Exercise can modulate many functions of the immune system and alter susceptibility to infection. Data generally support the hypothesis that exhaustive exercise stress may lead to immunosuppression and increased risk of infection (3,7,16), although there is some debate about the strength of evidence in this area (3). Several components of the innate immune system are compromised during single or repeated sessions of exercise stress. For example, alveolar macrophage antiviral resistance to herpes simplex virus (HSV) is reduced in mice after exercise to fatigue (7), as is macrophage antigen presentation (5), natural killer (NK) cell cytotoxicity (12), neutrophil oxidative burst (18), and the antigen specific cytokine response to HSV-induced upper respiratory infection (12). The effects associated with exercise stress are not unlike those that have resulted from other stress paradigms. For example, psychological stress has been shown to affect many components of the innate and adaptive immune responses to a variety of pathogens including HSV infection (27). Both epidemiological and controlled experimental studies in animals provide support for the relationship between exercise stress and infection (16).
Various nutritional strategies, including glutamine (4), zinc (20), antioxidants (17), and carbohydrates (14,15) have been investigated as possible countermeasures for immuno-suppression during periods of stressful exercise training and competition. Although carbohydrate replacement during prolonged intense exercise holds the most promise, there is little evidence that this or other nutritional strategies produce clinically significant changes in susceptibility to infection.
β-glucan, a polysaccharide derived from the cell wall of yeast, fungi, algae, and oats, has well-documented immunostimulant properties but has not been examined in the context of preventing exercise induced immunosuppression. β-glucan can enhance the activities of both the innate and specific immune system components via direct activation of β-glucan specific receptors on macrophage, neutrophils, and NK cells (6,25) or indirectly after activation of pinocytic M-cells located in the Peyer's patches of the small intestine (19,21,22). β-glucan has been shown to enhance the resistance to various viral (26), bacterial (8), protozoan (30), and fungal diseases (1) as well as to promote antitumor activity (23). Although few studies have been done on β-glucan derived from oats, an enhanced immune response to bacterial (8) and protozoan (30) challenges has been reported. There are, however, no reports of the effects of β-glucan on exercise-induced alterations in immune function and susceptibility to infection.
The purpose of this study was to determine the effects of oral feedings of the soluble oat fiber β-glucan on innate immune function and susceptibility to upper respiratory tract infection in mice after exercise stress. This was done using a murine model of exercise and respiratory infection (7) in which the exercise stress protocol was expanded from one to three consecutive days of prolonged treadmill running to fatigue in order to mimic a short period of severe exercise training. A β-glucan enriched oat bran concentrate was used because of its solubility, natural occurrence in the diet, Generally Recognized as Safe (GRAS) designation by the FDA, and documented health benefits as an important component of the Heart-Healthy diet in various pathological conditions, including diabetes and cardiovascular disease (10,29).
Male CD-1 mice, 4 wk of age, were purchased from Harlan Sprague Dawley Labs and acclimated to our facility for at least 3 d before any experimentation. Mice were purchased as pathogen-free stock, and periodic screening of sentinel mice yielded negative results for common murine viral or bacterial pathogens. Mice were housed, four per cage, and cared for in the animal facility located at the University of South Carolina School of Medicine. Mice were maintained on a 12:12 h light-dark cycle in a low stress environment (22°C, 50% humidity, low noise) and given food (Purina Chow) and water (or oat β-glucan dissolved in water) ad libitum. All experiments were performed at the beginning of the active dark cycle. The Institutional Animal Care and Usage Committee of the University of South Carolina approved all experiments.
Mice were randomly assigned to one of the following four groups: exercise water (Ex-H2O), exercise oat β-glucan (Ex-OβG), control water (Con-H2O), or control oat β-glucan (Con-OβG). Ex-H2O and Con-H2O received tap water for the 10 d before inoculation, whereas Ex-OβG and Con-OβG mice were fed a solution of oat β-glucan for the 10 d before inoculation. The oat β-glucan solution was made from an oat bran concentrate enriched to 68% soluble β-glucan (manufactured by Nurture, Inc., Devon, PA, and supplied by the Quaker Oats Co., Barrington, IL), which was dissolved in the drinking water at a concentration of 0.6 mg·mL−1 and made fresh daily. Soluble oat β-glucan is a structural polysaccharide (~2 × 106 molecular weight) found in the cell walls of the bran layer and endosperm fractions of the whole seed. Structurally, they are linear chains of β-D-glucopyranolsyl units in which ~70% of the units are linked (1–4), but which also consist of β-D-cellotriosyl and β-D-cellotetraosyl residues separated by (1–3) linkages arranged in a random manner (28). Daily consumption of fluid was measured to ensure there were no differences in fluid ingestion between the water and the oat β-glucan solution. Oat β-glucan was not fed to the animals during the 21 d after inoculation. Body weight of each animal was monitored throughout the supplementation and exercise period to ensure that no weight loss was experienced by any group.
