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Am J Physiol Regul Integr Comp Physiol 294: R1593-R1599, 2008. First published March 19, 2008; doi:10.1152/ajpregu.00562.2007
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ENVIRONMENTAL, EXERCISE AND RESPIRATORY PHYSIOLOGY

Benefits of oat β-glucan on respiratory infection following exercise stress: role of lung macrophages

E. A. Murphy,1 J. M. Davis,1 A. S. Brown,1 M. D. Carmichael,1 J. A. Carson,1 N. Van Rooijen,4 A. Ghaffar,3 and E. P. Mayer2

1Division of Applied Physiology, Department of Exercise Science, Arnold School of Public Health and the 2Department of Pathology, Microbiology and Immunology, School of Medicine, University of South Carolina, Columbia, South Carolina; 3Department of Microbiology and Immunology, University of Health Sciences, Lahore, Pakistan; and 4Department of Cell Biology and Immunology, Vrije Universiteit of Amsterdam, Amsterdam, The Netherlands

Submitted 3 August 2007 ; accepted in final form 18 March 2008


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Exercise stress is associated with an increased risk for upper respiratory tract infection (URTI). We have shown that consumption of the soluble oat fiber β-glucan (OβG) can offset the increased risk for infection and decreased macrophage antiviral resistance following stressful exercise; however, the direct role of macrophages is unknown. This study examined the effect of macrophage depletion on the benefits of orally administered OβG on susceptibility to infection (morbidity, symptom severity, and mortality) following exercise stress. CL2MDP (Ex- H2O-CL2MDP, Ex-OβG-CL2MDP, Con-H2O-CL2MDP, Con-OβG-CL2MDP)-encapsulated liposomes were administered intranasally to deplete macrophages, and PBS (Ex-H2O-PBS, Ex-OβG-PBS, Con-H2O-PBS, Con-OβG-PBS)-encapsulated liposomes were given to macrophage-intact groups. Ex mice ran to volitional fatigue on a treadmill for 3 consecutive days, and OβG mice were fed a solution of 50% OβG in their drinking water for 10 consecutive days before infection. Fifteen minutes following the final bout of Ex or rest, mice were intranasally inoculated with 50 µl of a standardized dose of herpes simplex virus-1. Ex increased morbidity (P < 0.001) and symptom severity (P < 0.05) but not mortality (P = 0.09). The increase in morbidity and symptom severity was blocked by OβG consumption for 10 consecutive days before exercise and infection [morbidity (P < 0.001) and symptom severity (P < 0.05)]. Depletion of macrophages negated the beneficial effects of OβG on reducing susceptibility to infection following exercise stress, as evidenced by an increase in morbidity (P < 0.01) and symptom severity (P < 0.05). Results indicate that lung macrophages are at least partially responsible for mediating the beneficial effects of OβG on susceptibility to respiratory infection following exercise stress.

mice; herpes simplex virus-1; clodronate liposomes; innate immunity; nutrition

EXERCISE CAN MODULATE MANY functions of the immune system and alter susceptibility to upper respiratory tract infection (URTI). Data generally support the hypothesis that exercise stress can lead to immunosuppression and increased risk of infection [morbidity (time to sickness), symptom severity, and mortality (time to death)] (3, 4, 10, 12, 27, 30). The mechanisms for the increased susceptibility to infection following intense exercise are thought to be mediated by alterations in the stress response. Treadmill exercise has been well characterized as a physical stress (23), and it has been shown to increase circulating levels of stress hormones, leading to an impaired innate (20) and adaptive immune response (14) and thus increased susceptibility to infection. The effects associated with exercise stress are not unlike those that have resulted in other stress paradigms such as psychological stress, which has been shown to alter infectivity of a variety of pathogens, including herpes simplex virus (HSV)-1 and influenza (1, 16, 45).

