Training Modifies Innate Immune Responses in Blood ... - ATS Journals

ORIGINAL RESEARCH Training Modifies Innate Immune Responses in Blood Monocytes and in Pulmonary Alveolar Macrophages Linda Frellstedt1,2,7*, Ingrid Waldschmidt2,7*, Philippe Gosset3–8, Christophe Desmet8,11, Dimitri Pirottin8,11, Fabrice Bureau8,11, Fred ´ eric ´ Farnir9, Thierry Franck10, Marie-Capucine Dupuis-Tricaud2,11, Pierre Lekeux1,11, 1,11 and Tatiana Art 1

Center of Equine Sports Medicine, University of Liege, Liege, Belgium; 2CIRALE, National Veterinary School of Alfort, Goustranville, France 3Institut Pasteur de Lille, Centre d’Infection et d’Immunite de Lille, Lille, France; 4Universite Lille Nord de France, Lille, France; 5Centre National de la Recherche Scientifique, Lille, France; 6Institut National de la Sante et de la Recherche Medicale, Lille, France; 7Institut Federatif de Recherche 142, Lille, France; 8Laboratory of Cellular and Molecular Immunology, GIGA-Research, 9 Department of Animal Production, and 10Centre for Oxygen, Research & Development (CORD), Institute of Chemistry, University of Liege, Liege, Belgium; and 11Hippolia Foundation, Caen, France

Abstract In humans, strenuous exercise causes increased susceptibility to respiratory infections associated with down-regulated expression of Toll-like receptors (TLRs) and costimulatory and antigen-presenting molecules. Lower airway diseases are also a common problem in sport and racing horses. Because innate immunity plays an essential role in lung defense mechanisms, we assessed the effect of acute exercise and training on innate immune responses in two different compartments. Blood monocytes and pulmonary alveolar macrophages (PAMs) were collected from horses in untrained, moderately trained, intensively trained, and deconditioned states before and after a strenuous exercise test. The cells were analyzed for TLR messenger ribonucleic acid (mRNA) expression by real-time PCR in vitro, and cytokine production after in vitro stimulation with TLR ligands was measured by ELISA. Our results showed that training, but not acute exercise, modified the innate immune responses in both compartments. The mRNA expression of TLR3 was down-regulated by training in both cell types, whereas the expression of TLR4 was up-regulated in monocytes. Monocytes

The relationship between exercise and infection has been explored extensively in animal models and in human studies (1–5), but information regarding the effect of exercise or regular training on

treated with LPS and a synthetic diacylated lipoprotein showed increased cytokine secretion in trained and deconditioned subjects, indicating the activation of cells at the systemic level. The production of TNF-a and IFN-b in nonstimulated and stimulated PAMs was decreased in trained and deconditioned horses and might therefore explain the increased susceptibility to respiratory infections. Our study reports a dissociation between the systemic and the lung response to training that is probably implicated in the systemic inflammation and in the pulmonary susceptibility to infection. Keywords: exercise; innate immunity; monocytes; pulmonary

alveolar macrophages; equine

Clinical Relevance This research describes innate immune responses to training in blood and in the lung and provides new knowledge about detraining and its residual effects on immunity.

the innate immune response is scarce. In general, it has been concluded that regular moderate exercise is beneficial and protects from respiratory inflammation and infection (3, 6). In

contrast, prolonged strenuous exercise disturbs the immune response and results in an increased risk of respiratory illnesses (3–5, 7). The underlying mechanisms remain unclear.

( Received in original form July 29, 2013; accepted in final form January 29, 2014 ) *These authors contributed equally to this study. This work was supported by the Hippolia Foundation, the Conseil Regional de Basse-Normandie, the European Regional Development Fund, and the Fonds unique interministeriel. Correspondence and requests for reprints should be addressed to Linda Frellstedt, D.V.M., M.Sc., Center of Equine Sports Medicine, Faculty of Veterinary Medicine, University of Liege, Boulevard de Colonster 20, Batiment 42, 4000 Liege, Belgium. E-mail: [email protected] This article has an online supplement, which is accessible from this issue’s table of contents at www.atsjournals.org Am J Respir Cell Mol Biol Vol 51, Iss 1, pp 135–142, Jul 2014 Copyright © 2014 by the American Thoracic Society Originally Published in Press as DOI: 10.1165/rcmb.2013-0341OC on February 4, 2014 Internet address: www.atsjournals.org

Frellstedt, Waldschmidt, Gosset, et al.: Training Modifies Innate Immune Responses

