β-Glucan, extracted from oat, enhances disease resistance against bacterial and parasitic infections

Authors

  • Cheol-Heui Yun,

    1. Animal Biotechnology Centre, Department of Animal and Poultry Science, 51 Campus Drive, University of Saskatchewan, Saskatoon, SK, Canada S7N 5A8
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    • 1

      Research Center of Bioscience and Biotechnology, CTC Bio, Inc., 93 Jung-An Bldg., Ohkeum-Dong, Songpa-Gu, Seoul 138–858, South Korea.

  • Alberto Estrada,

    1. Animal Biotechnology Centre, Department of Animal and Poultry Science, 51 Campus Drive, University of Saskatchewan, Saskatoon, SK, Canada S7N 5A8
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  • Andrew van Kessel,

    1. Animal Biotechnology Centre, Department of Animal and Poultry Science, 51 Campus Drive, University of Saskatchewan, Saskatoon, SK, Canada S7N 5A8
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  • Byung-Chul Park,

    1. Naomi Berrie Center, Department of Medicine, College of Physicians and Surgeons, Columbia University, New York, NY 10032, USA
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    • 1

      Department of Medical Microbiology and Immunology, Göteborg University, Guldhedsgatan 10, 41346 Göteborg, Sweden.

  • Bernard Laarveld

    Corresponding author
    1. Animal Biotechnology Centre, Department of Animal and Poultry Science, 51 Campus Drive, University of Saskatchewan, Saskatoon, SK, Canada S7N 5A8
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*Corresponding author. Tel.: +1 (306) 966 4972; Fax: +1 (306) 966 4151, E-mail address: b.laarveld@usask.ca

Abstract

The effect of β-glucan, extracted from oats, on the enhancement of resistance to infections caused by Staphylococcus aureus and Eimeria vermiformis was studied in mice. In vitro study using macrophages isolated from the peritoneal cavity showed that β-glucan treatment significantly enhanced phagocytic activity. In vivo study further demonstrated that β-glucan treatment induced a significant (P<0.05) protection against the challenge with 5×108 of S. aureus in mice. Fecal oocyst shedding in the C57BL/6 mice infected with E. vermiformis was diminished by β-glucan treatment by 39.6% in intraperitoneal and 28.5% in intragastric group compared to non-treated control. Patency period was shorter and antigen (sporozoites and merozoites) specific antibodies were significantly (P<0.05–0.01) higher in β-glucan-treated group compared to non-treated control group. There were an increasing number of splenic IFN-γ-secreting cells in glucan-treated group via intraperitoneal route, which might be responsible for the enhancement of the disease resistance. Glucan treatment was able to effectively change the lymphocytes population (Thy 1.2+, CD4+ and CD8+ cells) in the mesenteric lymph nodes and Peyer's patches in mice infected with E. vermiformis. In conclusion, the oral or parenteral oat β-glucan treatment enhanced the resistance to S. aureus or E. vermiformis infection in the mice.

1Introduction

β-Glucans, carbohydrates and major structural components of the cell wall of yeast, fungi and some cereals such as oat and barley have been extensively investigated for their immunomodulatory activities. The cell wall glucans of yeast and fungi consist of β-1,3-linked d-glucose backbone with β-1,6-linked side chains of glucopyranosyl residues [1]. β-Glucans with β-1,3 and β-1,4 linkages are present in the endosperm cell wall of oat and barley as a polysaccharide-protein polymer complex of approximately 2×106 molecular mass [2].

β-Glucans with variable molecular sizes and secondary structures, isolated from various natural sources, have been studied for their ability to activate host defense mechanisms against microbial and parasitic infections. Recent analysis on the response of human leukocytes to β-glucans has shown that the αMβ2 integrin CR3 [3] or newly found receptor dectin-1 [4] is primarily responsible for the binding of [5] and the phagocytic/cytotoxic responses by [6,7] the β-glucan. β-Glucans are known to act as adjuvants [8–10] and immunostimulants [10–12] enhancing the activities of leukocytes especially of macrophages and natural killer cells. It is likely that this broad spectrum of activities may be primarily due to macrophage activation. In vivo and in vitro studies have shown that β-glucans induce the activation of macrophage, such as phagocytosis [13,14], lysosomal enzyme activity [15,16] and the secretion of IL-1, IL-6 and TNF-α[17,18]. Most studies on β-glucans as immunostimulants have concentrated on extractions from yeast and fungus and there is limited information on the utilization of β-glucans from cereals as prophylactic or therapeutic agents. Czop and Austen [19] demonstrated that β-glucans from barley recognize the specific receptors on human macrophages. Although no studies have been carried out to define this nature from oat β-glucan, it is certain that they recognize the same receptors of the human and mouse macrophages due to the identical chemical structure of barley and oat β-glucan.