Studies have focused on alterations in macrophage function as a mechanism for the detrimental effects of exercise on susceptibility to viral infection (10, 12). Macrophages act as a first line of defense in eliminating viral pathogens by inhibiting virus growth within itself, as well as to inactivate extracellular virus, suppress virus replication in adjacent cells, and destroy infected cells (46). Alveolar and peritoneal macrophage antiviral resistance to HSV is reduced in mice following exercise to fatigue (10, 12), as is macrophage antigen presentation (7). However, a direct role of macrophages on the increased susceptibility to infection following exercise stress has not been addressed.

Various nutritional strategies, including glutamine (6), zinc (33), antioxidants (31), and carbohydrates (28, 29), have been investigated as possible countermeasures for immunosuppression during periods of stressful exercise training and competition. However, there is no direct evidence from controlled experimental animal studies that any of these nutritional strategies can counteract the exercise-induced increase in susceptibility to infection or decrease in macrophage antiviral resistance of macrophages. Recent work in our laboratory has focused on the possible role of β-glucan (soluble oat fiber) feedings on the exercise-induced alterations on susceptibility to infection and proposed immune mechanisms (12).

β-Glucan is a polysaccharide derived from the cell wall of yeast, fungi, algae, and oats that has well-documented immunostimulant properties. β-Glucan can enhance the activities of both the innate and specific immune system components via direct activation of β-glucan-specific receptors on macrophages, neutrophils, and natural killer cells (9, 41) or indirectly following activation of pinocytic M cells located in the Peyer's patches of the small intestine (32, 34, 36). β-Glucan in general has been shown to enhance the resistance to various viral (12, 44), bacterial (13, 49), protozoan (48), and fungal diseases (2) and to promote antitumor activity (24, 35). Although fewer studies have been done on β-glucan derived from oats, a decrease in susceptibility to infection following viral (12), bacterial (13, 49), and protozoan (48) challenges has been reported.

We have found that 10 consecutive days of oat β-glucan feedings can offset the increased risk for URTI associated with stressful exercise (12), which may be mediated by its ability to block the decrease in macrophage antiviral resistance (12). However, a direct test of the role of macrophages in this response has not been done. Depletion of macrophages in various tissues in vivo is a technique that has been used to determine the role of macrophages in various infection models. The clodronate-filled liposome (CL2MDP-lip)-mediated suicide approach should be effective for this purpose because it can eliminate macrophage function selectively at various sites of the body (39). It has been shown that no other cell populations except macrophages are affected by this treatment (38). After phagocytosis of the CL2MDP-lip, the phospholipid bilayers of the liposomes are disrupted, and clodronate (CL2MDP) is released in the cytoplasm, which causes apoptotic cell death of macrophages (40). This technique has not yet been used in studies involving exercise stress or any form of β-glucan. However, using this technique, we have recently shown an important role of macrophages on the beneficial effects of moderate exercise on susceptibility to infection (25).

The purpose of this study was to determine the role of lung macrophages on the benefits of oral feedings of the soluble oat fiber β-glucan in terms of susceptibility to URTI following stressful exercise. Altered susceptibility to infection will be measured as changes in morbidity (time to sickness), symptom severity, and mortality (time to death). This was done using a murine model of exercise, respiratory infection (3, 4, 10, 12), and in vivo lung macrophage depletion (25). The exercise protocol consisted of three consecutive days of prolonged treadmill running to fatigue designed 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 designation, and documented health benefits as an important component of the Heart-Healthy diet in various pathological conditions, including diabetes and cardiovascular disease (19, 47). Macrophage depletion was done in the lungs using CL2MDP-lip; this technique has been shown to eliminate 75% of lung macrophages (21).


   METHODS
TOP
 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Animals. Male CD-1 mice, 4 wk of age, were purchased from Harlan Sprague Dawley Labs and acclimated to our facility for at least 3 days 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, 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 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. Figure 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. The Institutional Animal Care and Usage Committee of the University of South Carolina approved all experiments.


Figure 1
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  		Fig. 1. Experimental design schematic.

