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

Question

1. What are the distinct transcriptional profiles of macrophage clusters in skeletal muscle during muscular dystrophy?

2. How can flow cytometry panel distinguish macrophage populations?

3. What is the largest variance in PCA?

4. What is the role of galectin-3 + macrophages in degenerative lesions?

Accepted Answer

Single-cell RNA sequencing revealed six macrophage clusters in skeletal muscle during muscular dystrophy. Cluster 1 macrophages, referred to as SkMRMs, expressed genes associated with skeletal muscle-resident macrophages, such as Folr2, Mt2, Lyve1, Gas6, and Cbr2. Cluster 0 macrophages, known as galectin-3 + macrophages, expressed genes implicated in fibrosis, including Spp1, Fabp5, Gnmbp, Trem2, Lgals3, and various cathepsin genes. Cluster 2 macrophages, referred to as MDMs, expressed genes associated with monocyte-derived macrophages, such as Cd52, Plac8, Prdx5, and Hp. Cluster 3 macrophages expressed genes associated with dendritic cells, including Cd74, MHC II genes, and dendritic cell markers. Cluster 4 macrophages expressed genes associated with chromatin and cell cycle regulation, while cluster 5 macrophages expressed genes associated with interferon signaling. These distinct transcriptional profiles suggest the presence of different macrophage populations in skeletal muscle during muscular dystrophy.

Accepted Answer

The flow cytometry panel distinguishes macrophage populations based on the expression of galectin-3, Folr2, and CD52. Galectin-3, Folr2, and CD52 are used as markers to identify gal-3 + macrophages, SkMRMs, and MDMs. In healthy tissues, Folr2 + macrophages are most abundant in skeletal muscle, followed by the heart, while nearly absent in the brain, bone marrow, and blood. A gal-3 hi Folr2 lo macrophage population is identified in dystrophic muscle, corresponding to gal-3 + macrophages in scRNAseq analysis. CD52 is highly expressed in gal-3 -Folr2 -(WT) or gal-3 lo Folr2 -(mdx) macrophages, likely corresponding to MDMs in scRNAseq analysis. CD52 + monocytes or macrophages are present in all WT tissues, with the highest proportion in the blood, lung, liver, and adipose tissue. The flow cytometry panel discriminates three muscle macrophage populations based on galectin-3, Folr2, and CD52 expression. Bulk RNAseq was performed on FACS-sorted populations to link the flow cytometry macrophage populations to the scRNAseq profiles and establish their full transcriptomes. The top 100 scDEGs from each cluster were used to match transcriptomes to the sorted populations, confirming their match to the single cell populations. The gal-3 + scDEGs indicated partial overlapping profiles with the MDM scDEGs, suggesting a cell state transition. The gal-3 + population downregulated resident and monocyte-derived macrophage marker genes, suggesting a terminal transition state of either resident or monocyte-derived macrophages. Principal component analysis (PCA) and classification of DEGs using publicly available datasets further supported the distinct transcriptomes of SkMRMs, gal-3 + macrophages, and MDMs.

Accepted Answer

The largest variance (PC1) separated normal and dystrophic macrophages and contained genes enriched in the innate immune response. This suggests that gal-3 + macrophages exhibit enhanced phagocytosis of muscle debris and lipid membranes. The second largest (PC2) variation was enriched for genes in pathways associated with the lysosome and lipid metabolism. By directly comparing DEGs between the gal-3 + macrophages and SkMRMs, ECM and inflammation-related pathways were further identified as highly altered by the dystrophic milieu. Overall, the PCA helped classify the variation between sorted gal-3 hi macrophages, MDMs, and SkMRMs.

