Spatiotemporal transcriptomic mapping of regenerative inflammation in skeletal muscle reveals a dynamic multilayered tissue architecture
Andreas Patsalos, … , H. Lee Sweeney, Laszlo Nagy
Andreas Patsalos, … , H. Lee Sweeney, Laszlo Nagy
Published August 27, 2024
Citation Information: J Clin Invest. 2024;134(20):e173858. https://doi.org/10.1172/JCI173858.
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Research Article Inflammation

Spatiotemporal transcriptomic mapping of regenerative inflammation in skeletal muscle reveals a dynamic multilayered tissue architecture

Abstract

Tissue regeneration is orchestrated by macrophages that clear damaged cells and promote regenerative inflammation. How macrophages spatially adapt and diversify their functions to support the architectural requirements of actively regenerating tissue remains unknown. In this study, we reconstructed the dynamic trajectories of myeloid cells isolated from acutely injured and early stage dystrophic muscles. We identified divergent subsets of monocytes/macrophages and DCs and validated markers (e.g., glycoprotein NMB [GPNMB]) and transcriptional regulators associated with defined functional states. In dystrophic muscle, specialized repair-associated subsets exhibited distinct macrophage diversity and reduced DC heterogeneity. Integrating spatial transcriptomics analyses with immunofluorescence uncovered the ordered distribution of subpopulations and multilayered regenerative inflammation zones (RIZs) where distinct macrophage subsets are organized in functional zones around damaged myofibers supporting all phases of regeneration. Importantly, intermittent glucocorticoid treatment disrupted the RIZs. Our findings suggest that macrophage subtypes mediated the development of the highly ordered architecture of regenerative tissues, unveiling the principles of the structured yet dynamic nature of regenerative inflammation supporting effective tissue repair.

Authors

Andreas Patsalos, Laszlo Halasz, Darby Oleksak, Xiaoyan Wei, Gergely Nagy, Petros Tzerpos, Thomas Conrad, David W. Hammers, H. Lee Sweeney, Laszlo Nagy

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Figure 4

GPNMB is a marker and component of GFEMs, and its deficiency impairs regeneration.

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GPNMB is a marker and component of GFEMs, and its deficiency impairs reg...
(A) Upper: Brightfield images of day 4 after CTX Ly6CloF4/80hiGPNMB– and Ly6CloF4/80hiGPNMB+ muscle-infiltrating MFs ex vivo after 12 hours in culture (equal number of cells were seeded). Lower: apoptosis was assessed by cleaved caspase-3 immunostaining. Scale bars: 100 μm. (B) Percentage of apoptotic (CASP3+) Ly6CloF4/80hiGPNMB+ and Ly6CloF4/80hiGPNMB– MFs (unpaired t test with a P = 0.0007; n = 3). (C) Effect of GPNMB– and GPNMB+ MF-derived conditioned media on the proliferation and differentiation of C2C12 myoblasts. Scale bars: 100 μm. (D) Effect of GPNMB– and GPNMB+ MF-derived conditioned media on C2C12 myoblasts (n = 3). Proliferation index as percentage of Ki67+ cells (n = 10 fields/group; unpaired t test, P = 0.0008). Fusion index as percentage of myotubes (visualized by heavy chain of myosin II) with more than 3 nuclei (n = 10 fields/experiment/group; unpaired t test, P = 0.002). (E) Spatial cell proximity quantification of GFEMs (CD68+GPNMB+) versus other MF subtypes to regenerating eMyHC+ fibers at day 4 after CTX in C57BL/6J animals (n = 3; >200 mm2 tissue area/sample; unpaired t test, P = 0.0267). (F) Detection of GFEMs by CD68 and GPNMB, in relation to eMyHC+ fibers in C57BL/6J animals at day 4 after CTX injury. Insets indicate the split channels. Scale bars: 100 μm (left); 20 μm (insets). Lower panel indicates high-resolution volume projection confocal images (3D reconstruction distances are shown). (G) Left: H&E images of D2.Gpnmb– (KO) and D2.Gpnmb+ (WT) TAs at day 8 after CTX injury. Note the near complete absence of regenerating fibers (highlighted in white) and extensive inflammation areas (highlighted in red) in the D2.Gpnmb–. Right: IF detection of newly formed fibers by eMyHC in D2.Gpnmb– and D2.Gpnmb+ TAs at day 8 after CTX injury. Scale bars: 1 mm (left H&E); 500 μm (right H&E); 100 μm (IF panels). (H) IF detection of mature MFs by CD68 in KO and WT at day 8 after CTX injury, correlating to the extent of unresolved inflammation. Scale bars: 100 μm (main); 1 mm (insets). (I) H&E images of regenerating TAs (day 8 after CTX injury) from WT (C57BL/6J) and D2.Gpnmb– animals used for ST. Insets show magnified H&E areas (green rectangles). Scale bars: 500 μm (middle panels); 50 μm (far left and right panels). (J) Enhanced subspot resolution clustering of regenerating TAs (day 8 after CTX injury) from WT and D2.Gpnmb– identified 5 spatial clusters (n spots/group are indicated). Scale bars: 500 μm. (K) Top marker gene expression after z score transformation for each spatial cluster. Dot size represents the percentage of subspots expressing the gene. (L) Spatial expression of representative healthy muscle (Myh4), differentiating myoblasts (Myog), and persistent inflammation/mature MF (Cd68) genes in WT and D2.Gpnmb–. Note the loss of the distinct structure of regenerative zones in the KO. Scale bars: 500 μm. (M) Gpnmb and Myh3 spatial expression patterns in the C57BL/6J day 8 after CTX ST sample confirm the proximity of GPNMB+ MFs to early stage regenerating fibers and distinct tissue organization around lesions. Scale bars 500 μm (upper); 50 μm (lower). H&E previously presented in I. This duplication is intended to provide the location and context for the presented magnified feature plots. In all bar graphs, bars represent mean ± SD. *P < 0.05; **P < 0.01; ***P < 0.001.