Protozoa of the genus Eimeria cause coccidiosis and parasitize the intestinal epithelium of numerous animals, including rodents and domestic animals. Eimeria spp. is highly host specific and is often the cause of not only growth retardation but also high rates of morbidity and mortality [20,21]. Infections with Staphylococcus aureus, an important causative agent in animal and human, give rise to life-threatening endocarditis, septic arthritis, septicemia, surgical site infections and catheter-related bacteremia [22–25]. Anti-coccidial/bacterial drugs have been used effectively to treat coccidiosis and bacterial outbreaks; however, they provided temporary control only while the pathogen develops the drug resistance rendering further treatments ineffective [26–28]. Increasing public awareness of chemical-based drug use in domestic animals with associated concerns of residues in animal products being transferred to mankind as consumed are added to the problems of chemotherapy. Therefore, other means of disease prevention and treatment such as using immunostimulants in order to boost the host defense mechanisms are receiving more attention. The objective of the present study was to examine the immunostimulating activities of oat β-glucan in vitro and in vivo and to assess the potential of oat β-glucan as an alternative means of disease prevention or treatment.

2Materials and methods

2.1Oat β-glucan

Oat β-(1,3)(1,4)-linked glucan (ObG) prepared from endosperm was obtained from Ceapro Inc. (Edmonton, AB, Canada). The preparation consisted of 68.2%β-glucan based on the McCleary method (Megazyme, Sydney, Australia) and consisted of 1–3 μm glucan particles as visualized by light microscopy. The molecular mass of ObG was 1.1×106 Da as determined by a TSK-60 high-performance liquid chromatography column (Bio-Rad, Mississauga, ON, Canada). The endotoxin contamination of this preparation, determined by a Limulus amebocyte lysate chromogenic specific assay (BioWhittaker, Inc., Walkersville, MD, USA), was less than 5 pg mg−1. The β-glucan suspension was prepared in sterile, pyrogen-free phosphate-buffered saline (PBS). The ObG was administered either intragastrically (IG; 3 mg in 300 μl per mouse) or intraperitoneally (IP; 500 μg in 100 μl per mouse) to mice every other day since 10 days before challenge with Eimeria vermiformis. Control mice received equivalent volumes of sterile PBS. It appears that IP challenge of ObG resulted in formation of negligible granuloma at the site of injection.

2.2Macrophage isolation and phagocytic activity

Peritoneal cells were harvested by washing the peritoneal cavity of five BALB/c mice with 5 ml of cold RPMI with gentle massage to dislodge any loosely adherent cells. Lavaged cells were centrifuged at 300×g for 10 min and pooled. Pooled cells were washed once with RPMI–10% fetal calf serum (FCS). The cells were counted and adjusted to 1×106 cells ml−1 in RPMI–10% FCS. Macrophage monolayers were established by seeding 106 cells in 16-well chamber slides (Corning Glass Works, Corning, NY, USA), incubating them in a 5% CO2 atmosphere at 37°C for 4 h, and washing them with RPMI to remove non-adherent cells. The cells in RPMI–10% were incubated at 37°C in a 5% CO2 atmosphere for 2 h, at this time, the medium was removed and replaced with RPMI–10% FCS containing ObG at pre-determined optimal concentration of 100 μg ml−1. Micrococcus lysodeikticus bacteria, labelled with fluorescein isothiocyanate (FITC), were added and phagocytosis allowed to proceed for another 15 min. The cells were then fixed and counted by fluorescent microscope. Total number of infiltrated cells into IP cavity was counted in mice treated IP with ObG and compared with saline-treated control. The data are expressed as mean and individual total number of macrophages or phagocytosed macrophages from 10 different experiments.

2.3Animals

Female C57BL/6 mice, 6–7 weeks old, were purchased from Charles River Laboratories (St-Constant, QC, Canada). The experimental protocol used in this experiment was approved by the Animal Care Committee, University of Saskatchewan, in accordance with the requirements of the Canadian Council on Animal Care.

2.4Infectious challenge

S. aureus (ATCC-25923) were grown in tryptic soy broth (Becton Dickinson, Cockeysville, MD, USA) at 37°C for 18 h, diluted in fresh nutrient broth immediately before challenge, and viable colony counts were determined. Serial dilutions were made and 20 μl from each dilution was plated in triplicate on nutrient agar to determine the viable cell density for the inoculum. The bacterial colony counts were reported as the log10 colony-forming units (CFU) ml−1. Groups of 10 mice were treated IP with 200 μl of PBS alone or PBS containing 500 μg of glucose, ObG or zymosan 3 days before challenge with S. aureus. Challenge was performed by the IP injection of 100 μl of bacterial suspensions diluted in sterile PBS containing 1×1010, 5×109 or 5×108 CFU. The mice were observed four times per day for the first 72 h and twice per day thereafter. Moribund animals were killed by halothane inhalation. Clinical signs including mortality were scored and live mice 10 days after bacterial challenge were recorded as survivors.

