- Email: [email protected]
Beneficial role of simvastatin in experimental autoimmune myositis
International Immunopharmacology 79 (2020) 106051
Contents lists available at ScienceDirect
International Immunopharmacology journal homepage: www.elsevier.com/locate/intimp
Beneficial role of simvastatin in experimental autoimmune myositis a
G. Maalouly , J. Hajal ⁎ C. Irania, N. Faresb, a b
b,1
, Y. Saliba
b,1
, G. Rached
b,1
, H. Layoun
b,1
a
T a
, V. Smayra , G. Sleilaty ,
Faculty of Medicine, CHU Hotel Dieu de France Hospital, Saint Joseph University, Beirut, Lebanon Physiology and Pathophysiology Research Laboratory, Pole of Technology and Health, Faculty of Medicine, Saint Joseph University, Beirut, Lebanon
A R T I C LE I N FO
A B S T R A C T
Keywords: Myositis Statin Simvastatin Auto-immunity
Objective: Statins have immunomodulatory potential in autoimmune diseases but had not been studied as a disease-modifying agent in inflammatory myopathies. The objective of this study is to assess the effect of simvastatin in an experimental model of autoimmune myositis in mice on muscle strength and histopathology. Methods: Four groups of mice (n = 5 per group) were selected for experimentally induced myositis. Mice were immunized with 1.5 mg myosin in complete Freund’s adjuvant weekly for two times and injected with 500 ng pertussis toxin twice immediately after each immunization. From day 1 before immunization to 10 days after the last immunization, mice were treated with oral simvastatin (10 or 20 or 40 mg/kg) diluted in DMSO. The control group mice were injected with complete Freund’s adjuvant weekly for two times and did not receive treatment. Non-immunized mice (n = 5 per group) were treated either with simvastatin (5 mg/kg or 20 mg/kg or 40 mg/kg of simvastatin diluted in DMSO) or with DMSO. Results: Inflammation was observed in myositis groups with positive myositis-specific antibodies. Muscle strength dropped significantly after immunization. Immunized simvastatin 20 mg/kg treated group had significantly higher muscle strength versus non-treated myositis mice and versus other simvastatin doses. Besides, a trend toward higher serum Th17 percentage population was found in immunized non-treated mice, versus immunized simvastatin- treated mice, without significant difference. Conclusion: Simvastatin at 20 mg/kg decreases the severity of myositis in experimental autoimmune myositis and is a candidate of being a disease-modifying agent in inflammatory myopathies.
1. Introduction Statins have been proposed as immunomodulatory agents due to their potent effects both on T lymphocytes and on antigen presenting cells. Accumulating evidence has highlighted two CD4(+) T cell subsets, the Th17 and Treg cells, as important disease-related targets of statins [1]. Data from multiple sclerosis and rheumatoid arthritis studies suggest that statin might selectively restore the homeostasis of Th17 and Treg populations [2,3]. The evidence that statins inhibit Th17 differentiation and IL-17 secretion has prompted the assessment of the therapeutic potential of statins in animal models of Th17-mediated autoimmune diseases. The idiopathic inflammatory myopathies are a heterogeneous group of autoimmune muscle disorders with distinct features [4]. The presence of IL-17A producing cells in the inflamed muscle tissue of myositis patients and the results of in vitro studies suggest that the Th17 pathway may also have a key role in these diseases [5]. In addition,
experimental myositis is more severe in mice that were depleted of Tregs [6]. While statin could alter muscle metabolism, statin myopathy is dose-related. An increase in statin dose, and statin potency magnifies the risk of muscle toxicity. [7]. To the best of our knowledge, statins had not been studied as disease modifying agent in inflammatory myopathies. The objective of this study is to assess the effect of different doses of simvastatin in an experimental model of autoimmune myositis in mice on muscle strength and histopathology with concomitant measures of serum lymphocytes population and myositis related auto-antibodies to evaluate the diseasemodifying potential of statins in inflammatory myopathies.
⁎
Corresponding author. E-mail address: [email protected] (N. Fares). 1 Contributed equally to this work. https://doi.org/10.1016/j.intimp.2019.106051 Received 21 August 2019; Received in revised form 8 November 2019; Accepted 11 November 2019 1567-5769/ © 2019 Elsevier B.V. All rights reserved.
International Immunopharmacology 79 (2020) 106051
G. Maalouly, et al.
2. Materials and methods
2.4. Histological grading of inflammation
2.1. Animals and experimental protocol
The muscle tissue sections (10 µm) from each block were randomly selected and stained with hematoxylin and eosin. The degrees of inflammatory infiltrates were qualitatively assessed, graded into four categories and expressed as a mean score for each group: grade 1: < 5 muscle fibers involved; grade 2: a lesion involving 5–30 muscle fibers; grade 3: a lesion involving a muscle fasciculus; grade 4: diffuse extensive lesions. When multiple lesions were found in one section of muscle, 0.5 was added to the score.
