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[Preprint]. 2023 Apr 18:2023.04.18.537253.
doi: 10.1101/2023.04.18.537253.

Single-cell and spatial transcriptomics identify a macrophage population associated with skeletal muscle fibrosis

Affiliations

Single-cell and spatial transcriptomics identify a macrophage population associated with skeletal muscle fibrosis

Gerald Coulis et al. bioRxiv. .

Update in

  • Single-cell and spatial transcriptomics identify a macrophage population associated with skeletal muscle fibrosis.
    Coulis G, Jaime D, Guerrero-Juarez C, Kastenschmidt JM, Farahat PK, Nguyen Q, Pervolarakis N, McLinden K, Thurlow L, Movahedi S, Hughes BS, Duarte J, Sorn A, Montoya E, Mozaffar I, Dragan M, Othy S, Joshi T, Hans CP, Kimonis V, MacLean AL, Nie Q, Wallace LM, Harper SQ, Mozaffar T, Hogarth MW, Bhattacharya S, Jaiswal JK, Golann DR, Su Q, Kessenbrock K, Stec M, Spencer MJ, Zamudio JR, Villalta SA. Coulis G, et al. Sci Adv. 2023 Jul 7;9(27):eadd9984. doi: 10.1126/sciadv.add9984. Epub 2023 Jul 7. Sci Adv. 2023. PMID: 37418531 Free PMC article.

Abstract

The monocytic/macrophage system is essential for skeletal muscle homeostasis, but its dysregulation contributes to the pathogenesis of muscle degenerative disorders. Despite our increasing knowledge of the role of macrophages in degenerative disease, it still remains unclear how macrophages contribute to muscle fibrosis. Here, we used single-cell transcriptomics to determine the molecular attributes of dystrophic and healthy muscle macrophages. We identified six novel clusters. Unexpectedly, none corresponded to traditional definitions of M1 or M2 macrophage activation. Rather, the predominant macrophage signature in dystrophic muscle was characterized by high expression of fibrotic factors, galectin-3 and spp1. Spatial transcriptomics and computational inferences of intercellular communication indicated that spp1 regulates stromal progenitor and macrophage interactions during muscular dystrophy. Galectin-3 + macrophages were chronically activated in dystrophic muscle and adoptive transfer assays showed that the galectin-3 + phenotype was the dominant molecular program induced within the dystrophic milieu. Histological examination of human muscle biopsies revealed that galectin-3 + macrophages were also elevated in multiple myopathies. These studies advance our understanding of macrophages in muscular dystrophy by defining the transcriptional programs induced in muscle macrophages, and reveal spp1 as a major regulator of macrophage and stromal progenitor interactions.

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Conflict of interest statement

Competing interests: Dr. Mozaffar discloses an advisory role for and/or receiving research funds from Alexion, Amicus, Argenx, Arvinas, Audentes, AvroBio, Horizon Therapeutics, Immunovant, Maze Therapeutics, Momenta (now Janssen), Sanofi-Genzyme, Sarepta, Spark Therapeutics, UCB, and Modis/Zogenix. Dr. Mozaffar also serves on the data safety monitoring board for Acceleron, Avexis, and Sarepta. Drs. Golann, Si and Stec are employees and shareholders of Regeneron Pharmaceuticals. Dr. Spencer is a co-founder of MyoGene Bio and SkyGene Bio. All other authors declare that they have no competing interests.

