The Impaired Function of Macrophages Induced by Strenuous Exercise Could Not Be Ameliorated by BCAA Supplementation

The aim of this study was to evaluate the effect of strenuous exercise on the functions of peritoneal macrophages in rats and to test the hypothesis that branched-chain amino acid (BCAA) supplementation will be beneficial to the macrophages of rats from strenuous exercise. Forty male Wistar rats were randomly divided into five groups: (C) Control, E) Exercise, (E1) Exercise with one week to recover, (ES) Exercise + Supplementation and (ES1) Exercise + Supplementation with 1 week to recover. All rats except those of the sedentary control were subjected to four weeks of strenuous exercise. Blood hemoglobin, serum testosterone and BCAA levels were tested. Peritoneal macrophages functions were also determined at the same time. The data showed that hemoglobin, testosterone, BCAA levels, and body weight in group E decreased significantly as compared with that of group C. Meanwhile, phagocytosis capacity (decreased by 17.07%, p = 0.031), reactive oxygen species (ROS) production (decreased by 26%, p = 0.003) and MHC II mRNA (decreased by 22%, p = 0.041) of macrophages decreased in the strenuous exercise group as compared with group C. However, the chemotaxis of macrophages did not change significantly. In addition, BCAA supplementation could slightly increase the serum BCAA levels of rats from strenuous exercise (increased by 6.70%, p > 0.05). Moreover, the body weight, the blood hemoglobin, the serum testosterone and the function of peritoneal macrophages in group ES did not change significantly as compared with group E. These results suggest that long-term intensive exercise impairs the function of macrophages, which is essential for microbicidal capability. This may represent a novel mechanism of immunosuppression induced by strenuous exercise. Moreover, the impaired function of macrophage induced by strenuous exercise could not be ameliorated by BCAA supplementation in the dosing and timing used for this study.


Introduction
Exercise enhances or reduces immune functions depending on its frequency, duration and intensity. Regular physical activity is known to enhance immune functions leading to a decrease in the occurrence of infections. On the other hand, heavy or exhaustive exercise increases the susceptibility to infections [1][2][3]. Exercise increases or decreases the occurrence of infections that may be related to the changes of macrophage functions [4,5]. Monocytes/macrophages are considered to be the frontline of immunological defense against pathogens. These cells have prominent roles such as Ag presentation, chemotaxis, phagocytic, microbicidal, tumoricidal and secretory functions, as well as, innate immunity, by initiating inflammatory and immune responses [6]. Although some studies focus on the relationship between exercise and macrophages, little attention has been paid to the effects of long-term intensive exercise on macrophage functions. post-exercise and maintained for 4 weeks. The dose and delivery method of BCAA was determined based on previous studies [18,19].
Nutrients 2015, 7, page-page post-exercise and maintained for 4 weeks. The dose and delivery method of BCAA was determined based on previous studies [18,19].  3 Figure 1. Schematic representation of the experimental program. Group C, Control; E, Exercise; E1, Exercise with 1 week to recover; ES, Exercise + Supplementation; ES1, Exercise + Supplementation with 1 week to recover. T, testosterone; W, weight; Hb, hemoglobin; BCAA, branched-chain amino acid; ROS, Reactive oxygen species; AP, antigen presentation.

Biochemical Analyses
Blood sample (3 mL) was taken from the fossa orbitalis venous plexus of rats. Blood hemoglobin, serum testosterone and serum BCAA level were determined. Blood hemoglobin was measured by hematology analyzer (Sysmex, Japan). Serum testosterone level was assessed using a commercial ELISA kit (AssayPro, St. Charles, MO, USA) according to the manufacturer's instructions. All samples and standards were measured in duplicate. BCAA levels in the serum were measured using high-performance liquid chromatography (HPLC, Hitachi, Ltd., Tokyo, Japan) according to the method by Deyl et al. [20].