The present study was approved by the Ethical Committee of the Saint Joseph University of Beirut. The protocols were designed according to the Guiding Principles in the Care and Use of Animals approved by the Council of the American Physiological Society and were in adherence to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication no. 85-23, revised 1996). The animals were kept at a stable temperature (22 ± 3 °C) and humidity (50 ± 5%) and were exposed to 12:12 h light-dark cycle. They were fed ordinary rodent chow and were acclimatized at least one week under these conditions before the start of the study. Forty-five adult female BALB/c mice weighing 24 ± 2 g were used in this study. The mice were divided into nine groups: Freund Adjuvant group (SF), DMSO group (SD), Sham simvastatin treated groups (SS10, 20 and 40) and immunized treated and non-treated groups (MS10, 20, 40 and MYO respectively). Mice in the myositis group were immunized with 1.5 mg myosin in CFA (100 µl of 50% complete Freund’s adjuvant) weekly for two times and injected with 500 ng PT (pertussis toxin 500 ng in 200 µl saline) twice immediately after each immunization. Skeletal muscle myosin was partially purified, as described previously [8]. The control group mice were injected with CFA weekly for two times (Fig. 1). Four groups of mice (n = 5 per group) were selected for experimentally induced myositis. The first group (Myo) did not receive any treatment. From day 1 before immunization to 10 days after the last immunization, 3 groups of mice (MS10, MS20 and MS40) were treated with oral simvastatin (Simvastatin® Normon) (10 mg/kg or 20 mg/kg or 40 mg/kg) diluted in DMSO, respectively. Control group mice (SF) were injected with CFA without any treatment. Finally, non-immunized mice (SS10, SS20, SS40 and SD) were treated respectively either with simvastatin (10 mg/kg or 20 mg/ kg or 40 mg/kg of simvastatin diluted in DMSO) or with DMSO (Fig. 1).
2.5. Flow cytometric analysis Peripheral blood mononuclear cells (PBMCs) were prepared within 30 min from blood samples collected in EDTA tubes. Lymphocyte Th17 and Treg subsets determination in erythrocyte-lysed blood samples was performed using mouseTh17/Treg Phenotyping Kit (BD Pharmingen™, San Jose, USA) according to the manufacturer’s protocol. The antibodies cocktail contained: Mouse CD4 PerCP-Cy5.5, Mouse IL-17A PE and Mouse Foxp3 Alexa Fluor® 647. IL-17A (for Th17) and Foxp3 (for Treg) cells were expressed as percentages. 3. Statistical analysis Results are presented as the mean ± SD for the number of samples indicated in the figure legends. The data were analyzed using a repeated measures (Baseline, Week 2, Week 10) 2-way ANOVA model with Myositis (Yes, No) and statin dose (0, 10, 20, 40) as grouping factors, including interaction terms. The statistical analysis pre-specified a linear contrast for statin dose. All values with p < 0.05 are considered significant. 4. Results 4.1. Muscle strength
2.2. Evaluation of muscle strength in mice
The 2-way repeated measures ANOVA revealed a significant time effect (p < 0.001), time by myositis effect (p = 0.001), time by statin dose effect (p = 0.019) and a time by myositis by statin dose effect (p = 0.006). There was no linear effect for statin dose (p for prespecified linear contrast = 0.989). However, post hoc analysis showed a significant effect for statin dose 20 for animals (p for time by myositis by statin dose interaction = 0.008). That is, grip test for animal having received statin dose 20 showed a reversal of the week 2 effect upon week 10 assessment (Fig. 2).
Muscle strength was evaluated one day before immunization, seven days after the first immunization, and ten days after the last immunization. A dynamometer was used for measuring mouse forelimb muscle strength, as previously described [9].
2.3. Antibodies detection assay A qualitative in vitro assay was conducted to detect the different antibodies using the EUROLINE Test Kit (EUROIMMUN, Medizinische Labordiagnostika AG, Germany). This kit was used to detect different antibodies of the IgG class using 11 different antigens: Mi2ß, Ku, PMScl100, PM-Scl75, Jo-1, SRP, PL-7, PL-12, EJ, OJ and Ro-52, according to the manufacturer’s protocol.
4.2. Histopathology Obvious inflammatory infiltrates in the muscle tissues were observed in myositis groups (treated and non-treated immunized mice, Fig. 3A, B) with necrotic fibers in 2 mice in immunized non-treated group (Myo group, data not shown). Inflammation score was significantly decreased in MS20 group. No inflammation was found in
Fig. 1. Time line illustration of the experimental protocol. FA: Freund Adjuvant; PT: Pertussis Toxin. 2
International Immunopharmacology 79 (2020) 106051
G. Maalouly, et al.
Fig. 2. Muscle strength (as mean grip strength) (A) at baseline, 2 weeks and 10 weeks in all groups. (B) Percentage of Skeletal Muscle strength variation in all groups. The values are expressed as mean ± SD for each group of mice (n = 5/group). All values with p < 0.05 are considered significant. *p < 0.05 vs SF; #p < 0.05 vs Myo; +p < 0.05 vs baseline. SF: Sham Freund’s adjuvant; SD: sham DMSO; SS 10, 20 and 40: Sham Simvastatin with 10, 20, and 40 mg/kg respectively; Myo: immunized non-treated group; MS 10, 20 and 40: immunized treated groups with 10, 20, and 40 mg/kg of simvastatin respectively.
found in Myo group (Fig. 5). Simvastatin at 10 and 20 mg/kg seems to dampen this increase without reaching statistical significance. The FOXP3/Th17 ratio was enhanced in MS20 group without statistical signification.
control group. Average diameter of muscle fibers was significantly decreased in immunized non-treated group (38.48 μm ± 2.2. p < 0.05). Interestingly, simvastatin at 10 and 20 mg/kg was able to reverse muscle fiber atrophy (45 μm ± 2.7; 63.65 μm ± 6.8, respectively. p < 0.05) with greater recovery in MS20 group. This positive impact was lost in 40 mg/kg immunized treated groups (38.55 μm ± 2.3).
