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Acute effects of aerobic intensities on the cytokine response in women with mild multiple sclerosis
Multiple Sclerosis and Related Disorders 31 (2019) 82–86
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Clinical trial
Acute effects of aerobic intensities on the cytokine response in women with mild multiple sclerosis Shani Berkowitza,b, Anat Achironb,c,d, Michael Gurevichb, Polina Sonisb, Alon Kalrona,d,
T
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a
Department of Physical Therapy, School of Health Professions, Sackler Faculty of Medicine, Tel-Aviv University, Tel Aviv, Israel Multiple Sclerosis Center, Sheba Medical Center, Tel Hashomer, Israel c Sackler Faculty of Medicine, Tel-Aviv University, Tel Aviv, Israel d Sagol School of Neuroscience, Tel-Aviv University, Tel Aviv, Israel b
A R T I C LE I N FO
A B S T R A C T
Keywords: Multiple sclerosis Aerobic Exercise Cytokines Inflammation
Background: There is limited information as to the effects of aerobic exercise on the immune system in people with multiple sclerosis (PwMS). One of the unresolved questions is which training intensity level is optimal for PwMS. Objective: The purpose of this study is to compare the effects of moderate intensity with the effects of vigorous intensity on cytokine levels in women with mild MS. Methods: The study included 15 women with MS and a control of 10 healthy women. The initial session determined the value of the maximum oxygen consumption and was carried out on an instrumented treadmill with a portable system for monitoring gases. During the second session, the participant walked on the treadmill for 15-minutes at a moderate capacity level (∼50% VO2 peak). Blood samples were taken at baseline, immediately after exercise and two hours later. IL-4, IL-6, IL-10, IL-17A, TNF-α and IFN-ɣ cytokines were tested. The third session was identical to the second except for a vigorous training intensity session (∼80% VO2 peak). Results: IL-6 increased after moderate exercise for both the MS (p = 0.02) and control group (p = 0.02). IL-10 decreased during the vigorous session only in the MS group (p = 0.02). No other differences were seen over time in either group for all other cytokine measurements. Conclusions: With the exception of IL-10, women with mild MS have similar inflammatory reactions to moderate and high intensity exercise as do healthy women.
1. Introduction Exercise, a beneficial rehabilitation strategy for people with multiple sclerosis (PwMS) manages symptoms, restores function, optimizes quality of life, promotes wellness and encourages participation in activities of daily living (Motl et al., 2017). Previous studies have demonstrated that aerobic activity is inversely correlated with symptoms in PwMS and may be protective and possibly restorative (Dalgas, 2012; Giesser, 2015). Dalgas et al. (2012) reported that higher rates of exercise correspond with decreased rates of functional deterioration (Dalgas, 2012; Stuifbergen et al., 2006). Furthermore, there is growing evidence that exercise could be considered a disease-modifying therapy in PwMS (Giesser, 2015). Cytokines, a significant component of MS pathophysiology (Patel, 2013) are protein hormones that regulate communication between the innate and adaptive branches of the immune system
(Alexsander, 2002). A sense of balance exists between T helper cytokines in the healthy population, whereas in PwMS, this balance shifts and reflects increased pro-inflammatory cytokines (i.e., interleukin (IL)−1, IL-6) and decreased anti-inflammatory cytokines (i.e., IL-10). These deviations can accelerate the destruction of myelin in the brain and spinal cord of PwMS (Turner et al., 2009). There are limited data regarding the effects of physical activity on the immune system in PwMS. The cytokines that are of primary focus in MS studies involving exercise, include a diverse spectrum of pro-inflammatory cytokines such as IL-1, IL-2, IL-6, IL-17, IFN-γ and TNF-α, and anti-inflammatory cytokines such as IL-1ra, IL-4 and IL-10 (Negaresh et al., 2018). Importantly, many of these cytokines are known to contribute to the pathogenesis of MS (Palle et al., 2017). Several studies have examined the effects of aerobic exercise on selected cytokines (Heesen et al., 2003; Castellano et al., 2008; Shulz, 2004; Golzari et al., 2010) yet, according to a recent systematic review,
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Corresponding author at: Department of Physical Therapy, Sackler Faculty of Medicine, Tel-Aviv University, Tel Aviv, Israel. E-mail addresses: [email protected] (S. Berkowitz), [email protected] (A. Achiron), [email protected] (M. Gurevich), [email protected] (P. Sonis), [email protected] (A. Kalron). https://doi.org/10.1016/j.msard.2019.03.025 Received 20 December 2018; Received in revised form 17 February 2019; Accepted 31 March 2019 2211-0348/ © 2019 Elsevier B.V. All rights reserved.
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a maximal aerobic intensity test (incremental exercise test) was performed on an instrumented treadmill (Zebris® Medical GmbH, Germany) using the Balke protocol (Balke, 1959). Participants performed the maximal aerobic capacity test while fitted to the COSMED K5 portable metabolic cart with breath-by-breath technology. The COSMED K5 is a reliable and valid method of analyzing gasses (Baldari et al., 2015). O2, CO2, pulse, respiratory exchange ratio, O2 cost and O2 rate were continuously measured. Outcome measures derived from cardiopulmonary exercise testing were found to be a valid measure of cardio-respiratory fitness in PwMS with low to mild levels of disability (EDSS <4) (Heine et al., 2014). The second and third sessions (7–14 days in between) included 15 min of aerobic exercise performed on the treadmill. Sessions differed as to the aerobic intensity level. The first session was performed at a moderate intensity, defined as fairly-light or somewhat hard, calculated as a 50% VO2 peak (46–60% VO2 peak). The second was performed at a vigorous intensity defined as somewhat hard to very hard, calculated as a 80% VO2 peak (64–90% VO2 peak) (Pescatello et al., 2014). To ensure that the exercise was performed at predetermined levels, the Borg Rating of Perceived Exertion (RPE) was periodically recorded and intensity adjusted if necessary, in order to obtain moderate and vigorous intensities (Scherr et al., 2013; Borg, 1982). Prior to the aerobic second and third exercise sessions, the patient rested for five minutes. Subsequently, there was a two minute warm up at 5.3 km per hour (kph) according to the standard Balke protocol. After the initial warm up, the incline was increased every minute until the subject reached the predetermined intensity level (moderate/vigorous).
