Pathology – Research and Practice 211 (2015) 435–443

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Original Article

Exercise modulates the expression of IL-1␤ and IL-10 in the articular cartilage of normal and osteoarthritis-induced rats Mariel Rojas-Ortega a,1 , Raymundo Cruz a , Marco Antonio Vega-López a , Moisés Cabrera-González a , José Manuel Hernández-Hernández b , Carlos Lavalle-Montalvo c , Juan B. Kouri a,∗ a Departamento de Infectómica y Patogénesis Molecular, Centro de Investigación y de Estudios Avanzados del Instituto Politécnico Nacional (CINVESTAV-IPN), México D.F., Mexico b Departamento de Biología Celular, Centro de Investigación y de Estudios Avanzados del Instituto Politécnico Nacional (CINVESTAV-IPN), México D.F., Mexico c Facultad de Medicina, Universidad Nacional Autónoma de México (UNAM), México D.F., Mexico

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Article history: Received 12 September 2014 Received in revised form 2 January 2015 Accepted 21 January 2015 Keywords: Cartilage IL-1␤ IL-10 Exercise Osteoarthritis

a b s t r a c t After a joint lesion, high-impact exercise is a risk factor for the development of osteoarthritis (OA). The degradation of articular cartilage in OA has been associated with the activation of inflammatory cytokine signaling pathways. However, differences in cytokine expression in healthy and injured cartilage after exercise have not yet been analyzed. We used immunofluorescence and Western blot to study the expression of IL-1␤ and IL-10 in the articular cartilage of normal (N), sham-operated (S), and menisectomized (OA) rats subjected or not to high-impact exercise (E) for 3, 6, and 10 days (N, NE, S, SE, and OA groups). Cartilage integrity and proteoglycan content were only affected in the OA groups. Exercise increased the amount of IL-1␤ and IL-10 positive chondrocytes in NE and SE groups compared with non-exercised groups (N and S). The expression of IL-1␤ was up-regulated over time in the NE and OA groups, although in the late stages the increase was higher in the OA groups. In contrast, the expression of anti-inflammatory IL-10 was low in the OA group, whereas in the NE groups expression levels were higher at each time point analyzed. These results suggest that anti- and pro-inflammatory molecules in the cartilage might be tightly regulated to maintain the integrity of the tissue and that when this equilibrium is broken (when the meniscus is removed), the pro-inflammatory cytokines take over and OA develops. © 2015 Elsevier GmbH. All rights reserved.

Introduction Osteoarthritis (OA) is a chronic, degenerative, and incapacitating disease, characterized by deterioration of the articular cartilage, synovitis and alteration of the peri-articular structures and the subchondral bone [1]. The chondrocyte, the only cell type present in the cartilage, is responsible for remodeling and maintaining the structural and functional integrity of the cartilage matrix. During OA pathogenesis, chondrocytes respond to mechanical and biochemical insults, causing structural changes in the cartilage such

∗ Corresponding author at: Departamento de Infectómica y Patogénesis Molecular, Centro de Investigación y de Estudios Avanzados del Instituto Politécnico Nacional, Av. Instituto Politécnico Nacional, 2508, Col. San Pedro Zacatenco Apdo, Postal 14740, México D.F. 07360, Mexico. Tel.: +52 55 57473343. E-mail address: [email protected] (J.B. Kouri). 1 R.I.P. http://dx.doi.org/10.1016/j.prp.2015.01.008 0344-0338/© 2015 Elsevier GmbH. All rights reserved.