Figures

Fig. 1.
Fig. 1.. Identification of novel transcriptional programs in skeletal muscle macrophages by single-cell RNA sequencing.
(A) Muscle macrophages were isolated from 4-wk-old WT (healthy) and mdx (dystrophic) mice and analyzed by scRNAseq. n= 1 (B) Dimensionality reduction via UMAP of healthy or dystrophic muscle macrophages. (C) The proportion of WT or mdx muscle macrophage clusters. (D) Identities classified by genotype. (E) Differential gene expression analysis showing the top ten most differentially expressed genes for each cluster.
Fig. 2.
Fig. 2.. Muscle macrophages express novel transcriptomes, distinct from M1 and M2 macrophages.
(A-C) Heatmap showing the expression of the top 100 scRNAseq differentially expressed genes from the scRNAseq analysis (scDEGs) in FACS-sorted SkMRM (A), gal-3+ Mφ (B) and MDM (C). n= 3 per population. (D) Principal component analysis (PCA) applied to the FACS-sorted macrophage populations in A-C. (E) Pathway analysis of top gene sets enriched in FACS-sorted gal-3+ Mφ compared to SkMRM. (F) Pairwise comparison of gene expression between FACS-sorted gal-3+ Mφ and SkMRM. Colored points indicate DEGs from ECM-related gene sets. Lgals3 and Spp1 are highlighted by arrows. (G) Venn Diagrams of upregulated and downregulated DEGs in FACS-sorted gal-3+ Mφ compared to SkMRM (Gal-3+/SkMRM) and MDMs compared to SkMRMs (MDMs/SkMRMs). (H) Correlation analysis of DEGs from Gal-3+/SkMRM or MDM/SkMRM comparisons with M1 or M2 polarized Mφ. (I) Expression of M1 and M2 macrophage markers in gal-3+ Mφ (0), SkMRMs (1) and MDMs (2) from scRNAseq data. (J) Violin plots of M2 markers in gal-3+ Mφ, SkMRMs and MDMs from scRNAseq data.
Fig. 3.
Fig. 3.. Spatial transcriptomics reveals that Gal-3+ macrophages are associated with stromal cells and extracellular matrix.
(A and B) Spatially resolved gene expression of Lgals3 (galectin-3) (A) and H&E staining of D2-mdx quadriceps (B). Shown is one of 5 representative D2-mdx quadriceps. (C) Heatmap showing DEGs between Lgals3hi and Lgals3lo spots. Shown are genes with a fold change >= 1.5, FDR < 0.01. All spots in a section, including those with and without pathology, were unbiasedly analyzed. (D and E) Gene ontology/pathway analysis showing the enrichment of GO terms associated with Collagen/ECM (D) and fibroblasts (E) in galectin-3hi spots. (F) Expression of DEGs associated with fibrosis. (G) Spatially resolved gene expression of Pdgfra in mdx quadriceps. (H) Immunofluorescence staining of 4-wk-old mdx quadriceps reveals colocalization of PDGFRa and galectin-3 in dystrophic muscle.
Fig. 4.
Fig. 4.. Spp1 mediates FAP and macrophage interactions in dystrophic muscle.
(A) reference-based integration of skeletal muscle mononucleated cell datasets prepared from 3-mon-old WT and mdx mice. (B-G) Visualization and analysis of cell-cell communication using CellChat. Circle plots placing FAPs subsets as the central nodes of analysis in the mdx dataset (B-D). An interaction between a pair of cell types is depicted by a line connecting two cell types. The thickness of the line depicts the strength of the interaction between two cell types. A similar analysis applying macrophages as the central node of communication is shown (E-G). (H) Pathways enriched in the stromal cell and macrophage network of WT and mdx mice. (I) Relative contribution of Spp1 ligand (L)-receptor (R) pairs. (J) Expression of Spp1 receptors was measured in WT and mdx PDGFRα+Sca1+ FAPs by flow cytometry. Plots shown were gated on CD45CD31 live cells. (K-M) Representative histograms and quantification of the MFI of Spp1 receptors. 4-wk-old mice were analyzed. n= 3–4. *p<0.05, **p<0.01, ***p<0.001 using an unpaired Welch’s t-test. (N-P) RNAscope multiplexed with immunofluorescence staining showing the co-localization of macrophages (N) and Spp1 mRNA (P) in areas enriched with collagen (O).
Fig. 5.
Fig. 5.. Chronic activation of galectin-3+ macrophages in dystrophic muscle.
(A) The number of gal-3+ macrophages in B10.mdx hind limb muscle, normalized to muscle mass (g). n= 6–19 per time point (B and C) Representative histograms and quantitative analysis of the geometric mean fluorescence intensity (MFI) of Gal-3 in 4 wk- (B) and 52-wk-old (C) WT and mdx mice. n= 3–16 per group. 4-wk-old mice were analyzed. (D) Sirius red staining of WT and mdx quadriceps cryosections from 4 wk- and 52-wk-old mice. (E-F) Enumeration of gal-3+ macrophages in the valosin-containing protein (VCP)-associated inclusion body myopathy mouse model (E) and in the Facioscapulohumeral Muscular Dystrophy (TIC-DUX4) mouse model (F). n= 4–6, 10-mon-old mice (E); n= 5, 10-w-kold mice (F). (G and H) The regulation of gal-3+ macrophages frequency (G) and number (H) after injury. n= 7–9 per time point (G, H). *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 using an unpaired Welch’s t-test (B, C, E, F) or 2-way ANOVA with Sidak’s multiple comparison test (A, G, H).
Fig. 6.
Fig. 6.. Peripheral monocytes and skeletal muscle-resident macrophages give rise to galectin-3+ macrophages.
(A and B) Adoptive transfer of monocytes into 4-wk-old mdx mice. Graphical abstract of the workflow and representative flow plots of monocytes before and after transfer (A). Frequency of the donor monocytes that converted to gal-3+ macrophages at 2- and 7-days post-transfer (B). n= 3–13 per group. (C and D) Adoptive transfer of SkMRMs into 4-wk-old mdx mice. Schematic of the workflow and representative flow plots (C). Frequency of SkMRMs that converted to gal-3+ macrophages (D). n= 3–5 per group. (E-G) RT-qPCR quantification of the expression of cluster 1 (E), 2 (F) and 0 genes (G) in FACS-sorted SkMRMs from WT and mdx muscle, and *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 using an unpaired Welch’s t-test (B, C, E, F) or 2-way ANOVA with Sidak’s multiple comparison test (A, G, H). gal-3+ Mφ and MDMs from mdx muscle.
Fig. 7.
Fig. 7.. Galectin-3+ macrophages are elevated in human chronic muscle disease.
(A) Immunofluorescence staining of gal-3+ macrophages in human dystrophic muscle. CD68 (red), gal-3 (green), nuclei (blue). (B) Representative images of immunohistochemical staining of galectin-3 in control and myopathic patients. (C-E) Quantification of gal-3+ cells in the interstitial space (C), the perivascular area (D) or infiltrating the myofiber (E). n= 3–8 frozen sections per patient type. (F and G) Expression of human SPP1 (F) and COL1A (G) mRNA in control, DMD and LGMD biopsies. n= 6–8 patients were used to measure RNA expression. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 using a 2-way ANOVA with Kruskal-Wallis multiple comparison test (C-G).

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