Treadmill acclimation and exercise protocol.
The University's Institutional Animal Care and Use Committee approved the protocol described. After 4 d of oat β-glucan/water consumption, mice (Ex-H2O and Ex-OβG) were acclimated to the treadmill for a period 20 min·d−1 for the 3 d before the experimental exercise bouts. The exercise protocol consisted of an exhaustive exercise bout of treadmill running (performed in the evening, 6 p.m.) for three consecutive days. Mice in the exercise groups ran on the treadmill (two per lane) at a speed of 36 m·min−1 and a grade of 8% until they reached volitional fatigue. Fatigue was defined as the inability of the mouse to maintain the appropriate pace despite continuous hand prodding for 1 min at which time the mouse was removed from the treadmill. Electric shock was never used in these experiments as mice readily respond to a gentle tap of the tail or hindquarters encouraging them to maintain pace with the treadmill. Mice rarely required this type of continual prodding until they approached the point of fatigue. Mice in the control groups (Con-H2O and Con-OβG) remained in their cages in the treadmill room throughout the exercise bouts. These mice were exposed to similar handling and noise in an attempt to control for extraneous stresses that may be associated with treadmill running. Control mice were deprived of food and water during the exercise sessions.
In vivo titration of HSV-1.
Intranasal inoculation of Herpes simplex virus Type 1 (HSV-1) VR strain in the mouse is an established experimental model of respiratory infection. Although HSV-1 is not a common respiratory virus in humans, it can cause various pathological conditions in humans such as meningo-encephalitis, hepatitis, esophagitis, tracheobronchitis, and pneumonia as well as being associated with cases of adult respiratory distress syndrome (13). The intranasal route was chosen to mimic the typical route of entry of a viral challenge. HSV-1 was propagated in Vero cells and stored at −70°C in medium supplemented with 10% fetal bovine serum and 2% penicillin, streptomycin, and L-glutamine (PSG). The virus was titrated by administering 50 μL of various stock viral dilutions to additional mice in an initial experiment to determine the lethal dose. Morbidity and mortality were monitored for 21 d.
Intranasal infection with HSV-1.
On the day of the experiment, mice (N = 24 per group) were exposed to either control treatment or exercise to the point of volitional fatigue. Immediately after the control or exercise session, mice were returned to their cages. Fifteen minutes after exercise or rest, mice were lightly anesthetized with halothane and inoculated intranasally with 50 μL of HSV-1 VR strain. The dose yielded a 20–40% mortality rate among control mice in preliminary dose-response experiments. The actual dose (PFU·mL−1) of this virus was not specifically determined in this experiment. However, a similar LD 20 preparation of this virus strain contained 1.7 × 105 plaque-forming units (PFU·mL−1) (7). A dose of 1.28 × 106 PFU of a similar preparation of this virus was found to yield an average of 1.55 × 106 PFU per lung in a small sample (N = 5) of mice at 3 d after intranasal inoculation as described here. The pathogenesis and symptomatology of infection after intranasal inoculation of HSV have been well characterized (7,13,27). After inoculation, the mice were returned to their respective cages and housed in an isolated P2 facility. All animals were monitored twice daily for a period of 21 d for signs of morbidity and mortality. Several typical symptoms of illness were used to identify morbidity, including ruffled fur, redness around the eyes, nose or mouth, hunched back, and decreased activity. Mice that did not display any of these symptoms were considered healthy. These easily identifiable symptoms generally develop simultaneously with a quick onset and death follows within a day or two. It is rare for animals at this age to recover once they exhibit these symptoms. Under these circumstances, it is very difficult and usually not very informative to be more specific about the development and severity of symptoms. However, it was important to at least document the possibility that the experimental treatments, especially β-glucan, may have either delayed time to death once animals were sick and/or allowed for full recovery. This would obviously have important immunological implications.
