- Email: [email protected]
Vitamin D sufficiency associates with an increase in anti-inflammatory cytokines after intense exercise in humans
Cytokine 65 (2014) 134–137
Contents lists available at ScienceDirect
Cytokine journal homepage: www.journals.elsevier.com/cytokine
Short Communication
Vitamin D sufficiency associates with an increase in anti-inflammatory cytokines after intense exercise in humans Tyler Barker a,⇑, Thomas B. Martins b, Harry R. Hill b,c, Carl R. Kjeldsberg b,c, Brian M. Dixon d, Erik D. Schneider d, Vanessa T. Henriksen a, Lindell K. Weaver e,f,g a
The Orthopedic Specialty Hospital, Murray, UT 84107, USA ARUP Laboratories, Institute for Clinical and Experimental Pathology, Salt Lake City, UT 84108, USA Department of Pathology, University of Utah, Salt Lake City, UT 84132, USA d USANA Health Sciences, Inc., Salt Lake City, UT 84120, USA e Hyperbaric Medicine, Intermountain Medical Center, Murray, UT 84107, USA f Hyperbaric Medicine, LDS Hospital, Salt Lake City, UT 84143, USA g University of Utah, School of Medicine, Salt Lake City, UT 84132, USA b c
a r t i c l e
i n f o
Article history: Received 26 August 2013 Received in revised form 7 November 2013 Accepted 9 December 2013 Available online 31 December 2013 Keywords: Vitamin D Cytokines Skeletal muscle strength
a b s t r a c t The purpose of this study was to identify the influence of vitamin D status (insufficient vs. sufficient) on circulating cytokines and skeletal muscle strength after muscular injury. To induce muscular injury, one randomly selected leg (SSC) performed exercise consisting of repetitive eccentric–concentric contractions. The other leg served as the control. An averaged serum 25(OH)D concentration from two blood samples collected before exercise and on separate occasions was used to establish vitamin D insufficiency (<30 ng/mL, n = 6) and sufficiency (>30 ng/mL, n = 7) in young, adult males. Serum cytokine concentrations, single-leg peak isometric force, and single-leg peak power output were measured before and during the days following the exercise protocol. The serum IL-10 and IL-13 responses to muscular injury were significantly (both p < 0.05) increased in the vitamin D sufficient group. The immediate and persistent (days) peak isometric force (p < 0.05) and peak power output (p < 0.05) deficits in the SSC leg after the exercise protocol were not ameliorated with vitamin D sufficiency. We conclude that vitamin D sufficiency increases the anti-inflammatory cytokine response to muscular injury. Ó 2013 Elsevier Ltd. All rights reserved.
1. Introduction Maintaining a sufficient vitamin D status (i.e., serum 25hydroxyvitamin D (25(OH)D) concentration) has many health benefits, such as reducing the risk of cardiovascular disease, autoimmune diseases, and some forms of cancer. Vitamin D also regulates a variety of physiological events, including cytokine production. Pro-inflammatory cytokines, such as INF-c and IL-1b, promote inflammation and repair processes, but an excessive production of pro-inflammatory cytokines is detrimental to cellular homeostasis. Anti-inflammatory cytokines, such as IL-10 and IL-13, down regulate pro-inflammatory cytokine production. Although results are inconsistent in humans [1–5], increasing vitamin D increases the production of anti-inflammatory cytokines from immune cells [6,7]. In addition to its cytokine-modulating property, vitamin D moderates skeletal muscle function [for review, see Ref. [8]]. Vita⇑ Corresponding author. Address: The Orthopedic Specialty Hospital, 5848 S. Fashion Blvd., Murray, UT 84107, USA. Tel.: +1 801 314 4951; fax: +1 801 314 4862. E-mail address: [email protected] (T. Barker). 1043-4666/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.cyto.2013.12.004
min D insufficiency, or decreases in serum 25(OH)D, exacerbate muscular weakness induced by soft tissue injuries [9,10], which importantly, could cause a predisposition to additional trauma, such as stress fractures or falls. Therefore, understanding the role of vitamin D status on inflammatory cytokines and skeletal muscle strength following muscular injury could have significant implications on physical rehabilitation and the protection against further insult. Elucidating this relationship could establish vitamin D status as an attractive therapeutic target when considering the ease of increasing serum 25(OH)D concentrations. It is unknown, however, if vitamin D status influences antiinflammatory cytokines and skeletal muscle strength following muscular injury. Therefore, the purpose of this study was to identify the influence of vitamin D status (insufficient vs. sufficient) on circulating anti-inflammatory cytokines and muscle strength following muscular injury. We hypothesized that vitamin D sufficiency (serum 25(OH)D > 30 ng/mL) increases circulating anti-inflammatory cytokines and ameliorates muscular weakness following injury.
