Human epidemiological studies support the hypothesis that moderate exercise decreases the risk for upper respiratory tract infection (URTI), whereas intense exercise is associated with increased risk (5,9,20,24). However, the strength of this evidence has been debated based on, among other things, a lack of controlled virus challenge studies (5). We have used intranasal inoculation of herpes simplex virus-type one (HSV-1) as a model of respiratory infection to further evaluate the hypothesized exercise effects. The pathogenesis and symptomatology of infection in this model have been well characterized (3,9,11,12,23). Using this model, it has been shown that repeated days of fatiguing exercise increases susceptibility to respiratory infection (3,9), whereas repeated days of short-term moderate exercise decreases susceptibility in male mice (11). The negative effects of fatiguing exercise on infection have also been recently shown in female mice (3). The immune mechanisms could include, at least in male mice, altered macrophage antiviral resistance (9,18), antigen presentation (7), antigen-specific cytokine response, and natural killer cell cytotoxicity (17). The possible immune mechanisms, however, have not been studied in female mice.
Gender differences are clearly evident in other models of infection, including HSV-1 (2,3,14) and other infectious agents (13,27,30), with females generally having reduced susceptibility as compared with males. The gender differences are often studied with a focus on sex hormones (e.g., estrogen, progesterone, and testosterone), but this research is conflicting because of differing species, hormone dosages, and experimental conditions (6,25,29).
Our work on the effects of repeated sessions of prolonged treadmill exercise to fatigue on susceptibility to HSV-1 infection in females shows that females are similar to males in terms of morbidity (sickness symptoms), but have lower overall mortality (3). We hypothesize that differences in macrophage function could be involved in this gender difference in HSV-1 infection. Although other immune mechanisms are also likely to be involved, macrophages have received the most recent attention in animal models. Recent evidence supports a direct role of macrophages in exercise effects on susceptibility to both infection and cancer (9,10,21,22). Although gender differences are evident in some macrophage functions (1,26), no studies have been conducted of macrophage resistance to HSV-1 in female mice at rest or following exercise.
The primary purpose of the present study was to determine if gender differences exist in peritoneal macrophage antiviral resistance to HSV-1 at rest and following repeated daily sessions of moderate and exhaustive exercise. It is hypothesized that alterations in macrophage antiviral resistance to HSV-1 will be associated with gender differences in susceptibility to HSV-1 respiratory infection as previously reported (3).
Male (N = 36) and female (N = 36) CD-1 mice, 8 wk of age were purchased from Harlan Sprague Dawley Labs and acclimated to our facility for at least 1 wk 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. The university's institutional animal care and use committee approved the protocol as described below. Mice were housed three per cage and cared for in the animal facility 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 ad libitum. All experiments were performed at the end of the active dark cycle.
Treadmill acclimation and exercise protocol.
Animals were randomly assigned to control (C), moderate exercise (Mod), or exhaustive exercise (Exh) groups. Male (M-Mod, N = 12 and M-Exh, N = 12) and female (F-Mod, N = 12 and F-Exh, N = 12) exercise mice were acclimated to the treadmill for a period of 20 min·d−1 at 18 m·min−1, 5% grade for the 3 d before experimental exercise bouts. Males and females ran separately. Mice in the moderate exercise groups ran on the treadmill (three per lane) at a speed of 36 m·min−1 and a grade of 8% for 90 min·d−1 for three consecutive days. Mice in the exhaustive exercise groups also ran for 3 d (three per lane) at the same speed and grade, but they ran until they reached volitional fatigue, which typically takes about 150 min (3). Fatigue was defined as the inability to keep up with the speed of the treadmill for 1 min with continual mild hand prodding. This protocol was designed to elicit approximately 70-80% V̇O2max (12,28). Our model, which includes three consecutive days of prolonged treadmill running to fatigue, mimics a short period of severe exercise training similar to what may occur in athletes. The 3 d of 90-min running was chosen as a short period of moderate exercise because this running duration was easily attained by all mice, unlike the run to fatigue. Additionally, 3 d of exercise was chosen because of our experience that the effects of a single session of such exercise on macrophage function and susceptibility to HSV-1 infection are less robust and often require a larger sample size. We interpret the effects of this protocol to be a cumulative effect of repeated daily sessions of exercise. No shock was used because mice readily run with little extraneous motivation. Mice rarely required hand prodding until they approached fatigue. Mice in the control groups (F-C, N = 12 and M-C, N = 12) 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 can be associated with treadmill running. Control and exercise mice were deprived of food and water during the exercise sessions.
Peritoneal macrophage antiviral resistance.
