OP0247 IN MYOSITIS MUSCLE FIBRE PLAYS A DIRECT AND CRITICAL ROLE IN THERAPEUTIC RESPONSE TO GLUCOCORTICOIDS

Abstract

Background:Myositis are rare autoimmune diseases, affecting more women than men, characterized by chronic inflammation of skeletal muscle causing muscle weakness, decreased quality of life and increased mortality.Glucocorticoids (GC) are potent anti-inflammatory drugs, and are the first line treatment of myositis. They improve muscle strength of myositis patients (therapeutic effect), yet muscle recovery is generally only partial. Moreover, GC have an iatrogenic effect on skeletal muscle fibre leading to steroid myopathy. Thus myositis care has to be improved. Despite the autoimmune terrain of myositis, our team has recently shown that muscle fibres themselves develop immuno-metabolic modifications that participate to muscle weakness and perpetuation of the disease1. GC effects are mediated by the glucocorticoid receptor (GR), which is expressed in various cell types including immune cells and myofibres, but the cells mediating therapeutic responses remain to be determined.Objectives:Unravel the mechanisms underlying the therapeutic effect of GC in myositis, particularly elucidate the role of skeletal muscle fibres.Methods:Experimental myositis was induced in eight to ten week-old C57BL/6J female mice by a single intradermal injection of part of skeletal muscle fast-type C protein along with Freund’s adjuvant and an intraperitoneal (IP) injection of pertussis toxin, as previously described2. Prednisone (PDN) was administered 14 days (D) after the immunization at 1 mg/kg/day for 7 days by gavage. Mice were euthanized 21 days after myositis induction. Muscle strength was assessed by grip test at D 0, before the 1st PDN administration (D 14) and the day before sacrifice (D 20). To investigate whether the PDN effects are mediated by myofibre, we generated transgenic mice carrying two LoxP sites within the GR gene in muscle, expressing the tamoxifen-inducible Cre-ERT2 recombinase selectively in skeletal muscle fibre (HSA-CreERT2/GR L2/L2). Tamoxifen (1 mg/day for 5 days by IP injection) was administered 9 days after immunization to induce GR ablation selectively in skeletal muscle fibres (GR(i)skm-/- mice). Similar treatments were applied to GR L2/L2 that do not express Cre-ER(T2), and served as controls.We compared 4 groups of myositis mice, GR L2/L2 treated by PDN (n=9) or vehicle (n=9) and GR(i)skm-/- treated by PDN (n=10) or vehicle (n=10), by grip test and at the histological level (hematoxylin-eosin (HE) and Gomori trichrome (GT) staining). Moreover, LC3 expression was studied by RTqPCR and western blot.Results:Muscle strength was decreased in both GR L2/L2 and GR(i)skm-/- myositis mice from D 14 to D 20. GR L2/L2 myositis mice recovered muscle strength after PDN treatment; no significant difference compared to D 0 was detected. In contrast, PDN did not improve muscle strength in GR(i)skm-/- myositis mice (Figure 1).HE and GT staining did not reveal quantitative differences in inflammatory infiltrate. Necrotic and degenerative fibres were detected in the 4 groups. At RTqPCR, LC3, an autophagy marker, was upregulated in PDN-treated GR L2/L2 myositis mice compared to untreated GR L2/L2 myositis mice; moreover it was 2-fold more expressed in PDN-treated GR L2/L2 myositis mice compared to PDN-treated GR(i)skm-/- mice.Conclusion:GR in skeletal muscle fibre is crucial to mediate the therapeutic response to GC in a murine model of myositis. Autophagy is one of the candidate pathways controlled by myofibre GR underlying this effect.