Regular participation in vigorous physical activity presents a unique set of challenges, as acute exercise induces inflammation and the formation of reactive oxygen species (ROS) (32). These effects are clearly seen in the gastrointestinal compartment, which is densely populated with immunologically active cells (i.e., lamina propria, intraepithelial, and intestinal lymphocytes). Consequent to oxidant stress, intense exercise elevates plasma concentration of the proinflammatory cytokines tumor necrosis factor (TNF)-α and interleukin (IL)-1β (26). In contrast, exercise training confers several positive health effects, many of which are independent of age. These include restoration of muscle mass and function, improved cellular respiratory capacity, and elevated endogenous antioxidant concentration (4). Moreover, regular exercise may alleviate oxidant stress by decreasing and increasing proinflammatory and anti-inflammatory cytokine production, respectively. In young C57BL/6 mice, baseline lymphocyte expression of proinflammatory TNF-α decreases (13) and concentration of anti-inflammatory IL-10 increases (14) with voluntary freewheel running. This anti-inflammatory effect of training occurs in nonlymphoid tissues as well. Gomez-Merino et al. (8) found that training decreases IL-1β in adipose tissue of young male Wistar rats. Indeed, regular activity has been consistently shown to improve the cytokine profile across all ages, which may abrogate (at least in part) immunoscenescent changes (4).
Aging is accompanied by varied functional declines that complicate the exercise response (4). These include heightened as well as blunted physiologic and immunologic responses to stress (10,39). For example, old (21–24 months) Fisher 344/N rats had an adrenocorticotropic hormone (ACTH) response to hemorrhagic stress that was <50% of that demonstrated in young (3–4 months) animals (11). This is important given that acute exercise is a stressful event that can promote inflammation by activating the hypothalamic–pituitary–adrenal axis (24). Specifically, catecholaminergic (β-adrenergic) receptors on lymphocytes can induce IL-6 and TNF-α production through NF-κB activation (24). Huang et al. (16) demonstrated experimentally (in rat spleen) that norepinephrine exposure (dose range: 10−6 to 10−4 M) induces IL-6 production from lymphocytes. Finally, elderly individuals also show reduced protein synthesis in response to heavy exercise (36). Dennis et al. (6) found significant increases in TIMP1 and α-cardiac actin (ACTC1) mRNA in the vastus lateralis muscle after resistance exercise in young individuals (32 ± 7 yr); in contrast, no significant postexercise changes in gene expression of either TIMP1 or ACTC1 were reported for elderly individuals (72 ± 5 yr). In response to acute exercise, no change in skeletal muscle IL-1β gene expression was observed in healthy seniors (62–72 yr), whereas a 3.5-fold increase was observed in younger subjects (20–34 yr) (17). These findings suggest that older individuals may have a blunted protein synthesis response to acute exercise stress.
Many cellular declines begin in early to late middle age; in fact, loss of muscle mass/decreased muscle proliferation occurs in people as young as 25 yr (20). These changes accumulate, becoming observable in late middle age or old age because the physiologic “threshold” for cellular damage is exceeded (22). In the elderly, the culmination of these changes is observed as broad functional declines. Middle adulthood may thus be the “ideal” age for preventative health or lifestyle interventions. King et al. (18) demonstrated that the protective effect of regular exercise on functional capacity in late life is promptly recognized if physical activity is begun in middle adulthood. Accordingly, understanding the effects of exercise-associated cytokine and apoptotic protein production in older individuals is important for several reasons. First, there is a growing segment of highly active older adults who are involved in moderate-to-high-intensity exercise (31). Second, with advancing age, there is an increase in basal inflammation (4) and an impaired stress response (10). Third, training is associated with many positive health effects and may slow the rate of immunosenescence (13).
