Background/Purpose: In polymyositis (PM), CD8⁺ cytotoxic T lymphocytes (CTLs) are assumed to induce muscle cell death. We presumed that the injured muscle cells release pro-inflammatory molecules including damage associated molecular patterns (DAMPs), which would accelerate further CTL-mediated muscle injury. Inhibition of muscle cell death could be a novel therapeutic strategy in PM. We have previously shown that the suppressive effects of perforin deficiency on the severity and incidence of myositis were partial in C protein induced-myositis (CIM), suggesting the involvement of another pathway in addition to the perforin/granzyme pathway. The aims of this study are to clarify the mechanisms of CTL-mediated muscle injury, the pattern of cell death of muscle cells, and the therapeutic effects of inhibition of muscle cell death on in vitro and in vivo models of PM.

Methods: OT-I CTLs, and their mutants lacking perforin 1 or granzyme B were cocultured with myotubes differentiated from C2C12 cells that were retrovirally transduced with the genes encoding MHC class I (H2K^b) and SIINFEKL peptide derived from ovalbumin (H2K^bOVA-myotubes) to discern how muscle cell death is mediated in vitro by CTL. CIM was used to study the effect of necrostatin-1s (nec1s) on the severity of myositis in vivo. The levels of high mobility group box-1 protein (HMGB1), one of the DAMPs, in the supernatant of the coculture and the serum of CIM were measured by ELISA. Muscle biopsy specimens of PM patients were examined with terminal deoxynucleotidyl transferase nick-end labeling (TUNEL) assay, and histologically for the expression of the necroptosis associated proteins including receptor-interacting serine-threonine kinase (RIPK3) and mixed lineage kinase domain-like pseudokinase (MLKL).

Results: OT-I CTLs lacking perforin 1 or granzyme B were as cytotoxic to H2K^bOVA-myotubes as wild type OT-I CTLs. Inhibition of Fas ligand by the Fas-Fc chimera protein reduced cytotoxicity of CTLs against the myotubes. The TUNEL assay and time-lapse imaging of cell death visualized by Annexin V and PI revealed that the cell death of the myotubes was non-apoptotic. The CTL-mediated cell death of myotubes was inhibited by nec1s, a necroptosis inhibitor, or Ripk3 silencing with siRNA, but not by benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone, an apoptosis inhibitor. Treatment of myotubes with nec1s inhibited the CTL-induced increase of HMGB1 level in the supernatant of the coculture. Therapeutic administration of nec1s on CIM improved the grip strength of mice and reduced the severity of myositis histologically. The serum level of HMGB1 was lower in nec1s-treated CIM mice compared to that in the untreated mice. Immunohistological staining of muscle tissue of PM patients revealed the expression of Fas, RIPK3, and phosphorylated MLKL on injured muscle cells.

Conclusion: Necroptosis is involved in muscle cell death in PM. Inhibition of necroptosis should be a novel therapeutic strategy in PM.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/inhibition-of-necroptosis-suppresses-muscle-cell-death-and-inflammatory-infiltrate-and-improves-muscle-strength-in-experimental-polymyositis/