Impact of aerobic water running training on peripheral immune-endocrine markers of overweight-obese women

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SCISPO-3007; No. of Pages 8 Science & Sports (2016) xxx, xxx—xxx

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ORIGINAL ARTICLE

Impact of aerobic water running training on peripheral immune-endocrine markers of overweight-obese women Impact de la formation en cours d’eau sur les paramètres immunitaires endocriniens des femmes en surpoids obèses A. Colato a, L. Fraga a, G. Dorneles b, P. Vianna c, J.A.B. Chies c, A. Peres a,b,∗ a

Research center, Methodist university center IPA, Porto Alegre, Brazil Laboratory of cellular and molecular immunology, Federal university of health sciences of Porto Alegre, Porto Alegre, Brazil c Laboratory of immunogenetics, Federal university of Rio Grande do Sul, Porto Alegre, Brazil b

Received 25 March 2016; accepted 12 April 2016

KEYWORDS Overweight-obesity; Women; Exercise; Immune; T cells

Summary Objective. — The purpose of this study was to evaluate the effect of water running training on immune-endocrine parameters evaluated in peripheral blood of overweight-obese women. Methods. — Eleven sedentary overweight-obese women (age 48.81 ± 12.87 years; body mass index 34.56 ± 4.08 kg/m2 ) participated of 12-week of aerobic water running training in a heated swimming pool (70 minutes/session; 3×/week). In addition, nine women matched to age and body mass index were recruited to sedentary control group (age 49.9 ± 10.5 years; body mass index 33.05 ± 3.45 kg/m2 ). Serum levels of interleukin-6 (IL-6), interleukin-10 (IL10), interleukin-17a (IL-17a), interferon-gamma (INF-␥), tumor necrosis factor-alpha (TNF-␣), salivary cortisol, and the peripheral frequency of CD3+CD4+ and CD3+CD8+ T cells were evaluated before and after 12 weeks in exercised individuals and after 12 weeks in control subjects. In addition, anthropometric measurements (body mass, % of body fat and trunk circumferences) and functional capacity (VO2Peak and time to exhaustion) were also evaluated. Results. — After 12 weeks of exercise training, serum adiponectin (P = 0.030), IL-10 (P = 0.01) and TNF-␣ (P = 0.004), and CD8+ T cells frequency (P = 0.03) increased significantly, while salivary cortisol reduced (P = 0.003) in exercised women. After 12 weeks of training, exercised

∗

Corresponding author. Laboratório de Imunologia Celular e Molecular, Universidade Federal de Ciências da Saúde de Porto Alegre, Rua Sarmento Leite, 245, sala 206, 90050-170 Porto Alegre, RS, Brazil. E-mail address: [email protected] (A. Peres). http://dx.doi.org/10.1016/j.scispo.2016.04.003 0765-1597/© 2016 Elsevier Masson SAS. All rights reserved.

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A. Colato et al. individuals had higher TNF-␣ levels (P = 0.02), CD4+ (P = 0.03) and CD8+ (P = 0.04) T cells than control group. In addition, exercised women reduced the waist circumference (P = 0.030) and improved the time to exhaustion (P = 0.003). Conclusion. — In conclusion, deep water running was able to modulate immune-endocrine parameters in absence of significant improvements in body composition or VO2Peak in overweightobesity women. This training could be an interesting tool to control the obesity-related chronic low-grade inflammation. © 2016 Elsevier Masson SAS. All rights reserved.

MOTS CLÉS Surpoids-obésité ; Les femmes ; L’exercice ; Immunitaire ; Les cellules T