the effect remains inconclusive (Negaresh et al., 2018). Moreover, the results of the studies contradict each other, making it more difficult to determine a definitive conclusion. Heterogeneity of the exercise protocols is a notable limitation of previous studies investigating the cytokine response to aerobic exercise in PwMS. Previous trials have examined different types of exercises (cycling, walking and running) with different training durations, frequencies and intensities (Negaresh et al., 2018). Training intensity, the focus of the present study, is an important parameter of aerobic exercise. The most common aerobic intensity modes are moderate and vigorous. Although, there is evidence supporting the beneficial effects of both training intensities for symptomatic relief in PwMS (Sandroff et al., 2015), to the best of our knowledge, the effects of different aerobic intensity levels on the immune response of PwMS has never been investigated. Clinicians engaged in physical rehabilitation in the MS population are striving to provide a personalized exercise regimen for PwMS (Motl et al., 2018). Therefore, addressing this query is worthwhile. Hopefully, this information can help adjust the proper intensity level in aerobic programs directed at the MS population. Therefore, the goal of our study was to compare the responses of selected cytokines at vigorous and moderate intensities in women with MS. Results of the MS participants were compared with those of healthy age-matched women. We focused on women only, due to fact that MS is more common in women and sex differences exist in the cytokine response (Benini et al., 2015). Our hypothesis was that the cytokine response found in the MS participants would differ from the healthy women's responses to the aerobic effort depending on the intensity level.
2.3. Blood samples and cytokine measures 2. Methods Blood samples were obtained on the second and third visit by venipuncture before a single session of exercise, immediately after and two hours post exercise. This blood collection protocol was chosen due to the variation in cytokine dynamics (Pederson, 2001; Ostrowski et al., 1999). All blood samples were drawn from the antecubital vein in a seated position and collected for at least 48 h after any MS-related interferon drug administration in order to control for the possible impact of the drug on cytokine regulation (Duddy et al., 1999). Blood samples remained at room temperature for 30 min. Once the blood had clotted, it was centrifuged at 3000 rpm for 15 min. Serum was then quickly removed, frozen at 20 °C and preserved for assessment. Two weeks later, it was moved to a deep freeze at −80 °C where it was kept until needed. Prior to running the analysis, samples were defrosted on ice. Multiple cytokine expression was measured by Luminex technology. Primary cytokine measures were interferon gamma (IFN- ɣ) and interleukin- 6, based on the literature (Negaresh et al., 2018; Castellano et al., 2008; Cullen et al., 2016). Secondary measures were tumor necrosis factor alpha (TNF-α), interleukin-4, interleukin-10 and interleukin-17A cytokine levels.
2.1. Study design and participants This is a case control study. Fifteen women with MS, mean age 33.8 (S.D. = 7.8) years, recruited from the Sheba Multiple Sclerosis Center, Tel-HaShomer, Israel, participated in the study. Ten age-matched healthy women, recruited through social media and local hospital advertisement facilities, served as controls. Inclusion criteria included: (1) a neurologist-confirmed diagnosis of definite MS according to the revised McDonald criteria (Thompson et al., 2018); (2) <4.5 score on the Expanded Disability Status Scale (EDSS) (Kurtzke, 1983), the ability to walk an equivalent of at least 500 m without a walking aid; (3) relapse-free for at least 90 days prior to testing; (4) 20 to 45 years of age; (5) patients receiving disease modifying drugs (e.g. Interferon β−1a) for at least 3 months; and (6) willing and able to wear an oxygen analyzer while walking. Exclusion criteria included: (1) pregnancy; (2) body-mass index ≥30; (3) cardiovascular disorders; (4) respiratory disorders; (5) unable to walk on a treadmill for 15 min. Furthermore, women were instructed to not to schedule the exercise sessions during menstruation. The Sheba Institutional Review Board, consistent with the Declaration of Helsinki, approved the study. Informed consent was obtained from all participants. Initially, the participants completed the Godin-Shephard Leisure-Time Physical Activity Questionnaire (Amireault, 2015) and subsequent anthropometric measures were taken. Once the intake was complete, each participant underwent a maximal aerobic intensity test to quantify aerobic capabilities. At the second session, the participant was randomized into a moderate or vigorous exercise group. On the third visit, the participants exercised at other levels of intensity.
2.4. Post-acquisition cytokine data processing Quantitative, multiplexed protein measurements from the serum samples to detect specific cytokines were performed by the ProcartaPlex High Sensitivity Immunoassay Mix & the Max panel kit from the eBioscience division of Affymetrix. ProcartaPlex immunoassays employs magnetic microsphere technology which allows for capture and detection of specific analysts from a serum sample. By using the Luminex MAGPIX® compact analyzer, simultaneous detection from a single sample of up to 50 protein targets, was possible. Analysis was carried out per the manufacturer's protocol. Concentration of samples was calculated by plotting the expected concentration of the standards against the median fluorescent intensity generated by each standard. A 5PL algorithm was used for the best curve fit. Assayed samples were analyzed in accordance with the operation manual of the Luminex MAGPIX® instrument.
2.2. Aerobic exercise protocol All study measurements were performed at the Sheba MS Center. Subjects completed the exercise testing in the morning (9–12 am) under the supervision of an accredited exercise physiologist. At the first visit, 83
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17A, IFN- ɣ and TNF- α in both groups during vigorous intensity exercise. The cytokine scores over time for aerobic exercise at moderate and vigorous intensities are presented in Tables 2 and 3.
