Peroxisome Proliferator Activated Receptor Beta (PPARβ) activity increases the immune response and shortens the early phases of skeletal muscle regeneration

Accepted Manuscript Peroxisome Proliferator Activated Receptor Beta (PPARβ) activity increases the immune response and shortens the early phases of skeletal muscle regeneration I. Mothe-Satney, J. Piquet, J. Murdaca, B. Sibille, P.A. Grimaldi, J. Neels, A.S. Rousseau PII:

S0300-9084(16)30295-4

DOI:

10.1016/j.biochi.2016.12.001

Reference:

BIOCHI 5105

To appear in:

Biochimie

Received Date: 24 October 2016 Revised Date:

29 November 2016

Accepted Date: 2 December 2016

Please cite this article as: I Mothe-Satney, J Piquet, J Murdaca, B Sibille, P. Grimaldi, J Neels, A. Rousseau, Peroxisome Proliferator Activated Receptor Beta (PPARβ) activity increases the immune response and shortens the early phases of skeletal muscle regeneration, Biochimie (2017), doi: 10.1016/j.biochi.2016.12.001. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Peroxisome Proliferator Activated Receptor Beta (PPARβ) activity increases ACCEPTED MANUSCRIPT the immune response and shortens the early phases of skeletal muscle regeneration

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I Mothe-Satney1, J Piquet1, J Murdaca1, B Sibille1, PA Grimaldi1†, J Neels1, AS Rousseau1*

Affiliations: Université Côte d’Azur, Inserm, C3M, Nice, France.

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Deceased May 16, 2016.

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*Corresponding author:

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Address correspondence to: Anne-Sophie ROUSSEAU

INSERM U 1065, Mediterranean Centre for Molecular Medicine (C3M) Team 9: Adaptive responses to immuno-metabolic dysregulations Université de Nice-Sophia Antipolis

151 Route de Ginestière BP 2 3194 06204 Nice Cedex 3 FRANCE Phone: ++ 33 (0) 4 89 06 43 46

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Fax: ++ 33 (0) 4 89 06 42 21

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Bâtiment Universitaire Archimed

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E-mail : [email protected]

Abstract

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Peroxisome Proliferator-Activated Receptor Beta (PPARβ) is a transcription factor playing an important role in both muscle myogenesis and remodeling, and in inflammation. However, its role in the coordination of the transient muscle inflammation and reparation process following muscle injury has not yet been fully determined. We postulated that activation of the PPARβ pathway alters the early phase of the muscle

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regeneration process, i.e. when immune cells infiltrate in injured muscle. Tibialis anteriors of C57BL6/J mice treated or not with the PPARβ agonist GW0742 were injected with cardiotoxin (or with physiological serum for the contralateral muscle). Muscle regeneration was monitored on days 4, 7, and 14 post-injury.

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We found that treatment of mice with GW0742 increased, at day 4 post-damage, the recruitment of immune cells (M1 and M2 macrophages) and upregulated the expression of the anti-inflammatory cytokine IL-10

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and TGF-β mRNA. Those effects were accompanied by a significant increase at day 4 of myogenic regulatory factors (Pax7, MyoD, Myf5, Myogenin) mRNA in GW0742-treated mice. However, we showed an earlier return (7 days vs 14 days) of Myf5 and Myogenin to basal levels in GW0742- compared to DMSO-treated mice. Differential effects of GW0742 observed during the regeneration were associated with

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variations of PPARβ pathway activity. Collectively, our findings indicate that PPARβ pathway activity shortens the early phases of skeletal muscle regeneration by increasing the immune response.

Highlights

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Receptor

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Key-words: Inflammation, muscle injury, macrophage, reparation, Peroxisome Proliferator Activated

1. PPARβ agonist increases macrophage infiltration 2. PPARβ agonist increases the anti-inflammatory response of muscle injury 3. PPARβ agonist promotes satellite cell proliferation and differentiation 4. PPARβ agonist shortens the early phases of regeneration 5. PPARβ agonist has no effect on physical capacity during the early period of regeneration 2

ACCEPTED MANUSCRIPT 1. Introduction 1

The ability of skeletal muscle to regenerate is altered in many diseases such as myopathy [1], diabetes [2, 3] and sarcopenia [4, 5]. This complication contributes to the progression of these diseases,

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because skeletal muscle integrity is crucial for the maintenance of metabolic homeostasis. Muscle injury triggers different signals (oxidative stress, inflammation) that act on molecular pathways necessary for the setup of the muscle regeneration process characterized by destruction, repair and remodeling phases. The

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main players are the satellite cells, localized between the basal lamina and the muscle fiber membrane [6]. After skeletal muscle damage, inflammation is triggered by resident macrophages. The latter will secrete

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cytokines, chemokines and growth factors, leading to the recruitment of circulating immune cells and to the activation of the satellite cells [7, 8]. The satellite cells express the paired-box protein Pax7 which regulates target genes involved in cell growth and proliferation. Pax7 also plays a key role in maintaining the proliferation of progenitors and in preventing early myogenic differentiation [9]. Upon activation, satellite

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cells will proliferate with upregulation of the myogenic determination factors Myf5 and MyoD, followed by downregulation of Pax7. The initiation of terminal differentiation starts with the expression of Myogenin and MRF4 [9]. It has been shown that activated M1 macrophages promote early pro-inflammatory events