as fibrillation, modification of the amount or composition of matrix proteins, development of cell “clusters” and phenotypic variability of chondrocytes, including programmed death [2–4]. Osteoarthritis is generally considered to be a non-inflammatory joint disease due to the absence of neutrophils in the synovial fluid and the lack of systemic manifestations of inflammation [1]. However, synovitis involving infiltration of activated CD4+T cells and CD68+ macrophages and the overexpression of proinflammatory mediators is common in OA [5]. Consequently, synovial inflammation is a factor that contributes to the deregulation of chondrocyte function during the remodeling of the cartilage extracellular matrix [6,7]. In addition to synovial cells and infiltrating activated mononuclear cells, chondrocytes are well known to secrete pro-inflammatory cytokines, including interleukin-1 beta (IL-1␤) and tumor necrosis factor-alpha (TNF)-␣, that can act autocrinally/paracrinally, leading to a significant breakdown of the cartilage macromolecules [8–12]. These cytokines directly inhibit the expression of cartilage-specific, extracellular matrix

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genes such as aggrecan and collagen type II [13–15] and up-regulate various catabolic genes such as matrix-degrading proteases [matrix metalloproteinase (MMP)-1, -3, -13, and ADAMTS-4, -5] [15–18]. Furthermore, anti-inflammatory cytokines are also produced by chondrocytes [12,19–22]. Interleukin-10 (IL-10) and transforming growth factor-␤ (TGF-␤) act as chondroprotectors, stimulating the expression of collagen type II and proteoglycan [23–26], and they counteract the deleterious effects of IL-1␤ and TNF-␣ through the inhibition of metalloproteinase expression and pro-inflammatory cytokines or nitric oxide (NO) production [27–30]. Cytokine IL-10 also antagonizes intrinsic apoptotic pathways induced by TNF-␣ or IL-1␤ in chondrocytes [24,28]. There are several risk factors associated with OA onset, including age, genes and obesity, but joint injuries (anterior cruciate ligament transection and meniscus tear) caused by the practice of high-impact exercises are key factors for the development of OA [31–33]. Most studies of OA pathogenesis have been conducted in surgically induced OA animal models, which closely resemble posttraumatic OA in humans [34]. In some studies, high-impact exercise was also used to accelerate the disease in OA models, because it takes months to develop without exercise [35,36]. Even though inflammatory and anti-inflammatory elements are undoubtedly involved in OA, they have not been thoroughly studied in humans following joint injuries due to the difficulty of obtaining cartilage samples for study. Indeed, very little is known concerning the activation of inflammatory pathways in cartilage after joint injury, when therapeutic intervention may be most useful for stopping or delaying OA onset. In addition, it is not yet known how inflammatory elements are adapted in healthy cartilage during high-impact exercise training to prevent the development of OA. Therefore, the aim of this study was to evaluate the effect of high-impact exercise on the expression of IL-1␤ and IL-10 in articular cartilage from normal and menisectomized OA rats. Our results show that exercise, but not sham surgery, modifies the expression of cytokines in healthy groups. However, differences in levels of cytokine expression between OA and normal groups suggest that, in healthy cartilage, anabolic proteins can efficiently counteract the action of catabolic proteins, while in injured cartilage, the activity of catabolic proteins is stronger than that of anabolic proteins. This imbalance might play an important pathogenic role in OA.

rats in which the capsule and synovial membrane were cut but the meniscus was not removed. These sham-operated rats were divided into non-exercised rats (S) and exercised rats (SE). Normal rats without any surgical procedures were subjected to the same protocol of high-impact exercise and were designed as NE. As a control group, we included normal rats without surgery or highimpact exercise (N) but following the same time sequences as the sham and OA rats. In western blot studies, we used a single group of normal rats as control (also designated N). Exercise protocol High-impact exercise was performed as follows: nine rats were placed in a cardboard box (surface of 60 × 30 cm) and subjected to 2 min of lateral movements with up and down displacements (approximately 100 displacements per animal). Rats were then grasped and released from a height of 30 cm (15 times per rat, approximately 2 min), and lastly, by continuously shaking the box, they were forced to jump for 1 min (approximately 80 continuous jumps up to 10 cm in height). This protocol was repeated three times to complete 15 min of exercise. This type of exercise stimulated the muscles, joints and bones by working flexion, extension and compression of the limbs. Exercise training began 2 days after surgery and was performed daily for 3, 6, or 10 days (3, 6, 8, or 10 days for the Western blot studies). Cartilage samples For histological studies, medial femoral condyles from the right knee were removed and fixed for 12 h at 4 ◦ C in 4% paraformaldehyde in PBS, pH 7.2. After three washes with PBS, samples were incubated in sucrose 10% in PBS, pH 7.2 for 12 h at 4 ◦ C. Samples were then embedded in tissue freezing medium (Leica Microsystems, Wetzlar, DE) and immediately frozen at −20 ◦ C. For the Western blot (WB) studies, we pooled the articular cartilage of 10 rats from each experimental group, thus 10 rats per group were considered n = 1. Cartilage was removed from the medial femoral condyles from the right knee and immediately frozen and stored at −80 ◦ C until processed to extract the proteins. Immunofluorescence

