You are on page 1of 10

Scand J Med Sci Sports 2011 doi: 10.1111/j.1600-0838.2010.01288.

& 2011 John Wiley & Sons A/S

Effect of exercise training intensity on murine T-regulatory cells and vaccination response
J. Wang1,2,*,w, H. Song3,*, X. Tang3, Y. Yang2, V. J. Vieira4, Y. Niu3, Y. Ma1,5
Key Laboratory of Cellular and Molecular Immunology, Henan University, Kaifeng, China, 2Institute of Molecular Medicine, Henan University, Kaifeng, China, 3College of Physical Education, Henan University, Kaifeng, China, 4Obesity and Metabolism Laboratory, Jean Mayer USDA Human Nutrition Research Center on Aging at Tufts Universit, Boston, Massachusetts, USA, 5Institute of Immunology, College of Medicine, Henan University, Kaifeng, China
1

Corresponding author: Junpeng Wang, PhD, Institute of Molecular Medicine, College of Medicine, Henan University, Kaifeng 475004, China. Tel/fax:186 378 388 0398. E-mail: jpwangchina@henu.edu.cn
Accepted for publication 13 December 2010

To understand the underlying mechanism(s) for the eect of exercise at dierent intensities on T cell and DNA vaccination responses, we treated mice in a training protocol with regular moderate-intensity exercise (MIE) or prolonged, exhaustive high-intensity exercise (HIE). After 6 weeks of training, splenocytes were isolated to evaluate cytokine expression and T-regulatory (Treg) cell proportion by RTPCR and FACS, respectively. Another set of mice that completed the same training protocol were used to determine DNA vaccination responses. These mice were immunized three times with HBV DNA vaccine at 2-week intervals and euthanized on day 14 after the last immunization. Serum and splenocytes were isolated to determine humoral and cell-mediated immunity (CMI). Results

showed that HIE increased anti-inammatory cytokine expression and CD41CD251 Treg cell proportion. Further, HIE decreased IFN-c expression, T-lymphocyte proliferation, and antigen-specic cytotoxic response in HBV DNA vaccine-immunized mice. MIE did not change anti-inammatory cytokine expression or CD41CD251 Treg cell proportion but increased pro-inammatory cytokine expression and augmented antigen-specic CMI. Thus, MIE lower the risk of cancer and infectious illness through enhancing the pro-inammatory responses. By contrast, HIE might increase the risk of common infections, such as upper respiratory tract infection, due to an upregulation of CD41CD251 Treg cells and anti-inammatory responses.

Exercise, depending on its intensity, can have either positive or negative eects on immune function and general health (Pedersen & Homan-Goetz, 2000). Regular moderate-intensity exercise (MIE) enhances immune functions compared with the typical sedentary individuals in terms of potentiation of T cellmediated immunity (CMI), NK cell cytotoxicity, and Th1 response in human or animal models (Sugiura et al., 2001; Davis et al., 2004; Murphy et al., 2004). However, prolonged, exhaustive high-intensity exercise (HIE) and intensive periods of endurance exercise training may impair immune function, increasing susceptibility to infections and decreasing NK cell activity, pro-inammatory cytokine (Interleukin [IL2]) expression, and IFN-g production (Davis et al., 1997; Davis et al., 1998; Steensberg et al., 2001). The
*Contributed equally.

Current address: Nutrition Immunology Laboratory, Jean Mayer USDA Human Nutrition Research Center on Aging at Tufts University, 711 Washington Street, Boston, Massachusetts 02111, USA. J. W. and Y. M. share senior authorship.

negative eects of HIE and positive eects of MIE, however, do not extend equally to all aspects of immune function. Interestingly, a recent study of the patients with type 2 diabetes mellitus (DM) showed that regular Tai Chi Chuan (TCC), a type of traditional Chinese martial art exercise, alters the Th1/Th2/ T-regulatory (Treg) balance by increasing the gene encoding forkhead/winged helix transcription factor (Foxp3) but not TGF-b expression in patients with type 2 DM or health middle-aged volunteers (Yeh et al., 2006; Yeh et al., 2009). CD41CD251 Treg cells, a CD41 T cell subtype, specically express Foxp3, a master specic and functional marker of Treg cells (Sakaguchi, 2005). In addition, these cells constitutively express IL-2R a-chain (CD25) and suppress immune responses via cell contact-dependent mechanisms (i.e., natural Treg cells). Studies show that the depletion of these cells causes autoimmune disease and enhances the immune response to foreign antigens (Furuichi et al., 2005; Sakaguchi, 2005; Fernandez et al., 2008). However, whether prolonged, exhaustive HIE may change Treg cell number and suppressive function,