Peritoneal Macrophages Preparation
As reported in our previous study [5], the MΦs (peritoneal macrophages) were removed by peritoneal lavage using RPMI 1640 (GIBCO, Carlsbad, CA, USA). The cells were washed by centrifugation, resuspended in RPMI 1640 with 10% fetal bovine serum (GIBCO, Carlsbad, CA, USA), plus 1% penicillin-streptomycin solution, and then placed in 6-well tissue culture microplates. Plates were incubated for 2 h at 37˝C in a humidified atmosphere of 5% CO 2 . After the removal of non-adherent cells, the adherent cells were detached by treatment with 0.25% Trypsin and suspended in RPMI 1640 at a concentration of 2ˆ10 6 cells/mL. Cell viability was checked with the Trypan blue dye and was >96%. Cell purity checked by the Giemsa dye test was >98%.

Chemotaxis Assay
Following Yang et al. [21] and Novak et al. [22], with little modification, the macrophages were washed twice in serum-free RPMI 1640 and resuspended at 1ˆ10 6 cells/mL. 100 µL cells were added into the upper chambers of a 24-well transwell plate with 8-µm pore size polycarbonate filters (Costar, Corning, NY, USA). The plate was equilibrated at 37˝C in a 5% CO 2 cell culture incubator for 30 min. 600 µL of the serum-free RPMI 1640 (MCP-1, 10 ng/mL, Sigma-Aldrich, St. Louis, MO, USA) was added into the lower chambers of the transwells to induce migration. After 2 h at 37˝C in a 5% CO 2 cell culture incubator, the cells remaining in the upper chambers were wiped off with a cotton swab. Migrated cells attached to the lower surface of the filters were fixed with 75% ethanol for 30 min, washed with water, and stained with hematoxylin. The number of migrated cells was counted under microscope. For each sample, cells in 5 randomly picked fields under 200ˆmagnification were counted. Data were expressed relative to control group cell migration.

Phagocytosis Assay
The uptake of the neutral red by macrophages was measured following Long et al. [23] with the following modifications. The cell suspension (2ˆ10 6 cells/mL) was incubated in a 96-well flat-bottomed microtiter plate 100 µL/well for 2 h at 37˝C in a 5% CO 2 cell incubator. After one wash with warm PBS (pH 7.2 to 7.4), 200 µL of 0.1% neutral red (Amersco, Solon, OH, USA) solution in PBS was added. To minimize crystal formation during the neutral red assay, the dye solution was incubated overnight at 37˝C and sterile filtered before use. After 30 min of incubation of the culture plates at 37˝C, neutral red solution was aspirated, and each well was thrice carefully rinsed with PBS. Finally, the intracellular dye was extracted with 200 µL of a mixture of 100% ethanol and 99.9% acetic acid (1:1 v/v). The mixtures were mixed fully and evaluated at a wavelength of 550 nm on a Bio-Rad 550 microplate reader (Bio-Rad Laboratories, Hercules, CA, USA). The absorbance represented phagocytosis by macrophages.

Reactive Oxygen Species Determination
Following Bae et al. [24], with little modification (no stimulus was added to the cell suspension to induce macrophage activation), the macrophages (5ˆ10 5 cells) were incubated with 2 1 ,7 1 -dichlorofluorescein diacetate (DCFH-DA; Molecular Probes) for 20 min. The fluorescence intensity was analyzed by flow cytometry using a Coulter EPICS XL TM flow cytometer with the System II TM software (Beckman Coulter, Fullerton, CA, USA). The level of ROS was expressed as relative fluorescence intensities generated by counting 10,000 cells.

