The Relationship of IL-8 and IL-10 Myokines and Performance in Male Marathon Runners Presenting Exercise-Induced Bronchoconstriction

unclear which exercise-induced factors, such as myokines, could diminish the negative impact of the reduction in pulmonary function imposed by the exercise in question. In this study, we aim to evaluate the prevalence of exercise-induced bronchoconstriction (EIB) and also to investigate the e ect

4 March 2020; Accepted: 26 March 2020; Published: 11 April 2020 Abstract: At present, it is unclear which exercise-induced factors, such as myokines, could diminish the negative impact of the reduction in pulmonary function imposed by the exercise in question. In this study, we aim to evaluate the prevalence of exercise-induced bronchoconstriction (EIB) and also to investigate the e ect of myokines in the performance of marathon runners presenting EIB or not. Thirty-eight male recreational marathon runners (age 38.8 [33–44], height 175.7 [172.0–180.3]; weight 74.7 [69.3–81.6]) participated in this study, and through spirometry tests, a prevalence of 23.6% of EIB was found, which is in agreement with the literature. The volunteers who tested positive to EIB (EIB+) presented lower maximum aerobic capacity compared to those who tested negative (EIB ) (EIB+44.02 [39.56–47.02] and EIB 47.62 [44.11–51.18]p=0.03). The comparison of plasma levels of IL-1 (EIB+p=0.296, EIB p=0.176, EIB+vs. EIB baselinep=0.190 immediately after p=0.106), IL-4 (undetectable), IL-6 (EIB+p=0.003, EIB p 0.001, EIB+vs. EIB baselinep=0.301 immediately afterp=0.614), IL-8 (EIB+p=0.003, EIB p 0.001, EIB+vs. EIB baselinep=0.110 immediately afterp=0.453), IL-10 (EIB+p=0.003, EIB p 0.001, EIB+vs. EIB baselinep=0.424 immediately afterp=0.876) and TNF- (EIB+p=0.003, EIB p 0.001, EIB+vs. EIB baseline p=0.141 immediately afterp=0.898) were similar in both groups 24 h before and immediately after Int. J. Environ. Res. Public Health2020,17, 2622; doi:10.3390 /ijerph17082622 /journal/ijerph

Int. J. Environ. Res. Public Health2020,17, 2622 2 of 15 the marathon. However, negative correlations were found between the marathon nishing time and the levels of IL-8 (r= 0.81,p=0.022), and IL-10 (r= 0.97,p 0.001) immediately after completing the marathon. In conclusion, for the rst time, it is shown that the myokines IL-8 and IL-10 are related to improvement of the performance of marathon runners presenting EIB. Keywords:pulmonary function; endurance exercise; EIB; cytokines; FEV1; FVC; aerobic capacity 1. Introduction Exercise-induced bronchoconstriction (EIB) is de ned as a transient narrowing of the airways that occurs after exercise in individuals with (EIBA) or without underlying asthma (EIBwA) [1,2]. Both EIBA and EIBwAhave peculiarities in pathogenic mechanisms, diagnostic criteria, and responses to treatment and prevention [3]. Regarding the pathogenic mechanisms, although at present, the cause of EIB is not entirely understood, two classical theories, i.e., the osmotic and the thermal theories, were purposed to explain its occurrence. In relation to the latter, the occurrence of vasodilation, associated with airway rewarming, plays a role in the induction of bronchial obstruction after exercise [4]. Regarding the osmotic theory, which is currently the most accepted, the hyperventilation through the mouth associated with intense exercise requires the humidi cation and heating of large volumes of air in a short period of time, leading to airway dryness. Water loss by evaporation in airway surfaces is associated with events that can trigger the contraction of bronchial smooth muscle, such as mast cell degranulation [5], which releases pro-in ammatory mediators that are involved in smooth muscle contractions, mucus production, and microvascular permeability, leading to airway edema and bronchoconstriction [5,6]. Furthermore, as mentioned in the review from Couto et al. [7], epithelial damage can also be involved in EIB, and several studies have demonstrated increased in ltration of eosinophils, neutrophils, and/or epithelial cells associated with EIB, as well as an increase in airway in ammatory markers. More speci cally, Seys et al. [8] demonstrated an increased presence of damage-associated molecular patterns (DAMPS) in athletes with EIB. In terms of EIB diagnosis, it is important to clarify that the clinical history and the