135

ORIGINAL RESEARCH The innate immunity represents the first line of defense and is effective immediately after exposure to pathogens. In the airways, pulmonary alveolar macrophages (PAMs) with airway epithelial cells represent the sentinels of this system. In immune cells, a variety of pathogen recognition receptors, located on the surface or in intracellular compartments, interact with pathogen-associated molecular patterns (PAMPs) and control the host defense mechanisms. Toll-like receptors (TLRs) represent important signaling pathogen recognition receptors that recognize PAMPs. They are essential for the initiation of the immune response against bacteria, viruses, and fungi (8, 9). Once TLRs are stimulated by PAMPs, a downstream cascade is activated, which results in the production of interferons, proinflammatory cytokines, chemokines, and cytotoxic activities (8, 9). This first response is essential for an immediate protection against viral and bacterial infection. It has been widely recognized that alteration of this response promotes respiratory infection and lung inflammation, as described in athletes after prolonged intense exercise and strenuous competition (3, 7, 10–12). The effect of exercise on the different leukocyte populations, phagocytosis, and oxidative burst activity has been examined, and these data revealed an increased phagocytosis associated with a defect in oxidative burst activity in granulocytes (13, 14). Researchers in human sports medicine have compared populations of sedentary and regularly exercising people after a single cycling exercise. They showed that the protein expression of TLR1, -2, -3, and -4 measured by flow cytometry is decreased in peripheral blood monocytes in regularly exercising subjects (8, 15). This altered expression of TLRs is also associated with a lower production of proinflammatory cytokines (IL-6, IL-1a, TNF-a) in response to stimulation with LPS (8). Furthermore, regular exercise induces circulating antiinflammatory cytokines (IL-10, IL-6) (8, 16) and thereby limits the effects of proinflammatory cytokines. Sport horses, and especially racing horses, suffer frequently from respiratory inflammation and infection (17–22), as do human athletes. They have a greater athletic potential than humans, evidenced by a variety of physiological parameters. Compared with humans, horses have 136

a greater capacity to develop physical efforts as evidenced by their maximum speeds, maximal oxygen consumption (23, 24), blood oxygen content (25), cardiac output due to a higher maximal heart rate (26, 27), and a larger heart size in proportion to their body weight. Therefore, horses represent a unique model for the study of exercise and training on immune function. Furthermore, easy sampling of peripheral blood and bronchoalveolar lavages (BALs) allows researchers to perform longitudinal studies in horses participating in a training program. To investigate the effects of acute (single strenuous exercise) and chronic (training) exercise on the innate immunity, we analyzed TLR messenger ribonucleic acid (mRNA) expression and function in the local and in the systemic compartment in a group of horses submitted to a training program. In this study, we focused on TLR2 and TLR4, which are mainly involved in the recognition of gram-positive and gramnegative bacteria, and TLR3, which is implicated in the recognition of respiratory viruses. For this study, the innate immune response was investigated in peripheral blood monocytes and PAMs from untrained, trained, and deconditioned equine athletes. We examined (1) the mRNA expression of TLRs in monocytes and PAMs, (2) the baseline cytokine production in monocytes and PAMs cultured ex vivo, (3) the cytokine production after stimulation of TLRs ex vivo, and (4) the relationship with serum concentrations of stress markers such as cortisol.

Materials and Methods A full description of the materials and methods can be found in the online supplement. Our protocol was approved by the Animal Ethics Committee of the National Veterinary School of Alfort (agreement number 13/12/11–9). Eight healthy untrained Standardbred horses were enrolled in this longitudinal study. The horses were examined and sampled at several time points during the study. After the initial sampling (i.e., at the untrained stage), horses underwent 8 weeks of moderate training followed by 8 weeks of intensive training. The horses were kept in a grass pasture for a period of

deconditioning of 3 months. The horses underwent a standardized exercise test (SET) on a treadmill until fatigue before and at the end of the training period and at the end of the deconditioning period. The SET was performed as previously published (21). A fourth step was performed at 11.0 m/s until fatigue. Blood samples and BAL fluid (BALF) were collected before and after SET, before and at the end of the moderate and the intensive training periods, and at the end of the deconditioning period to assess the effect of the training status on the innate immune response. The horses were sampled in the morning after an overnight fast, and the sampling procedure was always conducted in the same way to avoid the effects of circadian rhythm. Blood was collected aseptically for hematology, monocyte isolation, and serum cortisol measurements. PBMCs were isolated by Histopaque density gradient (density, 1.077 g/ml) centrifugation, and monocytes were recovered by adherence and treated with TLR ligands as described in the online supplement. The horses were sedated, and BAL was performed with a nasobronchial tube (Bivona; MILA International, Inc., Erlanger, KY). Three hundred milliliters of sterile 0.9% NaCl solution were delivered to a pulmonary site and were retrieved gently by aspiration. The BALF was homogenized, total BALF cells were recovered by centrifugation, and PAMs were isolated by adherence. PAM culture and activation with TLR ligands were performed as described in the online supplement. Cell culture supernatants were assayed for TNF-a (R&D Systems, Abingdon, UK) and IFN-b (USCN Life Science Inc., Wuhan, China) concentrations with commercially available equine ELISA kits. RNA was isolated from nonstimulated control cells, and cDNA was generated as previously described (28). Concentration and quality of RNA were assessed by UV spectrophotometry (Nanodrop ND2000; Thermo Scientific, Aalst, Belgium) and by agarose gel electrophoresis. The mRNA expression of TLR2, TLR3, TLR4, and TLR6 was quantified by real-time PCR. Cortisol concentrations in serum were determined by solid-phase, competitive chemiluminescent immunoassay (IMMULITE 2000 Cortisol; Siemens Healthcare Diagnostics Products Ltd., Erlangen, Germany). Blood cell counts,

American Journal of Respiratory Cell and Molecular Biology Volume 51 Number 1 | July 2014

ORIGINAL RESEARCH BALF cell counts, TNF-a and IFN-b concentrations, and relative gene expression data were analyzed by a general linear mixed model (SAS, Cary, NC). All data are reported as least square means 6 associated SE. Differences were considered to be significant at P , 0.05.

