http://informahealthcare.com/arp ISSN: 1381-3455 (print), 1744-4160 (electronic) Arch Physiol Biochem, Early Online: 1–9 ! 2015 Informa UK Ltd. DOI: 10.3109/13813455.2014.1003566
ORIGINAL ARTICLE
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An acute bout of exercise modulate the inflammatory response in peripheral blood mononuclear cells in healthy young men Stine M. Ulven1, Silje Stange Foss2#, Anne Marie Skjølsvik2#, Hans Kristian Stadheim3, Mari CW. Myhrstad1, Ellen Raael1, Marit Sandvik2, Ingunn Narverud1,2, Lene Frost Andersen2, Jørgen Jensen3, and Kirsten B. Holven2 1
Department of Health, Nutrition and Management, Faculty of Health Sciences, Oslo and Akershus University College of Applied Sciences, St Olavsplass, Oslo, Norway, 2Department of Nutrition, Institute for Basic Medical Sciences, University of Oslo, Blindern, Oslo, Norway, and 3 Norwegian School of Sports Science, Oslo, Norway Abstract
Keywords
Context: Exercise increases the levels of circulating inflammatory mediators. Objective: Does an acute bout of exercise affect the mRNA gene expression level of inflammatory markers in peripheral blood mononuclear cells (PBMCs) and contribute to the circulating levels of inflammatory mediators? Materials and methods: Ten healthy, non-smoking men (22–28 years old) performed 1-hour cycling at 70% of VO2 max. Results: The gene transcripts of CXCL16, IL-1b, IL-8, COX-2, TXB21 and GATA3 were significantly up-regulated in PBMCs. Serum levels of CXCL16, IL-6, TNFa and IL-10 were also significantly increased after exercise. Discussion and conclusion: Increased mRNA transcription of inflammatory genes in PBMCs may contribute to increased level of inflammatory markers after an acute bout of exercise. The increased mRNA levels of GATA-3 and TXB21 may indicate that T cell lymphocytes are activated and secrete cytokines into the circulation. It needs to be further investigated if exercise changes the Th1/Th2 balance.
Acute exercise, gene expression, inflammation, peripheral blood mononuclear cells, T helper cell response
Introduction Physical activity is associated with a decreased risk for premature morbidity and regular exercise has been shown to prevent chronic diseases such as cardiovascular diseases (CVD), type 2 diabetes (T2D), certain cancers, obesity, dementia and hypertension (Blair et al., 2001; Rovio et al., 2005; Warburton et al., 2006). Low-grade chronic inflammation is suggested to be a key factor in the pathogenesis of several chronic diseases, and long-term effect of physical activity has been showed to reduce the levels of inflammatory markers (Geffken et al., 2001; King et al., 2003; Mattusch et al., 2000). On the contrary, it is well known that a bout of exercise increases the circulating levels of a number of inflammatory mediators, including interleukin (IL)-6, whereas tumour necrosis factor (TNF)-a is only stimulated by very intense exercise (Pedersen et al., 2007). The increase in IL-6 during exercise is followed by an increase in the antiinflammatory mediators IL-1 receptor antagonist (IL-1ra), soluble TNF-Receptors (sTNFRs) and IL-10 (Petersen & Pedersen, 2005; Pedersen et al., 2007).
#Contributed equally. Correspondence: Stine Marie Ulven, Department of Health, Nutrition and Management, Faculty of Health Sciences, Oslo and Akershus University College of Applied Sciences, PO Box 4, St Olavsplass, 0130 Oslo, Norway. Tel: +47 67236348. E-mail: [email protected]
History Received 25 September 2014 Revised 4 December 2014 Accepted 27 December 2014 Published online 27 February 2015
Cytokines are secreted from many tissues and origins are not always well-defined. Muscle is considered the major contributor to secretion of IL-6 during exercise (Petersen & Pedersen, 2005), but also adipose tissue can produce and secrete IL-6 during exercise (Lyngso et al., 2002). However, the circulating levels of inflammatory mediators do not necessarily reflect the levels in source tissues. Peripheral blood mononuclear cells (PBMCs) consist of monocytes and lymphocytes and have important function in secretion of cytokines and immune responses (Holven et al., 2002, 2003, 2006; Pasterkamp & Daemen, 2008; Visvikis-Siest et al., 2007). Previous studies have shown that PBMCs may contribute to the inflammatory response after exercise (Connolly et al., 2004; Radom-Aizik et al., 2009a, 2009b). Several inflammatory response genes including chemokine (C-C motif) ligand 4 (MIP-1b), CD69 antigen (p60, early T cell activation antigen) and chemokine (C motif) ligand 2 were up-regulated after a 30-minute cycle test (80% peak O2 uptake) (Connolly et al., 2004). A brief bout of exercise has also been shown to alter the expression of inflammatory genes in PBMCs in early- and late-pubertal males and females (Radom-Aizik et al., 2009a). Cytokines produced by T lymphocytes may be involved in the host immunity against infection. T lymphocytes can differentiate into T helper cells type 1 (Th1) and T helper cells type 2 (Th2). Th1 cells are characterized by production of interferon (IFN)-g, TNF-a and IL-2 (Mosmann et al., 1986; Seder & Paul, 1994), while Th2 cells are characterized by production of IL-4, IL-5, IL-6 and
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IL-10 (Mosmann et al., 1986; Seder & Paul, 1994). Th1 and Th2 cell responses are mutually inhibitory, and therefore the Th1/Th2 balance has been used as an indicator of the changes in immune function (Jager & Kuchroo, 2010; Mosmann & Sad, 1996). Recently it was shown that strenuous exercise increases the expression of genes in PBMCs related to Th2 cell function (Xiang et al., 2014), while moderate exercise has been shown to increase expression of genes related to Th1 cell function (Yeh et al., 2009). Whether PBMCs are activated and contribute to the circulating levels of inflammatory mediators after an acute bout of exercise is incompletely understood and remains to be elucidated. It is well known that acute exercise causes acute transient increased level of inflammatory markers in circulation. However, circulating markers may be derived from many metabolic organs, such as skeletal muscle and adipose tissue. It is therefore not clear if the immune cells contribute to increased level of inflammatory markers after an acute bout of exercise. The aim of the present study was therefore to examine the serum levels and PBMC gene expression levels of inflammatory mediators after a standardized and controlled acute bout of exercise.