 
Nutrient treatment. Mice were randomly assigned to one of the following eight groups (n = 19–30/group): exercise-water-macrophage intact (Ex-H2O-PBS; n = 27), exercise-water-macrophage depletion (Ex-H2O-CL2MDP; n = 25), exercise-oat β-glucan-macrophage intact (Ex-OβG-PBS; n = 22), exercise-oat β-glucan-macrophage depletion (Ex-OβG-CL2MDP; n = 19), control-water-macro phage intact (Con-H2O-PBS; n = 26), control-water-macrophage depletion (Con-H2O- CL2MDP; n = 26), control-oat β-glucan-macrophage intact (Con-OβG-PBS; n = 27), and control-oat β-glucan-macrophage depletion (Con-OβG-CL2MDP; n = 25). H2O mice received tap water for the 10 days before inoculation, whereas OβG mice were fed a solution of oat β-glucan for the 10 days before 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) that was dissolved in the drinking water at a concentration of 0.8 mg/ml and made fresh daily. 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 days following 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 days of oat β-glucan/water consumption, Ex mice were acclimated to the treadmill for a period of 20 min/day for the 3 days before the experimental exercise bouts. The exercise protocol consisted of an exhaustive exercise bout of treadmill running (performed in the evening, 6:00 P.M.) for three consecutive days. Mice in the exercise groups ran on the treadmill (2/lane) at a 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 the mouse was removed from the treadmill. Electric shock was never used in these experiments, since 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 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.

CL2MDP administration. CL2MDP-lip were a kind gift from Roche Diagnostics (Mannheim, Germany). CL2MDP-lip were prepared as previously described (39). CL2MDP-lip were administered to mice 2 days before infection with HSV-1 (following day 1 of exercise), as well as on day 4 postinfection to deplete lung macrophages. Lung macrophage depletion was done via intranasal administration: mice received 100 µl of CL2MDP. This procedure results in the selective depletion of almost all lung macrophages while other cells remain unaffected after the elimination procedure (37, 42, 43). Some macrophages begin to return after a period of 5 days, but complete repopulation does not occur until around day 18 (37). Mice were briefly anesthetized using halothane for this procedure. Macrophage-intact mice received similar doses of PBS-filled liposomes. CL2MDP-lip are engulfed by macrophages via endocytosis. After disruption of the phospholipid bilayers of the liposomes under the influence of the lysosomal phospholipases in the macrophage, CL2MDP, which is dissolved in the aqueous compartments between the liposomal bilayers, is released in the cell. The CL2MDP is accumulated intracellularly, and, after exceeding a threshold concentration, the cell is irreversibly damaged and dies by apoptosis (36).

In vivo titration of HSV-1. Intranasal inoculation of HSV-1 VR strain in the mouse is an established experimental model of respiratory infection (3, 4, 10–12, 25, 26). Although HSV-1 is not a common respiratory virus in humans, it can cause various pathological conditions in humans such as meningoencephalitis, hepatitis, esophagitis, tracheobronchitis, and pneumonia and is associated with cases of adult respiratory distress syndrome (26). 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% FBS and 2% penicillin, streptomycin, and L-glutamine. 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, mortality, and symptom severity were monitored for 21 days.

Intranasal infection with HSV-1. On the day of the experiment, mice were exposed to either control treatment or exercise. Immediately following exercise or control treatment, mice were returned to their cages, and 15 min later they were lightly anesthetized with halothane and inoculated intranasally with 50 µl of HSV-1 VR strain. The dose yielded a 30–40% mortality rate among control mice in preliminary dose-response experiments. The actual dose [plaque-forming unit (PFU)/ml] of this virus was determined by plaque titration to be 1.28 X 105 PFUs/mouse. Lung viral titers were not measured in this study; however, administration of this dose typically results in a range of 1.2–2.6 X 105 PFUs in the lungs 2 days following infection. The pathogenesis and symtomatology of infection following intranasal inoculation of HSV have been well characterized (3, 4, 10–12, 25, 26). Following infection, the mice were returned to their respective cages and housed in an isolated P2 facility. All animals were monitored two times daily by an investigator blinded to the treatments for a period of 21 days for signs of morbidity, symptom severity, and mortality. Several typical symptoms of illness were included in the symptom severity scale (see Table 1), including ruffled fur, redness around the eyes, nose, or mouth, hunched back, and unresponsiveness. Each symptom was given a score from one to three depending on the severity of that symptom. Symptoms that generally appear at onset of sickness were weighted less (ruffled fur, redness around the eyes) than symptoms appearing as the illness became more severe (hunched back pose, unresponsiveness). In this model, symptoms such as ruffled fur, lesions, and red eyes will appear first (days 3–4) followed by neurological symptoms or the mouse displaying a "hunched back" pose (days 5–6); mice that are severely ill will be unresponsive (after day 6). Mice that displayed any of these sickness symptoms were considered morbid.


