16 | Immunoproteasome inhibition positively impacts the gut-muscle axis in Duchenne muscular dystrophy

Debora Mostosi¹, A. Farini², F. Strati³, M. Molinaro², S. Saccone², C. Amoroso³, B. Cassani⁴, E. Leonetti¹, F. Caprioli⁵, F. Facciotti³, Y. Torrente^1|2 | ¹Stem Cell Laboratory, Dino Ferrari Center, Department of Pathophysiology and Transplantation, University of Milan, Milan, Italy; ²Neurology Unit, Fondazione IRCCS Ca' Granda Ospedale Maggiore Policlinico, Milan, Italy; ³Department of Biotechnology and Biosciences, University of Milano-Bicocca, Milan, Italy; ⁴Department of Medical Biotechnologies and Translational Medicine, University of Milan, Italy; ⁵Department of Pathophysiology and Transplantation, University of Milan, Unit of Gastroenterology and Endoscopy, Fondazione IRCCS Ca’ Granda, Ospedale Policlinico di Milano, Italy.

Abstract

Duchenne muscular dystrophy (DMD), a fatal muscle-wasting disorder resulting from mutations in the DMDgene, is often compounded by secondary non-muscle complications. A key factor in DMD progression is chronic inflammation, triggered by the release of Danger Associated Molecular Patterns (DAMPs) following myofiber necrosis, which attracts proinflammatory cells to dystrophic muscle. Notably, gastrointestinal (GI) dysfunction, common in DMD patients, can also contribute to systemic inflammation via the gut microbiota. The immunoproteasome (IP) plays a crucial role in regulating antigen presentation, cytokine production, and immune cell responses, and acts as a first line of defense in the gut by modulating the NF-κB pathway. Our prior research demonstrated that IP inhibition alleviates molecular, histological, and functional deficits in mdx mice, a well-established DMD model. Here, we investigated the impact of IP inhibition on the gut-muscle axis in these mice. Treatment with the IP inhibitor ONX-0914 increased the presence of anti-inflammatory CD206+ M2 macrophages in both the colon and muscle of mdx mice, while partially reduced pro-inflammatory CD68+ M1 cells. This treatment also improved muscular mitochondrial activity, enhanced muscle performance, and decreased serum creatine phosphokinase (CPK) levels and fibrosis. Utilizing microbiota perturbation models, including broad-spectrum antibiotics and fecal microbiota transplantation from IP-inhibited mdx mice, we demonstrated that ONX-0914's beneficial effects on muscle function are mediated through microbiotadependent mechanisms. Our findings contribute to understanding the role of IP inhibition as a potential therapeutic strategy for modulating the dystrophic gut-muscle axis, suggesting new avenues for microbiota-targeted therapies.