Wang et al.
which is regarded as a factor that increases the risk for upper respiratory tract infection (URTI) (Curotto de Lafaille & Lafaille, 2009), is still unclear. The purpose of the present study was to examine whether regular MIE and prolonged, exhaustive HIE aect CD41CD251 Treg cell proportion and DNA vaccination responses and if so, through what mechanism(s), utilizing a mouse model of exercise. Our objectives were to (1) measure pro- and anti-inammatory cytokines and CD41CD251 Treg cells in exercise-trained mice; (2) determine whether regular MIE and prolonged, exhaustive HIE aect humoral and cellular immune responses in exercised mice vaccinated with the HBV DNA vaccine encoding hepatitis B surface antigen (HBsAg, pVAX-S2), as a model DNA vaccine. Materials and methods
Experimental animals and reagents
Six- to 8-week-old syngeneic, female C57BL/6 mice were purchased from the Animal Institute of the Chinese Medical Academy (Beijing, China) and housed under a 12 h light cycle. The mice were provided with pathogen-free water and food, and were randomly distributed among the experimental groups. All management procedures were approved by the Institutional Animal Care and Use Committee of Henan University. Chinese hamster ovary cells, mammalian cells for transfection, expression, and large-scale recombinant protein production, expressing HBsAg (rHBsAg) were purchased from China North Pharmaceutical Group Corp. (NCPC, Hebei, China). The HBsAg-derived peptide S208-215 (ILSPFLPL; H-2Kb-restricted) and ovalbumin peptide 257264 (SIINFEKL; H2Kb-restricted) were synthesized by GL Biochem Co. Ltd. (Shanghai, China). Plasmid pVAX-S2, encoding the HBV surface antigens preS2 and S, was constructed using pVAX1 as described previously (Wang et al., 2008). The plasmid was maxi-prepared, puried by PEG8000 precipitation, and then dissolved in saline at a concentration of 1 mg/mL.

Exercise program
Seventy-two mice were divided into four groups: na ve, MIE, HIE, and treadmill control (TC). The exercise regimen was 5 days a week for 6 weeks. It has been shown that the percentage of maximal oxygen consumption (%VO2max) of small rodents running on the treadmill can be predicted by their speed or relative intensity of exercise (Fernando et al., 1993). Based on this study, the following exercise protocols were used for mice to achieve MIE and HIE (about 70% and 491% of %VO2max, respectively). Two dierent experiments were performed, shown schematically in Fig. 1. The mice in the MIE group were subjected to treadmill exercise on a level belt without any slope for 30 min at 818 m/min during the rst week and for 60 min at 18 m/min during the next 5 weeks. The mice in the HIE group trained ve times for 30 min each on a treadmill set at 15 m/min (5% gradient) during the rst week, ve times for 60 min each at 1526.8 m/min (5% gradient) during the second week, and ve times for 60 min each at 26.8 m/min (10% gradient) during the next 4 weeks. To control for any non-exercise eects of treadmill running such as handling, novel environment, noise, and vibration, animals of the TC group were kept in the same room and placed on a slow-moving treadmill (5 m/min, 5 min/day,

Fig. 1. Experimental design for evaluation of the eects of MIE and HIE on inammatory cytokine production, Tregulatory (Treg) cell proportion, and HBV DNA vaccine responses. Thirty-two mice were randomized to four groups (8/group, high-intensity exercise [HIE], moderate-intensity exercise [MIE], treadmill control [TC], and na ve) as described in the Materials and methods. Following 6 weeks of prescribed exercise, mice were sacriced and splenocytes were collected to determine the eects of MIE and HIE on cytokine production and Treg cell proportion (a). Forty mice in four groups (n 5 10/group) were given the same exercise-training as mentioned above and then immunized with HBV DNA vaccine three times to assess the eects of MIE and HIE on HBV DNA vaccine responses (b). Serum was collected at time points as indicated for Ab analysis and splenocytes were collected to determine T cell proliferation, intracellular cytokines, and cytotoxicity.

5 days/week). Within 1620 h after the completion of 6 weeks of exercise according to their respective program, 32 mice were euthanized by the inhalation of carbon dioxide. Splenocytes were isolated as required for the evaluation of cellular surface markers, intracellular staining, and RT-PCR. In addition, after the completion of 6 weeks of exercise according to their respective program, 40 mice were immunized with HBV DNA vaccine pVAX-S2 on days 0, 14, and 28 as described in our previous study (Wang et al., 2008), during which time they continued running. Serum samples were collected from the retro-orbital sinus before and after immunization at 2 week intervals. The mice were euthanized by the inhalation of carbon dioxide on day 14 after the third immunization, and splenocytes were isolated to evaluate T cell proliferation, cytotoxic activity, and intracellular staining. Twenty mice were used to evaluate T cell proliferation and cytokine production and the remaining 20 mice were used to determine in vivo cytotoxic activity.

Exercises, Treg cells and vaccination


Analysis of cell surface markers and intracellular cytokine expression
Fluorescent-conjugated monoclonal antibodies recognizing CD4, CD25, IFN-g, IL-10, Foxp3, and isotype controls were purchased from Biolegend (San Diego, California, USA). Puried antibodies to CD16/CD32 were purchased from BD Biosciences Pharmingen (San Jose, California, USA). Within 1620 h after the completion of 6 weeks of exercise, single-cell splenocyte suspensions were harvested and 2 106 cells were restimulated for 4 h with 50 ng/mL PMA and 500 ng/mL ionomycin (both from Sigma, St. Louis, Missouri, USA) in the presence of monensin (GolgiStop, BD Pharmingen, San Jose, California, USA), and then 1 106 cells were stained as described previously (Tai et al., 2008). Cellular uorescence was detected and 2 104 cells were acquired using the FACSCalibur system. The data were analyzed using FlowJo software.

