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J Appl Physiol 116: , First published February 20, 2014; doi: /japplphysiol Influence of oxidative stress, diaphragm fatigue, and inspiratory muscle training on the plasma
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J Appl Physiol 116: , First published February 20, 2014; doi: /japplphysiol Influence of oxidative stress, diaphragm fatigue, and inspiratory muscle training on the plasma cytokine response to maximum sustainable voluntary ventilation Dean E. Mills, 1 Michael A. Johnson, 1 Martin J. McPhilimey, 1 Neil C. Williams, 1 Javier T. Gonzalez, 2 Yvonne A. Barnett, 1 and Graham R. Sharpe 1 1 Sport, Health and Performance Enhancement (SHAPE) Research Group, School of Science and Technology, Nottingham Trent University, Nottingham, United Kingdom; and 2 School of Life Sciences, Northumbria University, Newcastle upon Tyne, United Kingdom Submitted 18 November 2013; accepted in final form 14 February 2014 Mills DE, Johnson MA, McPhilimey MJ, Williams NC, Gonzalez JT, Barnett YA, Sharpe GR. Influence of oxidative stress, diaphragm fatigue, and inspiratory muscle training on the plasma cytokine response to maximum sustainable voluntary ventilation. J Appl Physiol 116: , First published February 20, 2014; doi: /japplphysiol The influence of oxidative stress, diaphragm fatigue, and inspiratory muscle training (IMT) on the cytokine response to maximum sustainable voluntary ventilation (MSVV) is unknown. Twelve healthy males were divided equally into an IMT or placebo (PLA) group, and before and after a 6-wk intervention they undertook, on separate days, 1hof(1) passive rest and (2) MSVV, whereby participants undertook volitional hyperpnea at rest that mimicked the breathing and respiratory muscle recruitment patterns commensurate with heavy cycling exercise. Plasma cytokines remained unchanged during passive rest. There was a main effect of time (P 0.01) for plasma interleukin-1 (IL-1 ) and interleukin-6 (IL-6) concentrations and a strong trend (P 0.067) for plasma interleukin-1 receptor antagonist concentration during MSVV. Plasma IL-6 concentration was reduced after IMT by 27 18% (main effect of intervention, P 0.029), whereas there was no change after PLA (P 0.753). There was no increase in a systemic marker of oxidative stress [DNA damage in peripheral blood mononuclear cells (PBMC)], and diaphragm fatigue was not related to the increases in plasma IL-1 and IL-6 concentrations. A dose-response relationship was observed between respiratory muscle work and minute ventilation and increases in plasma IL-6 concentration. In conclusion, increases in plasma IL-1 and IL-6 concentrations during MSVV were not due to diaphragm fatigue or DNA damage in PBMC. Increases in plasma IL-6 concentration during MSVV are attenuated following IMT, and the plasma IL-6 response is dependent upon the level of respiratory muscle work and minute ventilation. cytokine; respiratory muscles; ventilation Address for reprint requests and other correspondence: D. Mills, Queensland Children s Medical Research Institute, The Univ. of Queensland, Royal Children s Hospital, Brisbane, Australia, 4029 ( WE RECENTLY DEMONSTRATED THAT plasma interleukin-6 (IL-6) concentration increased when young, healthy adults undertook 1 h of volitional hyperpnea at rest that mimicked the breathing and respiratory muscle recruitment patterns commensurate with cycling exercise undertaken at an estimated maximum lactate steady-state intensity (42). This finding supports the notion that the respiratory muscles may contribute to systemic increases in plasma IL-6 concentration observed when the intensity of respiratory muscle work is increased such as in strenuous whole-body exercise, asthma attacks, and exacerbations of chronic obstructive pulmonary disease (COPD) (34, 48, 71). We also demonstrated that volitional hyperpnea elevated plasma IL-6 concentrations in the absence of diaphragm fatigue, and that inspiratory muscle training (IMT) reduced the plasma IL-6 response to exercise but not volitional hyperpnea (42). Whether other plasma cytokines may be affected by such interventions is unknown. Whole-body exercise and inspiratory resistive loading (IRL) can increase the plasma concentration of the proinflammatory cytokine interleukin-1 (IL-1 ) and IL-6 (45, 66, 67). Within the diaphragm of rats exposed to IRL, IL-1 and IL-6 are stimulated by oxidative stress and possibly diaphragm fatigue (56, 57, 65). IL-1 can impair striated muscle function and potentially reduce muscle contractility (37). IL-1 may also have a role within muscle repair and regeneration following injury. The IL-1 gene is activated with strenuous muscular contraction, and the associated micro injury that occurs within the muscle stimulates quiescent resident macrophages to secrete IL-1 (11, 17, 21, 51, 64). IL-1 also stimulates proteolysis and acts a chemoattractant for macrophages which can phagocytize cellular debris and provide a large source of growth factors which may stimulate myogenesis (11, 17, 21, 51, 