Regulatory T cells suppress novel skeletal muscle macrophages identified by single-cell RNA sequencing

Abstract

Abstract Duchenne muscular dystrophy (DMD) is a lethal X-linked disorder that results from mutations in the dystrophin gene causing necrosis, inflammation and ultimately fibrosis. In DMD, muscle macrophages are multifaceted effector immune cells that regulate multiple pathogenic processes, including myofiber injury, inflammation and fibrosis, but also promote regeneration. Previously, we demonstrated that regulatory T cells (Tregs) ameliorated the severity of muscular dystrophy by regulating M1-like and M2-like macrophage activation, and more recently, a subset of undefined macrophages. Herein, we used single-cell RNA sequencing (scRNAseq) approaches to define the phenotypic complexity and transcriptional profile of muscle macrophages in healthy and dystrophic muscle. A T-distributed Stochastic Neighbor Embedding (t-SNE) analysis of gene expression data revealed that muscle macrophages clustered into six novel populations defined by unique transcriptional programs. We observed that a population expressing high levels of galectin-3 was absent in wildtype and dystrophic muscle before the onset of disease, but was increased at the onset of acute pathology and further expanded when Tregs were depleted in the mdx mouse model of DMD. In light of emerging evidence in the literature suggesting a role for galectin-3 and Tregs in fibrosis, we performed in vivo functional assays to underscore the role of the Treg-Macrophages axis in the control of fibrosis in DMD. Our data support a new paradigm, in which unique transcriptional programs define novel macrophage populations likely adapted to the diseased muscle milieu, and suggest that therapies focused on augmenting Tregs function in dystrophic muscle may be beneficial to treat fibrosis.