The purpose of this study was to describe the effects of training on lymphocyte expression of 1) proinflammatory and anti-inflammatory cytokines and 2) proapoptotic and anti-apoptotic proteins in response to an acute exercise challenge. We hypothesized that older (15–16 months) mice exposed to long-term freewheel running would have increased expression of proinflammatory (TNF-α, IL-1β) cytokines and proapoptotic (caspase-3, caspase-7) proteins and decreased expression of anti-inflammatory (IL-10) cytokines and anti-apoptotic (Bcl-2) proteins in intestinal lymphocytes after acute treadmill exercise. In other words, trained older mice would parallel the intestinal lymphocyte cytokine and apoptotic protein expression observed in young mice after acute treadmill exercise (13,14). We also hypothesized that older untrained mice would be less responsive in their cytokine and apoptotic protein response to acute exercise stress, suggestive of immunosenescence.
Female C57BL/6 mice (n = 80; Harlan, Indianapolis, IN), 11 months old at the start of the study, were individually housed at 21°C ± 1°C on a 12:12-h reversed light–dark cycle for 4 months until study termination (i.e., at 15–16 months of age). At 16 months, female C57BL/6 mice are not capable of maintaining viable pregnancies (29). Mice had ad libitum access to standard rodent diet (Lab Rodent Chow; PMI Feeds, Richmond, IN) and tap water throughout the experiments. All animals were housed and cared for in conformance with the policy of the American College of Sports Medicine on research with experimental animals, the Canadian Council on Animal Care, and the University animal research ethics committee.
We specifically selected 15- to 16-month-old C57BL/6 mice for this study because of the potential for preventative exercise intervention at middle age. We recognize that this age is not reflective of truly old animals. However, the life expectancy for C57BL/6 mice is quite variable with reported 20–22 (1) and 24 (35) months under standard housing and breeding conditions. Moreover, 15- to 16-month-old C57BL/6 mice already have several senescent changes including thymic involution (21), elevated ROS in visceral adipose tissue (40), and inflammatory phenotypes in the kidney (12). From an exercise perspective, inclusion of very old animals presents significant methodological and interpretation challenges. For example, sharp declines in locomotor behavior have been reported at 18 months in C57BL/6 mice (2).
Mice were matched by weight and randomized to exercise training conditions: access to in-cage running wheels (WR or “trained,” n = 40) or to a no-running-wheel (NWR or “untrained,” n = 40) condition for 4 months. An automated computer monitoring system (Vital View Application software; Mini-Mitter, Sunriver, OR) captured the number of completed revolutions via a magnetic switch attached to each wheel (23 cm in diameter). Activity during the dark cycle was recorded as the number of revolutions completed per 15-min interval, converted to distance run (km), and summed by day, week, and month.
Acute treadmill exercise
WR and NWR mice were randomly assigned to an acute exercise condition: 1) a single bout of treadmill running with sacrifice immediately (i.e., WR IMM, n = 20; NWR IMM, n = 20) after completion of exercise or 2) a no-treadmill sedentary group (i.e., WR SED, n = 20; NWR SED, n = 20) exposed only to treadmill noise and vibrations for 90 min before sacrifice. At sacrifice, mice were screened for the presence of gastrointestinal tumors and pathologies (i.e., bleeding) and animals with intestinal tumors were excluded. The exercise protocol consisted of a single, 90-min bout of intense treadmill exercise (10 min warm-up, 30 min at 22 m·min−1, 30 min at 25 m·min−1, and 30 min at 28 m·min−1, 2° slope; Panlab Mouse Treadmill; Harvard Apparatus Canada Saint-Laurent, QC, Canada) during the dark cycle. To avoid carryover effects of freewheel running, in-cage running wheels were locked 24 h before the treadmill exercise bout. Mice were motivated to run by gentle prodding with a nylon test tube brush until the end of the run-to-exhaustion protocol or until reaching volitional fatigue.