5. Discussion To the best of our knowledge, it is the first study to evaluate statins as disease-modifying agents in inflammatory myopathies. Herein, we demonstrate that simvastatin, could modulate clinical, histological and biological features in experimental autoimmune myositis. Statins demonstrated their potential of reducing inflammation, oxidative stress and fibrosis in many studies. However, because of the rare but widely publicized skeletal muscle related symptoms of these drugs, they were not sufficiently studied in muscle inflammatory diseases. One study demonstrated a therapeutic effect of simvastatin (5–10 mg/kg per day) in Mdx dystrophic mice, ameliorating diaphragmatic strength and hindlimb force and reduced muscle inflammation and fibrosis, with reduced NADPH Oxidase 2 protein expression and autophagy activation [10]. In a pilot trial of simvastatin in
4.3. Detection of serum autoantibodies Ro-52, Jo-1, and PM-Scl100 antibodies were not detected in myositis group nor in statin treated myositis groups. OJ, EJ, PL-12, PL-7, SRP, PM-Scl75, Ku, and Mi-2β antibodies were found moderately to strongly positive in the myositis group and in statin treated myositis groups. OJ and Ku antibodies were significantly decreased in MS40 group (Fig. 4). 4.4. Lymphocyte population in serum A trend toward increased serum Th17 lymphocyte population was
Fig. 3. Histological analysis of skeletal muscle tissue sections stained with hematoxylin and eosin showing inflammatory infiltrates in (A) immunized non-treated and treated with 10, 20 and 40 mg/kg of simvastatin groups (magnification× 200). (B) Histogram showing semi-quantitative score of infiltrating cells per field (a.u.) in Myo, MS 10, MS 20 and MS 40 groups. The values are expressed as mean ± SD for each group of mice (n = 5/group). All values with p < 0.05 are considered significant. *p < 0.05 vs Myo. Myo: myositis; MS: myositis simvastatin; a.u.: arbitrary unit. 3
International Immunopharmacology 79 (2020) 106051
G. Maalouly, et al.
Our study follows the protocol of the improved animal model of experimental autoimmune myositis [8]. 20 mg/kg of simvastatin reversed myositis-related drop in muscle strength with a statistically significant improvement in grip test. This improvement is lost with higher dose (40 mg/kg) and may be due to dose-related toxicity. Besides, this strength muscle improvement was associated with increasing of muscle fiber diameter suggesting significant recovery from myositisrelated atrophy. Muscle specific antibodies found in myositis patients were found previously only in T-reg depleted mice in a study using the traditional protocol of immunization [6]. In our study, immunized tested mice, were moderately to strongly positive for OJ, EJ, PL-12, PL-7, SRP, PMScl75, Ku, and Mi-2β antibodies. Human studies in inflammatory myositis had noted an expansion of peripheral proinflammatory T cells, Th17, as well as a deficiency of suppressor populations of Tregs [12]. Besides recent data show that high ROCK activity exhibited by Th17 cells in lupus patients is inhibited by simvastatin [13]. The statin effect on Treg kinetics seems to be complex: statins can directly promote the migration of Treg to inflamed tissues [14]; nevertheless, a negative dose-related effect on circulatory Treg was suspected in one systematic review and meta-analysis of statins effect in acute coronary syndrome patients [15]. In our study, simvastatin treatment at 20 mg/kg was associated with a trend toward an increasing in FOXP3/Th17 ratio expressed by decreasing of circulating Th17 in immunized mice. However, as we did not studied recruitment of Th17 and Treg in inflamed muscles, solid conclusion cannot be drawn.
Fig. 4. Semi-qualitative score of serum autoantibodies in tested mice of immunized non-treated and treated with 10, 20 or 40 mg/kg of simvastatin groups. Evaluation of serum autoantibodies were graded on a subjective scale of 0–3, corresponding to negative, week, moderate or strong color saturation, respectively. The values are expressed as mean ± SD for each group of mice (n = 5/group). All values with p < 0.05 are considered significant. *p < 0.05 vs Myo. Myo: myositis; MS: myositis simvastatin; a.u.: arbitrary unit.
sporadic inclusion-body myositis, simvastatin appeared safe and well tolerated with stability of clinical condition in most treated patients, in contrast with the trend of clinical worsening observed in non-treated patients [11].