have demonstrated increased in ltration of eosinophils, neutrophils, and/or epithelial cells associated with EIB, as well as an increase in airway in ammatory markers. More speci cally, Seys et al. [8] demonstrated an increased presence of damage-associated molecular patterns (DAMPS) in athletes with EIB. In terms of EIB diagnosis, it is important to clarify that the clinical history and the presence of typical symptoms (dyspnea, chest tightness, cough, and wheeze) can be used only as a complement to determine the occurrence of EIB [9], since valid diagnoses of EIB should be established though direct or indirect tests. For instance, the methacholine challenge is direct, whereas the eucapnic voluntary challenge as well as the standardized treadmill exercise test—whereby exercise may be followed by a decrease of 10 % or more in forced expiratory volume (FEV1) compared to pre-exercise levels—are indirect tests [6,7,9]. It is worthy to mention that, for some authors [10,11], the standard treadmill exercise test is preferred for EIB due to its resemblance to real-life exercise. The diagnosis of EIBAand EIBwAis of great importance for people who exercise or practice sport; the disorder can manifest during/after any physical activity, and shows similar prevalence in both genders [1,2]. It is important to emphasize that this condition is frequently not recognized, often being attributed to fatigue, and, even when recognized, it does not receive the attention it deserves [12,13]. It is important to note that EIB is quite common among practitioners of endurance sports [1]. The marathon, a sport that requires great physical effort, has significantly increased in popularity in recent decades [13,14]. Several publications, including from our group, have shown a significant increase in the production of pro-inflammatory cytokines, both systemic and in the upper airways [ Based on this information, we aimed to evaluate the prevalence of EIB in a group of recreational marathon runners without asthma, as well as to investigate both systemic and upper airway in ammatory responses and their correlation with marathon performance.

information, we aimed to evaluate the prevalence of EIB in a group of recreational marathon runners without asthma, as well as to investigate both systemic and upper airway in ammatory responses and their correlation with marathon performance.

Int. J. Environ. Res. Public Health2020,17, 2622 3 of 15 2. Methods 2.1. Subjects and Study Design Fifty recreational male athletes registered for the S¢o Paulo International Marathon to be held on 17 June 2012, in the city of S¢o Paulo, were recruited. Athletes had to meet the inclusion criteria (i.e., train at least three times per week and have completed a marathon in the last twelve months). Exclusion criteria were a history of cardiopulmonary, respiratory, or metabolic disease, the use of medication for chronic diseases, or any factor that did not permit them to run the race or nish the International Marathon of Sao Paulo. One week later, the athletes who consented were contacted to ll in a questionnaire about smoking habits and general health conditions such as the presence/absence of cardiopulmonary or metabolic diseases. The time they usually need to complete a 10 km training run was also asked, and this was used to determine the initial treadmill speed in the cardiopulmonary test (as will be described below) and estimate the time they would need to complete the marathon. In this contact, runners had baseline measurements (see below) and were also informed about the peripheral blood sample collection, as well as the ergospirometric and spirometry tests, planned for thirty days after the marathon. From the 50 athletes recruited, two did not complete the marathon and were therefore excluded from the study; 10 athletes were excluded because they did not complete the exams, leaving the present study with a total of 38 athletes. All subjects gave their informed consent for inclusion before they participated in the study. The study was conducted in accordance with the Declaration of Helsinki, and the protocol was approved by the Ethics Committee of Federal University of S¢o Paulo (0573/11). It was an analytical cross-sectional study, with three visits on di erent days (Figure). At the rst visit, all participants were submitted to baseline peripheral blood sampling and measurement of physical characteristics (weight, height, and body composition). Immediately after they completed the marathon, a second collection of peripheral blood was performed. In the last visit

