Abstract
Following acute injury, stromal cells promote tissue regeneration by a diversity of mechanisms. Time-resolved single-cell RNA sequencing of muscle mesenchymal stromal cells (MmSCs) responding to acute injury identified an ‘early-responder’ subtype that spiked on day 1 and expressed a notable array of transcripts encoding immunomodulators. IL-1β, TNF-α and oncostatin M each strongly and rapidly induced MmSCs transcribing this immunomodulatory program. Macrophages amplified the program but were not strictly required for its induction. Transfer of the inflammatory MmSC subtype, tagged with a unique surface marker, into healthy hindlimb muscle induced inflammation primarily driven by neutrophils and macrophages. Among the abundant inflammatory transcripts produced by this subtype, Cxcl5 was stroma-specific and highly upregulated with injury. Depletion of this chemokine early after injury revealed a substantial impact on recruitment of neutrophils, a prolongation of inflammation to later times and an effect on tissue regeneration. Mesenchymal stromal cell subtypes expressing a comparable inflammatory program were found in a mouse model of muscular dystrophy and in several other tissues and pathologies in both mice and humans. These ‘early-responder’ mesenchymal stromal cells, already in place, permit rapid and coordinated mobilization and amplification of critical cell collaborators in response to injury.
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Data availability
New data generated in this paper were deposited in Gene Expression Omnibus database under accession no. GSE205738.
Code availability
No custom code was generated for this study. Algorithms used for data processing and analysis are referenced in the Methods.
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Acknowledgements
We thank T. Korn and S. Heink for the IL-6 reporter/depleter line; A. Muñoz-Rojas, G. Wang, T. Xiao, S. Galván-Peña, K. Hattori, A. Ortiz-Lopez, V. Piekarsa, N. Asinovski, F. Chen and S. Nepal for experimental assistance; K. Seddu, A. Baysoy, J. Lee, I. Magill and staff at the Broad Genomics Platform for RNA-seq; A. Muñoz-Rojas, L. Yang, B. Vijaykumar and N. Patel for computational help; C. Laplace for graphics; the Harvard Medical School (HMS) Immunology Flow Core; the HMS MiCRoN Core; the HMS Rodent Histopathology Core; and R.G. Spallanzani, A. Muñoz-Rojas, A. Mann, D. Owen, J. Leon and G. Wang for insightful discussions. This work was funded by National Institutes of Health (NIH) grant R01 AR070334 (D.M.), the JPB Foundation (D.M.) and NIH training grant T32GM007753 (O.K.Y., D.A.M. and T.J.). B.S.H. was partially supported by a Deutsche Forschungsgemeinschaft fellowship (HA 8510/1) and P.K.L. by F32 AG072874.
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O.K.Y., B.S.H. and D.M. conceptualized the study. O.K.Y., B.S.H., P.K.L., D.A.M. and M.M.R. designed and performed experiments. O.K.Y., B.S.H., P.K.L, D.A.M., T.J. and M.M.R. analyzed and interpreted data. O.K.Y., B.S.H. and D.M. wrote the paper, which all authors reviewed. D.M. and C.B. provided supervision. D.M. obtained funding.
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Extended data
Extended Data Fig. 1 Identification of an inflammatory MmSC subtype after acute skeletal-muscle injury.
a, Gating strategy for the cytofluorimetric sorting of MmSCs from hindlimb muscles. b, Heat map of the top 20 differentially expressed genes in MmSCs from D1 following CTX-induced injury comparing each cluster vis-à-vis all other clusters. c, Violin plot of the expression of the early signature (top 100 transcripts distinguishing early time points from the rest) across all MmSCs clusters on D1 following CTX-induced injury. d, Density plot of the expression of the indicated genes and gene signatures in MmSCs from D1 following CTX-induced injury. e, Violin plots of the expression of the signatures differentiating the four muscle stromal subtypes from Scott et al.44 across all MmSCs clusters on D1 following CTX-induced injury.
Extended Data Fig. 2 Role of IL-1β, TNFα, OSM in inducing MmSC inflammatory program.
a, Population-level RNA-seq of MmSCs isolated at 2 (n = 2), 4 (n = 1) or 8 (n = 2) hrs after ip co-injection of IL-1β, TNFα, OSM and IL-17A vs after PBS injection (n = 2). Expression levels across time points of the top 20 transcripts from the 100-gene inflammatory signature most differentially expressed. Y-axes plot values in arbitrary units. Each data point represents an individual mouse. b, Violin plots of Il1b, Tnf and Osm expression across cell populations in skeletal muscle on D0.5 after CTX-induced injury. scRNA-seq dataset as per16. Cell nomenclature as per original dataset. c, Gating strategy for the cytofluorimetric analysis of diverse immunocyte populations from hindlimb muscles. AU, arbitrary units; APC, antigen-presenting cells; FAPs, fibro/adipogenic progenitors; M1, inflammatory MFs; MuSC, muscle stem cells; PBS, phosphate-buffered saline; FC, fold change.
Extended Data Fig. 3 Impact of the loss of IL-1β, TNFα and OSM on MmSC inflammatory phenotypes.
a, Population-level RNA-seq of MmSCs isolated from Il1b knock-out (KO) (n = 3) and wild-type (WT) (n = 2) littermates at D1 following CTX-induced injury. Volcano plot comparison of the different conditions. The 100-gene inflammatory signature is shown in red, with numbers at the top indicating up- and downregulated transcripts (in comparison with total transcript numbers in black). b, Same as (a) except B6 mice were treated with a combination of IL-1β, TNFα and OSM neutralizing antibodies or IgG isotype controls. P determined by Chi-squared test.
Extended Data Fig. 4 Il6 and Cxcl5 expression in MmSCs at D1 and across skeletal muscle cell populations at homeostasis and various time points after injury.
a, Violin plot of Il6 expression across all MmSCs clusters on D1 following CTX-induced injury. b, Violin plot of Il6 expression across cell populations in skeletal muscle at homeostasis, as per16. c, Violin plot of Cxcl5 expression across all MmSCs clusters on D1 following CTX-induced injury. d, Violin plot of Cxcl5 expression across cell populations in skeletal muscle at homeostasis, as per16. M2, reparative MFs; Proliferating IC, proliferating immune cells; other abbreviations as per Extended Data Fig. 2. Cell nomenclature as per original dataset.
Extended Data Fig. 5 Expression of 100-gene inflammatory signature in immunocytes and MmSCs at homeostasis and upon acute injury.
Fold change vs fold change plot of population-level RNA-seq of CD45+ cells and MmSCs at D0 and D1 following CTX-induced injury (n = 3 per group). The 100-gene inflammatory signature is shown in red. Abbreviations as per Extended Data Fig. 1.
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Yaghi, O.K., Hanna, B.S., Langston, P.K. et al. A discrete ‘early-responder’ stromal-cell subtype orchestrates immunocyte recruitment to injured tissue. Nat Immunol 24, 2053–2067 (2023). https://doi.org/10.1038/s41590-023-01669-w
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DOI: https://doi.org/10.1038/s41590-023-01669-w