Methods Subjects Sixteen healthy, non-smoking men were considered for participation in the study. Five were excluded due to the eligibility criteria and one did not conduct the pre-test according to protocol and was therefore excluded from the study. The inclusion criteria were healthy males between 18 and 40 years old. Exclusion criteria included smoking, prevalence of chronic disease such as Type 1 and 2 diabetes mellitus, rheumatism, serious illness during the past five years such as myocardial infarction, stroke and cancer, hypertension, blood lipids outside the reference values, prevalence of milk allergy, lactose- and/or gluten intolerance, the use of anti-inflammatory medications, and not being able to perform the exercise according to the protocol. The study was conducted at the Norwegian School of Sport Science in 2011. The study protocol was approved by The Regional Committee of Medical Ethics and The Norwegian Directorate of Health and the study complies with the Declaration of Helsinki. Informed consent was obtained from all subjects. Exercise intervention The subjects refrained from alcohol consumption and vigorous physical activity the day prior to the exercise test days. At each test day, the subjects visited the study centre and body composition, blood pressure and venous blood samples were taken after an overnight fast (12 h). A standardized breakfast composed of two slices (80 g) of oatmeal bread, 10 g margarine (40% fat), four slices (30 g) of cheese (27% fat), 28 g red peppers, 40 g cucumber and 0.5 L of apple-grape juice was served between 08.30 am and 8.45 am, 1 h before the cycle test started. The total energy and macronutrient composition of the meal is shown in Table 1. After breakfast, the participants waited for 1 h before they started the exercise test at the ergometer cycle (Monark 839E, Varberg, Sweden).
Arch Physiol Biochem, Early Online: 1–9
Table 1. Total energy and macronutrient composition of the breakfast. Energy, KJ Protein, E% Total fat, E% Saturated fat, E% Trans fat, E% Mono-unsaturated fat, E% Poly-unsaturated fat, E% Carbohydate, E% Fibre, gram
1886 16 28 15 1 8 3 56 6,7
The participants cycled for 1 hour at 70% of VO2 max. During the test, measurements such as lactate, heart rate (HR), VO2 and Borg’s scale was conducted after 4 min, 15 min, 30 min, 45 min and 60 min. The exercise test day was repeated twice, separated by 1 week. Prior to the two test days, the participants had to meet at the Norwegian School of Sport Science twice in the same week to determine their VO2 max and to perform a pre-test (separated by 1 week from the first test day). In total, the duration of the study was 3 weeks. In week 1, the participants visited the study centre to determine their VO2 max and to perform the pre-test. In week 2 they performed the first exercise test and in week 3 they performed the second cycle test. VO2 max determination and pre-test Prior to the VO2 max test, a standardized warm-up was performed by each test subject. The standardized warm-up was performed as a continuous incremental test where the workload was increased each fifth minute with 25 watts. During each workload oxygen consumption was measured on average every 3 or 4 min using an Oxycon Pro (Jaeger-Toennis Instruments, Hochberg, Germany), and air was collected using a mouth V2-mask (Hans Rudolph Instruments, USA) in combination with a nose bracket. The relationship between VO2 and Watt from the warm-up was later used to make a linear regression for estimating the workload equivalent of 70% of VO2 max. After each 5-min workload a capillary sample from the fingertip (Accu-Check, Safe-T-Pro Plus, Roche, Germany) was taken for measuring lactate using an electro-enzymatic lactate analyser (1500 Sport, YSI Inc., USA). The incremental test was stopped when capillary lactate had increased 1.5 mmol above the concentration at the first workload (75 watts). Between the standardized warm-up and the VO2 max test subjects were given a 5-min break where they cycled at a workload of 50 watts. The VO2 max test was designed with an increase in intensity of 25 watts every 30 sec. Subjects started the VO2 max test with a workload, which was 25 watts higher than they ended the standardized warm-up. All subjects had to meet point one and at least two of the three other criteria so that they reached VO2 max: (1) Oxygen consumption reached a plateau, meaning VO2 increased less than 1 mL kg1 min1, while the workload was increased twice with 25 watts. (2) Respiratory exchange ratio values were above 1.10. (3) Post blood lactate measurements were above 7.0 mmol/L.
Exercise and inflammation
DOI: 10.3109/13813455.2014.1003566
(4) Rated perceived exertion was 19 on the Borg Scale 6-20. VO2 max-run was based on average of the two highest measurements. Measurements of oxygen consumption were taken every 30 sec. After the VO2 max test, a pre-test at 70% of VO2 max for 40 min was performed to verify that the calculated workload was correct and to ensure that the subjects could accomplish the exercise intervention.
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Measurements of body composition and food intake Anthropometrical measures like weight and height were determined by use of standard calibrated scales. Waist circumference was measured at the level midway between the lowest rib margin and the iliac crest. The reading of waist circumference in centimetres was done when the tape measure was snug, but not causing compression on the subjects’ skin. Hip circumference was measured over the widest part of the hip. Body composition was measured by BIA on a Tanita Scale (BC-418MA, Tanita Corporation, Japan). The participants were scanned using the athlete function on the scale and 0.5 kg was subtracted for compensation for light clothing. Food intake was registered once using validated food diary for four consecutive days of which one was a weekend day. The subjects received the food diaries at the pre-test day (the week before the first test day) and they received instructions from a clinical nutritionist on how to register food intake the week before the first test day. In addition, the subjects were told not to change their food intake during the study period. Blood sampling Venous blood samples were drawn after an overnight fast (12 hours) (at 8.00 am) and immediately after the cycle test was finished (at 10.45 am). Serum was obtained from silica gel tubes (Becton Dickinson (BD) vacutainer) and kept at room temperature for at least 30 min, until centrifugation (1500 g, 12 min), and immediately prepared for subsequent analysis of routine laboratory analyses or aliquoted and stored at 80 C until further analyses. Plasma was obtained from EDTA tubes (BD vacutainer), immediately placed on ice and centrifuged within 10 min (1500 g, 4 C, 10 min) and aliquoted and stored at 80 C until further analysis. Routine laboratory analysis Fasting serum hsCRP, total cholesterol, low density lipoprotein (LDL) cholesterol, high density lipoprotein (HDL) cholesterol, triacylglycerides (TAG), glucose and insulin were measured by standard methods at a routine laboratory (Fu¨rst Medical Laboratory, Norway).