Results

lymphocyte count was significantly decreased after moderate (P , 0.001) and intensive training (P , 0.001) and remained decreased after deconditioning (P , 0.001). In contrast, acute exercise had no effect on the total lymphocyte count. The peripheral blood neutrophil count was only increased after acute exercise in the intensively trained condition (P , 0.001).

Training Induces Airway Inflammation

To study the effects of acute exercise and regular training on the innate immune response in the lung, we first examined the cells in the BALF. There was a significant increase in total BAL cells in the intensively trained condition at rest and after strenuous exercise compared with total BAL cell counts in the untrained (P = 0.0071) and moderately trained (P = 0.0037) conditions (Table 1). No significant difference in BAL macrophage numbers was determined. The number of BAL lymphocytes was significantly increased after intensive training (P = 0.029) and deconditioning (P = 0.018) in comparison to the moderate training. The BAL neutrophils were significantly increased after intensive training when compared with the untrained (P = 0.015) and moderately trained (P = 0.025) conditions and may indicate the development of airway inflammation after a period of intensive training. Concurrently, we examined the distribution of cells in peripheral blood to determine whether a similar effect could be found in the systemic compartment. Training or acute exercise had no significant effect on the complete white blood cell or monocyte counts (Table 2). The total

Training Leads to Decreased Expression of TLR3 in Monocytes and PAMs

To characterize the effects on the innate immune system more specifically, we examined the mRNA expression of TLRs in monocytes and PAMs. We focused on the mRNA expression of TLR2/6, which recognizes molecular patterns specific for gram-positive bacteria; TLR4, which recognizes bacterial LPS specific for gramnegative bacteria; and TLR3, which mediates the response to double-stranded RNA, a marker of viral infection (8, 9), to account for the most common respiratory pathogens in horses. In monocytes, the mRNA expression of TLR3 (P , 0.015) and TLR6 (P , 0.025) was significantly decreased after training and deconditioning compared with the untrained condition (Figure 1A). The mRNA expression of TLR2 was significantly increased after a period of deconditioning when compared with the untrained (P = 0.0024) and intensively trained conditions (P = 0.0028). Training (P = 0.0004), and to a lower degree deconditioning (P = 0.03), resulted in an up-regulated TLR4 mRNA expression when compared with the untrained condition. Acute exercise had no significant

effect on the TLR mRNA expression in monocytes (data not shown). In PAMs, moderate (P , 0.0001) and intensive (P , 0.0001) training induced a significant decrease in TLR3 mRNA expression (Figure 1B), but acute exercise had no effect on TLR3 mRNA expression (data not shown). Training or acute exercise had no effect on the mRNA expression of TLR2 and TLR4 in PAMs. However, deconditioning induced a significant decrease in TLR4 mRNA expression (P = 0.0004). In contrast, TLR6 mRNA expression in PAMs was upregulated by moderate (P = 0.0126) and intensive training (P = 0.0005) and continued to increase during the deconditioning period (P , 0.0001). Training Results in a Proinflammatory Response after Ex Vivo Treatment with TLR Agonists in Monocytes

We next investigated the effect of ex vivo treatment with TLR agonists to assess whether the changes in TLR mRNA expression were associated with an altered cytokine secretion after training and/or acute exercise. Baseline TNF-a (Figure 2A) and IFN-b (Figure 2B) secretion in monocytes with medium alone did not differ at any time point. Treatment with synthetic diacylated lipoprotein (FSL) and LPS resulted in significantly increased TNF-a production (P , 0.009) at all time points in comparison to cells in medium alone, whereas Poly(I:C) did not. The secretion of TNF-a induced by FSL and LPS was significantly higher after training and deconditioning (P , 0.0001) when compared with the untrained condition. In addition, LPS-stimulated cells after

Table 1. Effect of Training and Exercise on Bronchoalveolar Lavage Fluid Cell Counts Total cell count (cells/mm3) Untrained Untrained-PE Moderate training Intensive training Intensive training-PE Deconditioned Deconditioned-PE

72.5 79.29 58.28 113.58 97.97 89.45 90.45

6 6 6 6 6 6 6

Macrophages/mm3

40.39* 36.58 16.47 53.14†,‡ 40.65†,‡ 31.76 15.38

34.34 35.1 31.91 40.54 39.47 34.97 31.34

6 6 6 6 6 6 6

5.83 5.48 6.29 5.56 5.37 7.31 6.64

Lymphocytes/mm3 33.18 39.78 22.28 55.11 38.71 47.34 50.86

6 6 6 6 6 6 6

5.83 6.15 6.23 8.09‡ 6.86‡ 20.55‡ 8.5‡

Neutrophils/mm3 3.13 3.04 2.6 5.4 4.78 4.73 4.67

6 6 6 6 6 6 6

0.64 0.87 0.96 0.72†,‡ 1.01 1.05 0.97

Definition of abbreviation: PE, posteffort. *Data are expressed as least square means 6 SE. † Significant differences (P , 0.05) in cell counts from untrained horses. ‡ Significant differences (P , 0.05) in cell counts from moderate training.


