Single-cell analysis of skeletal muscle macrophages reveals age-associated functional subpopulations

Tissue-resident macrophages represent a group of highly responsive innate immune cells that acquire diverse functions by polarizing towards distinct subgroups. The subgroups of macrophages that reside in skeletal muscle (SKM) and their changes during aging are not fully understood. By single-cell transcriptomic analysis, we found that mouse SKM macrophages comprise two large populations, Lyve1+ and Lyve1-. Lyve1+ macrophages expressed high levels of mRNAs encoding angiogenesis and tissue repair proteins, while Lyve1- macrophages expressed high levels of mRNAs encoding proteins with functions in translation, antigen presentation, and the proinflammatory response. SKM macrophages were further classified into four functional subgroups based on the expression levels of another cell surface marker MHCII: Lyve1+/MHCII-lo (similar to alternatively activated M2), Lyve1-/MHCII-hi (similar to classically activated M1), and two new subgroups, Lyve1+/MHCII-hi and Lyve1-/MHCII-lo, all displayed strong phagocytic capacities. Flow cytometric analysis validated the presence of the four macrophage subgroups in SKM. The new subgroup Lyve1+/MHCII-hi had traits of both M2 and M1 macrophages, while the other subgroup, Lyve1-/MHCII-lo, expressed high levels of mRNAs encoding cytotoxicity proteins. In old SKM, Lyve1+/MHCII-hi macrophages were less abundant and Lyve1-/MHCII-hi macrophages were more abundant. Furthermore, complementary unsupervised classification revealed the emergence of small clusters expressing proinflammatory markers including S100a8 and S100a9 mRNAs in aged SKM. In sum, our study has identified dynamically polarized mouse SKM macrophages and a shift toward a proinflammatory status during aging. Resident macrophages were isolated from mouse skeletal muscle from young and old C57BL/6JN mice by FACS. Single-cell libraries were prepared with 10x Genomics and sequenced with Illumina Nova-sequencer.