64). Therefore, oxidative stress, respiratory muscle fatigue/injury, and repair/regeneration may be stimuli for the production of IL-1 by human respiratory muscles. In addition, since respiratory muscle training attenuates exercise-induced diaphragm fatigue during whole-body exercise (68), IMT may also reduce IL-1 production by the inspiratory muscles. Whole-body exercise also increases the plasma concentration of the anti-inflammatory cytokine interleukin-1 receptor antagonist (IL-1ra) (45, 54, 72). IL-1ra is stimulated by IL-6 and IL-1 and acts to restrict and limit the extent of the inflammatory response by inhibiting signaling transduction of interleukin-1 through the interleukin-1 receptor complex (18, 62). Recent evidence has demonstrated that the plasma IL-1ra response to exercise is attenuated following whole-body endurance training (72). This may be due to a training-induced reduction in IL-6 and/or IL- which may also occur following IMT. The maximum lactate steady state represents the highest work rate at which a physiological steady state can be achieved and, therefore, marks the boundary between sustainable and nonsustainable work rates (30, 31). The minute ventilation (V E) associated with the maximum lactate steady state is known to be well below an individual s maximum sustainable /14 Copyright 2014 the American Physiological Society voluntary ventilation (MSVV) (31) and, therefore, our previously reported breathing-induced plasma IL-6 response may have been submaximal (42). The notion that inflammatory responses to respiratory muscle work and V E may be dose dependent is supported by the observation that increases in plasma IL-6 concentration are intensity dependent during whole-body exercise (47, 54). Therefore, an aim of this study was to examine the response of plasma cytokines IL-1, IL-1ra, and IL-6 to MSVV, whereby participants undertook volitional hyperpnea at rest that mimicked the breathing and respiratory muscle recruitment patterns commensurate with heavy cycling exercise. We also sought to examine the mechanism(s) for cytokine production by measuring diaphragm fatigue [known to be elicited by MSVV (3)] and a systemic marker of oxidative stress. Finally, we examined whether these responses were altered by IMT. METHODS Plasma Cytokine Response to Maximum Sustainable Ventilation Mills DE et al. Participants. Twelve nonsmoking recreationally active males provided written, informed consent to participate in the study which was approved by the Nottingham Trent University Human Ethics Committee. The characteristics, pulmonary function, maximal inspiratory pressure (MIP), maximum power output, and peak oxygen uptake of the study participants has been described previously (42). A selfreported medical questionnaire confirmed that participants were free from illness and injury and not taking any medication and/or antioxidant supplements during the study. Each participant completed a 24-h diet record prior to their first trial, which was then replicated prior to all subsequent trials. Throughout the study, participants were instructed to adhere to their habitual training regimen and not to engage in any strenuous exercise the day preceding, the day of, and in the 24 h following each trial. Participants arrived at the laboratory 4 h postprandially having abstained from alcohol and caffeine in the 24 h before testing. 971 Experimental design. Participants attended the laboratory on three separate occasions, before and after a 6-wk intervention (Fig. 1). Each laboratory visit was separated by 48 h and took place at the same time of day. During the first visit, participants were familiarized with all testing procedures and pulmonary function, and MIP were measured. During the second visit, participants performed a maximal incremental cycling test. The subsequent two experimental trials were randomized, lasted 1 h and comprised (1) passive rest (PASSIVE) and (2) MSVV. Thereafter, participants were randomly, and equally, divided into an IMT or placebo (PLA) group and completed the intervention. At least 48-h postintervention, participants repeated the maximal incremental cycling test and experimental trials in the same order as preintervention. All trials were performed on an electromagnetically braked cycle ergometer (Excalibur Sport, Lode, Groningen, The Netherlands). Pulmonary function and maximal inspiratory mouth pressure. Pulmonary function was assessed according to published guidelines (41) using a pneumotachograph (Pneumotrac, Vitalograph, Buckingham, UK) calibrated with a 3-l syringe. A hand-held mouth pressure meter (MicroRPM, CareFusion, Basingstoke, UK) measured MIP as an index of global inspiratory muscle strength. The mouthpiece assembly incorporated a 1-mm orifice to prevent glottic closure during inspiratory efforts. Maneuvers were performed while standing, initiated from residual volume, and sustained for at least 1 s. Repeat efforts separated by 30 s were performed until three serial measures differed by no more than 10% or 10 cmh 2O, whichever was smallest (8, 10, 28). The highest value recorded was used for subsequent analysis. Maximum incremental cycling test. Cycling began at 0 W, and power was subsequently increased by 10 W every 15 s in order to result in exercise intolerance within 10 min. This rapid incremental protocol was selected to maximize V E at the cessation of the test and, therefore, reflect heavy exercise. The power at which exercise intolerance ensued defined maximum power output, and the highest V E and oxygen uptake recorded in any 30 s period defined peak V E (V E peak) and peak oxygen uptake, respectively. Fig. 1. Schematic of experimental design. 