Exercise deactivates leukocytes in asthma

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Exercise Deactivates Leukocytes in Asthma

DOI 10.1055/s-0033-1358477 Int J Sports Med For personal use only. No commercial use, no depositing in repositories.

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Immunology

Exercise Deactivates Leukocytes in Asthma

Authors Affiliations

Key words ▶ asthma ● ▶ exercise ● ▶ immunology ● ▶ allergy ● ▶ cytokines ●

R. P. Vieira1, R. A. Silva3, M. C. Oliveira-Junior1, F. R. Greiffo1, A. P. Ligeiro-Oliveira1, M. A. Martins2, C. R. F. Carvalho3 1

Nove de Julho University, São Paulo, Brazil Clinical Medicine (LIM 20), University of São Paulo, Brazil 3 Physical Therapy (LIM 34), University of São Paulo, Brazil 2

Abstract

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Leukocytes play a central role in asthma physiopathology. Aerobic training (AT) reduces leukocytes recruitment to the airways, but the effects of AT on some aspects of leukocytes activation in asthma are unknown. Therefore, the effects of 4 weeks of AT on airway inflammation, pulmonary and systemic Th2 cytokines levels, leukocytes expression of pro and anti-inflammatory, profibrotic, oxidants and anti-oxidants mediators in an experimental model of asthma was investigated. AT reduced the levels of IL-4, IL-5, IL-13 in bronchoalveolar lavage fluid (BALF) (p < 0.001), serum levels of IL-5, while increased BALF and serum levels of IL-10 (p < 0.001). In addition, AT

Introduction

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accepted after revision September 23, 2013 Bibliography DOI http://dx.doi.org/ 10.1055/s-0033-1358477 Published online: 2013 Int J Sports Med © Georg Thieme Verlag KG Stuttgart · New York ISSN 0172-4622 Correspondence Dr. Rodolfo Paula Vieira Nove de Julho University Rua Vergueiro 235/249 São Paulo 01504-001 Brazil Tel.: + 55/11/3061 7180 Fax: + 55/11/3061 7180 [email protected]

A growing number of studies point out the beneficial effects of regular practice of aerobic training (AT) for the management of asthmatic individuals [2, 5–7, 9, 13, 19, 23–26, 30–32, 36]. In summary, these studies demonstrate that AT significantly improves asthma symptoms, including dyspnea and exercise-induced bronchoconstriction (EIB), health-related quality of life, and also reduces corticosteroid needing as well as reduces the levels of exhaled nitric oxide, suggesting a possible anti-inflammatory effects of AT for the airways [7, 9, 23–26, 30, 31]. More recently, a study from Mendes et al. (2011) demonstrated for the first time that AT reduces eosinophilic inflammation in asthmatic patients, confirming the antiinflammatory effects of AT [24]. However, the mechanisms involved in the anti-inflammatory effects of AT for asthma remains not fully elucidated. In this way, a growing number of experimental studies have been performed aiming to investigate the possible cellular and molecular mechanism underlying the anti-inflammatory effect of

reduced leukocytes activation, showed through decreased expression of Th2 cytokines (IL-4, IL-5, IL-13; p < 0.001), chemokines (CCL5, CCL10; p < 0.001), adhesion molecules (VCAM-1, ICAM1; p < 0.05), reactive oxygen and nitrogen species (GP91phox and 3-nitrotyrosine; p < 0.001), inducible nitric oxide synthase (iNOS; p < 0.001), nuclear factor kB (NF-kB; p < 0.001) while increased the expression of anti-inflammatory cytokine (IL-10; p < 0.001). AT also decreased the expression of growth factors (TGF-beta, IGF-1, VEGF and EGFr; p < 0.001). We conclude that AT reduces the activation of peribronchial leukocytes in a mouse model of allergic asthma, resulting in decreased airway inflammation and Th2 response.

AT in animals’ models of asthma, currently experimental models of acute and chronic allergic airway inflammation [11, 12, 27–29, 34, 35, 37–39]. In general, these studies have demonstrated that AT reduces eosinophilic and lymphocytic airway inflammation, Th2 cytokines production, nuclear factor kB (NF-kB) activation, while increases the expression of anti-inflammatory cytokines IL-1ra and IL-10 [11, 12, 27–29, 34, 35, 37–39]. From these studies, some initial evidences of the cellular and molecular effects of AT in experimental models of acute and chronic allergic airway inflammation were identified. For instance, Pastva et al., 2004 and 2005 demonstrated that part of the anti-inflammatory effects of AT could be attributed to reduced NF-kB activation and glucocorticoid receptor expression in peribronchial leukocytes and also in airway epithelium [28, 29]. Following Pastva’s study, Vieira et al., 2007 demonstrated that AT also induces the production of anti-inflammatory cytokine IL-10 [37], a finding that was further confirmed by Silva et al., 2010, that elegantly added the stimulatory effect of AT on IL-1ra expression [35].

