.[CrossRef][Medline]
    23. 23
  23. Minakami, H., E. Entani, K. Tayama, S. Fujiyama, and H. Masai. 1984. Isolation and characterization of a new polysaccharide-producing Acetobacter sp. Agric. Biol. Chem. 48:2405-2414.
    24. 24
  24. Mizutani, Y., Y. Nio, and O. Yoshida. 1992. Effect of PSK and its subfractions on peripheral blood lymphocytes mediated cytotoxicity against urinary bladder tumor cells. J. Urol. 148:1571-1576.[Medline]
    25. 25
  25. Nagata, J., Y. Higashiuesato, G. Maeda, I. Chinen, M. Saito, K. Iwabuchi, and K. Onoe. 2001. Effects of water-soluble hemicellulose from soybean hull on serum antibody levels and activation of macrophages in rats. J. Agric. Food Chem. 49:4965-4970.[CrossRef][Medline]
    26. 26
  26. Ohmura, Y., K. Matsunaga, I. Motokawa, K. Sakurai, and T. Ando. 2001. Protective effects of a protein-bound polysaccharide, PSK, on Candida albicans infection in mice via tumor necrosis factor-alpha induction. Int. Immunopharmacol. 1:1797-1811.[CrossRef][Medline]
    27. 27
  27. Ozinsky, A., D. M. Underhill, J. D. Fontenot, A. M. Hajjar, K. D. Smith, C. B. Wilson, L. Schroeder, and A. Aderem. 2000. The repertoire for pattern recognition of pathogens by the innate immune system is defined by cooperation between Toll-like receptors. Proc. Natl. Acad. Sci. USA 97:13766-13771.[Abstract/Free Full Text]
    28. 28
  28. Pope, C., S. K. Kim, A. Marzo, D. Masopust, K. Williams, J. Jiang, H. Shen, and L. Lefrancois. 2001. Organ-specific regulation of the CD8 T-cell response to Listeria monocytogenes infection. J. Immunol. 166:3402-3409.[Abstract/Free Full Text]
    29. 29
  29. Rau, D. C., and V. A. Parsegian. 1990. Direct measurement of forces between linear polysaccharides xanthan and schizophyllan. Science 249:1278-1281.[Abstract/Free Full Text]
    30. 30
  30. Ray, N. B., and A. M. Krieg. 2003. Oral pretreatment of mice with CpG DNA reduces susceptibility to oral or intraperitoneal challenge with virulent Listeria monocytogenes. Infect. Immun. 71:4398-4404.[Abstract/Free Full Text]
    31. 31
  31. Rogers, H. W., C. S. Tripp, and E. R. Unanue. 1995. Different stages in the natural and acquired resistance to an intracellular pathogen. Immunologist 3:152-156.
    32. 32
  32. Saito, K., T. Yajima, H. Nishimura, K. Aiba, and Y. Yoshikai. 2003. Soluble branched ß-(1,4)glucans from Acetobacter species show strong activities to induce interleukin-12 in vitro and inhibit T-helper 2 cellular response with immunoglobulin E production in vivo. J. Biol. Chem. 278:38571-38578.[Abstract/Free Full Text]
    33. 33
  33. Suzuki, I., K. Hashimoto, N. Ohno, H. Tanaka, and T. Yadomae. 1989. Immunomodulation by orally administered beta-glucan in mice. Int. J. Immunopharmacol. 11:761-769.[CrossRef][Medline]
    34. 34
  34. Tayama, K., H. Minakami, E. Entani, S. Fujiyama, and H. Masai. 1985. Structure of an acidic polysaccharide from Acetobacter sp. Agric. Biol. Chem. 49:959-966.
    35. 35
  35. Tayama, K., H. Minakami, E. Entani, S. Fujiyama, and H. Masai. 1986. Structure of an acidic polysaccharide elaborated by Acetobacter sp. Agric. Biol. Chem. 50:1271-1278.
    36. 36
  36. Taylor, P. R., G. D. Brown, D. M. Reid, J. A. Willment, L. Martinez-Pomares, S. Gordon, and S. Y. C. Wong. 2002. The ß-glucan receptor, dectin-1, is predominantly expressed on the surface of cells of the monocyte/macrophage and neutrophil lineages1. J. Immunol. 169:3876-3882.[Abstract/Free Full Text]
    37. 37
  37. Trinchieri, G., and P. Scott. 1994. The role of interleukin 12 in the immune response, disease and therapy. Immunol. Today 15:460-463.[CrossRef][Medline]
    38. 38
  38. Trinchieri, G. 2003. Interleukin-12 and the regulation of innate resistance and adaptive immunity. Nat. Rev. Immunol. 3:133-146.[CrossRef][Medline]
    39. 39
  39. Tripp, C. S., M. K. Gately, J. Hakimi, P. Ling, and E. R. Unanue. 1994. Neutralization of IL-12 decreases resistance to Listeria in SCID and CB-17 mice: reversal by IFN-{gamma}. J. Immunol. 152:1883-1887.[Abstract]
    40. 40
  40. Tripp, C. S., S. F. Wolf, and E. R. Unanue. 1993. Interleukin 12 and tumor necrosis factor alpha are costimulators of interferon gamma production by natural killer cells in severe combined immunodeficiency mice with listeriosis, and interleukin 10 is a physiologic antagonist. Proc. Natl. Acad. Sci. USA 90:3725-3729.[Abstract/Free Full Text]
    41. 41
  41. Unanue, E. R. 1997. Why listeriosis? A perspective on cellular immunity to infection. Immunol. Rev. 158:5-9.[CrossRef][Medline]
    42. 42
  42. Unanue, E. R. 1997. Studies in listeriosis show the strong symbiosis between the innate cellular system and the T-cell response. Immunol. Rev. 158:11-25.[CrossRef][Medline]
    43. 43
  43. Unanue, E. R. 1997. Interrelationship among macrophages, NK cells and neutrophils in early stage of Listeria resistance. Curr. Opin. Immunol. 9:35-43.[CrossRef][Medline]
    44. 44
  44. Vetvieka, V., B. P. Thornton, and G. D. Ross. 1996. Soluble ß-glucan polysaccharide binding to the lectin site of neutrophil or NK cell complement receptor type 3 (CD11b/CD18) generates a primed state of the receptor capable of mediating cytotoxicity of iC3b-opsonized target cells. J. Clin. Investig. 98:50-61.[Medline]
    45. 45
  45. Williams, D. L., I. W. Browder, and N. R. Di Luzio. 1983. Immunotherapeutic modification of Escherichia coli-induced experimental peritonitis and bacteremia by glucan. Surgery 93:448-454.[Medline]
    46. 46
  46. Xia, Y., V. Vetvicka, J. Yan, M. Hanikyrova, T. Mayadas, and G. D. Ross. 1999. The beta-glucan-binding lectin site of mouse CR3 (CD11b/CD18) and its function in generating a primed state of the receptor that mediates cytotoxic activation in response to iC3b-opsonized target cells. J. Immunol. 162:2281-2290.[Abstract/Free Full Text]