Peritoneal macrophage antiviral resistance to HSV-1.
On the day of the experiment, mice (N = 18 per group) were exposed to either control treatment or exercise treatment. Immediately after exercise or rest mice were euthanized in a bell jar by halothane overdose. Death occurred within <1 min. Peritoneal macrophages were collected, prepared, and infected with HSV-1 as previously described (7). Briefly, peritoneal macrophages were obtained by lavage of the peritoneal cavity with 5 mL of culture media. Peritoneal lavage cells were washed and red blood cells were lysed with tris (hydroxymethyl) aminomethane-ammonium chloride, pH 7.2. Cells from three animals of the same group were pooled in order to obtain enough cells. Cells in each pool were adjusted to a concentration of 2 × 106 cells·mL−1 in cell culture media. Viability was determined using trypan blue exclusion >90%. Subsequently, 200 μL of the cell preparation was added to the wells of a 96-well microtiter plate and allowed to adhere at 37°C, and 5% CO2. After 12 h, each well was washed gently to remove nonadherent cells. The adherent macrophages were infected with HSV-1 KOS strain virus contained in 50 μL of medium. The virus was allowed to absorb for 90 min. Prewarmed RPMI-1640 supplemented with 10% fetal bovine serum was added to each well (to a final volume of 250 μL), and the plates were incubated at 37°C and 5% CO2 for 72 h. The HSV-1 virus used had been propagated in Vero cells and titrated on macrophages. A dose that resulted in a macrophage viability of 50% was chosen for this experiment. Aliquots of the virus were stored at −80°C. Seventy-two hours after infection with HSV-1, a cytopathic effect was observed in the macrophages and was quantified by a neutral red dye uptake assay as previously described (7).
NK flow cytometric assay.
On the day of the experiment, mice (N = 12 per group) were either exposed to control treatment or exercise treatment. Mice were euthanized 30 min after treatment by halothane overdose. Spleens were removed, weighed, and immediately homogenized in RPMI-1640, and the homogenate was centrifuged at 1100 rpm for 10 min. Blood cells were lysed with tris (hydroxymethyl) aminomethane-ammonium chloride, pH 7.2, and washed once in RPMI-1640. Remaining lymphocytes were placed in a T75 flask in an incubator at 37°C, and 5% CO2 for 1 h to eliminate any adherent cells (macrophages). Lymphocytes were adjusted to a concentration of 5 × 106 cells·mL−1 and put into 12 × 75 mm polystyrene tubes to yield effector: target ratios of 80:1, 20:1, 5:1, and 1:1 in addition to an effector cell control tube that contained effector cells only. Cells were serofuged for 60 s, and the supernatant was decanted by inversion. RPMI-1640 supplemented with 10% fetal bovine serum was added to each tube in the amount of 130 μL. After this, 10 μL of DiO (3 mM solution of 3,3′-dioctadecyloxacarbocyanine perchlorate) dissolved in DMSO (dimethyl sulfoxide) stained YAC cells were added to all tubes except the effector cell control tube. In addition one tube contained DiO stained YAC cells only and served as a target background control. YAC cells were stained by adding 10 μL of DiO stain forcefully into 1 mL of YAC cells concentrated at 1 × 106 cells·mL−1 in a 12 × 75 mm polystyrene tube. The tubes were incubated for 20 min at 37°C, and 5% CO2. After the incubation, cells were washed three times in RPMI-1640 using a serofuge. Cells were readjusted to a concentration of 5 × 106 cells·mL−1 and stored in an incubator at 37°C and 5% CO2 until use. Stained cells were used within 2 h of staining.
After addition of YAC cells to the effectors, 130 μL of propidium iodide (PI) working solution (10 mg of PI in 100 mL of RPMI-1640 supplemented with 10% fetal bovine serum) were added to each tube. Tubes were vortexed and serofuged for 30 s before being placed in the incubator at 37°C and 5% CO2 for 2 h. At the end of the incubation, the cell button was dislodged by vortexing and the tubes were placed in an ice bath. Specimens were evaluated within 30 min.