Vieira RP et al. Exercise Deactivates Leukocytes in Asthma. Int J Sports Med

Immunology

However, the literature demonstrates that the leukocytes are responsible also for the release of Th2 cytokines, growth factors and oxidants, which play a central role in the inflammatory and remodeling process in asthma [3, 17, 18, 20–22, 33]. Therefore, the present study investigate the effects of AT on chronic allergic airway inflammation, focusing on the effects of AT on peribronchial leukocytes activation (i. e., expression of pro-inflammatory, anti-inflammatory, pro-fibrotic, oxidants and anti-oxidants and growth factors by leukocytes) involved in the inflammatory and remodeling process in asthma.

and the differential cell counts (300 cells per lamina) were performed using cytospins preparations stained with May-Grunwald-Giemsa [27, 34, 37]. We clarify that the results of total and differential cell count was already presented in the following previous study [39].

Materials and Methods

Lung histology, immunohistochemistry and morphometic analysis

This study was approved by the ethical committee of the School of Medicine of the University of Sao Paulo. The “Guide for care and use of laboratory animals” was followed (NIH publication 85-23, revised 1996). In addition, we state that the present manuscript is in accordance to the IJSM’s ethical standard [10].

Lungs were fixed in formalin and embedded in paraffin. 5-micrometer thick sections were stained with hematoxylin and eosin for lung structure and inflammation analysis [37]. Immunohistochemistry was performed with anti-IL-4, anti-IL-5, antiIL10, anti-IL-13, anti-CCL5, anti-CCL10, anti-VCAM-1, anti-ICAM-1, anti-GP91phox, anti-3-nitrotyrosine, anti-NF-kB, anti-iNOS, anti-TGF-beta, anti-IGF-1, anti-VEGF and anti-EGFr antibodies (Santa Cruz Biotechnology, Santa Cruz, CA), using a biotinstreptavidin-peroxidase method. With a 50-line, 100-point grid connected to the ocular of the microscope, we assessed the peribronchial density of positive leukocytes for the markers described above, using a point-counting technique [37]. Counting was performed in 5 complete airways for each animal at 1 000 × magnification. Results were expressed as positive cells per square millimeter [37].

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Animals and experimental groups 32 BALB/c male mice (20–25 g) were distributed in control (Control; n = 8), aerobic training (AT; n = 8), ovalbumin sensitized (OVA; n = 8) and ovalbumin sensitized + aerobic training (OVA + AT; n = 8) groups. We state that the immunohistochemical and the cytokines measurements in bronchoalveolar lavage fluid (BALF) were performed in the samples of previous study [37–39].

Cytokines measurements The levels of IL-4, IL-5, IL-10 and IL-13 were quantified in bronchoalveolar lavage and in serum by ELISA using commercial kits (BD Elispot kit, CA, USA) according to the manufacturer recommendation.

Treadmill training and test protocol Animals were adapted to treadmill training (15 min, 25 % inclination and 0.2 km/h) during 3 days. In the following day, all animals were submitted to maximal exercise test, as previously described [37, 38]. The physical test was repeated 30 days after the beginning of AT. The results from physical test were presented in the previous study [39]. The treadmill physical training was performed during 4 weeks, 5×/week, 60 min per session, at low intensity (corresponding to 50 % of maximal exercise capacity reached in the maximal exercise test). The exercise has started one day after the first OVA or saline inhalation exposure [39].

Statistical analysis

Chronic model of allergic asthma

BALF levels of pro-inflammatory Th2 and antiinflammatory cytokines profile

4 intra-peritoneal (i.p.) injections of OVA (20ug per mouse) adsorbed with aluminum hydroxide or saline solution for control groups (non-sensitized mice) were performed on days 0, 14, 28 and 42. 21 days after the first i.p. injection, mice were challenged with aerosolized OVA (1 %) or with a saline solution 3 times a week until the 50th day [37–39].

