Oat β-glucan can counteract the exercise-induced increased risk for upper respiratory tract infection (URTI) in mice, which is at least partly mediated by its effects on lung macrophages. Substantial evidence in humans indicates that carbohydrate-containing sports drinks can offset the decreased immune function associated with stressful exercise. However, no studies in animals or humans have directly examined their effects on URTI using a controlled virus-challenge model. We examined the effects of sucrose feedings alone and in combination with oat β-glucan on susceptibility to infection and on macrophage antiviral resistance in mice following stressful exercise. These effects were also examined in rested, nonimmunocompromised control mice. Mice were assigned to one of four groups: H2O (water), sucrose (S), oat β-glucan (OβG), and sucrose + oat β-glucan (S+OβG). OβG and S treatments consisted of a solution of 50% OβG and 6% sucrose, respectively, and were administered in drinking water for 10 consecutive days. Exercise consisted of a treadmill run to fatigue performed on three consecutive days. Mice were then intranasally inoculated with a standardized dose of herpes simplex virus 1 (HSV-1) and monitored for morbidity and mortality for 21 days. Additional mice were used to determine macrophage antiviral resistance. In the exercise experiment, S, OβG, and S+OβG all reduced morbidity (P < 0.05), while only S+OβG reduced mortality (P < 0.05). Macrophage antiviral resistance was also increased in S, OβG, and S+OβG treatments (P < 0.05). In resting controls, S and S+OβG reduced morbidity and mortality (P < 0.05) and showed a trend toward increased macrophage antiviral resistance. There was no significant additive effect of S and OβG in either control or exercised animals. These data extend our previous work on the benefits of oat β-glucan to show that sucrose feedings have similar effects on susceptibility to respiratory infection and macrophage antiviral resistance in both resting controls and following exercise stress.
- immune function
data from controlled experimental animal studies support the hypothesis that exercise stress can increase susceptibility to upper respiratory tract infection (URTI) (4, 5, 8, 10, 18, 20). However, evidence from human studies, primarily based on epidemiological and observational data, is less clear (1). Our laboratory has used both a herpes simplex virus 1 (HSV-1) and an influenza [A/Puerto Rico/8/34(H1N1)] mouse model of respiratory infection to examine the effects of exercise stress on susceptibility to infection (4, 5, 8, 10, 11, 24, 27). We have reported that exercise stress can increase susceptibility to both HSV-1 and influenza infection [morbidity (time-to-sickness), symptom severity, and mortality (time to death)] (4, 5, 8, 10, 11, 27), which is associated with a decrease in macrophage intrinsic antiviral resistance (8, 10). A clear understanding of the mechanisms whereby stressful exercise can decrease immune function and increase susceptibility to infection have yet to be elucidated; however, it is thought that an exercise-induced increase in stress hormones may play a necessary role (19, 31).
Various nutritional strategies have been examined to counteract the immunosuppression and associated increase in incidence of URTI during periods of stressful training and competition (10, 11, 24, 31, 32, 34). Among these, carbohydrate-containing sports drinks have received the most interest, largely for their ability to offset the exercise-induced alterations in immune system components via suppression of the stress hormone response (16, 29, 31, 34). For example, they have been shown to attenuate increases in blood neutrophil, natural killer cell and monocyte counts, inflammatory cytokines, neutrophil oxidative burst, and the mitogen-induced proliferation of T cells (14, 17, 29, 31, 34, 35), although their effects on salivary IgA output and natural killer cell function have been inconsistent (2, 23, 30, 31). These data are often used to support the hypothesis that carbohydrate feedings can decrease susceptibility to infection. The proposed mechanism for these effects is thought to be due to attenuation of the stress response (31); carbohydrate ingestion during intense exercise has been shown to decrease levels of plasma cortisol and catechalomines (16, 22, 31, 34). However, to our knowledge there have been no controlled virus challenge studies in animals or humans to determine whether these changes in immune function affect resistance to infection. In addition, there are no data on carbohydrate feedings affecting macrophage function. β-glucan derived from oats has also been investigated for its ability to counteract the exercise-induced immune suppression (9). We have shown that oat β-glucan can reduce incidence of morbidity and mortality to HSV-1 respiratory infection following three consecutive days of stressful exercise in mice, the mechanisms of which we attributed, at least in part, to enhanced macrophage function (10, 24). Macrophages contain a receptor for β-glucan on their cell surface membrane that, when bound, can initiate a cascade of immune defenses leading to enhanced resistance to infection (5, 6, 12, 25, 26, 37, 40).