T. Barker et al. / Cytokine 65 (2014) 134–137
2. Materials and methods The Urban Central Region Institutional Review Board at Intermountain Healthcare (Salt Lake City, UT, USA) approved this study. Subjects were informed of and provided written and verbal consent to the experimental protocol and procedures. Reportedly healthy and recreationally-active (i.e., 30 min of physically active at least 3 times per week) males participated in this study. Subject characteristics were similar between groups (Supplemental Table). Subject inclusion and exclusion criteria has been reported previously [2]. Subjects were asked to keep their diet consistent with their eating habits during the previous year and to refrain from any dietary supplements. Subjects were asked to refrain from physical activity and anti-inflammatory agents 72-h prior to a blood draw procedure. Data was collected during the winter.
135
greater in the vitamin D sufficient than those in the insufficient group (Supplemental Table). Serum IFN-c, IL-1b, IL-10, and IL-13 were quantitated (pg/mL) using the multiplex technology of Luminex (Austin, TX, USA), as described (ARUP Laboratories – Institute for Clinical and Experimental Pathology, University of Utah, Salt Lake City, UT, USA) [11]. Cytokine concentrations were measured in the Pre, 1-h, 24-h, 48-h, 72-h, and 168-h blood samples. Plasma PTH (pg/mL) and calcium (mg/dL) clinical chemistries were measured in the Bsl blood sample (ARUP Laboratories). Single-leg peak isometric force (performed in triplicate; CON CV = 3.40%; SSC CV = 4.10%) and peak power output were performed on a horizontal Plyo-Press (Athletic Republic, Park City, UT, USA), as described [2,9]. 2.4. Statistical analyses
2.1. Study protocol The first-two blood samples were obtained 28-d (Baseline, [Bsl]) and immediately (Pre) before the exercise protocol (see below). The remaining blood samples were obtained: immediately (Post), 1-h, 24-h, 48-h, 72-h, and 168-h after the exercise protocol. Blood draws were performed prior to strength testing. Familiarization with the single-leg strength testing procedure was performed at Bsl. Thereafter, single-leg strength testing was performed immediately before (Pre) and immediately (Post), 24-h, 48-h, 72-h, and 168-h after the exercise protocol.
Data were checked for normality prior to all statistical analyses with a Shapiro–Wilk test. Statistical significance of data were assessed with a repeated-measures analysis of variance (ANOVA) followed by a post hoc t-tests with a Bonferroni correction for multiple pairwise comparisons when appropriate. Due to non-normally distributed data, statistical significance of cytokine data were assessed with separate Friedman ANOVA tests. All statistical analyses were performed with SYSTAT (version 13.1, Chicago, IL, USA). Statistical significance was set at p < 0.05. Data presented as mean ± SEM.
2.2. Exercise protocol
3. Results and discussion
Twenty-eight days after strength testing familiarization, each subject had one randomly selected leg (SSC) perform an intense exercise protocol to induce muscle injury. The other leg served as the contralateral control (CON). The intense exercise protocol consisted of 10 sets of 10 repetitive eccentric-concentric jumps through a full range of motion (90° of knee flexion to full extension) at 75% of body mass with a 20 s rest between each set on a horizontal Plyo-Press (Athletic Republic, Park City, UT, USA), as described [9]. If subjects were no longer able to complete two successive jumps, they were allowed to perform presses. If unable to complete presses, the exercise protocol was terminated. The mean number of jumps and presses completed during the exercise protocol were not statistically different between the vitamin D insufficient (jumps, 69 ± 12; presses 44 ± 8) and sufficient (jumps, 56 ± 13; presses, 18 ± 4) groups. Of the 13 subjects, 6 subjects (3 insufficient and 3 sufficient) were able to complete the exercise protocol. Blood chemistries and leg strength data were not significantly different between those who finished and those who did not finish the exercise protocol.
