Inhibiting trained immunity in Duchenne muscular dystrophy enhances muscle regeneration and reduces fat deposition

<title>Abstract</title> <p> Background Trained immunity (TI) refers to a hyperresponsive functional state of innate immune cells, such as inflammatory monocytes, induced in response to a variety of stimuli including signals released from damaged tissue, and characterised by increased cytokine production. Inflammatory monocytes play a critical role in Duchenne muscular dystrophy (DMD) by driving chronic muscle inflammation and disease progression. In this study we examined the role of trained immunity in dystrophic muscle pathology by specifically targeting this process in monocytes. Methods We used the <italic>mdx</italic> mouse model of DMD to investigate TI induction in splenic and bone marrow monocytes isolated during the peak of muscle injury and inflammation. Transcriptional changes in monocytes were examined by performing RNA-Seq, while cytokine production following stimulation with LPS was examined by multiplex ELISA. Mice were treated for two weeks with twice weekly i.v. injections of apolipoprotein A1–based, rapamycin-loaded nanobiologics (mTORi-NB) to specifically target TI in monocytes within the bone marrow and the spleen through the inhibition of the mTOR pathway which plays a central role in TI induction. Inhibition of TI in monocytes was assessed <italic>in vitro</italic> by measuring cytokine production following stimulation with LPS. Satellite cells and FAPs were sorted from limb muscles and cultured <italic>in vitro</italic> to assess the effect of the treatment on their proliferation and differentiation properties. Results Dystrophic muscle injury was associated with transcriptional changes in <italic>mdx</italic> monocytes linked to chromatin and metabolic remodelling as well as with hyperproduction of proinflammatory cytokines, consistent with TI induction. Treatment of <italic>mdx</italic> mice with mTORi-NB resulted in improved skeletal muscle regeneration and reduced fat deposition. <italic>In vitro</italic> experiments confirmed the enhanced myogenic properties in satellite cells and reduced adipogenesis in FAPs mediated by the inhibition of TI in monocytes. Conclusions Dystrophic muscle injury induces TI in both bone marrow and splenic monocytes characterised by hyperproduction of proinflammatory cytokines. Targeting TI specifically in monocytes through mTOR inhibition enhances muscle regeneration and reduces intramuscular fat deposition. Overall, our findings suggest that inhibition of TI represents a promising therapeutic strategy to slow DMD progression. </p>