Résumé Objectif. — L’objectif de cette étude était d’évaluer l’effet de la formation d’eau courante sur les paramètres immunitaires et endocriniens du sang périphérique chez les femmes en surpoids obèses. Méthodes. — Onze femmes en surpoids obèses sédentaires (âge 48,81 ± 12,87 années ; indice de masse corporelle 34,56 ± 4,08 kg/m2 ) ont participé de 12 semaines de formation en cours d’exécution de l’eau dans la piscine chauffée (70 minutes/session ; 3×/semaine). En outre, neuf femmes appariés aux l’âge et l’indice de masse corporelle ont été recrutés pour groupe témoin sédentaire (âge 49,9 ± 10,5 ans ; indice de masse corporelle 33,05 ± 3,45 kg/m2 ). Les niveaux de l’interleukine-6 (IL-6), l’interleukine-10 (IL-10), interleukine-17a (IL-17 a), l’interférongamma (IFN-␥), facteur de nécrose tumorale alpha (TNF-␣), cortisol salivaire et la fréquence périphérique de CD3+CD4+ et CD3+ cellules T CD8+ ont été évaluées avant et au bout de 12 semaines chez des personnes exercées et au bout de 12 semaines chez les sujets témoins. En outre, les mesures anthropométriques (masse corporelle, pourcentage de graisse corporelle et circonférences du tronc) et la capacité fonctionnelle (VO2 Peak et le temps de l’épuisement) ont également été évalués. Résultats. — Après 12 semaines de formation d’exercice, l’adiponectine sérique (p = 0,030), IL10 (p = 0,01) et le TNF-␣ (p = 0,004), et les lymphocytes T CD8+ (p = 0,03) ont augmenté de manière significative, alors que le cortisol salivaire réduit (p = 0,003) chez les femmes exercées. Après 12 semaines de formation, les individus avaient exercé des niveaux de TNF-␣ plus élevés (p = 0,02), CD4+ (p = 0,03) et CD8+ (p = 0,04) des lymphocytes T que le groupe témoin. En outre, les femmes exercées réduit la circonférence de la taille (p = 0,030) et d’améliorer le temps de l’épuisement (p = 0,003). Conclusion. — En conclusion, l’eau courante profonde était capable de moduler les paramètres immunitaires endocriniens en l’absence d’améliorations significatives dans la composition corporelle ou de la consommation d’oxygène. Cette formation pourrait être un outil intéressant pour contrôler l’inflammation de bas grade chronique liée à l’obésité. © 2016 Elsevier Masson SAS. Tous droits r´ eserv´ es.

1. Introduction Chronic low-grade inflammation, an increase in order to 2—3 fold of pro-inflammatory mediators such as cytokines, is implicated in the physiopathology of obesity and associated diseases, like type 2 diabetes and atherosclerosis [1]. While the exact molecular mechanisms of inflammation is controversy, metabolic disruption due to weight gain have been shown to affect adaptive immunity and neuroendocrine systems [2,3]. Impairment in T cell function, reduced peripheral frequency of cytotoxic T cell (CD8+) and an increase in Th1 T helper phenotype in detriment of Th2 phenotype are some changes visualized in obesity [3]. Due to these alterations, obesity has been identified as independent risk factor for cancer, cardiovascular diseases and virus infection [4]. In the recent H1N1 influenza epidemic, obesity was associated with increased hospitalization and infection severity [5]. Obesity also presents elevated circulating cortisol levels due to two mechanisms:

• dysfunction in hypothalamic-pituitary-adrenal axis with block of negative feedback; • high glucosteroids synthesis by adipocytes [6]. Collectively, these findings suggest that obesity may cause impaired immune-neuroendocrine responsiveness. Systematic physical training is able to improve a series of metabolic health and decreases the risk of numerous diseases in overweight-obese individuals [7]. In the past years, studies suggested that exercise have immunoregulatory effects. Some of these effects are likely attributable to reduction of adipose tissue, since that adipocytes are the main sites of release of inflammatory cytokines in obesity [8,9]. For the other hand, recent evidences shows that exercise, in absence of weight loss, can influence phenotype of immune cells and inflammatory mediators [10]. However, few studies have focused on peripheral T cells or immune-endocrine communication [11—13]. Recently, we demonstrated that 12 weeks of concurrent training was able

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Water training and immunity in obesity to increase T helper lymphocyte (CD4+) and T cytotoxic lymphocyte (CD8+) concomitant with a decrease in frequency of HLA-DR+ on lymphocytes and monocytes of overweightobese individuals [11]. It is important to salient that traditional aerobic exercises are often associated with increased risk of injuries due to mechanical stress in lower-joint extremities [14]. Because of this, the American College Sports Medicine (ACSM) has recommended aquatic aerobic exercise for obese individuals to reduce the injury risk and increase adherence to exercise training [15,16]. The practice of water-based exercise may be an interesting strategy of exercise training for obese individuals, improving body composition [15,17] and the functional capacity [15,18] besides reducing injury risks. In this exercise method, the subject uses a floater attached to the trunk allowing the running movements without contacting the pool floor [16]. Overweight-obese women present higher proinflammatory status compared to matched-paired men [19] and increased susceptibility to the development of obesity-related diseases [2,5]. While previous studies demonstrated that water-based training could improve the functional capacity and quality of life in overweight-obese individuals [15,17,18], no previous studies focused in immune system of overweight-obese women. Thus, the aim of this study was to evaluate the impact of water running training on the systemic inflammatory markers, T cells subsets and salivary cortisol levels in overweight-obese women.