Skeletal muscle enzyme activity
Cytochrome c oxidase (CO) activity was measured in sedentary (no treadmill challenge) mice (WR and NWR) as an indicator of training status; after 10 wk of exercise training, CO has been shown to be increased by 38% in skeletal muscle (quadriceps) of young mice (5). After sacrifice by sodium pentobarbital (0.6–0.8 mL per mouse, intraperitoneally) overdose, soleus and plantaris muscles were isolated from all non–treadmill running (i.e., WR SED and NWR SED) mice. Samples were frozen in liquid nitrogen and stored at −80°C until assayed. Muscles were cut into 5- to 10-mg segments, mashed, and homogenized in buffer (glycerol (50%), sodium phosphate buffer (20 mM), 2-mercaptoethanol (5 mM), EDTA (0.5 mM), and bovine serum albumin (10%)) to yield a 50:1 dilution and sonicated (using a 3-mm tip, 2 s on, 5 s off for a total of 20 s at 60 Hz; Vibra Cell (Sonics and Materials, Danbury, CT)). Protein content was determined by the Lowry assay. Muscle homogenates were diluted to 1:500 dilutions in 10 mM potassium phosphate buffer. Reduced cytochrome c (20 μL) and diluted homogenate (10 μL) were combined with warmed (37°C) phosphate buffer (970 μL). The decrease in cytochrome c absorbance was determined spectrophotometrically at 550 nm.
The stress response of older mice to acute treadmill exercise was measured by plasma 8-iso-PGF2α and corticosterone. In brief, a 1-mL syringe containing heparin was used to collect blood via cardiac puncture immediately after sacrifice. Plasma was separated by centrifugation (6 min at 400 rpm) and frozen at −80°C before it was assessed by direct enzyme immunoassay using a commercially available kit as per the manufacturer’s specifications (Cayman Chemical, Ann Arbor, MI). Samples (100 μL sample, 25 μL of 10N NaOH) were hydrolyzed for 2 h at 45°C, neutralized to pH 6–8 with 12N HCl, centrifuged at 14,000g for 5 min, and incubated with 8-iso-PGF2α antibody for 24 h at 4°C. Percent absorbance was read at 412 nm at room temperature using a Power Wave 340 microplate spectrophotometer (Biotek Instruments, Winooski, VT). The intra-assay coefficient of variation (%CV) was 11.7%. Corticosterone was measured using a commercially available enzyme immunoassay kit according to the manufacturer’s instructions (Cayman Chemical). The cold spike protocol was used for the purification of plasma samples, and corticosterone concentrations was measured using a Power Wave 340 microplate spectrophotometer (Biotek Instruments) at 412 nm. The intra-assay coefficient of variation (%CV) was 12.5%.
Intestinal lymphocyte isolation
Isolation of intestinal lymphocytes was performed as described elsewhere (13). Cold phosphate-buffered saline was used to wash the excised intestinal compartment; Peyer patches and visible fat were removed, and a single-cell suspension was prepared by isolation over a prewashed nylon wool (0.3 g) column. The eluted lysate was layered over a Lympholyte-M density gradient medium (Cedarlane Laboratories, Mississauga, Ontario, Canada) and centrifuged to remove debris. The remaining pellet (containing intraepithelial and lamina propria lymphocytes) was suspended in 400 μL of phosphate-buffered saline. Turk staining solution (99 μL) was used to enumerate intestinal lymphocytes (1 μL) by light microscopy. This procedure yields a high lymphocyte recovery (i.e., 90.9% ± 0.5% CD45+ cells by flow analysis).