E. vermiformis was obtained from the Health of Animals Laboratory, Agriculture and Agri-Food Canada (Saskatoon, SK, Canada). Methods for its propagation in mice have been previously described [29].

2.5Collection of samples

The fecal oocysts shedding for each group were monitored daily until no further shedding was detected for two consecutive days. The oocysts, diluted 1:10 to 1:4000 in a saturated sodium chloride, were counted microscopically using a McMaster chamber. Sera were prepared from blood obtained at day 0 and days 17 and 25. All sera were clarified by centrifugation and stored at −20°C until analysis by enzyme-linked immunosorbent assay (ELISA). Mice were killed at the end of experiment (day 25 post-infection), and the entire small intestine, spleen and mesenteric lymph nodes (MLN) were removed. The small intestine was cut longitudinally and incubated in 3 ml of PBS containing 0.05 TIU ml−1 Aprotinin (Sigma), 5 mM EDTA, 2mM phenylmethylsulfonyl fluoride, and 0.02% NaN3, for 4 h at 4°C. The samples were centrifuged at 3000×g for 15 min at 4°C and the supernatants stored at −20°C until used. Spleen and MLN tissues were homogenized in RPMI-1640 medium containing 10% FCS (Gibco BRL, Life Technologies Inc., NY, USA) and the cells collected using a 70-μm cell strainer (Falcon, Becton Dickinson Labware, Franklin Lakes, NJ, USA). Spleen cells were treated with a 1% ammonium chloride solution to lyse erythrocytes. The cells were washed three times by centrifugation with RPMI-1640 medium. The cells were resuspended in RPMI-1640–10% FCS, counted and adjusted to 107 cells ml−1.

2.6Sporozoite and merozoite antigen preparation

To prepare merozoite antigens, oocysts were disrupted by vigorous vortexing at the maximum speed with glass beads and the suspensions were centrifuged through a Percoll gradient (density 1.11 g ml−1) at 4500×g for 20 min. Sporocysts were washed three times and sporozoites excysted by incubation for 2 h at 37°C in Hanks’ balanced salt solution (calcium and magnesium free) supplemented with 0.25% trypsin and 0.1% deoxycholic acid. In order to prepare merozoite antigens, infected mice were killed at day 4 post-infection, the ileum was removed and incubated in PBS for 30 min at 37°C in a CO2 incubator to release the merozoites. Sporozoites and merozoites were separated from debris by passage through a glass wool column and discontinuous Percoll density gradient centrifugation at 600×g for 25 min at 24°C. The collections were washed and then ruptured using ultrasonication (Sonifier 450; Branson Ultrasonics Corporation, Danbury, CT, USA) on ice. The suspensions were centrifuged at 3500×g for 20 min at 4°C and the supernatants were collected for use as antigens. Protein concentrations were estimated from the absorbance ratios at A260/A280 wavelength in a spectrophotometer (Spectronic 601, Milton Roy Co. Analytical Products, Rochester, NY, USA).

2.7Measurement of antigen specific antibodies

ELISA was used to measure specific anti-sporozoite and anti-merozoite antibodies in serum and intestinal washes. For specific antibodies, each well of 96-well microtiter plates (Immulon 2; Dynatec, Laboratories Inc., Chantilly, VA, USA) was coated with 20 μg ml−1 of sporozoite or merozoite antigen and incubated for 18 h at 4°C. The wells were blocked with PBS containing 2% bovine serum albumin (BSA) for 30 min at 37°C. Hundred μl of serum samples (1:10 dilution) were added to each well and incubated for 2 h at 37°C. As a secondary antibody, 100 μl of biotinylated goat anti-mouse IgG, IgM, IgG, IgG1 or IgG2a (Southern Biotechnology Associates, Birmingham, AL, USA) diluted 1:1000 in PBS–0.2% BSA were added and incubated for 1 h at 37°C. Hundred μl of alkaline phosphatase-conjugated streptavidin (Gibco BRL, Life Technologies Inc., MD, USA) were added and incubated for 30 min at 37°C. Between the steps, the wells of all plates were washed three times with PBS containing 0.05% Tween 20 (PBS–T). The substrate consisted of 1 mg ml−1 of p-nitrophenyl phosphate (104 phosphatase substrate tablets; Sigma) in 1.0 M diethanolamine buffer, pH 9.8, was added and the reaction stopped. The absorbance of each well at 405 nm was measured using an automated spectrophotometer (Molecular Devices Vmax Kinetic Microplate Reader; Molecular Devices, Menlo Park, CA, USA). Antibody levels were reported as the optical density (OD) readings, after the subtraction of the OD read-out of the mean plus 3 S.E.M. of a series of control wells with normal mouse serum added, and expressed as means±S.E.M. for each group.