Materials and methods Animals Male Wistar rats weighing 130–150 g were housed on a 12h light/dark schedule and allowed free access to food and water. All surgical procedures were carried out in rats anesthetized with an intraperitoneal injection of 60 mg/kg ketamine and 4 mg/kg xylazine solution. Animals were euthanized by CO2 inhalation, and cartilage samples were removed under aseptic conditions. We used 3 rats per experimental group (81 rats) for histopathology and immunofluorescence studies, and 30 rats per experimental group (270 rats) for western blot studies. All procedures for animal care and use were approved by our institutional ad hoc committee and performed following the animal facility’s regulations and Mexican official regulatory guideline NOM-062-ZOO-1999.

Frozen condyles were cryosectioned in the coronal plane of the tissue (cryostat Leica CM1100, Leica Microsystems) to obtain 6 ␮m thick slices, which were mounted on gelatin-coated slides. The sections were hydrated for 15 min in PBS, pH 7.2 and permeabilized with 0.2% Tween-20 in PBS for 10 min at room temperature. Then they were pre-incubated with 0.2% IgG-free BSA for 20 min at room temperature. To identify the proteins, the samples were incubated overnight at 4 ◦ C with polyclonal rabbit anti-rat IL-1␤ antibodies (1:100) and polyclonal goat anti-rat IL-10 antibodies (1:100, all from Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA), followed by incubation with FITC conjugated anti-rabbit IgG antibodies or FITC-conjugated anti-goat IgG antibodies (1:100, Jackson Immunoresearch Inc., West Grove, PA, USA) for 1 h at room temperature. Cartilage samples with the corresponding primary antibodies replaced with non-immune goat serum (1:100) were used as negative controls. Nuclei were counterstained with propidium iodide (10 ␮g/ml, Sigma–Aldrich Inc., St. Louis, MO, USA) for 5 min.

Experimental animal models Safranin-O-fast green staining and OA grading The experimental surgical menisectomy procedure to induce OA has been reported in detail elsewhere [35] and entails a unilateral menisectomy of the medial meniscus from the right knee (OA groups). To control for the acute inflammation of the synovial membrane resulting from the surgery, we included sham-operated

Based on the Osteoarthritis Research Society International (OARSI) recommendations for the histopathological assessment of OA in rat articular cartilage, serial sectioning of tissue was performed in the coronal plane (Fig. 1, black dashed lines) to obtain

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Fig. 1. Macroscopic view of rat articular cartilage. On the experimental days indicated, rats were euthanized and condyles were carefully dissected and photographed. The figure shows condyles from normal rats without exercise (N), rats with exercise (NE), sham-operated rats without exercise (S), sham-operated rats with exercise (SE) and menisectomy-operated rats with exercise (OA). Dashed lines denote the zones in which condyles were divided. A, anterior; M, middle; and P, posterior. Scale bar = 2 mm.