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(removal of cellular debris) and myoblast proliferation [10, 11], whereas activated anti-inflammatory M2 macrophages are associated with advanced stages of tissue repair and stimulate myoblast differentiation and

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fusion [12]. T lymphocytes are also transiently recruited into the skeletal muscle after an acute injury [13]. The regulatory T (Treg) cell (CD4+Foxp3+) subset is mainly represented and is shown to increase as the innate immune cells shift from a pro-inflammatory (M1) to anti-inflammatory phenotype (M2) [14, 15]. During skeletal muscle regeneration, the pathway involving the transcription factor peroxisome proliferator-activated receptor beta (PPARβ) - which is also known as PPARδ or PPARβ/δ - seems to be of

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Abbreviations: Pax, paired-box; Myf, myogenic factor; MyoD, myogenic differentiation; MRF, myogenic regulatory factor; PPAR, peroxisome proliferator activated receptor; Treg, regulatory T cells; TLA, tibialis lateralis anterior; NF-κB, nuclear factor kappa B; CTX, cardiotoxin; PDK4, pyruvate deshydrogenase kinase 4; IL-, interleukin; TNF, tumor necrosis factor; TGF, transforming growth factor; MCP-1, monocyte chemoattractant protein 1; Q-PCR, quantitative PCR, JNK, c-Jun N-terminal kinase.

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particular importance for the normal sequence of events following injury, as shown by using a PPARβ-null

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mouse model [16]. PPARβ is known to regulate metabolism and inflammation [17]. This nuclear receptor has been involved in myogenesis, favoring proliferation and differentiation of satellites cells [18]. Our team previously showed that PPARβ is highly expressed in skeletal muscle and even more in skeletal muscle of physically trained rodents [19; 20]. PPARβ has the particularity to be activated by a broad spectrum of

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endogenous and exogenous ligands. The activation of the PPARβ pathway in skeletal muscle, by overexpressing PPARβ protein and/or through pharmacological ligand binding (GW0742), induces an oxidative remodeling of skeletal muscles. This remodeling is characterized by an increase in the number of

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oxidative fibers [19] and in capillary numbers per fiber [21]. These effects are accompanied by a hyperplasia corresponding to the generation of 600 new myofibers in the tibialis lateralis anterior (TLA) muscle [21].

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We have also shown that PPARβ activation leads to the estimated addition of about 1 million new myonuclei in TLA muscle. This occurs without any signal of hypertrophy or regeneration [22]. Collectively, findings from our team indicate that PPARβ activation leads to some skeletal muscle adaptations that are similar to those observed in response to both endurance and resistance training [19; 22; 23]. Inversely, the

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muscle-specific knockout of PPARβ in mice has led to a lower skeletal muscle oxidative capacity which precedes the development of metabolic disorders [24]. Together, scientific data suggest that the maintenance of PPARβ activity is crucial to prevent age and lifestyle-related metabolic disorders. PPARβ also plays an

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important role in immune cell functions. Indeed, it has been shown that PPARβ is necessary for the differentiation of monocytes into M2 macrophages in adipose tissue [25, 26]. We also recently demonstrated

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that PPARβ activation or overexpression might affect inflammation by altering T cell development in the thymus [27]. Moreover, in cardiomyocytes and microglial cells, PPARβ regulates inflammation by controlling NF-κB activity. Indeed, an increase in PPARβ expression can decrease the NF-κB activity in a trans-repressive manner by physically interacting with the p65 subunit [28; 29]. Until now, studies have focused on the effect of PPARβ activation on myogenesis by using a myoblast C2C12 in vitro culture model treated with a PPARβ agonist [30] or in vivo by using a PPARβ-null mice model in the context of muscle regeneration [16]. Chandrashekar and colleagues [16] have shown that inactivating the PPARβ pathway increased macrophage infiltration and reduced myoblast proliferation in 4

regenerating muscle. However, it remains to be determined whether in vivo PPARβ activation by agonists

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results in opposite effects, leading to a reduced inflammation and to an improved process of regeneration. In this study, we investigated this hypothesis by treating adult mice with a diet enriched with a PPARβ agonist. The inflammatory and anti-inflammatory responses to a cardiotoxin-induced injury in TLA and the resulting expression of myogenic regulatory factors were studied during the first 14 days of skeletal muscle

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regeneration. We report here a potential role for PPARβ pathway activators in skeletal muscle regeneration.

2. Material and methods

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2.1 Animal studies and treatments. Animals were maintained in a 12-h light, 12-h dark cycle and received food [UAR (Usine d’Alimentation Rationnelle), Villemoisson sur Orge, France] and water ad libitum

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(agreement number of the animal facility: A 06-088-014). All experimental procedures were conducted according to French legislation and to the EU Directive 2010/63 for animal experiments and were approved by the Institutional Ethic Committee for the Use of Laboratory Animals (CIEPAL-AZUR) (N°2015120415185104). Seventy two C57Bl/6J wild-type mice (8 to 10-weeks old) were purchased from

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Charles River (France). A first group of 24 mice was used to evaluate the effects of an oral supplementation with the specific PPARβ agonist GW0742 on PPARβ pathway activation. After one week of acclimatization, the mice were divided in two groups (n=12 per group): one received standard chow

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supplemented with GW0742 (3mg/kg/day) and the other a vehicle-supplemented food (DMSO 1%). After two weeks, the mice were sacrificed and their skeletal muscle harvested and processed for Q-PCR analyses