This report provides the first data demonstrating an increase in serum IL-10 and IL-13 concentrations after muscular insult with vitamin D sufficiency and without an alteration in pro-inflammatory cytokines (Fig. 1A–D), which could have significant implications on physical rehabilitation. Evidence from experimental animal models of muscular dystrophy [12] and sepsis [13] show that IL-10 and IL-13 promote muscle regeneration and abrogate skeletal muscle weakness. Muscular weakness, however, was not attenuated in the vitamin D sufficient subjects with concurrent increases in IL-10 and IL-13 (Fig. 2A and B). Therefore, the physiological impact of increasing anti-inflammatory cytokines following muscle injury with vitamin D sufficiency remains unknown. The reason for the conflicting results between vitamin D and muscle strength following injury is unknown, but it could relate to the narrow range (13.4–43.9 ng/mL) in serum 25(OH)D concentrations between vitamin D groups. It is also unclear if the serum 25(OH)D concentration demarcating vitamin D sufficiency from insufficiency was appropriate for this population and experimental modality (i.e., contraction-induced injury) as PTH and calcium were similar between groups (Supplemental Table). Considering the wealth of data identifying the influence of vitamin D on skeletal muscle strength [for review, see Ref. [8]], future research is encouraged to include subjects with a wide range of serum 25(OH)D concentrations when investigating the role of vitamin D status on skeletal muscle strength in healthy adults following injury. In addition to those discussed above, there are several limitations with this study. First, although the criteria for subject participation was strict, this study consisted of a small sample size. Second, the amount of work performed during the exercise protocol was crudely assessed by recording the number of repetitions executed. Future investigations would benefit from a more stringent approach when assessing the amount of work performed during exercise and a larger sample size. Third, this study was conducted in young, reportedly healthy adult males. Therefore,
2.3. Analytical procedures Serum 25(OH)D concentrations (ng/mL) were measured in duplicate (CV = 3.60%) from the blood samples collected at Bsl and Pre (USANA Health Sciences, Inc., Salt Lake City, UT, USA), and as procedurally described elsewhere [1]. In brief, serum 25(OH)D concentrations were detected using high performance liquid chromatography (Agilent, Series 6460, Model G6460A, Santa Clara, CA, USA) – tandem mass spectrometer (Agilent, Series 6410, Model G6410B, Santa Clara, CA, USA) and corrected for recovery of the 25(OH)D3 internal standard. The averaged serum 25(OH)D concentration from Bsl and Pre was used to establish vitamin D status (insufficient < 30; sufficient > 30 ng/mL). Thus, by design, serum 25(OH)D concentrations were significantly (p < 0.05)
10
A
Insufficient Sufficient
5 0 -5 -10
Change in Serum IL-1β (pg/mL) From Pre
T. Barker et al. / Cytokine 65 (2014) 134–137
Change in Serum IFN-γ (pg/mL) From Pre
136
30 20 10 0 -10 -20 -30
10
C
1
Pre Post 1-h 24-h 48-h 72-h 168-h
Change in Serum IL-13 (pg/mL) From Pre
Change in Serum IL-10 (pg/mL) From Pre
Pre Post 1-h 24-h 48-h 72-h 168-h
1,*
0 -10 -20
B
10
D
2,*
5
*
*
0 -5 -10 Pre Post 1-h 24-h 48-h 72-h 168-h
Pre Post 1-h 24-h 48-h 72-h 168-h
10
A 1
0 -10
1,2,3
1,3
1
-20 -30
Insufficient Sufficient
-40
Pre
Post 24-h 48-h 72-h 168-h
SSC Leg Peak Power Output (% of CON Leg)
SSC Leg Peak Isometric Force (% of CON Leg)
Fig. 1. The change in serum IFN-c, IL-1b, IL-10, and IL-13 concentrations (pg/mL) from Pre. The change in serum IFN-c (A) and IL-1b (B) concentrations (pg/mL) were not significantly different within or between vitamin D groups. (C) The increase in serum IL-10 concentrations were significantly greater at Post and 1-h (1p < 0.05 vs. 48-h) in the vitamin D sufficient group. The change in serum IL-10 concentrations at 1-h were significantly (p < 0.05) different between vitamin D insufficient and sufficient groups. (D) Serum IL-13 changes (pg/mL) were significantly (2p < 0.05) different at Post compared to those at 168-h in the vitamin D sufficient group. The IL-13 changes at Post, 1-h, and 48-h were significantly (p < 0.05) different between vitamin D insufficient and sufficient groups. Figure legend provided in ‘A’. Data presented as mean ± SEM.