Fig. 5. (A) Representative flow cytometry dot plots of serum Th17 lymphocyte population determined by IL17 and CD4+ staining in immunized non-treated and treated with 10, 20 or 40 mg/kg of simvastatin groups. (B) Histogram of IL17 + CD4+ population in SS 10, 20 and 40, Myo, MS 10, 20, and 40 groups. (C) Histogram of FOXP3/IL 17 ratio in SS 10, 20 and 40, Myo, MS 10, 20, and 40 groups. (D) Histogram of FOXP3 + CD4+ population in SS 10, 20 and 40, Myo, MS 10, 20, and 40 groups. The values are expressed as mean ± SD for each group of mice (n = 5/group). All values with p < 0.05 are considered significant. SS 10, 20 and 40: Sham Simvastatin with 10, 20, and 40 mg/kg respectively; Myo: immunized non-treated group; MS 10, 20 and 40: immunized treated groups with 10, 20, and 40 mg/kg of simvastatin respectively. a.u.: arbitrary unit. 4
International Immunopharmacology 79 (2020) 106051
G. Maalouly, et al.
Taken together, these data suggest that simvastatin at a dose of 20 mg/kg seems to have a promising therapeutic effect in experimental autoimmune myositis with reversal of myositis-related drop in muscle strength. Reduced inflammation and decreased Th17 population could be one possible mechanism of this protective effect. Further studies are warranted to investigate the difficult equilibrium between immunomodulation and toxicity of simvastatin in autoimmune myositis.
[5]
[6]
[7]
Acknowledgments
[8]
This work was supported by the Research Council of Saint Joseph University of Beirut, Project FM306.
[9]
Appendix A. Supplementary material
[10]
Supplementary data to this article can be found online at https:// doi.org/10.1016/j.intimp.2019.106051.
[11]
[12]
References [1] C. Ulivieri, C.T. Baldari, Statins: from cholesterol-lowering drugs to novel immunomodulators for the treatment of Th17-mediated autoimmune diseases, Pharmacol. Res. 88 (2014) 41–52. [2] X. Zhang, Y. Tao, J. Wang, R. Garcia-Mata, S. Markovic-Plese, Simvastatin inhibits secretion of Th17-polarizing cytokines and antigen presentation by DCs in patients with relapsing remitting multiple sclerosis, Eur. J. Immunol. 43 (1) (2013 Jan) 281–289. [3] T.T. Tang, Y. Song, Y.J. Ding, Y.H. Liao, X. Yu, R. Du, et al., Atorvastatin upregulates regulatory T cells and reduces clinical disease activity in patients with rheumatoid arthritis, J. Lipid Res. 52 (2011) 1023–1032. [4] E.M. Moran, F.L. Mastaglia, The role of interleukin-17 in immune-mediated
[13]
[14] [15]
5
inflammatory myopathies and possible therapeutic implications, Neuromuscul. Disord. 24 (11) (2014 Nov) 943–952. A. Tournadre, M. Porcherot, P. Chérin, I. Marie, E. Hachulla, P. Miossec, Th1 and Th17 balance in inflammatory myopathies: interaction with dendritic cells and possible link with response to high-dose immunoglobulins, Cytokine 46 (3) (2009 Jun) 297–301. Y. Allenbach, S. Solly, S. Grégoire, O. Dubourg, B. Salomon, G. Butler-Browne, et al., Role of regulatory T cells in a new mouse model of experimental autoimmune myositis, Am. J. Pathol. 174 (3) (2009 Mar) 989–998. T.A. Jacobson, Toward “pain-free” statin prescribing: clinical algorithm for diagnosis and management of myalgia, Mayo Clin. Proc. 83 (2008) 687–700. J. Kang, H.Y. Zhang, G.D. Feng, D.Y. Feng, H.G. Jia, Development of an improved animal model of experimental autoimmune myositis, Int. J. Clin. Exp. Pathol. 8 (11) (2015) 14457–14464. J.P. Smith, P.S. Hicks, L.R. Ortiz, M.J. Martinez, R.N. Mandler, Quantitative measurement of muscle strength in the mouse, J. Neurosci. Methods 62 (1–2) (1995 Nov) 15–19. N.P. Whitehead, M.J. Kim, K.L. Bible, M.E. Adams, S.C. Froehner, A new therapeutic effect of simvastatin revealed by functional improvement in muscular dystrophy, Proc. Natl. Acad. Sci. USA 112 (41) (2015 Oct 13) 12864–12869. C. Sancricca, M. Mora, E. Ricci, P.A. Tonali, R. Mantegazza, M. Mirabella, Pilot trial of simvastatin in the treatment of sporadic inclusion-body myositis, Neurol. Sci. 32 (5) (2011) 841–847. F. Espinosa-Ortega, D. Gómez-Martin, K. Santana-De Anda, J. Romo-Tena, P. Villaseñor-Ovies, J. Alcocer-Varela, Quantitative T cell subsets profile in peripheral blood from patients with idiopathic inflammatory myopathies: tilting the balance towards proinflammatory and pro-apoptotic subsets, Clin. Exp. Immunol. 179 (3) (2015) 520–528. C. Rozo, Y. Chinenov, R.K. Maharaj, S. Gupta, L. Leuenberger, K.A. Kirou, et al., Targeting the RhoA-ROCK pathway to reverse T-cell dysfunction in SLE, Ann. Rheum. Dis. 76 (4) (2017) 740–747. D.A. Forero-Peña, F.R. Gutierrez, Statins as modulators of regulatory T-cell biology, Mediators Inflamm. 2013 (2013) 167086. N. Sorathia, H. Al-Rubaye, B. Zal, The effect of statins on the functionality of CD4+CD25+FOXP3+ regulatory T-cells in acute coronary syndrome: a systematic review and meta-analysis of randomised controlled trials in asian populations, Eur. Cardiol. 14 (2) (2019 Jul 11) 123–129.