Intestinal lymphocyte fractionation in lysis buffer (on ice; 45 min) was followed by centrifugation (15 min at 10,000g) of lysates (1 × 105 cells). The supernatant was extracted, and protein concentration was determined by bicinchoninic acid assay. Protein (40 μg) and selected molecular weight markers (Full Range Rainbow; Amersham Biosciences, Piscataway, NJ) were electrophoresed on a 12% SDS–PAGE gel before transferring to the polyvinylidene fluoride membrane. Ponceau S was used to stain membranes to confirm the quality of transfer and equal loading. After electrophoresis, membranes were incubated for 1 h with primary antibody (1:200 in 10% milk–tris-buffered saline tween-20): TNF-α (clone: N-19; goat anti–human polyclonal IgG), IL-1β (clone: F × 02l; mouse anti–rat monoclonal), IL-10 (clone: JES5-2A5; rat anti–mouse monoclonal IgG1), caspase-3 (clone: H-277; rabbit anti–human polyclonal IgG), caspase-7 (clone: 10-1-62; mouse anti–human monoclonal IgG1), and Bcl-2 (clone: C-2; mouse anti–human monoclonal IgG1) (Santa Cruz Biotechnology, Santa Cruz, CA). Membranes were incubated for 1 h with secondary antibody: biotin-conjugated rabbit anti–goat IgG-B (TNF-α), and horseradish peroxidase–conjugated goat anti–mouse IgG–HRP (IL-1β, IL-10, caspase-7, and Bcl-2) or goat anti–rabbit IgG–HRP (caspase-3) IgG at a concentration of 1:2000 in 10% milk–tris-buffered saline tween-20. ECL Plus detection reagent (Amersham Biosciences), Piscataway, NJ and the ChemiGenius 2 Bio-imaging System (Syngene, Cambridge, UK) were used for protein determination. A biotinylated protein ladder was used to identify the molecular weight of selected proteins (Cell Signalling Technology, Pickering, Ontario, Canada). Recombinant standards (Cedarlane Laboratories) were run on each gel. Samples from each experimental condition were run on each immunoblot, and band densities were normalized to control bands on each immunoblot (units reported as arbitrary densitometric units [A.U.]) for each group.
Cytokine and apoptotic protein expression of WR and NWR mice were analyzed by two-way ANOVA with freewheel training (two levels: wheel running, no wheel running) and acute treadmill exercise (two levels: sedentary, immediate) conditions as the independent factors. Intestinal lymphocyte cytokine and apoptotic protein expression and plasma concentration of corticosterone and 8-iso-PGF2α were the dependent factors (SPSS for Windows Version 19; SPSS, Inc., Chicago, IL). One-way ANOVA was used to analyze the main effect of training (two levels of independent factor: wheel running, no wheel running) on cytochrome c oxidase activity in skeletal muscles. Homogeneity of variance was confirmed by the Levene test. Significant differences were accepted if P < 0.05; values are group means ± 1 SEM for respective units (e.g., μmol·min−1·g−1; arbitrary [densitometric] units; g).
Physiological indicators of training
The training characteristics of older C57BL/6 female mice are shown in Table 1. WR and NWR mice did not differ in initial body weights. Not unexpectedly, after 4 months of freewheel activity, WR mice were significantly lighter than NWR mice (F 1,84 = 15.902, P < 0.001). WR mice accumulated an average of 573.91 km during 4 months; the pattern of running volume was steady for the first 2 months before decreasing by 20–30 km per month thereafter. Skeletal muscle cytochrome c oxidase activity was significantly higher in the soleus (F 1,37 = 19.443, P < 0.001) and plantaris (F 1,37 = 50.559, P < 0.001) muscles of (trained) WR compared to (untrained) NWR mice.
Plasma concentrations of 8-iso-PGF2α and corticosterone for older WR and NWR mice in response to acute treadmill exercise are shown in Table 2. Acute treadmill exercise was associated with significantly increased plasma 8-iso-PGF2α concentration in both WR (F 1,28 = 38.732, P < 0.001) and NWR (F 1,30 = 16.865, P < 0.001) mice relative to their respective sedentary (no treadmill) controls. Acute treadmill exercise led to significant elevations in plasma corticosterone in trained (F 1,30 = 46.091, P < 0.001) and untrained (F 1,31 = 67.751, P < 0.001) mice. Given the role of plasma corticosterone and 8-iso-PGF2α as indicators of physiological and oxidative stress, it can be inferred that the acute exercise bout was similarly stressful for both trained and untrained older mice.