972 Plasma Cytokine Response to Maximum Sustainable Ventilation Mills DE et al. Experimental trials. During experimental trials, the configuration of the cycle ergometer and the body position adopted by each participant were identical to those adopted during the maximal incremental cycling test. MSVV was preceded by a 5-min rest period, and during PASSIVE participants remained seated on the cycle ergometer for the duration of the trial. During MSVV participants undertook volitional hyperpnea at rest that mimicked the breathing [tidal volume (V T); breathing frequency (f B); and duty cycle (T I/T TOT)] and respiratory muscle recruitment [peak transdiaphragmatic pressure (P dipeak)] patterns commensurate with heavy cycling exercise. These variables were determined from the average values obtained between 70 80% of the V E peak attained during the preintervention maximal incremental cycling test; pilot work showed that this represented the maximum voluntary ventilation that could be sustained for 1 h. During the postintervention MSVV trial, participants mimicked the breathing and respiratory muscle recruitment patterns performed during the preintervention MSVV trial. An audio metronome paced f B and T I/T TOT, and real-time visual feedback of V T and P dipeak was provided throughout. The use of P dipeak targets allowed us to more precisely control the mechanical work of breathing during MSVV (3, 35). All experimental trials were performed in an environmental chamber (Design Environmental WIR52-20HS, Design Environmental, Ebbw Vale, UK) at 20 C and 90% relative humidity to minimize mucosal drying during MSVV (42). Flow, pulmonary gas exchange, and pressure measurements. During all trials participants wore a facemask (model 7940, Hans Rudolph, Kansas City, MO) connected to a flow sensor (ZAN variable orifice pneumotach, Nspire Health, Oberthulba, Germany) that was calibrated using a 3-l syringe. Gas concentrations were measured using fast responding laser diode absorption spectroscopy sensors, which were calibrated using gases of known (5% CO 2, 15% O 2 and balance N 2) concentrations (BOC, Guildford, UK), and ventilatory and pulmonary gas exchange variables were determined breath-bybreath (ZAN 600USB, Nspire Health). During experimental trials a two-way nonrebreathing valve (model 2730, Hans Rudolph) was attached distally to the pneumotachograph, and a 1.5-m length of wide-bore tubing was connected to the inspiratory port. During MSVV, CO 2 was added into this tubing to increase F ICO 2 and thus retain end-tidal and, consequently, blood PCO 2 at levels commensurate with rest (9, 31). On the expiratory port of the two-way valve a Fleisch no. 3 pneumotachograph was attached and connected to a differential pressure transducer ( 2.5 cmh 2O) (TSD160A, BIOPAC Systems, Goleta, CA) and differential bridge amplifier (DA100C, BIOPAC Systems) to allow alignment of flow and pressure signals. Esophageal (P e) and gastric (P ga) pressures were measured and calibrated as described previously (42). Transdiaphragmatic pressure (P di) was calculated by subtracting P e from P ga. As an estimate of respiratory muscle work, P di and P e were integrated over the period of inspiratory flow and multiplied by f B and labeled the diaphragm pressure-time product (PTP di) and the inspiratory muscle pressure-time product (PTP e), respectively. Nonphysiological flows and pressures that resulted from swallowing, coughing, and breath holding were identified by visual inspection and removed. Breathing mechanics data were ensemble averaged into six 10-min blocks and used for subsequent analysis. Bilateral anterior magnetic phrenic nerve stimulation. Bilateral anterior magnetic phrenic nerve stimulation (BAMPS) (43) was applied using two double 25-mm coils connected to two Magstim stimulators (Magstim, Whitland, UK) as described previously (42). To determine supramaximal phrenic nerve stimulation, three single twitches were obtained every 30 s at intensities of 50, 60, 70, 80, 85, 90, 95, and 100% of maximal stimulator output. A plateau in transdiaphragmatic twitch pressure (P ditw) and M-wave responses with increasing stimulation intensities indicated maximum depolarization of the phrenic nerves. P ditw was assessed every 30 s using eight single stimuli at 100% of maximal stimulator output. P ditw was measured at baseline and within 15 ( 15 min), 35 ( 35 min), and 60 ( 60 min) min after each experimental trial. Additionally, P