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(described below). Then, a second group of 48 mice was subjected to the same diets as those described above (24 with the vehicle- and 24 with the GW0742-supplemented diet). These oral treatments began two weeks before muscle injury and were applied throughout the procedure. Mice were injured by injection of 50µl of cardiotoxin (CTX) from Naja pallida at 0.03 mg/ml (L8102, Latoxan, France) in the left TLA, under gas anesthesia (5 % Vetflurane). The right TLA received 50µl of saline (control leg). In order to avoid excessive pain, the mice received a subcutaneous injection of Buprenorphine (100 µl at 30µg/ml) 20 minutes before gas anesthesia. Twelve mice (6 from each group of supplementation) were sacrificed by cervical dislocation at day 4, 7, 9 and 14 post-injury. TLA were harvested, weighed and used for further analysis (see 5

below). Moreover, some mice were subjected to physical tests 2 days before muscle injury and the day

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before sacrifice for day 7 and 14 post-injury. The strength of upper limbs was measured using a grip test equipped with a bar (Bio-GS3, Bioseb). After several measurements the best value was recorded and the maximal strength was expressed in Newton per gram (N/g). The endurance of the mice was evaluated using a treadmill (five-lane motorized treadmill, LE8710M, Bioseb) at 18m.min-1 for one hour, with a slope of 5

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degrees. The rear of the treadmill was equipped with a low-voltage electric stimulating bar to encourage each mouse to run. The bar was set to deliver 0.2 mA at a frequency of 0.25 Hz, which caused an uncomfortable shock but did not injure the animal. Number of shocks was recorded and the electric delivery

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was stopped if 50 shocks were reached. The mice were previously familiarized with the tests one week before the evaluation.

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2.2 Muscle dissociation and flow cytometry. Two-third of each TLA (injured and control) were washed with phosphate-buffered saline (PBS), minced and digested with 2mg/ml of type A collagenase in 1.5ml of DMEM medium supplemented with 10 % fetal bovine serum (FBS) for 60 min at 37°C. The muscle was further dissociated by performing 5 passages through a 3-ml syringe with an 18G needle. After an additional

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15 min of digestion and a second round in the syringe, the homogenate was diluted 3 times in DMEM, filtrated (70µm filter) and centrifuged at 300g for 20 minutes. The cell pellet was resuspended in PBS supplemented with 0.5 % FBS at a final concentration of 5x106 cells/ml. Cell suspensions were incubated

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with Fc Block (anti-mouse CD16/CD32 monoclonal antibody, BD Biosciences) for 15 min at 4°C before

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staining with fluorescently labeled primary antibodies for 20 min at 4°C in PBS, 0.5% FBS. Cells were washed twice and resuspended in PBS, 0.5% FBS with DAPI. Stained cell preparations were analyzed using a BD FACSCanto II flow cytometer (BD Biosciences). Percentages of DAPI negative cells were not different between experimental conditions (DMSO and GW0742) in control and injured TLA (44.5% vs 44.1% in control TLA and 49.1% vs 45.2% in injured TLA, for DMSO and GW0742 respectively). T lymphocytes and macrophages were analyzed by flow cytometry at different times post-injury (4, 7 and 14). F4/80-allophycocyanin, CD11b- phycoerythrin-Cy7, CD11c- phycoerythrin, CD3-phycoerythrin and CD4allophycocyanin were purchased from eBioscience. CD8-Peridinin chlorophyll antibody was from BD Biosciences. 6

2.3 RNA extraction and quantitative real-time PCR. One third of each TLA (injured and control) was used

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for RNA extraction. Total RNA was extracted with Trizol reagent following the supplier's protocol (Invitrogen). 1 µg of RNA was reverse-transcribed using a QuantiTect Reverse Transcription Kit (Qiagen) on a Qcycler II (Quanta Biotech). Quantitative PCR was performed using SYBR Premix Takara (Tli RNase H Plus, Clontech) on a StepOne machine (Life Technologies). The relative amount of all mRNAs was

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calculated using the comparative ∆∆Ct method and 36B4 was used as the invariant gene control for all conditions. The mice used for the reference corresponded to the DMSO group at day 14 post-injury. We checked that the values obtained for the genes studied at this day were not different from the values obtained

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in the first group (14-day GW0742-supplemented diet study) in which mice received a DMSOsupplemented diet and were not injured (control group). The value obtained for this first group is

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represented as the baseline in each graph. Primer sequences used are presented in Table 1.

2.4 Statistical analysis. Paired comparisons between injured and control TLAs have been performed using Wilcoxon signed rank test and permutation exact test. The effect of GW0742 treatment was evaluated with the Mann-Whitney test. The kinetics of regeneration was tested using Kruskal-Wallis test. All data were

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analyzed using Statview (v4.57) and StatXact softwares (v8). Statistical significance was accepted at

3. Results

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p<0.05. The results are presented as means ± standard errors.

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3.1 15-days dietary GW0742 treatment increases PPARβ activity but does not significantly alter the potential of proliferation and differentiation of TLA muscle cells. Firstly, we have verified that a 2-week treatment with GW0742 administrated orally through food, effectively led to an increase in PPARβ pathway activity in TLA muscle. We investigated the effect of the treatment on the level of PDK4 mRNA, previously described as a main PPARβ-regulated target gene in various tissues including skeletal muscle [22]. As shown in Figure 1, two weeks of oral supplementation with GW0742 promoted a significant 2.5 fold induction of PDK4 mRNA in TLA compared to DMSO control mice. We also examined in these mice whether the GW0742 treatment altered the expression of 7

genes used in the characterization of the different stages of the regeneration process. In uninjured mice, the

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mRNA level of Pax-7 - which is known to regulate target genes involved in cell growth and proliferation was significantly lower in GW0742 treated mice compared to DMSO control mice. While this decrease of Pax-7 seemed to be accompanied by a decrease in Myf5 mRNA level, a target gene of Pax-7, the latter decrease does not reach statistical significance due to a high degree in variation of expression of Myf5 in the

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samples analyzed (Table 2).