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approximately 80% of the cells are lymphocytes and 12% are monocytes (http://www.bd.com/vacutainer/pdfs/bd_cpt_ VDP40105.pdf). Pellets were frozen and stored at 80 C prior to RNA isolation. Total RNA was isolated from all PBMC samples using RNeasy mini kit (Qiagen, Hilden, Germany) with lysis buffer added b-mercaptoethanol according to the manufacturer’s instructions, and stored at 80 C. RNA quantity and quality measurements were performed using the ND 1000 Spectrophotometer (Saveen Werner, Malmo¨, Sweden) and Agilent Bioanalyser (Agilent Technologies, Santa Clara, CA, USA), respectively. All samples had RNA integrity number 47; 200 ng RNA was reverse transcribed by high-capacity RNA-to-cDNA kit (Applied Biosystems, Foster City, CA, USA). Custom TaqMan Array micro Fluidic cards (Applied Biosystems) were used for RT-qPCR amplification of the target genes. TATA binding protein (TBP) was selected as endogenous control based on running a TaqMan Human Endogenous Control Plate-test (data not shown) (TelleHansen et al., 2012). A list of the inflammatory genes analysed in the present study are given in Supplementary Table 1. Primer sequences can be provided by request. A total of hundred ml of cDNA (1:10 fold dilution), RNase-free water and TaqMan gene expression master mix were applied to the micro Fluidic card and centrifuged according to the manufacturer’s protocol. The DCt values were calculated as Ct values of each target gene minus the Ct values of the endogenous control, TBP (housekeeping). Enzyme immunoassay (EIA) Serum, soluble TNF receptor (sTNFR)I, sTNFRII, TNF-a, IL-10, IL-6, CXCL16, soluble intracellular adhesion molecule 1 (sICAM-1) and soluble vascular cellular adhesion molecule1 (sVCAM-1) were measured by EIA from R&D Systems (Minneapolis, MN) according the manufacturer’s instructions. Statistics The present study was designed as a pilot study and no adjustment for multiple testing and power calculations were performed. The significance before and after exercise was assessed by Wilcoxon signed rank test (using DCt when comparing mRNA levels). All values are presented as median (min-max). Probability values (asymptotic) were considered statistically significant at a value of 0.05. All statistical analyses were performed using the Statistical Package for the Social Sciences (SPSS) version 19 for Microsoft (SPSS, Inc., USA).
Results PBMC and RNA isolation and reverse transcription real-time quantitative polymerase chain reaction (RT-qPCR) After blood collection, PBMC were isolated using the BD Vacutainer Cell Preparation tubes according to the manufacturer’s instructions (Becton, Dickinson and Company, NJ 07417, USA) within 2 h. This is a well-documented and standardized method to collect mononuclear cells with high purity (above 90%) and, according to the manufacturer,
Characterization of the 10 male participants is shown in Table 2. The median age for the group was 25 years (22–28, min-max). The median value for the VO2 max (62 mL kg1 min1) was above the reference value compared to an average population of men between 20 and 29 years old indicating that the participants had a very good physical fitness. This is also shown by the low median value of fat mass, 7.3 kg (3.9–14.0 kg, min-max) and of the fat percent, 9.6% (4.8–16.6%, min-max) compared with reference values
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Arch Physiol Biochem, Early Online: 1–9
(Table 2). The participants also had a high median value of fat free mass, 70 kg (65–77 kg, min-max), compared to median weight, 79 kg (69–88 kg, min-max) (Table 2). Food intake of the participant is shown in Table 3. The intake of fat and protein was within, while the intake of carbohydrates was below, the new Nordic dietary recommendations.
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Table 2. Baseline characteristics. (n ¼ 10) Age (y) Weight (kg) Height (cm) BMI (kg/m2) Waist:hip ratio Heart rate (beat/min) Systolic BP (mm/Hg) Diastolic BP (mm/Hg) VO2 max (ml/kg/min) Fat percent (%) Fat free mass (kg) Fat mass (kg) Total cholesterol (mmol/L) Low density lipoprotein cholesterol (mmol/L) High density lipoprotein cholesterol (mmol/L) Triglycerides (mmol/L) Glucose (mmol/L) HbA1c (%) Insulin (pmol/L) hsCRP (mg/L)
25 79 184 23.3 0.84 46 131 71 62 9.6 70 7.3 3.7 2.4
Reference value
(22–28) (69–88) (180–194) (21.3–25.3) (0.79–0.89) (42–54) (120–144) (61–96) (53.6–70.6) (4.8–16.6) (65–77) (3.9–14.0) (3.4–4.9) (1.7–3.4)
1.4 (1.1–1.7) 0.73 4.9 5.4 47 0.4
51.0 5130 585 48.5 10.0–20.0 8.9–17.9 2.9–6.1 1.2–4.3
52.60 4.0–4.6 56.1 18–173 55.0
Data are given as an average of two test days and are given as median (min-max). BP, blood pressure; BMI, body mass index; n indicates number of individuals.