200-␮m slices through the whole cartilage [37]. For illustrative purpose, the cartilage was divided into anterior, middle, and posterior zones, corresponding to the sections at approximately 600 ␮m (Supplementary Fig. 1), 1800 ␮m (Fig. 2) and 3000 ␮m (Supplementary Fig. 2), respectively. Cartilage cryosections were hydrated with PBS for 5 min and stained to assess proteoglycan content with the safranin-O-fast green technique following standard procedures. Supplementary material related to this article can be found, in the online version, at http://dx.doi.org/10.1016/j.prp.2015.01.008. Cartilage degeneration was graded from 0 to 5, where 0 was no degeneration, 1 was minimal degeneration (5–10% of the total projected cartilage area was affected by matrix or chondrocyte loss), 2 indicated mild degeneration (11–25% affected); 3 was moderate (26–50% affected); 4 was marked degeneration (51–75% affected) and 5 was severe degeneration (more than 75% affected) [37]. Western blot Cartilage samples (approximately 0.2 g per experimental group) were homogenized in polytron (Kinematica Inc., Bohemia, NY,

USA) in 500 ␮l lysis buffer [25 mM Tris–HCl, pH 7.6, 150 mM NaCl, 0.2 mM EDTA, 0.5 mM dithiothreitol, 1% Triton X-100 and enzyme inhibitors cocktail (Complete, Roche Applied Science, Manheim, DE)] and then clarified by centrifugation for 5 min at 10,000 × g. The protein concentration was determined using the Bradford procedure. SDS–PAGE was performed using 15% gels and 40 ␮g protein per gel lane. Proteins were transferred by wet transfer for 2.5 h at 350 mA to nitrocellulose membrane (Bio-Rad Laboratories, Hercules, CA, USA). Membranes were blocked with 5% nonfat dry milk and 0.5% BSA in Tris-buffered saline, pH 7.5, containing 0.1% Tween 20 (TBS-T) for 2 h at 37 ◦ C with gentle shaking, and then incubated overnight at 4 ◦ C with the following antibodies: rabbit polyclonal anti-rat IL-1␤ (1:800; Santa Cruz Biotechnology Inc.) and goat polyclonal anti-rat IL-10 (1:600; Santa Cruz Biotechnology Inc.). The immunoreactions were observed after 1 h of incubation with horseradish peroxidase-labeled anti-rabbit or anti-goat secondary antibodies (1:40,000, Jackson Immunoresearch Inc.), using the chemiluminescence ECL Plus Western blotting detection system (GE healthcare, Buckinghamshire, UK). The expression of ␤-actin (1:1000; Santa Cruz Biotechnology Inc.) was used as

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Fig. 2. Proteoglycan content in the middle zone of rat cartilage. On the experimental days indicated, rats were euthanized and articular cartilage was processed for safranin-O fast green staining. The figure shows cartilage from normal rats without exercise (N), rats with exercise (NE), sham-operated rats without exercise (S), sham-operated rats with exercise (SE) and menisectomy-operated rats with exercise (OA). Scale bar = 50 ␮m. Arrows show fibrillation, arrowheads indicate chondrocyte clusters and asterisk indicate loss of proteoglycan.

an internal control for each experimental group. The protein bands were quantified by densitometry with Image J software (http://imagej.nih.gov/ij/).

Microsystems) coupled to a microscope (DMLS, Leica Microsystems), using a 40×/0.65 C Plan lens. Microscope and software settings were maintained for all captured images.