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  		Table 1. Symptom severity scale for herpes simplex virus-1 infection

 
Statistical analysis. Statistical analyses were performed using a commercially available statistical package 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 symptom severity over the 21-day postinfection period, body weights, and fluid consumption were done using a three-way Analysis of Variance in SAS with the Student-Newman-Keul's post hoc analysis (P < 0.05) (exercise X oat β-glucan X macrophage). Differences in treadmill run times were analyzed using a two-way Analysis of Variance in SigmaStat with the Student-Newman-Keul's post hoc analysis (P < 0.05) (oat β-glucan X macrophage).


   RESULTS
TOP
 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Exercise treatment. Run time to fatigue was not significantly different between the exercise groups. Average run time to fatigue over the three exercise days was 149 ± 6 min for Ex-H2O-PBS, 143 ± 5 min for Ex-H2O-CL2MDP, 149 ± 7 min for Ex-OβG-PBS, and 149 ± 4 min for Ex-OβG-CL2MDP. Run times were also not different on days 1–3 of exercise, which indicates that the protocol was relatively well tolerated by the mice and that there was no apparent training affect that occurred over this time period.

Nutrient consumption and weight gain. There were no differences in the average amount of fluid consumed by the oat β-glucan, exercise, or macrophage depletion groups. Over the course of the 10-day ingestion period, mice consumed ~6.2 ml/day of fluid that resulted in a daily dose of oat β-glucan of ~2.5 mg/mouse; this dose of oat β-glucan is similar to the recommended daily dose in humans that is necessary to conform to a "heart healthy diet" as recommended by the Food and Drug Administration (FDA). There was also no difference in body weight across the groups over time.

Morbidity. Data from exercise and nonexercise control groups are graphed separately for morbidity, mortality, and symptom severity to more clearly present the most important results of this study. Group differences in morbidity were evident over the 21-day postinfection period. Intranasal administration of HSV-1 following 3 days of exercise stress resulted in an increase in morbidity (i.e., time to sickness) compared with resting controls (P < 0.001), exercise mice drinking water with intact macrophages (Ex-H2O-PBS) experienced an 89% incidence in morbidity while only 46% of control mice drinking water with intact macrophages (Con-H2O-PBS) got sick [comparison not made graphically, since the data from exercise and nonexercise groups are presented separately; these results are consistent with our previous findings (10, 12)]. Consumption of oat β-glucan for 10 consecutive days before infection offset this increase in morbidity (P < 0.001); only 55% of exercise mice drinking oat β-glucan with intact macrophages (Ex-OβG-PBS) displayed symptoms of sickness (Fig. 2A); this was not significantly different from the control mice drinking water with intact macrophages (Con-H2O-PBS). Administration of CL2MDP-lip to exercise mice negated the beneficial effects of oat β-glucan (P < 0.01); exercise mice drinking oat β-glucan with depleted macrophages (Ex-OβG-CL2MDP) experienced an 84% morbidity compared with 55% for mice with intact macrophages (Ex-OβG-PBS) (Fig. 2A). There was no significant difference between exercise mice drinking water with intact macrophages (Ex-H2O-PBS) (89%) and those with depleted macrophages (Ex-H2O-CL2MDP) (96%). Control mice with depleted macrophages experienced greater morbidity than control mice with intact macrophages, Con-H2O-CL2MDP experienced 88% morbidity compared with 46% for Con-H2O-PBS (P < 0.001), and Con-OβG-CL2MDP experienced 92% morbidity compared with only 37% for Con-OβG-PBS (P < 0.001) (Fig. 2B).