The purpose of this study was to examine the effects of sucrose feedings alone and in combination with oat β-glucan on susceptibility to HSV-1 respiratory infection using a well-characterized mouse model in both exercise stressed and rested mice. Since sucrose and oat β-glucan treatments have been shown to modulate different aspects of the immune system it is possible that their combination treatment may result in an additive or even synergistic effect. We were also interested in whether any benefit of sucrose alone or in combination with oat β-glucan was associated with altered macrophage antiviral resistance.
Male CD-1 mice, 4 wk of age, were purchased from Harlan Labs and acclimated to our facility for at least 3 days prior to 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 five 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 administered a Purina Chow diet. All experiments were performed at the beginning of the active dark cycle. Fig. 1 is a schematic representation of the experimental design for this study. Animals were removed from the experiment if they refused to run on the treadmill, did not survive the inoculation, or if they expelled the inocula by sneezing. Typically, in our hands this results in elimination of < 10% of animals. To avoid a ceiling or floor effect of the various treatments, it was necessary to use a different dose of virus for both the exercise and rested control groups; therefore the exercise and rested control experiments were carried out separately. 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: water (H2O), sucrose (S), oat β-glucan (OβG), or sucrose+oat β-glucan (S +OβG). Mice in the H2O group received tap water for the 10 days prior to inoculation. The S group consumed 6% sucrose (Sigma Aldrich, St. Louis, MO) in their drinking water, and the OβG group was fed a solution of oat β-glucan for the 10 days prior to inoculation. The oat β-glucan solution was made from an oat bran concentrate enriched to 50% soluble β-glucan (Oatvantage manufactured by Nurture, Devon, PA), which was dissolved in the drinking water at a concentration of 0.8 mg/ml and made fresh daily. The S+OβG mice consumed both treatments in their drinking water. Preliminary evidence in our laboratory has shown that mice fed sucrose in their drinking water consume more fluid than those drinking only water. Therefore, mice were, on average, only allowed to consume a maximum volume of 8 ml of each treatment daily; this was done using two water bottles for the S groups, one that contained a maximum of 40 ml of S or S+OβG and one containing just plain water. The treatments were administered to mice at 7 PM daily. The study was designed to examine only the prophylactic effects of these treatments. Therefore, treatments were not fed to the mice during the 21 days following inoculation.
Treadmill acclimation and exercise protocol.
After 4 days of nutrient consumption, exercise mice were acclimated to the treadmill for a period of 20 min a day for the 3 days prior to the experimental exercise bouts. The exercise protocol consisted of an exhaustive exercise bout of treadmill running (performed in the evening, 9 PM, 2 h following administration of the nutrient treatment) for three consecutive days. Mice in the exercise experiment ran on the treadmill (2 per lane). Following a brief warm-up period (5 min at 20 m/min, 5 min at 24 m/min, 5 min at 28 m/min, and 5 min at 32 m/min) mice were run at a constant speed of 36 m/min 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 they were 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.
Intranasal infection with HSV-1.
Exercise and control experiments were carried out separately as it was necessary to use different doses of virus to avoid a ceiling or floor effect of the various treatments. On the day of the experiment, mice (n = 20/group) were exposed to either control treatment or exercise. Fifteen minutes following the final bout of exercise or rest mice were lightly anesthetized with isoflurane and intranasally inoculated with 50 μl of HSV-1 VR strain. The actual dose plaque-forming units/ml (PFU/ml) of this virus was determined by plaque titration to be 1.28 × 105 PFUs per mouse for the exercise experiment and 2.56 × 105 PFUs for the control experiment. The pathogenesis and symptomatology of infection following intranasal inoculation of HSV have been well characterized (3, 5, 8, 26, 28). Following infection, the mice were returned to their respective cages and housed in an isolated P2 facility. All animals were monitored twice daily by an investigator blinded to the treatments for a period of 21 days for signs of morbidity (ruffled fur; redness around the eyes, nose, or mouth; hunched back; and unresponsiveness) and mortality. Mice that displayed any symptom of morbidity were considered morbid.
Intrinsic antiviral resistance of macrophages.