Proinflammatory and anti-inflammatory cytokines
The effects of freewheel training and acute treadmill exercise on the expression of proinflammatory and anti-inflammatory cytokines in mouse intestinal lymphocytes are shown in Figure 1 (A–C). There was no significant main effect of long-term training on the expression of the proinflammatory cytokines TNF-α (F 1,69 = 0.01, P = 0.30) and IL-1β (F 1,70 = 1.43, P = 0.24) or the anti-inflammatory cytokine IL-10 (F 1,73 = 0.05, P = 0.83). There was a significant main effect of acute treadmill exercise on intestinal lymphocyte expression of TNF-α (F 1,69 = 29.60, P < 0.001) and IL-1β (F 1,70 = 9.02, P < 0.01): the expression of these inflammatory cytokines was higher immediately (TNF-α = 1.24 ± 0.05 [A.U.]; IL-1β = 1.09 ± 0.05 [A.U.]) after treadmill challenge compared to the sedentary (TNF-α = 0.82 ± 0.05; IL-1β = 0.90 ± 0.05 [A.U.]) condition. There was also a nonsignificant trend toward greater IL-10 expression immediately after acute exercise (F 1,73 = 3.52, P = 0.07).
There was a significant interaction between freewheel training and acute treadmill exercise for the expression of TNF-α in intestinal lymphocytes of older mice (F 1,69 = 7.01, P < 0.05). This was due to a greater increase in proinflammatory cytokine expression in WR mice (SED = 0.68 ± 0.08 [A.U.] vs IMM = 1.30 ± 0.08 [A.U.]; 91% increase) compared to NWR mice (SED = 0.96 ± 0.08 [A.U.] vs IMM = 1.18 ± 0.07 [A.U.]; 23% increase). There was no statistically significant interaction between freewheel training and acute exercise for IL-1β expression in intestinal lymphocytes from older mice (F 1,70 = 0.60, P = 0.44). In contrast, a significant interaction effect was seen for intestinal lymphocyte expression of IL-10 (F 1,73 = 4.13, P < 0.05). WR (SED = 0.84 ± 0.07 [A.U.] vs IMM = 1.09 ± 0.06 [A.U.]; 30% increase) but not NWR (SED = 0.98 ± 0.06 [A.U.] vs IMM = 0.97 ± 0.06 [A.U.]; 1% decrease) mice had higher IL-10 expression immediately after acute treadmill exercise compared with the sedentary condition.
Proapoptotic and anti-apoptotic proteins
The effects of freewheel training and acute treadmill exercise on the expression of proapoptotic and anti-apoptotic proteins in intestinal lymphocytes from older C57BL/6 mice are shown in Figure 2 (A–C). There was no significant main effect of long-term exercise training on the expression of the proapoptotic proteins caspase-3 (F 1,70 = 0.85, P = 0.36) and caspase-7 (F 1,70 = 1.29, P = 0.26) or the anti-apoptotic protein Bcl-2 (F 1,75 = 0.02, P = 0.90). There was a significant main effect of acute treadmill exercise on mouse intestinal lymphocyte expression of caspase-3 (F 1,70 = 4.66, P < 0.05) and caspase-7 (F 1,70 = 23.79, P < 0.001): the expression of these proapoptotic proteins was higher immediately (caspase-3 = 1.08 ± 0.06 [A.U.]; caspase-7 = 1.13 ± 0.05 [A.U.]) after treadmill challenge compared to the sedentary (no treadmill) condition (caspase-3 = 0.89 ± 0.06; caspase-7 = 0.77 ± 0.05 [A.U.]). Acute treadmill exercise did not alter intestinal lymphocyte expression of Bcl-2 (F 1,75 = 1.91, P = 0.17) across the training conditions.