T cell proliferation
Fourteen days after the third immunization, single-cell splenocyte suspensions were harvested and the cells stained with 1 mM CFSE as described previously (Wang et al., 2008). CFSE is an ester that can diuse into cells, where it reacts with amine groups to become uorescent. The uorescence is stably retained in cells and equally distributed between daughter cell populations with every cell division. Cell cultures were prepared by inoculating triplicate 96-well round-bottom plates with 5 105 cells/mL prepared in RPMI 1640 medium containing 5% FCS. The plates were incubated at 37 1C in a 5% CO2 incubator. T cell proliferation was evaluated by a FACS assay in cells re-stimulated with rHBsAg. The data were analyzed using FlowJo software.

In vivo cytotoxic assay


In vivo cytotoxic assays were carried out as described previously (Wang et al., 2008). In brief, single-cell splenocyte suspensions from na ve mice were pulsed with HBsAg CTL peptide S208215 and labeled with a high concentration (5 mM) of CFSE (CFSEhigh); these cells served as the target cells. An equal fraction of splenocytes was pulsed with OVA peptide (negative control) and labeled with a low concentration (0.5 mM) of CFSE (CFSElow); these cells served as the non-target control. The target and control cells were mixed together and injected into immunized mice at 2 107 total cells in 200 mL PBS via tail vein on day 14 after the third immunization. Four hours after injection, the mice were euthanized by CO2 inhalation and their splenocytes isolated. The labeled cells were analyzed based on their dierential CFSE uorescence intensities using the FACSCalibur system. Specic lysis was calculated according to the following formula: % specic lysis 5 [1 (% specic peptide loaded target cells/% control peptide loaded target cells)] 100%.

Detection of antigen-specic cytokine production


Fourteen days after the third immunization, single-cell splenocyte suspensions were harvested as described previously (Wang et al., 2008) and these cells at 2 106 cells in a 24 well plate were re-stimulated with HBsAg-derived peptide S208 215 (10 mg/mL) and anti-CD28 (5 mg/mL) mAb for 6 h at 37 1C and 5% CO2. For intracellular staining of IFN- g and IL-4, cells were re-stimulated for the last 4 h with 50 ng/mL PMA and 500 ng/mL ionomycin in the presence of monensin. One million cells were harvested and stained as described in the above-mentioned methods. Cellular uorescence was detected and 2 104 cells were acquired using the FACSCalibur system. The data were analyzed using FlowJo software.

RT-PCR
Total RNA was extracted from the splenocytes and reversetranscribed. The resulting cDNAs were PCR-amplied with the primers listed in Table 1, separated on a 1.5% agarose gel, and visualized by staining with ethidium bromide.

Statistical analysis
Results are expressed as means SEM. Statistical analysis was conducted using Sigmastat software. Signicant dierences were determined using ANOVA for dierent exercises eect and was followed by Fishers least signicance dierence post hoc test for individual comparisons. Po0.05 indicated statistical signicance.

Antibody ELISA
Serum samples were analyzed by ELISA on day 14 after the third immunization. Total IgG binding to HBsAg was measured with an ELISA kit according to the manufacturers instructions (SIIC Kinghaw Biotech Co. Ltd., Beijing, China) and expressed in international units. The amount of total antiHBsAg antibody was calculated as described previously (Wang et al., 2008).
Table 1. The Primers of target genes

Results Effect of MIE and HIE on anti- and pro-inammatory cytokine expression Cytokines play important roles in the polarization of immune responses. To determine the cytokine proles of C57BL/6 mice subjected to an MIE/HIE program, the genes, IL-12p40 (for IL-12), TGF-b,

Target gene b-actin IL-12p40 IL-2 TGF-b Ebi3

Primers sequences Sense: 5 0 -GGGACCTGACAGACTACCTCAT-3 0 Anti-sense: 5 0 -CAAGAAGGAAGGCTGGAAA-3 0 Sense: 5 0 -CTGGCCAGTACACCTGCCAC-3 0 Anti-sense: 5 0 -GTGCTTCCAACGCCAGTTCA-3 0 Sense: 5 0 -TCCACTTCAAGCTCTACAG-3 0 Anti-sense: 5 0 -GAGTCAAATC CAGAACATGC C-3 0 Sense: 5 0 -CTCCCACTCCCGTGGCTTCTAG-3 0 Anti-sense: 5 0 -GTTCCACATGTTGCTCCACACTT-3 0 Sense: 5 0 -CTTTGTGGCTGAGCGAAT-3 0 Anti-sense: 5 0 -CAGTCACTTGGTTTCCCATA-3 0

Annealing temperatures (1C) 60 59 55 60 58

Wang et al.
(CD25) expression in splenocytes were examined. FACS analysis showed that both HIE and MIE increased CD25 expression in total splenocytes (Fig. 4a and b).