137

ORIGINAL RESEARCH Table 2. Effect of Training and Exercise on Blood Cell Counts WBC (103 cells/mm3) Untrained Untrained-PE Moderate training Intensive training Intensive training-PE Deconditioned Deconditioned-PE

8.67 9.05 9.08 8.63 9.61 8.39 8.35

6 6 6 6 6 6 6

2.38* 2.36 0.4 1.48 1.07 1.19 0.83

Monocytes (103/mm3) 0.39 0.4 0.46 0.34 0.28 0.44 0.38

6 6 6 6 6 6 6

0.16 0.25 0.11 0.14 0.1 0.13 0.12

Lymphocytes (103/mm3) 4.6 4.4 3.65 3.62 3.31 3.35 3.45

6 6 6 6 6 6 6

0.73 0.82 0.47† 0.43† 0.7† 0.92† 0.68†

Neutrophils (103/mm3) 3.29 3.94 4.35 4.06 5.45 4.25 4.04

6 6 6 6 6 6 6

1.9 2.16 0.45 1.5 1.57†,‡,x 1.89 1.27

Definition of abbreviations: PE, post-effort; WBC, white blood cell count. *Data are reported as least square means 6 SE. † Significant differences (P , 0.05) in cell counts from untrained horses. ‡ Significant differences (P , 0.05) in cell counts from moderate training. x Significant differences (P , 0.05) in cell counts from intensive training.

deconditioning produced significantly more TNF-a than cells from horses after moderate and intensive training (P , 0.0001).

No significant increase of IFN-b levels was detected with the TLR ligands in cells from horses in the untrained condition. Stimulation with LPS of cells

from horses after intensive training (P , 0.025) and deconditioning (P , 0.001) induced significantly higher secretion of IFN-b compared with cells collected in the untrained condition. This secretion was also significantly different after deconditioning when compared with cells collected during moderate (P , 0.0001) and intensive training (P = 0.0024). Acute exercise did not affect the secretion of TNF-a or IFN-b in monocytes. Training Inhibits Ex Vivo TNF-a and IFN-b Secretion in PAMs after Stimulation with TLR2 and TLR3 Agonists

Figure 1. Relative messenger ribonucleic acid (mRNA) expression of Toll-like receptor (TLR)2, TLR3, TLR4, and TLR6 in monocytes (A) and pulmonary alveolar macrophages (B) from untrained, moderately trained, intensively trained, and deconditioned horses. †Significant differences (P , 0.05) in gene expression for untrained horses. ‡Significant differences (P , 0.05) in gene expression for moderately trained horses. xSignificant differences (P , 0.05) in gene expression for intensively trained horses.

138

To determine whether the antiviral and antibacterial defense mechanisms in the lung are impaired by training or acute exercise, we treated PAMs ex vivo with TLR agonists and assessed the production of TNF-a and IFN-b. The baseline production of TNF-a (Figure 2C) and IFN-b (Figure 2D) in nonstimulated PAMs was significantly decreased by moderate (P , 0.05) and intensive training (P , 0.001) and remained significantly decreased after deconditioning (P , 0.001). Stimulation with the three TLR ligands significantly increased TNF-a production in cells from horses in the untrained condition (P , 0.03). TNF-a production induced by FSL and Poly(I:C) was decreased by intensive training (P , 0.005) and was not restored after a period of deconditioning when treated with FSL (P = 0.0172). No significant effect of training or deconditioning on the production of TNF-a was observed in LPS-treated cells. Acute exercise had no effect on the secretion of TNF-a in nontreated or treated PAMs.


ORIGINAL RESEARCH

Figure 2. Ex vivo production of TNF-a (A, C) and IFN-b (B, D) in monocytes (A, B) and pulmonary alveolar macrophages (C, D) in medium alone and treated with synthetic diacylated lipoprotein (FSL), Poly(I:C), or LPS after 6 hours of incubation. *Significant differences (P , 0.05) from nontreated cells at the corresponding time point. †Significant differences (P , 0.05) from untrained horses. ‡Significant differences (P , 0.05) from moderately trained horses. xSignificant differences (P , 0.05) from intensively trained horses. NC, nonstimulated control cells.

