Angiotensin-(1-7)/Mas receptor 

Life Sciences 282 (2021) 119792

 journal homepage: www.elsevier.com/locate/lifescie

Angiotensin-(1-7)/Mas receptor modulates anti-inflammatory effects of exercise training in a model of chronic allergic lung inflammation

Juliana Fabiana Grego ́rio a, Giselle Santos Magalh ̃aes a, b, Maria Glo ́ria Rodrigues-Machado b, K ́ezia Emanoeli Ramos Gonzagaa,b, Daisy Motta-Santosa,c, Puebla Cassini-Vieiraa,

Lucíola Silva Barcelos a, Maria Aparecida Ribeiro Vieira a, Robson Augusto Souza Santos a, Maria Jose Campagnole-Santosa,*

a Department of Physiology and Biophysics, National Institute of Science and Technology in Nanobiopharmaceutics (INCT-Nanobiofar), Federal University of Minas Gerais, Belo Horizonte, MG, Brazil

b Post-Graduate Program in Healthy Sciences of Faculty of Medical Sciences of Minas Gerais, Belo Horizonte, MG, Brazil

c Sports Department, School of Physical Education, Physiotherapy and Occupational Therapy, Federal University of Minas Gerais, Belo Horizonte, MG, Brazil

 ARTICLEINFO

Keywords:

Renin-angiotensin system Pulmonary remodeling Immunoglobulin E

IL-10

IL-13 A779 Asthma

ABSTRACT

Aims: Exercise training increases circulating and tissue levels of angiotensin-(1-7) [Ang-(1-7)], which was shown to attenuate inflammation and fibrosis in different diseases. Here, we evaluated whether Ang-(1-7)/Mas receptor is involved in the beneficial effects of aerobic training in a chronic model of asthma.

Material and methods: BALB/c mice were subjected to a protocol of asthma induced by ovalbumin sensitization (OVA; 4 i.p. injections) and OVA challenge (3 times/week for 4 weeks). Simultaneously to the challenge period, part of the animals was continuously treated with Mas receptor antagonist (A779, 1 μg/h; for 28 days) and trained in a treadmill (TRE; 60% of the maximal capacity, 1 h/day, 5 days/week during 4 weeks). PGC1-α mRNA expression (qRT-PCR), plasma IgE and lung cytokines (ELISA), inflammatory cells infiltration (enzymatic activity assay) and airway remodeling (by histology) were evaluated.

Key findings: Blocking the Mas receptor with A779 increased IgE and IL-13 levels and prevented the reduction in extracellular matrix deposition in airways in OVA-TRE mice. Mas receptor blockade prevented the reduction of myeloperoxidase activity, as well as, prevented exercise-induced IL-10 increase. These data show that activation of Ang-(1-7)/Mas receptor pathway is involved in the anti-inflammatory and anti-fibrotic effects of aerobic training in an experimental model of chronic asthma.

Significance: Our results support exercise training as a non-pharmacological tool to defeat lung remodeling induced by chronic pulmonary inflammation. Further, our result also supports development of new therapy based on Ang-(1-7) or Mas agonists as important tool for asthma treatment in those patients that cannot perform aerobic training.

   1. Introduction

Several clinical studies suggest that regular physical exercise, as part of a pulmonary rehabilitation program, provides better control of asthma reducing hospital admissions [1,2]. Studies in murine models showed that exercise training may interfere with the migration pattern of Th2 cells after allergen challenge, reduces the sensitivity of these cells to chemoattractants [3], attenuates the release of cytokine Th2- associated [4,5], and reduces the expression of adhesion molecules

that facilitate migration of eosinophils and neutrophils to the airways [3]. Moreover, prevention of the increase in immunoglobulin E (IgE) levels and inhibition of signaling pathways that support leukocyte sur- vival in the inflammatory tissue were also described [5,6]. Clinical ev- idence showed that a regular physical activity program reduces the count of eosinophils in sputum, attenuates systemic inflammation and reduces bronchial hyperresponsiveness [1,7].

Physical exercise also downregulates the angiotensin converting enzyme (ACE)/angiotenisn II/angiotensin type 1 receptor (ACE/AngII/

 * Corresponding author at: Department of Physiology and Biophysics, Federal University of Minas Gerais, Av. Antoˆnio Carlos, 6627 – ICB, 31270-901 Belo Horizonte, MG, Brazil.

E-mail address: mjcampagnole@ufmg.br (M.J. Campagnole-Santos).

https://doi.org/10.1016/j.lfs.2021.119792

Received 6 April 2021; Received in revised form 16 June 2021; Accepted 29 June 2021

Available online 3 July 2021

0024-3205/© 2021 Elsevier Inc. All rights reserved.


J.F. Greg ́orio et al.