3.2 Expression and transcriptional activity of PPARβ vary during the regeneration period.

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We examined the expression and transcriptional activity of PPARβ during muscle regeneration after an acute injury. In order to induce muscle injury and study the subsequent muscle regeneration, we used the

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standardized method of CTX injection in TLA muscle, which is a highly reproducible model [31]. Muscles were harvested and compared 4, 7 and 14 days following CTX-induced injury. At day 4, PPARβ and PDK4 mRNA were significantly lower in the injured compared to uninjured TLA. This difference was no longer significant at day 7 and later (Figure 2A and B). Moreover, at day 4, PPARβ mRNA was significantly lower

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in the injured TLA of GW0742-treated mice compared to control DMSO-treated mice. No difference between groups was observed at day 7 and 14 for PPARβ mRNA but its transcriptional activity, as reflected by PDK4 mRNA level, was significantly higher at day 7 in the GW0742-treated mice compared to DMSO-

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treated mice (Figure 2A and B). Compared to the baseline (which corresponds to the values obtained in the first group in which mice received a DMSO-supplemented diet and were not injured), expression level of

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PPARβ tended to be lower at day 4 (Figure 2A).

3.3 Immune cell infiltration into injured TLA muscle is increased in GW0742 treated mice at day 4 post-injury. At various time points (day 4, day 7 and day 14) after the acute injury induced by injection of CTX in the TLA of healthy C57BL/6J mice, we collected and digested TLA (injured and uninjured (control)) and retrieved the stromal vascular fraction, which was then characterized by flow cytometry. Cytometry analysis 8

showed that relatively few macrophages and T lymphocytes were detected in uninjured TLA (Figure 3A,

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Table 3) but that macrophage (M1 and M2) presence was increased at day 4 in the injured compared to uninjured TLA (Figure 3A). The difference with the uninjured TLA remained significant at day 14 for M1 macrophages (Figure 3A). Our results showed a different kinetic of M1 macrophage infiltration between GW0742 and DMSO treated mice. Early influx of M1 macrophages was observed in GW0742-treated mice

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at day 4 in injured TLA muscle whereas M1 macrophages were peaking at day 7 in control mice. M1 macrophages were significantly higher (4-fold) in GW0742- compared to DMSO- treated mice at day 4 (Figure 3A). No differences in M1 macrophage number between groups were apparent at day 7 and 14. M2

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macrophages peaked relatively early (four days post-injury) in both groups. Interestingly, the magnitude of the response was 4-fold higher in GW0742-treated mice compared to their DMSO-treated counterparts

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(Figure 3A). In both groups, the number of M2 macrophages was dramatically decreased at day 7. At day 4, CD3+, CD4+ and CD8+ T cells number (per 10 mg of TLA) was significantly increased in injured TLA compared to uninjured TLA, with similar magnitude with GW0742 treatment (Table 3). Compared to uninjured TLA, the difference remained significant at day 7 for CD4+ in both GW0742 and DMSO mice but

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was no longer significant at day 14 (Table 3). For technical reasons (low number of cells for immunolabelling) we were unable to characterize the Treg subtype of CD4+ cells. Next, we performed RT-qPCR analysis of selected cytokines or markers characterizing M1 inflammatory or M2 anti-inflammatory

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responses, such as CD68, MCP-1, IL-6, TNF-α, IL-10 and TGF-β. These markers were all significantly increased in injured compared to uninjured TLA. Among those markers, IL-10 and TGF-β mRNA, exhibited

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a significantly higher expression in injured TLA of GW0742-treated compared to DMSO-treated mice (Figure 3B). The difference between groups for CD68, MCP-1, IL-6 and TNF-α mRNA levels was not significant (Table 4). However, we observed at day 4, significant negative correlations between PDK4 and TNF-α mRNA (R= - 0.76; p = 0.028) and between PDK4 and CD68 mRNA (R = - 0.53; p = 0.04), suggesting that markers of inflammation negatively correlated with PPARβ activity.

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3.4 GW0742 treatment improves the potential of satellite cell activation, proliferation and

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differentiation at day 4 and shortens the early phase of skeletal muscle regeneration. As shown in Figure 4A, in GW0742 treated mice, Pax 7 mRNA level was significantly higher at day 4 in injured compared with uninjured TLA. This difference was transient as it was no longer significant at day 7. In contrast, in the DMSO group, the difference between uninjured and injured TLA was not significant at

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day 4 but was significant at day 7 (Figure 4A). For GW0742- and DMSO-treated mice, the same kinetics of expression was observed regarding the myogenic regulatory factor Myf5 which is a Pax-7 target gene involved in proliferation of satellites cells (Figure 4B). Significant higher mRNA level for Myf5 compared

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with uninjured TLA was shown at day 4 for GW0742 treated mice and at day 7 for DMSO control mice (Figure 4B). Our results showed a significant increase in mRNA level of MyoD in the injured TLA at day 4

