A novel mouse model of myositis-associated interstitial lung disease was established by using TLR9 agonist combined with muscle homogenate.

Our group previously demonstrated that NETs were involved in interstitial lung diseases (ILD) among patients with idiopathic inflammatory myopathies (IIM) and the experimental autoimmune myositis (EAM) mouse model and that NETs activated lung fibroblasts through the TLR9-miR7-Smad2 axis. This study aimed to establish a novel mouse model of myositis-associated interstitial lung disease (MAILD) by using a TLR9 agonist (ODN2395). ODN2395 and muscle homogenate were used to induce MAILD in BALB/c mice. MAILD was evaluated using histopathology, immunohistochemistry, serum NETs determination, and myositis-specific antibody profile. Furthermore, TLR9 and IRF3 were examined in a lung biopsy tissue from a dermatomyositis patient with ILD. MAILD mice developed inflammatory myopathy with positive expression of myositis-specific antibodies. ILD occurred in all mice of the MAILD group. ODN2395 at doses of 5 μg, 10 μg, or 20 μg induced ILD, with increasing severity as the dose increased, but 20 μg ODN2395 was not recommended due to non-specific damage to the lungs. ILD could occur as early as one week after immunization and was most pronounced by the fourth/fifth week. MAILD process was accompanied by NETs infiltration and TLR9 activation. TLR9 activation was demonstrated in the patient with DM-ILD. Serum levels of Cit-H3 were elevated in the MAILD group. Skeletal muscle homogenate and ODN2395 induced neutrophils to form NETs in vitro. Combined with muscle homogenate, ODN2395 induced a novel MAILD mouse model with NETs infiltration and TLR9 activation, which are similar to pathogenesis of IIM-ILD, suggesting that MAILD model could replace EAM model in IIM-ILD research.