Our experimental protocol has allowed us to determine the effect of exercise and/or training on innate immune responses in horses over a period of 10 months. We demonstrate that the mRNA expression of TLR3 and TLR4 in monocytes was modified in an opposite manner by training. The observed down-regulation of TLR3 has also been reported in regularly exercising humans (8, 15). In contrast, the amplified mRNA expression of TLR4 in monocytes after a period of training and deconditioning is described for the first time. Previous cross-sectional studies have examined TLR4 cell-surface expression on monocytes by flow cytometry (29–34). Several researchers reported a lower cellsurface expression of TLR4 in physically active individuals when compared with physically inactive subjects (29, 30, 34). Stewart and colleagues described a lower TLR4 expression after a period of training in inactive individuals (33), and Oliviera and colleagues discovered a short-lived decrease in TLR4 immediately after exercise in endurance-trained subjects (31). McFarlin and colleagues (29) and Stewart

and colleagues (33) showed that the observed effect is not due to age but is related to the status of physical activity. In horses, it has been reported that young athletes are more susceptible to respiratory diseases than older athletes (18, 19, 35), but it remains unclear whether age plays an important role in the alteration of the innate immune response or if this is related to differences in exercise intensity. In addition to species-related discrepancies, the type and intensity of the training and physical activity might explain the differences between the reported studies in humans and our study in horses. Resistance exercise was performed on alternate days for 3 days per week in the human studies, whereas our racing horses worked for 5 consecutive days per week during the intensive endurance training. Finally, our data pointed out that mRNA expression of TLR was not always associated with a parallel profile in the cytokine response to TLR ligands. Monocytes are very sensitive to bacterial endotoxin and can be easily stimulated with LPS. Moreover, the increased secretion of TNF-a in LPSstimulated cells from trained and deconditioned individuals might be related


139

Only treatment with Poly(I:C) induced a significantly increased IFN-b secretion (P , 0.014) when compared with baseline. The IFN-b production induced by Poly(I:C) was decreased by intensive training (P = 0.0009) and remained decreased after a period of deconditioning (P = 0.0006). Acute exercise increased the secretion of IFN-b induced by LPS treatment (P = 0.0174) in the trained condition only (397.61 6 229.4 pg/ml before exercise versus 1,028.07 6 240.17 pg/ml after exercise). Cortisol Is Elevated after Acute Exercise but Is Not Associated with Cytokine Modulation

To explore the relationship between cortisol, which is released during physical stress, and the innate immune response, we examined serum cortisol concentrations in association with the different training statuses and acute strenuous exercise. Cortisol levels showed a significant increase 1 hour after acute exercise in the untrained (P , 0.001), intensively trained (P , 0.001), and deconditioned (P , 0.001) conditions but were not influenced by the training status itself (Figure 3). Serum cortisol levels were not correlated with cytokine production (data not shown).

Discussion

ORIGINAL RESEARCH

Figure 3. Serum cortisol concentrations in untrained, moderately trained, intensively trained, and deconditioned horses at rest and after a single strenuous exercise. *Significant differences (P , 0.001) for differences in horses at rest and horses after a single strenuous exercise. PE, posteffort.

to the up-regulated mRNA expression of TLR4 or to a more efficient recognition of PAMPs by TLRs at the systemic level. When TLR4 is activated it signals via MyD88 and TRIF, resulting in the production of TNF-a but also type I IFNs (9). An increased responsiveness of TLR4 might therefore explain the increased IFN-b secretion in the trained and deconditioned states. Down-regulated mRNA expression of TLR1, -2, -3, and -4 in monocytes has been reported in crosssectional studies (8, 15). In our study, no modulation of TLR2 mRNA expression was detected, whereas the response of monocytes to the TLR2/6 ligand FSL increased. We could not determine the function of TLR3 because no increased secretion of TNF-a and IFN-b was detected at the dose tested. The difference between our study and the data obtained in humans might be related to the species but also to the type and intensity of training applied. The response within the lungs differs from the response at the systemic level. Training induced mild airway inflammation, which was evidenced by an increased total BAL cell count and a significantly increased number of neutrophils and lymphocytes in the BALF after intensive training. Although the number of PAMs was not affected, their function was modified by training. Indeed, the mRNA expression of TLR3 is decreased in PAMs after moderate and intensive training and remains decreased after a period of deconditioning. The poly(I:C)induced secretion of TNF-a and IFN-b in PAMs was significantly impaired. In 140

addition, the response to the TLR2 ligand was decreased, but its expression was not modified. This may suggest a local suppression of innate immune defense mechanisms not only related to the modulation of TLR mRNA expression. The recruitment of lymphocytes detected after intense training might lead to the generation of a suppressive environment in the lung, potentially explaining the defect in macrophage function (36, 37). The phenotype and the function analysis of the cells recruited in the lung (particularly lymphocytes) during training might help to decipher this mechanism. Nevertheless, the decrease in IFN-b production by PAMs might be involved in the susceptibility to respiratory viral infections reported in regularly exercising humans (13) and horses (18, 38). Previous animal studies have demonstrated that strenuous exercise impairs PAM viral defense mechanisms (4, 39), decreases oxidative burst activity (40), impairs phagocytosis (41), and results in an increased susceptibility to viral infection (4, 39, 42). The underlying mechanisms have not been elucidated. Our study concurrently examines cells in two different compartments and reports a dissociation between the immune response in the lungs and in the systemic compartment. Our unique experimental protocol allows us to show that regular intense exercise, but not single bouts of exercise, leads to a local decreased secretion of IFN-b, a major antiviral cytokine, in the lungs. This decreased pulmonary production of IFN-b is associated with an inflammatory response in ex vivo treated