Accepted Answer

Galectin-3 + macrophages play a crucial role in degenerative lesions by interacting with stromal cells, such as FAPs, to promote fibrosis. Spatial transcriptomic analysis using Visium technology revealed that areas with high galectin-3 expression were confined to regions with active pathology and densely populated by mononuclear cells. The differential gene expression analysis showed that genes associated with phagocytosis, endocytosis, leukocyte migration, regeneration, repair, ECM, and fibrosis were enriched in galectin-3 high areas. Notably, genes encoding ECM components, growth factor pathways inducing fibrosis, and PDGFRa were also increased in these areas. Immunofluorescence assays further confirmed the juxtaposition of galectin-3 + macrophages with PDGFRa + stromal cells in pathological lesions, suggesting a functional and spatial interfacing between these cell types in the dystrophic environment.

Accepted Answer

The Spp1 pathway plays a crucial role in intercellular communication between FAPs and gal-3 + macrophages during muscular dystrophy. It is involved in promoting muscle fibrosis and regulating macrophage and FAP interactions. The Spp1 pathway is characterized by the highest relative contribution to the Spp1 pathway being attributed to the Spp1-CD44 ligand (L)-receptor pair, followed by several integrin heterodimer receptors. CD44, Itgav, and Itgb3 are increased in mdx stromal cells, indicating their involvement in the Spp1 pathway. The expression of Spp1 receptors in WT and mdx PDGFRa + Sca1 + stromal cells was measured by flow cytometry. Degenerative lesions with increased macrophages and collagen contain elevated levels of Spp1, suggesting its role in the communication between FAPs and gal-3 + macrophages in muscular dystrophy. Overall, the Spp1 pathway is a key signaling axis controlling the interactions between FAPs and gal-3 + macrophages during muscular dystrophy, with Spp1 signals primarily through CD44 and a subset of integrin heterodimers.

Accepted Answer

Muscle damage expands a population of galectin-3 macrophages that is chronically activated in muscular dystrophy. Galectin-3 protein is upregulated in dystrophic muscle macrophages as early as 4 weeks and remains elevated at 52 weeks of age. The chronic activation of gal-3 + macrophages is associated with increased collagen deposition at 52 weeks. The induction of a gal-3 + transcriptional program is conserved in other forms of muscle disease, such as limb girdle muscular dystrophy 2B and valosin-containing protein (VCP)-associated inclusion body myopathy (VIBM). Gal-3 + macrophages are also elevated in mouse models of Facioscapulohumeral Muscular Dystrophy (FSHD). The expansion of gal-3 + macrophages at day 5 after BaCl2-induced acute injury coincides with the transition of muscle towards the repair phase and increased collagen deposition, suggesting their involvement in remodeling of the extracellular matrix during muscle regeneration.

Accepted Answer

SkMRMs and monocytes differentiate into galectin-3 hi Folr2 lo macrophages through adoptive transfer assays. SkMRMs or bone marrow monocytes from CD45.1 + congenic WT mice are injected into the quadriceps of 4-wk-old mdx.CD45.2+ recipient mice. Prior to the adoptive transfer, bone marrow monocytes do not express Folr2 or galectin-3. However, after transfer, CD45.1 + transferred monocytes upregulate galectin-3 and Folr2, leading to an increased proportion of cells with the gal-3 hi Folr2 lo phenotype as early as day 2 and remain elevated 7 days after transfer. The adoptive transfer of CD45.1 + SkMRMs from WT muscle results in a downregulation of Folr2 and an upregulation of galectin-3, resembling the galectin-3 hi Folr2 lo phenotype by the second day after transfer. However, CD45.1 + SkMRMs decline substantially 7 days after transfer, suggesting that galectin-3 hi Folr2 lo emerging from the SkMRM pool are short-lived. The regulation of SkMRMs, MDMs, and gal-3 + macrophage marker genes in endogenous macrophages FACS-sorted from 4-wk-old WT and mdx muscle was assessed by RT-qPCR.