Microscopy

Statistical analysis

Fluorescence analyses were performed with an inverted Confocal microscope (LMS700, Carl Zeiss AG, Oberkochen, DE) using a 40× plan neofluor oil immersion lens. Fluorochromes were excited with 488 nm argon laser and 543 nm helium–neon laser lines. Images from three different fields of each section (three sections per slide) were captured and processed using the ZEN 2011 Confocal software (Carl Zeiss AG). Macroscopic images were captured with a digital camera and LAZ EZ image manager software coupled to a stereoscopic microscope (EZ4D, Leica Microsystems). Images of safranin-O stained cartilages (anterior, middle, and posterior zones) were captured with a digital camera (DFC320, Leica Microsystems) and LAS image manager software (Leica

For immunofluorescence studies, cell counts were performed on recorded images; for each protein we scored nine randomly picked microscopic fields per animal from the cartilage slides. Total chondrocyte counts from images of the whole cartilage were taken as 100%. ZEN 2011 confocal microscope (Carl Zeiss AG) software was used to obtain the data from the positive cells. Data are shown as mean ± standard deviation. Statistical significance is represented as *p < 0.05, **p < 0.01 or ***p < 0.001 when NE groups are compared to N groups, and # p < 0.05, ## p < 0.01, or ### p < 0.001 when SE groups are compared to S groups. Three independent experiments were performed (n = 3). For Western blot studies, protein expression values are shown as the ratio of cytokine/actin. Data are shown as mean ± standard

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deviation. Statistical significance is represented as *p < 0.05 or **p < 0.01 when values are compared to N group and # p < 0.05 or ## p < 0.01 when NE groups are compared to OA groups. Three independent experiments were performed (n = 3). Statistical analyses were performed using the Graph Pad prism 5 program (Graph Pad Software Inc., San Diego, CA, USA). One-way ANOVA analysis with Bonferroni’s multiple comparison test was used to compare means among the experimental groups.

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Results Effect of exercise on articular cartilage integrity and proteoglycan content Neither sham surgery nor high-impact exercise for 10 days affected the integrity of cartilage (Fig. 1) or its proteoglycan content (Fig. 2, Supplementary Figs. 1 and 2). Moreover, we did not find any

Fig. 3. IL-1␤ expression in rat cartilage. On the experimental days indicated, rats were euthanized and articular cartilage was processed for indirect immunofluorescence for IL-1␤. Upper panel shows a representative immunofluorescence experiments for cartilage samples from N, NE, S, and SE groups at 3, 6, and 10 days are shown. IL-1␤ was labeled with FITC (green) and nuclei were counterstained with propidium iodide (red). Scale bar = 50 ␮m. In lower panel data are mean number of IL-1␤ positive cells ± standard deviation for N, NE, S, and SE groups. Statistical significance is represented as *p < 0.05, **p < 0.01, or ***p < 0.001 when NE groups are compared to N groups and # p < 0.05, ## p < 0.01, or ### p < 0.001 when SE groups are compared to S groups. Data from three independent experiments (n = 3). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

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Fig. 4. IL-10 expression in rat cartilage. On the experimental days indicated, rats were euthanized and articular cartilage was processed for indirect immunofluorescence for IL-10. Upper panel shows a representative immunofluorescence experiments for cartilage samples from N, NE, S, and SE groups at 3, 6, and 10 days are shown. IL-10 was labeled with FITC (green) and nuclei were counterstained with propidium iodide (red). Scale bar: 50 ␮m. In lower panel data are mean number of IL-10 positive cells ± standard deviation for N, NE, S, SE and OA groups. Statistical significance is represented as *p < 0.05, **p < 0.01, or ***p < 0.001 when NE groups are compared to N groups and # p < 0.05, ## p < 0.01, or ### p < 0.001 when SE groups are compared to S groups. Data from three independent experiments (n = 3). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

hallmarks of OA in any of the cartilage from N, NE, S or SE groups (Fig. 2, Supplementary Figs. 1 and 2). As expected, menisectomy and exercise altered the macroscopic appearance of articular cartilage (Fig. 1). While the cartilage from N, NE, S, and SE groups had a bright, glossy surface, the cartilage from OA groups was opalescent, with an increasingly rough surface over time (Fig. 1). Furthermore, menisectomy, and exercise induced histological OA hallmarks, including a loss of proteoglycan

content (asterisks in Fig. 2), fibrillation (arrows in Fig. 2) and the formation of chondrocyte clusters (arrowheads in Fig. 2), as previously reported in detail by our group [35]. The degeneration score for OA cartilage was classified as grade 1 for 3 days, as we found minimal degeneration, then grade 2 for 6 days, and finally grade 3 for 10 days. Therefore, our results suggest that at least over a short period of time, high-impact exercise alone did not affect the overall structure of articular cartilage.