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in the GW0742-treated group. However, values of expression levels returned rapidly to basal level at day 7 (Figure 4C). Myogenin mRNA level was significantly higher at day 4 post-injury in injured TLA compared to uninjured TLA and compared to other days, with no difference observed between treatment groups at day 4 (Figure 4D). However, at day 7, Myf5 and myogenin mRNA levels were significantly lower in GW0742-

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treated mice than in DMSO-treated mice and were not different from the baseline level (Figure 4B,4D). In DMSO-treated mice, those values returned to baseline level later (observable at day 14). The fusion myogenic factor MRF4 was shown to be down-regulated at day 4 in both treatment groups in injured TLA

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with level significantly lower in GW0742 treated mice compared to DMSO mice. Values return to baseline level in both groups at day 7 without differences between treatment groups (Figure 4E). Myostatin signaling

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inhibition has been shown to be involved in muscle growth [32]. The mRNA level of myostatin was dramatically lower in injured compared to uninjured TLA (Figure 4F). This difference was still significant at day 14. Mice treated with GW0742 exhibited lower myostatin mRNA in response to injury at day 4 compared to DMSO treated mice. However, there was no difference between groups for the other days (Figure 4F).

3.5 Exercise capacity of mice was not improved with GW0742 treatment during the early phase of regeneration. 10

Total weight of mice was not significantly altered by GW0742 treatment and/or by CTX-induced TLA

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injury (Table 5). However, injured TLA muscle weight was significantly lower than uninjured contralateral TLA at day 4. The difference was still significant at day 7 post-injury but not at day 14 (Table 5). Moreover, CTX injection has no effect on the uninjured TLA weight (data not shown). A grip-test was performed to determine non-specific potential force loss in response to injury but we did not see any differences during

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the regeneration phase and in regard to the GW0742 treatment (Table 6). As mice did not present any signs of mechanical problems and pain at day 6 and 13, we performed a running to exhaustion protocol on a treadmill at day 6 and day 13 to determine endurance performance capacity of mice. Compared to before the

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CTX-induced TLA injury, running performances were significantly decreased at day 6 and 13. The loss of running exercise capacity was higher, but not significantly different, at day 13 than at day 6. No difference

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of performance loss was observed between GW0742- and DMSO-treated mice (Table 6).

4. Discussion

PPARβ presents an interesting therapeutic target in a large variety of inflammatory diseases. PPARβ

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activation or overexpression leads to a decrease in inflammation, and deletion of PPARβ leads to an aggravation of the inflammatory state [17]. In this study, we highlight a role for PPARβ as a regulator of the immune response in the context of skeletal muscle regeneration by treating mice with GW0742, a potent and

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specific PPARβ agonist [33]. Our results support the fact that GW0742 exerts differential effects at the different time-points of the muscle regeneration process.

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In accordance with other studies [13, 34, 35], the number of macrophages in the injured TLA is high at day 4 and 7 post injury and then decreases. Our most important finding is that both M1 and M2 macrophages are markedly increased in the injured TLA of GW0742-treated mice, compared to DMSOtreated mice at day 4. This also tends to be the case in the uninjured TLA of GW0742-treated mice but it does not reach statistical significance. Mechanisms involved in the increase in immune cell infiltration in skeletal muscle in response to muscle injury are not clear. The stability of the microvasculature is important because it ensures both the provision of macrophages and the delivery of nutrients and oxygen for the support of the metabolically intense phase of regeneration. We used an experimental model of injury with 11

CTX which presents the advantage to be specific to muscle fibers, thereby eliminating complicating factors

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of damage to motor nerves and their associated blood vessels [31]. Since we previously showed that a 48-hrs treatment of mice with GW0742 increased the capillary number by 1.5 fold in TLA [21], we might expect the same effect in our study, which could explain the increase in the infiltration of immune cells. We have analyzed the mRNA expression of several markers of blood vessels (PECAM, VEGF, TIE-1, TIE-2).

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However, there was no difference between GW0742 and DMSO groups in both injured and uninjured TLA (data not shown). The fact that we don’t observe any difference in the expression of these endothelial markers after a two-week treatment with GW0742 suggests that the pro-angiogenic effects observed after

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48-hrs of GW0742 treatment are transient. The increase in macrophage infiltration is also explained by an increase in chemoattractants in the injury environment. Interestingly, the migration and accretion of

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macrophages to the site of injury is increased with the inhibition of myostatin, therefore improving muscle regeneration [34, 35]. We reported that at day 4 myostatin expression in the injured TLA of GW0742-treated mice was further decreased compared to injured TLA of DMSO-treated mice (Fig.4F). However, whether this effect is linked to the higher macrophage infiltration in the injured TLA, shown in GW0742-treated

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mice, remains to be investigated.