2.8Detection of IFN-γ and IL-4 cytokines

An enzyme-linked immunospot assay was used for the enumeration of IFN-γ- and IL-4-secreting cells in spleen and MLN. Ninety-six-well nitrocellulose plates (Millipore, Millipore Ltd, Mississauga, ON, Canada) were coated with 5 μg of primary monoclonal antibody, IFN-γ and IL-4 (PharMingen, San Diego, CA, USA) in 50 μl of sterile PBS and incubated for 18 h at 4°C. The wells were washed three times with sterile PBS–T and then rinsed with sterile PBS alone. The wells were blocked with 200 μl of RPMI–5% FCS and incubated for 1 h at 37°C. Effector cell (spleen or MLN) suspension consisted of 1×106 cells ml−1 and 20 μg ml−1 of stimulant solution, either sporozoite or merozoite, diluted in RPMI–5% FCS. The wells were filled with 100 μl of cell suspension and 100 μl of stimulant solution. The plates were incubated, without disturbing, in a humid atmosphere containing 5% CO2 for 48 h at 37°C. After extensive washing with cold sterile PBS–T to remove adherent cells, 5 μg of biotin-conjugated anti-IFN-γ or anti-IL-4 monoclonal antibodies (PharMingen) in 50 μl of PBS–T were added to wells and incubated for 2 h at 37°C. The wells were washed with PBS–T and 50 μl of streptavidin–alkaline phosphate conjugate, diluted 1:1000 in PBS–T, were added to the wells and incubated for 1 h at 37°C. The wells were washed twice with PBS–T and twice with borate buffer (15 mM disodium tetraborate, 15 mM boric acid, 5 mM MgCl2, pH 9.8). Hundred μl of alkaline phosphatase chromogen substrate, which consisted of 0.15 mg ml−1 of 5-bromo-4-chloro-3-indolylphosphate toluidine (Sigma) and 0.3 mg ml−1 of p-nitroblue tetrazolium chloride (Sigma) in borate buffer, was added to each well. The plates were incubated for 20 min at room temperature. The substrate solution was decanted and the wells were thoroughly rinsed with tap water, dried and examined for the presence of blue spots. The spots were enumerated microscopically under low magnification and expressed, relative to 105 cells plated, as mean±S.E.M. for each group.

2.9Flow cytometry analysis

Subpopulations of leukocytes in spleen, MLN and Peyer's patches (PPs) was tested in mice infected with E. vermiformis and/or treated with a single dose of ObG (500 μg). ObG was injected IP at day 0 and groups of six mice were inoculated with 2.5×103 oocysts of E. vermiformis 12 h after glucan administration. The organs were taken at days 5, 12 and 19 post-inoculation (p.i.), and leukocytes, adjusted to 2×106 cells ml−1, were placed in 100 μl of fluorescence-activated cell sorter (FACS) buffer (PBS, 2% BSA and 0.05% NaN3) for staining. Previously evaluated dilutions of the following FITC-labeled monoclonal antibodies were used: anti-CD4, anti-CD8 and anti-Thy 1.2 (PharMingen). FITC-labeled irrelevant monoclonal antibody with no specific binding ability was used as a negative control. Cells were incubated with the antibodies at 4°C for 30 min, washed three times with FACS buffer by centrifugation, resuspended in 100 μl of FACS buffer and fixed with 2% formaldehyde solution at 4°C for 18 h. The cells were analyzed in single-color mode on a FACScan flow cytometer (Becton Dickinson, Mountain View, CA, USA), linked to a Hewlett-Packard computer using LYSIS II software. From each preparation 104 cells were acquired. Data are presented as the percentage of positive cells following subtraction of the background staining produced by the irrelevant monoclonal antibody control.

2.10Statistical analysis

Results were expressed as means±S.E.M. and compared by the Student's t-test or by analysis of variance using the Statistical Analysis System (SAS Institute Inc., Carry, NC). Differences were considered statistically significant when P<0.05. Survival test was analyzed using chi-square test.

3Results

3.1Macrophage activity

The ability of ObG to stimulate cells isolated from the peritoneal cavity was assessed. As shown in Fig. 1 in vitro stimulation of peritoneal cells with ObG induced proliferation resulting in significantly (P<0.01) higher number of cells after 24-h culture compared to saline-treated control. It is worthwhile to point out that macrophages that phagocytosed bacteria were also significantly (P<0.01) higher than in control.