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Effect of exercise on IL-1ˇ and IL-10 expression in healthy cartilage As pro-inflammatory IL-1␤ expression [8–12] and high-impact exercise [31–33] have been associated with the development of OA, we evaluated the effect of high-impact exercise on the expression of this cytokine in chondrocytes from healthy articular cartilage. Compared to N groups, sham surgery (S) did not affect the number of IL-1␤ positive cells during the 10-day experiment, which was steady at approximately 35% in both groups (Fig. 3), thus acute inflammation did not modify the percentage of IL-1␤ positive chondrocytes. On the other hand, exercise significantly increased the number of chondrocytes expressing IL-1␤ in both NE groups (approximately 1.7-fold vs. N; p < 0.05, 0.01, and 0.01 at 3, 6, and 10 days, respectively) and SE groups (approximately 2-fold vs. S; p < 0.05, 0.001, and 0.05 at 3, 6, and 10 days, respectively), from the third day of exercise, and these values were maintained throughout the 10-day experiment (Fig. 3). Positive cells were mainly localized in the superficial and middle zones of the cartilage for all experimental groups (Fig. 3). Current evidence suggests that anti-inflammatory cytokine IL-10 counteracts the catabolic activities of pro-inflammatory cytokines IL-1␤ and TNF-␣ in articular chondrocytes [24,28]. However, the effect of exercise on the expression of IL-10 in healthy articular cartilage is currently unknown. When compared to N groups, sham surgery (S) did not affect the number of IL-10 positive cells over the 10-day experimental period, as they were about 34% in both groups (Fig. 4). Acute inflammation, then, did not modify the percentage of IL-10 positive chondrocytes. In contrast, exercise significantly increased the number of chondrocytes expressing IL10 in both NE groups (approximately 1.75-fold vs. N; p < 0.05 and

Fig. 5. Pro-inflammatory IL-1␤ levels in rat cartilage. On the experimental days indicated, rats were euthanized and articular cartilage was processed for western blot. Data are the mean ratio of IL-1␤/actin ± standard deviation of N, NE and OA groups. Representative Western blot experiments are shown. Statistical significance is represented as *p < 0.05 or **p < 0.01 when values are compared to N group and # p < 0.05 or ## p < 0.01 when NE groups are compared to OA groups. Data from three independent experiments (n = 3).

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0.001 at 6 and 10 days, respectively) and SE groups (approximately 2.49-fold vs. S; p < 0.001, 0.01, and 0.001 at 3, 6, and 10 days, respectively). As with the IL-1␤ positive cells, the IL-10 positive cells were mainly localized in the superficial and middle zones of the cartilage for all experimental groups (Fig. 4). Our results thus suggest that exercise, but not sham surgery, induces the expression of IL-1 ␤ and IL-10 in chondrocytes from healthy articular cartilage. Effect of exercise on IL-1ˇ and IL-10 expression in injured cartilage We performed Western blot studies to accurately evaluate the effects of exercise on IL-1␤ and IL-10 protein levels in healthy cartilage and compare their expression levels with those from injured cartilage. Consistent with the increase in IL-1␤ positive chondrocytes, IL1␤ protein levels in the cartilage from NE groups were up-regulated during the experiment in comparison with the N group (Fig. 5). In the OA groups, which included both menisectomy and exercise, levels of IL-1␤ were also up-regulated in a time-dependent manner, up to 2.3-fold (p < 0.05) at 10 days in comparison with the N group (Fig. 5). These numbers are higher than those for the NE groups, which at 8 and 10 days IL-1␤ levels were 2.1-fold (p < 0.01) and 1.6fold (p < 0.05), lower respectively, that the levels in OA cartilage on the same days (Fig. 5). As with the IL-1␤ protein levels and consistent with the increase in IL-10 positive chondrocytes, IL-10 protein levels in the cartilage from NE groups were also up-regulated over the course of the experiment in comparison with the N group (p < 0.05 for all time points). However, in menisectomized cartilage (OA groups)