However, there was a significant interaction between freewheel training and acute treadmill exercise for caspase-3 (F 1,70 = 4.06, P < 0.05) because of the greater increase in proapoptotic protein expression in intestinal lymphocytes of WR mice in response to acute exercise (SED = 0.84 ± 0.09 [A.U.] vs IMM = 1.20 ± 0.09 [A.U.]; 43% increase) compared to NWR mice (SED = 0.94 ± 0.09 [A.U.] vs IMM: 0.95 ± 0.08 [A.U.]; 1% increase). There was a borderline significant interaction between freewheel training and acute exercise for caspase-7 expression in mouse IL (F 1,70 = 3.85, P = 0.054); this was due to a post-acute exercise increase in the expression of caspase-7 in WR mice (SED = 0.66 ± 0.08 [A.U.] vs IMM = 1.16 ± 0.08 [A.U.]; 76% increase) compared to little change in NWR mice (SED = 0.89 ± 0.07 [A.U.] vs IMM = 1.10 ± 0.07 [A.U.]; 24% increase). There was also a significant interaction effect (F 1,75 = 8.37, P < 0.01) for intestinal lymphocyte expression of Bcl-2. WR mice (SED = 1.11 ± 0.06 [A.U.] vs IMM = 0.87 ± 0.06 [A.U.]; 22% decrease) but not NWR mice (SED = 0.95 ± 0.06 [A.U.] vs IMM = 1.04 ± 0.05 [A.U.]; 9% increase) had a lower expression of anti-apoptotic Bcl-2 immediately after acute treadmill exercise.
The effect of exercise training in older (15–16 months) mice on intestinal lymphocyte cytokine and apoptotic protein expression in response to acute exercise challenge was investigated. No cumulative effect of freewheel training alone on protein expression in the intestinal lymphocytes of these older animals was found. Acute exercise was associated with a higher expression of TNF-α, IL-1β, caspase-3, and caspase-7 in mouse IL. There were interesting significant interactions between freewheel training and acute treadmill exercise for TNF-α, IL-10, caspase-3, and Bcl-2. This was demonstrated by changes in intestinal lymphocyte expression of pro-(TNF-α) and anti-(IL-10) inflammatory cytokines and proapoptotic (caspase-3) and anti-apoptotic (Bcl-2) proteins in response to acute exercise in WR compared to NWR mice. These findings support the hypothesis that older mice undergoing voluntary exercise training have an intestinal lymphocyte cytokine and apoptotic protein response similar to those reported in the literature for young animals. Older untrained mice showed few changes in the expression of these cytokines and apoptotic proteins after an acute exercise challenge, suggestive of either less responsiveness or a blunted response in line with immunosenescence.
Proinflammatory and anti-inflammatory cytokines
Older WR mice had higher TNF-α, IL-1β, and IL-10 expression in intestinal lymphocytes immediately after exercise. This mirrors findings describing the effect of oxidant stress on lymphocyte cytokine expression in young animals reported previously. In response to acute exercise challenge, Rosa Neto et al. (33) found increased TNF-α expression in rat adipose tissue and Hoffman-Goetz et al. (15) showed a 48% increase in mouse intestinal IL-10. Although inflammation is harmful to intestinal tissue, there may be potential physiologic “benefits” to the proinflammatory and anti-inflammatory cytokine responses to oxidant stress. Suzuki et al. (38) reported that acute exercise induces an immediate increase in proinflammatory cytokines (IL-6, IL-1β); this is followed by an anti-inflammatory response (increased IL-10, IL-1ra, and IL-4). This secondary, anti-inflammatory cytokine response was suggested to counter intestinal inflammation and prevent traumatic tissue damage. Petersen and Pedersen (30) proposed that acute exercise induces an anti-inflammatory environment in three possible ways. First, in response to acute exercise, there are elevations in muscle-derived IL-6 that stimulate the production of the anti-inflammatory cytokines IL-1ra and IL-10. Second, IL-6 inhibits the production of the proinflammatory cytokine TNF-α. Finally, acute exercise increases circulating epinephrine, which in vivo prevents TNF-α production (30). Some of the long-term benefits of regular exercise may thus arise from these secondary anti-inflammatory cytokine responses to acute exercise (23). This compensatory cytokine response may shift lymphocytes toward an anti-inflammatory milieu and protect against chronic inflammation, a key chronic disease risk factor.