Contents lists available at ScienceDirect
International Immunopharmacology journal homepage: www.elsevier.com/locate/intimp
Beneficial role of simvastatin in experimental autoimmune myositis a
G. Maalouly , J. Hajal ⁎ C. Irania, N. Faresb, a b
b,1
, Y. Saliba
b,1
, G. Rached
b,1
, H. Layoun
b,1
a
T a
, V. Smayra , G. Sleilaty ,
Faculty of Medicine, CHU Hotel Dieu de France Hospital, Saint Joseph University, Beirut, Lebanon Physiology and Pathophysiology Research Laboratory, Pole of Technology and Health, Faculty of Medicine, Saint Joseph University, Beirut, Lebanon
A R T I C LE I N FO
A B S T R A C T
Keywords: Myositis Statin Simvastatin Auto-immunity
Objective: Statins have immunomodulatory potential in autoimmune diseases but had not been studied as a disease-modifying agent in inflammatory myopathies. The objective of this study is to assess the effect of simvastatin in an experimental model of autoimmune myositis in mice on muscle strength and histopathology. Methods: Four groups of mice (n = 5 per group) were selected for experimentally induced myositis. Mice were immunized with 1.5 mg myosin in complete Freund’s adjuvant weekly for two times and injected with 500 ng pertussis toxin twice immediately after each immunization. From day 1 before immunization to 10 days after the last immunization, mice were treated with oral simvastatin (10 or 20 or 40 mg/kg) diluted in DMSO. The control group mice were injected with complete Freund’s adjuvant weekly for two times and did not receive treatment. Non-immunized mice (n = 5 per group) were treated either with simvastatin (5 mg/kg or 20 mg/kg or 40 mg/kg of simvastatin diluted in DMSO) or with DMSO. Results: Inflammation was observed in myositis groups with positive myositis-specific antibodies. Muscle strength dropped significantly after immunization. Immunized simvastatin 20 mg/kg treated group had significantly higher muscle strength versus non-treated myositis mice and versus other simvastatin doses. Besides, a trend toward higher serum Th17 percentage population was found in immunized non-treated mice, versus immunized simvastatin- treated mice, without significant difference. Conclusion: Simvastatin at 20 mg/kg decreases the severity of myositis in experimental autoimmune myositis and is a candidate of being a disease-modifying agent in inflammatory myopathies.
1. Introduction Statins have been proposed as immunomodulatory agents due to their potent effects both on T lymphocytes and on antigen presenting cells. Accumulating evidence has highlighted two CD4(+) T cell subsets, the Th17 and Treg cells, as important disease-related targets of statins [1]. Data from multiple sclerosis and rheumatoid arthritis studies suggest that statin might selectively restore the homeostasis of Th17 and Treg populations [2,3]. The evidence that statins inhibit Th17 differentiation and IL-17 secretion has prompted the assessment of the therapeutic potential of statins in animal models of Th17-mediated autoimmune diseases. The idiopathic inflammatory myopathies are a heterogeneous group of autoimmune muscle disorders with distinct features [4]. The presence of IL-17A producing cells in the inflamed muscle tissue of myositis patients and the results of in vitro studies suggest that the Th17 pathway may also have a key role in these diseases [5]. In addition,
experimental myositis is more severe in mice that were depleted of Tregs [6]. While statin could alter muscle metabolism, statin myopathy is dose-related. An increase in statin dose, and statin potency magnifies the risk of muscle toxicity. [7]. To the best of our knowledge, statins had not been studied as disease modifying agent in inflammatory myopathies. The objective of this study is to assess the effect of different doses of simvastatin in an experimental model of autoimmune myositis in mice on muscle strength and histopathology with concomitant measures of serum lymphocytes population and myositis related auto-antibodies to evaluate the diseasemodifying potential of statins in inflammatory myopathies.
⁎
Corresponding author. E-mail address: [email protected] (N. Fares). 1 Contributed equally to this work. https://doi.org/10.1016/j.intimp.2019.106051 Received 21 August 2019; Received in revised form 8 November 2019; Accepted 11 November 2019 1567-5769/ © 2019 Elsevier B.V. All rights reserved.
International Immunopharmacology 79 (2020) 106051
G. Maalouly, et al.
2. Materials and methods
2.4. Histological grading of inflammation
2.1. Animals and experimental protocol
The muscle tissue sections (10 µm) from each block were randomly selected and stained with hematoxylin and eosin. The degrees of inflammatory infiltrates were qualitatively assessed, graded into four categories and expressed as a mean score for each group: grade 1: < 5 muscle fibers involved; grade 2: a lesion involving 5–30 muscle fibers; grade 3: a lesion involving a muscle fasciculus; grade 4: diffuse extensive lesions. When multiple lesions were found in one section of muscle, 0.5 was added to the score.
