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  1. Evaluation Summary:

    Krasniewski and colleagues leveraged single-cell transcriptomics to identify subpopulations of macrophages in the skeletal muscle of aging male mice. They identify several new resident subpopulations of skeletal muscle macrophages, spanning a range of polarization states using novel markers, and they identify a shift in relative abundances of these subpopulations with age, leading to a functional shift in inflammatory marker expression and phagocytic capacity. The study overall is an interesting and timely investigation of skeletal muscle macrophage populations and transcriptomics in both healthy young and old mice, and should be a valuable resource for the inflammaging and muscle biology field.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)

  2. Reviewer #1 (Public Review):

    Krasniewski et al. investigated the varying populations of mouse skeletal muscle resident macrophages with age using single-cell transcriptomics. They find that mouse SKM macrophages can be divided into two subgroups, anti-inflammatory LYVE+ and proinflammatory LYVE- macrophages. They further classify these macrophages into LYVE1+/MHCII-lo (similar to classical M2 activation), LYVE1-/MHCII-hi (similar to classical M1 activation), LYVE1+/MHCII-hi, and LYVE1-/MHCII-lo, with LYVE1+/MHCII-hi macrophages expressing traits of both the classical M1 and M2 macrophages, and LYVE1-/MHCII-lo macrophages being enriched with mRNAs encoding cytotoxicity proteins. Using a phagocytosis assay, they show that LYVE1-/MHCII-lo macrophages have a reduced % of phagocytosing cells, notably while having a higher gMFI. Interestingly, the data also indicate that commonly used polarization markers CD206 and CD86 are widely and simultaneously expressed, suggesting they are not best suited to represent polarization. By performing an unsupervised classification of macrophage clusters, the authors find smaller macrophage subpopulations that suggest increased expression of inflammatory and cell-cycle related mRNAs in old SKM. These data expand and enhance what the field uses as markers for macrophage polarization and suggests and age-dependent change in macrophage populations, with older SKM containing a higher percentage of proinflammatory M1-like macrophages.

    The manuscript presents interesting findings and provides an important resource to better understand innate immune shifts with aging as well as their functional consequences. Although most conclusions are well supported by data, clarifications are needed to fully support the study and improve its long-term reproducibility.

    Since the study is performed exclusively on male animals and aging is extremely sex-dimorphic, it would be important for the authors to contextualize the findings in terms of how they may be affected as a function of sex.

  3. Reviewer #2 (Public Review):

    The manuscript by Krasniewski et al., utilizes single cell RNA seq analysis to investigate skeletal muscle macrophage populations in healthy young and old mice. Utilizing transcriptomic analysis and further validation via immune-staining and flow cytometry, the authors show that multiple sub-populations of macrophage subsets exist in healthy skeletal muscle, and are characterized by differential expression of Lyve and MHCII. Additionally, utilizing further analysis comparing skeletal muscle macrophage subpopulations in young vs old mice, the authors show that during aging these macrophage subpopulations skew from an M2-like state to a pro-inflammatory M1-like state. Lastly, unsupervised classification of old vs young macrophages revealed novel subsets of macrophages that increase during aging, suggesting a role for skeletal muscle macrophages in aging related muscle pathophysiology.

    The study overall is an interesting and timely investigation of skeletal muscle macrophage populations and transcriptomics in both healthy young and old mice, and would be a valuable resource to the inflammaging and muscle biology field. However, the manuscript is largely descriptive and does not provide any mechanistic or functional insight or test what role these macrophage subsets play in muscle physiology of young or old mice.

  4. Reviewer #3 (Public Review):

    In this article, the authors sought to dissect the heterogeneity of skeletal muscle macrophages and catalogue the changes occurring with age. To do so, they performed a scRNAseq of CD11b+ cells in skeletal muscles in 3 and 23 mo old male mice. They describe four different subsets defined by MHC-II and Lyve1 expression displaying distinct transcriptional profiles. They confirm the existence of these subsets using flow cytometry and immunofluorescence imaging of skeletal muscle tissue. They show that these subsets display different phagocytic capacity, notably in accordance with GO annotation suggesting enriched expression of phagocytic genes in LnHl. Comparisons of macrophage subsets between young and old animals revealed a decrease of Lyve1+ and increase of Lyve1- macrophages in old. Analysis of differentially expressed genes revealed an overall increase of pro-inflammatory genes in old macrophages. Finally, the authors performed an unbiased analysis to confirm the subsets described with the supervised analysis. In general, and as presented scRNA analyses are superficial that provide scant new insights into macrophage biology and aging.