Figure 1.

Effect of ObG on total number of macrophages. M. lysodeikticus bacteria were labelled with FITC and inoculated into macrophage cultures. After 24 h incubation the macrophages were fixed and counted by fluorescent microscope. Total number of infiltrated cells into IP cavity was counted in mouse treated IP with ObG and compared with saline-treated control. The data are expressed as mean of total number of infiltrated cells or phagocytosed macrophages from 10 different experiments. Open bar indicates the group treated with ObG and closed bar indicates glucose- or PBS-treated control. **Significantly different from control (P<0.01).

3.2IP challenge with S. aureus

To examine whether the administration of ObG could influence the course of S. aureus infection, we assessed the survival of mice IP challenged with different doses of S. aureus 3 days after the IP administration of PBS and glucose as control, zymosan as a crude form of glucan or ObG. The results shown in Fig. 2 indicated that both ObG and zymosan treatments induced a significant (P<0.05) protection to the lethal challenge of 5×108 bacteria when compared to the PBS-treated control group.

Figure 2.

Protection against S. aureus by ObG in mice. Groups of 10 mice were treated IP with 200 μl of PBS alone (⋄) or PBS containing 500 μg of glucose (Δ), ObG (▴) or zymosan (•) 3 days before challenge with IP injection of 100 μl of bacterial suspensions diluted in sterile PBS. The mice were observed four times per day for the first 72 h and twice per day thereafter. Clinical signs including mortality were scored and live mice 10 days after bacterial challenge were recorded as survivors.

3.3Oocysts shedding

The effect of ObG administration in parasitic infection was evaluated in mice inoculated with oocysts of E. vermicormis. Oocysts shedding dramatically decreased in ObG administered groups at the peak of the shedding (days 10–13 p.i.) compared to non-treated control group (Fig. 3). Total oocysts shedding was reduced by 39.6% in IG and 28.5% in SC compared to non-treated. Patency period in ObG-treated groups was 2 days shorter than non-treated control group (P<0.05).

Figure 3.

Effect of ObG treatment on the discharge of oocysts in mice inoculated with E. vermiformis. ObG was administered every other day by either IG or IP route and the shedding of oocysts from each group of mice was monitored daily from 5 days p.i. The graph is plotted by mean values. Δ, IG β-glucan-treated group; ◯, IP β-glucan-treated group; □, non-treated group.

3.4Induction of antigen specific immunoglobulins

Table 1 shows the effect of ObG on anti-sporozoite and anti-merozoite antibodies in mice inoculated with E. vermiformis. All examined serum antibodies in the infected groups were higher than non-infected naïve control group. Serum anti-sporozoite IgG in IP was significantly higher (P<0.05) than non-treated control group. Intestinal anti-merozoite IgA in the ObG-treated group was significantly (P<0.05) higher compared to non-treated control. There is no significant difference in intestinal anti-sporozoite IgA between β-glucan-treated and non-treated groups.

Table 1.  Levels of anti-sporozoite or anti-merozoite antibodies from the serum and intestinal fluids in mice inoculated with E. vermiformis
  1. Groups of six mice were inoculated with 2.5×104 oocysts of E. vermiformis and treated with β-glucan (3 mg by IG or 500 μg by IP or none) every 48 h. Infected non-treated control group was administered PBS. Samples were taken at day 25 p.i. and OD of antigen specific antibodies in the serum was detected. Results are expressed as mean values of groups of six mice±S.E.M.

  2. *Significantly (P<0.05) different from non-treated control.

GroupAnti-sporozoiteAnti-merozoite
 Serum IgGIntestinal IgASerum IgGIntestinal IgA
IG0.221±0.010.113±0.020.314±0.010.197±0.01*
IP0.281±0.02*0.104±0.010.432±0.01*0.181±0.01*
NT0.219±0.010.103±0.000.303±0.020.167±0.01

3.5Antigen specific cytokine secretion

Table 2 shows the effect of ObG on numbers of IFN-γ- and IL-4-secreting cells in response to the sporozoite antigen in spleen and MLN. IP administration of ObG showed a tendency toward high expression of IFN-γ-secreting cells in the spleen and MLN.

Table 2.  Effect of β-glucan administered by different routes on the numbers of cytokine-secreting cells from spleen or MLN in mice inoculated with E. vermiformis
  1. Groups of six mice were inoculated with 2.5×104 oocysts of E. vermiformis and treated with β-glucan (3 mg by IG or 500 μg by IP or none) every 48 h. Infected non-treated (NT) control group was administered PBS. Results are expressed as mean values of groups of six mice. ND, not detectable.