2.5. Statistical analysis Descriptive statistics were performed to determine distributions of demographic, clinical and cytokine serum levels and to compare the characteristics of the women with MS and the controls. Following scanning of the cytokines, the raw data was normalized. The empirical Bayes approach was used to address the working batch effect in the data. Data for cytokine outcome measures were not successfully transformed to form a normal distribution and were therefore analyzed with a non-parametric two directional Friedman's repeated measure test (baseline, immediately after and two hours later). The Wilcoxon signed rank two-tailed pairwise test determined the differences between significant outcomes and the differences between intensities (moderate vs. vigorous) for each group. The Mann-Whitney signed rank two-tailed independent test determined the differences between groups. To determine effects of time and group, a repeated measures ANOVA model was separately implemented for each training intensity. All analyses were performed using SPSS software (Version 25.0 for Windows, SPSS Inc., Chicago, IL). Reported p values were 2-tailed and p < 0.05 was considered as statistically significant.
4. Discussion The present study discovered several novel findings. Firstly, both the MS and control group illustrated a similar cytokine pattern in response to exercise of both intensities, with the exception of IL-10 during the vigorous intensity exercises. This main result is in line with a recent study reporting that levels of IL-10 and TNF-α in response to exercise were similar in healthy and MS remitting subjects (Majdinasab et al., 2018). We believe that the current findings enhance the evidence of the benefits of exercise for the MS population. According to our results, even high intensity exercise does not influence the inflammatory response of the cytokine profile in women with mild MS. Secondly, moderate exercise caused significant increases in IL-6 over time in both the women with mild MS and the healthy controls. This finding is in partial agreement with Castellano et al. (2008) and Majdinasab et al. (2018)'s studies. They who found an increase in IL-6 immediately after a moderately intense aerobic exercise session. Importantly, IL-6 is a cytokine implicated in both pro-inflammatory and anti-inflammatory processes. Trans-signaling processes are critical for the pro-inflammatory properties of IL-6 while IL-6R signaling is considered to cause an anti-inflammatory response (Gobel, 2018). Exercise triggers the release of IL-6 in the skeletal muscle, brain and peri‑tendinous tissues (Peake et al., 2015). In MS, CD4+ T cells mediate pathogenesis. TH1 cells produce IFN- ɣ which plays a role in tissue injury. IL-6 has been found to suppress TH1 activity which then interferes with IFN- ɣ production. Moreover, IL-6 has been shown to induce both IL-4 and IL-10, which generates anti-inflammatory effects (Gleeson et al., 2011; Janssens et al., 2015). The present results suggest that aerobic exercise possesses anti-inflammatory properties through the upregulation of IL-6. Interestingly, the cytokine response was similar between groups, despite the higher fitness level of the healthy women compared with the mild MS participants. The issue whether cardiorespiratory fitness influences the acute inflammatory response to exercise is still controversial. Kleiven stated that recreational cyclists self-reported superior fitness, thereby associating a lower CRP response to prolonged competitive exercise (Kleiven et al., 2017). In contrast, a recent study comparing master athletes with lower-fit untrained adults, reported no difference in the anti-inflammatory IL-10 and TGF-β responses to a single bout of exercise (Minuzzi et al., 2017). Similarly, another study investigating the cytokine responses (IL-6, IL-10, TNF-α) to acute exercise in healthy adults found the response was similar between participants with higher and lower levels of cardiorespiratory fitness (Windsor et al., 2018). Future research are encouraged to examine whether levels of fitness are related to the acute cytokine response in MS. Another interesting finding of our study concerns IL-10. Following the vigorous exercise session, IL-10 immediately decreased post-exercise compared to two hours post- exercise solely in the MS group. The IL-10 is classified as an important anti-inflammatory cytokine, functioning as a key immune-regulator, decreasing excessive TH1. The response of IL-10 is still controversial. Majdinasab et al., found a significant decrease of IL-10 immediately after an aerobic exercise session in a PwMS group tested during the time of relapse, whereas, a significant increase was seen one hour post-exercise in the MS group who were in remission (Majdinasab et al., 2018). In contrast, no changes over time was found for IL-10 following 30 min of aerobic exercise (Heesen et al., 2003). Worth noting, healthy adults showed no differences in the IL-10 response between low, moderate and high exercise intensities (Cullen et al., 2016). Additionally, the IL-10 value recorded at baseline in the MS group during the vigorous session was almost 3 times higher
3. Results Out of the total 25 subjects, one woman from the MS group and one healthy woman dropped out due to illness, therefore, a total of 14 women with mild MS and 9 healthy controls were included in the final analysis. The MS group exhibited a median duration of 7.6 (IQR=12.7) years since diagnosis. The median EDSS score was 1.5 (IQR=1.3) indicating a mild neurological disability. No differences between groups were found for age, body mass index, body fat percentage and leisure time activity level (based on the Godin questionnaire). VO2 peak was significantly lower in the women with MS compared to the healthy controls. The characteristics relating to demographics, clinical characteristics and aerobic fitness are presented in Table 1. Compared to baseline, after 2 h of exercising at a moderate intensity level, IL-6 significantly increased in both women (p = 0.02). The IL-6 increase was similar between groups (p = 0.33). No differences were found over time for IL-4, IL-10, IL-17A, IFN- ɣ and TNF- α in both groups during moderate intensity exercise. As for vigorous intensity exercise, IL-10 significantly decreased over time solely in the PwMS group (p = 0.02). No differences were found over time for IL-4, IL-6, ILTable 1 Demographic and clinical characteristics of the study sample. Mean (SD) Age (y) MS 33.8 (7.8) Controls 28.3 (6.2) Height (cm) MS 165.8 (8.1) Controls 160.3 (5.3) Weight (kg) MS 65.6 (16.2) Controls 58.2 (8.7) Body mass index (kg/m2) MS 23.3 (4.2) Controls 22.7 (3.4) Body fat percentage (%) MS 35.4 (6.1) Controls 32.2 (8.2) Leisure time activity level (score) MS – Controls – VO2 peak (ml/kg/min) MS 23.0 (7.4) Controls 36.9 (10.6) ⁎
Median
Min-Max
p-value
34.0 26.0
20.0–44.0 21.0–40.0
0.09
164.0 158.5
153.0–182.0 155.0–171.0
0.07
60.75 56.45
50.0–100.8 46.7- 74.5
0.35
22.2 22.1
19.5–30.0 18.7–28.9
0.77
35.6 34.0
28.8–48.8 17.8–43.9
0.29
34.0 37.0
0.0–49.0 0.0–75.0
0.48
22.5 36.3
12.3–34.6 21.2–53.1
0.02*
p < 0.05. 84
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Table 2 Cytokine scores (pg/ml) over time for exercise at a moderate intensity according to study groups.