Anesthesia and animals’ euthanasia 72h after the last inhalation day and exercise test, animals were anesthetized by intramuscular injection of ketamine (50 mg/kg) and xylazine (40 mg/kg), and tracheostomized to collect bronchoalveolar lavage fluid (BALF). The blood was collected through the abdominal vein for the cytokines quantification, followed by euthanasia through exsanguinations.

Bronchoalveolar Lavage Fluid (BALF) procedures Lungs were gently washed with 1.5 ml of saline (administered as three 0.5 ml volumes) via the tracheal cannula. Total cell counts were performed using a hematocytometer (Neubauer chamber) Vieira RP et al. Exercise Deactivates Leukocytes in Asthma. Int J Sports Med

Parametric and nonparametric data were expressed as means ± SD and as medians ± 95 % confidence interval (95 % CI), respectively. Comparisons among groups were performed by one-way analysis of variance followed by the Student-NewmanKeuls post hoc test (parametric data) or by one-way analysis of variance on ranks followed by Dunn’s post-hoc test (nonparametric data); the significance level was adjusted to 95 % (p < 0.05).

Results

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The levels of pro-inflammatory Th2 cytokines (IL-4, IL-5, IL-13) and anti-inflammatory cytokine (IL-10) in BALF are presented ▶ Fig. 1a–d, respectively. The results demonstrated that AT in ● significantly reduced the levels of IL-4, IL-5 and IL-13 when compared with OVA group (p < 0.01). The results also demonstrated that AT significantly increased the levels of IL-10 in both nonsensitized (AT) and sensitized (OVA + AT) groups (p < 0.05).

Systemic Th2 (IL-5) and anti-inflammatory (IL-10) response The levels of pro-inflammatory Th2 cytokine IL-5 and anti▶ Fig. 2a, b, inflammatory cytokine IL-10 are presented in ● respectively. The results demonstrated that AT significantly ▶ Fig. 2a; reduced the levels of IL-5 compared with OVA group (● p < 0.01). The results also demonstrated that AT significantly increased the levels of IL-10 in both non-sensitized (AT) and sen▶ Fig. 2b; p < 0.05). sitized (OVA + AT) groups (●

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Fig. 1 Figure shows the levels of pro and anti-inflammatory cytokines in BALF. In a, b and c, *p < 0.01 when compared with all groups. In d, *p < 0.05 when compared with Control group.

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Peribronchial leukocytes expression of Th2 and Th1 cytokines, chemokines and adhesion molecules The expression of Th2 cytokines, Th1 cytokines, chemokines and ▶ Fig. 3a–d, respectively. adhesion molecules are presented in ● The results demonstrated that AT significantly reduced the expression of Th2 cytokines (IL-4, IL-5 and IL-13) by leukocytes ▶ Fig. 3a; p < 0.001). The when compared with OVA group (● expression of Th1 cytokines (IL-2 and IFN-gamma) were not

▶ Fig. 3b; changed when compared all experimental groups (● p > 0.05). The results also demonstrated that AT significantly reduced the expression of chemokines (CCL11 and CCL5) when ▶ Fig. 3c; p < 0.01). In addition, AT compared with OVA group (● also significantly reduced the expression of adhesion molecules (VCAM-1 and ICAM-1) when compared with OVA group ▶ Fig. 3d; p < 0.01). (●

Vieira RP et al. Exercise Deactivates Leukocytes in Asthma. Int J Sports Med

Immunology

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Expression of oxygen and nitrogen reactive species, anti-inflammatory cytokine and NF-kB by peribronchial leukocytes ▶ Fig. 4a), The expression of Gp91Phox and 3-nitrotyrosine (● ▶ Fig. 4b), IL-10 (● ▶ Fig. 4c) and NF-kB (● ▶ Fig. 4d) are iNOS (● ▶ Fig. 4. The results demonstrated that AT signifipresented in ● cantly reduced the expression of Gp91Phox and 3-nitrotyrosine ▶ Fig. 4a; p < 0.001). The results also demonstrated that AT sig(● ▶ Fig. 4b; p < 0.001). On nificantly reduced the iNOS expression (● the other hand, AT in sensitized mice significantly increased the ▶ Fig. 4c; expression of anti-inflammatory cytokine IL-10 (● p < 0.01). In addition, AT also significantly reduced the NF-kB ▶ Fig. 4d; p < 0.001). expression (●

Peribronchial leukocytes derived growth factors ▶ Fig. 5a–d shows the expression of growth factors TGF-beta, ● IGF-1, VEGF and EGFr, respectively. The results demonstrated Vieira RP et al. Exercise Deactivates Leukocytes in Asthma. Int J Sports Med

that AT significantly reduced OVA-induced the expression of all growth factors investigated, as TGF-beta (p < 0.01), IGF-1 (p < 0.001), VEGF (p < 0.001) and EGFr (p < 0.01).