10
B 3
0 -10
1,3
-20 -30 -40 -50
Pre
Post 24-h 48-h 72-h 168-h
Fig. 2. The peak isometric force and peak power output in the SSC leg as a % of the CON leg. (A) Peak isometric force was significantly decreased after the exercise protocol (1p < 0.05 vs. Pre; 2p < 0.05 vs. 72-h; 3p < 0.05 vs. 168-h) in the SSC leg. Differences between vitamin D groups were not significant. (B) Peak power output was significantly decreased at Post (1p < 0.05 vs. Pre) and 48-h (3p < 0.05 vs. 168-h) in the SSC leg. Differences between vitamin D groups were not significant. Figure legend provided in ‘A’. Data presented as mean ± SEM.
caution is recommended when translating the present findings to other populations with diverse clinical ailments. Finally, it is unknown if fatigue- or damage-related mechanisms were contributing to muscular weakness after the imposed exercise protocol. It is likely that both fatigue- and damage-related mechanisms were contributing to muscular weakness; with the former dominating the immediate deficit and the latter mediating the persistent deficits in muscle strength.
4. Conclusion Based on the new data in this communication, we conclude that vitamin D sufficiency is associated with an increase in anti-inflammatory cytokines after a muscle injury. Understanding the influence of vitamin D on anti-inflammatory cytokines could have broad implications on the rehabilitation from muscular insult and the subsequent predisposition to further trauma.
Acknowledgements This study was funded in part by the Intermountain Research and Medical Foundation (Intermountain Healthcare, Salt Lake City, UT, USA) and the ARUP Institute for Clinical and Experimental Pathology (Salt Lake City, UT, USA).
Appendix A. Supplementary material Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.cyto.2013.12.004. References [1] Barker T, Martins TB, Hill HR, Kjeldsberg CR, Henriksen VT, Dixon BM, et al. Different doses of supplemental vitamin D maintain interleukin-5 without altering skeletal muscle strength: a randomized, double-blind, placebocontrolled study in vitamin D sufficient adults. Nutr Metab (Lond) 2012;9:16. [2] Barker T, Martins TB, Hill HR, Kjeldsberg CR, Dixon BM, Schneider ED, et al. Circulating pro-inflammatory cytokines are elevated and peak power output correlates with 25-hydroxyvitamin D in vitamin D insufficient adults. Eur J Appl Physiol 2013;113:1523–34. [3] Kelly P, Suibhne TN, Morain O, Sullivan O. Vitamin D status and cytokine levels in patients with Crohn’s disease. Int J Vitam Nutr Res 2011;81: 205–10. [4] Schleithoff SS, Zittermann A, Tenderich G, Berthold HK, Stehle P, Koerfer R. Vitamin D supplementation improves cytokine profiles in patients with congestive heart failure: a double-blind, randomized, placebo-controlled trial. Am J Clin Nutr 2006;83:754–9. [5] Shab-Bidar S, Neyestani TR, Djazayery A, Eshraghian MR, Houshiarrad A, Kalayi A, et al. Improvement of vitamin D status resulted in amelioration of biomarkers of systemic inflammation in the subjects with type 2 diabetes. Diabetes Metab Res Rev 2012;28:424–30. [6] Khoo AL, Chai LY, Koenen HJ, Kullberg BJ, Joosten I, van der Ven AJ, et al. 1,25dihydroxyvitamin D3 modulates cytokine production induced by Candida albicans: impact of seasonal variation of immune responses. J Infect Dis 2011;203:122–30.