Real Time reverse-transcription polymerase chain reaction (Real Time RT-PCR)
Total RNA of macrophages was isolated using a modified guanidinium isothiocyanate-CsCl method [25]. RNA was reverse transcribed into cDNA using the Revertaid TM First Strand cDNA Synthesis Kit from Fermentas. Quantitative PCR was carried out in triplicates in reactions consisting of 12.5 µL 2ˆMaxima SYBR Green/ROX qPCR Master mix (Thermo Scientific), 1 µL cDNA, nuclease-free water and 300 nM of each primer [26]. Using Primer Express software (Applied Biosystems), we designed the following primers for the present study: β-actin (forword: 5'-GGA GAT TAC TGC CCT GGC TCC TA-3'; reverse: 5'-GAC TCA TCG TAC TCC TGC TTG CTG-3') and MHC II α chain (forword: 5'-AGA GAC CAT CTG GAG ACT TG-3'; reverse: 5'-CAT CTG GGG TGT TGT TGG A-3'). Amplifications were performed on a StepOne Plus™ PCR-Cycler (Life Technologies) with the following parameters: activation at 95˝C for 10 min, 40 cycles of denaturation at 95˝C for 15 s, and annealing/extension at 60˝C for 1 min. The threshold cycle (CT, the number of cycles to reach threshold of detection) was determined for each reaction, and the levels of the target mRNAs were quantified relatively to the level of the housekeeping gene β-actin using 2´∆ ∆CT method [27].

Statistical Analysis
All values are expressed as mean˘SD( Standard Deviation), and statistical significance was set at p < 0.05. Mean values were compared between groups by ANOVA(Analysis of Variance) with the LSD(Least Significant Difference) method as a post hoc test. Data were analyzed using SPSS 19.0 for windows.

Body Weight, Hemoglobin and Testosterone Levels
The mean final body weight and the mean concentrations of blood hemoglobin and serum testosterone are presented in Table 2. The weight of the rats of the strenuous exercise group was significantly lower than that of the sedentary control group (decreased by 13.86%, p = 0.000). In addition, blood assay showed that blood hemoglobin and serum testosterone in strenuous exercise group decreased significantly as compared with the sedentary control group (decreased by 9.27%, p = 0.005; 31.40%, p = 0.001; respectively). Furthermore, body weight, blood hemoglobin and serum testosterone in group ES were still significantly lower than that of group C. There was no significant difference between group ES and E (p > 0.05).  Figure 2 shows changes in serum BCAA levels after strenuous exercise and BCAA supplementation. The data showed that serum BCAA levels of rats from strenuous exercise group decreased significantly as compared with the rats from the sedentary group (decreased by 11.71%, p = 0.007). On the other hand, BCAA supplementation could recover serum BCAA levels. There was no significant difference between group ES and C. After seven days of recovery, no difference was observed between group E1 and C.

Serum BCAA Levels
Nutrients 2015, 7, page-page no significant difference between group ES and C. After seven days of recovery, no difference was observed between group E1 and C.     Nutrients 2015, 7, page-page no significant difference between group ES and C. After seven days of recovery, no difference was observed between group E1 and C.    Figure 4 shows changes in chemotaxis of MÔs after strenuous exercise and BCAA supplementation. In this study, we added MCP-1 into the lower chambers of the transwells to induce migration. Data  Figure 4 shows changes in chemotaxis of MΦs after strenuous exercise and BCAA supplementation. In this study, we added MCP-1 into the lower chambers of the transwells to induce migration. Data showed that the migration capacity of MΦs from the strenuous exercise group has a tendency to increase as compared with the cells from the sedentary control. However, there was no significant difference between the two groups. Furthermore, BCAA supplementation could not change the chemotaxis of MΦs.