AT1) axis and upregulates the ACE2/angiotensin-(1-7)/Mas receptor [ACE2/Ang-(1-7)/Mas] pathway, currently known as the counter- regulatory pathway of the renin-angiotensin system (RAS) [8–12]. Aerobic training attenuated arterial hypertension by decreasing the content of Ang II and enhancing Ang-(1-7) in both left ventricle, aorta and rostral ventrolateral medulla of spontaneously hypertensive rats [8,10,12–14]. To the contrary, animals with genetic deletion of the Mas receptor, presented inappropriate response to swimming training, higher levels of circulating Ang II and increased deposition of type I and III collagen in the left ventricle [9]. Further, in a recent clinical study with healthy subjects, our group showed that oral administration of Ang- (1-7) attenuated skeletal muscle damage, reducing creatinine kinase levels and improving physical performance following eccentric exercise [15].

It has been shown that activation of Ang-(1-7)/Mas receptor pathway exerts an inhibitory effect in neutrophilic and eosinophilic inflammatory diseases, as well as anti-fibrotic and pro-resolutive actions [16–22]. In lung inflammation models, such as asthma and pulmonary emphysema, treatment with Ang-(1-7) decreased the synthesis of cytokines and chemokines, the migration of inflammatory cells to the lung, pulmonary fibrosis and airway hyperresponsiveness [17,18,23]. More recently, we have also described that Ang-(1-7) triggers pro-resolutive mechanisms in inflammatory processes [16,19]. In the lung, Mas receptor is expressed in the smooth muscle of the epithelium and airways, alveolar cells, vascular smooth muscle cells and endothelium [12,16,18]. Mas receptor has also been identified in cells of the immune system, such as dendritic cells, lymphocytes, macrophages, eosinophils, neutrophils and alveolar macrophages, indicating a cellular mechanism for Ang-(1-7) actions in the immune system [12,16,19]. Therefore, the objective of the present study was to evaluate whether part of the beneficial effects triggered by aerobic training in asthma could be related to Ang-(1-7)/ Mas receptor pathway.

2. Material and methods

2.1. Animals

Male BALB/c mice (8 weeks of age, weighing 25–30 g) from the Animal Facility of our Institution (Centro de Bioterismo - CEBIO, Bio- logical Sciences Institute, Federal University of Minas Gerais were housed under a 12/12-hour light-dark cycle with free access to standard chow and water. Animals were randomly assigned to four groups: (i) saline-sensitized and saline-challenged, control group (CTRL-SED; n =

Life Sciences 282 (2021) 119792

8); (ii) ovalbumin (OVA)-sensitized and OVA-challenged (OVA-SED; n = 8); (iii) OVA-sensitized and OVA-challenged and trained in a treadmill (OVA-TRE; n = 8); (iv) OVA-sensitized and OVA-challenged and trained in a treadmill and treated with selective Mas receptor antagonist, A779 (OVA-TRE-A779; n = 8). The animal use ethics committee (CEUA) of the Federal University of Minas Gerais, Brazil approved all animal care and experimental procedures in this study (protocol 11/2014).

2.2. Chronic allergic lung inflammation

Male BALB/c mice (8 weeks of age) were sensitized by intraperito- neal (i.p) injections of ovalbumin (OVA; 20 μg/animal) diluted in saline (0.5 ml NaCl 0.9%) on days 0, 14, 28 and 42 (Fig. 1), as previously described [17,22]. At the 21st day animals were subjected to OVA (1%) inhalation diluted in NaCl (0.9%), three times a week, with a duration of 30 min per session, until the 46th day. Inhalation was performed in acrylic boxes (30 cm × 15 cm × 20 cm) attached to an Ultrasonic nebulizer (Soniclear Pulmosonic Star; Frequency: 50/60 Hz). Non- sensitized animals (controls) received intraperitoneal saline (0.5 ml, 0.9% NaCl) and inhaled saline (NaCl, 0.9%), at the same days and duration as asthmatics animals. Seventy-two hours after the last inha- lation animals were euthanized, tissues and blood samples were collected, frozen and stored at − 80 ◦C for further analysis.

2.3. Aerobic training

Aerobic training (TRE) and maximum effort test (MET) were per- formed as previously described [4]. Briefly, animals were initially adapted to a treadmill for 3 days (15 min at a speed of 0.2 km/h). After the adaptation period and, to establish the intensity of training, animals were subjected to a MET. The test was consisted in 5 min adaptation to the treadmill (0.2 km/h) followed by a progressive increase in treadmill speed (0.1 km/h every 2.5 min). The test was ended when the animals reached fatigue, characterized by inability to continue running even after light mechanical stimulus on the rear back of the animals. MET was repeated after the 2nd week of training to adjust the exercise load and at the end of the protocol, 47th day. Exercise training was performed at an intensity corresponding to 60% of maximum speed reached at MET. Thus, animals were trained at moderate intensity for 1 h/day, 5 days a week, beginning on the 21st day of the protocol along with the start of nebulization with OVA, until the 46th day of the protocol. Training was always performed in the morning (8 a.m.–12 p.m.) and the inhalation in the afternoon (2 p.m.–6 p.m.), to insure an interval of at least 4 h