2. Methods 2.1. Subjects Eleven overweight and obese women (height 1.63 ± 0.06 meters; body mass 94.65 ± 13.29 kg; BMI 35.62 ± 2.34 kg/m2 ) were enrolled for exercise group. We recruited ten self-reported sedentary women with a maximum of 150 minutes of physical activity (analyzed by questionnaires) in the previous six months for the control group. All participants had a BMI greater than 25 kg/m2 but were otherwise healthy and they were not engaged in structured physical training in a period of 6 months previous to study. G Power (version 3.1.3; Franz Faul, Kiel, Germany) was used to calculate the sample size of the study and indicated that a minimum of 10 exercised participants would be enough to detect differences in IL-10 (a primary outcome variable) with a power of 75% and 5% significance level. Exclusion criteria included metabolic syndrome, diabetes, uncontrolled hypertension (blood pressure > 130/90), and history of cardiovascular, endocrine or autoimmune diseases, musculoskeletal injury. Also, participants that followed any physical exercise within the last 6 months prior to this study as well as those with prior major illness or intake of medications or supplements were also excluded. This study was approved by the Ethics Research Committee of Methodist University Center IPA (protocol number 278/2011) and all participants read and signed the informed consent. The participants were recruited from posters and lectures about the study. Initially, a total of 30 participants

3 were screened and 11 were eligible and enrolled to the physical training.

2.2. Study design After inclusion in the study, all eligible participants (11 women, aged 48.77 ± 12.87 years) were engaged to supervised deep water running training (DWR) at Methodist University Center IPA (Porto Alegre, Brazil). Before the beginning of the training period and 48 hours after the last training session, subjects were evaluated through the cardiorespiratory test, anthropometric measurements, and blood analysis. In addition, sedentary matched-control group were evaluated only after 12 weeks corresponding to the intervention period of the exercised group. The subjects were instructed to avoid physical exertion, consumption of painkillers or anti-inflammatory drugs, alcohol or caffeine and maintain a normal diet during the 24 hours pretest.

2.3. Body composition Body weight and height were determined by a semianalytical scale (Welmy, Santa Barbara do Oeste, São Paulo, Brazil) with a capacity for 200 kg and an attached stadiometer, with accuracies of 0.1 kg and 0.005 cm, respectively. Body composition was assessed by the bioelectrical impedance method (Byodinamics, BIA 310e, Seattle, Washington, USA), following all guidelines pretest, and considering the values of mass percentage fat and free fat mass (kg). Waist (WC, cm), abdomen (AC, cm) and hip (HC, cm) circumferences were measured through inelastic measuring tape. The BMI of each participant was calculated by dividing the weight (kg) by the height squared (m). All assessments were performed by a trained exercise physiologist.

2.4. Peak oxygen consumption Peak oxygen consumption (VO2Peak ) was assessed through an incremental test until exhaustion on a motorized treadmill (Inbramed Millennium ATL, Porto Alegre, Brazil) as previously reported [11]. Expired gases were collected using an ergoespirometer (V02000, Medgraphics, St Paul, MN, USA). Heart rate (HR) was monitored during the test using a frequency counter (POLAR FT7, Tempere, Finland) and blood pressure was checked with a sphygmomanometer (Solidor, Hebei Medicinis and Health Prod., China). Throughout the test, the participants used a mask (Medgraphics, St. Paul, Minnesota, USA) connected to a gas analyzer, which allowed the collection and storage of data for each breath (breath by breath) using Aerograph (St. Paul, Minnesota, USA) software. The test was conducted according to the modified Bruce protocol [20]. After each stage the participant reported their perception of exertion based on the Borg scale so that the intensity of the stage could be reproduced during the period of physical training [21]. The test was interrupted in the presence of any of the following events: • HR with values above 10 bpm of the maximum predicted for age; • signs and symptoms of angina;

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• decrease or absence of significant increase in systolic blood pressure with increased intensity of training; • respiratory quotient above 1.1; • voluntary exhaustion. The VO2Peak was defined as the highest value obtained after the treatment of data by three researchers independent to the study. In addition, the time to exhaustion was recorded as a variable of endurance performance. Verbal encouragement was provided to ensure that the maximum values were reached.