Chemotaxis
Nutrients 2015, 7, page-page showed that the migration capacity of MÔs from the strenuous exercise group has a tendency to increase as compared with the cells from the sedentary control. However, there was no significant difference between the two groups. Furthermore, BCAA supplementation could not change the chemotaxis of MÔs.  Figure 5 shows changes in ROS generation of MÔs after strenuous exercise and BCAA supplementation. The production of ROS in MÔs from the strenuous exercise group decreased significantly as compared with the cells from the sedentary group (Control vs. Strenuous exercise, 1.00 ± 0.14 vs. 0.74 ± 0.16; decreased by 26%, p = 0.003). After seven days of recovery, the ROS generation of MÔs from group E1 was significantly higher than that of group E (p = 0.000), and no difference was observed between group E1 and C. In addition, the ROS generation of MÔs from group ES did not change as compared with group E. It was still significantly lower than that of group C (p = 0.020). Similarly, the ROS generation of MÔs from group ES1 was significantly higher than that of group ES (p = 0.012), and there was no difference as compared with group E1 or C (p > 0.05).  Figure 5 shows changes in ROS generation of MΦs after strenuous exercise and BCAA supplementation. The production of ROS in MΦs from the strenuous exercise group decreased significantly as compared with the cells from the sedentary group (Control vs. Strenuous exercise, 1.00˘0.14 vs. 0.74˘0.16; decreased by 26%, p = 0.003). After seven days of recovery, the ROS generation of MΦs from group E1 was significantly higher than that of group E (p = 0.000), and no difference was observed between group E1 and C. In addition, the ROS generation of MΦs from group ES did not change as compared with group E. It was still significantly lower than that of group C (p = 0.020). Similarly, the ROS generation of MΦs from group ES1 was significantly higher than that of group ES (p = 0.012), and there was no difference as compared with group E1 or C (p > 0.05).

ROS Generation
Nutrients 2015, 7, page-page showed that the migration capacity of MÔs from the strenuous exercise group has a tendency to increase as compared with the cells from the sedentary control. However, there was no significant difference between the two groups. Furthermore, BCAA supplementation could not change the chemotaxis of MÔs.  Figure 5 shows changes in ROS generation of MÔs after strenuous exercise and BCAA supplementation. The production of ROS in MÔs from the strenuous exercise group decreased significantly as compared with the cells from the sedentary group (Control vs. Strenuous exercise, 1.00 ± 0.14 vs. 0.74 ± 0.16; decreased by 26%, p = 0.003). After seven days of recovery, the ROS generation of MÔs from group E1 was significantly higher than that of group E (p = 0.000), and no difference was observed between group E1 and C. In addition, the ROS generation of MÔs from group ES did not change as compared with group E. It was still significantly lower than that of group C (p = 0.020). Similarly, the ROS generation of MÔs from group ES1 was significantly higher than that of group ES (p = 0.012), and there was no difference as compared with group E1 or C (p > 0.05).  Figure 6 shows changes in MHC II mRNA level of MΦs after strenuous exercise and BCAA supplementation. Data showed that MHC II, the key molecule mediated macrophage antigen presentation [28][29][30][31], decreased significantly in the MΦs of strenuous exercise rats (Control vs. Strenuous exercise, 1.00˘0.19 vs. 0.78˘0.09; decreased by 22%, p = 0.041). After seven days of recovery, MHC II mRNA of MΦs from group E1 was significantly higher than group E (p = 0.017), and no difference was observed between group E1 and C. In addition, MHC II mRNA of MΦs from group ES did not change as compared with that of group E. It was still significantly lower than that of group C (p = 0.047). Similarly, MHC II mRNA of MΦs from group ES1 was significantly higher than that of group ES (p = 0.003), and there was no difference as compared with group E1 or C (p > 0.05).

