Spatiotemporal transcriptomic mapping of regenerative inflammation in skeletal muscle reveals a dynamic multilayered tissue architecture

Tissue regeneration is orchestrated by macrophages that clear damaged cells and promote regenerative inflammation. How macrophages spatially adapt and diversify their functions to support the architectural requirements of actively regenerating tissue remains unknown. In this study, we reconstructed the dynamic trajectories of myeloid cells isolated from acutely injured and early-stage dystrophic muscles. We identified divergent subsets of monocytes/macrophages and dendritic cells (DCs) and validated markers (e.g., GPNMB) and transcriptional regulators associated with defined functional states. In dystrophic muscle, specialized repair-associated subsets exhibited distinct macrophage diversity and reduced DC heterogeneity. Integrating spatial transcriptomics analyses with immunofluorescence uncovered the ordered distribution of subpopulations and multilayered regenerative inflammation zones (RIZs) where distinct macrophage subsets are organized in functional zones around damaged myofibers supporting all phases of regeneration. Importantly, intermittent glucocorticoid treatment disrupted the RIZs. Our findings suggest that macrophage subtypes mediated the development of the highly ordered architecture of regenerative tissues, unveiling the principles of the structured yet dynamic nature of regenerative inflammation supporting effective tissue repair. Overall design: Single-cell trancriptomic profiling of C57 mouse CD45+ cells from TA, after one (D1), two (D2) and four days (D4 - see GSE161467) after CTX-induced acute sterile muscle injury and CD45+ cells from 2 month old D2.mdx GAST using high throughput sequencing (scRNA-seq, 10x Chromium V3 chemistry). Spatial trancriptomic profiling of D4 and D8 post-CTX TA muscles from WT and DBA2/J as well as 2 month old Vehicle and Prednisone-treated GAST from D2.mdx (stRNA-seq 10x Visium).