Fifteen minutes following the final bout of exercise or rest, mice (n = 16/group) were killed in a bell jar by isoflurane overdose. Death occurred in < 1 min. Peritoneal macrophages were collected, prepared, and infected with HSV-1 as previously described (8). Briefly, peritoneal macrophages were attained by lavage of the peritoneal cavity with culture media (RPMI 1640 supplemented with 10% fetal bovine serum and 2% penicillin, streptomycin, and l-glutamine). Peritoneal lavage cells were washed, and red blood cells were lysed with Tris (hydroxymethyl) aminomethane-ammonium chloride, pH 7.2 (prepared in the laboratory from chemicals supplied by Sigma Aldrich). Cells from two animals of the same group were pooled to obtain enough cells to perform the assay. Cells in each pool were adjusted to a concentration of 2 × 106 cells/ml. For each cell preparation, 200 μl aliquots were added to the wells of a 96-well microtiter plate and allowed to adhere at 37°C and 5% CO2. Following a 12-h incubation period, each well was washed gently to remove nonadherent cells. Adherent macrophages were infected with HSV-1 KOS strain contained in 50 μl of culture medium. The virus was allowed to absorb for 90 min. Prewarmed RPMI 1640 was added to each well (200 μl), and the plates were incubated for 72 h at 37°C and 5% CO2. Seventy-two hours after infection with HSV-1, antiviral resistance was determined by a neutral red dye uptake assay as previously described (8).
Plasma analysis of glucose.
Blood was collected from the inferior vena cava in a heparinized syringe and centrifuged at 4°C and 4,000 rpm for 10 min and stored at −80°C until analysis. Plasma was analyzed for glucose by using a commercially available kit (Randox Laboratories, Oceanside, CA). All samples were run in duplicate.
Statistical analyses were performed using commercially available statistical software from SigmaStat (version 2.03, SigmaStat; SPSS, Chicago, IL). Differences in morbidity and mortality between groups across the 21-day postinfection period were determined using a Lifetest Survival Analysis program (P < 0.05). Differences in macrophage antiviral resistance and plasma glucose were analyzed using a two-way ANOVA in SigmaStat with Student-Neuman-Keuls post hoc analysis (P < 0.05, S+OβG). Any “trend” toward a difference among treatments is defined as P ≤ 0.1.
Body weight and run times.
Run time-to-fatigue was not significantly different between the exercise groups. Average run time-to-fatigue over the three exercise days was 97 ± 32 min. In addition, run times were not different on days 1 through 3 of exercise, which indicates that there was no apparent training effect that occurred over this time period. Body weight was also recorded during the treatment period in both the exercise and control experiment. There was no difference in body weight across the groups, indicating that the short-term treatment administration was generally well tolerated among the groups.
Data from exercise and nonexercise control experiments are graphed separately throughout the results section. In the exercise experiment group, differences were evident over the 21-day postinfection period. S, OβG, and S+OβG resulted in a decrease in morbidity compared with H2O treatment (P < 0.05) (Fig. 2A). Mean ± SE time-to-sickness was 9.9 ± 2.0 days for H2O mice, 15.1 ± 1.8 days for S mice, 14.5 ± 1.9 days for OβG mice, and 18.6 ± 1.6 days for S+OβG. S, OβG, and S+OβG ingestion resulted in 50, 47, and 25% morbidity, respectively, whereas mice consuming only H2O experienced 73% morbidity. The combined treatment of S and OβG appeared to further enhance the benefits of the independent treatments; however, this did not reach statistical significance (P = 0.1). In the control experiment, S and S+OβG resulted in a decrease in morbidity compared with H2O treatment (P < 0.01) (Fig. 2B). Mean ± SE time-to-sickness was 8.3 ± 1.9 days for H2O mice, 16.2 ± 1.9 days for S mice, 11.3 ± 2.0 days for OβG mice, and 17.6 ± 1.9 days for S+OβG. H2O, S, OβG, and S+OβG ingestion resulted in 80, 37, 63, and 26% incidence in morbidity, respectively.
In the exercise experiment group, differences were evident over the 21-day postinfection period. The combination treatment of S+OβG resulted in a decrease in mortality compared with H2O treatment (P < 0.05) (Fig. 3A). S and OβG treatments resulted in a trend toward decreased mortality; however, these did not reach statistical significance. Mean ± SE time-to-death was 14.7 ± 2.0 days for H2O mice, 17.6 ± 1.7 days for S mice, 17.4 ± 1.6 days for OβG mice, and 18.9 ± 1.6 days for S+OβG. H2O, S, OβG, and S+OβG treatments resulted in 53, 35, 37, and 20% incidence in mortality, respectively. In the control experiment, S and S+OβG resulted in a decrease in mortality compared with H2O and OβG treatment (P < 0.05) (Fig. 3B). Mean ± SE time-to-death was 13.7 ± 2.2 days for H2O mice, 19.4 ± 1.4 days for S mice, 13.7 ± 1.9 days for OβG mice, and 18.6 ± 1.7 days for S+OβG. H2O, S, OβG, and S+OβG ingestion resulted in 53, 21, 53, and 21% incidence in mortality, respectively.