MHC II mRNA Level
Nutrients 2015, 7, page-page 3.6. MHC II mRNA Level Figure 6 shows changes in MHC II mRNA level of MÔs after strenuous exercise and BCAA supplementation. Data showed that MHC II, the key molecule mediated macrophage antigen presentation [28][29][30][31], decreased significantly in the MÔs of strenuous exercise rats (Control vs. Strenuous exercise, 1.00 ± 0.19 vs. 0.78 ± 0.09; decreased by 22%, p = 0.041). After seven days of recovery, MHC II mRNA of MÔs from group E1 was significantly higher than group E (p = 0.017), and no difference was observed between group E1 and C. In addition, MHC II mRNA of MÔs from group ES did not change as compared with that of group E. It was still significantly lower than that of group C (p = 0.047). Similarly, MHC II mRNA of MÔs from group ES1 was significantly higher than that of group ES (p = 0.003), and there was no difference as compared with group E1 or C (p > 0.05). Total RNA of macrophages was isolated and reverse transcribed into cDNA. Quantitative PCR was carried out using β-actin as the housekeeping gene. Data are means ± SD. * p < 0.05; ** p < 0.01.

Discussion
In order to investigate the effects of strenuous exercise and BCAA supplementation on blood index of rats, blood hemoglobin and serum testosterone were tested. The data showed that blood hemoglobin and serum testosterone in the strenuous exercise group decreased significantly as compared with the control group. Furthermore, the body weight of the strenuous exercise group reduced significantly than that of the sedentary control. In addition, most of experimental groups could not keep up with the velocity of treadmill and had to be assisted by hand to complete the job at the last week of training. This means that the high-intensity exercise induced them to approach exhaustion. The data showed that the unbalanced condition was induced by four weeks of highintensity training. Moreover, BCAA supplementation could not change the body weight, the blood hemoglobin and the serum testosterone as compared with group E, which means that the unbalanced condition induced by strenuous exercise could not be improved by BCAA supplementation.
Phagocytosis, chemotaxis and antigen presentation are very important for macrophages in the removal of potentially pathogenic microorganisms [32]. Consequently, we tested the effects of strenuous exercise on the functions of MÔs. The results indicated that phagocytosis capacity (decreased by 17.07%, p < 0.05) and major histocompatibility complex (MHC) II antigen (decreased by 22%, p < 0.05) of MÔs from the strenuous exercise group was significantly lower than that of the control group. MHC II is a key molecule mediated macrophage antigen presentation [28][29][30][31]; meaning that MHC II mediated antigen presentation of MÔs will be inhibited by strenuous exercise. Our results are similar to others. For instance, it has been reported that the phagocytosis of pulmonary alveolar macrophages (PAM) was impaired after seven weeks of strenuous exercise [33]. In the study 8 Figure 6. Effects of strenuous exercise and BCAA supplementation on MHC II mRNA level of MΦs. Total RNA of macrophages was isolated and reverse transcribed into cDNA. Quantitative PCR was carried out using β-actin as the housekeeping gene. Data are means˘SD. * p < 0.05; ** p < 0.01.