peripheral blood monocytes after exposure to PAMPs. The stimulation with TLR agonists illustrated the potential of PAMs and monocytes to respond to viral or bacterial stimuli at rest or after acute exercise in untrained, trained, and deconditioned horses. Acute exercise induces no significant differences in the innate immune response, but regular intense exercise results in impaired immune responses in PAMs and an enhanced proinflammatory potential in peripheral blood monocytes, both after treatment with PAMPs. Earlier studies have suggested differences between the innate immune response of peripheral blood monocytes and tissue macrophages in response to training (13), although this is not supported by scientific data. Monocytes are part of a sterile compartment in the peripheral blood; therefore, they are not exposed to pathogens or debris unless a pathologic process is in progress. On the contrary, PAMs are located on the surface of the airway mucosa. This environment is free of large particles due to the airway structure but does not eliminate the passage of pathogens and debris into the lower airways. Therefore, PAMs are commonly exposed to a number of foreign particles and are likely less responsive to stimuli in comparison to monocytes to avoid the development of inappropriate airway inflammation. During intensive exercise, PAMs are also exposed to mechanical as well as heat and cold stress caused by inspiratory and expiratory forces. These factors could explain, at least partially, the differences that we observed between the two investigated compartments and the functional alteration of these cells. Similarly, Raidal and colleagues (40) revealed impaired oxidative burst activity in PAMs after a period of intensive training in horses. The underlying mechanisms were not further explored but might implicate the previously described interactions of oxidative burst with TLR signaling. The response of monocytes in trained and deconditioned subjects to a potential bacterial infection could imply beneficial effects on the immune system after a period of regular training. After exposure to pathogens, monocytes can be mobilized from the systemic compartment into the lungs, where they could compensate the impaired response of PAMs once located within the lung (43, 44).


ORIGINAL RESEARCH The effects observed in association with the deconditioning period represent novel scientific data. Although it has been explored in many human studies in relation to injuries and illnesses requiring bed rest, no data have been published examining the effect of deconditioning on innate or adaptive immune responses. In this study, we show that a period of deconditioning of 3 months does not reverse the effects of intensive training on innate immune responses. Considering the short lifetime of monocytes, we can suspect that such a prolonged effect probably implicates epigenetic modifications, which have been reported to induce delayed effects. Further research is needed to explore the underlying mechanisms in the different compartments. Previous reports have suggested that cortisol alters the immune response after exercise because increased serum cortisol levels after strenuous exercise have been reported in horses (45–47). We showed that only acute exercise, but not training status, had an effect on cortisol concentrations. As reported by Kohut and colleagues (42),

no correlation between the changes in cytokine production and serum cortisol concentrations could be evidenced. This is in contrast to a longitudinal study that was recently performed in human athletes (11). Morgado and colleagues reported that plasma cortisol levels were associated with the training status of swimmers. This discrepancy might be explained by differing species sensitivity to corticosteroids (48). Horses are considered to be corticosteroid resistant, and it has been shown that even experimental increases in corticosteroids fail to induce immunosuppression if the levels remain within the physiological range (49, 50). In summary, cortisol is useful as a marker of a single strenuous exercise and acute stress but probably not as a mechanism involved in the control of the innate immune response influenced by training. In conclusion, we show that training induces mild airway inflammation and a decreased mRNA expression of TLR3 in monocytes and PAMs. The low mRNA expression of TLR3 in trained individuals is associated with impaired

References 1. Nieman DC. Is infection risk linked to exercise workload? Med Sci Sports Exerc 2000; 32(7 Suppl):S406–S411. 2. Gleeson M, Walsh NP; British Association of Sport and Exercise Sciences. The BASES expert statement on exercise, immunity, and infection. J Sports Sci 2012;30:321–324. 3. Pedersen BK, Toft AD. Effects of exercise on lymphocytes and cytokines. Br J Sports Med 2000;34:246–251. 4. Davis JM, Kohut ML, Colbert LH, Jackson DA, Ghaffar A, Mayer EP. Exercise, alveolar macrophage function, and susceptibility to respiratory infection. J Appl Physiol (1985) 1997;83:1461–1466. 5. Kohut ML, Boehm GW, Moynihan JA. Prolonged exercise suppresses antigen-specific cytokine response to upper respiratory infection. J Appl Physiol (1985) 2001;90:678–684. 6. Nieman DC. Exercise immunology: practical applications. Int J Sports Med 1997;18:S91–S100. 7. Nieman DC. Risk of upper respiratory tract infection in athletes: an epidemiologic and immunologic perspective. J Athl Train 1997;32:344–349. 8. Gleeson M, McFarlin B, Flynn M. Exercise and Toll-like receptors. Exerc Immunol Rev 2006;12:34–53. 9. Akira S, Uematsu S, Takeuchi O. Pathogen recognition and innate immunity. Cell 2006;124:783–801. 10. Suzuki K, Yamada M, Kurakake S, Okamura N, Yamaya K, Liu Q, Kudoh S, Kowatari K, Nakaji S, Sugawara K. Circulating cytokines and hormones with immunosuppressive but neutrophil-priming potentials rise after endurance exercise in humans. Eur J Appl Physiol 2000;81:281–287. 11. Morgado JM, Rama L, Silva I, de Jesus Inacio ´ M, Henriques A, Laranjeira P, Pedreiro S, Rosado F, Alves F, Gleeson M, et al. Cytokine production by monocytes, neutrophils, and dendritic cells is hampered by long-term intensive training in elite swimmers. Eur J Appl Physiol 2012;112:471–482. 12. Pyne DB, Gleeson M. Effects of intensive exercise training on immunity in athletes. Int J Sports Med 1998;19(Suppl 3):S183–S191; discussion S191–S194.