Accepted Answer

The genes preferentially expressed in gal-3 + macrophages, SkMRMs, and MDMs are spp1, Fabp5, Gpnmb, Folr2, Mt2, Lyve1, Plac8, Chil3, and Prdx5. These genes were interrogated due to their specific expression patterns in these cell types. The expression of SkMRM genes in Folr2 hi macrophages isolated from dystrophic muscle (mdx SkMRM) was observed to decrease, while gal-3 + macrophage genes were upregulated. Similarly, MDMs in dystrophic muscle showed an increase in gal-3 + macrophage gene expression, while MDM genes were lowly expressed in gal-3 + macrophages. This regulation of gene expression is consistent with adoptive transfer assays and supports the interpretation that SkMRM and recruited CD52 + monocytes are activated and differentiate into galectin-3 hi Folr2 lo macrophages within the dystrophic milieu.

Accepted Answer

To determine the presence of gal-3 + macrophages in human dystrophic muscle, researchers quantified their numbers in archived muscle biopsies. Immunofluorescence assays revealed that galectin-3 was expressed in a subset of CD68 + macrophages. Immunohistochemical examination showed that the number of gal-3 + macrophages in interstitial or perivascular regions did not differ significantly between control and myopathic patients, except for an increase in gal-3 + perivascular macrophages in LGMD2A. However, the number of gal-3 + myofiber-invading macrophages was significantly elevated in DMD, ASS, and LGMD2A. This suggests that gal-3 + macrophages are present in human dystrophic muscle and interact with FAPs.

Accepted Answer

Single-cell RNA sequencing (scRNAseq) is used in this study to define macrophage diversity in healthy and diseased muscle. By analyzing the gene expression profiles of individual macrophages, researchers can identify distinct populations and their characteristics. In healthy muscle, scRNAseq helps to establish a homeostatic signature of macrophages, which serves as a baseline for comparison. In diseased muscle, scRNAseq reveals alterations in macrophage populations, indicating a transition from a homeostatic state to a diseased state. This information is crucial for understanding the role of macrophages in muscle diseases and their potential as therapeutic targets.

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

Chat with Paper

AI Agents for this Paper

Most frequently asked questions

1. What are the distinct transcriptional profiles of macrophage clusters in skeletal muscle during muscular dystrophy?

2. How can flow cytometry panel distinguish macrophage populations?

3. What is the largest variance in PCA?

4. What is the role of galectin-3 + macrophages in degenerative lesions?

5. What is the role of Spp1 pathway in intercellular communication between FAPs and gal-3 + macrophages in muscular dystrophy?

6. How does muscle damage affect galectin-3 macrophages?

7. How do SkMRMs and monocytes differentiate into galectin-3 hi Folr2 lo macrophages?

8. What genes are preferentially expressed in gal-3 + macrophages, SkMRMs, and MDMs?

9. Are gal-3 + macrophages present in human dystrophic muscle?

10. How does single-cell RNA sequencing define macrophage diversity in healthy and diseased muscle?

Citations

Macrophage phenotypes and functions: resolving inflammation and restoring homeostasis.

Muscle inflammation is regulated by NF-κB from multiple cells to control distinct states of wasting in cancer cachexia

Bioactive Nanofiber‐hydrogel Composite Regulates Regenerative Microenvironment for Skeletal Muscle Regeneration After Volumetric Muscle Loss

Influences of Aged Bone Marrow Macrophages on Skeletal Health and Senescence.

Mapping Cell Atlases at the Single-Cell Level.

References

Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2

HTSeq—a Python framework to work with high-throughput sequencing data

TopHat: discovering splice junctions with RNA-Seq

Comprehensive Integration of Single-Cell Data.

The Molecular Signatures Database Hallmark Gene Set Collection

Related Papers (5)

Absence of a Calmitine-Specific Protease Inhibitor in Skeletal Muscle Mitochondria of Patients with Duchenne's Muscular Dystrophy

Serum Osteopontin as a Novel Biomarker for Muscle Regeneration in Duchenne Muscular Dystrophy

Jagged 1 Rescues the Duchenne Muscular Dystrophy Phenotype

ACE2 Is Augmented in Dystrophic Skeletal Muscle and Plays a Role in Decreasing Associated Fibrosis

A-utrophin up-regulation in mdx skeletal muscle is independent of regeneration