Effect of exercise on CD41CD251 Treg cells To further investigate whether HIE/MIE aects the CD41CD251 Treg cells, splenocytes from exercisetrained mice were isolated and T cell surface makers were co-stained and detected by FACS. Our results indicated that the proportion of CD41CD251 T cells was signicantly higher in the HIE group than in either of the control groups, whereas there was no signicant dierence between the MIE group and the controls (Fig. 4c). In addition, HIE but not MIE markedly increased Foxp3 expression, a CD41 CD251 Treg cell-specic marker (Fig. 4d). These results suggest that HIE, but not MIE, may impair immune response in mice by increasing Treg cells. IL-10 is a key immunomodulatory factor that inhibits the release of pro-inammatory cytokines by innate immune cells (Maynard et al., 2007). Because IL-10 is produced by CD41 T cells, we asked whether exercise of varying intensities alters the production of IL-10. Our results showed that IL10 expression by CD41 T cells was signicantly higher in HIE compared with control groups (Fig. 4e), whereas no eect was seen for MIE (Fig. 4e). These results also suggest that HIE may impair immune responses possibly through an increase in IL-10 production by CD41 T cells.

Fig. 2. Eects of exercise on pro- and anti-inammatory cytokine expression, as determined by RT-PCR. Total RNA was collected from the splenocytes of mice subjected to moderate- or high-intensity exercise (MIE/HIE) or from na ve or TC mice after 6 weeks. The mRNA levels of IL-2, IL-12p40, EBi3, and TGF-b were analyzed by RT-PCR and electrophoresis (a) and were normalized according to b-actin to evaluate the relative expression of the target (b). Results means SEM, n 5 8. *Po0.05 compared with the na ve and TC group.

and Ebi3 (for IL-35) were detected by RT-PCR, while IL-10 expression was examined by FACS analysis. As shown in Figs 2 and 3, HIE up-regulated TGF-b, Ebi3, and IL-10 expression and down-regulated IL-12p40 expression compared with the na ve and TC groups. By contrast, MIE increased the expression of IL-12p40 but had no eect on the expression of TGF-b, Ebi3, and IL-10. HIE increases IL-2R but reduces IL-2 and IFN-g expression IL-2 is a key cytokine promoting T cell proliferation, and changes in its production are often associated with T cell activation and proliferation. We therefore examined whether exercise of varying intensities alters IL-2 production. Our results showed that, compared with the two control groups, IL-2 mRNA expression was lower in the HIE group and higher in the MIE group (Fig. 2). By contrast, production of the T cell cytokine IFN-g was inhibited by HIE (Fig. 3a and c), consistent with nding in the expression of pro-inammatory cytokine IL-12 (Fig. 2). Because IL-2 up-regulates IL-2R expression and exerts its eects on T cells through the high-anity IL-2R, the eects of HIE and MIE on IL-2Ra

Effect of exercise on HBV DNA vaccination DNA vaccines have a number of advantages over conventional vaccines, including the ability to induce strong humoral and cellular immune responses. We therefore examined whether exercise of varying intensities dierentially aects humoral and cellular immunity, specically, in terms of the immune response to HBV DNA vaccination, as a model DNA vaccine. We rst analyzed the ability of HIE/MIE to elicit a humoral and cellular immune response to HBV DNA vaccine. The running C57BL/6 mice were vaccinated intramuscularly with 100 mg of pVAX-S2. Total serum IgG against HBsAg was determined by quantitative ELISA. As shown in Fig. 5, signicant dierences in total anti-HBsAg IgG were not detected among the four groups. To further determine the eects of HIE/MIE on CMI responses, T cell proliferation was evaluated in vitro. Figure 6 shows that the MIE group exhibited a higher level of T cell proliferation, whereas the HIE group exhibited a lower T cell proliferation compared with the two control groups.

Exercises, Treg cells and vaccination

Fig. 3. Eects of exercise on IL-10 and IFN-g expression, as determined by FACS analysis. Splenocytes isolated from C57BL/ 6 mice on week 6 after moderate- or high-intensity exercise were stained for IL-10 and IFN-g and analyzed by FACS. Representative results are shown. The percent expression of IL-10 and IFN-g in splenocytes from each group (a and b) and the per cent expression of IL-10 and IFN-g, summarized as the means of three independent experiments (c), are indicated. Results are means SEM, n 5 8. Means without a common letter dier, Po0.05.

Next, we wanted to determine the eects of HIE/ MIE on HBsAg-specic Th cell responses. Cytokine expression of CD41 and CD81 T cells were examined by intracellular staining. The data showed that MIE increased the IFN-g expression for antigenspecic CD41 and CD81 T cells (Fig. 7). By contrast, HIE reduced the IFN-g expression in antigenspecic CD41 and CD81 T cells (Fig. 7). In addition, neither HIE nor MIE had an eect on IL-4 expression in total CD41 T cells, indicating an unaltered Th2 response (Fig. 7). Specic cytotoxic responses have been demonstrated as another key eector required to clear HBV-infected cells at the carrier stage (Davis et al., 1996). To conrm the eect of HIE/MIE on CMI, the specic cytotoxic response was tested in vivo on day 14 after the third immunization. As shown in Fig. 8, the MIE groups had a signicantly augmented eect on HBsAg-specic cytotoxic responses compared with the other groups. By contrast, HIE impaired antigen-specic cytotoxic activities. Overall, the magnitude of impairment of the HBsAgspecic cytotoxic response corresponded with the

amounts of HBsAg-specic IFN-g-positive CD81 T cells observed in Fig. 6 in the same group.

