The Effect of Long-Term Exercise on the Production of Osteoclastogenic and Antiosteoclastogenic Cytokines by Peripheral Blood Mononuclear Cells and on Serum Markers of Bone Metabolism

Although it is recognized that the mechanical stresses associated with physical activity augment bone mineral density and improve bone quality, our understanding of how exercise modulates bone homeostasis at the molecular level is lacking. In a before and after trial involving 43 healthy adults, we measured the effect of six months of supervised exercise training on the spontaneous and phytohemagglutinin-induced production of osteoclastogenic cytokines (interleukin-1α, tumor necrosis factor-α), antiosteoclastogenic cytokines (transforming growth factor-β1 and interleukins 4 and 10), pleiotropic cytokines with variable effects on osteoclastogenesis (interferon-γ, interleukin-6), and T cell growth and differentiation factors (interleukins 2 and 12) by peripheral blood mononuclear cells. We also measured lymphocyte phenotypes and serum markers of bone formation (osteocalcin), bone resorption (C-terminal telopeptides of Type I collagen), and bone homeostasis (25 (OH) vitamin D, estradiol, testosterone, parathyroid hormone, and insulin-like growth factor 1). A combination of aerobic, resistance, and flexibility exercises done on average of 2.5 hours a week attenuated the production of osteoclastogenic cytokines and enhanced the production of antiosteoclastogenic cytokines. These changes were accompanied by a 16% reduction in collagen degradation products and a 9.8% increase in osteocalcin levels. We conclude that long-term moderate intensity exercise exerts a favorable effect on bone resorption by changing the balance between blood mononuclear cells producing osteoclastogenic cytokines and those producing antiosteoclastogenic cytokines. This trial is registered with Clinical Trials.gov Identifier: NCT02765945.


Introduction
Interest in cytokines as regulators of bone metabolism began with the experiments of Horton and associates who, in 1972, found that conditioned medium from PHA-stimulated peripheral blood mononuclear cells contained bone resorbing activity [1]. This activity was eventually found to be due to interleukin-(IL-) 1 and tumor necrosis factor-(TNF-) [2,3], prompting a series of studies examining the role of these and other proinflammatory cytokines as mediators of bone resorption in periodontal disease, rheumatoid arthritis, osteolytic malignancies, and osteoporosis [4][5][6][7][8]. In addition to IL-1 and TNF-, the spectrum of cytokines with effects on bone metabolism now includes the antiosteoclastogenic cytokines IL-4 and IL-10 and transforming growth factor-(TGF-) , and two pleiotropic cytokines, interferon- (IFN-) and IL-6, whose effects on bone vary depending on experimental conditions [9,10].
Along with fall prevention and calcium and vitamin D supplementation, the Surgeon General has recommended regular physical activity as the first line in fracture prevention in persons with low bone density [11]. Evidence indicates that the mechanical stresses associated with physical activity augment bone mineral density and improve bone quality by promoting adaptive changes in its geometry and architecture [12]. In the microgravity of space, however, bone loss continues at an average rate of one to two percent per month despite the institution of vigorous exercise programs designed to duplicate conditions on Earth [13,14], indicating that there remains much to be learned about the mechanism(s) whereby physical exercise (and gravity) influences the ontogeny and functioning of hematopoietic and bone cells involved in the maintenance of bone health. We have investigated the possibility that long-term moderate intensity exercise improves bone health by favorably altering the production of cytokines with osteoclastogenic and antiosteoclastogenic properties by peripheral blood mononuclear cells.

Subjects.
This before and after clinical study was approved by the Institutional Review Board of East Tennessee State University. Each subject read and signed the informed consent in the presence of an investigator.
Subjects aged 30 to 60 were recruited from the general population by placing an outline of the study and a request for volunteers in three local newspapers. A total of 77 persons responded and all agreed to be screened for eligibility (see Figure 1). Fifty-two volunteers met eligibility criteria and were enrolled in a hospital-based wellness center where they underwent 6 months of supervised training. Subjects were required to exercise for a minimum of 30 minutes twice a week. Each training session consisted of a combination of aerobic (60%), resistance (weightlifting) (30%), and flexibility (stretching) (10%) exercises. Aerobic exercise choices included walking, running, cycling, rowing, climbing, skiing, and aerobics.
Forty-three subjects (25 women, average age 48 years [range , and 18 men, average age 49 years [range ) successfully completed the study; their risk factors for osteoporosis are listed in Table 1. Nine subjects were excluded: 8 did not meet attendance requirements and 1 moved to another location.
Supervisors kept detailed records documenting attendance, the duration and type of each exercise, weights, changes in medications, changes in dietary or smoking habits, and state of health. These data, along with risk factors for osteoporosis, were analyzed for their potential effects on outcome measurements (cytokine production, C-terminal telopeptides of Type I collagen, osteocalcin levels, hormonal levels, lymphocyte phenotypes, and mitogen responses) and for group and within-group differences.
Immunologic studies were done at baseline and after completion of 6 months of training. In order to minimize seasonal influences on the results, 2-3 subjects were enrolled each month over an eleven-month period. Subjects were instructed not to exercise outside of the supervised exercise program and for at least 24 hours prior to blood drawing. All blood samples were drawn in the morning at approximately the same time of day.