Fig. 6. Anti-inflammatory IL-10 levels in rat cartilage. On the experimental days indicated, rats were euthanized and articular cartilage was processed for Western blot. Data are the mean ratio of IL-10/actin ± standard deviation of N, NE, and OA groups. Representative Western blot experiments are shown. Statistical significance is represented as *p < 0.05 or **p < 0.01 when values are compared to N group and # p < 0.05 or ## p < 0.01 when NE groups are compared to OA groups. Data from three independent experiments (n = 3).

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IL-10 protein levels were only temporarily up-regulated, increasing about 4.9-fold at 3 days (p < 0.01 compared to N group) and then dropping to baseline at 6–10 days (p < 0.05 compared to NE groups, Fig. 6). Our results thus suggest that in injured cartilage, exercise induces the expression of pro-inflammatory IL-1␤, but the expression of anti-inflammatory IL-10 is maintained at low levels.

Discussion To the best of our knowledge, this is the first report to demonstrate the role of high-impact exercise on the expression of specific pro- and anti-inflammatory cytokines in healthy and injured cartilage. Our results suggest that in healthy cartilage, anabolic proteins (IL-10) might directly or indirectly counteract the action of exercise-induced catabolic proteins (IL-1␤), while in injured cartilage, the activity of catabolic proteins is stronger than that of anabolic proteins – an imbalance leading to OA in these experimental conditions. Although, the development of knee OA has been associated with sports-related joint injuries [31–33], the effect of regular physical activity on healthy articular cartilage remains a subject of debate. In normal rats, strenuous running for up to 12 weeks induced the development of OA, with a concomitant increase in IL-␣ and TNF␣ levels and chondrocytes death [38]. In comparison, our results show that high-impact exercise for up to 10 days up-regulated proinflammatory IL-1␤ levels without changes in the overall structure of healthy articular cartilage, while the same exercise protocol in menisectomized rats provoked OA hallmarks, including increased levels of IL-1␤. Therefore, these results suggest that strenuous and impact exercise induced the expression of pro-inflammatory cytokines, but the up-regulation of IL-1␤ alone was not enough to induce OA. The central role of pro-inflammatory cytokines in OA pathogenesis is widely acknowledged [5,12]; however, this is the first report to describe the increased expression of IL-1␤ when structural damage is still absent in the articular cartilage (3 days after the start of high-impact exercise). Furthermore, these changes cannot be attributed to acute inflammation, as demonstrated by the results from the sham surgery groups, in which the capsule and synovial membrane were cut. These results may explain the onset of cartilage deterioration at advanced OA stages, since IL-1␤ directly inhibits the expression of cartilage-specific extracellular matrix genes [13–15], up-regulates matrix-degrading proteases [15–17], stimulates the production of reactive oxygen species (ROS) and induces chondrocyte death [39,40]. Low levels of IL1␤ have been detected in the synovial fluid of patients with OA [41], suggesting that IL-1␤ acts locally to alter the metabolism of chondrocytes during OA pathogenesis. In fact IL-1␤ is able to autocrinally/paracrinally induce its own secretion to enhance its catabolic activity, but, significantly, it can also stimulate the synthesis of other pro-inflammatory cytokines, such as TNF-␣, IL-6, IL-8, and CCL5 chemokine [12], strengthening the destruction of articular cartilage by activating several ligand–receptor signaling pathways in chondrocytes. Although the number of pro-inflammatory IL-1␤ positive chondrocytes also increased in the NE groups, semi-quantitative analysis shows that IL-1␤ expression levels at late stages (6–10 days) were lower in NE groups than in OA groups. Therefore, it seems that in healthy cartilage, exercise-induced overexpression of IL-1␤ may be sufficiently regulated by anti-inflammatory elements, preventing the degradation of articular cartilage [42]. The strong down-regulation of IL-10 in the OA groups, then, may account for the progression of the disease, since IL-10 may reduce the