T. Barker et al. / Cytokine 65 (2014) 134–137 [7] Khoo AL, Chai LY, Koenen HJ, Oosting M, Steinmeyer A, Zuegel U, et al. Vitamin D(3) down-regulates proinflammatory cytokine response to Mycobacterium tuberculosis through pattern recognition receptors while inducing protective cathelicidin production. Cytokine 2011;55:294–300. [8] Ceglia L, Harris SS. Vitamin D and its role in skeletal muscle. Calcif Tissue Int 2013;92:151–62. [9] Barker T, Henriksen VT, Martins TB, Hill HR, Kjeldsberg CR, Schneider ED, et al. Higher serum 25-hydroxyvitamin D concentrations associate with a faster recovery of skeletal muscle strength after muscular injury. Nutrients 2013;5:1253–75. [10] Barker T, Martins TB, Hill HR, Kjeldsberg CR, Trawick RH, Weaver LK, et al. Low vitamin D impairs strength recovery after anterior cruciate ligament surgery. J Evidence-Based Compl Altern Med 2011;16:201–9.
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[11] Martins TB, Pasi BM, Pickering JW, Jaskowski TD, Litwin CM, Hill HR. Determination of cytokine responses using a multiplexed fluorescent microsphere immunoassay. Am J Clin Pathol 2002;118:346–53. [12] Villalta SA, Rinaldi C, Deng B, Liu G, Fedor B, Tidball JG. Interleukin-10 reduces the pathology of mdx muscular dystrophy by deactivating M1 macrophages and modulating macrophage phenotype. Hum Mol Genet 2011;20:790–805. [13] Takahashi Y, Katayose D, Shindoh C. Interleukin-13 prevents diaphragm muscle deterioration in a septic animal model. Tohoku J Exp Med 1999;189:191–202.
Contents lists available at ScienceDirect
Cytokine journal homepage: www.journals.elsevier.com/cytokine
Short Communication
Vitamin D sufficiency associates with an increase in anti-inflammatory cytokines after intense exercise in humans Tyler Barker a,⇑, Thomas B. Martins b, Harry R. Hill b,c, Carl R. Kjeldsberg b,c, Brian M. Dixon d, Erik D. Schneider d, Vanessa T. Henriksen a, Lindell K. Weaver e,f,g a
The Orthopedic Specialty Hospital, Murray, UT 84107, USA ARUP Laboratories, Institute for Clinical and Experimental Pathology, Salt Lake City, UT 84108, USA Department of Pathology, University of Utah, Salt Lake City, UT 84132, USA d USANA Health Sciences, Inc., Salt Lake City, UT 84120, USA e Hyperbaric Medicine, Intermountain Medical Center, Murray, UT 84107, USA f Hyperbaric Medicine, LDS Hospital, Salt Lake City, UT 84143, USA g University of Utah, School of Medicine, Salt Lake City, UT 84132, USA b c
a r t i c l e
i n f o
Article history: Received 26 August 2013 Received in revised form 7 November 2013 Accepted 9 December 2013 Available online 31 December 2013 Keywords: Vitamin D Cytokines Skeletal muscle strength
a b s t r a c t The purpose of this study was to identify the influence of vitamin D status (insufficient vs. sufficient) on circulating cytokines and skeletal muscle strength after muscular injury. To induce muscular injury, one randomly selected leg (SSC) performed exercise consisting of repetitive eccentric–concentric contractions. The other leg served as the control. An averaged serum 25(OH)D concentration from two blood samples collected before exercise and on separate occasions was used to establish vitamin D insufficiency (<30 ng/mL, n = 6) and sufficiency (>30 ng/mL, n = 7) in young, adult males. Serum cytokine concentrations, single-leg peak isometric force, and single-leg peak power output were measured before and during the days following the exercise protocol. The serum IL-10 and IL-13 responses to muscular injury were significantly (both p < 0.05) increased in the vitamin D sufficient group. The immediate and persistent (days) peak isometric force (p < 0.05) and peak power output (p < 0.05) deficits in the SSC leg after the exercise protocol were not ameliorated with vitamin D sufficiency. We conclude that vitamin D sufficiency increases the anti-inflammatory cytokine response to muscular injury. Ó 2013 Elsevier Ltd. All rights reserved.