Discussion Most studies on the eects of exercise on immunity have shown that habitual, moderate physical activity augments immune responses by increasing the expression of pro-inammatory cytokines, including IL-2, IL-1b, and TNF-a (Haahr et al., 1991; Zaldivar et al., 2006) while exhaustive exercise tends to be immunosuppressive. In the current study, MIE was shown to increase both IL-2 production and the expression of pro-inammatory cytokine IL-12 (IL12p40), which promotes Th1-type immunity and decreases the risk of URTI. The increased expression of IL-12 was consistent with that of IFN-g. Moreover, regular MIE did not aect the anti-inammatory cytokines TGF-b and IL-10 but increased IL2Ra (CD25) expression. In our study, we did not determine IL-2/IL-2R activity. However, we speculate that IL-2/IL-2R signaling is involved in inducing

Wang et al.

Fig. 4. Eect of exercise on CD41CD251 T-regulatory cells and IL-10 expression. Splenocytes isolated from C57BL/6 mice on week 6 after exercise of moderate or high intensity were stained either for surface markers or intracellularly with anti-CD4 jointly with anti-CD25, anti-Foxp3, or anti-IL-10 antibodies and then analyzed by FACS. Representative results showing the per cent expression of CD25 in splenocytes from each group (a) and the per cent expression of CD25 are included. The results in (b) and the abundance of CD251 (c), Foxp3 (d), and IL-10 (e) in CD41 T cells means SEM of eight independent experiments. Means without a common letter dier, Po0.05.

IFN-g production by regular MIE. In addition, IL-2/ IL-2R signaling is essential for maintaining selftolerance, as mice decient in either IL-2 or IL-2R exhibit lethal autoimmunity (Nelson, 2004; Antony et al., 2006). These in vivo studies strongly favored a

model whereby IL-2 controls autoimmunity through the production of CD41CD251 Treg cells. In the current study, MIE did not alter the proportion of CD41CD251 Treg cells, thus supporting recent studies in which regular moderate exercise (TCC)

Exercises, Treg cells and vaccination


had no eect on the CD41CD251 Treg cells of healthy humans (Yeh et al., 2009). In a recent human study, regular TCC exercise increased the T-bet expression, a specic transcription factor for Th1 cells, and Foxp3 expression in patients with type 2 DM, but not in normal age-matched adults, suggesting that the immunological eects of MIE may be modulated by health status. Unlike MIE, prolonged, exhaustive HIE has been shown to suppress immune responses and increase the risk of URTI (Nieman, 1997; Nieman et al., 2006). Although the plasma level of IL-6, an inammation-responsive cytokine, increases following an acute bout of prolonged, exhaustive exercise, antiinammation cytokines (e.g., IL-1 receptor antagonist [IL-1ra], IL-4, IL-10, and cortisol) also increase, which may minimize the inammatory process (Homan-Goetz., 1996; Steensberg et al., 2003). Furthermore, IL-6 is regarded as anti- and pro-inammatory depending on the tissues where it is secreted (Pedersen & Bruunsgaard, 2003; Calle & Fernandez, 2010). Lastly, studies demonstrate that an acute bout of intensity exercise can increase the production of proinammatory cytokines in response to exercise, but these cytokines decline post-exercise (Pedersen & Homan-Goetz, 2000; Petersen & Pedersen, 2005).Our results suggest that the mechanism by which HIE increases the risk of URTI may be

Fig. 5. Eect of exercise on humoral responses in immunized running mice. Mice subjected to moderate- or highintensity exercise were subsequently immunized with HBV DNA vaccine on days 0, 14, and 28. Serum samples from C57BL/6 mice were collected for ELISA on day 14 after the nal boost. Total anti-HBsAg antibodies were quantitated with a commercial kit, and values were determined relative to the standard concentration of anti-HBsAg antibody. Mean titers are expressed in milli-international units per milliliter. Total anti-HBsAg IgG in serum was detected as described in the Materials and methods.

Fig. 7. Eect of exercise on antigen-specic cytokine productions in T cells. T cells isolated from the spleen of C57BL/6 mice on day 14 after the nal boost were stimulated with HBsAg-derived peptide S208-215 for 6 h in culture. Intracellular staining for IL-4 or IFN-g in CD41 T cells, and IFN-g in CD81 T cells was performed by FACS. The summaries of percentage of IL-4 in CD41, IFN-g in CD41 and CD81 T cells are shown. Results are means SEM, n 5 5. *Po0.05 compared with the na ve and TC group.

Fig. 6. Eect of exercise on T cell proliferation in immunized running mice. To conrm whether HIE/MIE impaired/enhanced cell-mediated immune responses to the HBV DNA vaccine, the T cells were isolated from animals of all groups on day 14 after the nal immunization and then the T cell proliferation was performed as described in Materials and methods. The histograms show a presentative result (a) and the percentage of divided cells was summarized in the means of ve independent experiments (b). Means without a common letter dier, Po0.05.