Discussion

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The present study showed for the first time that AT inhibit the lung leukocytes activation seen in an experimental model of allergic asthma, demonstrated through the reduced expression of cytokines, chemokines, adhesion molecules, reactive oxygen and nitrogen species, NF-kB and growth factors by peribronchial leukocytes, while increases the expression of the anti-inflammatory cytokine IL-10. Leukocytes play a central role in the pathophysiology of asthma [3, 17, 18, 20–22, 33]. Leukocytes, especially Th2 leukocytes are differentiated leukocytes responsible for release of Th2

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Fig. 4 Figure shows the expression of oxygen and nitrogen reactive species, anti-inflammatory cytokine and NF-kB by peribronchial leukocytes. In a, b and d, *p < 0.001 when compared with all groups. In c, *p < 0.01 when compared with all groups.

cytokines, i. e., IL-4, IL-5 and IL-13, which exert pro-inflammatory and pro-fibrotic effects on asthma [3, 17, 18, 20–22, 33]. In summary, IL-4, IL-5 and IL-13 are involved in eosinophils, dendritic cells and T-lymphocytes differentiation, proliferation and activation, exerting their effects both in the lungs as systemically [3, 17, 18, 20–22, 33]. In the present study we found that AT significantly reduced not only the expression of IL-4, IL-5 and IL-13 by leukocytes but also the BALF levels of IL-4, IL-5 and IL-13, strongly suggesting that the reduced expression of IL-4, IL-5 and IL-13 by lung leukocytes reflect leukocytes deactivation. Furthermore, the results also demonstrated that AT significantly reduced the serum levels of IL-5, showing that the effects of AT on allergic response is not limited to the lungs, but also may involve a systemic component. However, the results found in the present study may not be applied to circulating leukocytes, an issue that should be investigated in further studies. Beyond Th2 cytokines, chemokines, as CCL5 and CCL11 present an important role in chronic allergic airway inflammation [4]. These chemokines regulates eosinophils trafficking to the airways and are present at increased levels in the asthmatic airways and also related with late onset asthmatic response [4]. In the present study, we demonstrate that AT significantly reduced the expression of CCL5 and CCL11 by leukocytes, reinforcing the anti-inflammatory milieu induced by exercise. However, many other mediators are involved in the eosinophilic trafficking to the airways, as adhesion molecules. Adhesion molecules, i. e.,

VCAM-1 and ICAM-1 are well studied molecules in inflammatory diseases and are found abundantly in the airways of asthmatic patients and in animal models of asthma [8, 18, 40]. Increased expression of these molecules is thought to exert a central role in the eosinophils adhesion and transmigration during asthmatic inflammation [3]. Again, the present study shown that AT significantly reduced the expression of ICAM-1 and VCAM-1 by peribronchial leukocytes, accounting to the antiinflammatory effects of AT. Following unresolved chronic allergic airway inflammation, airway remodeling is a key feature of asthma and is thought to be irreversible and the main component of airway hyperresponsiveness and obstruction [21]. Airway remodeling is characterized by hypertrophy and hyperplasia of airway epithelial cells and smooth muscle, mucus hypersecretion and increased deposition of extra-cellular matrix proteins in airway walls [21]. Different proteins families are involved in the remodeling process in asthma, as growth factors (TGF-beta, IGF-1, VEGF and EGF), matrix metalloproteases (MMPs) and tissue inhibitor of matrix metalloproteases (TIMPs) [1, 16]. Of note, growth factors stimulate the synthesis of extra-cellular matrix proteins and are accredited to be the main mediators involved in remodeling [1, 16]. In the present study we show for the first time that AT inhibited the lung expression of TGF-beta, IGF-1, VEGF and EGFr in OVA-sensitized animals. Therefore, these results explicitly show that AT may inhibit the airway remodeling process. Vieira RP et al. Exercise Deactivates Leukocytes in Asthma. Int J Sports Med

Immunology

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Fig. 5 Figure shows the expression of growth factors by peribronchial leukocytes. In a and d, *p < 0.01 when compared with all groups. In b and c, *p < 0.001 when compared with all groups.