Table 3. Energy and macronutrient intake. (n ¼ 10) Energy (KJ) Protein (E%) Fat (E%) Carbohydrates (E%) Added sugar Alcohol (E%) Fibre (g/d)
13 450 16.7 36.2 42.0 9.9 2.3 33.7
Reference value*
(10 791–16 538) (15.4–17.9) (30.2–37.3) (37.9–46.3) (6.4–14.9) (0.0–5.5) (21.9–37.8)
The characterization of the exercise intervention is presented in Table 4. The workload at both test days was set to the load calculated to correspond to 70% of VO2 max (44.97 ml/kg/min). Median oxygen uptake at the tests was 45.0 ml/kg/min (43.5–46.4 ml/kg/min min-max). The level of lactate increased during the first 15 min, from 0.98 mmol/L (0.87– 1.09 mmol/l) at baseline to 2.97 mmol/L, but then decreased again throughout the exercise. Heart rate and Borg scale significantly increased during exercise. No significant changes were found in the level of plasma glucose, hsCRP and insulin from pre- to post-exercise (data not shown). The effect of exercise on circulating inflammatory markers
0.8–2.1
(0.58–1.26) (4.7–5.4) (5.0–5.6) (30–69) (0.05–2.1)
Characterization of the exercise
13 800a 10–20 25–40 45–60 510 25–35
Data are mean of 4 days food records; n is number of individuals. Data is given as median (IQR). *Norwegian reference values. a Reference value of energy intake of male 18–30 years in Norway with a PAL of 1.8.
The exercise-induced response on the circulating levels of inflammatory markers is shown in Figure 1. The serum levels of sTNFRI, IL-6, IL-10, TNFa, CXCL16, sICAM-1 and sVCAM-1 significantly increased by physical activity (p 0.05 for all), whereas the level of sTNFRII was unchanged by physical activity. The effect of exercise on PBMC gene expression of inflammatory mediators To further elucidate if PBMC gene expression of inflammatory mediators are influenced by exercise we analysed the mRNA level of 18 inflammatory markers. The mRNA levels of IL-1b, CXCL16, IL-8, cyclo-oxygenase (COX)-2, T-box 21 (TBX21) and trans-acting T-cell-specific transcription factor (GATA-3) (p50.05 for all) were significantly upregulated by exercise (Figures 2 and 3). The toll-like receptor (TLR)-2 mRNA was significantly down-regulated after exercise (p50.05) (Figure 3). In addition, the gene transcripts of TNF-a, CD40, CD40L, transforming growth factor (TGF)-b, IFN-g, IL-18, toll-like receptor (TLR)-4, toll-like receptor (TLR-6), CD3e molecule epsilon (CD3E), cluster of differentiation 8a (CD8A) and forkhead box P3 (FOXP3) were all unchanged by exercise (Figure 2 and 3).
Discussion In the present study, we observed a significant increase in serum levels of CXCL16, sTNFRI, IL-6, IL-10, TNF-a, sICAM-1 and sVCAM-1 in healthy, athletic males after an acute bout of exercise (cycling 1 h at 70% of VO2 max). At the same time, the mRNA level of CXCL16, IL-1b, IL-8, COX-2, GATA3 and TXB21 were significantly up-regulated by exercise, while TLR-2 mRNA was significantly downregulated.
Table 4. Characteristics of exercise intervention. 4 min (n ¼ 10) VO2 (L/min) HR (beat/min) Lactate (mmol/L) Borg scale
42.14 133 2.71 13.4
(39.52–45.55) (126–147) (2.13–3.22) (7.9–14.5)
15 min (n ¼ 10) 43.30 154 2.97 14.1
(41.29–47.80)* (138–168)* (2.34–3.74)* (12.3–15.6)*
30 min (n ¼ 10) 44.18 157 2.91 15.0
(41.54–47.97)* (137–170)* (2.26–3.79) (13.0–16.5)*
45 min (n ¼ 10) 44.49 161 2.62 15.6
(41.77–48.33)* (139–172)* (2.17–4.28)** (14.0–16.5)*
n Indicates number of individuals. Data are given as an average of two test days and given as median (min-max). Significant different from 4 min; *p50.01; **p50.05. Wilcoxon signed Rank test.
60 min (n ¼ 10) 44.97 163 2.40 15.4
(43.40–51.30)* (139–172)* (1.85–3.60) (14.0–17.0)*
Exercise and inflammation
DOI: 10.3109/13813455.2014.1003566
P=0.01 10
(B) 2.0
8
1.5
IL-10 [pg/ml]
(A) IL-6 [pg/ml]
Figure 1. The effect of an acute bout of exercise on serum IL-6 (A), IL-10 (B), TNFa (C), CXCL16 (D), sTNFR1 (E), sTNFR2 (F), sICAM-1 (G) and sVCAM-1 (H). Data are calculated as an average of two test days and is given as mean (SEM). Wilcoxon signed rank test was used to calculate the difference before and after exercise.
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P=0.02 (G) 150 sICAM-1 [ng/ml]
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It is well known that physical activity increases the circulating level of IL-6 (Bernecker et al., 2013; Langberg et al., 2002; Nybo et al., 2002; Moldoveanu et al., 2000) and the increase in serum IL-6 has been suggested to be mainly caused by increased secretion from skeletal muscle (Steensberg et al., 2002). In the present study, we observed a two-fold increase in serum level of IL-6, which is lower compared with most previous studies (Connolly et al., 2004; Nieman et al., 2001; Ostrowski et al., 1998; Ostrowski et al., 1999; Rohde et al., 1997). One explanation could be that the subjects in the present study were served a breakfast containing 59 g of carbohydrates (56% of total energy intake) prior to the exercise test. Carbohydrate loading before exercise has been shown to diminish the exercise induced increase in circulating levels of IL-6 (NehlsenCannarella et al., 1997). It has also recently been shown that plasma levels of IL-6 and TNF-a were reduced after consumption of a carbohydrate rich meal (79% of total energy intake) (Meher et al., 2014). In contrast, a post-prandial
Physical exercise
Baseline
Physical exercise
increase in IL-6 has been observed after intake of high carbohydrate (76% of total energy intake) (Gregersen et al., 2012). A possible pro-inflammatory post-prandial effect, however, caused by the served meal in the present study may be less likely due to the lower level of IL-6 after exercise compared with other studies. We also observed an increase in circulating level of TNF-a and our finding is in agreement with others who have reported small, but significant increase in the circulating level of TNFa after exercise (Dufaux & Order, 1989; Ostrowski et al., 1998; Steensberg et al., 2002). This increase is in contrast to others who have reported no significant change in the circulating leval of TNF-a after exercise (Drenth et al., 1995; Rohde et al., 1997; Steensberg et al., 2002). The fact that we also observed a significant increase in sTNFR1 strengthens our results that the TNF pathway is induced by exercise and sTNFR1 may attenuate the potent inflammatory reaction induced by TNF-a (Moldoveanu et al., 2001), thereby balancing the inflammatory effect. We also observed
