Single-cell and spatial transcriptomics identify a macrophage population associated with skeletal muscle fibrosis

The monocytic/macrophage system is essential for skeletal muscle homeostasis, but its dysregulation contributes to the pathogenesis of muscle degenerative disorders. Despite our increasing knowledge of the role of macrophages in degenerative disease, it still remains unclear how macrophages contribute to muscle fibrosis. Here, we used single-cell transcriptomics to determine the molecular attributes of dystrophic and healthy muscle macrophages. We identified six novel clusters. Unexpectedly, none corresponded to traditional definitions of M1 or M2 macrophage activation. Rather, the predominant macrophage signature in dystrophic muscle was characterized by high expression of fibrotic factors, galectin-3 and spp1. Spatial transcriptomics and computational inferences of intercellular communication indicated that spp1 regulates stromal progenitor and macrophage interactions during muscular dystrophy. Galectin-3+ macrophages were chronically activated in dystrophic muscle and adoptive transfer assays showed that the galectin-3+ phenotype was the dominant molecular program induced within the dystrophic milieu. Histological examination of human muscle biopsies revealed that galectin-3+ macrophages were also elevated in multiple myopathies. These studies advance our understanding of macrophages in muscular dystrophy by defining the transcriptional programs induced in muscle macrophages, and reveal spp1 as a major regulator of macrophage and stromal progenitor interactions. ### Competing Interest Statement Dr. Mozaffar discloses an advisory role for and/or receiving research funds from Alexion, Amicus, Argenx, Arvinas, Audentes, AvroBio, Horizon Therapeutics, Immunovant, Maze Therapeutics, Momenta (now Janssen), Sanofi-Genzyme, Sarepta, Spark Therapeutics, UCB, and Modis/Zogenix. Dr. Mozaffar also serves on the data safety monitoring board for Acceleron, Avexis, and Sarepta. Drs. Golann, Si and Stec are employees and shareholders of Regeneron Pharmaceuticals. Dr. Spencer is a co-founder of MyoGene Bio and SkyGene Bio. All other authors declare that they have no competing interests.