As part of the anti-inflammatory and anti-fibrotic effects of AT in chronic allergic airway inflammation, we have investigated the effects of AT on the expression of reactive oxygen species (ROS), reactive nitrogen species (RNS), anti-inflammatory cytokine IL-10 and nuclear transcription factor NF-kB. Regarding the role of ROS and RNS in the pathogenesis of asthma, the literature clearly demonstrates that increased ROS and RNS production modulates Th2 inflammatory and fibrotic response in asthma [33, 41]. In agreement with the current literature, the present study demonstrated that OVA sensitized animals presented increased expression of 3-nitrotyrosine and Gp91phox [33, 41]. On the other hand, AT significantly reduced their expression, confirming the inhibitory effects of AT on reactive oxygen and nitrogen species synthesis by leukocytes, which may be involved in these anti-inflammatory and anti-fibrotic effects of AT in asthma. In addition, a growing number of studies demonstrates that IL-10 present anti-inflammatory properties, by inhibiting the eosinoplilic inflammation and Th2 cytokines release, notably IL-4, IL-5 and IL-13 [17, 22]. In this way, the present results demonstrated that AT training significantly increased the expression of IL-10 by leukocytes as well as increased the levels IL-10 in the lung and also systemically. However, although we observe a strong stimulus from AT on IL-10 release by leukocytes, the exact molecular mechanisms of IL-10 mediating the anti-inflammatory effects of AT in asthma remains to be further investigated. Finally, we also investigated the effects of AT on NF-kB expression by peribronchial leukocytes. Several studies show increased NF-kB expression in airways of asthmatic

Vieira RP et al. Exercise Deactivates Leukocytes in Asthma. Int J Sports Med

patients and in animal models of asthma [8, 14, 15, 35, 38, 39]. These studies show that NF-kB controls not only the expression of pro-inflammatory cytokines, but also the expression of profibrotic mediators [8, 14, 15, 35, 38, 39]. Our study has confirmed that ovalbumin-induced chronic allergic lung inflammation is followed by increased expression of NF-kB in leukocytes. However, again, the results of the present study showed that AT significantly reduced NF-kB expression, possibly accounting as part of mechanisms involved in the anti-inflammatory and antifibrotic effects of AT in a mouse model of asthma. In conclusion, the present study shows that aerobic training reduces chronic allergic airway inflammation and remodeling in a mouse model of asthma and these results seem to be partially mediated by deactivation of peribronchial leukocytes.

Acknowledgements

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This study was supported by Fundação de Amparo à Pesquisa do Estado de São Paulo – FAPESP (Process 05/04413-1 and 07/01026-2) Laboratórios de Investigação Médica da Faculdade de Medicina da USP – LIM FMUSP, Conselho Nacional de Pesquisa e Desenvolvimento – CNPq and Coordenação de Pessoal de Nível Superior – CAPES.