Increased M1 and M2 macrophage infiltration at day 4 was concomitant, but the resulting response was mostly anti-inflammatory. Indeed, anti-inflammatory cytokines and growth factors (IL-10 and TGF-β)

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were further increased with GW0742 treatment in the injured TLA compared to DMSO, whereas proinflammatory cytokines were not differently expressed in both groups. Even if macrophages are the

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predominant immune cells in the regenerative muscle, T cells are also present. Like macrophages, we showed that CD3+ cells (and the CD4+ and CD8+ subsets) peaked at day 4 post-injury and their number was strongly increased in the injured TLA compared to the non-injured TLA. However, GW0742 treatment had no significant effect on T cell subpopulations. Interestingly, regulatory T (Tregs) cells are known to accumulate in acutely injured muscle and were shown to express high levels of IL-10 [14]. Due to the low number of stromal vascular cells retrieved from the muscle samples, we were not able to analyze the Treg population in our study. However, this subset of T lymphocytes might be involved in the regulation of the anti-inflammatory response that we observed. 12

Taken together, the global result of the increase in immune cells infiltration with GW0742 treatment

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was an increase in the anti-inflammatory response, despite M1 macrophages that remain elevated. Therefore, we were wondering whether the increased number in M1 macrophages was beneficial or deleterious for the regeneration process. Evidence from the literature demonstrated that M1 macrophages are actually beneficial for the cell proliferation phase. Indeed, macrophage-satellite cell co-cultures showed that M1

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macrophages are essential for facilitating muscle fiber reconstitution by enhancing satellite cell proliferation [10] and M1 macrophage deficiency severely impairs satellite cell proliferation and skeletal muscle regeneration [38]. However, it has been previously shown that M1 macrophage presence delays satellite cell

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differentiation [10]. One of the master regulators of myogenic differentiation is MyoD. In our study, increased M1 macrophage infiltration in the injured TLA of GW0742-treated mice at day 4 is associated

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with a significantly higher MyoD mRNA level. On the contrary, in PPARβ-null mice, MyoD expressing cells were reduced at day 3 and 7 of muscle regeneration [16]. Our result suggests that GW0742 treatment did not impair the differentiation process but on the contrary seemed to improve it. This may be due, at least partly, to a preservation of the pro-inflammatory signal in the presence of GW0742, since cytokines such as

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TNF-α are expressed at day 4. TNF-α is a target-gene of NFκB known to be necessary for the normal muscle regeneration process [39]. Interestingly, PPARβ can interact with the p65 subunit of NFκB leading to a decrease in NFκB transcriptional activity [28, 29]. We observed at day 4 a downregulation of PPARβ in

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response to GW0742 and its target-gene PDK4. The lack of PDK4 induction at this time might be due the decrease in PPARβ level itself, since it is known that PPARβ expression is critical for its level of activity.

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Stress-activated pathways such as JNK pathway are induced during the early phase of regeneration to promote proliferation [40]. Interestingly PPARβ expression is negatively linked to an increase in JNK pathway activity [20]. Transient variations of PPARβ expression in skeletal muscle is also shown to be due to epigenetic processes, also highly sensitive to the redox environment [41]. Together, these arguments led us to assume that PPARβ downregulation seen at day 4 could be an adaptive response to allow the induction of the inflammatory response necessary to initiate the regeneration. This is supported by the strong negative correlations between PDK4 expression and the inflammatory markers TNF-α and CD68, supporting the existence of a regulatory process involved in the modulation of PPARβ activity. Interestingly, PPARβ 13

expression is also lower at day 4 in uninjured contralateral TLA which was injected with PBS compared to

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its expression quantified at day 7 and 14 and compared to the level determined in mice that were not subjected to TLA PBS or CTX injections. Even if we cannot exclude that the injection of PBS has induced a slight muscle fiber lesion in the contralateral TLA, we rather think that this effect is due to an increase in systemic inflammatory and oxidative stress exposition induced by the injury.

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While pro-inflammatory M1 macrophages are critical for the early phase of satellite cell activation and proliferation, anti-inflammatory M2 macrophages are necessary for the normal myoblast differentiation and fusion [12]. It is noteworthy that the most determinant effect on muscle differentiation may not be

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directly linked to the number of immune cells but to the global pro-/anti-inflammatory balance. IL-10 has been shown to play a significant regulatory role in muscular dystrophy by reducing M1 macrophage

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activation and toxicity, increasing M2 macrophage activation and modulating muscle differentiation [42]. As IL-10 is increased with GW0742 treatment in our study, this suggests that IL-10 might permit the differentiation process, despite the presence of a high number of pro-inflammatory macrophages at day 4. This is supported by the fact that the early (myogenin) and late (MRF4) differentiation markers were

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expressed. However, even if the expression of the myogenic regulatory factors studied did not differ between GW0742- and DMSO-treated groups at day 4, this was not the case at day 7. Indeed, Myf5 and Myogenin returned to their basal level at day 7 in GW0742-treated mice and only at day 14 for DMSO-

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treated mice. This result suggests that GW0742 treatment has induced either a faster return to basal level or an earlier kinetics of myogenic response. It is noteworthy that this effect was concomitant with a return of

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PPARβ to its basal level which was associated with an increase in PPARβ transcriptional activity (Fig. 2A and 2B). We were unable to study the consequences of a shortened proliferation and differentiation on the muscle integrity recovery. Indeed, the regeneration process is not finished at day 14 in both DMSO and GW0742-treated mice, as shown by the myostatin expression level that was still lower than in the uninjured TLA. Moreover, performance capacity of mice, measured by a running test until exhaustion on a treadmill, was still altered at day 14, without any sign of biomechanical dysfunction. Further studies are necessary to analyze the evolution of both proliferation and differentiation markers at earlier and later time-points after injury and the consequences on muscle remodeling, metabolism and on performance capacity. 14

ACCEPTED MANUSCRIPT In conclusion, we have shown that GW0742 treatment induced changes in PPARβ activity by modulating both PPARβ activation and PPARβ expression during the regeneration process. We highlight for the first time a role for increase PPARβ activity as a regulator of the immune response in the context of skeletal muscle regeneration. Our most important finding is an increase in macrophage infiltration (both M1

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and M2) in injured muscle of mice treated with a PPARβ agonist. This effect was accompanied by a higher anti-inflammatory response and an earlier return to basal levels of some myogenic regulatory factors. Consequently, GW0742 treatment might improve the adaptive response to the regeneration process, but the

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consequences on the recovery of physical performances remain to be determined.