Macrophage antiviral resistance to HSV-1.
Peritoneal macrophages were isolated from mice following 10 days of H2O, S, OβG, or S+OβG ingestion in exercise and control experiments, and their intrinsic antiviral resistance to HSV-1 was examined in culture. Peritoneal macrophages were used because they generally reflect changes in alveolar macrophages and prevent the need to pool large numbers of mice to obtain enough cells. Figure 4 compares the antiviral resistance (expressed as a viability index) of peritoneal macrophages following incubation with HSV-1 KOS. In the exercise experiment (Fig. 4A) S, OβG, and S+OβG increased the intrinsic antiviral resistance of macrophages (P < 0.05); however, there was no further benefit of the combination treatment. In the control experiment there were no statistically significant differences among the groups (Fig. 4B). However, there was a trend toward a beneficial effect of S, OβG, and S+OβG; mice on these treatments had an ∼20% increase in viability of macrophages compared with the H2O group.
Plasma glucose was measured to determine its role on the benefits of S and OβG on susceptibility to infection and macrophage antiviral resistance. In the exercise experiment, there was a main effect of sucrose (S and S+OβG groups) on glucose levels (P < 0.01); sucrose groups had significantly higher glucose levels vs. H2O and OβG groups (Fig. 5A). Similarly, in the control experiment, there was a main effect of sucrose (P < 0.001); sucrose feedings (S and S+OβG groups) resulted in significantly higher glucose levels (Fig. 5B) vs. H2O and OβG groups.
Various nutritional strategies, including carbohydrate-containing sports drinks, β-glucan, and quercetin have been examined for their ability to offset the immune suppression and increased susceptibility to infection that has been associated with stressful exercise (10, 11, 24, 31, 32, 34). Among these, carbohydrate-containing sports drinks have received the most attention, primarily for their ability to offset the exercise-induced alterations in the immune system through suppression of the stress hormone response (16, 29, 31, 34). However, there have been no controlled virus challenge studies in humans or animals that have reported a beneficial effect of carbohydrate-containing sports drinks on susceptibility to respiratory infection. This study used an established mouse model of HSV-1 respiratory infection to examine the effects of sucrose feedings alone and in combination with oat β-glucan on susceptibility to respiratory infection and in vitro macrophage antiviral resistance in both exercise-stressed and rested control mice. The primary findings indicate that sucrose feedings can offset the increase in susceptibility to infection (morbidity) following exercise stress that may be due in part to an increase in macrophage antiviral resistance. These data also support our previous findings of a beneficial effect of oat β-glucan on susceptibility to infection and antiviral resistance of macrophages following exercise stress. However, the combined effect of sucrose and oat β-glucan on susceptibility to infection (morbidity and mortality), while greater than the independent treatments did not reach statistical significance, and there was no further benefit on macrophage function.
Nutritional interventions have been evaluated as possible countermeasures to immune dysfunction following stressful exercise training. Carbohydrate-containing sports drinks have provided the most consistent benefits in human investigations. Studies have reported an attenuation in the increase in blood neutrophil, natural killer cell and monocyte counts, inflammatory cytokines, neutrophil oxidative burst, and the mitogen-induced proliferation of T cells (14, 17, 29, 31, 34, 35) when carbohydrate drinks are administered with exercise. These data are often used to support the hypothesis that carbohydrate feedings can decrease susceptibility to infection. However, this is the first study to use a controlled experimental virus challenge model to show that carbohydrate (sucrose) supplementation can reduce susceptibility to infection (morbidity) following stressful exercise. It is important to note that sucrose feedings in this study were administered prior to the exercise treatment and not during and after exercise training as described in many of the aforementioned human studies. It is possible that the beneficial effects of sucrose may have been even larger had the treatment been administered during and after the exercise bouts.