Mitogenic
Assays. Mitogenic responses were measured by adding methyl-3 H-thymidine (20 Ci/mL, 50 L) to culture samples removed 6 hours prior to supernatant harvesting. Labeled samples were incubated under 5% CO 2 at 37 ∘ C for an additional 6 hours, and the cells were collected on glass fiber filter paper using a Mash II Cell Harvester. Samples were air-dried, placed in vials containing Scinti-Verse II, and assayed with a Beckman LS 9800 liquid scintillation counter. Proliferative responses are expressed as net counts per minute (Δcpm), calculated as cpm in PHA-stimulated cultures minus cpm in unstimulated cultures.

Statistical Analysis.
Statistical analysis was done using STATISTICA (Statsoft, Inc., Tulsa, OK). The two-sided ttest for dependent samples was used to determine the significance of differences between measurements. The Pearson correlation test was used to quantify the relationship between two variables and linear regression was used to determine the predictability of Y from X. One-way analysis of variance (ANOVA) was used to compare measurements of three or more groups. Unless stated otherwise, results are expressed as the mean ± (SEM).

Cytokine Production by PBMCs.
In this analysis, we have identified IFN-as an osteoclastogenic cytokine and IL-6 as an antiosteoclastogenic cytokine. In our opinion, this most accurately reflects their effects on bone formation and resorption. The reader is referred to Table 2 for a summary of the effects of these and the other studied cytokines on bone homeostasis and immune cell function.

Mitogenic Responses.
PHA-induced proliferative responses of T cells fell from 9,142 ± 760 Δcpm to 3,155± 584 Δcpm following exercise ( < .0001). The reason for this change is not known, although it may reflect the change in CD4/CD8 ratios and an increased activity of suppressor cell populations (e.g., M2 monocytes, B1 cells, and T repressor cells).

Serum
Factors. CTXI levels fell by 16% ( = .0128) and osteocalcin levels increased by 9.8% ( > .05) in response to the exercise program (Figure 7). There was no significant change in serum levels of estradiol, testosterone, parathyroid hormone, or insulin-like growth factor-1 following exercise.

Group and Within-Group Variations
3.9.1. Cytokines. PBMCs taken from men prior to exercise spontaneously produced more IFN-and TNF-than mononuclear cells taken from women ( ≤ .04). Following exercise, men spontaneously produced more IL1-and TNFthan women ( ≤ .004).
In both men and women, exercise attenuated the production of TNF-and IFN-( ≤ .0062) and enhanced the production of IL-6 and TGF-1 ( ≤ .0028) in PHA-stimulated cultures. Postexercise levels of TGF-also increased in PHA − cultures in both groups ( ≤ .0049) (two-sided t-test for dependent samples) (Figures 9 and 10).

Habits and Body
Metrics. Four subjects (9.3%) changed their diet to one that was lower in energy intake and animal fat, and 2 of the 5 tobacco users discontinued smoking during the study. There were no changes in medications or alcohol consumption.
By completion of the study, 32% of the women and 27% of the men had lost weight, and 4% of the women and the 11% of men had gained weight; there was no significant change in the mean weight of either group.
No group or within-group differences could be demonstrated as a result of weight change, menopause, use of medications, alcohol consumption, or smoking (one-way ANOVA).