 Fig. 1. Experimental protocol. In order to induce chronic allergic lung inflammation, animals were sensitized by intraperitoneal (i.p.) injections of ovalbumin (OVA; 20 μg/animal diluted in saline) on days 0, 14, 28 and 42. Challenges with ovalbumin (OVA, 1%) were performed 3 times per week starting on day 21 until day 46. Chronic inhibition of the Mas receptor was made by chronic infusion of the Mas receptor antagonist, A779 (1 μg/h for 28 days with osmotic mini-pumps). Maximal effort test (MET) was performed on 19th, 34th and 47th day of the protocol. Aerobic training on a treadmill (1 h a day, 5 days/week) was performed at 60% of the maximum effort time, starting on day 21st (a total of 20 sessions). Animals were euthanized 72 h after the last challenge with OVA.

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J.F. Greg ́orio et al. between procedures. 2.4. A779 treatment

To achieve chronic inhibition of the Mas receptor, on the 19th day of the experimental protocol, animals were anesthetized (i.p.) with keta- mine solution (60–80 mg/kg) associated with xylazine (8–15 mg/kg). Next, an osmotic mini-pump (Alzet® model 1004; ALZET, CA, USA) filled with the selective Mas antagonist, A779, was implanted in the interscapular region of the back of animal, accordingly to manufacturer instructions. A779 infusion rate was 1 μg/h and lasted for 28 days in OVA-TRE-A779 group. Animals from OVA-TRE group received mini- pumps filled with 0.9% NaCl solution. Sedentary groups (CTRL-SED and OVA-SED) were subjected to a sham surgery consisting of all sur- gical procedures except pump implantation.

2.5. Blood and tissue collection

Seventy-two hours after the last inhalation of OVA, animals were deep anesthetized (i.p.) with ketamine (60–80 mg/kg) associated with xylazine (8–15 mg/kg). Next, the trachea was exposed and isolated with a suture thread. Blood was collected from the jugular vein and imme- diately after, animals were subjected to sternal thoracotomy to lungs removal. Left lung was fixed in 10% buffered formalin. The soleus muscle of the right paw and the lungs were collected, placed into polyethylene tubes, and immediately frozen on dry ice and then stored at− 80◦C.

2.6. Levels of serum antibody

Serum samples were obtained on 49th day (at the end of protocol). Anti-OVA IgE antibodies were measured by capture-ELISA (IgE Mouse Elisa kit, Shibayagi, Shibukawa, Gunna, Japan) using plates coated with antibody anti-IgE specific for OVA.

2.7. Cytokines evaluation

The level of cytokines was measured in right lung using commercial ELISA kits (R&D Systems, Minneapolis, MN, USA). Briefly, the right lung was homogenized with phosphate buffered saline (PBS) that contained proteases inhibitors: 0.1 mM of Phenylmethanesulfonyl fluoride, 0.1 nM of Benzethonium chloride, 10 mM of EDTA, 20 units of Kallikrein in- hibitor, Aprotinin A and 0.05% Tween 20. Samples were centrifuged for 10 min at 10,000 rpm and 4 ◦C. After centrifugation, the supernatant was properly collected. Cytokines concentration was measured accord- ing to the manufacturer procedures (R&D Systems, Minneapolis, MN, USA). Colorimetric reactions were analyzed with a spectrophotometer at a wavelength of 492 nm.

2.8. Morphometric analysis

Lungs were fixed in formalin and embedded in paraffin as previously described [17,20]. Briefly, lung sessions (4 μm) were stained with hae- matoxylin and eosin (H&E) for structural analysis or stained with Gomori's trichrome to evaluate extracellular matrix deposition in the lungs.

2.9. PGC1-α mRNA expression

Total RNA from soleus muscle was extracted using TRIzol reagent (Invitrogen, San Diego, CA, USA), treated with DNAse (RNase-free; Invitrogen®) and reverse transcribed with Moloney Murine Leukemia Virus Reverse Transcriptase (M-MLV RT; Promega, Madison, WI, USA). The endogenous GAPDH (internal control), PGC1-α cDNA was amplified using specific primers and SYBR green reagent (Applied Biosystems, Foster City, CA, USA) in ViiATM 7 System (Applied Biosystems ®). The

Life Sciences 282 (2021) 119792

relative comparative CT method was used to verify gene expression levels using the equation 2− ΔΔCT [24].