ditw at each measurement point was followed by the assessment of the potentiated P ditw response. Participants performed a 3-s maximal Müeller maneuver and 5 s later a single stimuli was delivered. This procedure was repeated six times with each measure separated by 30 s. The average of the median three individual P ditw responses were used for analysis. Diaphragm fatigue was defined as a 15% reduction in P ditw compared with the baseline value (36, 42). Measurement of systemic oxidative stress. Arterialized venous blood was sampled from a dorsal hand vein via an indwelling 21-G cannula (40). Arterialization was ensured by immersing the hand in water at 40 C for 10 min prior to cannulation and by warming the hand during trials using an infrared lamp. Levels of systemic oxidative stress were determined using the Comet Assay, which can measure oxidative DNA damage in peripheral blood mononuclear cells (PBMC). Previously, data has reported an increase in systemic DNA damage following heavy cycling exercise using the Comet Assay (49, 69). During the experimental trials, 5-ml blood samples were taken at rest, immediately following the experimental trial (0 h) and 1 day into recovery ( 24 h). Blood was immediately transferred into precooled tubes containing lithium heparin (SARSTEDT, Leicester, UK). PBMC were isolated using density gradient centrifugation. The heparinized venous blood was mixed with 5 ml of phosphate buffered saline and layered onto a lymphocyte separation medium (Ficoll- Paque Plus, Griener Bio-One, Stonehouse, UK) inside a Leucosep tube (Griener Bio-One) and centrifuged for 15 min at 800 g and room temperature. The opaque mononuclear cell layer was aspirated and washed 3 times in phosphate buffered saline and centrifuged for 10 min at 200 g and room temperature. The cells were cryopreserved in liquid nitrogen for subsequent analysis in a medium consisting of 10% Dimethyl sulfoxide (Sigma-Aldrich, Gillingham, UK), 20% fetal bovine serum (Invitrogen, Paisley, UK) and 70% X-Vivo 10 media (Lonza, Wokingham, UK). Levels of DNA damage (DNA single-strand breaks and alkali labile lesions) in PBMC were determined in duplicate using the alkaline Comet Assay (59) and the modified alkaline Comet Assay (15) as described by Marthandan et al. (38). In the modified Comet Assay, PBMC embedded on slides were treated with either formamidopyrimidineglycosylase (FPG) which recognizes oxidatively modified purines (6), or with endonuclease III (ENDO III) which recognizes oxidatively modified pyrimidines (2). These enzymes nick DNA at the sites of oxidatively damaged nucleotides, creating single-strand breaks which can be detected with the alkaline Comet Assay. PBMC treated with 150 M of hydrogen peroxide for 5 min at 4 C (to induce oxidative damage) were used as internal positive controls in the modified alkaline Comet Assay. The Comet Assays were performed at 4 C to minimize the repair of existing basal levels of DNA damage present in PBMC. PBMC ( cells/gel) were embedded in a 1% agarose gel on frosted microscope slides and lysed for 1 h in a high salt alkaline buffer [2.5 M sodium chloride, 0.1 M EDTA, 0.01 M Tris, 1% (v/v), Triton X-100, ph 10]. For the modified Comet Assays, slides were equilibrated in enzyme buffer (0.04 M HEPES, 0.1 M potassium chloride, 0.5 mm EDTA, 0.2 mg/ml bovine serum albumin, ph 8.0) prior to the application of FPG or ENDO III. Slides treated with the lesion specific enzymes were incubated at 37 C in a humid, dark chamber for 45 min. Following enzyme treatment (or immediately after alkaline lysis for the alkaline Comet Assay), the slides were placed in electrophoresis buffer (0.3 M sodium hydroxide, 1 mm EDTA, ph 13) for 20 min, to allow alkaline unwinding of the DNA, and then electrophoresed at 25 V and 300 ma for 30 min. Subsequently, slides were neutralized (0.4 M Tris, ph 7.5) and stained (50 g/ml ethidium bromide) to visualize DNA. Stained slides were digitally analyzed using ultraviolet microscopy (Carl Zeiss, Welwyn Garden City, UK) and scored (50 PBMC per slide) using analysis software (Komet 5.5, Andor Bio Imaging, Nottingham, UK) by the same investigator. The interassay coefficient of variation (CV) was 10%. Plasma Cytokine Response to Maximum Sustainable Ventilation Mills DE et al. 973 Cytokines and additional blood analyses and measurements. At rest, immediately after (0 h) and 1 ( 1 h),2( 2 h), and 24 ( 24 h) h after each experimental trial, 3 5 ml blood samples were taken for the measurement of plasma IL-1, IL-1ra, and IL-6 concentrations. Blood was transferred into precooled tubes containing 1.6 mg/ml of K 3E EDTA (SARSTEDT) and immediately centrifuged for 15 min at 1,000 g and 5 C. The plasma supernatant was subsequently removed and stored at 80 C until further analysis. Commercial solid phase sandwich ELISA were used to measure plasma concentrations of IL-1, IL-6 (Quantikine HS, R&D Systems Abingdon, UK), and IL-1ra (Quantikine, R&D Systems) in duplicate. The assays have a detection limit of 0.1 p
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