A Becton Dickinson FACScan flow cytometer equipped with an argon ion laser operating at 488 nm, and Research software was used to assess two parameter flow histograms. DiO labeled target cell membranes emit a green fluorescence (FL1) and PI labeled compromised cells emit a red fluorescence (FL3). Five thousand events were acquired with “high” running speed on each sample. The target background control was used to set quadrants for data analysis. The two controls one consisting of Ficoll separated lymphocytes/effector cells and one consisting of DiO stained YAC cells both containing PI were used to determine the amount of spontaneous cell death in both populations. The run results were acceptable if the percentage of these controls were <5%. Percent lysis of DiO stained YAC cells was evaluated.
Statistical analyses were performed using commercially available statistical packages from the SAS system (version 8.2, SAS Institute Inc., Cary, NC) and SigmaStat (version 2.03, SigmaStat, SPSS, Chicago, IL). Differences in morbidity and mortality between groups across the 21-d postinfection period were determined using a Lifetest Survival Analysis program in SAS (P < 0.05). Differences in treadmill run times were analyzed using the Student's t-test in SigmaStat (P < 0.05). Differences in NK cell activity, macrophage antiviral resistance, weight gain, and fluid consumption were compared using a two-way ANOVA in SigmaStat with Tukey post hoc analysis (P < 0.05).
Run time to fatigue was not significantly different between the exercise groups. Average run time to fatigue over the three exercise days was 145 ± 45 min for Ex-H2O and 136 ± 37 min for Ex-OβG. There was no difference in run times to fatigue over the 3 d of exercise, which indicates that the protocol was relatively well tolerated by the mice and that there was no apparent training effect that occurred over this time period.
Nutrient consumption and weight gain.
There were no differences in the average amount of fluid consumed by each of the experimental groups. Over the course of the 10-d ingestion period, mice consumed approximately 6.2 mL·d−1 of fluid that resulted in a daily dose of approximately 3.7 mg per mouse of oat β-glucan. Fluid consumption was 5.9 ± 0.9 mL, 6.4 ± 0.7 mL, 6.4 ± 0.9 mL, and 6.2 ± 0.7 mL·d−1 for Ex-H2O, Ex-OβG, Con-H2O, and Con-OβG, respectively. These results indicate that 24-h fluid consumption was not affected by the dissolved oat β-glucan or the exhaustive exercise. This is also reflected by a lack of difference in body weight across the groups. Weight gain over the course of the acclimation phase and 3-d exercise period was 2.1 ± 1.7 g in Ex-H2O, 2.7 ± 1.9 g in Ex-OβG, 2.9 ± 1.4 g in Con-H2O, and 3.3 ± 1.6 g in Con-OβG. Although food intake was not measured in this study, it seems unlikely that it was significantly different since fluid intake and body weight were not different among the groups.
The results of the experiment showed differences in morbidity across the four groups. Figure 1 illustrates the time course in morbidity for the four groups over the 21-d postinfection period. Intranasal administration of HSV-1 after fatiguing exercise resulted in greater morbidity than in resting controls (P = 0.036). Fatigue mice (Ex-H2O) experienced a 57% incidence of morbidity compared with only 29% of control mice (Con-H2O). The 25% morbidity quartile point estimate for Ex-H2O was 6.0 ± 0.10 d whereas that of Con-H2O was 7.5 ± 0.09 d. However, consumption of oat β-glucan for 10 consecutive days before infection offset this increase in morbidity (P = 0.048). Only 37% of Ex-OβG mice displayed symptoms of sickness; this was not significantly different from the Con-H2O (29%) mice. The 25% morbidity quartile point estimate for Ex-OβG was 15.5 ± 0.09 d. As expected, oat β-glucan also protected the control animals. Con-OβG mice experienced only an 8% incidence in morbidity (P = 0.05) vs Con-H2O.