S. M. Ulven et al. (B) P
ORIGINAL ARTICLE
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An acute bout of exercise modulate the inflammatory response in peripheral blood mononuclear cells in healthy young men Stine M. Ulven1, Silje Stange Foss2#, Anne Marie Skjølsvik2#, Hans Kristian Stadheim3, Mari CW. Myhrstad1, Ellen Raael1, Marit Sandvik2, Ingunn Narverud1,2, Lene Frost Andersen2, Jørgen Jensen3, and Kirsten B. Holven2 1
Department of Health, Nutrition and Management, Faculty of Health Sciences, Oslo and Akershus University College of Applied Sciences, St Olavsplass, Oslo, Norway, 2Department of Nutrition, Institute for Basic Medical Sciences, University of Oslo, Blindern, Oslo, Norway, and 3 Norwegian School of Sports Science, Oslo, Norway Abstract
Keywords
Context: Exercise increases the levels of circulating inflammatory mediators. Objective: Does an acute bout of exercise affect the mRNA gene expression level of inflammatory markers in peripheral blood mononuclear cells (PBMCs) and contribute to the circulating levels of inflammatory mediators? Materials and methods: Ten healthy, non-smoking men (22–28 years old) performed 1-hour cycling at 70% of VO2 max. Results: The gene transcripts of CXCL16, IL-1b, IL-8, COX-2, TXB21 and GATA3 were significantly up-regulated in PBMCs. Serum levels of CXCL16, IL-6, TNFa and IL-10 were also significantly increased after exercise. Discussion and conclusion: Increased mRNA transcription of inflammatory genes in PBMCs may contribute to increased level of inflammatory markers after an acute bout of exercise. The increased mRNA levels of GATA-3 and TXB21 may indicate that T cell lymphocytes are activated and secrete cytokines into the circulation. It needs to be further investigated if exercise changes the Th1/Th2 balance.
Acute exercise, gene expression, inflammation, peripheral blood mononuclear cells, T helper cell response
Introduction Physical activity is associated with a decreased risk for premature morbidity and regular exercise has been shown to prevent chronic diseases such as cardiovascular diseases (CVD), type 2 diabetes (T2D), certain cancers, obesity, dementia and hypertension (Blair et al., 2001; Rovio et al., 2005; Warburton et al., 2006). Low-grade chronic inflammation is suggested to be a key factor in the pathogenesis of several chronic diseases, and long-term effect of physical activity has been showed to reduce the levels of inflammatory markers (Geffken et al., 2001; King et al., 2003; Mattusch et al., 2000). On the contrary, it is well known that a bout of exercise increases the circulating levels of a number of inflammatory mediators, including interleukin (IL)-6, whereas tumour necrosis factor (TNF)-a is only stimulated by very intense exercise (Pedersen et al., 2007). The increase in IL-6 during exercise is followed by an increase in the antiinflammatory mediators IL-1 receptor antagonist (IL-1ra), soluble TNF-Receptors (sTNFRs) and IL-10 (Petersen & Pedersen, 2005; Pedersen et al., 2007).
#Contributed equally. Correspondence: Stine Marie Ulven, Department of Health, Nutrition and Management, Faculty of Health Sciences, Oslo and Akershus University College of Applied Sciences, PO Box 4, St Olavsplass, 0130 Oslo, Norway. Tel: +47 67236348. E-mail: [email protected]
History Received 25 September 2014 Revised 4 December 2014 Accepted 27 December 2014 Published online 27 February 2015
Cytokines are secreted from many tissues and origins are not always well-defined. Muscle is considered the major contributor to secretion of IL-6 during exercise (Petersen & Pedersen, 2005), but also adipose tissue can produce and secrete IL-6 during exercise (Lyngso et al., 2002). However, the circulating levels of inflammatory mediators do not necessarily reflect the levels in source tissues. Peripheral blood mononuclear cells (PBMCs) consist of monocytes and lymphocytes and have important function in secretion of cytokines and immune responses (Holven et al., 2002, 2003, 2006; Pasterkamp & Daemen, 2008; Visvikis-Siest et al., 2007). Previous studies have shown that PBMCs may contribute to the inflammatory response after exercise (Connolly et al., 2004; Radom-Aizik et al., 2009a, 2009b). Several inflammatory response genes including chemokine (C-C motif) ligand 4 (MIP-1b), CD69 antigen (p60, early T cell activation antigen) and chemokine (C motif) ligand 2 were up-regulated after a 30-minute cycle test (80% peak O2 uptake) (Connolly et al., 2004). A brief bout of exercise has also been shown to alter the expression of inflammatory genes in PBMCs in early- and late-pubertal males and females (Radom-Aizik et al., 2009a). Cytokines produced by T lymphocytes may be involved in the host immunity against infection. T lymphocytes can differentiate into T helper cells type 1 (Th1) and T helper cells type 2 (Th2). Th1 cells are characterized by production of interferon (IFN)-g, TNF-a and IL-2 (Mosmann et al., 1986; Seder & Paul, 1994), while Th2 cells are characterized by production of IL-4, IL-5, IL-6 and
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IL-10 (Mosmann et al., 1986; Seder & Paul, 1994). Th1 and Th2 cell responses are mutually inhibitory, and therefore the Th1/Th2 balance has been used as an indicator of the changes in immune function (Jager & Kuchroo, 2010; Mosmann & Sad, 1996). Recently it was shown that strenuous exercise increases the expression of genes in PBMCs related to Th2 cell function (Xiang et al., 2014), while moderate exercise has been shown to increase expression of genes related to Th1 cell function (Yeh et al., 2009). Whether PBMCs are activated and contribute to the circulating levels of inflammatory mediators after an acute bout of exercise is incompletely understood and remains to be elucidated. It is well known that acute exercise causes acute transient increased level of inflammatory markers in circulation. However, circulating markers may be derived from many metabolic organs, such as skeletal muscle and adipose tissue. It is therefore not clear if the immune cells contribute to increased level of inflammatory markers after an acute bout of exercise. The aim of the present study was therefore to examine the serum levels and PBMC gene expression levels of inflammatory mediators after a standardized and controlled acute bout of exercise.
