Single-cell analysis of skeletal muscle macrophages reveals age-associated functional subpopulations
- National Institute on Aging, United States
Abstract
Tissue-resident macrophages represent a group of highly responsive innate immune cells that acquire diverse functions by polarizing towards distinct subpopulations. The subpopulations of macrophages that reside in skeletal muscle (SKM) and their changes during aging are poorly characterized. By single-cell transcriptomic analysis with unsupervised clustering, we found eleven distinct macrophage clusters in male mouse SKM with enriched gene expression programs linked to reparative, proinflammatory, phagocytotic, proliferative, and senescence-associated functions. Using a complementary classification, membrane markers LYVE1 and MHCII identified four macrophage subgroups: LYVE1-/MHCIIhi (M1-like, classically activated), LYVE1+/MHCIIlo (M2-like, alternatively activated), and two new subgroups, LYVE1+/MHCIIhi and LYVE1-/MHCIIlo. Notably, one new subgroup, LYVE1+/MHCIIhi, had traits of both M2 and M1 macrophages, while the other new subgroup, LYVE1-/MHCIIlo, displayed strong phagocytotic capacity. Flow cytometric analysis validated the presence of the four macrophage subgroups in SKM, and found that LYVE1- macrophages were more abundant than LYVE1+ macrophages in old SKM. A striking increase in proinflammatory markers (S100a8 and S100a9 mRNAs) and senescence-related markers (Gpnmb and Spp1 mRNAs) was evident in macrophage clusters from older mice. In sum, we have identified dynamically polarized SKM macrophages and propose that specific macrophage subpopulations contribute to the proinflammatory and senescent traits of old SKM.
Data availability
The single-cell RNA-seq analysis was uploaded to GEO with identifier GSE195507.
-
Single-cell analysis of skeletal muscle macrophages reveals age-associated functional subpopulationsNCBI Gene Expression Omnibus, GSE195507.
Article and author information
Author details
Funding
National Institutes of Health (Z01-AG000511)
- Linda K Krasniewski
- Papiya Chakraborty
- Chang-Yi Cui
- Krystyna Mazan-Mamczarz
- Christopher Dunn
- Yulan Piao
- Jinshui Fan
- Changyou Shi
- Tonya Wallace
- Cuong Nguyen
- Isabelle A Rathbun
- Rachel Munk
- Dimitrios Tsitsipatis
- Supriyo De
- Payel Sen
- Luigi Ferrucci
- Myriam M Gorospe
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Ethics
Animal experimentation: All animal study protocols were approved by the NIA Institutional Review Board (Animal Care and Use Committee). (ASP #476-LGG-2023)
Reviewing Editor
- Bérénice A Benayoun, University of Southern California, United States
Version history
- Received: February 17, 2022
- Preprint posted: February 24, 2022 (view preprint)
- Accepted: October 10, 2022
- Accepted Manuscript published: October 19, 2022 (version 1)
- Version of Record published: November 2, 2022 (version 2)
Copyright
This is an open-access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication.
Metrics
-
- 3,530
- Page views
-
- 470
- Downloads
-
- 10
- Citations
Article citation count generated by polling the highest count across the following sources: Crossref, PubMed Central, Scopus.
Download links
A two-part list of links to download the article, or parts of the article, in various formats.
Downloads (link to download the article as PDF)
Open citations (links to open the citations from this article in various online reference manager services)
Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)
Single-cell analysis of skeletal muscle macrophages reveals age-associated functional subpopulations
eLife 11:e77974.
https://doi.org/10.7554/eLife.77974
Further reading
-
- Cell Biology
- Developmental Biology
Cylicins are testis-specific proteins, which are exclusively expressed during spermiogenesis. In mice and humans, two Cylicins, the gonosomal X-linked Cylicin 1 (Cylc1/CYLC1) and the autosomal Cylicin 2 (Cylc2/CYLC2) genes, have been identified. Cylicins are cytoskeletal proteins with an overall positive charge due to lysine-rich repeats. While Cylicins have been localized in the acrosomal region of round spermatids, they resemble a major component of the calyx within the perinuclear theca at the posterior part of mature sperm nuclei. However, the role of Cylicins during spermiogenesis has not yet been investigated. Here, we applied CRISPR/Cas9-mediated gene editing in zygotes to establish Cylc1- and Cylc2-deficient mouse lines as a model to study the function of these proteins. Cylc1 deficiency resulted in male subfertility, whereas Cylc2-/-, Cylc1-/yCylc2+/-, and Cylc1-/yCylc2-/- males were infertile. Phenotypical characterization revealed that loss of Cylicins prevents proper calyx assembly during spermiogenesis. This results in decreased epididymal sperm counts, impaired shedding of excess cytoplasm, and severe structural malformations, ultimately resulting in impaired sperm motility. Furthermore, exome sequencing identified an infertile man with a hemizygous variant in CYLC1 and a heterozygous variant in CYLC2, displaying morphological abnormalities of the sperm including the absence of the acrosome. Thus, our study highlights the relevance and importance of Cylicins for spermiogenic remodeling and male fertility in human and mouse, and provides the basis for further studies on unraveling the complex molecular interactions between perinuclear theca proteins required during spermiogenesis.
-
- Cell Biology
- Structural Biology and Molecular Biophysics
Previously we showed that 2D template matching (2DTM) can be used to localize macromolecular complexes in images recorded by cryogenic electron microscopy (cryo-EM) with high precision, even in the presence of noise and cellular background (Lucas et al., 2021; Lucas et al., 2022). Here, we show that once localized, these particles may be averaged together to generate high-resolution 3D reconstructions. However, regions included in the template may suffer from template bias, leading to inflated resolution estimates and making the interpretation of high-resolution features unreliable. We evaluate conditions that minimize template bias while retaining the benefits of high-precision localization, and we show that molecular features not present in the template can be reconstructed at high resolution from targets found by 2DTM, extending prior work at low-resolution. Moreover, we present a quantitative metric for template bias to aid the interpretation of 3D reconstructions calculated with particles localized using high-resolution templates and fine angular sampling.