2.10. Histological score of inflammation

Inflammatory cells were assessed in 10 fields from peribronchial, perivascular and parenchymal lung regions and estimated by a semi- quantitative method, as previously described [17,20]. The following score was used in this evaluation: 1 = no inflammation, 2 = mild inflammation, 3 = moderate inflammation, 4 = severe inflammation. For this analysis, slides were randomly numbered and evaluated by a different researcher, to assure a double-blind assessment.

2.11. Determination of EPO, MPO and NAG enzymatic activity

Content of eosinophils, neutrophils and macrophages in the lung was assessed by enzymatic assays measuring eosinophil peroxidase (EPO), myeloperoxidase (MPO) and N-acetyl-b-D-glycosaminidase (NAG) ac- tivity, respectively, as previously described [16,25]. Absorbance was read in an ELISA reader (Expert Plus ASYS Hitech GmbH, Eugenorf, Austria) at 492 nm (EPO), 450 nm (MPO) and 400 nm (NAG). Values are expressed in optical density (O.D.).

2.12. Statistical analysis

Data were expressed as mean ± standard error of the mean (SEM). One-way ANOVA followed by Tukey's multiple comparison test and, for parametric data, Kruskal-Wallis ANOVA followed by multiple compar- isons of Dunn test were used as appropriated and denoted in figure legends. The significance level was set at p < 0.05. Graphics and analysis were performed with Graphpad Prism software (CA, USA; Version 9.2.1).

3. Results

3.1. Markers of exercise training

Fig. 2 shows mRNA expression of PGC1-α in the soleus muscle. PGC1- α is a transcriptional co-activator described as a master regulator of biogenesis and mitochondrial function, being considered a marker of exercise training [26]. Trained groups, OVA-sensitized and challenge, treated (1.88 ± 0.53 a.u; Fig. 2A) or not with A779 (1.85 ± 0.15 a.u; Fig. 2A) showed a significant increase in PGC1-α gene expression in comparison to CTRL-SED (1.21 ± 0.22 a.u; Fig. 2A). OVA-SED mice did not present alteration in PGC1-α mRNA levels (0.97 ± 0.16 a.u; Fig. 2A). Physical performance was evaluated in mice comparing the maximal effort time achieved before and at the end of the protocol (47th day). There was no significant difference among the groups in the 1st test, before OVA challenge/exercise training (Fig. 2B). Exercise training increased the maximal effort time in OVA-TRE (41.18 ± 1.5 min vs 33.41 ± 2.2 min, before; Fig. 2B), which was 25% higher than the time achieved before training. OVA-SED, on the other hand, presented a decreased time at the end of the protocol (29.65 ± 2.31 min vs 33.44 ± 2.4 min, before; Fig. 2B) corresponding to a 11% fall in maximal effort time. CTRL-SED animals also presented an increase in maximum effort time (36.59 ± 2.4 min; Fig. 2B), around 11% of the time before protocol (32.93 ± 2.13 min), which was approximately half of the time induced by training. OVA-TRE mice treated with A779, however, presented a 16% increase in maximal effort time (43.65 ± 1.53 vs 38.28 ± 1.7 min; Fig. 2B), a value that was lower than that presented in OVA-TRE mice, but not significantly different from CRTL-SED (Fig. 2C).

3.2. Serum levels of IgE

As expected, plasma levels of OVA-specific IgE, a key factor in the onset and spread of the inflammatory cascade in asthma, were elevated

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J.F. Greg ́orio et al. Life Sciences 282 (2021) 119792

 Fig. 2. A- Expression of PGC1-α mRNA in the soleus muscle (a.u.); B- Maximal time (in minutes) and C- Alteration in maximal time (in %) obtained in the maximum effort test (minutes) in control sedentary (CTRL-SED), OVA groups (OVA-SED, OVA- (OVA-TRE) and OVA-TRE treated with A779 (OVA-TRE-A779). *p < 0.05 in comparison to CTRL-SED, #p < 0.05 in comparison to OVA-SED (One way ANOVA followed by Tukeys' multiple comparison test; n = 4–7; panels A and C). *p < 0.05 compared to the 1st test, before OVA challenge (Student t test for paired observations; n = 6–7; panel B).

in the OVA-SED group (13 ± 6.6 U/ml; Table 1). IgE levels in OVA-TRE mice (0.00 ± 0.0 U/ml) were similar to CTRL-SED (0.00 ± 0.0 U/ml). However, IgE levels were also surprisingly high in the OVA-TRE-A779 group (23 ± 9.6 U/ml). These data show that Mas receptor blockade prevented the effect of chronic aerobic exercise on the suppression of OVA-specific IgE synthesis in mice sensitized and exposed to OVA.