Fifteen minutes following the final bout of exercise or rest, mice were killed 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 (9). 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 two animals of the same group were pooled to obtain sufficient cells. Equal numbers of pools were used in all groups. Cells in each pool were adjusted to a concentration of 2 × 106 cells·mL−1 in cell culture media. Viability, determined using trypan blue exclusion, was typically 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 (about 7-10 plaque-forming units per cell) 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. 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 (9).
Following halothane overdose, blood was collected by draining the inferior vena cava with a heparinized sterile 1-mL syringe. Samples were then transferred to tubes, centrifuged at 3000 rpm, and the plasma was stored at −70°C until further analysis for plasma estrogen.
Statistical analyses were performed using commercial statistical packages from Sigma Stat (SPSS Inc., Chicago, IL). Differences in macrophage antiviral resistance and body weights were compared using a two-way ANOVA (P < 0.05) with Student-Neuman-Keuls post hoc comparisons. Run times to fatigue were compared using a Student's t-test (P < 0.05). Values are reported as mean ± standard error unless otherwise stated.
Body weights were obtained on all animals to ensure that no significant weight loss occurred during the course of the study. No significant body weight changes were found in any of the groups during the course of the exercise treatment period. Table 1 lists average final body weights across the six groups. As expected, males had significantly greater body weights than their female counterparts (P < 0.001); however, neither moderate nor exhaustive exercise had an effect on body weight.
Run time to fatigue.
Average run time to fatigue over the three exercise days was 155.28 ± 4.46 min for F-Exh and 144.64 ± 4.63 min for M-Exh (P < 0.05), but no differences were seen between males and females. This is inconsistent with a previous study using similar exercise protocols, where females ran longer, but this may be because of the lower statistical power in this study as a result of the smaller sample size (N = 12 vs 45 in the previous study) (3). No difference was noted in run times to fatigue during the 3 d of exercise, which indicates that the protocol was relatively well tolerated and that no apparent training affect occurred over this short time period.
Peritoneal macrophage antiviral function.
In this experiment, peritoneal macrophages (pooled cells N = 6 per group) were isolated from the six groups of mice and their intrinsic antiviral function was examined. Figure 1 compares the antiviral function (expressed as a viability index) of peritoneal macrophages from mice killed immediately after the exercise or control period. An overall significant gender difference (P < 0.05) was noted, with females having greater (~4%) viability compared with males. Post hoc analysis revealed that F-C had significantly greater (~10%) macrophage viability as compared with M-C (P < 0.05) and F-Mod had significantly greater (~7%) macrophage viability as compared with M-Mod (P < 0.05); however, no gender differences were observed in the exhaustive exercise groups. A significant exercise effect (P < 0.001) was found, with moderate exercise increasing viability (~9%) and exhaustive exercise reducing viability (~17%) as compared with control, which is consistent with our earlier work in males (12). Although no statistical interactions were found, exhaustive exercise appeared to suppress macrophage viability to a greater extent in females (~25%) than in males (~11%).
Gender differences in symptom severity and mortality have been reported in certain disease and infection models, but little information exists with respect to HSV-1 infection, especially as influenced by exercise. The immune mechanisms also have not been investigated. Given the observation by Han et al. (14) that males have greater mortality following HSV corneal inoculation and our previous observation that males have increased mortality than do females following intranasal inoculation with HSV-1, both at rest and following repeated exercise stress (3), we designed this study to test the hypothesis that these gender differences may be caused partly by differences in macrophage resistance to HSV-1. The results support the hypothesis in resting controls in which macrophage antiviral resistance was lower in males than in females. Following exhaustive exercise, however, macrophage function is similarly suppressed in both males and females, which may correlate with previous observations of increased mortality in males following exhaustive exercise (3). It is interesting that although the association between morbidity (sickness symptoms) and macrophage resistance is consistent with the hypothesis (i.e., morbidity is similarly increased in males and females following exhaustive exercise), more females recover.
Gender differences in susceptibility to many diseases are well characterized, although the direction and magnitude of differences often vary depending on the specific pathogen or disease state. In general, females appear less susceptible to many viral infections (4,8,15). We and others have shown that female mice have lower mortality following intranasal (3) and corneal (14) inoculation with HSV-1. Results from the present study suggest that gender differences are also evident in peritoneal macrophage resistance to HSV-1, with females having greater antiviral resistance than males at rest. Given the primary role of macrophages in resistance to infection, and in particular HSV-1 respiratory infection (24), these findings suggest that gender differences in macrophage antiviral resistance may play an important role in gender differences in susceptibility to HSV-1 in resting mice. The extent to which differences in macrophage antiviral resistance is important in morbidity and mortality following moderate and exhaustive exercise await further research. No data exist on in vivo resistance to HSV-1 infection following moderate exercise in males and females and, as the presents study points out, an apparent mismatch exists in the association between morbidity versus mortality and macrophage function in males and females following exhaustive exercise. It is certainly possible that alterations in macrophage resistance to infection could differentially affect morbidity and mortality. For example, the initial response to pathogens may alter inflammatory cytokine release, inflammation, and associated symptoms that may or may not affect overall survival. This acute increase in inflammation may initially protect from lethal effects of the pathogen, but ultimately may lead to chronic dysregulation of inflammation and an increased susceptibility to autoimmune diseases (16). And, of course, macrophages are not the only immune component involved in overall host defense against infection, including those of the adaptive immune system that could be differentially affected by exercise in males and females.
