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

Macrophages are essential for skeletal muscle homeostasis, but how their dysregulation contributes to the development of fibrosis in muscle disease remains unclear. Here, we used single-cell transcriptomics to determine the molecular attributes of dystrophic and healthy muscle macrophages. We identified six clusters and unexpectedly found that none corresponded to traditional definitions of M1 or M2 macrophages. Rather, the predominant macrophage signature in dystrophic muscle was characterized by high expression of fibrotic factors, galectin-3 (gal-3) and osteopontin (<i>Spp1</i>). Spatial transcriptomics, computational inferences of intercellular communication, and in vitro assays indicated that macrophage-derived Spp1 regulates stromal progenitor differentiation. Gal-3⁺ macrophages were chronically activated in dystrophic muscle, and adoptive transfer assays showed that the gal-3⁺ phenotype was the dominant molecular program induced within the dystrophic milieu. Gal-3⁺ macrophages were also elevated in multiple human myopathies. These studies advance our understanding of macrophages in muscular dystrophy by defining their transcriptional programs and reveal <i>Spp1</i> as a major regulator of macrophage and stromal progenitor interactions.