Methods Subjects Sixteen healthy, non-smoking men were considered for participation in the study. Five were excluded due to the eligibility criteria and one did not conduct the pre-test according to protocol and was therefore excluded from the study. The inclusion criteria were healthy males between 18 and 40 years old. Exclusion criteria included smoking, prevalence of chronic disease such as Type 1 and 2 diabetes mellitus, rheumatism, serious illness during the past five years such as myocardial infarction, stroke and cancer, hypertension, blood lipids outside the reference values, prevalence of milk allergy, lactose- and/or gluten intolerance, the use of anti-inflammatory medications, and not being able to perform the exercise according to the protocol. The study was conducted at the Norwegian School of Sport Science in 2011. The study protocol was approved by The Regional Committee of Medical Ethics and The Norwegian Directorate of Health and the study complies with the Declaration of Helsinki. Informed consent was obtained from all subjects. Exercise intervention The subjects refrained from alcohol consumption and vigorous physical activity the day prior to the exercise test days. At each test day, the subjects visited the study centre and body composition, blood pressure and venous blood samples were taken after an overnight fast (12 h). A standardized breakfast composed of two slices (80 g) of oatmeal bread, 10 g margarine (40% fat), four slices (30 g) of cheese (27% fat), 28 g red peppers, 40 g cucumber and 0.5 L of apple-grape juice was served between 08.30 am and 8.45 am, 1 h before the cycle test started. The total energy and macronutrient composition of the meal is shown in Table 1. After breakfast, the participants waited for 1 h before they started the exercise test at the ergometer cycle (Monark 839E, Varberg, Sweden).
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Table 1. Total energy and macronutrient composition of the breakfast. Energy, KJ Protein, E% Total fat, E% Saturated fat, E% Trans fat, E% Mono-unsaturated fat, E% Poly-unsaturated fat, E% Carbohydate, E% Fibre, gram
1886 16 28 15 1 8 3 56 6,7
The participants cycled for 1 hour at 70% of VO2 max. During the test, measurements such as lactate, heart rate (HR), VO2 and Borg’s scale was conducted after 4 min, 15 min, 30 min, 45 min and 60 min. The exercise test day was repeated twice, separated by 1 week. Prior to the two test days, the participants had to meet at the Norwegian School of Sport Science twice in the same week to determine their VO2 max and to perform a pre-test (separated by 1 week from the first test day). In total, the duration of the study was 3 weeks. In week 1, the participants visited the study centre to determine their VO2 max and to perform the pre-test. In week 2 they performed the first exercise test and in week 3 they performed the second cycle test. VO2 max determination and pre-test Prior to the VO2 max test, a standardized warm-up was performed by each test subject. The standardized warm-up was performed as a continuous incremental test where the workload was increased each fifth minute with 25 watts. During each workload oxygen consumption was measured on average every 3 or 4 min using an Oxycon Pro (Jaeger-Toennis Instruments, Hochberg, Germany), and air was collected using a mouth V2-mask (Hans Rudolph Instruments, USA) in combination with a nose bracket. The relationship between VO2 and Watt from the warm-up was later used to make a linear regression for estimating the workload equivalent of 70% of VO2 max. After each 5-min workload a capillary sample from the fingertip (Accu-Check, Safe-T-Pro Plus, Roche, Germany) was taken for measuring lactate using an electro-enzymatic lactate analyser (1500 Sport, YSI Inc., USA). The incremental test was stopped when capillary lactate had increased 1.5 mmol above the concentration at the first workload (75 watts). Between the standardized warm-up and the VO2 max test subjects were given a 5-min break where they cycled at a workload of 50 watts. The VO2 max test was designed with an increase in intensity of 25 watts every 30 sec. Subjects started the VO2 max test with a workload, which was 25 watts higher than they ended the standardized warm-up. All subjects had to meet point one and at least two of the three other criteria so that they reached VO2 max: (1) Oxygen consumption reached a plateau, meaning VO2 increased less than 1 mL kg1 min1, while the workload was increased twice with 25 watts. (2) Respiratory exchange ratio values were above 1.10. (3) Post blood lactate measurements were above 7.0 mmol/L.
Exercise and inflammation
DOI: 10.3109/13813455.2014.1003566
(4) Rated perceived exertion was 19 on the Borg Scale 6-20. VO2 max-run was based on average of the two highest measurements. Measurements of oxygen consumption were taken every 30 sec. After the VO2 max test, a pre-test at 70% of VO2 max for 40 min was performed to verify that the calculated workload was correct and to ensure that the subjects could accomplish the exercise intervention.
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Measurements of body composition and food intake Anthropometrical measures like weight and height were determined by use of standard calibrated scales. Waist circumference was measured at the level midway between the lowest rib margin and the iliac crest. The reading of waist circumference in centimetres was done when the tape measure was snug, but not causing compression on the subjects’ skin. Hip circumference was measured over the widest part of the hip. Body composition was measured by BIA on a Tanita Scale (BC-418MA, Tanita Corporation, Japan). The participants were scanned using the athlete function on the scale and 0.5 kg was subtracted for compensation for light clothing. Food intake was registered once using validated food diary for four consecutive days of which one was a weekend day. The subjects received the food diaries at the pre-test day (the week before the first test day) and they received instructions from a clinical nutritionist on how to register food intake the week before the first test day. In addition, the subjects were told not to change their food intake during the study period. Blood sampling Venous blood samples were drawn after an overnight fast (12 hours) (at 8.00 am) and immediately after the cycle test was finished (at 10.45 am). Serum was obtained from silica gel tubes (Becton Dickinson (BD) vacutainer) and kept at room temperature for at least 30 min, until centrifugation (1500 g, 12 min), and immediately prepared for subsequent analysis of routine laboratory analyses or aliquoted and stored at 80 C until further analyses. Plasma was obtained from EDTA tubes (BD vacutainer), immediately placed on ice and centrifuged within 10 min (1500 g, 4 C, 10 min) and aliquoted and stored at 80 C until further analysis. Routine laboratory analysis Fasting serum hsCRP, total cholesterol, low density lipoprotein (LDL) cholesterol, high density lipoprotein (HDL) cholesterol, triacylglycerides (TAG), glucose and insulin were measured by standard methods at a routine laboratory (Fu¨rst Medical Laboratory, Norway).