IL-4 IL-6 IL-10 IL-17A IFN-ɣ TNF-α ⁎ a b c
MS Baseline
Immediately after
Post 2 h
Controls Baseline
Immediately after
Post 2 h
a
p-value Time MS
a
3.38 4.84 1.86 0.50 0.71 1.61
2.50 4.15 0.54 0.43 0.66 1.21
3.41 (6.58) 13.91 (17.46) 1.93 (5.81) 0.39 (1.22) 1.00 (2.52) 1.34 (1.96)
5.79 3.99 3.21 1.13 1.75 4.33
6.96 2.85 3.50 1.12 1.68 3.49
4.95 6.97 5.93 1.24 2.01 1.69
0.43 0.02*,c 0.11 0.85 0.34 0.57
0.75 0.02*,c 0.69 0.96 0.76 0.54
(7.17) (10.42) (6.16) (1.33) (1.33) (2.19)
(5.41) (11.26) (4.44) (0.09) (1.90) (1.95)
(10.60) (6.43) (3.55) (1.84) (2.33) (5.82)
(13.22) (4.67) (4.77) (1.84) (2.14) (4.53)
(8.45) (6.22) (10.50) (2.10) (2.80) (1.11)
Time control
b
Time x group
0.24 0.33 0.44 0.23 0.99 0.21
p < 0.05; Results presented as mean (SD);. Comparison between scores at baseline, immediately after and post 2-hours are presented separately in the MS and control groups. Indicates whether changes over time were different between groups. Between baseline and post 2 h.
Table 3 Cytokine scores (pg/ml) over time for exercise at a vigorous intensity according to study groups.
IL-4 IL-6 IL-10 IL-17A IFN-ɣ TNF-α ⁎ a b c
MS Baseline
Immediately after
Post 2 h
Controls Baseline
Immediately after
Post 2 h
a
p-value Time MS
a
4.37 5.92 5.05 0.67 1.45 1.86
4.81 4.04 2.07 0.56 1.22 1.88
3.86 7.89 1.70 0.61 0.96 1.71
10.88 (17.71) 2.63 (3.17) 2.45 (3.44) 1.11 (1.65) 2.18 (2.43) 3.10 (3.78)
15.32 (27.26) 2.84 (3.97) 2.21 (3.76) 1.20 (0.16) 2.16 (2.15) 3.26 (5.13)
4.70 8.45 1.90 1.02 1.63 1.31
0.62 0.24 0.02*,c 0.63 0.51 0.75
0.54 0.61 0.58 0.45 0.69 0.17
(7.25) (10.44) (8.82) (1.21) (3.26) (3.42)
(9.38) (7.68) (4.95) (1.04) (1.98) (2.82)
(7.46) (12.43) (4.76) (1.30) (1.74) (2.40)
(9.12) (10.44) (2.69) (1.56) (1.74) (0.73)
Time control
b
Time x group
0.17 0.41 0.05*,c 0.49 0.88 0.38
p < 0.05; Results presented as mean ± standard deviation;. Comparison between scores at baseline, immediately after and post 2-hours are presented separately in the MS and control groups. Indicates whether changes over time were different between groups. Between baseline and post 2 h.
vigorous intensity level, although women with mild MS demonstrated a greater decrease in IL-10 compared with healthy women, all other cytokines had similar inflammatory reactions, indicating the safety and potential efficacy of exercise in PwMS. Nevertheless, future research is necessary in order to show the cytokine response of longer-term aerobic programs.
compared to the IL-10 value recorded at baseline in the moderate intensity session. We speculate that the relatively large distribution of IL10 values, together with the small sample size, contributed to our findings, therefore, we advise considering this finding with caution. The present study provides valuable information for the MS community, especially for PwMS involved in physical activity. However, we acknowledge several limitations. Firstly, a large variability was found in the cytokine level change from pre- to post- exercise, a phenomenon common in cytokine research (Peake et al., 2015). In the present study, cytokine measurements were taken at baseline, immediately after and two hours following the exercise session. There is a possibility that if further testing points would have been added (e.g., six hours after), the cytokine response would have been more distinct. It would be interesting for future research to evaluate the precise time period during which cytokine levels return to baseline values (i.e. IL-6 was still significantly elevated two hours post- exercise) and how it varies amongst subjects, patients and controls. Secondly, this study was performed mainly on women with mild MS and a relapsing-remitting type of disease. It would be worthwhile to investigate whether there would be differences according to gender in PwMS with a more severe disability and progressive MS. Furthermore, we did not monitor the use of contraceptives, although this issue is rather controversial. While some have shown an effect (Timmons et al., 2005), others claim that studies investigating the cytokine response post exercise, may not need to control oral contraception (Sim et al., 2015). Finally, although the present data set included the main cytokines involved in aerobic exercise, the investigation of additional cytokines may increase our understanding of how exercise impacts the pro-inflammatory/anti-inflammatory balance.