Figure 2
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  		Fig. 2. Time course of morbidity across the 21-day postinfection period for exercise (A) and nonexercise control (B) groups (n = 19–30 animals/group). A: *P < 0.05 for exercise oat β-glucan macrophage intact (Ex-OβG-PBS) vs. exercise water macrophage intact (Ex-H2O-PBS) and for exercise oat β-glucan macrophage intact (Ex-OβG-PBS) vs. exercise oat β-glucan macrophage depletion (Ex-OβG-CL2MDP). B: *P < 0.05 for control water macrophage intact (Con-H2O-PBS) vs. control water macrophage depletion (Con-H2O-CL2MDP) and for control oat β-glucan macrophage intact (Con-OβG-PBS) vs. control oat β-glucan macrophage depletion (Con-OβG-CL2MDP).

 
Symptom severity. Sickness symptoms were also graded to better address possible differences in severity of sickness (see Table 1). Morbidity data simply indicate the day on which the first symptom was observed. The mean time to death across all groups was day 10; symptom severity was analyzed across groups on days before this (days 1–9). Figure 3 shows the symptom severity score for the eight groups of mice for the 9 days following infection. Symptom severity was significantly different across the groups. Exercise mice drinking water with intact macrophages (Ex-H2O-PBS) experienced a significantly higher symptom severity score than control mice drinking water with intact macrophages (Con-H2O-PBS) on days 5–9 (P < 0.05) (comparison not made graphically, since the data from exercise and nonexercise groups are presented separately). Consumption of oat β-glucan for 10 consecutive days before infection offset this increased symptom severity (Fig. 3A); severity of symptoms in exercise mice drinking oat β-glucan with intact macrophages (Ex-OβG-PBS) was not different from the control mice drinking water with intact macrophages (Con-H2O-PBS). Administration of CL2MDP-lip to exercise mice negated the beneficial effects of oat β-glucan on reducing severity of symptoms following exercise stress, and exercise mice drinking oat β-glucan with depleted macrophages (Ex-OβG-CL2MDP) experienced significantly greater symptom severity scores on days 5–9 compared with mice with intact macrophages (Ex-OβG-PBS) (P < 0.05) (Fig. 3A). There was no significant difference between exercise mice drinking water with intact macrophages (Ex-H2O-PBS) and those with depleted macrophages (Ex-H2O-CL2MDP). Control mice with depleted macrophages experienced greater symptoms than control mice with intact macrophages, Con-H2O-CL2MDP experienced greater symptom severity on days 5–9 compared with Con-H2O-PBS (P < 0.05), and Con-OβG-CL2MDP experienced greater symptom severity on days 5–9 compared with Con-OβG-PBS (P < 0.05) (Fig. 3B).


Figure 3
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  		Fig. 3. Time course of symptom severity across the 21-day postinfection period for exercise (A) and nonexercise control (B) groups (n = 19–30/group). A: *P < 0.05 for Ex-OβG-PBS vs. Ex-H2O-PBS And for Ex-OβG-PBS vs. Ex-OβG-CL2MDP. B: *P < 0.05 for Con-H2O-PBS vs. Con-H2O-CL2MDP and for Con-OβG-PBS vs. Con-OβG-CL2MDP.