1. Introduction Maintaining a sufficient vitamin D status (i.e., serum 25hydroxyvitamin D (25(OH)D) concentration) has many health benefits, such as reducing the risk of cardiovascular disease, autoimmune diseases, and some forms of cancer. Vitamin D also regulates a variety of physiological events, including cytokine production. Pro-inflammatory cytokines, such as INF-c and IL-1b, promote inflammation and repair processes, but an excessive production of pro-inflammatory cytokines is detrimental to cellular homeostasis. Anti-inflammatory cytokines, such as IL-10 and IL-13, down regulate pro-inflammatory cytokine production. Although results are inconsistent in humans [1–5], increasing vitamin D increases the production of anti-inflammatory cytokines from immune cells [6,7]. In addition to its cytokine-modulating property, vitamin D moderates skeletal muscle function [for review, see Ref. [8]]. Vita⇑ Corresponding author. Address: The Orthopedic Specialty Hospital, 5848 S. Fashion Blvd., Murray, UT 84107, USA. Tel.: +1 801 314 4951; fax: +1 801 314 4862. E-mail address: [email protected] (T. Barker). 1043-4666/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.cyto.2013.12.004
min D insufficiency, or decreases in serum 25(OH)D, exacerbate muscular weakness induced by soft tissue injuries [9,10], which importantly, could cause a predisposition to additional trauma, such as stress fractures or falls. Therefore, understanding the role of vitamin D status on inflammatory cytokines and skeletal muscle strength following muscular injury could have significant implications on physical rehabilitation and the protection against further insult. Elucidating this relationship could establish vitamin D status as an attractive therapeutic target when considering the ease of increasing serum 25(OH)D concentrations. It is unknown, however, if vitamin D status influences antiinflammatory cytokines and skeletal muscle strength following muscular injury. Therefore, the purpose of this study was to identify the influence of vitamin D status (insufficient vs. sufficient) on circulating anti-inflammatory cytokines and muscle strength following muscular injury. We hypothesized that vitamin D sufficiency (serum 25(OH)D > 30 ng/mL) increases circulating anti-inflammatory cytokines and ameliorates muscular weakness following injury.
T. Barker et al. / Cytokine 65 (2014) 134–137
2. Materials and methods The Urban Central Region Institutional Review Board at Intermountain Healthcare (Salt Lake City, UT, USA) approved this study. Subjects were informed of and provided written and verbal consent to the experimental protocol and procedures. Reportedly healthy and recreationally-active (i.e., 30 min of physically active at least 3 times per week) males participated in this study. Subject characteristics were similar between groups (Supplemental Table). Subject inclusion and exclusion criteria has been reported previously [2]. Subjects were asked to keep their diet consistent with their eating habits during the previous year and to refrain from any dietary supplements. Subjects were asked to refrain from physical activity and anti-inflammatory agents 72-h prior to a blood draw procedure. Data was collected during the winter.
135
greater in the vitamin D sufficient than those in the insufficient group (Supplemental Table). Serum IFN-c, IL-1b, IL-10, and IL-13 were quantitated (pg/mL) using the multiplex technology of Luminex (Austin, TX, USA), as described (ARUP Laboratories – Institute for Clinical and Experimental Pathology, University of Utah, Salt Lake City, UT, USA) [11]. Cytokine concentrations were measured in the Pre, 1-h, 24-h, 48-h, 72-h, and 168-h blood samples. Plasma PTH (pg/mL) and calcium (mg/dL) clinical chemistries were measured in the Bsl blood sample (ARUP Laboratories). Single-leg peak isometric force (performed in triplicate; CON CV = 3.40%; SSC CV = 4.10%) and peak power output were performed on a horizontal Plyo-Press (Athletic Republic, Park City, UT, USA), as described [2,9]. 2.4. Statistical analyses
2.1. Study protocol The first-two blood samples were obtained 28-d (Baseline, [Bsl]) and immediately (Pre) before the exercise protocol (see below). The remaining blood samples were obtained: immediately (Post), 1-h, 24-h, 48-h, 72-h, and 168-h after the exercise protocol. Blood draws were performed prior to strength testing. Familiarization with the single-leg strength testing procedure was performed at Bsl. Thereafter, single-leg strength testing was performed immediately before (Pre) and immediately (Post), 24-h, 48-h, 72-h, and 168-h after the exercise protocol.