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approximately 80% of the cells are lymphocytes and 12% are monocytes (http://www.bd.com/vacutainer/pdfs/bd_cpt_ VDP40105.pdf). Pellets were frozen and stored at 80 C prior to RNA isolation. Total RNA was isolated from all PBMC samples using RNeasy mini kit (Qiagen, Hilden, Germany) with lysis buffer added b-mercaptoethanol according to the manufacturer’s instructions, and stored at 80 C. RNA quantity and quality measurements were performed using the ND 1000 Spectrophotometer (Saveen Werner, Malmo¨, Sweden) and Agilent Bioanalyser (Agilent Technologies, Santa Clara, CA, USA), respectively. All samples had RNA integrity number 47; 200 ng RNA was reverse transcribed by high-capacity RNA-to-cDNA kit (Applied Biosystems, Foster City, CA, USA). Custom TaqMan Array micro Fluidic cards (Applied Biosystems) were used for RT-qPCR amplification of the target genes. TATA binding protein (TBP) was selected as endogenous control based on running a TaqMan Human Endogenous Control Plate-test (data not shown) (TelleHansen et al., 2012). A list of the inflammatory genes analysed in the present study are given in Supplementary Table 1. Primer sequences can be provided by request. A total of hundred ml of cDNA (1:10 fold dilution), RNase-free water and TaqMan gene expression master mix were applied to the micro Fluidic card and centrifuged according to the manufacturer’s protocol. The DCt values were calculated as Ct values of each target gene minus the Ct values of the endogenous control, TBP (housekeeping). Enzyme immunoassay (EIA) Serum, soluble TNF receptor (sTNFR)I, sTNFRII, TNF-a, IL-10, IL-6, CXCL16, soluble intracellular adhesion molecule 1 (sICAM-1) and soluble vascular cellular adhesion molecule1 (sVCAM-1) were measured by EIA from R&D Systems (Minneapolis, MN) according the manufacturer’s instructions. Statistics The present study was designed as a pilot study and no adjustment for multiple testing and power calculations were performed. The significance before and after exercise was assessed by Wilcoxon signed rank test (using DCt when comparing mRNA levels). All values are presented as median (min-max). Probability values (asymptotic) were considered statistically significant at a value of 0.05. All statistical analyses were performed using the Statistical Package for the Social Sciences (SPSS) version 19 for Microsoft (SPSS, Inc., USA).
Results PBMC and RNA isolation and reverse transcription real-time quantitative polymerase chain reaction (RT-qPCR) After blood collection, PBMC were isolated using the BD Vacutainer Cell Preparation tubes according to the manufacturer’s instructions (Becton, Dickinson and Company, NJ 07417, USA) within 2 h. This is a well-documented and standardized method to collect mononuclear cells with high purity (above 90%) and, according to the manufacturer,
Characterization of the 10 male participants is shown in Table 2. The median age for the group was 25 years (22–28, min-max). The median value for the VO2 max (62 mL kg1 min1) was above the reference value compared to an average population of men between 20 and 29 years old indicating that the participants had a very good physical fitness. This is also shown by the low median value of fat mass, 7.3 kg (3.9–14.0 kg, min-max) and of the fat percent, 9.6% (4.8–16.6%, min-max) compared with reference values
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(Table 2). The participants also had a high median value of fat free mass, 70 kg (65–77 kg, min-max), compared to median weight, 79 kg (69–88 kg, min-max) (Table 2). Food intake of the participant is shown in Table 3. The intake of fat and protein was within, while the intake of carbohydrates was below, the new Nordic dietary recommendations.
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Table 2. Baseline characteristics. (n ¼ 10) Age (y) Weight (kg) Height (cm) BMI (kg/m2) Waist:hip ratio Heart rate (beat/min) Systolic BP (mm/Hg) Diastolic BP (mm/Hg) VO2 max (ml/kg/min) Fat percent (%) Fat free mass (kg) Fat mass (kg) Total cholesterol (mmol/L) Low density lipoprotein cholesterol (mmol/L) High density lipoprotein cholesterol (mmol/L) Triglycerides (mmol/L) Glucose (mmol/L) HbA1c (%) Insulin (pmol/L) hsCRP (mg/L)
25 79 184 23.3 0.84 46 131 71 62 9.6 70 7.3 3.7 2.4
Reference value
(22–28) (69–88) (180–194) (21.3–25.3) (0.79–0.89) (42–54) (120–144) (61–96) (53.6–70.6) (4.8–16.6) (65–77) (3.9–14.0) (3.4–4.9) (1.7–3.4)
1.4 (1.1–1.7) 0.73 4.9 5.4 47 0.4
51.0 5130 585 48.5 10.0–20.0 8.9–17.9 2.9–6.1 1.2–4.3
52.60 4.0–4.6 56.1 18–173 55.0
Data are given as an average of two test days and are given as median (min-max). BP, blood pressure; BMI, body mass index; n indicates number of individuals.
Table 3. Energy and macronutrient intake. (n ¼ 10) Energy (KJ) Protein (E%) Fat (E%) Carbohydrates (E%) Added sugar Alcohol (E%) Fibre (g/d)
13 450 16.7 36.2 42.0 9.9 2.3 33.7
Reference value*
(10 791–16 538) (15.4–17.9) (30.2–37.3) (37.9–46.3) (6.4–14.9) (0.0–5.5) (21.9–37.8)
The characterization of the exercise intervention is presented in Table 4. The workload at both test days was set to the load calculated to correspond to 70% of VO2 max (44.97 ml/kg/min). Median oxygen uptake at the tests was 45.0 ml/kg/min (43.5–46.4 ml/kg/min min-max). The level of lactate increased during the first 15 min, from 0.98 mmol/L (0.87– 1.09 mmol/l) at baseline to 2.97 mmol/L, but then decreased again throughout the exercise. Heart rate and Borg scale significantly increased during exercise. No significant changes were found in the level of plasma glucose, hsCRP and insulin from pre- to post-exercise (data not shown). The effect of exercise on circulating inflammatory markers
0.8–2.1
(0.58–1.26) (4.7–5.4) (5.0–5.6) (30–69) (0.05–2.1)
Characterization of the exercise
13 800a 10–20 25–40 45–60 510 25–35
Data are mean of 4 days food records; n is number of individuals. Data is given as median (IQR). *Norwegian reference values. a Reference value of energy intake of male 18–30 years in Norway with a PAL of 1.8.