The present study was approved by the Ethical Committee of the Saint Joseph University of Beirut. The protocols were designed according to the Guiding Principles in the Care and Use of Animals approved by the Council of the American Physiological Society and were in adherence to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication no. 85-23, revised 1996). The animals were kept at a stable temperature (22 ± 3 °C) and humidity (50 ± 5%) and were exposed to 12:12 h light-dark cycle. They were fed ordinary rodent chow and were acclimatized at least one week under these conditions before the start of the study. Forty-five adult female BALB/c mice weighing 24 ± 2 g were used in this study. The mice were divided into nine groups: Freund Adjuvant group (SF), DMSO group (SD), Sham simvastatin treated groups (SS10, 20 and 40) and immunized treated and non-treated groups (MS10, 20, 40 and MYO respectively). Mice in the myositis group were immunized with 1.5 mg myosin in CFA (100 µl of 50% complete Freund’s adjuvant) weekly for two times and injected with 500 ng PT (pertussis toxin 500 ng in 200 µl saline) twice immediately after each immunization. Skeletal muscle myosin was partially purified, as described previously [8]. The control group mice were injected with CFA weekly for two times (Fig. 1). Four groups of mice (n = 5 per group) were selected for experimentally induced myositis. The first group (Myo) did not receive any treatment. From day 1 before immunization to 10 days after the last immunization, 3 groups of mice (MS10, MS20 and MS40) were treated with oral simvastatin (Simvastatin® Normon) (10 mg/kg or 20 mg/kg or 40 mg/kg) diluted in DMSO, respectively. Control group mice (SF) were injected with CFA without any treatment. Finally, non-immunized mice (SS10, SS20, SS40 and SD) were treated respectively either with simvastatin (10 mg/kg or 20 mg/ kg or 40 mg/kg of simvastatin diluted in DMSO) or with DMSO (Fig. 1).
2.5. Flow cytometric analysis Peripheral blood mononuclear cells (PBMCs) were prepared within 30 min from blood samples collected in EDTA tubes. Lymphocyte Th17 and Treg subsets determination in erythrocyte-lysed blood samples was performed using mouseTh17/Treg Phenotyping Kit (BD Pharmingen™, San Jose, USA) according to the manufacturer’s protocol. The antibodies cocktail contained: Mouse CD4 PerCP-Cy5.5, Mouse IL-17A PE and Mouse Foxp3 Alexa Fluor® 647. IL-17A (for Th17) and Foxp3 (for Treg) cells were expressed as percentages. 3. Statistical analysis Results are presented as the mean ± SD for the number of samples indicated in the figure legends. The data were analyzed using a repeated measures (Baseline, Week 2, Week 10) 2-way ANOVA model with Myositis (Yes, No) and statin dose (0, 10, 20, 40) as grouping factors, including interaction terms. The statistical analysis pre-specified a linear contrast for statin dose. All values with p < 0.05 are considered significant. 4. Results 4.1. Muscle strength
2.2. Evaluation of muscle strength in mice
The 2-way repeated measures ANOVA revealed a significant time effect (p < 0.001), time by myositis effect (p = 0.001), time by statin dose effect (p = 0.019) and a time by myositis by statin dose effect (p = 0.006). There was no linear effect for statin dose (p for prespecified linear contrast = 0.989). However, post hoc analysis showed a significant effect for statin dose 20 for animals (p for time by myositis by statin dose interaction = 0.008). That is, grip test for animal having received statin dose 20 showed a reversal of the week 2 effect upon week 10 assessment (Fig. 2).
Muscle strength was evaluated one day before immunization, seven days after the first immunization, and ten days after the last immunization. A dynamometer was used for measuring mouse forelimb muscle strength, as previously described [9].
2.3. Antibodies detection assay A qualitative in vitro assay was conducted to detect the different antibodies using the EUROLINE Test Kit (EUROIMMUN, Medizinische Labordiagnostika AG, Germany). This kit was used to detect different antibodies of the IgG class using 11 different antigens: Mi2ß, Ku, PMScl100, PM-Scl75, Jo-1, SRP, PL-7, PL-12, EJ, OJ and Ro-52, according to the manufacturer’s protocol.
4.2. Histopathology Obvious inflammatory infiltrates in the muscle tissues were observed in myositis groups (treated and non-treated immunized mice, Fig. 3A, B) with necrotic fibers in 2 mice in immunized non-treated group (Myo group, data not shown). Inflammation score was significantly decreased in MS20 group. No inflammation was found in
Fig. 1. Time line illustration of the experimental protocol. FA: Freund Adjuvant; PT: Pertussis Toxin. 2
International Immunopharmacology 79 (2020) 106051
G. Maalouly, et al.
Fig. 2. Muscle strength (as mean grip strength) (A) at baseline, 2 weeks and 10 weeks in all groups. (B) Percentage of Skeletal Muscle strength variation in all groups. The values are expressed as mean ± SD for each group of mice (n = 5/group). All values with p < 0.05 are considered significant. *p < 0.05 vs SF; #p < 0.05 vs Myo; +p < 0.05 vs baseline. SF: Sham Freund’s adjuvant; SD: sham DMSO; SS 10, 20 and 40: Sham Simvastatin with 10, 20, and 40 mg/kg respectively; Myo: immunized non-treated group; MS 10, 20 and 40: immunized treated groups with 10, 20, and 40 mg/kg of simvastatin respectively.
found in Myo group (Fig. 5). Simvastatin at 10 and 20 mg/kg seems to dampen this increase without reaching statistical significance. The FOXP3/Th17 ratio was enhanced in MS20 group without statistical signification.
control group. Average diameter of muscle fibers was significantly decreased in immunized non-treated group (38.48 μm ± 2.2. p < 0.05). Interestingly, simvastatin at 10 and 20 mg/kg was able to reverse muscle fiber atrophy (45 μm ± 2.7; 63.65 μm ± 6.8, respectively. p < 0.05) with greater recovery in MS20 group. This positive impact was lost in 40 mg/kg immunized treated groups (38.55 μm ± 2.3).