GroupNumber of cells/105
 IFN-γ-secreting cellsIL-4-secreting cells
 SpleenMLNSpleenMLN
IG1.7ND5.0ND
IP8.316.71.7ND
NT1.7ND6.75.0
Naive controlNDNDNDND

3.6Effect of a single IP administration of ObG on lymphocyte populations

FITC-labeled antibodies were used to assess the effect of a single dose of ObG on lymphocyte populations (Table 3). FACS analysis of lymphocytes isolated from spleen, MLN and PP showed that mice inoculated with E. vermiformis had a significant decrease in the percentage of total Thy 1.2+ cells. The ObG treatments (infected and ObG treated (I–T) and non-infected and ObG treated (NI–T)) induced significant increases (P<0.05) on Thy 1.2+ cells when compared to non-treated (I–NT and NI–NT) groups.

Table 3.  Effect of a single administration of ObG by IP route on the percentage of total T lymphocytes (Thy 1.2+) from spleen, MLN and PP in mice inoculated with E. vermiformis on day 12 post-infection
  1. ObG was injected IP at day 0 and groups of six mice were inoculated with 2.5×103 oocysts of E. vermiformis 12 h after glucan administration. The organs were taken and the percentage levels of T (Thy 1.2+) lymphocytes were analyzed by FACS. Data represent the mean values of groups of six mice±S.E.M.

  2. aI, infected; NI, non-infected; T, treated with β-glucan; NT, not treated with β-glucan.

OrganTreatment groupsaProbabilitya (<)
 I–TI–NTNI–TNI–NTITI×T
Spleen10.2±0.610.9±1.923.0±2.623.4±4.50.001 0.05
MLN39.9±1.839.6±0.650.5±0.043.9±1.10.0010.050.05
PP14.6±0.513.4±0.613.8±0.39.5±0.4  0.05

Table 4 shows the percentages of CD4+ and CD8+ T cells in spleen, MLN and PP of infected and ObG-treated groups on days 5, 12 and 19 p.i. compared to those of controls. In the infected groups (I–T and I–NT), CD4+ T cells were significantly reduced in MLN at day 5 p.i. (P<0.001) and, in spleen (P<0.01) at day 12 p.i. compared to non-infected groups (NI–T and NI–NT). It appears that infection caused significantly (P<0.001) lower CD4+ T cells in MLN compared to non-infected groups and this reduction was marginally smaller, yet significant (P<0.005) at day 12 p.i., in ObG-treated groups. CD8+ T cell populations of infected groups (I–T and I–NT) were significantly increased in spleen (P<0.005) and PP (P<0.001) at day 5 p.i. compared to non-infected groups. In PP, ObG treatment effectively enhanced the CD4+ T cells and reduced the CD8+ T cells compared to non-treated control groups.

Table 4.  Effect of a single administration of ObG by IP route on the percentage of CD4+ and CD8+ T cells in spleen, MLN and PP from mice inoculated with E. vermiformis
  1. ObG was injected IP at day 0 and groups of six mice were inoculated with 2.5×103 oocysts of E. vermiformis 12 h after glucan administration. The organs were taken at days 5, 12 and 19 p.i., and the percentage levels of CD4+ and CD8+ T cells were analyzed by FACS. Data represent the mean values of groups of six mice±S.E.M.

  2. aI, infected; NI, non-infected; T, treated with β-glucan; NT, not treated with β-glucan.

Source of cellsDays post-infectionTreatment groupsaProbabilitya (<)
  I–TI–NTNI–TNI–NTITI×T
CD4, spleen526.8±0.424.6±0.525.8±1.324.0±1.1   
 1215.8±0.116.6±0.120.1±2.921.4±0.80.001  
 1915.4±0.316.4±0.518.7±0.219.8±0.1   
CD8, spleen511.8±0.111.6±0.39.2±0.67.9±0.30.005  
 125.5±0.35.9±0.26.4±0.66.8±0.2   
 195.8±0.16.7±0.15.3±0.56.4±0.4 0.001 
CD4, MLN526.7±0.824.3±0.638.0±0.237.6±0.80.001  
 1228.1±0.125.5±0.238.1±0.635.5±0.10.0010.010.005
 1924.3±0.325.2±0.635.9±0.136.1±0.10.001  
CD8, MLN510.6±0.19.6±0.412.8±0.013.0±0.10.001 0.005
 1210.5±0.313.1±0.514.2±0.412.6±0.2  0.05
 199.9±0.39.0±0.17.1±0.08.6±0.00.005 0.05
CD4, PP514.3±0.812.8±0.214.0±0.214.0±0.2 0.05 
 1212.4±0.410.7±0.712.3±0.612.2±0.2  0.05
 1913.1±0.411.1±0.210.0±0.110.4±0.1   
CD8, PP55.9±0.37.1±0.04.5±0.24.4±0.10.0010.050.05
 124.4±0.24.6±0.13.9±0.23.3±0.00.05  
 194.9±0.35.1±0.23.3±0.13.6±0.10.05  