2.5. Water running training Subjects completed three sessions of DWR per week for 12 weeks, with 1—2 days rest between each session. Each session had the duration of 70 minutes, including initial warm up, training (60 minutes) and final stretching. We used the perceived exertion as an intensity indicator of aerobic exercise; the same was recorded every 5 minutes through an analog scale. The DWR was conducted in a heated swimming pool (dimensions: 25 × 20 m and depth of 170 cm), temperature close to 28 ◦ C. All individuals used a flotation vest. In the first three weeks, the intensity of exercise session was 12 from Borg scale. In addition, the intensity of exercise was progressively increased every three weeks to stage ‘‘15’’ of Borg scale in the final three weeks of training.

2.6. Blood samples 15 mL of peripheral blood by venipuncture, and 2 mL of saliva samples after an overnight fast of 12 hours (between 7:00 AM and 10:00 AM) were collected. The blood samples were divided into tubes without anticoagulant to obtain the serum and into heparinized tubes for whole blood (BD Vacutainer heparin tubes). Serum samples were separated by centrifugation for 10 minutes at 1048 g, divided into aliquots, and frozen at —20 ◦ C for further analysis. The heparinized whole blood was analyzed within 3 hours of collection. Saliva samples were collected into specific tubes (Salivettes® , Sarstedt, Germany) and micro-centrifuged (500 g) to remove cells and insoluble matter before storing at —20 ◦ C. Forty-eight hours after the last concurrent training session, blood samples were collected again.

2.7. Enzyme-linked immunosorbent assay (ELISA) Commercially available ELISA kits were used to determine serum IL-6, TNF-␣, IL-10, INF-␥, and IL-17a (Mini ELISA Development Kit, 900-M21, PeproTech Inc, New Jersey, USA), adiponectin (MBL Adiponectin ELISA kit, USA), ghrelin (Millipore Corporation, USA) and salivary cortisol (DBC Labs, Montreal, Canada). The intra-assay coefficient variation was < 7.5% for all cytokines.

2.8. Flow cytometry Immunophenotyping was carried out using a direct immunofluorescence technique. Monoclonal antibodies specific for CD3-PE, CD4-FITC, CD8-FITC, as well as the

appropriate isotype controls, were purchased from Becton Dickinson (San Jose, CA, USA). We used a two-color staining method using monoclonal antibodies labeled with fluorescein isothiocyanate and antibodies labeled with phycoerythrin added to 12 × 75 mm test tubes. One hundred microliters of heparinized whole blood were added to 5 ␮L of appropriate antibodies in each test tube. The mixture was incubated in the dark at 4 ◦ C for 20 minutes. Phosphatebuffered saline (PBS 1×) (2 mL) was added to the mixture, which was then centrifuged for 5 minutes 167.7 g and the supernatant discarded. Red blood cells were lysed by adding 1 mL of lysing buffer (Cell-Lyse, Becton Dickinson, San Jose, CA, USA) for 15 minutes in the dark at room temperature. PBS (2 mL) was added to the samples, centrifuged for 5 minutes 167.7 g and the supernatant discarded. The pellets were resuspended in 1 mL of PBS/formaldehyde 1% and stored at 4 ◦ C in the dark until analysis. Flow cytometry was performed using a FACSCalibur instrument and CellQuest software (Becton Dickinson, San Jose, CA, USA). Compensation parameters for multiparametric studies were adjusted at the start of analysis and 10,000 events per sample were acquired with a live gate applied to the lymphocytes gate.

2.9. Statistical analysis All variables were tested for normality of distribution by the Shapiro Wilk test. For those that showed normality, we used the paired t-test for comparison of baseline and post-training (mean ± standard deviation). For the variables that deviated from normality, we used the nonparametric Wilcoxon test (median and interquartile range). Comparison with sedentary control group was performed through Independent Student t-test or Man-Whitney test. All analyses were performed by SPSS version 20.0 (SPSS Inc., Chicago, IL, USA). All statistical tests were two-tailed and were performed using a significance level of a P < 0.05.