Disclosure The authors have approved the final article Conflict of interest statement The authors confirm that there are no known conflicts of interest regarding the work described in the current manuscript. Funding This work was supported by an unrestricted [MS200136_0072] from the Merk KGaA, Damstadt, Germany.
grant
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Clinical trial
Acute effects of aerobic intensities on the cytokine response in women with mild multiple sclerosis Shani Berkowitza,b, Anat Achironb,c,d, Michael Gurevichb, Polina Sonisb, Alon Kalrona,d,
T
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a
Department of Physical Therapy, School of Health Professions, Sackler Faculty of Medicine, Tel-Aviv University, Tel Aviv, Israel Multiple Sclerosis Center, Sheba Medical Center, Tel Hashomer, Israel c Sackler Faculty of Medicine, Tel-Aviv University, Tel Aviv, Israel d Sagol School of Neuroscience, Tel-Aviv University, Tel Aviv, Israel b
A R T I C LE I N FO
A B S T R A C T
Keywords: Multiple sclerosis Aerobic Exercise Cytokines Inflammation
Background: There is limited information as to the effects of aerobic exercise on the immune system in people with multiple sclerosis (PwMS). One of the unresolved questions is which training intensity level is optimal for PwMS. Objective: The purpose of this study is to compare the effects of moderate intensity with the effects of vigorous intensity on cytokine levels in women with mild MS. Methods: The study included 15 women with MS and a control of 10 healthy women. The initial session determined the value of the maximum oxygen consumption and was carried out on an instrumented treadmill with a portable system for monitoring gases. During the second session, the participant walked on the treadmill for 15-minutes at a moderate capacity level (∼50% VO2 peak). Blood samples were taken at baseline, immediately after exercise and two hours later. IL-4, IL-6, IL-10, IL-17A, TNF-α and IFN-ɣ cytokines were tested. The third session was identical to the second except for a vigorous training intensity session (∼80% VO2 peak). Results: IL-6 increased after moderate exercise for both the MS (p = 0.02) and control group (p = 0.02). IL-10 decreased during the vigorous session only in the MS group (p = 0.02). No other differences were seen over time in either group for all other cytokine measurements. Conclusions: With the exception of IL-10, women with mild MS have similar inflammatory reactions to moderate and high intensity exercise as do healthy women.
1. Introduction Exercise, a beneficial rehabilitation strategy for people with multiple sclerosis (PwMS) manages symptoms, restores function, optimizes quality of life, promotes wellness and encourages participation in activities of daily living (Motl et al., 2017). Previous studies have demonstrated that aerobic activity is inversely correlated with symptoms in PwMS and may be protective and possibly restorative (Dalgas, 2012; Giesser, 2015). Dalgas et al. (2012) reported that higher rates of exercise correspond with decreased rates of functional deterioration (Dalgas, 2012; Stuifbergen et al., 2006). Furthermore, there is growing evidence that exercise could be considered a disease-modifying therapy in PwMS (Giesser, 2015). Cytokines, a significant component of MS pathophysiology (Patel, 2013) are protein hormones that regulate communication between the innate and adaptive branches of the immune system
(Alexsander, 2002). A sense of balance exists between T helper cytokines in the healthy population, whereas in PwMS, this balance shifts and reflects increased pro-inflammatory cytokines (i.e., interleukin (IL)−1, IL-6) and decreased anti-inflammatory cytokines (i.e., IL-10). These deviations can accelerate the destruction of myelin in the brain and spinal cord of PwMS (Turner et al., 2009). There are limited data regarding the effects of physical activity on the immune system in PwMS. The cytokines that are of primary focus in MS studies involving exercise, include a diverse spectrum of pro-inflammatory cytokines such as IL-1, IL-2, IL-6, IL-17, IFN-γ and TNF-α, and anti-inflammatory cytokines such as IL-1ra, IL-4 and IL-10 (Negaresh et al., 2018). Importantly, many of these cytokines are known to contribute to the pathogenesis of MS (Palle et al., 2017). Several studies have examined the effects of aerobic exercise on selected cytokines (Heesen et al., 2003; Castellano et al., 2008; Shulz, 2004; Golzari et al., 2010) yet, according to a recent systematic review,
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Corresponding author at: Department of Physical Therapy, Sackler Faculty of Medicine, Tel-Aviv University, Tel Aviv, Israel. E-mail addresses: [email protected] (S. Berkowitz), [email protected] (A. Achiron), [email protected] (M. Gurevich), [email protected] (P. Sonis), [email protected] (A. Kalron). https://doi.org/10.1016/j.msard.2019.03.025 Received 20 December 2018; Received in revised form 17 February 2019; Accepted 31 March 2019 2211-0348/ © 2019 Elsevier B.V. All rights reserved.
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a maximal aerobic intensity test (incremental exercise test) was performed on an instrumented treadmill (Zebris® Medical GmbH, Germany) using the Balke protocol (Balke, 1959). Participants performed the maximal aerobic capacity test while fitted to the COSMED K5 portable metabolic cart with breath-by-breath technology. The COSMED K5 is a reliable and valid method of analyzing gasses (Baldari et al., 2015). O2, CO2, pulse, respiratory exchange ratio, O2 cost and O2 rate were continuously measured. Outcome measures derived from cardiopulmonary exercise testing were found to be a valid measure of cardio-respiratory fitness in PwMS with low to mild levels of disability (EDSS <4) (Heine et al., 2014). The second and third sessions (7–14 days in between) included 15 min of aerobic exercise performed on the treadmill. Sessions differed as to the aerobic intensity level. The first session was performed at a moderate intensity, defined as fairly-light or somewhat hard, calculated as a 50% VO2 peak (46–60% VO2 peak). The second was performed at a vigorous intensity defined as somewhat hard to very hard, calculated as a 80% VO2 peak (64–90% VO2 peak) (Pescatello et al., 2014). To ensure that the exercise was performed at predetermined levels, the Borg Rating of Perceived Exertion (RPE) was periodically recorded and intensity adjusted if necessary, in order to obtain moderate and vigorous intensities (Scherr et al., 2013; Borg, 1982). Prior to the aerobic second and third exercise sessions, the patient rested for five minutes. Subsequently, there was a two minute warm up at 5.3 km per hour (kph) according to the standard Balke protocol. After the initial warm up, the incline was increased every minute until the subject reached the predetermined intensity level (moderate/vigorous).