These are also the first data to show a benefit of sucrose feedings on macrophage antiviral resistance. This, along with the other documented benefits on immune function, is a likely mechanism of protection against respiratory infection. Previous literature has demonstrated that macrophages play a necessary role in the elimination of virus in this model of respiratory infection (24, 26). The exact mechanisms whereby sucrose feedings may increase macrophage function have yet to be determined; however, it is likely that attenuation in stress hormones and maintenance of blood glucose play a role (16, 29, 31, 34). It is well documented that carbohydrate-containing sports drinks can offset the exercise-induced increase in cortisol, which has been shown to play a role in immune function suppression (16, 29, 31, 34), and we have shown that catecholamines play a role in the exercise-induced decrease in antiviral resistance (19). Similarly, the relationship between glucose and optimal immune function has been well established (39). Immune cells, including macrophages, neutrophils, and T and B lymphocytes have been shown to possess major glucose-transporter proteins that are required to support the large energy demands of these cells (13, 21).
These data also confirm our previous findings of a beneficial effect of oat β-glucan on susceptibility to HSV-1 respiratory infection and macrophage antiviral resistance following stressful exercise. Oral feedings of oat β-glucan for 10 consecutive days significantly decreased symptoms of morbidity and revealed a trend toward a decrease in mortality following three consecutive days of exercise stress. As previously demonstrated, oat β-glucan feedings also offset the exercise-induced decrease in macrophage antiviral resistance (9). In contrast, one study in humans did not find any beneficial effects of oat β-glucan feedings on URTI symptoms, natural killer cell activity, neutrophil respiratory burst, or phytohemagglutinin-stimulated lymphocyte proliferation (33). However, the findings of this study were based on self-reported symptomatology and there was no attempt made to identify the pathogenic origin. Furthermore, many immune system components like lung macrophages, which have been shown to play a necessary role in susceptibility to respiratory infection following exercise stress, were not measured in the aforementioned study. Given the difficulty in measuring lung macrophage activity, most human studies do not focus on this component of immune function.
The benefits of β-glucan on host defense have been attributed to activation of various immune system components (36–38). Macrophages, NK cells, and neutrophils contain specific β-glucan receptor sites on their cell membrane, such as complement receptor 3 and dectin-1 (6) that, when bound, result in increased functional activity (7, 38). Using macrophage depletion techniques we have demonstrated that the benefits of oat β-glucan on susceptibility to infection in this model are at least, in part, mediated by lung macrophages; depletion of macrophages using clodronate-filled liposomes negated the beneficial effects of oat β-glucan on susceptibility to HSV-1 respiratory infection (24).
While the independent treatments of sucrose and oat β-glucan feedings did not significantly decrease mortality in this study, the combined treatments did. The combined treatment of sucrose and oat β-glucan resulted in a statistically significant decrease in mortality over the water-treated group (∼35% reduction in mortality) and a nonsignificant reduction in mortality over the independently treated groups (∼15–20%). The failure to find a significant effect of the independent treatments on mortality may be due to the small sample size and resulting low power. While not statistically significant, the mortality effect sizes for oat β-glucan were similar to what we have previously reported for this model of infection and exercise stress (∼20% reduction in mortality following 10 days of oat β-glucan feedings). In addition, for morbidity there was a trend toward a further beneficial effect of the combined treatment over the independent treatments. These data indicate the potential for a combined treatment effect of oat β-glucan and sucrose feedings, this is certainly possible given that both have been shown to modulate different aspects of the immune system.
The effects of sucrose ingestion on susceptibility to respiratory infection and macrophage antiviral resistance were also examined in rested control mice. The control experiment was carried out independently of the exercise experiment to avoid a ceiling or floor effect; a stronger dose of virus was used in the rested control experiment. Sucrose feedings reduced infection rates (morbidity and mortality) in nonexercise control mice, which may have important implications for nonimmunosuppressed individuals. The mechanism for such an effect may be attributed to decreased stress hormones and increased blood glucose concentration, both of which have been associated with optimal immune function (15, 39). There was no effect of oat β-glucan on preventing susceptibility to infection in the control mice; however, the dose of virus used in this study may have been too strong to overcome any potential benefit of oat β-glucan.
Carbohydrate-containing sports drinks have well documented effects as a partial countermeasure for the immunosuppression associated with stressful exercise. However, these are the first data to indicate a beneficial effect of carbohydrate (sucrose) feedings on susceptibility to infection following exercise stress in a controlled mouse model of respiratory infection, the effects of which may be, in part, mediated by its benefits on macrophage antiviral resistance. These findings also confirm our previous report of a beneficial effect of oat β-glucan on susceptibility to HSV-1 respiratory infection and macrophage antiviral resistance following exercise stress. Furthermore, these data indicate a beneficial effect of sucrose feedings on respiratory infection in resting control mice, which may have important implications for nonimmunosuppressed individuals.
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