Immunology References 1 Al-Muhsen S, Johnson JR, Hamid Q. Remodeling in asthma. J Allergy Clin Immunol 2011; 128: 451–462 2 American Association of Cardiovascular & Pulmonary Rehabilitation. Guidelines of Pulmonary Rehabilitation Programmes. 2nd ed. Human Kinetics Publishers, Champaign, Illinois, USA: 1998 3 Brooks AM, Bates ME, Vrtis RF, Jarjour NN, Bertics PJ, Sedgwick JB. Urokinase-type plasminogen activator modulates airway eosinophil adhesion in asthma. Am J Respir Cell Mol Biol 2006; 35: 503–511 4 Coleman JM, Naik C, Holguin F, Ray A, Ray P, Trudeau JB, Wenzel SE. Epithelial eotaxin-2 and eotaxin-3 expression: relation to asthma severity, luminal eosinophilia and age at onset. Thorax 2012; 67: 1061–1066 5 Disabella V, Sherman C. Exercise for asthma patients: little risk, big rewards. Phys Sportsmed 1998; 26: 75–84 6 Engström I, Fällström K, Karlberg E, Sten G, Bjure J. Psychological and respiratory physiological effects of a physical exercise programme on boys with severe asthma. Acta Paediatr Scand 1991; 80: 1058–1065 7 Fanelli A, Cabral AL, Neder JA, Martins MA, Carvalho CR. Exercise training on disease control and quality of life in asthmatic children. Med Sci Sports Exerc 2007; 39: 1474–1480 8 Ferreira SC, Toledo AC, Hage M, Santos AB, Medeiros MC, Martins MA, Carvalho CR, Dolhnikoff M, Vieira RP. Creatine activates airway epithelium in asthma. Int J Sports Med 2010; 31: 906–912 9 Gonçalves RC, Nunes MPT, Cukier A, Stelmach R, Martins MA, Carvalho CRF. Effects of an aerobic physical training program on psychosocial characteristics quality-of-life, symptoms and exhaled nitric oxide in individuals with moderate or severe persistent asthma. Rev Bras Fisioter 2008; 12: 127–135 10 Harriss DJ, Atkinson G. Update – ethical standards in sport and exercise science research. Int J Sports Med 2011; 32: 819–821 11 Hewitt M, Creel A, Estell K, Davis IC, Schwiebert LM. Acute exercise decreases airway inflammation, but not responsiveness, in an allergic asthma model. Am J Respir Cell Mol Biol 2009; 40: 83–89 12 Hewitt M, Estell K, Davis IC, Schwiebert LM. Repeated bouts of moderate-intensity aerobic exercise reduce airway reactivity in a murine asthma model. Am J Respir Cell Mol Biol 2010; 42: 243–249 13 Huang SW, Veiga R, Sila U, Reed E, Hines S. The effect of swimming in asthmatic children-participants in a swimming program in the city of Baltimore. J Asthma 1989; 26: 117–121 14 Imanifooladi AA, Yazdani S, Nourani MR. The role of nuclear factorkappaB in inflammatory lung disease. Inflamm Allergy Drug Targets 2010; 9: 197–205 15 Janssen-Heininger YM, Poynter ME, Aesif SW, Pantano C, Ather JL, Reynaert NL, Ckless K, Anathy V, van der Velden J, Irvin CG, van der Vliet A. Nuclear factor kappaB, airway epithelium, and asthma: avenues for redox control. Proc Am Thorac Soc 2009; 6: 249–255 16 Kelly EA, Jarjour NN. Role of matrix metalloproteinases in asthma. Curr Opin Pulm Med 2003; 9: 28–33 17 Ligeiro de Oliveira AP, Peron JPS, Lino-dos-Santos-Franco A, Acceturi BG, Vieira RP, Ibanez OM, Cabrera WHK, Franco M, Rizzo LV, Vargafitg BB, Tavares-de-Lima W. Ovariectomized OVA-sensitized mice display increased frequency of CD4 + Foxp3 + T regulatory cells in the periphery. Plos One 2013; 8: e65674 18 Ligeiro de Oliveira AP, Lino-dos-Santos-Franco A, Acceturi BG, Hamasato EK, Machado ID, Gimenes Júnior JA, Vieira RP, Damazo AS, Farsky SH, Tavares-de-Lima W, Palermo-Neto J. Long-term amphetamine treatment exacerbates inflammatory lung reaction while decreases airway hyper-responsiveness after allergic stimulus in rats. Int Immunopharmacol 2012; 14: 523–529 19 Livingstone JL, Gillespie M. Value of breathing exercises in asthma. Lancet 1935; 2: 705 20 Lowder T, Dugger K, Deshane J, Estell K, Schwiebert LM. Repeated bouts of aerobic exercise enhance regulatory T cell responses in a murine asthma model. Brain Behav Immun 2010; 24: 153–159 21 Mauad T, Bel EH, Sterk PJ. Asthma therapy and airway remodeling. J Allergy Clin Immunol 2007; 120: 997–1009 quiz 1010–1011