5. Discussion To the best of our knowledge, it is the first study to evaluate statins as disease-modifying agents in inflammatory myopathies. Herein, we demonstrate that simvastatin, could modulate clinical, histological and biological features in experimental autoimmune myositis. Statins demonstrated their potential of reducing inflammation, oxidative stress and fibrosis in many studies. However, because of the rare but widely publicized skeletal muscle related symptoms of these drugs, they were not sufficiently studied in muscle inflammatory diseases. One study demonstrated a therapeutic effect of simvastatin (5–10 mg/kg per day) in Mdx dystrophic mice, ameliorating diaphragmatic strength and hindlimb force and reduced muscle inflammation and fibrosis, with reduced NADPH Oxidase 2 protein expression and autophagy activation [10]. In a pilot trial of simvastatin in
4.3. Detection of serum autoantibodies Ro-52, Jo-1, and PM-Scl100 antibodies were not detected in myositis group nor in statin treated myositis groups. OJ, EJ, PL-12, PL-7, SRP, PM-Scl75, Ku, and Mi-2β antibodies were found moderately to strongly positive in the myositis group and in statin treated myositis groups. OJ and Ku antibodies were significantly decreased in MS40 group (Fig. 4). 4.4. Lymphocyte population in serum A trend toward increased serum Th17 lymphocyte population was
Fig. 3. Histological analysis of skeletal muscle tissue sections stained with hematoxylin and eosin showing inflammatory infiltrates in (A) immunized non-treated and treated with 10, 20 and 40 mg/kg of simvastatin groups (magnification× 200). (B) Histogram showing semi-quantitative score of infiltrating cells per field (a.u.) in Myo, MS 10, MS 20 and MS 40 groups. The values are expressed as mean ± SD for each group of mice (n = 5/group). All values with p < 0.05 are considered significant. *p < 0.05 vs Myo. Myo: myositis; MS: myositis simvastatin; a.u.: arbitrary unit. 3
International Immunopharmacology 79 (2020) 106051
G. Maalouly, et al.
Our study follows the protocol of the improved animal model of experimental autoimmune myositis [8]. 20 mg/kg of simvastatin reversed myositis-related drop in muscle strength with a statistically significant improvement in grip test. This improvement is lost with higher dose (40 mg/kg) and may be due to dose-related toxicity. Besides, this strength muscle improvement was associated with increasing of muscle fiber diameter suggesting significant recovery from myositisrelated atrophy. Muscle specific antibodies found in myositis patients were found previously only in T-reg depleted mice in a study using the traditional protocol of immunization [6]. In our study, immunized tested mice, were moderately to strongly positive for OJ, EJ, PL-12, PL-7, SRP, PMScl75, Ku, and Mi-2β antibodies. Human studies in inflammatory myositis had noted an expansion of peripheral proinflammatory T cells, Th17, as well as a deficiency of suppressor populations of Tregs [12]. Besides recent data show that high ROCK activity exhibited by Th17 cells in lupus patients is inhibited by simvastatin [13]. The statin effect on Treg kinetics seems to be complex: statins can directly promote the migration of Treg to inflamed tissues [14]; nevertheless, a negative dose-related effect on circulatory Treg was suspected in one systematic review and meta-analysis of statins effect in acute coronary syndrome patients [15]. In our study, simvastatin treatment at 20 mg/kg was associated with a trend toward an increasing in FOXP3/Th17 ratio expressed by decreasing of circulating Th17 in immunized mice. However, as we did not studied recruitment of Th17 and Treg in inflamed muscles, solid conclusion cannot be drawn.
Fig. 4. Semi-qualitative score of serum autoantibodies in tested mice of immunized non-treated and treated with 10, 20 or 40 mg/kg of simvastatin groups. Evaluation of serum autoantibodies were graded on a subjective scale of 0–3, corresponding to negative, week, moderate or strong color saturation, respectively. The values are expressed as mean ± SD for each group of mice (n = 5/group). All values with p < 0.05 are considered significant. *p < 0.05 vs Myo. Myo: myositis; MS: myositis simvastatin; a.u.: arbitrary unit.
sporadic inclusion-body myositis, simvastatin appeared safe and well tolerated with stability of clinical condition in most treated patients, in contrast with the trend of clinical worsening observed in non-treated patients [11].