Similar effects of oat β-glucan were found for mortality (i.e., time to death) over the 21-d postinfection period. Figure 2 illustrates the time course in mortality across the four groups. Exercise resulted in greater mortality than in resting controls (39% for Ex-H2O vs 21% for Con-H2O), but this did not reach statistical significance (P = 0.15). However, oral administration of oat β-glucan offset this trend toward increased mortality, significantly prolonging the survival time of animals that exercised to fatigue (17% in Ex-OβG vs 39% in Ex-H2O; P = 0.05). No effect of oat β-glucan was found in control animals. Point estimates were not reported for mortality, because overall mortality rates did not reach 25%. This relatively small effect of this dose of HSV-1 (<LD 25) probably accounts for the unexpected failure to find a statistically significant beneficial effect of oat β-glucan in control animals in this experiment (i.e., ceiling effect).
Peritoneal macrophage antiviral function to HSV-1.
In this experiment peritoneal macrophages were isolated from the four groups of mice and their intrinsic antiviral resistance to HSV-1 was examined. Figure 3 compares the antiviral resistance (expressed as a viability index) of peritoneal macrophages from mice sacrificed immediately after exercise. Clearly, antiviral resistance in mice exercised to volitional fatigue for 3 d (Ex-H2O) is significantly lower than in control mice (Con-H2O), (P < 0.007). Oat β-glucan consumption for 10 consecutive days (Ex-OβG) offset this decrease in macrophage antiviral resistance associated with exercise stress (P < 0.001). Oat β-glucan also protected the control animals (P = 0.041).
NK cell cytotoxicity was examined 30 min after the last bout of exhaustive exercise. Figure 4 illustrates that splenic NK cytotoxicity was not affected by either exercise stress or oat β-glucan consumption. However, the specific percentages of NK cells in the splenocyte suspensions were not quantitated and therefore any change in NK function on a per cell basis was not determined.
Evidence of a possible benefit of nutrition on immune function and risk of infection during periods of exercise stress is limited and often contradictory, perhaps with the exception of carbohydrate feedings that show consistent effects on immune function (14,15), but there is no information on infection risk. Another carbohydrate molecule that has not been studied in this context, but has well established immunostimulant properties and wide spread health benefits is β-glucan (10,19,21,22,29). This study used an established animal model of exercise and respiratory infection to determine whether ingestion of the soluble oat fiber β-glucan could protect mice from upper respiratory tract infection after 3 d of exercise stress and to determine possible immune mechanisms. The data suggest that oat β-glucan can block the increase in morbidity and mortality after intranasal inoculation of a standard dose of HSV-1 in exercise-stressed mice. This was associated with an increase in macrophage antiviral resistance to HSV-1 but not NK cytotoxicity.
The specific pathogenesis and symptomatology of HSV infection via intranasal inoculation is well characterized in the literature (13). Studies have used this model to determine the effect of exercise on susceptibility to infection and possible immune mechanisms (7,11,12). For this experiment, we used a slight modification of this model in which the exercise stress was extended from one to three consecutive days to mimic a short period of heavy training similar to what may occur in athletes and military personnel. The results confirm the negative effect of exercise stress on morbidity after intranasal infection with HSV-1, but the increase in mortality did not reach statistical significance as reported in our previous study (7). This apparent discrepancy could be due to a number of factors including the use of a different exercise stress protocol but is more likely due to the smaller sample size and more conservative statistical analysis used here. Indeed, the effect of exercise-stress on mortality in the two experiments is very similar (40% and 39% in the exercise stress groups vs 17% and 21% in control groups).
Nutrition strategies such as the use of carbohydrates (14,15), zinc (20), glutamine (4), and vitamin C (17) have had limited success in altering the immune response and susceptibility to infection. For example, consumption of carbohydrate containing sports drinks during prolonged intense exercise can increase various aspects of the innate immune system (15) and lessen the inflammatory cytokine response (14), but there is no information on risk of infection. There is only one report of a small benefit of vitamin C on symptoms of URTI after an ultramarathon race (17), but in this case the potential immune mechanisms are poorly defined.
Previous work with primarily insoluble yeast or fungi derived β-glucan suggests that it can stimulate a wide range of immunological activities, including increased macrophage (19,21,22), NK (22,25), and neutrophil function (25), and can enhance host resistance to fungal (1) and viral (26) diseases, and cancer (23). Less is know about the immuno-stimulant properties of soluble oat β-glucan, which incidentally is an important fiber component of the “Heart-Healthy” diet as defined by the FDA (10). Oat β-glucan has been shown to stimulate macrophage function (8,30), and increase resistance to bacterial (8) and protozoan (30) infections in mice. However, these are the first data to show a benefit of β-glucan (in any form) on exercise-induced immune suppression and susceptibility to infection.