University of S¢o Paulo (0573/11). It was an analytical cross-sectional study, with three visits on di erent days (Figure). At the rst visit, all participants were submitted to baseline peripheral blood sampling and measurement of physical characteristics (weight, height, and body composition). Immediately after they completed the marathon, a second collection of peripheral blood was performed. In the last visit (30 days later), the pulmonary function was evaluated at rest and immediately after a maximal cardiopulmonary exercise test to detect EIB. The athletes were instructed to describe any respiratory symptom during the study period. All volunteers reported that they did not experience any respiratory symptoms during the study period (i.e., before, during. or after the race). The researchers were blinded for the results of the marathon during the whole period of data collection.Int. J. Environ. Res. Public Health 2020, 17, x 3 of 17 2. Methods 2.1. Subjects and Study Design Fifty recreational male athletes registered for the São Paulo International Marathon to be held on June 17th, 2012, in the city of São Paulo, were recruited. Athletes had to meet the inclusion criteria (i.e., train at least three times per week and have completed a marathon in the last twelve months). Exclusion criteria were a history of cardiopulmonary, respiratory, or metabolic disease, the use of medication for chronic diseases, or any factor that did not permit them to run the race or finish the International Marathon of Sao Paulo. One week later, the athletes who consented were contacted to fill in a questionnaire about smoking habits and general health conditions such as the presence/absence of cardiopulmonary or metabolic diseases. The time they usually need to complete a 10 km training run was also asked, and this was used to determine the initial treadmill speed in the cardiopulmonary test (as will be described below) and estimate the time they would need to complete the marathon. In this contact, runners had baseline measurements (see below) and were also informed about the peripheral blood sample collection, as well as the ergospirometric and spirometry tests, planned for thirty days after the marathon. From

determine the initial treadmill speed in the cardiopulmonary test (as will be described below) and estimate the time they would need to complete the marathon. In this contact, runners had baseline measurements (see below) and were also informed about the peripheral blood sample collection, as well as the ergospirometric and spirometry tests, planned for thirty days after the marathon. From the 50 athletes recruited, two did not complete the marathon and were therefore excluded from the study; 10 athletes were excluded because they did not complete the exams, leaving the present study with a total of 38 athletes. All subjects gave their informed consent for inclusion before they participated in the study. The study was conducted in accordance with the Declaration of Helsinki, and the protocol was approved by the Ethics Committee of Federal University of São Paulo (0573/11). It was an analytical cross-sectional study, with three visits on different days (Figure 1). At the first visit, all participants were submitted to baseline peripheral blood sampling and measurement of physical characteristics (weight, height, and body composition). Immediately after they completed the marathon, a second collection of peripheral blood was performed. In the last visit (30 days later), the pulmonary function was evaluated at rest and immediately after a maximal cardiopulmonary exercise test to detect EIB. The athletes were instructed to describe any respiratory symptom during the study period. All volunteers reported that they did not experience any respiratory symptoms during the study period (i.e., before, during. or after the race). The researchers were blinded for the results of the marathon during the whole period of data collection. Figure 1. Study design. 2.2. Cytokines Measurements and Cellular Counting Plasma samples were obtained from peripheral blood collected in tubes containing ethylenediamine tetraacetic acid [(EDTA) 2 tubes of 5 mL], which were centrifuged at 900 g for 10 min. Plasma samples was then stored at −80 °C for later determination of circulating cytokine concentration. Plasma IL-1β, IL-4, IL-6, IL-8, IL-10, and TNF-α concentrations were obtained by Multiplex MILLIPLEX (Merck Millipore, Burlington, MA, USA) analysis, according to manufacturer’s Figure 1.Study design. 2.2. Cytokines Measurements

acid [(EDTA) 2 tubes of 5 mL], which were centrifuged at 900 g for 10 min. Plasma samples was then stored at −80 °C for later determination of circulating cytokine concentration. Plasma IL-1β, IL-4, IL-6, IL-8, IL-10, and TNF-α concentrations were obtained by Multiplex MILLIPLEX (Merck Millipore, Burlington, MA, USA) analysis, according to manufacturer’s Figure 1.Study design. 2.2. Cytokines Measurements and Cellular Counting Plasma samples were obtained from peripheral blood collected in tubes containing ethylenediamine tetraacetic acid [(EDTA) 2 tubes of 5 mL], which were centrifuged at 900gfor 10 min. Plasma samples was then stored at 80 C for later determination of circulating cytokine concentration.