Discussion
The primary effect of our exercise program was to change the balance between PBMCs producing osteoclastogenic cytokines and those producing antiosteoclastogenic cytokines. It is likely that similar changes occurred in hematopoietic cells occupying the microenvironment of bone where they are ideally situated to influence the ontogeny and functioning of cells responsible for bone formation (osteoblasts), bone resorption (osteoclasts), and the transduction of bone loading signals (osteocytes) (see Table 2). This conclusion is supported by the finding that our exercise program caused significant reductions in serum levels of CTXI, a reliable marker of bone resorption, and modest rises in osteocalcin, a reliable marker of bone formation [53]. Postexercise levels of CTXI correlated inversely with estradiol levels, suggesting that estradiol enhanced the antiresorptive effects of exercise, a conclusion in keeping with the report of others that the  beneficial effect of exercise on bone density is blunted after menopause [54].
Our study participants averaged two and one-half hours of moderate intensity exercise per week: the same amount of exercise recommended for adult men and women by the World Health Organization for maintenance of health [55]. Participants divided their time between aerobic exercises (57%), resistance exercises (35%), and flexibility exercises (8%) and spent an average of 71 minutes in each training session. Extended over a six-month period, this amount of exercise was sufficient to reduce the spontaneous and PHA-induced production of osteoclastogenic cytokines by 24 and 59%, respectively, and to increase the spontaneous and PHA-induced production of antiosteoclastogenic cytokines by 89% and 50%, respectively. In PHA-stimulated cultures, osteoclastogenic cytokine production fell in proportion to the time subjects spent in each session doing aerobic exercises.
Cross-sectional studies involving adult subjects and using bone mineral density measurements have shown that exercises involving high impact (e.g., jumping) and high resistance (e.g., weightlifting) appear to be particularly effective in improving bone mass and content, especially when the intensity of the exercise is high and the speed of movement is elevated. Loaded (weight-bearing) aerobic exercises such as walking or running also have the potential to improve bone mass in adults and have the added benefit of improving cardiovascular conditioning [56,57]. Studies on the impact of different exercise modalities on biomarkers of bone formation and resorption support the importance of both resistance and aerobic training in maintaining bone health. Fujimura and associates found that high intensity resistance training (weightlifting) done three times weekly for four months by 17 young male subjects caused a sustained increase in serum calcitonin levels, although plasma procollagen Type I C-terminal levels, a marker of bone resorption, did not change [58]. In a study involving 56 previously inactive young women, Lester and associates found that 8 weeks of resistance or combined aerobicresistance training increased serum levels of osteocalcin but had no significant effect on markers of bone resorption [59]. In a study more closely resembling ours, Alghadir and associates found that 12 weeks of moderate intensity aerobic training done for 45 to 60 minutes three times weekly by 65 healthy subjects (36 males, 29 females) aged 30-60 increased serum osteocalcin levels and decreased serum levels of deoxypyridinoline, a marker of bone resorption [60]. In our study, it is of interest that postexercise culture levels of IL-6 were proportionate to weight, which is a measure of one's mass times the intensity of the gravity field (9.8 m/sec 2 on Earth). It is possible, therefore, that the failure of exercise programs to attenuate bone loss in the microgravity of space is related, at least in part, to suboptimal production of this pleiotropic cytokine. IL-6 exerts contextdependent effects on bone metabolism [5,30,31]. Although it can stimulate osteoclastogenesis and bone resorption by upregulating RANKL expression in osteoblasts [32], its effects on bone metabolism are predominantly osteogenic and antiresorptive. IL-6 enhances bone formation by promoting the differentiation of osteoblast precursors [33,34] and by protecting osteoblasts against apoptosis [5,35]; it can inhibit bone resorption directly by downregulating RANKL signaling pathways in osteoclasts [36] and indirectly by suppressing the production of TNF-and IL-1 and stimulating the production of IL-4, IL-10, and IL-1 receptor antagonist by immune cells [37,38]. It is also an essential growth factor for B cells, the primary source of OPG in bone marrow stroma [39], and can induce IL-2 production in T cells [38,40,41]. IL-6 is produced in osteoblasts and osteocytes in response to bone loading signals and, like TGF-, plays an important role in bone remodeling [42]. Thus, studies similar to that described in this report would be of particular interest if done on persons exercising in the microgravity of space.  Figure 9: Osteoclastogenic cytokines in women and men. Exercise attenuated the production of TNF-and IFN-in PHA + cultures in both women and men. In PHA − cultures, levels of TNF-(women) and IFN-(men) also fell. The white and black columns represent pre-and postexercise levels, respectively. Results are given as the mean ± SEM. * indicates a p value of <.05 (two-sided t-test for dependent samples). Note that men spontaneously produced more osteogenic cytokines than women both before and after exercise.  Figure 10: Antiosteoclastogenic cytokines in women and men. In PHA − cultures, exercise enhanced the production of TGF-in women and IL-10, IL-6, and TGF-in men. In PHA + cultures, exercise enhanced the production of IL-4, IL-6, and TGF-in women and IL-6 and TGFin men. The white and black columns represent pre-and postexercise levels, respectively. In both figures, IL-4 and IL-10 values are listed on the left Y axis and IL-6 and TGF-values on the right Y axis. Results are given as the mean ± SEM. * indicates a p value of <.05 (two-sided t-test for dependent samples). Note that, in PHA − cultures, postexercise production of IL-10 and IL-6 is greater in men than in women. This study would have benefitted from the inclusion of a nonexercising age-and sex-matched control group. Phenotypic analysis of Th17 and T regulatory cells and an assay for the mononuclear cell production of IL-17, a cytokine with osteoclastogenic activity, would also have been beneficial. Cardiorespiratory fitness measurements (VO 2max ) done before and after the exercise program was completed would have added potentially useful information.

Conclusions
Long-term moderate intensity exercise exerts a favorable effect on bone resorption by changing the balance between peripheral blood mononuclear cells producing osteoclastogenic cytokines and those producing antiosteoclastogenic cytokines. This beneficial effect may be enhanced by estradiol, emphasizing the importance of this hormone as a regulator of bone metabolism. The results provide a new insight as to how physical exercise contributes to the maintenance of bone health and suggest a possible molecular mechanism to explain the difference in the antiresorptive effects of exercise done on Earth as compared to exercise done in the microgravity of space.

Disclosure
The funders/supporters had no role in the design and conduct of the study; collection, management, analysis, or interpretation of the data; review or approval of the paper; or decision to submit the paper for publication.