3.3. Lung accumulation of leukocytes

To evaluate whether Mas receptor modulated the anti-inflammatory effects of exercise on inhibition and migration of inflammatory cells, we measured accumulation of neutrophils, eosinophils and macrophages in lung tissue, by determining the activity of the enzymes, MPO, EPO and NAG, respectively (Fig. 3). Aerobic training in OVA mice was able to inhibit the increase in MPO (1.02 ± 0.06 O.D./mg of tissue vs 1.55 ± 0.16 O.D./mg of tissue, in OVA SED group) and EPO activities (0.24 ± 0.03 O.D./mg of tissue vs 0.38 ± 0.03 O.D./mg of tissue, in OVA-SED group; Fig. 3A–B). OVA-TRE-A779 mice presented increased MPO ac- tivity (1.41 ± 0.12 O.D./mg of tissue vs 0.87 ± 0.12 O.D./mg of tissue, in CTRL-SED; Fig. 3A). Although we observed a trend, the enzymatic ac- tivity of EPO was not significantly changed in OVA-TRE-A779 (0.30 ± 0.04 O.D./mg of tissue vs 0.23 ± 0.04 O.D./mg of tissue, in CTRL-SED; Fig. 3B). There was no significant change in NAG activity in the lung in any group (Fig. 3C).

3.4. Pulmonary level of cytokines

Seventy-two hours after last OVA challenge IL-5 (825.5 ± 18.36 pg/ mg; Fig. 3A) was higher in OVA-SED mice. Although there was a trend to be increased, levels of IL-4 (35.46 ± 26.1 pg/mg vs 0.00 ± 0.0 pg/mg, in CTRL-SED; Fig. 3C) and IL-13 (11.18 ± 1.23 pg/mg vs 0.015 ± 0.015 pg/ mg, in CTRL-SED; Fig. 3B) were not significantly higher in the lung of OVA-SED mice. Exercise training did not significantly alter these cyto- kines (Fig. 4A–C), however, it increased the anti-inflammatory cytokine, IL-10 (81.7 ± 18.7 pg/mg; vs 20.6 ± 3.3 pg/mg, in CTRL-SED; Fig. 4D). Treatment with A779 did not significantly change IL-5 and IL-4, how- ever, OVA-SED-A779 mice presented a large increase in IL-13 (27.9 ± 6.28 pg/mg; Fig. 4 B) associated to a significant decrease in IL-10 (25.98 ± 9.75 pg/mg) in comparison to OVA-TRE mice (8.93 ± 4.78 pg/mg and 81.71 ± 18.72 pg/mg, respectively; Fig. 4B and D).

3.5. Pulmonary remodeling

Fig. 5 presents representative images showing extracellular matrix deposition in lung sections stained with Gomori's trichrome. Exercise training significantly prevented extracellular matrix deposition (17.41 ± 2.05% vs 34.37 ± 4.06% in OVA-SED; Fig. 5A–C). This effect was completely blocked by Mas antagonist (32.22 ± 0.96%; Fig. 5D and E). Further, as can be seen in Fig. 6, aerobic training prevented alveolar wall thickening induced by OVA challenge (1.87 ± 0.09 μm2 vs 2.60 ± 0.22 μm2 in OVA SED group), suggesting an anti-remodeling effect of exercise in the alveolar parenchyma (Fig. 6A–C and E). A779 prevented the effect of physical exercise in alveolar wall thickening (3.01 ± 0.32 μm2 vs 1.87 ± 0.09 μm2 in OVA-TRE group; Fig. 6D–E). Furthermore, exercise training significantly attenuated total cell infiltration in the lungs, evaluated by histological score (1.79 ± 0.18 a.u vs 3.38 ± 0.28 a.u in OVA-SED group). This attenuating effect of exercise was blocked by A779 (3.08 ± 0.23 a.u vs 1.79 ± 0.18 in OVA-TRE group; Fig. 6F).

4. Discussion

In the present study, we showed that pharmacological blockade of the Mas receptor attenuated beneficial effects of aerobic training in a model of chronic allergic lung inflammation. Treatment of trained OVA- sensitized and challenged mice with Mas receptor antagonist, A779, prevented the reduction in: (i) serum IgE levels; (ii) extracellular matrix deposition in airways; (iii) alveolar wall thickening; and (iv) MPO

Table 1

Serum levels of specific IgE to OVA.

Groups

CTRL-SED OVA-SED OVA-TRE OVA-TRE-A779

IgE, U/ml

0.0±00 13±6.6 0.0±00 23±10*

    Data are presented as mean ± SEM. CTRL = control, SED = sedentary, OVA = ovalbumin sensitized and challenge, TRE = trained and A779 = Mas receptor antagonist treated mice. n = 5–6.

* p < 0.05 in comparison to OVA-TRE (ANOVA Kruskal-Wallis followed by Dunn's multiple compari- son test).