Interleukin-10 inhibits the synthesis of proinflammatory cytokines TNF-α and IL-1β by regulating the transcription factor NF-κB (9). WR mice had a marked increase in IL-10 expression in response to oxidant stress associated with acute exercise, whereas NWR mice showed little change in IL-10 expression after acute exercise. This finding is novel. Interleukin-10 plays an important antioxidant role by inhibiting the oxidative respiratory burst and preventing ROS-mediated tissue damage (9). Our results suggest that training not only preserves the cytokine response to oxidant stress in older animals but also may bolster anti-inflammatory reserve or accelerate postexercise recovery as seen at younger ages.
Regular training of moderate intensity decreases the prevalence and severity of gastrointestinal distress after acute exercise in young athletes (3). It is unknown if this protective effect of training on gastrointestinal symptoms occurs in middle-aged adults. However, a recent prospective cohort study (n = 47,228; 18-yr follow-up) of male health professionals indicated that highly active individuals (≥57.4 MET·h·wk−1) had decreased risk of developing diverticulitis (relative risk (RR) = 0.75, 95% confidence interval (CI) = 0.58–0.95) or diverticular bleeding (RR = 0.54, 95% CI = 0.38–0.77) compared to sedentary (≤8.2 MET·h·wk−1) males (37). Another prospective cohort study (n = 8205; 3-yr follow-up) in elderly subjects (≥68 yr) showed that regular walking (three times a week) decreased the RR of gastrointestinal bleeding (RR = 0.60, 95% CI = 0.4–0.8) compared to elderly sedentary participants (27). The IL-10 results reported here may have implications for older recreational athletes as the incidence of inflammatory intestinal disorders and acute gastrointestinal distress increases with age (27).
Proapoptotic and anti-apoptotic proteins
Elevated inflammatory cytokine concentration triggers lymphocyte apoptosis as mitochondrial procaspases (i.e., caspase-3 and -7) undergo cleavage in response to oxidant stress (19). Active caspases move into the cytoplasm, degrade the plasma membrane, and induce lysis (19). Anti-apoptotic proteins, such as Bcl-2, antagonize these harmful effects and prevent the release and activation of mitochondrial caspases. Our results suggest that older WR mice seem to have a similar apoptotic response to oxidant stress as reported elsewhere for young mice (14). Casual consideration of this apoptotic response to acute exercise might be interpreted as maladaptive or harmful. However, apoptosis is an evolutionarily conserved means of host defense, and the capacity to induce apoptosis in response to stress suggests preserved immune function (19). In young mice, exhaustive treadmill exercise increases caspase-3 and caspase-7 and decreases Bcl-2 expression in intestinal lymphocytes (14). In healthy young people, caspase activation in cells with high levels of DNA damage decreases cancer risk (25). Thus, the apoptotic responsiveness of WR mice to acute exercise challenge might indicate better clearance potential of damaged lymphocytes; in contrast, older NWR mice had increased expression only of caspase-7 without any changes in caspase-3 or Bcl-2 expression in intestinal lymphocytes.
Physiological indicators of training and oxidative stress
Four months of freewheel running induced physiological changes indicative of “training” in older C57BL/6 mice. WR mice had increased cytochrome c oxidase enzyme activity in soleus and plantaris muscles. Long-term aerobic training has been shown to increase cytochrome c oxidase activity in young mice (5,14), but this has not been documented for older mice. However, resistance training increased CO levels in older adults (28). The finding is especially noteworthy because cytochrome c oxidase activity in skeletal muscle decreases with age and may be indicative of senescent changes in oxidative buffering capacity (28).