effects of IL-1␤ and TNF-␣ by decreasing the damage and enhancing the repair of the joint. In human chondrocyte cultures, IL-10 over-expression counteracts the increased activity of MMP-13 and down-regulates IL-1␤ levels, both induced by TNF-␣ [26]. In addition, IL-10 can decrease caspase activity and the bax/bcl-2 ratio in TNF-␣ stimulated chondrocytes, modulating the pro-apoptotic ability of TNF-␣ [24] and stimulating collagen type II and proteoglycan expression [23–26]. In patients with knee OA, moderate exercise increases the levels of IL-10 in synovial fluid and periarticular tissues, suggesting that this cytokine contributes to the beneficial effects of exercise on people with OA [43]. However, the low IL-10 levels following experimental OA induction may not be enough to overcome the pro-inflammatory activity and might be related to the increase in pro-inflammatory molecules. In mononuclear cells from synovial fluid, the neutralization of endogenously produced IL-10 results in increased production of IL-1␤, TNF-␣, and GM-CSF [42], and in OA cartilage, IL-1␤ activity may impair TGF-␤1 signaling primarily through down-regulation of TGF-␤RII, which is mediated by the p65/NF-kappa and the activator protein 1/JNK pathways, and secondarily through the upregulation of Smad7, which is an inhibitor of Smad, blocking the TGF-␤/Smad transduction pathway [44,45]. Moreover, some chondroprotector properties of IL-10 are related to the stimulation of the synthesis of IL-1␤ antagonist (IL-1Ra) and the tissue inhibitor of metalloproteinases-1 [46]. In addition, articular cartilage is a mechanosensitive tissue, able to perceive and respond to biomechanical signals to modify the structure and composition of the tissue. During exercise, then, biomechanical signals are perceived by cartilage in magnitude, frequency, and time-dependent manners, affecting chondrocyte metabolism and triggering intracellular signaling to regulate the expression of anabolic, catabolic, and structural genes [47]. In our rat model, the magnitude of biomechanical forces applied in exercise is quite different in the cartilage of menisectomized and non-menisectomized rats (because of lack of menisci), causing the differential expression pattern of pro- inflammatory (IL-1␤) and anti-inflammatory (IL-10) cytokines. Moreover, the magnitude of compressive forces can also modulate the response of articular cartilage during OA pathogenesis. Evidence suggests that moderate exercise, rather than strenuous exercise, is beneficial for cartilage. In anterior cruciate ligament transection (ACLT) and monosodium iodoacetate (MIA) OA rat models, moderate exercise (running for up to 28 days) reduced OA severity by decreasing apoptotic chondrocytes [36,48]. Furthermore, moderate exercise both up-regulated the expression of lubricin, a chondroprotective glycoprotein that serves as a critical lubricant between opposing cartilage surfaces, and increased the glycosaminoglycan content, which improves the viscoelasticity of cartilage, thereby protecting the collagen network [49]. Aside from cytokine expression, it would be interesting to know how chondroprotective elements are adjusted in non-menisectomized rats after impact exercise to prevent the development of OA. The expression of several pro- and anti-inflammatory cytokines in articular cartilage after exercise still requires further research. Early changes in cytokine concentration after joint injury indicate an important imbalance in the anabolic/catabolic equilibrium. Characterizing the molecules involved in this process might thus be of great importance in the search for target molecules for new therapeutic and early diagnostic methods for OA.

Conflict of interest The authors declare that there are no conflicts of interests.

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