Wang et al.

Fig. 8. Eect of exercise on antigen-specic cytotoxic responses in vivo. To analyze eect of exercise on HBsAg-specic cytotoxicity in vivo, a 1:1 mixture of the CSFEhigh labeled specic target and CSFElow labeled non-specic target cells were transferred into each group via i.v. After 4 h, these mice were killed and the specic lysis was analyzed by FACS and calculated as described in Materials and methods. The histograms show a presentative result (a) and the percentage of specic lysis was summarized in the means of ve independent experiments (b). Means without a common letter dier, Po0.05.

through a down-regulation of pro-inammatory cytokines (mainly IL-2, IFN-g, and IL-12) together with an increase in anti-inammatory cytokines (TGF-b, IL-10, and IL-35 [Ebi3]). In addition, HIE increased IL-2R expression relative to the control groups. Thus, the HIE-mediated impairment of immune responses may be due to a defective IL-2/IL2R signaling; however, this hypothesis needs to be validated. Whether an acute bout of high-intensity exercise could aect IL-12, IL-2, IL-2R, and IFNg expression is also yet to be determined. It has been demonstrated that the presence of higher numbers of Treg cells in the lungs of mice and humans during infection may lead to failure to clear the infection. This may contribute to the establishment of a chronic phase in which Treg cells are involved in limiting immune-mediated tissue damage (Curotto de Lafaille & Lafaille, 2009). We found that prolonged, exhaustive HIE increased the proportion of CD41CD251 T cells and the expression of Foxp3, a CD41CD251 Treg cell-specic marker, in spleen. It is imperative to further investigate whether prolonged or exhaustive HIE increases the risk of URTI through elevated number of Treg cells in infection models. It is well known that hormonal changes occur in response to exercise, including increases in the plasma concentration of epinephrine (adrenaline), cortisol, growth hormone, and prolactin, all of which have immunomodulatory eects (Pool & Axford, 2001; Venkatraman et al., 2001). Moreover, recent studies have shown that a novel anti-inammatory cytokine, IL-35, expands Treg cells and inhibits IFNg production. Thus, it is possible that HIE expands CD41CD251 Treg cells by increasing IL-35 production (Niedbala et al., 2007). Furthermore, cortisol and epinephrine suppress Th1 cell cytokine produc-

tion and Th1 response promotes CMI, which aords the host protection against viruses (Franchimont et al., 2000). In addition, cortisol, and anti-inammatory cytokines such as TGF-b, can also induce the expansion of CD41CD251 Treg cells (Horwitz et al., 2008; Kang et al., 2008). However, whether hormones or other immune-modulating factors are involved in expanded CD41CD251 Treg cells seen after HIE is worth further investigation. Compared with what we know about HIE, we know even less about how regular MIE augments immune responses. Studies have suggested that the density of b-adrenergic receptor, which is expressed on Th1, not Th2 cells, is involved in mediating the endurance exercise-training associated increase in Th1 response (Nieto et al., 1997; Kohut et al., 2004). Another mechanism that deserves further validation is that MIE may enhance T cell function by increasing CD28 expression, because CD28 has been shown to induce IL-2 production and IL-2R expression leading to T cell proliferation (Jenkins et al., 1991; Shimizu et al., 2008). DNA vaccines specic for HBV hold particular promise in the treatment of chronic HBV infections and have been shown to induce strong humoral immunity and CMI in animal models and healthy volunteers (Mancini-Bourgine et al., 2004). In addition, several studies have shown that MIE increases secondary antibody responses to antigens especially certain protein antigens (Sugiura et al., 2001). However, whether exercise of varying intensities has dierential eects on the immune response to DNAbased vaccinations had not been examined. In this study, we showed that MIE augmented CMI by increasing the production of Th1 cell cytokines and causing the proliferation of HBsAg-specic T cells,

Exercises, Treg cells and vaccination


thereby stimulating HBsAg-specic cytotoxic activities, which provide protection against viruses. In contrast, HIE led to impaired CMI in mice vaccinated with the HBV DNA vaccine. However, whether this eect is mediated by the HIE-specic expansion of CD41CD251 Treg cells or an increase in IL-10 expression by CD41 T cells remains to be investigated. Perspectives We report here that regular/habitual physical activity (i.e., MIE) enhances the pro-inammatory cytokine and CMI response in mice, which have an implication in lowering the risk of cancer and infectious illness in humans. However, as chronic low grade inammation is being increasingly associated with the risk of developing chronic cardiovascular and metabolic diseases, regular exercise that includes some high-intensity work can improve health benet probably via an altered Th1/Treg balance (Balducci et al., 2010). HIE training appears to suppress immune function by increasing Treg cells and results in a reduced pro-inammatory and an increased antiinammatory cytokine expression, which might increase risk of common infections such as URTI (Curotto de Lafaille & Lafaille, 2009). A recent study showed that the combination of high-intensity aerobic plus resistance exercise training, in addition to daily physical activity, is required to achieve a signicant anti-inammatory eect in type 2 diabetic patients (Balducci et al., 2010). However, it would be interesting to determine whether HIE, or its combination with resistance exercise training is more benecial than MIE in reducing risk of chronic cardiovascular and metabolic diseases via its antiinammatory eects, especially CD41CD251 Treg cells.
Key words: cell-mediated immunity, cytokines, lymphocytes.