3. Results As a result of the DWR there was a significant reduction in waist circumference (P = 0.030) and a tendency to decrease in percentage of fat mass (P = 0.061). No significant differences were founded in body mass, BMI, AC, HC or metabolic basal rate after the training period (P > 0.05) (Table 1). No statistical differences were observed in comparison with sedentary control group in neither of variables analyzed (P > 0.05). The participants improved the time to exhaustion on cardiopulmonary test (12.61 ± 1.39 to 13.97 ± 1.65 minutes; P = 0.003) but no statistical differences were found in comparison to control group. No significant difference was found in relative VO2Peak (P > 0.05) (Fig. 1). Table 2 shows the cytokine, adiponectin, ghrelin and salivary cortisol levels before and after DWR. After training, serum adiponectin (P = 0.030), IL-10 (P = 0.003), TNF-␣ (P = 0.010) significantly increased by approximately 4.95%, 17.6% and 47% respectively, with higher TNF-␣ levels after training compared to control group (P = 0.023). In addition, salivary cortisol levels reduced after 12 weeks of DWR Training (9.13%; P = 0.003), although there were no significant changes in IL-6, IL-17a, INF-␥ or ghrelin (P > 0.05).

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Water training and immunity in obesity Table 1

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Changes in body composition and oxygen consumption pre- and post-deep water running training. Exercise group

Control group

Before Body mass (kg) BMI (kg/m2 ) Fat mass (%) Waist circumference (cm) Abdominal circumference (cm) Hip circumference (cm) Metabolic Basal Rate (kcal)

92.46 34.76 39.23 100.65 109.88 117.26 1690.23

After ± ± ± ± ± ± ±

14.87 4.19 4.12 6.67 10.94 9.22 268.40

90.70 34.20 37.75 96.58 108.30 115.69 1714.00

± ± ± ± ± ± ±

15.20 4.79 5.29 4.83a 9.50 8.48 304.39

92.40 33.05 39.20 94.72 104.77 113.16 1618.88

± ± ± ± ± ± ±

5.35 3.45 1.75 5.41 6.24 7.75 117.81

Values are mean (SD). BMI: body mass index. a Significantly different compared with pretraining (P < 0.05).

Table 2

Changes in adiponectin, cytokines and salivary cortisol after12 weeks of deep water running training. Exercise group

Adiponectin (pg/mL) IL-6 (pg/mL) IL-10 (pg/mL) IL-17a (pg/mL) INF-␥ (pg/mL) TNF-␣ (pg/mL) Ghrelin (pg/mL) Salivary cortisol (pg/mL)

Control group

Before

After

35.53 ± 3.41 270.00 (145.00—410.00) 200.00 (140.00—251.00) 30.00 (20.00—60.00) 260.00 (100.00—370.00) 450.00 (230.00—590.00) 49.25 (19.25—54.25) 63.43 ± 15.41

37.29 ± 2.90a 240.00 (60.00—690.00) 380.00 (290.00—890.00)a 50.00 (40.00—70.00) 350.00 (200.00—490.00) 1010.00 (560.00—1680.00)a,b 34.25 (31.75—109.25) 56.97 ± 15.38a

34.40 ± 5.45 178.20 (141.80—893.30) 345.30 (189.40—420.30) 51.90 (31.25—87.45) 437.15 (212.47—555.37) 526.10 (313.10—607.10) 41.75 (23.00—51.75) 60.54 ± 9.87

Values are mean (SD). IL: interleukin; INF: interferon; TNF: tumor necrosis factor. a Significantly different compared with pretraining (P < 0.05). b Significantly different compare to control group (P < 0.05).

Regarding the peripheral frequency of T Lymphocytes before and after 12 weeks of DWR training, we observed a significantly increase of CD3+CD8+ T cells (Fig. 2b; P = 0.03), with no differences in CD3+CD4+ T cells (Fig. 2a; P = 0.06). CD8+ (P = 0.045) and CD4+ (P = 0.038) T cells were higher after DWR Training compared to Control Group (Fig. 2).