ex vivo TNF-a and IFN-b secretion in PAMs. Our findings may explain the increased susceptibility to pulmonary viral infections after prolonged intensive training. The impact of cell recruitment in the airways and the development of an immune-suppressive environment might be an interesting hypothesis. Finally, our model allowed us to examine specific innate immune responses in two different compartments in a longitudinal study and thereby offers new data that might add to the understanding of the effects of training and acute exercise on immunity. n Author disclosures are available with the text of this article at www.atsjournals.org. Acknowledgments: The authors thank the University of Liege, the GIGA, the CORD, the CIRALE, the Frank Duncombe Laboratory, Zoetis, Raja Fares, Ilham Sbai, Jean-Clement Bustin, Julien De Curraize, and Elodie Paumier-Andr e´ for their support during the study. The authors thank Julien De Curraize for training the horses that participated in the study.

13. Walsh NP, Gleeson M, Shephard RJ, Gleeson M, Woods JA, Bishop NC, Fleshner M, Green C, Pedersen BK, Hoffman-Goetz L, et al. Position statement. Part one: immune function and exercise. Exerc Immunol Rev 2011;17:6–63. 14. Nieman DC. Immune response to heavy exertion. J Appl Physiol (1985) 1997;82:1385–1394. 15. Lancaster GI, Khan Q, Drysdale P, Wallace F, Jeukendrup AE, Drayson MT, Gleeson M. The physiological regulation of toll-like receptor expression and function in humans. J Physiol 2005;563: 945–955. 16. Ostrowski K, Rohde T, Asp S, Schjerling P, Pedersen BK. Pro- and antiinflammatory cytokine balance in strenuous exercise in humans. J Physiol 1999;515:287–291. 17. Burrell MH, Wood JL, Whitwell KE, Chanter N, Mackintosh ME, Mumford JA. Respiratory disease in thoroughbred horses in training: the relationships between disease and viruses, bacteria and environment. Vet Rec 1996;139:308–313. 18. Wood JL, Newton JR, Chanter N, Mumford JA. Association between respiratory disease and bacterial and viral infections in British racehorses. J Clin Microbiol 2005;43:120–126. 19. Wood JL, Newton JR, Chanter N, Mumford JA. Inflammatory airway disease, nasal discharge and respiratory infections in young British racehorses. Equine Vet J 2005;37:236–242. 20. Allen KJ, Tremaine WH, Franklin SH. Prevalence of inflammatory airway disease in national hunt horses referred for investigation of poor athletic performance. Equine Vet J Suppl 2006;36:529–534. 21. Richard EA, Fortier GD, Pitel PH, Dupuis MC, Valette JP, Art T, Denoix JM, Lekeux PM, Erck EV. Sub-clinical diseases affecting performance in Standardbred trotters: diagnostic methods and predictive parameters. Vet J 2010;184:282–289. 22. Rossdale PD, Hopes R, Digby NJ, offord K. Epidemiological study of wastage among racehorses 1982 and 1983. Vet Rec 1985;116: 66–69. 23. Noakes TD. Implications of exercise testing for prediction of athletic performance: a contemporary perspective. Med Sci Sports Exerc 1988;20:319–330.