4Discussion

Immunomodulating effects of β-glucan, extracted from oat, were evaluated in vitro with macrophages and in vivo using the mice infected with S. aureus or E. vermiformis. Major findings of current study are the following: 1. The ObG induced proliferation and phagocytosis in peritoneal macrophages. 2. The ObG induced prolonged survival time in the mice challenged with S. aureus. 3. The ObG enhanced resistance against coccidiosis by inducing high level of antigen specific antibody and cellular responses in mice inoculated with E. vermiformis.

In vitro co-culture with ObG increased proliferation and phagocytic activity of macrophages isolated from the peritoneum. This result coincides with the previous finding that in vitro stimulation of macrophages with ObG resulted in the production of IL-1 in a dose- and time-dependent manner [30]. Further study showed that ObG was primarily recognized by macrophages, most likely through their receptors since without macrophages very little or no activity was revealed (data not shown). However, we are aware of the existence of the receptors for glucan on leukocytes other than macrophages. Thus, the contribution of ObG activation of these cells to enhanced macrophage activity remains to be determined.

The efficacy of ObG along with zymosan was monitored in mice challenged with S. aureus. It appears that ObG and zymosan were able to prolong the survival time after the infection as well as to induce low mortality. This finding coincides with the in vitro study that ObG enhanced the number and activity of peritoneal macrophages, which resulted in enhanced survival time and reducing mortality. These results also support the possibility that glucan may have used particular receptors on the surface of macrophages as a number of other studies suggested. Indeed β-glucan-binding site was mapped to a region of CD11b-located C-terminal to the I-domain [5]. A study examined mouse leukocyte for the presence and sugar specificity of a lectin site capable of recognizing β-glucans using leukocytes from normal or CD11b deficient mouse and showed similarity of mouse and human CR3 in response to β-glucans [3,7]. Recent exciting results show that macrophages express unique pattern-recognition receptor, dectin-1, which recognizes a variety of β-1,3-linked and β-1,6-linked glucans from fungi and plants [4]. However it is still an open question what major roles these two receptors (CR3 and dectin-1) play upon stimulation with not only glucan but also foreign carbohydrates from infectious pathogens. It is worthwhile to mention that dendritic cells may share the same receptors with macrophages, which was not tested in the present study.

Further studies were carried to examine the impact of β-glucan on the resistance to parasitic infection, coccidiosis, caused by E. vermiformis in the mouse. The ObG-treated mice, inoculated with oocysts of E. vermiformis, shed fewer oocysts with shorter patency period compared to those in the non-treated group. This result is well supported by previous findings where we showed significantly higher gamont counting in the non-treated control than ObG-treated groups [31], indicating that ObG was effective in decreasing the number of sporozoites and/or merozoites, therefore reducing gamonts and oocysts counts. It appears that the increased specific antibodies against asexual stages of E. vermiformis, both sporozoites and merozoites, in ObG-treated mice play a partial role in protecting them from the infection. The relatively low oocyst production in ObG-treated group is partially a result of cessation of sporozoite and merozoite and the development of reduced gametocytes. Others also showed that antibody against these two different stages of parasite reduced the number of parasites produced in the intestinal tract and partially prevented infection [32]. Other reports showed similar pattern that orally administered soluble β-d-glucan from Sclerotinia sclerotiorum stimulates not only systemic but also mucosal immunity in mice [33]. In addition, a number of studies demonstrated the ability of glucan to enhance resistance to parasitic infection and to potentiate non-specific and specific antibody responses [33,34].

Reynolds et al. [35] suggested that the efficacy of glucan treatment might vary depending upon its route of administration and the specific host–parasite interaction. In the E. vermiformis infection various defense mechanisms can play the key role, depending on the route of challenge. Our data showed that β-glucan treatments, both IG and IP routes, enhanced immunity probably through attracting antigen-presenting cells, especially macrophages, and further stimulated T cells. It should not be ignored, however, that innate immunity and non-immune-mediated mechanisms such as metabolism, motility and digestion process can be important, especially in the mice inoculated with the ObG by oral route since viscosity of β-glucan is a concern in animal diets. However significantly higher (P<0.01) intestinal anti-merozoite IgA than in non-treated controls suggests that there was enhanced specific immunity against coccidial infection by ObG. Augmentation of specific immune responsiveness might be more important than stimulation of non-specific effector mechanisms for the resistance [34,36]. It is likely that specific antibodies (anti-merozoites IgG and IgA in the intestine) were higher in glucan-treated groups and might play an important role in bacterial and parasitic infection, especially during the prepatency period. The level of anti-sporozoite antibody was not increased even during the early stage of infection (data not shown), indicating that the penetration and further development of sporozoites takes place for a very short time period [32]. Limited time of exposure may be one of the evading mechanisms for infectious organisms.