4. Discussion It is well known that a myriad of exercise regimens may improve the low-grade inflammatory process in obesity and associated diseases, including type 2 diabetes and cardiovascular diseases [10]. The results of our study indicate that, in overweight-obese women, a 12-week deep water running training regimen can increase circulating adiponectin, TNF-␣ and IL-10 levels, reduce salivary cortisol levels as well as induce a significant increase in CD8+ T cells. In addition to these immune-endocrine changes, the physical training was able to induce a reduction in waist circumference and increase time to exhaustion in cardiopulmonary test. The influence of obesity on CD4+ T cells was inconclusive, with some reports demonstrating higher peripheral frequency [3,4], and others reported that CD4+ T cells was lower in obese individuals [22]. Regarding to CD8+ T cells, the consensus is that circulating cytotoxic lymphocytes

are reduced in obesity [22,23]. The decreased peripheral frequency of CD8+ T cells is linked to a greater infiltration of CD8+ on adipose tissue in a co-stimulatory mechanism with pro-inflammatory M1 macrophages [24]. In this way, our study showed that 12 weeks of DWR training was able to increase CD4+ and CD8+ T cells in order of 12.80% and 27.18% in overweight-obese women, respectively. These changes may be related to the fact that moderate exercise training was associated with increase in the number and function of leukocytes and decreased disease susceptibility [25]. Despite of this, no significant changes in T lymphocytes subsets after intervention period were found on previous data [12,13]. For the other hand, we recently demonstrate a significant increase in peripheral CD4+ and CD8+ T cells after concurrent training in men and women overweight-obese adults [11]. In addition, the increased frequency of CD8+ T cells in peripheral blood after exercise training can be linked to the fact that exercise training attenuates the CD8+ T cells infiltration in the adipose tissue [8] and the expression of chemotactic factors in this site [26]. In our study, a decreased salivary cortisol levels is another possible mechanism for the increased T cells on peripheral circulation after exercise training. In this sense, cortisol has greater influence on lymphopenia [25] and changes in peripheral CD8+ T cells occurred in parallel to neuroendocrine changes during and after exercise training [27].

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Figure 1 The effect of twelve weeks of deep water running training on VO2Peak (A) and Time to Exhaustion (B). Paired t-test was used to calculate the difference before and after training (P ≤ 0.05).

Increase of caloric intake and lower physical activity levels leads to adipocyte hypertrophy and tissue hypoxia, thus triggering the infiltration of inflammatory leucocytes, mainly M1 macrophages, and increasing the expression of inflammatory cytokines and adipokines, such as IL-6, TNF-␣ and leptin, with concomitant decreasing in adiponectin levels leading to systemic chronic low-grade inflammation [1]. Several studies suggest that physical training can promote an anti-inflammatory environment, helping in the treatment of chronic diseases such as obesity and attenuating the immunological disturbance [9]. In this sense, the increasing of circulating adiponectin, IL-10 and TNF-␣ levels despite little changes in body composition in our study can confirm the anti-inflammatory effects of exercise without significant changes in adipose tissue [10]. IL-10 is a potent immunoregulatory cytokine produced mainly by regulatory T cells. The main functions of IL-10 are the downregulation of adaptive immune response and reduce of inflammation-induced tissue damage through the downregulation of MHC and co-stimulatory molecules (CD80 and CD86) on antigen-presenting cells [28]. In addition, IL10 inhibits partial or completely the expression of several cytokines with pro-inflammatory functions [29]. Moreover, IL-10 can increase insulin sensitivity and protect against M1 macrophage infiltration in obesity-skeletal muscle from rats

Figure 2 The effect of twelve weeks of deep water running training on peripheral frequency of CD4+ T cells (A) and CD8+ T cells (B). Data are displayed as mean ± standard deviation. Paired t-test was used to calculate the difference before and after training (P ≤ 0.05). Unpaired t-test was used to calculate the difference between groups (P ≤ 0.05).