Acknowledgements
This work was supported in part by the National Natural Science Foundation of China (30972687) (to Y. M) and a research initiation fund from Henan University to J. W.

References
Antony PA, Paulos CM, Ahmadzadeh M, Akpinarli A, Palmer DC, Sato N, Kaiser A, Heinrichs C, Klebano CA, Tagaya Y, Restifo NP. Interleukin-2dependent mechanisms of tolerance and immunity in vivo. J Immunol 2006: 176: 52555266. Balducci S, Zanuso S, Nicolucci A, Fernando F, Cavallo S, Cardelli P, Fallucca S, Alessi E, Letizia C, Jimenez A, Fallucca F, Pugliese G. Antiinammatory eect of exercise training in subjects with type 2 diabetes and the metabolic syndrome is dependent on exercise modalities and independent of weight loss. Nutr Metab Cardiovasc Dis 2010: 20: 608617. Calle MC, Fernandez ML. Eects of resistance training on the inammatory response. Nutr Res Pract 2010: 4: 259 269. Curotto de Lafaille MA, Lafaille JJ. Natural and adaptive Foxp31 regulatory T cells: more of the same or a division of labor? Immunity 2009: 30: 626635. Davis HL, McCluskie MJ, Gerin JL, Purcell RH. DNA vaccine for hepatitis B: evidence for immunogenicity in chimpanzees and comparison with other vaccines. PNAS 1996: 93: 7213 7218. Davis JM, Kohut ML, Colbert LH, Jackson DA, Ghaar A, Mayer EP. Exercise, alveolar macrophage function, and susceptibility to respiratory infection. J Appl Physiol 1997: 83: 14611466. Davis JM, Murphy EA, Brown AS, Carmichael MD, Ghaar A, Mayer EP. Eects of moderate exercise and oat {beta}-glucan on innate immune function and susceptibility to respiratory infection. Am J Physiol Regul Integr Comp Physiol 2004: 286: R366R372. Davis JM, Weaver JA, Kohut ML, Colbert LH, Ghaar A, Mayer EP. Immune system activation and fatigue during treadmill running: role of interferon. Med Sci Sports Exerc 1998: 30: 863868. Fernandez MA, Puttur FK, Wang YM, Howden W, Alexander SI, Jones CA. T regulatory cells contribute to the attenuated primary CD81 and CD41 T cell responses to herpes simplex virus type 2 in neonatal mice. J Immunol 2008: 180: 15561564. Fernando P, Bonen A, Homan-Goetz L. Predicting submaximal oxygen consumption during treadmill running in mice. Can J Physiol Pharmacol 1993: 71: 854857. Franchimont D, Galon J, Gadina M, Visconti R, Zhou YJ, Aringer M, Frucht DM, Chrousos GP, OShea JJ. Inhibition of Th1 immune response by glucocorticoids: dexamethasone selectively inhibits IL-12-induced Stat4 phosphorylation in T lymphocytes. J Immunol 2000: 164: 17681774. Furuichi Y, Tokuyama H, Ueha S, Kurachi M, Moriyasu F, Kakimi K. Depletion of CD251CD41T cells (Tregs) enhances the HBV-specic CD81T cell response primed by DNA immunization. World J Gastroenterol 2005: 11: 37723777. Haahr PM, Pedersen BK, Fomsgaard A, Tvede N, Diamant M, Klarlund K, Halkjaer-Kristens J, Bendtzen K. Eect of physical exercise on in vitro production of interleukin 1, interleukin 6, tumour necrosis factor-alpha, interleukin 2 and interferon-gamma. Int J Sports Med 1991: 12: 223 227. Homan-Goetz L. Exercise and cytokines: spontaneous and elicited responses. Boca Raton, Florida: CRC Press, 1996: 5577. Horwitz DA, Zheng SG, Wang J, Gray JD. Critical role of IL-2 and TGF-beta in generation, function and stabilization of Foxp31CD41Treg. Eur J Immunol 2008: 38: 912915. Jenkins MK, Taylor PS, Norton SD, Urdahl KB. CD28 delivers a costimulatory signal involved in antigen-specic IL-2 production by human T cells. J Immunol 1991: 147: 24612466. Kang Y, Xu L, Wang B, Chen A, Zheng G. Cutting edge: immunosuppressant