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enzymatic activity. In addition, Mas antagonist induced an increase in IL-13 and prevented the exercise-induced increase in the anti- inflammatory cytokine, IL-10 in the lung.

In this study, we demonstrated for the first time that activation of Mas receptor is necessary to modulate the effect of physical training on the reduction of circulating IgE levels in OVA-sensitized and challenged mice. In previous study, we showed that chronic infusion with Ang-(1-7) attenuated the increase in IgE levels [17,20] in a similar model of chronic asthma. Few studies have investigated the modulatory effect of physical training on IgE levels in asthma. Our result is in agreement with Pastva et al. [5], that showed reduced levels of OVA-specific IgE in challenged trained mice. Mechanisms triggered by chronic exercise to blunt IgE synthesis/release have yet to be elucidated. However, the data of the present study show that physical training is attenuating the installation of the Th2 immune response and Ang-(1-7)/Mas receptor pathway plays an important role in this process.

Aerobic training can modulate the immune response in models of chronic allergic lung inflammation, reducing the expression of Th2- related cytokines, such as IL-5, IL-4 and IL-13, and increasing levels of the anti-inflammatory cytokine, IL-10 [4,27]. Yeh et al. [28], showed that physical activity increases the circulating number of regulatory T cells (Treg), the major source of IL-10 [29], and their ability to release immunomodulatory cytokine IL-10 and the transforming growth factor beta (TGF-β) [28]. Herein, 72 h after the last of OVA challenge, OVA- TRE mice presented a trend to decrease IL-4 and IL-5, associated with a significant increase in pulmonary levels of IL-10, supporting an anti- inflammatory role induced by exercise in murine model of asthma. Mas receptor antagonist completely prevented IL-10 increase in OVA- TRE-A779 mice. In previous studies, we [22,23] and others [12,18,30] have shown that activation of Mas receptor produces anti-inflammatory effects that involve positive regulation of IL-10 in inflammatory disor- ders. In addition, blocking the Mas receptor, with A779, abolished the effects of Ang-(1-7) in mice with allergic pulmonary inflammation (35). Furthermore, exercise training favors the activation ACE2/Ang-(1-7)/ Mas pathway [8,10,11,31]. Together, these data indicate that Ang-(1- 7)/Mas receptor pathway can be involved in exercise-induced immu- noregulatory effects in allergic lung inflammation.

Additionally, we have also observed a significant increase in IL-13 levels in OVA-TRE mice treated with A779. This data is consistent with previous study showing an important increase in IL-13 in Mas KO mice [21]. The IL-13 is an important Th2-associated cytokine involved in pulmonary fibrosis [32]. It has been demonstrated that airway fi- broblasts increase their ability to release type I collagen in the presence of IL-13 [33]. Furthermore, this cytokine activates metalloproteinase 2 (MMP2), an important enzyme that participates in the airway remod- eling process [33]. IL-13 is also required for the heavy-chain class switching from IgM to IgE. In fact, the high levels of IgE in OVA-TRE- A779 mice correlate with a significant increase (~2 folds) in IL-13 in the lung of these mice. Thus, these data show that processes triggered by the activation of the Mas receptor might attenuate fibrotic pathways involved with airway remodeling in asthma. Moreover, activation of the Mas receptor in physical training is essential to mitigate the processes involved in pulmonary fibrosis due to allergic lung inflammation.

The release of cytokines and chemokines in asthma induces increased expression of several adhesion molecules, increasing adhesion interactions between endothelial cells and circulating inflammatory cells allowing their passage into inflamed lung tissue [34]. Here, we evaluated the migration of inflammatory cells to the airways by quan- tifying enzymes produced during their stay in the tissues, as a result of a constant inflammatory stimulus. Our data showed that the decrease in MPO activity in the lungs promoted by physical training was prevented by A779 treatment. There was a trend to prevent the effect of exercise on EPO activity; however, it did not reach statistical significance. In addi- tion, with histological score analysis we showed that the inhibition on lung cell infiltrate induced by exercise training was attenuated by Mas antagonist, A779. These data are in agreement with other [35] showing

 Fig. 3. Enzymatic activity of myeloperoxidase (MPO, O.D./mg wet tissue; A), Eosinophil Peroxidase (EPO, O.D./mg wet tissue; B) and N-acetyl-β-D-glyco- saminidase (NAG, nmol/mg tissue; C) in the lungs of control sedentary (CTRL- SED), OVA groups (OVA-SED, OVA- (OVA-TRE) and OVA-TRE treated with A779 (OVA-TRE-A779). *p < 0.05 significantly different from CTRL-SED groupand #p < 0.05 significantly different from OVA-SED group (=4–7) (One way ANOVA followed by Tukeys' multiple comparison test).