the effect remains inconclusive (Negaresh et al., 2018). Moreover, the results of the studies contradict each other, making it more difficult to determine a definitive conclusion. Heterogeneity of the exercise protocols is a notable limitation of previous studies investigating the cytokine response to aerobic exercise in PwMS. Previous trials have examined different types of exercises (cycling, walking and running) with different training durations, frequencies and intensities (Negaresh et al., 2018). Training intensity, the focus of the present study, is an important parameter of aerobic exercise. The most common aerobic intensity modes are moderate and vigorous. Although, there is evidence supporting the beneficial effects of both training intensities for symptomatic relief in PwMS (Sandroff et al., 2015), to the best of our knowledge, the effects of different aerobic intensity levels on the immune response of PwMS has never been investigated. Clinicians engaged in physical rehabilitation in the MS population are striving to provide a personalized exercise regimen for PwMS (Motl et al., 2018). Therefore, addressing this query is worthwhile. Hopefully, this information can help adjust the proper intensity level in aerobic programs directed at the MS population. Therefore, the goal of our study was to compare the responses of selected cytokines at vigorous and moderate intensities in women with MS. Results of the MS participants were compared with those of healthy age-matched women. We focused on women only, due to fact that MS is more common in women and sex differences exist in the cytokine response (Benini et al., 2015). Our hypothesis was that the cytokine response found in the MS participants would differ from the healthy women's responses to the aerobic effort depending on the intensity level.
2.3. Blood samples and cytokine measures 2. Methods Blood samples were obtained on the second and third visit by venipuncture before a single session of exercise, immediately after and two hours post exercise. This blood collection protocol was chosen due to the variation in cytokine dynamics (Pederson, 2001; Ostrowski et al., 1999). All blood samples were drawn from the antecubital vein in a seated position and collected for at least 48 h after any MS-related interferon drug administration in order to control for the possible impact of the drug on cytokine regulation (Duddy et al., 1999). Blood samples remained at room temperature for 30 min. Once the blood had clotted, it was centrifuged at 3000 rpm for 15 min. Serum was then quickly removed, frozen at 20 °C and preserved for assessment. Two weeks later, it was moved to a deep freeze at −80 °C where it was kept until needed. Prior to running the analysis, samples were defrosted on ice. Multiple cytokine expression was measured by Luminex technology. Primary cytokine measures were interferon gamma (IFN- ɣ) and interleukin- 6, based on the literature (Negaresh et al., 2018; Castellano et al., 2008; Cullen et al., 2016). Secondary measures were tumor necrosis factor alpha (TNF-α), interleukin-4, interleukin-10 and interleukin-17A cytokine levels.
2.1. Study design and participants This is a case control study. Fifteen women with MS, mean age 33.8 (S.D. = 7.8) years, recruited from the Sheba Multiple Sclerosis Center, Tel-HaShomer, Israel, participated in the study. Ten age-matched healthy women, recruited through social media and local hospital advertisement facilities, served as controls. Inclusion criteria included: (1) a neurologist-confirmed diagnosis of definite MS according to the revised McDonald criteria (Thompson et al., 2018); (2) <4.5 score on the Expanded Disability Status Scale (EDSS) (Kurtzke, 1983), the ability to walk an equivalent of at least 500 m without a walking aid; (3) relapse-free for at least 90 days prior to testing; (4) 20 to 45 years of age; (5) patients receiving disease modifying drugs (e.g. Interferon β−1a) for at least 3 months; and (6) willing and able to wear an oxygen analyzer while walking. Exclusion criteria included: (1) pregnancy; (2) body-mass index ≥30; (3) cardiovascular disorders; (4) respiratory disorders; (5) unable to walk on a treadmill for 15 min. Furthermore, women were instructed to not to schedule the exercise sessions during menstruation. The Sheba Institutional Review Board, consistent with the Declaration of Helsinki, approved the study. Informed consent was obtained from all participants. Initially, the participants completed the Godin-Shephard Leisure-Time Physical Activity Questionnaire (Amireault, 2015) and subsequent anthropometric measures were taken. Once the intake was complete, each participant underwent a maximal aerobic intensity test to quantify aerobic capabilities. At the second session, the participant was randomized into a moderate or vigorous exercise group. On the third visit, the participants exercised at other levels of intensity.
2.4. Post-acquisition cytokine data processing Quantitative, multiplexed protein measurements from the serum samples to detect specific cytokines were performed by the ProcartaPlex High Sensitivity Immunoassay Mix & the Max panel kit from the eBioscience division of Affymetrix. ProcartaPlex immunoassays employs magnetic microsphere technology which allows for capture and detection of specific analysts from a serum sample. By using the Luminex MAGPIX® compact analyzer, simultaneous detection from a single sample of up to 50 protein targets, was possible. Analysis was carried out per the manufacturer's protocol. Concentration of samples was calculated by plotting the expected concentration of the standards against the median fluorescent intensity generated by each standard. A 5PL algorithm was used for the best curve fit. Assayed samples were analyzed in accordance with the operation manual of the Luminex MAGPIX® instrument.
2.2. Aerobic exercise protocol All study measurements were performed at the Sheba MS Center. Subjects completed the exercise testing in the morning (9–12 am) under the supervision of an accredited exercise physiologist. At the first visit, 83
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17A, IFN- ɣ and TNF- α in both groups during vigorous intensity exercise. The cytokine scores over time for aerobic exercise at moderate and vigorous intensities are presented in Tables 2 and 3.