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Aknowledgments

The authors thank Veronique Corcelle and the animal facility staff (Unit1065, C3M, Institut National de la Santé et de la Recherche Médicale (INSERM)), for their excellent care of mice. This work was supported by the University of Nice Sophia Antipolis, the Institut National de la Santé et de la Recherche Médicale and by

2014).

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Conflict of interest

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Association Nationale pour le Traitement à Domicile des Insuffisants Respiratoires (Appel à projet SFNEP,

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The authors have no conflict of interest Author contributions

I.M.-S. assisted with mice experiments and participated to the writing of the paper; J.P performed qPCR analysis, prepared figures, analyzed data. J.M. performed the flow cytometry experiments, B.S. participated in the preparation of the manuscript. P.G. initiated the work on PPARβ and myogenesis. J.N. participated in the preparation of the manuscript. A.-S.R. designed and supervised the research, performed mice experiments, qPCR analysis, and wrote the paper.

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Figure legends

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Figure 1. mRNA level of PPARβ/δ target-gene PDK4 in TLA from mice treated or not with GW0742. Mice were fed with a standard chow supplemented with GW0742 (3mg/kg/day) or vehicle-supplemented food (DMSO 1%). After two weeks, the mice were sacrificed, their skeletal muscle harvested and processed for Q-PCR analyses. Data are expressed in arbitrary unit of expression (A.U). Data are shown as mean ± s.d

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(n=12 in each group). *: p < 0.05 vs vehicle.

Figure 2. Expression and transcriptional activity of PPARβ vary during the regeneration period. Mice

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were fed with a standard chow supplemented with GW0742 (3mg/kg/day) or vehicle-supplemented food (DMSO 1%). After two weeks, the left TLA was injured with CTX, while the right TLA received the same

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volume of saline (control leg). Mice were sacrificed at day 4, 7 and 14 post-injury. TLA were harvested, and processed for Q-PCR analyses. Effect of GW0742 on PPARβ/δ (A) and on PDK4 (B) mRNA levels. Data are shown as mean ± s.d (n=4 in each group). *: p < 0.05 vs DMSO; #: p < 0.05 vs non-injured TLA; §: p < 0.05 vs all other times. Gray bars: non-injured TLA; black bars: injured TLA. The baseline indicates the

DMSO.

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basal expression level of PPARβ/δ (A) or PDK4 (B) mRNA in the TLA of non-injured mice treated with

Figure 3. Macrophage infiltration and IL-10/TGFβ mRNA levels are increased in injured TLA from

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GW0742-treated mice at 4 days post-injury. Mice received oral supplementation and were injured as described in figure 2. The number of M1 and M2 macrophages was analyzed by flow cytometry at day 4, 7

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and 14 post-injury. (A) Histogram representing the number of M1 and M2 macrophages par 10 mg of TLA. (B) IL-10 and TGFβ mRNA levels. Data are expressed in arbitrary unit of expression (A.U). Data are shown as mean ± s.d (n=4 in each group). *: p < 0.05 vs DMSO; #: p < 0.05 vs non-injured TLA; §: p < 0.05 vs all other times. Figure 4. GW0742 treatment improves the potential of satellite cell activation, proliferation and differentiation at day 4 and shortens the early phase of skeletal muscle regeneration. Mice received oral supplementation and were injured as described in figure 2. TLA were harvested, and processed for 21

Q-PCR analyses. Effect of GW0742 treatment on Pax7 (A) Myf5 (B), MyoD (C), Myogenin (D) MRF4 (E)

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and Myostatin (F) mRNA levels. Data are shown as mean ± s.d (n=4 in each group) and are expressed in arbitrary unit of expression (A.U). *: p < 0.05 vs DMSO; #: p < 0.05 vs non-injured TLA; §: p < 0.05 vs all other times. Gray bars: non-injured TLA; black bars: injured TLA. The baseline indicates the basal expression level of Pax7 (A), Myf5 (B), MyoD (C) Myogenin (D) MRF4 (E) or Myostatin (F) mRNA in the

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TLA of non-injured mice treated with DMSO.

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Tables

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TABLE 1: Primer sequences used for RT-QPCR Genes