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 Fig. 4. Pulmonary levels of IL-5 (A), IL-13 (B), IL-4 (C) and IL-10 (D) in control sedentary (CTRL-SED), OVA groups (OVA-SED, OVA- (OVA-TRE) and OVA-TRE treated with A779 (OVA-TRE-A779) (n = 4–7). *p < 0.05 in comparison with CTRL-SED, and &p < 0.05 in comparison with OVA-TRE (One way ANOVA fol- lowed by Tukeys' multiple comparison test).

that in a short-term model of asthma, Ang-(1-7) via Mas receptor sup- pressed the effect of phytohaemagglutinin P (PHA) inducing cell pro- liferation in human peripheral blood mononuclear cells. In the same study, A779 treatment abolished the inhibitory effect of Ang-(1-7) on the accumulation of eosinophils, neutrophils and lymphocytes in the lung of OVA-mice [35]. In our study, we have to point out that tissue was collected 72 h after the last challenge in a long term model by OVA sensitization and challenge (4 weeks), which might have reflected in the number and profile of cells observed at the time point studied.

Physical exercise is associated with the RAS modulation, increasing the expression of components of the protective arm of the system, mitigating inflammation [31]. A limitation of our study was not to have measured plasma and pulmonary levels of Ang-(1-7). However, in pre- vious studies, it was shown that sensitization and challenge with OVA reduces the Ang-(1-7) levels and the expression of the receptor Mas in the lung [17,22]. Ang-(1-7)/Mas receptor pathway presents immune modulatory effects in different inflammatory models, as well as, atten- uation of skeletal muscle damage and improvement of physical perfor- mance in men subjected to eccentric exercise. However, mice with genetic deletion of Mas receptor presented inappropriate cardiac response to exercise training [9] and aggravated inflammation and remodeling induced by asthma [21]. Mas KO mice subjected to exercise training presented increased ventricular expression of extracellular matrix components, collagen I and collagen III [9]. Accumulated evi- dence suggests that in pathological conditions such as hypertension and heart failure, the beneficial effects of physical training are associated

with positive modulation of ACE2/Ang-(1-7)/Mas axis [8,10,11,31,36], together with a reduction in pro-inflammatory cytokines such as TNF-α and IL-1β [31]. These data demonstrate that exercise induced beneficial effects are mediated, at least in part, by Ang-(1-7)/Mas pathway. Data of the present study advances this knowledge by showing that one mech- anism by which exercise training may improve outcomes of asthma is related to Ang-(1-7)/Mas. It is important to point out that our study was based on the administration of A779, a selective antagonist of the Mas receptor [37,38]. A779 was not shown to antagonize the actions of Alamandine or C21 on MrgD and AT2 receptors, respectively [39,40]. Thus it is unlikely that the effects observed in our study are related to a non-specific effect of A779 on other angiotensin receptors.

Pulmonary remodeling is a key component of worsening asthma and reduced lung function over time [41]. A consistent finding in our study was that aerobic training prevented the deposition of extracellular ma- trix in OVA group and this anti-fibrotic effect was inhibited by A779 treatment. In keeping, studies have shown that airway remodeling can be attenuated, or even reversed, by aerobic training [4,27,42]. Our data advanced by showing that Ang-(1-7)/Mas receptor is importantly involved in attenuating inappropriate remodeling, induced by OVA challenge.

Physical training increases exercise capacity or performance while pulmonary inflammation/remodeling contributed to exercise intoler- ance and reduction in the aerobic capacity of asthmatic animals. In- dividuals with chronic lung disease are more susceptible to present reduction in exercise tolerance and skeletal muscle dysfunction [43–45],

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J.F. Greg ́orio et al. Life Sciences 282 (2021) 119792

 Fig. 5. Representative histological images of lung sections stained with Gomori's trichrome in control sedentary group (CTRL-SED; A), OVA groups: OVA-SED (B), OVA-TRE (C) and OVA-TRE treated with A779 (OVA-TRE-A779; D). As can be seen, OVA-SED mice presented a pronounced deposition of extracellular matrix (asterisks) in the peribronchial and perivascular regions compared to CTRL-SED, which was attenuated by physical training (OVA-TRE). Mas receptor antagonist prevented the effect of exercise on extracellular matrix deposition (OVA-TRE-A779). In E, percentage of extracellular matrix deposition in the lung parenchyma. Bars = 100 μm. n = 4–5; *p < 0.05 in comparison with CTRL-SED, #p < 0.05 in comparison with OVA-SED and &p < 0.05 in comparison with OVA-TRE (One way ANOVA followed by Tukeys' multiple comparison test).