2.5. Statistical analysis Descriptive statistics were performed to determine distributions of demographic, clinical and cytokine serum levels and to compare the characteristics of the women with MS and the controls. Following scanning of the cytokines, the raw data was normalized. The empirical Bayes approach was used to address the working batch effect in the data. Data for cytokine outcome measures were not successfully transformed to form a normal distribution and were therefore analyzed with a non-parametric two directional Friedman's repeated measure test (baseline, immediately after and two hours later). The Wilcoxon signed rank two-tailed pairwise test determined the differences between significant outcomes and the differences between intensities (moderate vs. vigorous) for each group. The Mann-Whitney signed rank two-tailed independent test determined the differences between groups. To determine effects of time and group, a repeated measures ANOVA model was separately implemented for each training intensity. All analyses were performed using SPSS software (Version 25.0 for Windows, SPSS Inc., Chicago, IL). Reported p values were 2-tailed and p < 0.05 was considered as statistically significant.
4. Discussion The present study discovered several novel findings. Firstly, both the MS and control group illustrated a similar cytokine pattern in response to exercise of both intensities, with the exception of IL-10 during the vigorous intensity exercises. This main result is in line with a recent study reporting that levels of IL-10 and TNF-α in response to exercise were similar in healthy and MS remitting subjects (Majdinasab et al., 2018). We believe that the current findings enhance the evidence of the benefits of exercise for the MS population. According to our results, even high intensity exercise does not influence the inflammatory response of the cytokine profile in women with mild MS. Secondly, moderate exercise caused significant increases in IL-6 over time in both the women with mild MS and the healthy controls. This finding is in partial agreement with Castellano et al. (2008) and Majdinasab et al. (2018)'s studies. They who found an increase in IL-6 immediately after a moderately intense aerobic exercise session. Importantly, IL-6 is a cytokine implicated in both pro-inflammatory and anti-inflammatory processes. Trans-signaling processes are critical for the pro-inflammatory properties of IL-6 while IL-6R signaling is considered to cause an anti-inflammatory response (Gobel, 2018). Exercise triggers the release of IL-6 in the skeletal muscle, brain and peri‑tendinous tissues (Peake et al., 2015). In MS, CD4+ T cells mediate pathogenesis. TH1 cells produce IFN- ɣ which plays a role in tissue injury. IL-6 has been found to suppress TH1 activity which then interferes with IFN- ɣ production. Moreover, IL-6 has been shown to induce both IL-4 and IL-10, which generates anti-inflammatory effects (Gleeson et al., 2011; Janssens et al., 2015). The present results suggest that aerobic exercise possesses anti-inflammatory properties through the upregulation of IL-6. Interestingly, the cytokine response was similar between groups, despite the higher fitness level of the healthy women compared with the mild MS participants. The issue whether cardiorespiratory fitness influences the acute inflammatory response to exercise is still controversial. Kleiven stated that recreational cyclists self-reported superior fitness, thereby associating a lower CRP response to prolonged competitive exercise (Kleiven et al., 2017). In contrast, a recent study comparing master athletes with lower-fit untrained adults, reported no difference in the anti-inflammatory IL-10 and TGF-β responses to a single bout of exercise (Minuzzi et al., 2017). Similarly, another study investigating the cytokine responses (IL-6, IL-10, TNF-α) to acute exercise in healthy adults found the response was similar between participants with higher and lower levels of cardiorespiratory fitness (Windsor et al., 2018). Future research are encouraged to examine whether levels of fitness are related to the acute cytokine response in MS. Another interesting finding of our study concerns IL-10. Following the vigorous exercise session, IL-10 immediately decreased post-exercise compared to two hours post- exercise solely in the MS group. The IL-10 is classified as an important anti-inflammatory cytokine, functioning as a key immune-regulator, decreasing excessive TH1. The response of IL-10 is still controversial. Majdinasab et al., found a significant decrease of IL-10 immediately after an aerobic exercise session in a PwMS group tested during the time of relapse, whereas, a significant increase was seen one hour post-exercise in the MS group who were in remission (Majdinasab et al., 2018). In contrast, no changes over time was found for IL-10 following 30 min of aerobic exercise (Heesen et al., 2003). Worth noting, healthy adults showed no differences in the IL-10 response between low, moderate and high exercise intensities (Cullen et al., 2016). Additionally, the IL-10 value recorded at baseline in the MS group during the vigorous session was almost 3 times higher
3. Results Out of the total 25 subjects, one woman from the MS group and one healthy woman dropped out due to illness, therefore, a total of 14 women with mild MS and 9 healthy controls were included in the final analysis. The MS group exhibited a median duration of 7.6 (IQR=12.7) years since diagnosis. The median EDSS score was 1.5 (IQR=1.3) indicating a mild neurological disability. No differences between groups were found for age, body mass index, body fat percentage and leisure time activity level (based on the Godin questionnaire). VO2 peak was significantly lower in the women with MS compared to the healthy controls. The characteristics relating to demographics, clinical characteristics and aerobic fitness are presented in Table 1. Compared to baseline, after 2 h of exercising at a moderate intensity level, IL-6 significantly increased in both women (p = 0.02). The IL-6 increase was similar between groups (p = 0.33). No differences were found over time for IL-4, IL-10, IL-17A, IFN- ɣ and TNF- α in both groups during moderate intensity exercise. As for vigorous intensity exercise, IL-10 significantly decreased over time solely in the PwMS group (p = 0.02). No differences were found over time for IL-4, IL-6, ILTable 1 Demographic and clinical characteristics of the study sample. Mean (SD) Age (y) MS 33.8 (7.8) Controls 28.3 (6.2) Height (cm) MS 165.8 (8.1) Controls 160.3 (5.3) Weight (kg) MS 65.6 (16.2) Controls 58.2 (8.7) Body mass index (kg/m2) MS 23.3 (4.2) Controls 22.7 (3.4) Body fat percentage (%) MS 35.4 (6.1) Controls 32.2 (8.2) Leisure time activity level (score) MS – Controls – VO2 peak (ml/kg/min) MS 23.0 (7.4) Controls 36.9 (10.6) ⁎
Median
Min-Max
p-value
34.0 26.0
20.0–44.0 21.0–40.0
0.09
164.0 158.5
153.0–182.0 155.0–171.0
0.07
60.75 56.45
50.0–100.8 46.7- 74.5
0.35
22.2 22.1
19.5–30.0 18.7–28.9
0.77
35.6 34.0
28.8–48.8 17.8–43.9
0.29
34.0 37.0
0.0–49.0 0.0–75.0
0.48
22.5 36.3
12.3–34.6 21.2–53.1
0.02*
p < 0.05. 84
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Table 2 Cytokine scores (pg/ml) over time for exercise at a moderate intensity according to study groups.