22 Mays LE, Ammon-Treiber S, Mothes B, Alkhaled M, Rottenberger J, Müller-Hermelink ES, Grimm M, Mezger M, Beer-Hammer S, Von Stebut E, Rieber N, Nürnberg B, Schwab M, Handgretinger R, Idzko M, Hartl D, Kormann MS. Modified Foxp3 mRNA protects against asthma through an IL-10-dependent mechanism. J Clin Invest 2013; 123: 1216–1228 23 Mendes FA, Almeida FM, Cukier A, Stelmach R, Jacob-Filho W, Martins MA, Carvalho CR. Effects of aerobic training on airway inflammation in asthmatic patients. Med Sci Sports Exerc 2011; 43: 197–203 24 Mendes FA, Gonçalves RC, Nunes MP, Saraiva-Romanholo BM, Cukier A, Stelmach R, Jacob-Filho W, Martins MA, Carvalho CR. Effects of aerobic training on psychosocial morbidity and symptoms in patients with asthma: a randomized clinical trial. Chest 2010; 138: 331–337 25 Moreira A, Delgado L, Haahtela T, Fonseca J, Moreira P, Lopes C, Mota J, Santos P, Rytilä P, Castel-Branco MG. Physical training does not increase allergic inflammation in asthmatic children. Eur Respir J 2008; 32: 1570–1575 26 Neder JA, Nery LE, Silva AC, Cabral AL, Fernandes AL. Short-term effects of aerobic training in the clinical management of moderate to severe asthma in children. Thorax 1999; 54: 202–206 27 Olivo CR, Vieira RP, Arantes-Costa FM, Perini A, Martins MA, Carvalho CRF. Effects of aerobic exercise on chronic allergic airway inflammation and remodeling in guinea pigs. Resp Physiol Neurobiol 2012; 182: 81–87 28 Pastva A, Estell K, Schoeb TR, Atkinson TP, Schwiebert LM. Aerobic exercise attenuates airway inflammatory responses in a mouse model of atopic asthma. J Immunol 2004; 172: 4520–4526 29 Pastva A, Estell K, Schoeb TR, Schwiebert LM. RU486 blocks the antiinflammatory effects of exercise in a murine model of allergen-induced pulmonary inflammation. Brain Behav Immun 2005; 19: 413–422 30 Pearson RS. The management of asthma. Br Med J 1950; 1: 1311–1314 31 Ram FS, Robinson SM, Black PN, Picot J. Physical training for asthma. Cochrane Database Syst Rev 2005 CD001116 32 Ramazanoglu YM, Kraemer R. Cardiorespiratory response to physical conditioning in children with bronchial asthma. Pediatr Pulmonol 1985; 1: 272–277 33 Sevin CM, Newcomb DC, Toki S, Han W, Sherrill TP, Boswell MG, Zhu Z, Collins RD, Boyd KL, Goleniewska K, Huckabee MM, Blackwell TS, Peebles RS Jr. Deficiency of gp91phox inhibits allergic airway inflammation. Am J Respir Cell Mol Biol 2013 [Epub ahead of print] 34 Silva ACD, Vieira RP, Nisiyama M, Santos ABG, Perini A, Mauad T, Dolhnikoff M, Martins MA, Carvalho CRF. Exercise inhibits allergic lung inflammation. Int J Sports Med 2012; 33: 402–409 35 Silva RA, Vieira RP, Duarte AC, Lopes FD, Perini A, Mauad T, Martins MA, Carvalho CR. Aerobic training reverses airway inflammation and remodeling in asthma murine model. Eur Respir J 2010; 35: 994–1002 36 Szentágothai K, Gyene I, Szócska M, Osváth P. Physical exercise program for children with bronchial asthma. Pediatr Pulmonol 1987; 3: 166–172 37 Vieira RP, Claudino RC, Duarte AC, Santos AB, Perini A, Faria Neto HC, Mauad T, Martins MA, Dolhnikoff M, Carvalho CR. Aerobic exercise decreases chronic allergic lung inflammation and airway remodeling in mice. Am J Respir Crit Care Med 2007; 176: 871–877 38 Vieira RP, de Andrade VF, Duarte AC, Dos Santos AB, Mauad T, Martins MA, Dolhnikoff M, Carvalho CR. Aerobic conditioning and allergic pulmonary inflammation in mice II: effects on lung vascular and parenchymal inflammation and remodeling. Am J Physiol 2008; 29: L670–L679 39 Vieira RP, Toledo AC, Ferreira SC, Santos ABG, Medeiros MC, Hage M, Mauad T, Dolhnikoff M, Martins MA, Carvalho CRF. Airway epithelium mediates the anti-inflammatory effects of exercise on asthma. Respir Physiol Neurobiol 2011; 175: 383–389 40 Walsh GM. Antagonism of eosinophil accumulation in asthma. Recent Pat Inflamm Allergy Drug Discov 2010; 4: 210–213 41 Yamamoto M, Tochino Y, Chibana K, Trudeau JB, Holguin F, Wenzel SE. Nitric oxide and related enzymes in asthma: relation to severity, enzyme function and inflammation. Clin Exp Allergy 2012; 42: 760–768

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