Data were checked for normality prior to all statistical analyses with a Shapiro–Wilk test. Statistical significance of data were assessed with a repeated-measures analysis of variance (ANOVA) followed by a post hoc t-tests with a Bonferroni correction for multiple pairwise comparisons when appropriate. Due to non-normally distributed data, statistical significance of cytokine data were assessed with separate Friedman ANOVA tests. All statistical analyses were performed with SYSTAT (version 13.1, Chicago, IL, USA). Statistical significance was set at p < 0.05. Data presented as mean ± SEM.
2.2. Exercise protocol
3. Results and discussion
Twenty-eight days after strength testing familiarization, each subject had one randomly selected leg (SSC) perform an intense exercise protocol to induce muscle injury. The other leg served as the contralateral control (CON). The intense exercise protocol consisted of 10 sets of 10 repetitive eccentric-concentric jumps through a full range of motion (90° of knee flexion to full extension) at 75% of body mass with a 20 s rest between each set on a horizontal Plyo-Press (Athletic Republic, Park City, UT, USA), as described [9]. If subjects were no longer able to complete two successive jumps, they were allowed to perform presses. If unable to complete presses, the exercise protocol was terminated. The mean number of jumps and presses completed during the exercise protocol were not statistically different between the vitamin D insufficient (jumps, 69 ± 12; presses 44 ± 8) and sufficient (jumps, 56 ± 13; presses, 18 ± 4) groups. Of the 13 subjects, 6 subjects (3 insufficient and 3 sufficient) were able to complete the exercise protocol. Blood chemistries and leg strength data were not significantly different between those who finished and those who did not finish the exercise protocol.
This report provides the first data demonstrating an increase in serum IL-10 and IL-13 concentrations after muscular insult with vitamin D sufficiency and without an alteration in pro-inflammatory cytokines (Fig. 1A–D), which could have significant implications on physical rehabilitation. Evidence from experimental animal models of muscular dystrophy [12] and sepsis [13] show that IL-10 and IL-13 promote muscle regeneration and abrogate skeletal muscle weakness. Muscular weakness, however, was not attenuated in the vitamin D sufficient subjects with concurrent increases in IL-10 and IL-13 (Fig. 2A and B). Therefore, the physiological impact of increasing anti-inflammatory cytokines following muscle injury with vitamin D sufficiency remains unknown. The reason for the conflicting results between vitamin D and muscle strength following injury is unknown, but it could relate to the narrow range (13.4–43.9 ng/mL) in serum 25(OH)D concentrations between vitamin D groups. It is also unclear if the serum 25(OH)D concentration demarcating vitamin D sufficiency from insufficiency was appropriate for this population and experimental modality (i.e., contraction-induced injury) as PTH and calcium were similar between groups (Supplemental Table). Considering the wealth of data identifying the influence of vitamin D on skeletal muscle strength [for review, see Ref. [8]], future research is encouraged to include subjects with a wide range of serum 25(OH)D concentrations when investigating the role of vitamin D status on skeletal muscle strength in healthy adults following injury. In addition to those discussed above, there are several limitations with this study. First, although the criteria for subject participation was strict, this study consisted of a small sample size. Second, the amount of work performed during the exercise protocol was crudely assessed by recording the number of repetitions executed. Future investigations would benefit from a more stringent approach when assessing the amount of work performed during exercise and a larger sample size. Third, this study was conducted in young, reportedly healthy adult males. Therefore,