[30]. Many of the beneficial induced by physical training in obese individuals, such as ameliorate in cardiac dysfunction and insulin resistance, were associated with increase in levels of IL-10 [31,32]. On the other hand, the present finding of higher serum TNF-␣ is inconsistent with literature. While the general evidence point to decrease in TNF-␣ and others proinflammatory cytokines after exercise training [10,31], some reports appoints to the fact that moderate endurance exercise, like deep water running, can stimulate Th1 phenotype of T cells. In this sense, Balducci and co-workers [33] demonstrated that the inflammatory adaptations is intensity dependent in type 2 diabetes patients, and moderate training induces a significant increase of TNF-␣, INF-␥, IL-1␤ and IL-10. Corroborating to this, elderly people submitted to moderate exercise training presented increase in CD28 expression on T cells and Th1/Th2 balance, analyzed by the increased INF-␥ levels and maintenance of IL-4 levels [34]. For the other hand, significant reductions on TNF-␣ levels were found due to improvements in adipose tissue in past studies [35,36]. Recently, Stensvold et al. [35] showed that after 12 weeks of interval training the levels of TNF-␣

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Water training and immunity in obesity were lower when compared to control group in individuals with metabolic syndrome, this changes occurred in parallel to reductions in total body fat mass (from 33.96 ± 7.3% to 32.26 ± 7.9%). In accordance, Ho et al. [36] found that continuous aerobic exercise, strength training, and concurrent training decreases systemic TNF-␣ levels and fat mass in overweight-obese adults compared to control group. In this sense, the enhancement on the adiponectin levels can reflect that DWR training induces immunoregulatory effects in the adipose tissue in our study. Adiponectin is a 30 kDa protein hormone synthesized in adipose tissue and has been postulated that it improves insulin sensitivity, inflammation, glucose use and protects against cardiovascular disease [37]. While Kraemer and Castracane [38] reported the necessity that significant body weight reduction after exercise training for improvements of adiponectin levels, we demonstrated that 12 weeks of DWR training could increase adiponectin levels despite the small body composition changes in women. In addition, adiponectin and IL-10 plays a key role in the control of low-grade inflammation in both systemic and lean adipose tissue, and we demonstrated that regular moderate physical training can stimulate an increase in anti-inflammatory mediators by down regulate or completely controls the expression of several pro-inflammatory cytokines [10]. One focus of aerobic exercise training is the enhancement of functional capacity. Overweight-obese individuals have lower VO2Peak , which positively correlate with a series of cardiovascular diseases [39]. Despite no changes were observed in VO2Peak after the period, however an individual analysis has demonstrated responders and non-responders to training. Our results are similar to Rica et al. [18], with no effects of water running training in anthropometric variables, but significant changes in functional parameters, such as time to exhaustion in our study, after intervention period. Moreover, water running with greater intensity than used in this study showed significant improvement in oxygen consumption [15]. However, Ross and co-workers [40] showed that low moderate-intensity exercise may not be enough to improve cardiorespiratory fitness for many sedentary obese adults, but increasing intensity can eliminate the non-responders to exercise. Previous studies showed that long-term water-based training was effective for improve body composition, functional capacity and metabolic parameters of obese individuals [15,17,18]. In this sense, Boidin et al. [17] showed that a 9-month of high-intensity interval training in a immersed ergocycle combined with Mediterranean diet was able to modify fasting glycemia and triglycerides levels with improved body composition variables (i.e. body mass, waist circumference and body fat). Contributing to this, we demonstrated that DWR training was also effective for improve the inflammatory status of overweight-obese women in the present study. Therefore, the improvement on the metabolic and lipid profiles of obese individuals can be related to immunoregulatory effects of water-based training.

5. Conclusion In conclusion, our data suggest that 12 weeks of moderateintensity deep water running training significantly improved

7 peripheral frequency of T cells, serum cytokines and adipokines, and reduced salivary cortisol in a group of inactive overweight-obese women. These findings support the idea that the immunoregulatory effects of exercise training occurs independently of significant alterations in weight loss, once that slightly changes in waist circumference and fat mass were founded after the intervention. We suggest that the practice of regular, supervised, and moderate physical exercise may act as a protective factor against the immunological changes of obesity. Indeed, further studies are needed to standardize the appropriate type, frequency, and intensity of exercise applied to obese individuals.

Disclosure of interest The authors declare that they have no competing interest.

Acknowledgements ¸ão de The authors thanks to the Brazilian agencies Coordenac Aperfeic ¸oamento de Pessoal de Nível Superior (CAPES) (PROSUP/CAPES model), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq MCTI 2014) and Fundac ¸ão de Amparo à Pesquisa do Estado do Rio Grande do Sul (FAPERGS) for their financial support.

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Please cite this article in press as: Colato A, et al. Impact of aerobic water running training on peripheral immuneendocrine markers of overweight-obese women. Sci sports (2016), http://dx.doi.org/10.1016/j.scispo.2016.04.003