 
Mortality. Similar treatment effects were found for mortality (i.e., time to death) over the 21-day postinfection period (Fig. 4, A and B). Intranasal administration of HSV-1 following 3 days of exercise stress resulted in a nonsignificant 21% increase in mortality compared with resting controls (P = 0.09); exercise mice drinking water with intact macrophages (Ex-H2O-PBS) experienced a 44% incidence in mortality, whereas only 23% of control mice drinking water with intact macrophages (Con-H2O-PBS) exhibited symptoms of mortality [comparison not made graphically, since the data from exercise and nonexercise groups are presented separately; these results are consistent with our previous findings (10, 12)]. Consumption of oat β-glucan for 10 consecutive days before infection resulted in a 17% decrease (nonsignificant) in mortality compared with exercise mice drinking water (P = 0.1); only 27% of exercise mice drinking oat β-glucan with intact macrophages (Ex-OβG-PBS) experienced mortality. This was not significantly different from control mice drinking water with intact macrophages (Con-H2O-PBS) (23%). Administration of CL2MDP-lip to exercise mice negated the effect of oat β-glucan (P < 0.05); exercise mice drinking oat β-glucan with depleted macrophages (Ex-OβG-CL2MDP) experienced a 53% mortality compared with mice with intact macrophages (Ex-OβG-PBS) (27%). There was no significant difference between exercise mice drinking water with intact macrophages (Ex-H2O-PBS) (44%) and those with depleted macrophages (Ex-H2O-CL2MDP) (50%). Control mice with depleted macrophages experienced greater mortality than control mice with intact macrophages, Con-H2O-CL2MDP experienced 50% mortality compared with 23% for Con-H2O-PBS (P = 0.07), and Con-OβG-CL2MDP experienced 32% mortality compared with only 11% for Con-OβG-PBS (P = 0.08). The relatively small sample size for survival analysis and resulting relatively low-power probably accounts for the failure to find statistically significant differences among some groups that are likely to be different (i.e., P values >0.05 to <0.1); however, the magnitude of the differences in mortality for exercise and oat β-glucan are similar to those that have been reported previously (10, 12) and follow similar trends to the highly significant effects on morbidity and symptom severity.


Figure 4
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  		Fig. 4. Time course of mortality across the 21-day postinfection period for exercise (A) and nonexercise control (B) groups (n = 19–30/group). A: *P < 0.05 for Ex-OβG-PBS vs. Ex-OβG-CL2MDP, #P < 0.1 for Ex-OβG-PBS vs. Ex-H2O-PBS. B: #P < 0.1 for Con-H2O-PBS vs. Con-H2O-CL2MDP and for Con-OβG-PBS vs. Con-OβG-CL2MDP.

 

   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
Evidence of a possible benefit of nutrition on macrophage function and risk of infection during periods of exercise stress is limited. Our laboratory has examined the effect of oat β-glucan on preventing the exercise-induced alterations in macrophage function and susceptibility to infection. Previously, we have reported that oat β-glucan can offset the negative effects of exercise on susceptibility to infection; oat β-glucan was also shown to counteract the decrease in intrinsic macrophage antiviral resistance following exercise stress (12). This study used an established animal model of exercise, respiratory infection, and in vivo macrophage depletion to determine the specific role of macrophages on the benefits of oral feedings of the soluble oat fiber β-glucan on susceptibility to URTI (morbidity, symptom severity, and mortality) following stressful exercise in mice. The primary results indicate that depletion of lung macrophages using CL2MDP-lip negates the beneficial effects of oat β-glucan on susceptibility to infection (morbidity and symptom severity) following stressful exercise. Overall, the data suggest that lung macrophages play a direct role on the benefits of oat β-glucan in offsetting the increased risk for infection following exercise stress.

Animal models have been used to examine the effects of exercise stress on susceptibility to infection and possible immune mechanisms (3, 4, 8, 10, 12, 17, 18, 22). The specific pathogenesis and symtomatology of HSV-1 infection via intranasal inoculation is well characterized in the literature (26) and has been used in other stress paradigms, including psychological stress (45). The exercise protocol in this experiment is designed to mimic a short period of heavy training similar to what may occur in athletes and military personnel. The results of this study generally support the negative effects of this type of exercise stress on susceptibility to infection (3, 4, 10, 12). Macrophages have been implicated in the detrimental effects of exercise stress on infection risk because of their clear role as a first line of defense against most infectious agents (3, 10, 12). Two types of macrophage-mediated resistance mechanisms have been described, which include intrinsic and extrinsic resistance (46). Intrinsic resistance refers to the capacity of the macrophage to inhibit virus growth within the cell itself, and extrinsic resistance refers to the macrophages ability to inactivate extracellular virus, suppress virus replication in adjacent cells, and destroy infected cells. Both alveolar and peritoneal intrinsic antiviral resistance of macrophages is reduced following exercise stress (10, 12, 20), as is macrophage antigen presentation (7).