Intense treadmill exercise induced oxidative stress in WR and NWR mice, demonstrated by elevated plasma corticosterone and 8-iso-PGF2α immediately after the acute exercise bout. Plasma 8-iso-PGF2α is a biomarker of oxidative stress and is derived from arachidonic acid through a reaction catalyzed by oxidative intermediates. Acute exercise induces tissue hypoxia and inhibits SOD2 activity. This leads to increased 8-iso-PGF2α production from multiple cellular origins due to ROS generation. In response to cellular exposure to SRM 1648 (an inducer of oxidant stress), alveolar macrophages generate increased glutathione and 8-iso-PGF2α (34). Given ischemia reperfusion in the intestinal–splanchnic interface in response to acute exercise, lymphocyte-derived ROS may contribute to the elevated plasma 8-iso-PGF2α observed in this study. Corticosterone is produced by the adrenal gland in response to physiological or psychological stress. This hormone plays a crucial role in the sympathetic response by stimulating gluconeogenesis and accessing energy reserves (7). Similar elevations in plasma 8-iso-PGF2α and corticosterone in trained (WR) and untrained (NWR) older C57BL/6 mice indicate that animals responded to the acute-exercise challenge with equivalent oxidant stress responses.
This study is not without limitations. First, we measured intestinal lymphocyte protein for the cytokines and apoptotic outcomes of interest and not mRNA expression. We did not measure cytokine transcription in response to acute exercise but only whole protein in the sample. Second, although we have previously shown high leukocyte concentration in intestinal lymphocyte lysates, some proteins may not have originated from lymphocytes but from other intestinal cells. This means that not all of the measured proteins definitively originated from intestinal lymphocytes. Third, our study only examined the effect of one bout of acute treadmill exercise on the intestinal lymphocyte cytokine and apoptotic protein response. Future research might use repeated exercise bouts to determine whether the oxidant stress response differs between training groups. It is possible that exercise training might protect against repeated acute exercise-induced inflammation or, alternatively, that WR and NWR mice do not differ in their intestinal lymphocyte response when faced with multiple exercise exposures. Fourth, we did not include young mice for direct comparisons with older animals. Thus, we cannot draw inferences about an “aging” effect as opposed to an effect observed in older animals. Fifth, animals were 15–16 months at the time of sacrifice. As noted previously, this is not extremely old because C57BL/6 mice have a life-expectancy ranging from 19 to 24 months. Nevertheless, this age cohort provides a useful basis for comparison with healthy late–middle age or young-elderly adults, when exercise interventions may be most beneficial to slow immunosenescence. Sixth, we did not perform a full necropsy on the mice, and it is possible that the presence of tumors in peripheral organs or tissues could affect cytokine expression in intestinal lymphocytes. However, none of the mice included in the statistical analysis had intestinal tumors as determined by gross visual inspection. Finally, we only included one postexercise time point for analysis; additional time points would be necessary to determine the kinetics of the intestinal lymphocyte cytokine and apoptotic protein responses to acute exercise.
In summary, 4 months of freewheel running in healthy older (15–16 months) female C57BL/6 mice was associated with a conserved intestinal lymphocyte response to exercise oxidative stress as demonstrated by a higher expression of the proinflammatory cytokines TNF-α and IL-1β, the anti-inflammatory cytokine IL-10, and the apoptotic proteins caspase-3 and caspase-7 and by a lower expression of the anti-apoptotic protein Bcl-2. In contrast, untrained mice showed little change in intestinal lymphocyte expression of cytokine or apoptotic proteins in response to an acute exercise stimulus. This was although acute exercise was associated with increased plasma corticosterone and 8-iso-PGF2α. This study used an animal model to explore differences in the acute exercise–induced oxidant stress response between trained and untrained older mice. We cautiously propose that voluntary exercise training preserves cytokine and apoptotic responses in the intestinal tract of older animals and may help to counter blunted or senescent changes in the immune system.
This research was supported by the Natural Sciences and Engineering Research Council of Canada. N. Packer is the holder of a Canadian Institutes of Health Research postgraduate award.
The authors thank J. Guan for technical assistance.
The authors report no conflicts of interest.
The results of the present study do not constitute endorsement by the American College of Sports Medicine.
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