primers (sense/antisense) F-AGATGGTGGCAGAGCTATGACC/R-TCCTCCTGTGGCTGTTCC

PDK4

F-GGGTCTCAATAGTGTCACC/R-GTGGGCCTGGGCATTTAGCA

IL-10

F-GGTTGCCAAGCCTTATCGGA/R-ACCTGCTCCACTGCCTTGCT

TGFβ

F-CAGTGGCTGAACCAAGGAGACGG/R-CATGGATGGTGCCCAGGTCGC

IL-6

F-TCCAGTTGCCTTCTTGGGAC/R-TGTAATTAAGCCTCCGACTTG

TNFα

F-GGCAGGTCTACTTTGGAGTCATTGC/R-ACATTCGAGGCTCCAGTGAATTCGG

MCP-1

F-AGCACCAGCCAACTCTCAC/R-TCTGGACCCATTCCTTCTTG

CD68

F-AGCTTCTGCTGTGGAAATGC/R-AAGAGGGACTGGTCACGGT

Pax7

F-CGATTAGCCGAGTGCTCAGA/R-GGAGGTCGGGTTCTGATTCC

MyoD

F-AGCACRACAGTGGCGACTCA/R-GCTCCACTATGCTGGACAGG

Myf5

F-TGAGGGAACAGGTGGAGAAC/R-AGCTGGACACGGAGCTTTT

MRF4

F-AGAGGGCTCTCCTTTGTATCC/R-CTGCTTTCCGACGATCTGTGG

Myogenin

F-AACTACCTTCCTGTCCACCTTCA/R-GTCCCCAGTCCCTTTTCTTCCA

Myostatin

F-CAGCCATGGTAGTAGACCGC/R-TACAGCCTGTGGTGCTTGAA

36B4

F-TCCAGGCTTTGGGCATCA/R-CTTTATCAGCTGCACATCACTCAGA

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PPARβ

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TABLE 2: Effects of 2-week GW0742 treatment on mRNA levels of genes involved in skeletal muscle regeneration

[mRNA] Pax7 Myf5 MyoD Myogenin MRF4 Myostatin

DMSO

GW0742

0.83 ± 0.08 1.00 ± 0.43 1.19 ± 0.42 1.07 ± 0.67 0.93 ± 0.11 0.791 ± 0.44

0.47 ± 0.23 * 0.54 ± 0.32 0.77 ± 0.56 0.91 ± 0.77 0.92 ± 0.13 0.788 ± 0.68

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in TLA of mice.

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Mice treated with GW0742 (n=12) or vehicle DMSO (n=12). Values are presented as means +/- SD. * p < 0.05 is significant in comparison to control DMSO value

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TABLE 3: Number of CD3+, CD4+ and CD8+ T cells for 10mg of TLA

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DMSO

4d GW

DMSO

7d GW

DMSO

14d GW

CD3+ number/10mg TLA Uninjured Injured 6.7 ± 1.1 403 ± 365# 80 ± 127 507 ± 365§# 4.3 ± 0.9 61.8 ± 25.2# 28.6 ± 38.5 73.4 ± 17.3 2.2 ± 1.3 27.8 ± 28.0# 2.7 ± 3.1 8.8 ± 7.0

CD4+ number/10mg TLA Uninjured Injured 2.9 ± 1.8§ 147 ± 136§# 25 ± 42§ 122 ± 114§# 2.25 ± 0.80 26.8 ± 17.6# 6.12 ± 6.42 36.5 ± 8.3# 0.46 ± 0.40 3.4 ± 2.0 0.55 ± 0.62 0.89 ± 0.34

CD8+ number/10mg TLA Uninjured Injured 0.98 ± 0.63 82.0 ± 72.5# 22 ± 40 102.4 ± 73.7# 0.86 ± 0.65 6.5 ± 4 1.13 ± 1.32 10.6 ± 5.5 0.98 ± 0.87 14.9 ± 19.7 1.23 ± 1.56 4.3 ± 4.5

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Values are presented as means ± SD. #p < 0.05 is significant in comparison to uninjured TLA; § p < 0.05 in comparison to 7d and 14d post-injury. N=4 per time and group.

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TABLE 4: Effect of GW0742 treatment on mRNA levels of genes involved in inflammation in TLA of mice at day 4,

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relative to uninjured TLA of DMSO mice.

DMSO CD68 MCP-1 TNF-α IL-6

1 ± 0.90 1 ± 0.69 1 ± 0.40 1 ± 0.60

Injured #

34.6 ± 29.5 13.9 ± 11.0# 2.37 ± 2.01# 2.37 ± 1.59#

Uninjured

Injured

3.05 ± 2.05 0.95 ± 0.59 2.34 ± 2.34 0.51 ± 0.38

44.2 ± 16.9# 21.4 ± 11.6# 4.14 ± 1.39 # 2.39 ± 1.41#

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GW0742

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TABLE 5: Effects of GW0742 treatment on body and TLA weight loss after 4, 7 and 14 day post- injury. Values

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represent the difference between injured and uninjured TLA weights.

0d to 4d post-injury 0d to 7d post-injury 0d to 14d post-injury

∆ weight (g) DMSO GW0742 0.05 ± 0.30 -1.08 ± 2.05 0.06 ± 2.43 -0.62 ± 2.36 1.36 ± 0.68 -0.28 ± 1.65

∆ TLA weight (vs control leg) (mg) DMSO GW0742 -8.61 ± 1.38 * -13.2 ± 2.01 * -6.9 ± -2.59 * -6.36 ± 3.58 * 7.14 ± 2.63 1.31 ± 5.27

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* p < 0.0005 vs 0 (non parametric univariate test).

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TABLE 6: Effects of GW0742 treatment on endurance and strength performance variations after TLA injury. Values

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represent the difference between performance measured after (at day 7 and 14) and before injury.

7d 14d

∆ Running time (min) DMSO GW0742 -13.94 ± 14.69 -7.5 ± 8.54 -18.04 ± 22.15 -20.35 ± 13.92

∆ Grip strength (N/g) DMSO GW0742 -0.005 ± 0.007 0.002 ± 0.006 -0.004 ± 0.003 -0.003 ± 0.006

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