Fig. 5. (A) Representative flow cytometry dot plots of serum Th17 lymphocyte population determined by IL17 and CD4+ staining in immunized non-treated and treated with 10, 20 or 40 mg/kg of simvastatin groups. (B) Histogram of IL17 + CD4+ population in SS 10, 20 and 40, Myo, MS 10, 20, and 40 groups. (C) Histogram of FOXP3/IL 17 ratio in SS 10, 20 and 40, Myo, MS 10, 20, and 40 groups. (D) Histogram of FOXP3 + CD4+ population in SS 10, 20 and 40, Myo, MS 10, 20, and 40 groups. The values are expressed as mean ± SD for each group of mice (n = 5/group). All values with p < 0.05 are considered significant. SS 10, 20 and 40: Sham Simvastatin with 10, 20, and 40 mg/kg respectively; Myo: immunized non-treated group; MS 10, 20 and 40: immunized treated groups with 10, 20, and 40 mg/kg of simvastatin respectively. a.u.: arbitrary unit. 4
International Immunopharmacology 79 (2020) 106051
G. Maalouly, et al.
Taken together, these data suggest that simvastatin at a dose of 20 mg/kg seems to have a promising therapeutic effect in experimental autoimmune myositis with reversal of myositis-related drop in muscle strength. Reduced inflammation and decreased Th17 population could be one possible mechanism of this protective effect. Further studies are warranted to investigate the difficult equilibrium between immunomodulation and toxicity of simvastatin in autoimmune myositis.
[5]
[6]
[7]
Acknowledgments
[8]
This work was supported by the Research Council of Saint Joseph University of Beirut, Project FM306.
[9]
Appendix A. Supplementary material
[10]
Supplementary data to this article can be found online at https:// doi.org/10.1016/j.intimp.2019.106051.
[11]
[12]
References [1] C. Ulivieri, C.T. Baldari, Statins: from cholesterol-lowering drugs to novel immunomodulators for the treatment of Th17-mediated autoimmune diseases, Pharmacol. Res. 88 (2014) 41–52. [2] X. Zhang, Y. Tao, J. Wang, R. Garcia-Mata, S. Markovic-Plese, Simvastatin inhibits secretion of Th17-polarizing cytokines and antigen presentation by DCs in patients with relapsing remitting multiple sclerosis, Eur. J. Immunol. 43 (1) (2013 Jan) 281–289. [3] T.T. Tang, Y. Song, Y.J. Ding, Y.H. Liao, X. Yu, R. Du, et al., Atorvastatin upregulates regulatory T cells and reduces clinical disease activity in patients with rheumatoid arthritis, J. Lipid Res. 52 (2011) 1023–1032. [4] E.M. Moran, F.L. Mastaglia, The role of interleukin-17 in immune-mediated
[13]
[14] [15]
5
inflammatory myopathies and possible therapeutic implications, Neuromuscul. Disord. 24 (11) (2014 Nov) 943–952. A. Tournadre, M. Porcherot, P. Chérin, I. Marie, E. Hachulla, P. Miossec, Th1 and Th17 balance in inflammatory myopathies: interaction with dendritic cells and possible link with response to high-dose immunoglobulins, Cytokine 46 (3) (2009 Jun) 297–301. Y. Allenbach, S. Solly, S. Grégoire, O. Dubourg, B. Salomon, G. Butler-Browne, et al., Role of regulatory T cells in a new mouse model of experimental autoimmune myositis, Am. J. Pathol. 174 (3) (2009 Mar) 989–998. T.A. Jacobson, Toward “pain-free” statin prescribing: clinical algorithm for diagnosis and management of myalgia, Mayo Clin. Proc. 83 (2008) 687–700. J. Kang, H.Y. Zhang, G.D. Feng, D.Y. Feng, H.G. Jia, Development of an improved animal model of experimental autoimmune myositis, Int. J. Clin. Exp. Pathol. 8 (11) (2015) 14457–14464. J.P. Smith, P.S. Hicks, L.R. Ortiz, M.J. Martinez, R.N. Mandler, Quantitative measurement of muscle strength in the mouse, J. Neurosci. Methods 62 (1–2) (1995 Nov) 15–19. N.P. Whitehead, M.J. Kim, K.L. Bible, M.E. Adams, S.C. Froehner, A new therapeutic effect of simvastatin revealed by functional improvement in muscular dystrophy, Proc. Natl. Acad. Sci. USA 112 (41) (2015 Oct 13) 12864–12869. C. Sancricca, M. Mora, E. Ricci, P.A. Tonali, R. Mantegazza, M. Mirabella, Pilot trial of simvastatin in the treatment of sporadic inclusion-body myositis, Neurol. Sci. 32 (5) (2011) 841–847. F. Espinosa-Ortega, D. Gómez-Martin, K. Santana-De Anda, J. Romo-Tena, P. Villaseñor-Ovies, J. Alcocer-Varela, Quantitative T cell subsets profile in peripheral blood from patients with idiopathic inflammatory myopathies: tilting the balance towards proinflammatory and pro-apoptotic subsets, Clin. Exp. Immunol. 179 (3) (2015) 520–528. C. Rozo, Y. Chinenov, R.K. Maharaj, S. Gupta, L. Leuenberger, K.A. Kirou, et al., Targeting the RhoA-ROCK pathway to reverse T-cell dysfunction in SLE, Ann. Rheum. Dis. 76 (4) (2017) 740–747. D.A. Forero-Peña, F.R. Gutierrez, Statins as modulators of regulatory T-cell biology, Mediators Inflamm. 2013 (2013) 167086. N. Sorathia, H. Al-Rubaye, B. Zal, The effect of statins on the functionality of CD4+CD25+FOXP3+ regulatory T-cells in acute coronary syndrome: a systematic review and meta-analysis of randomised controlled trials in asian populations, Eur. Cardiol. 14 (2) (2019 Jul 11) 123–129.