Macrophages are a vital part of the initial defense against viral infection by limiting viral replication by inactivating extracellular virus and suppression virus replication in adjacent cells, and by destroying infected cells. Macrophages also play a key role in upregulation of the adaptive immune system to further fight infection. Our finding of greater intrinsic macrophage resistance to HSV-1 in females is consistent with other reports of greater phagocytic function (26) and increased superoxide dismutase and catalase activities in females (1). Together, these findings suggest that increased macrophage function may partially account for previous observations that females are less susceptible to the lethal effects of in vivo HSV-1 infection.
It is also likely that other components of the innate and adaptive immune system (e.g., natural killer cells, neutrophils, and lymphocytes) contribute to the gender differences in resistance to infection in this model. Much less is known, however, about these other immune parameters, especially in conjunction with exercise effects. We have previously shown that macrophage antiviral resistance plays an important role in our model of HSV-1 infection, both at rest and following moderate and exhaustive exercise, and in response to nutritional immunomodulators (9,11).
Results from the present study and previous ones from our laboratory suggest that macrophage antiviral resistance to HSV-1 increases following moderate treadmill exercise (11,21) and decreases following an acute bout or 3 d of prolonged treadmill running to exhaustion (9) in male mice, and that this is associated with decreased and increased susceptibility to HSV-1 infection in vivo (11,21). We and others have shown that peritoneal and alveolar macrophage functions likely play an important role in this model of HSV-1 infection (9,18,19,21). This is the first experimental study to report similar macrophage responses in female and male mice following both moderate and exhaustive treadmill exercise. Clearly, however, the relationship between macrophage function and overall host defense is more tenuous in female mice, given the lack of association between decreased macrophage function and increased mortality following exhaustive exercise and the absence of data on the potential benefits of moderate exercise on HSV-1 mortality in females.
The present study used a shorter moderate exercise protocol consisting of only 3 d of 90 min·d−1 and older animals (8-9 wk) as compared with our previous study that used 6 d of 1 h·d−1 and younger animals (4-5 wk) (11). We also assessed resident peritoneal macrophages instead of alveolar macrophages because of their relative abundance that reduces the need for pooling. Although measurement of alveolar macrophages would have more direct relevance to resistance to respiratory infection, we have found that resistance to HSV-1 and cytotoxicity to B16 melanoma are similar in peritoneal and alveolar macrophages and they respond similarly to moderate and exhaustive exercise (data not shown). In the present study, we chose initially to examine peritoneal macrophages because HSV-1 infection is not isolated to the lungs and death occurs following its spread to other tissues (e.g., the adrenals and brain). Future studies will more specifically address the relative importance of lung and peritoneal macrophages in this model. Further research is warranted to specifically address the relative importance of lung and peritoneal macrophages in this model and the more specific threshold at which exercise enhances or impairs macrophage function in female and male mice along with the mechanisms responsible for the differences. Most likely contributing to increased macrophage function in females are differences in sex hormones (6), but this could not be evaluated in this study because plasma hormones, testosterone and progesterone, were not measured, and estrus cycle was not controlled. In fact, it is certainly possible that bigger differences would have been found if females had all been tested in the proestrus or estrus phase when estrogen and progesterone are at their highest concentration.
To our knowledge, this is the first evidence of a gender difference in macrophage antiviral resistance to HSV-1. It is also the first study to evaluate the effects of repeated daily sessions of moderate and exhaustive exercise on macrophage function in female mice. The data suggest that female mice have greater macrophage antiviral resistance to HSV-1 than males, but respond similarly to short-term moderate and exhaustive exercise, which we interpret to be a cumulative effect of repeated daily sessions. These data, in conjunction with our previous evidence that females are less susceptible to the lethal effects of HSV-1 infection, suggest that the protection in females may be mediated, in part, by greater macrophage antiviral resistance to HSV-1, at least in resting mice. Further research is necessary to better understand the relationship between altered macrophage function and host defense to HSV-1 in females following repeated daily sessions of moderate and exhaustive exercise, the role of inflammation and sex hormones on these observed gender differences, and to address the more specific threshold at which exercise modifies macrophage function and susceptibility to respiratory infection in male and female mice.
This study was supported by a doctoral student grant from the American College of Sports Medicine Foundation.
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