Int. J. Environ. Res. Public Health2020,17, 2622 4 of 15 Plasma IL-1 , IL-4, IL-6, IL-8, IL-10, and TNF- concentrations were obtained by Multiplex MILLIPLEX (Merck Millipore, Burlington, MA, USA) analysis, according to manufacturer's instructions. The concentration of high-sensitivity C-reactive protein (HSCRP) protein and creatine phosphokinase (CPK) were determined spectrophotometrically. 2.3. Evaluation of Aerobic and Pulmonary Capacity Thirty days after the marathon, all 38 participants underwent pulmonary function testing with a computerized pneumotachograph spirometer (SpiroPro, SensorMedics, Yorba Linda, CA, USA) pre- and post- treadmill exercise test, according to the recommendations of the American Thoracic Society [17], to diagnosis of EIB. Immediately afterwards (0 min), and 5, 10, 15, and 20 min after the end of the test, the volunteers performed spirometry tests. EIB was considered positive (EIB+) when FEV1 decreased by 10% or more in relation to the baseline test in any of the spirometric tests performed after the treadmill test [17]. A maximal effort test was accomplished by all volunteers in order to determine their aerobic capacity. Cardiopulmonary exercise testing was performed on a treadmill coupled with a gas analyzer (FitMate—, Cosmed, Rome, Italy) and with an electrocardiography software (TEB ® APEX 2000, S¢o Paulo, Brazil). The analysis was performed using the maximal incremental treadmill protocol with a xed slope of 1% and a 1 km/h increase every minute, until the maximum treadmill speed (18 km/h) was reached. The rst minute on the treadmill was the same for all subjects, i.e., 5 km/h. In the second minute, a di erent speed based on the time needed to complete a 10 km run was used as described: those who reported taking less than 35 min to run 10 km started the second minute of the test at 9 km/h (group 1: 8 volunteers); those who reported needing between 36 and 45 min started the second minute of the test at 8 km/h (group 2: 8 volunteers); those who reported taking more than 46 min (maximum 1 h and 10 min) to run the 10 km started the second minute of the test at 7 km/h (group 3: 22 volunteers) [18].

1: 8 volunteers); those who reported needing between 36 and 45 min started the second minute of the test at 8 km/h (group 2: 8 volunteers); those who reported taking more than 46 min (maximum 1 h and 10 min) to run the 10 km started the second minute of the test at 7 km/h (group 3: 22 volunteers) [18]. When the maximum treadmill speed was reached (after 10, 11, or 12 min), a 2% inclination was added every minute (the treadmill maximum inclination capacity was 26%) until completion of test. The test was interrupted when the volunteer reported intense fatigue or exhaustion, or by the physician responsible for monitoring the tests when abnormalities on the electrocardiogram and oxygen consumption were observed. Oxygen uptake (VO2) was considered maximum (VO2 max) when a respiratory exchange ratio (RER)>1.1 L was reached, or when the report of exhaustion was associated with reaching the maximum predicted heart rate and an RER greater than 1 L. In addition, the lactate threshold was also determined during the cardiorespiratory test based on the nonlinear increases in ventilation and changes in respiratory exchange ratio (RER) parameter. 2.4. Statistical Analysis All results were assessed for testing the null hypothesis of normality using the Shapiro-Wilks test. The nonparametric baseline prerace and the postrace values from blood in ammatory markers were compared using the Wilcoxon test. The EIB negative ( ) and positive (+) subjects were compared using the Mann-Whitney U test at signi cance level ofp<0.05. Spearman's correlation was applied to analyze the correlations within each group, and the results were considered statistically signi cant at p<0.02. Parameters such as anthropometric value, oxygen consumption, pulmonary capacity, and in ammatory marker concentrations are presented as median and percentiles. A statistical analysis was performed using software GraphPad Prism 8 (version 8.1.2, GraphPad Software, San Diego, CA, USA). 2.5. Data Availability Statement The datasets generated and/or analyzed during the current study are available from the corresponding author upon request.

was performed using software GraphPad Prism 8 (version 8.1.2, GraphPad Software, San Diego, CA, USA). 2.5. Data Availability Statement The datasets generated and/or analyzed during the current study are available from the corresponding author upon request.

Investigates myokines' impact on performance in marathon runners with exercise-induced bronchoconstriction.