Infection and Immunity, December 2004, p. 7005-7011, Vol. 72, No. 12
0019-9567/04/$08.00+0     DOI: 10.1128/IAI.72.12.7005-7011.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Aziz, A., Gauthier, A., Bezier, A., Poinssot, B., Joubert, J.-M., Pugin, A., Heyraud, A., Baillieul, F. (2007). Elicitor and resistance-inducing activities of {beta}-1,4 cellodextrins in grapevine, comparison with {beta}-1,3 glucans and {alpha}-1,4 oligogalacturonides. J Exp Bot 58: 1463-1472 [Abstract] [Full Text]  
  • Banus, H. A., Vandebriel, R. J., de Ruiter, H., Dormans, J. A. M. A., Nagelkerke, N. J., Mooi, F. R., Hoebee, B., van Kranen, H. J., Kimman, T. G. (2006). Host Genetics of Bordetella pertussis Infection in Mice: Significance of Toll-Like Receptor 4 in Genetic Susceptibility and Pathobiology.. Infect. Immun. 74: 2596-2605 [Abstract] [Full Text]  

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowReprints and Permissions
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Li, W.
Right arrow Articles by Yoshikai, Y.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Li, W.
Right arrow Articles by Yoshikai, Y.

Copyright © 2009 by the American Society for Microbiology.   For an alternate route to Journals.ASM.org, visit: http://intl-journals.asm.org | More Info»