Evidence of a possible benefit of nutrition on macrophage function and risk of infection during periods of exercise stress is limited. 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 function (11, 12, 24, 32, 34, 36), and can enhance host resistance to fungal (2) and viral (12, 44) diseases and cancer (24, 35). Less is know about the immunostimulant properties of soluble oat β-glucan, which incidentally is an important fiber component of the "Heart-Healthy" diet as defined by the FDA (19). Oat β-glucan has been shown to stimulate macrophage function (12, 13, 48) and increase resistance to viral (12), bacterial (13, 49) and protozoan (48) infections in mice.

The benefits of β-glucan on host defense have been attributed to activation of various immune system components (34, 36, 41). Macrophages, natural killer (NK) cells, and neutrophils contain specific β-glucan receptor sites on their cell membrane, such as complement receptor 3 and dectin-1 (5), that when bound results in increased functional activity (9, 41). 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). Following 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, which results in release of cytokines that are responsible for initiating an extensive cascade of systemic immune responses (15, 32, 34). It is also possible for very small β-glucan particles to be absorbed directly in the lymphatic and vascular systems where they can interact directly with circulating immune cells via their β-glucan receptors (19, 47). Previously, in our laboratory, we have reported a beneficial effect of oat β-glucan on offsetting the increased risk for infection following exercise stress. Oral feedings of oat β-glucan for 10 consecutive days significantly decreased symptoms of morbidity and mortality following 3 consecutive days of exercise stress (12); however, there was no direct evidence that lung macrophages were playing a role in this response. The beneficial effects of oat β-glucan in this model of infection and exercise stress are confirmed in this study. The most important finding of this study is that lung macrophages are necessary mediators for the beneficial effects of oral feedings of oat β-glucan on reducing susceptibility to infection following exercise stress, as evidenced by the fact that depletion of lung macrophages negated the beneficial effects of oat β-glucan.

Selective depletion of macrophages in vivo through administration of liposomes containing dichloromethylene diphosphonate is a technique that has been used to determine the role of macrophages in various infection models. This study does not elicit the precise activities of macrophages that are upregulated by oat β-glucan. However, previously in our laboratory, we have reported that oral feedings of oat β-glucan enhanced macrophage intrinsic resistance to HSV-1 in control mice and blocked the suppression associated with exercise stress (12). 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 cytokine release, acid phosphatase activity, phagocytosis, and H2O2 production (32, 36), as reported by others. Similar effects of macrophage depletion were found in nonexercise control mice following oat β-glucan feedings. However, it appears that macrophage depletion in nonexercise control mice consuming oat β-glucan had less severe effects on susceptibility to infection than in nonexercise control mice drinking water. This indicates that macrophages are not the only cell responsible for the beneficial effects of oat β-glucan, which is not surprising, since other immune cells have been reported to be activated by oat β-glucan, for example, neutrophils and NK cells may play an important role in this model of infection.

The results from this study demonstrate that macrophages are at least partially responsible for the benefits of oat β-glucan on reducing susceptibility to URTI (based primarily on the highly significant effects of morbidity and symptom severity, along with similar trends in mortality) following short-term exercise stress in mice. However, the exact mechanisms whereby oat β-glucan activates macrophages and the specific functions of macrophages that are enhanced by oat β-glucan in this model of infection and exercise stress have yet to be elucidated.


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This work was funded by a grant from the American College of Sports Medicine.


   FOOTNOTES
 

Address for reprint requests and other correspondence: J. M. Davis, PHRC, #301, 921 Assembly St., Univ. of South Carolina, Columbia, SC 29208 (e-mail: jmdavis{at}sc.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


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E. A. Murphy, J. M. Davis, M. D. Carmichael, E. P. Mayer, and A. Ghaffar
Benefits of oat {beta}-glucan and sucrose feedings on infection and macrophage antiviral resistance following exercise stress
Am J Physiol Regulatory Integrative Comp Physiol, October 1, 2009; 297(4): R1188 - R1194.
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