141

ORIGINAL RESEARCH 24. Rose RJ, Hodgson DR, Kelso TB, McCutcheon LJ, Reid TA, Bayly WM, Gollnick PD. Maximum O2 uptake, O2 debt and deficit, and muscle metabolites in Thoroughbred horses. J Appl Physiol (1985) 1988;64: 781–788. 25. Snow DH, Vogel CJ. Equine fitness: the care and training of the athletic horse. Newton Abbott, Devon, UK: David and Charles, Inc.; 1987. 26. Evans DL, Rose RJ. Cardiovascular and respiratory responses in Thoroughbred horses during treadmill exercise. J Exp Biol 1988;134: 397–408. 27. Noakes TD. Lore of running. Cape Town, South Africa: Oxford University Press; 1992. 28. Waldschmidt I, Pirottin D, Art T, Audigie´ F, Bureau F, Tosi I, El Abbas S, Farnir F, Richard E, Dupuis MC. Experimental model of equine alveolar macrophage stimulation with TLR ligands. Vet Immunol Immunopathol 2013;155:30–37. 29. McFarlin BK, Flynn MG, Campbell WW, Craig BA, Robinson JP, Stewart LK, Timmerman KL, Coen PM. Physical activity status, but not age, influences inflammatory biomarkers and toll-like receptor 4. J Gerontol A Biol Sci Med Sci 2006;61:388–393. 30. McFarlin BK, Flynn MG, Campbell WW, Stewart LK, Timmerman KL. TLR4 is lower in resistance-trained older women and related to inflammatory cytokines. Med Sci Sports Exerc 2004;36: 1876–1883. 31. Oliveira M, Gleeson M. The influence of prolonged cycling on monocyte Toll-like receptor 2 and 4 expression in healthy men. Eur J Appl Physiol 2010;109:251–257. 32. Timmerman KL, Flynn MG, Coen PM, Markofski MM, Pence BD. Exercise training-induced lowering of inflammatory (CD141CD161) monocytes: a role in the anti-inflammatory influence of exercise? J Leukoc Biol 2008;84:1271–1278. 33. Stewart LK, Flynn MG, Campbell WW, Craig BA, Robinson JP, McFarlin BK, Timmerman KL, Coen PM, Felker J, Talbert E. Influence of exercise training and age on CD141 cell-surface expression of toll-like receptor 2 and 4. Brain Behav Immun 2005; 19:389–397. 34. Flynn MG, McFarlin BK, Phillips MD, Stewart LK, Timmerman KL. Tolllike receptor 4 and CD14 mRNA expression are lower in resistive exercise-trained elderly women. J Appl Physiol (1985) 2003;95: 1833–1842. 35. Christley RM, Hodgson DR, Rose RJ, Hodgson JL, Wood JL, Reid SW. Coughing in thoroughbred racehorses: risk factors and tracheal endoscopic and cytological findings. Vet Rec 2001;148:99–104. 36. Semple PL, Binder AB, Davids M, Maredza A, van Zyl-Smit RN, Dheda K. Regulatory T cells attenuate mycobacterial stasis in alveolar and blood-derived macrophages from patients with tuberculosis. Am J Respir Crit Care Med 2013;187:1249–1258.

142

37. Kirby AC, Newton DJ, Carding SR, Kaye PM. Pulmonary dendritic cells and alveolar macrophages are regulated by gammadelta T cells during the resolution of S. pneumoniae-induced inflammation. J Pathol 2007;212:29–37. 38. Wilsher S, Allen WR, Wood JL. Factors associated with failure of thoroughbred horses to train and race. Equine Vet J 2006;38:113–118. 39. Kohut ML, Davis JM, Jackson DA, Jani P, Ghaffar A, Mayer EP, Essig DA. Exercise effects on IFN-beta expression and viral replication in lung macrophages after HSV-1 infection. Am J Physiol 1998;275: L1089–L1094. 40. Raidal SL, Rose RJ, Love DN. Effects of training on resting peripheral blood and BAL-derived leucocyte function in horses. Equine Vet J 2001;33:238–243. 41. Raidal SL, Love DN, Bailey GD, Rose RJ. The effect of high intensity exercise on the functional capacity of equine pulmonary alveolar macrophages and BAL-derived lymphocytes. Res Vet Sci 2000;68: 249–253. 42. Kohut ML, Davis JM, Jackson DA, Colbert LH, Strasner A, Essig DA, Pate RR, Ghaffar A, Mayer EP. The role of stress hormones in exercise-induced suppression of alveolar macrophage antiviral function. J Neuroimmunol 1998;81:193–200. 43. Chen L, Zhang Z, Barletta KE, Burdick MD, Mehrad B. Heterogeneity of lung mononuclear phagocytes during pneumonia: contribution of chemokine receptors. Am J Physiol Lung Cell Mol Physiol 2013;305: L702–L711. 44. Goodyear A, Troyer R, Bielefeldt-Ohmann H, Dow S. MyD88dependent recruitment of monocytes and dendritic cells required for protection from pulmonary Burkholderia mallei infection. Infect Immun 2012;80:110–120. 45. Horohov DW, Dimock A, Guirnalda P, Folsom RW, McKeever KH, Malinowski K. Effect of exercise on the immune response of young and old horses. Am J Vet Res 1999;60:643–647. 46. Robson PJ, Alston TD, Myburgh KH. Prolonged suppression of the innate immune system in the horse following an 80 km endurance race. Equine Vet J 2003;35:133–137. 47. Cywinska A, Szarska E, Degorski A, Guzera M, Gorecka R, Strzelec K, Kowalik S, Schollenberger A, Winnicka A. Blood phagocyte activity after race training sessions in Thoroughbred and Arabian horses. Res Vet Sci 2013;95:459–464. 48. Bamberger CM, Schulte HM, Chrousos GP. Molecular determinants of glucocorticoid receptor function and tissue sensitivity to glucocorticoids. Endocr Rev 1996;17:245–261. 49. Jefferies WM. Cortisol and immunity. Med Hypotheses 1991;34: 198–208. 50. Magnuson NS, McGuire TC, Banks KL, Perryman LE. In vitro and in vivo effects of corticosteroids on peripheral blood lymphocytes from ponies. Am J Vet Res 1978;39:393–398.