Wang et al.
as adjuvant for tolerogenic immunization. J Immunol 2008: 180: 51725176. Kohut ML, Thompson JR, Lee W, Cunnick JE. Exercise training-induced adaptations of immune response are mediated by {beta}-adrenergic receptors in aged but not young mice. J Appl Physiol 2004: 96: 13121322. Mancini-Bourgine M, Fontaine H, ScottAlgara D, Pol S, Brechot C, Michel ML. Induction or expansion of T-cell responses by a hepatitis B DNA vaccine administered to chronic HBV carriers. Hepatology 2004: 40: 874882. Maynard CL, Harrington LE, Janowski KM, Oliver JR, Zindl CL, Rudensky AY, Weaver CT. Regulatory T cells expressing interleukin 10 develop from Foxp31and Foxp3-precursor cells in the absence of interleukin 10. Nat Immunol 2007: 8: 931941. Murphy EA, Davis JM, Brown AS, Carmichael MD, Van Rooijen N, Ghaar A, Mayer EP. Role of lung macrophages on susceptibility to respiratory infection following shortterm moderate exercise training. Am J Physiol Regul Integr Comp Physiol 2004: 287: R1354R1358. Nelson BH. IL-2, Regulatory T Cells, and Tolerance. J Immunol 2004: 172: 39833988. Niedbala W, Wei XQ, Cai B, Hueber AJ, Leung BP, McInnes IB, Liew FY. IL-35 is a novel cytokine with therapeutic eects against collageninduced arthritis through the expansion of regulatory T cells and suppression of Th17 cells. Eur J Immunol 2007: 37: 30213029. Nieman DC. Risk of upper respiratory tract infection in athletes: an epidemiologic and immunologic perspective. J Athl Train 1997: 32: 344349. Nieman DC, Henson DA, Dumke CL, Lind RH, Shooter LR, Gross SJ. Relationship between salivary IgA secretion and upper respiratory tract infection following a 160-km race. J Sports Med Phys Fitness 2006: 46: 158162. Nieto JL, Diaz-Laviada I, Malpartida JM, Galve-Roperh I, Haro A. Adaptations of the b-adrenoceptoradenylyl cyclase system in rat skeletal muscle to endurance physical training. Pugers Arch Eur J Physiol 1997: 434: 809814. Pedersen BK, Bruunsgaard H. Possible benecial role of exercise in modulating low-grade inammation in the elderly. Scand J Med Sci Sports 2003: 13: 5662. Pedersen BK, Homan-Goetz L. Exercise and the immune system: regulation, integration, and adaptation. Physiol Rev 2000: 80: 10551081. Petersen AMW, Pedersen BK. The antiinammatory eect of exercise. J Appl Physiol 2005: 98: 11541162. Pool AJ, Axford JS. The eects of exercise on the hormonal and immune systems in rheumatoid arthritis. Rheumatology 2001: 40: 610614. Sakaguchi S. Naturally arising Foxp3expressing CD251CD41regulatory T cells in immunological tolerance to self and non-self. Nat Immunol 2005: 6: 345352. Shimizu K, Kimura F, Akimoto T, Akama T, Tanabe K, Nishijima T, Kuno S, Kono I. Eect of moderate exercise training on T-helper cell subpopulations in elderly people. Exerc Immunol Rev 2008: 14: 2437. Steensberg A, Fischer CP, Keller C, Moller K, Pedersen BK. IL-6 enhances plasma IL-1ra, IL-10, and cortisol in humans. Am J Physiol Endocrinol Metab 2003: 285: E433E437. Steensberg A, Toft AD, Bruunsgaard H, Sandmand M, Halkjar-Kristensen J, Pedersen BK. Strenuous exercise decreases the percentage of type 1 T cells in the circulation. J Appl Physiol 2001: 91: 17081712. Sugiura H, Sugiura H, Nishida H, Inaba R, Mirbod SM, Iwata H. Eects of dierent durations of exercise on macrophage functions in mice. J Appl Physiol 2001: 90: 789794. Tai P, Wang J, Jin H, Song X, Yan J, Kang Y, Zhao L, An X, Du X, Chen X, Wang S, Xia G, Wang B. Induction of regulatory T cells by physiological level estrogen. J Cell Physio 2008: 214: 456464. Venkatraman JT, Feng X, Pendergast D. Eects of dietary fat and endurance exercise on plasma cortisol, prostaglandin E2, interferon-{gamma} and lipid peroxides in runners. J Am Coll Nutr 2001: 20: 529536. Wang J, Su B, Ding Z, Du X, Wang B. Cimetidine enhances immune response of HBV DNA vaccination via impairment of the regulatory function of regulatory T cells. Biochem Biophys Res Commun 2008: 372: 491496. Yeh SH, Chuang H, Lin LW, Hsiao CY, Eng HL. Regular tai chi chuan exercise enhances functional mobility and CD4CD25 regulatory T cells. Br J Sports Med 2006: 40: 239243. Yeh SH, Chuang H, Lin LW, Hsiao CY, Wang PW, Liu RT, Yang KD. Regular Tai Chi Chuan exercise improves T cell helper function of patients with type 2 diabetes mellitus with an increase in T-bet transcription factor and IL-12 production. Br J Sports Med 2009: 43: 845850. Zaldivar F, Wang-Rodriguez J, Nemet D, Schwindt C, Galassetti P, Mills PJ, Wilson LD, Cooper DM. Constitutive pro- and anti-inammatory cytokine and growth factor response to exercise in leukocytes. J Appl Physiol 2006: 100: 11241133.

10