IL-4 IL-6 IL-10 IL-17A IFN-ɣ TNF-α ⁎ a b c
MS Baseline
Immediately after
Post 2 h
Controls Baseline
Immediately after
Post 2 h
a
p-value Time MS
a
3.38 4.84 1.86 0.50 0.71 1.61
2.50 4.15 0.54 0.43 0.66 1.21
3.41 (6.58) 13.91 (17.46) 1.93 (5.81) 0.39 (1.22) 1.00 (2.52) 1.34 (1.96)
5.79 3.99 3.21 1.13 1.75 4.33
6.96 2.85 3.50 1.12 1.68 3.49
4.95 6.97 5.93 1.24 2.01 1.69
0.43 0.02*,c 0.11 0.85 0.34 0.57
0.75 0.02*,c 0.69 0.96 0.76 0.54
(7.17) (10.42) (6.16) (1.33) (1.33) (2.19)
(5.41) (11.26) (4.44) (0.09) (1.90) (1.95)
(10.60) (6.43) (3.55) (1.84) (2.33) (5.82)
(13.22) (4.67) (4.77) (1.84) (2.14) (4.53)
(8.45) (6.22) (10.50) (2.10) (2.80) (1.11)
Time control
b
Time x group
0.24 0.33 0.44 0.23 0.99 0.21
p < 0.05; Results presented as mean (SD);. Comparison between scores at baseline, immediately after and post 2-hours are presented separately in the MS and control groups. Indicates whether changes over time were different between groups. Between baseline and post 2 h.
Table 3 Cytokine scores (pg/ml) over time for exercise at a vigorous intensity according to study groups.
IL-4 IL-6 IL-10 IL-17A IFN-ɣ TNF-α ⁎ a b c
MS Baseline
Immediately after
Post 2 h
Controls Baseline
Immediately after
Post 2 h
a
p-value Time MS
a
4.37 5.92 5.05 0.67 1.45 1.86
4.81 4.04 2.07 0.56 1.22 1.88
3.86 7.89 1.70 0.61 0.96 1.71
10.88 (17.71) 2.63 (3.17) 2.45 (3.44) 1.11 (1.65) 2.18 (2.43) 3.10 (3.78)
15.32 (27.26) 2.84 (3.97) 2.21 (3.76) 1.20 (0.16) 2.16 (2.15) 3.26 (5.13)
4.70 8.45 1.90 1.02 1.63 1.31
0.62 0.24 0.02*,c 0.63 0.51 0.75
0.54 0.61 0.58 0.45 0.69 0.17
(7.25) (10.44) (8.82) (1.21) (3.26) (3.42)
(9.38) (7.68) (4.95) (1.04) (1.98) (2.82)
(7.46) (12.43) (4.76) (1.30) (1.74) (2.40)
(9.12) (10.44) (2.69) (1.56) (1.74) (0.73)
Time control
b
Time x group
0.17 0.41 0.05*,c 0.49 0.88 0.38
p < 0.05; Results presented as mean ± standard deviation;. Comparison between scores at baseline, immediately after and post 2-hours are presented separately in the MS and control groups. Indicates whether changes over time were different between groups. Between baseline and post 2 h.
vigorous intensity level, although women with mild MS demonstrated a greater decrease in IL-10 compared with healthy women, all other cytokines had similar inflammatory reactions, indicating the safety and potential efficacy of exercise in PwMS. Nevertheless, future research is necessary in order to show the cytokine response of longer-term aerobic programs.
compared to the IL-10 value recorded at baseline in the moderate intensity session. We speculate that the relatively large distribution of IL10 values, together with the small sample size, contributed to our findings, therefore, we advise considering this finding with caution. The present study provides valuable information for the MS community, especially for PwMS involved in physical activity. However, we acknowledge several limitations. Firstly, a large variability was found in the cytokine level change from pre- to post- exercise, a phenomenon common in cytokine research (Peake et al., 2015). In the present study, cytokine measurements were taken at baseline, immediately after and two hours following the exercise session. There is a possibility that if further testing points would have been added (e.g., six hours after), the cytokine response would have been more distinct. It would be interesting for future research to evaluate the precise time period during which cytokine levels return to baseline values (i.e. IL-6 was still significantly elevated two hours post- exercise) and how it varies amongst subjects, patients and controls. Secondly, this study was performed mainly on women with mild MS and a relapsing-remitting type of disease. It would be worthwhile to investigate whether there would be differences according to gender in PwMS with a more severe disability and progressive MS. Furthermore, we did not monitor the use of contraceptives, although this issue is rather controversial. While some have shown an effect (Timmons et al., 2005), others claim that studies investigating the cytokine response post exercise, may not need to control oral contraception (Sim et al., 2015). Finally, although the present data set included the main cytokines involved in aerobic exercise, the investigation of additional cytokines may increase our understanding of how exercise impacts the pro-inflammatory/anti-inflammatory balance.
Disclosure The authors have approved the final article Conflict of interest statement The authors confirm that there are no known conflicts of interest regarding the work described in the current manuscript. Funding This work was supported by an unrestricted [MS200136_0072] from the Merk KGaA, Damstadt, Germany.
grant
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