2.3. Analytical procedures Serum 25(OH)D concentrations (ng/mL) were measured in duplicate (CV = 3.60%) from the blood samples collected at Bsl and Pre (USANA Health Sciences, Inc., Salt Lake City, UT, USA), and as procedurally described elsewhere [1]. In brief, serum 25(OH)D concentrations were detected using high performance liquid chromatography (Agilent, Series 6460, Model G6460A, Santa Clara, CA, USA) – tandem mass spectrometer (Agilent, Series 6410, Model G6410B, Santa Clara, CA, USA) and corrected for recovery of the 25(OH)D3 internal standard. The averaged serum 25(OH)D concentration from Bsl and Pre was used to establish vitamin D status (insufficient < 30; sufficient > 30 ng/mL). Thus, by design, serum 25(OH)D concentrations were significantly (p < 0.05)
10
A
Insufficient Sufficient
5 0 -5 -10
Change in Serum IL-1β (pg/mL) From Pre
T. Barker et al. / Cytokine 65 (2014) 134–137
Change in Serum IFN-γ (pg/mL) From Pre
136
30 20 10 0 -10 -20 -30
10
C
1
Pre Post 1-h 24-h 48-h 72-h 168-h
Change in Serum IL-13 (pg/mL) From Pre
Change in Serum IL-10 (pg/mL) From Pre
Pre Post 1-h 24-h 48-h 72-h 168-h
1,*
0 -10 -20
B
10
D
2,*
5
*
*
0 -5 -10 Pre Post 1-h 24-h 48-h 72-h 168-h
Pre Post 1-h 24-h 48-h 72-h 168-h
10
A 1
0 -10
1,2,3
1,3
1
-20 -30
Insufficient Sufficient
-40
Pre
Post 24-h 48-h 72-h 168-h
SSC Leg Peak Power Output (% of CON Leg)
SSC Leg Peak Isometric Force (% of CON Leg)
Fig. 1. The change in serum IFN-c, IL-1b, IL-10, and IL-13 concentrations (pg/mL) from Pre. The change in serum IFN-c (A) and IL-1b (B) concentrations (pg/mL) were not significantly different within or between vitamin D groups. (C) The increase in serum IL-10 concentrations were significantly greater at Post and 1-h (1p < 0.05 vs. 48-h) in the vitamin D sufficient group. The change in serum IL-10 concentrations at 1-h were significantly (p < 0.05) different between vitamin D insufficient and sufficient groups. (D) Serum IL-13 changes (pg/mL) were significantly (2p < 0.05) different at Post compared to those at 168-h in the vitamin D sufficient group. The IL-13 changes at Post, 1-h, and 48-h were significantly (p < 0.05) different between vitamin D insufficient and sufficient groups. Figure legend provided in ‘A’. Data presented as mean ± SEM.
10
B 3
0 -10
1,3
-20 -30 -40 -50
Pre
Post 24-h 48-h 72-h 168-h
Fig. 2. The peak isometric force and peak power output in the SSC leg as a % of the CON leg. (A) Peak isometric force was significantly decreased after the exercise protocol (1p < 0.05 vs. Pre; 2p < 0.05 vs. 72-h; 3p < 0.05 vs. 168-h) in the SSC leg. Differences between vitamin D groups were not significant. (B) Peak power output was significantly decreased at Post (1p < 0.05 vs. Pre) and 48-h (3p < 0.05 vs. 168-h) in the SSC leg. Differences between vitamin D groups were not significant. Figure legend provided in ‘A’. Data presented as mean ± SEM.
caution is recommended when translating the present findings to other populations with diverse clinical ailments. Finally, it is unknown if fatigue- or damage-related mechanisms were contributing to muscular weakness after the imposed exercise protocol. It is likely that both fatigue- and damage-related mechanisms were contributing to muscular weakness; with the former dominating the immediate deficit and the latter mediating the persistent deficits in muscle strength.
4. Conclusion Based on the new data in this communication, we conclude that vitamin D sufficiency is associated with an increase in anti-inflammatory cytokines after a muscle injury. Understanding the influence of vitamin D on anti-inflammatory cytokines could have broad implications on the rehabilitation from muscular insult and the subsequent predisposition to further trauma.
Acknowledgements This study was funded in part by the Intermountain Research and Medical Foundation (Intermountain Healthcare, Salt Lake City, UT, USA) and the ARUP Institute for Clinical and Experimental Pathology (Salt Lake City, UT, USA).
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