The exercise-induced response on the circulating levels of inflammatory markers is shown in Figure 1. The serum levels of sTNFRI, IL-6, IL-10, TNFa, CXCL16, sICAM-1 and sVCAM-1 significantly increased by physical activity (p 0.05 for all), whereas the level of sTNFRII was unchanged by physical activity. The effect of exercise on PBMC gene expression of inflammatory mediators To further elucidate if PBMC gene expression of inflammatory mediators are influenced by exercise we analysed the mRNA level of 18 inflammatory markers. The mRNA levels of IL-1b, CXCL16, IL-8, cyclo-oxygenase (COX)-2, T-box 21 (TBX21) and trans-acting T-cell-specific transcription factor (GATA-3) (p50.05 for all) were significantly upregulated by exercise (Figures 2 and 3). The toll-like receptor (TLR)-2 mRNA was significantly down-regulated after exercise (p50.05) (Figure 3). In addition, the gene transcripts of TNF-a, CD40, CD40L, transforming growth factor (TGF)-b, IFN-g, IL-18, toll-like receptor (TLR)-4, toll-like receptor (TLR-6), CD3e molecule epsilon (CD3E), cluster of differentiation 8a (CD8A) and forkhead box P3 (FOXP3) were all unchanged by exercise (Figure 2 and 3).
Discussion In the present study, we observed a significant increase in serum levels of CXCL16, sTNFRI, IL-6, IL-10, TNF-a, sICAM-1 and sVCAM-1 in healthy, athletic males after an acute bout of exercise (cycling 1 h at 70% of VO2 max). At the same time, the mRNA level of CXCL16, IL-1b, IL-8, COX-2, GATA3 and TXB21 were significantly up-regulated by exercise, while TLR-2 mRNA was significantly downregulated.
Table 4. Characteristics of exercise intervention. 4 min (n ¼ 10) VO2 (L/min) HR (beat/min) Lactate (mmol/L) Borg scale
42.14 133 2.71 13.4
(39.52–45.55) (126–147) (2.13–3.22) (7.9–14.5)
15 min (n ¼ 10) 43.30 154 2.97 14.1
(41.29–47.80)* (138–168)* (2.34–3.74)* (12.3–15.6)*
30 min (n ¼ 10) 44.18 157 2.91 15.0
(41.54–47.97)* (137–170)* (2.26–3.79) (13.0–16.5)*
45 min (n ¼ 10) 44.49 161 2.62 15.6
(41.77–48.33)* (139–172)* (2.17–4.28)** (14.0–16.5)*
n Indicates number of individuals. Data are given as an average of two test days and given as median (min-max). Significant different from 4 min; *p50.01; **p50.05. Wilcoxon signed Rank test.
60 min (n ¼ 10) 44.97 163 2.40 15.4
(43.40–51.30)* (139–172)* (1.85–3.60) (14.0–17.0)*
Exercise and inflammation
DOI: 10.3109/13813455.2014.1003566
P=0.01 10
(B) 2.0
8
1.5
IL-10 [pg/ml]
(A) IL-6 [pg/ml]
Figure 1. The effect of an acute bout of exercise on serum IL-6 (A), IL-10 (B), TNFa (C), CXCL16 (D), sTNFR1 (E), sTNFR2 (F), sICAM-1 (G) and sVCAM-1 (H). Data are calculated as an average of two test days and is given as mean (SEM). Wilcoxon signed rank test was used to calculate the difference before and after exercise.
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P=0.02 (G) 150 sICAM-1 [ng/ml]
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It is well known that physical activity increases the circulating level of IL-6 (Bernecker et al., 2013; Langberg et al., 2002; Nybo et al., 2002; Moldoveanu et al., 2000) and the increase in serum IL-6 has been suggested to be mainly caused by increased secretion from skeletal muscle (Steensberg et al., 2002). In the present study, we observed a two-fold increase in serum level of IL-6, which is lower compared with most previous studies (Connolly et al., 2004; Nieman et al., 2001; Ostrowski et al., 1998; Ostrowski et al., 1999; Rohde et al., 1997). One explanation could be that the subjects in the present study were served a breakfast containing 59 g of carbohydrates (56% of total energy intake) prior to the exercise test. Carbohydrate loading before exercise has been shown to diminish the exercise induced increase in circulating levels of IL-6 (NehlsenCannarella et al., 1997). It has also recently been shown that plasma levels of IL-6 and TNF-a were reduced after consumption of a carbohydrate rich meal (79% of total energy intake) (Meher et al., 2014). In contrast, a post-prandial
Physical exercise
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Physical exercise
increase in IL-6 has been observed after intake of high carbohydrate (76% of total energy intake) (Gregersen et al., 2012). A possible pro-inflammatory post-prandial effect, however, caused by the served meal in the present study may be less likely due to the lower level of IL-6 after exercise compared with other studies. We also observed an increase in circulating level of TNF-a and our finding is in agreement with others who have reported small, but significant increase in the circulating level of TNFa after exercise (Dufaux & Order, 1989; Ostrowski et al., 1998; Steensberg et al., 2002). This increase is in contrast to others who have reported no significant change in the circulating leval of TNF-a after exercise (Drenth et al., 1995; Rohde et al., 1997; Steensberg et al., 2002). The fact that we also observed a significant increase in sTNFR1 strengthens our results that the TNF pathway is induced by exercise and sTNFR1 may attenuate the potent inflammatory reaction induced by TNF-a (Moldoveanu et al., 2001), thereby balancing the inflammatory effect. We also observed
S. M. Ulven et al. (B) P