contributing to a sedentary lifestyle and reduction in aerobic capacity. In agreement, in our study OVA-SED mice presented pulmonary inflam- mation and remodeling and worsening of functional capacity evaluated by a 11% decrease in maximum effort time at the end of the protocol. Exercise training improved physical capacity and, OVA-TRE showed an increase in maximal effort time around 25% of the initial time. Blocking Mas receptor in training mice attenuated maximal effort time and, OVA- TRE-A779 maximal effort time at the end of the protocol was increased in 16%. This value was lower than that achieved by OVA-TRE and not significantly different from CTRL-SED. These data showed that A779 did not completely prevent the increase in animal performance in the

physical test, despite these mice having important pulmonary inflam- mation and airway remodeling. Ang-(1-7)/Mas pathway is an important anti-inflammatory and anti-fibrotic mechanism triggered by exercise to fight allergic pulmonary inflammation, however, other mechanisms induced by training, such as increased levels of hormones and adrena- line, muscle adaptations, factors released by the muscle [46–48] can have accounted for the improvement in physical capacity and have prevented the fall in maximal effort time in OVA-TRE-A779. We also observed that CTRL-SED mice showed an increase in 11% in the maximum effector time at the end of the protocol probably due to the natural gain in muscle mass with growth, since protocol was started in

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J.F. Greg ́orio et al. Life Sciences 282 (2021) 119792

 Fig. 6. (A–D) Representative histological images of lung sections stained with H&E from group control sedentary (CRTL-SED), OVA groups (OVA-SED), OVA- (OVA- TRE) and OVA-TRE treated with A779 (OVA-TRE-A779). OVA induced significant infiltration of inflammatory cells in the peribronchial, perivascular and alveolar regions (B), which was prevented by physical training (OVA-TRE; C). A779 treatment prevented exercise training effect (OVA-TRE-A779; D). The accumulation of cells in the lung tissue resulted in thickening of interalveolar interstitial area (asterisk), arrows show vascular remodeling (B and D). Measurement of alveolar wall thickening (E, μm2). Bars = 100 μm. n = 5 each. Panel F presents box plot with mean and 10–90 percentile of histological scores of pulmonary tissue showing inflammatory cell infiltrate in all groups (n = 4–5). *p < 0.05 significantly different from CTRL-SED group, #p < 0.05 significantly different from OVA-SED group and &p < 0.05 significantly different from OVA-TRE group (One way ANOVA followed by Tukeys' multiple comparison test).

animals with 8 weeks old and finished with 12 weeks old. Despite having a longer time in the test, the levels of PGC1-α data show that these an- imals were not trained. On the other hand, both OVA-TRE and OVA- TRE-A779 mice presented increase in PGC1-α levels.

In summary, the present study demonstrated for the first time, that Mas receptor activation is necessary for the manifestation of immune modulatory events induced by exercise training in an experimental model of chronic asthma. Mas receptor blockade in these animals pre- vented the attenuation in inflammation, lung remodeling and the reduction of IgE induced by aerobic training. Our data advanced in the understanding of the mechanisms triggered by physical exercise, showing that activation of the RAS protective arm is essential to mediate

the anti-inflammatory and anti-fibrotic effects of exercise in experi- mental asthma. Our results reinforce once more that regular practice of physical exercise is an important adjunct in the treatment of asthmatic patients. Furthermore, it opens the perspective that development of new therapy based on Ang-(1-7) or Mas agonists may be beneficial for treatment of asthmatic patients that cannot perform aerobic training.

Data availability statement

The data that support the findings of this study are available from the corresponding authors upon reasonable request.

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J.F. Greg ́orio et al. Funding

This work was supported by Fundaça ̃o de Amparo `a Pesquisa do Estado de Minas Gerais (FAPEMIG), Conselho Nacional de Desenvolvi- mento Científico e Tecnolo ́gico (CNPq) Universal grants to M.J.C.-S. and Coordenaça ̃o de Aperfeiçoamento de Pessoal de Nível Superior (CAPES). JFG was a recipient of FAPEMIG MSc and CNPq PhD fellowships at Post- Graduate Program in Biological Sciences: Physiology and Pharma- cology, ICB, UFMG. M.J.C.-S., L.S.B. and R.A.S.S. are recipients of Research Fellowships from CNPq.

CRediT authorship contribution statement

J.F.G: Conception and design, acquisition of data, analysis and interpretation of data, drafting and revising the manuscript. GSM: Conception and design, acquisition of data, analysis and interpretation of data. K.E.R.G., D.M-S., P.C-V., L.B. and M.A.R.V.: Acquisition of data, analysis and interpretation of data. M.G.R-M. and R.A.S.S.: Analysis and interpretation of data and project funding. M.J.C-S.: Conception and design, analysis and interpretation of data, drafting, editing and revising manuscript and project funding. All authors approved the final version of the manuscript.

Declaration of competing interest

Nothing to declare.

Acknowledgements

The authors are thankful to Jose Roberto da Silva and Alessa Flavia da Silva for technical assistance.

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