The β-glucan treatment enhanced Th1 cell activities through increased IFN-γ secretion. IFN-γ is an important factor in the early expression of resistance to E. vermiformis[37]. Since C57BL/6 susceptible mice may be deficient in developing an adequate immunity against the parasite, the immunostimulation provided by β-glucan may have enhanced the specific cell-mediated immune response against E. vermiformis. IFN-γ and IL-4 interaction did not show a typical Th1/Th2 pattern and it is probable that equilibrium is established to ensure elimination of pathogen, while at the same time minimizing immune-mediated tissue damage.

We conducted an experiment to investigate further the dynamics of cell population as infection progressed in mice inoculated with oocysts of E. vermiformis with/without single β-glucan treatment. FACS analysis of lymphocytes from infected mice showed a significant decrease in the percentage of total Thy 1.2+ cells (P<0.005) during the peak of infection and recovered to normal level during the final stage of infection. In the I–T group, however, the percentage of Thy 1.2+ cells in MLN was significantly (P<0.05) increased at day 5 p.i. compared to I–NT group (data not shown) indicating the importance of early changes of cell population for the activation of local immunity. In addition, the ObG induced significant increases (P<0.05 in I×T) of Thy 1.2+ cells in spleen, MLN and PP at the peak of infection day 12 p.i. In the infected groups, both with/without ObG treatment, number of CD4+ T cells were significantly decreased in MLN at day 5 p.i. (P<0.001) and, in spleen (P<0.01) at day 12 p.i. compared to non-infected groups (Table 4). It appears that MLN was the most effective target area where the ObG treatment affected the population changes of CD4 and CD8 T lymphocytes the most. In PP ObG treatment appeared to be effective to modulate the changes of T lymphocytes in an early stage (day 5 p.i.) of infection (Table 4). There appeared to be a general, yet distinct, tendency of increasing CD4 T lymphocytes during the course of infection in ObG-treated group compared to non-treated control group. It is interesting to mention that ObG treatment appeared to be able to change the population of mucosal lymphocytes in the animals infected with intestinal pathogen more effectively than those in the non-infected one.

It is worthwhile to note that present results did not follow the typical Th1/Th2 scheme. It is probable that the simultaneous increase in CD4+ and CD8+ cells perhaps in response to parasite invasion and tissue damage may act to counterbalance cytokine production, which functions to eliminate infected cells but also contains enough to prevent damage to healthy uninfected cells. The known distribution of the putative β-glucan receptors [3,4] would suggest that stimulation of IFN-γ secretion is due to the activation of cells such as macrophages NK (CD56+ CD3−) and/or NKT (CD56+ CD3+) cells. Similar mechanisms likely mediate the increase of the total number of T (Thy 1.2+) lymphocytes in spleen and MLN observed following β-glucan treatment. CD4+ lymphocytes have been demonstrated to play an important role in the control of primary E. vermiformis infection [38]. Together with other studies our findings suggest that the immunomodulatory effects of ObG induced the up-regulation of T cells. The current study showed the changes of humoral and cell-mediated immunities upon the challenge of ObG against bacterial and parasitic infections in mice. It is likely that macrophage activation and humoral immunity contributed to the early stage of infection followed by cell-mediated immunity as infection/disease progressed.

In conclusion, the ObG increased resistance of susceptible C57BL/6 mice to E. vermiformis infection via enhancing cellular and antigen specific humoral immunity. These studies suggest that immune functions may be up-regulated by both oral and parenteral administration of ObG, and these enhanced responses may play an important role in providing resistance to bacterial and parasitic infections. Therapy for a number of infections caused by bacteria and parasitic pathogens in animals and humans has only partial efficacy due to the development of drug resistance rendering the treatments ineffective, and standard management practices have not been satisfactory in controlling such diseases. Therefore, alternative approaches to prevention and treatment deserve examination. The enhancement of the specific and non-specific defense mechanisms against infectious diseases is of enormous importance in both human and veterinary medicine. Current pharmacological treatments for the pathogenic infections may be enhanced when combined with ObG administration.

Acknowledgements

This work was supported in part by the Saskatchewan Agriculture Development Fund and Natural Sciences and Engineering Research Council of Canada.

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