The precise mechanisms for the apparent beneficial effects of oat β-glucan on susceptibility to infection after exercise stress cannot be determined in this study. Previous evidence suggests that multiple immune mechanisms may contribute to the negative effects of exercise stress on infection risk, including decreased macrophage resistance to HSV-1 (7,11) and antigen presentation (5), NK cell cytotoxicity (12), and neutrophil oxidative burst (18). Interestingly, the benefits of β-glucan on host defense have been attributed to activation of these same immune system components (21,22,25). Macrophages, NK cells, and neutrophils contain specific β-glucan receptor sites on their cell membrane, such as complement receptor 3 (CR3) and dectin-1 (2) that when bound results in increased functional activity (6,25). However, the mechanisms of stimulation can be dependent on the route of administration (e.g., intravenous, intraperitoneal, or oral) and specific characteristics of the β-glucan, including the source (e.g., oats, yeast, fungi, etc.), solubility, molecular mass, degree of branching, and conformation (ratio of 1→3 to 1→4 and 1→6 glucopyranolsyl linkages) (28). After oral administration of soluble β-glucan, pinocytic M-cells located in Peyer's patches of the small intestine can ingest the β-glucan via phagocytosis that results in release of cytokines that are responsible for initiating an extensive cascade of systemic immune responses (9,19,22). It is also possible for very small β-glucan particles to be absorbed directly into the lymphatic and vascular systems where they can interact directly with circulating immune cells via their β-glucan receptors (10,29).
In this study, oral feedings of oat β-glucan enhanced macrophage intrinsic resistance to HSV-1 in control mice and block the suppression associated with exercise stress. This may help to explain the benefits of oat β-glucan on resistance to infection in this model. Other mechanisms could include enhancement of related macrophage functions including acid phosphatase activity, phagocytosis, H2O2 production, and IL-1 production (19,21) as reported by others, albeit not in association with exercise stress. Suzuki et al. (22) showed that oat β-glucan can also increase NK cytotoxicity that can play an important role in susceptibility to infection, but this was not found in this study. This may be due to differences in the source (fungal vs oat β-glucan), and feeding schedule (1 vs 10 d) of the β-glucan.
β-glucans, especially water-soluble β-glucans from oat products, generally do not trigger serious side effects and carry a GRAS designation from the U.S. Food and Drug Administration (FDA). In fact, the health benefits of oat β-glucan are well documented in terms of lowering serum lipid risk factors for cardiovascular disease (10) and metabolic control of diabetes (29). Although the oral dose of oat β-glucan used in this mouse study is approximately twice the minimum daily human dose that the FDA requires for the “Heart Healthy” claim (10), it is similar to that used in human studies with no reported side effects. We found no side effects in this study (unpublished data), including no increase in the plasma concentration of tumor necrosis factor-alpha that was measured to evaluate the possibility that oat β-glucan could exaggerate the inflammatory response to severe exercise. This is relevant since recent evidence shows that yeast or fungi β-glucan administered i.p. in combination with the nonsteroidal anti-inflammatory drug (NSAID), indomethacin administered orally, can have a lethal interaction in mice (24). The mechanisms were traced to an exaggerated cytokine-driven inflammatory response to bacterial flora translocated from the gut that resulted in peritonitis, endotoxemia, and death. Individuals commonly take NSAIDs during and after periods of heavy training and bacterial translocation from the gut can occur during heavy exercise in the heat (24). Whether this is a real concern with soluble oat β-glucan given orally remains to be determined.
In conclusion, this is the first evidence of a possible health benefit of daily consumption of soluble oat β-glucan (in an oat bran concentrate) on the risk of URTI during a short period of severe exercise stress. The beneficial effects of oat β-glucan may result, at least in part, from increased macrophage resistance to HSV-1. If our data can be clinically translated and the potential side effects carefully evaluated, they may lead to an important new nutritional strategy to boost the immune system and decrease the risk of infection that can be a problem in athletes and military personnel who are often exposed to combinations of severe physical, psychological, and environmental stressors.
This work was funded by a grant from Quaker Oats Company and the Gatorade Sports Science Institute.
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