Discussion
In order to investigate the effects of strenuous exercise and BCAA supplementation on blood index of rats, blood hemoglobin and serum testosterone were tested. The data showed that blood hemoglobin and serum testosterone in the strenuous exercise group decreased significantly as compared with the control group. Furthermore, the body weight of the strenuous exercise group reduced significantly than that of the sedentary control. In addition, most of experimental groups could not keep up with the velocity of treadmill and had to be assisted by hand to complete the job at the last week of training. This means that the high-intensity exercise induced them to approach exhaustion. The data showed that the unbalanced condition was induced by four weeks of high-intensity training. Moreover, BCAA supplementation could not change the body weight, the blood hemoglobin and the serum testosterone as compared with group E, which means that the unbalanced condition induced by strenuous exercise could not be improved by BCAA supplementation.
Phagocytosis, chemotaxis and antigen presentation are very important for macrophages in the removal of potentially pathogenic microorganisms [32]. Consequently, we tested the effects of strenuous exercise on the functions of MΦs. The results indicated that phagocytosis capacity (decreased by 17.07%, p < 0.05) and major histocompatibility complex (MHC) II antigen (decreased by 22%, p < 0.05) of MΦs from the strenuous exercise group was significantly lower than that of the control group. MHC II is a key molecule mediated macrophage antigen presentation [28][29][30][31]; meaning that MHC II mediated antigen presentation of MΦs will be inhibited by strenuous exercise.
Our results are similar to others. For instance, it has been reported that the phagocytosis of pulmonary alveolar macrophages (PAM) was impaired after seven weeks of strenuous exercise [33]. In the study of Woods et al., exhaustive exercise can negatively affect macrophages expression of MHC II, which may be detrimental to the ability of MΦs to present antigen to T lymphocytes [34]. In addition, in a previous study we found that 11 weeks of overload training decreased the phagocytosis and chemotaxis of MΦs [4]. However, in this study we did not find the decrease of chemotaxis of MΦs after four weeks of strenuous exercise. The varying results may be influenced by the quality and/or quantity of exercise applied in the studies.
In addition, we measured the production of intracellular reactive oxygen species (ROS) in macrophages. To investigate the effect of strenuous exercise on the production of ROS, no stimulus (e.g., Lipopolysaccharides) was added to the cell suspension to induce macrophage activation. The data showed that the production of ROS in macrophages from strenuous exercise group was significantly lower than that of the control group (decreased by 26%, p < 0.01). The result is similar to our previous study, in which we found that ROS production of macrophages was inhibited by 11 weeks of overload training [4]. ROS are generally considered cytotoxic. However, intracellular ROS also serves as important second messengers in cell signaling [35]. A number of studies have shown that ROS play important roles in regulating macrophages' survival [36], differentiation [37] and secretion of inflammatory cytokines [38]. Therefore, the ROS level of macrophages from strenuous exercise group was lower than physiological levels, which would impair the function of macrophages mediated by ROS.
These results indicate that the functions (i.e., phagocytosis capacity, MHC II-mediated antigen presentation and ROS generation) of macrophages were inhibited by four weeks of high-intensity exercise, which could impair the removal capability of potentially pathogenic microorganisms. This may be a mechanism that explains why long-term intensive exercise induces immunodepression and increases the susceptibility to infections. Although the functions of macrophages were impaired after strenuous exercise, these functions were nearly recovered after one week recovery. It means that the hindering functions of macrophages induced by strenuous exercise, was non-permanent and reversible.
Taking account of the good effect of BCAA supplementation on immune system [11,14,15], we speculated that BCAA supplementation would be beneficial to the macrophages of rats from strenuous exercise. The data showed that the phagocytosis, the ROS production and the MHC II mRNA of MΦs from group ES was still significantly lower than that of group C. Moreover, there was no significant difference between that of groups ES and E. It means that the hindering function of MΦs induced by strenuous exercise could not be ameliorated by BCAA supplementation. The results may be explained by the serum BCAA levels. In this study, we found that serum BCAA level decreased significantly in the rats of strenuous exercise. BCAA supplementation could slightly increase the serum BCAA level of rats from strenuous exercise; however, there was no significant difference between groups ES and E (increased by 6.70%, p > 0.05). Few studies have evaluated the roles of BCAA on macrophage function. For instance, Petro and Bhattacharjee reported that the ability of peritoneal macrophages to phagocytose and to kill S. typhimurium was not affected by BCAA restriction [39]. Kitagawa et al. also reported that BCAAs have protective effects on hepatic ischemia-reperfusion-induced liver injury through the attenuation of Kupffer cell (macrophage) activation [40]. In vitro experiments showed that high-BCAA medium increased IL-10 expression and phagocytic activity of microglial cells (macrophages) but did not affect the migration ability of these cells [41]. In spite of all these, our results are still difficult to compare with that of other studies owing to the fact that little attention has been paid to the effects of BCAA supplementation on peritoneal macrophage functions, especially in the model of strenuous exercise. If the data from rats are similar to human beings, then our results suggest that dietary BCAA supplementation is not useful to improving the macrophages function of people who engage in strenuous exercise. However, the effects of BCAA supplementation may be affected by many factors such as the dosing and timing.