A discrete 'early-responder' stromal-cell subtype orchestrates immunocyte recruitment to injured tissue, thereby promoting regeneration [RNA-seq]

Following acute injury, stromal cells promote tissue regeneration by diverse mechanisms. Time-resolved single-cell RNA sequencing of muscle mesenchymal stromal cells (MmSCs) responding to cardiotoxin (CTX)-induced injury identified an “early-responder” subtype that spiked on day 1 and expressed a striking array of transcripts encoding immunomodulators. IL-1b, TNFa and oncostatin M, primarily produced by myeloid cells, each strongly and rapidly induced MmSCs transcribing most of these immunomodulators. 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 non-redundant impact on recruitment of neutrophils, a prolongation of inflammation at later times, and ineffectual tissue regeneration. MmSC 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” MmSCs, already in place, permit rapid and coordinated mobilization and amplification of critical cell collaborators during tissue regeneration. To study the impact of different cytokines on MmSCs in vivo, we intraperitoneally (i.p.) co-injected four cytokines (Il-1b, TNFa, oncostatin M, and Il-17A) into uninjured mice, and flow-cytometrically double-sorted MmSCs from the hindlimb muscles 2, 4 and 8 hrs later for population-level RNA-seq. 2 hrs was selected as the ideal timepoint. To deconvolute the significance of the individual cytokines, we repeated the experiment using the 2 hrs timepoint but injected cytokines individually and as pairs. In these experiments, MmSCs from mice i.p. injected with PBS was used as the control. To assess the transcriptional inductions in the major muscle-cell compartments with CTX-induced injury, we flow-cytometrically double-sorted CD31+, CD45+, MmSCs, and satellite cells (MuSCs) at homeostasis and 1 day after injury for population-level RNAseq. To validate that the IL-6 reporter/depleter system tagged MmSCs enriched for inflammatory transcripts, we flow-cytometrically double-sorted Thy1.1+ and Thy1.1- MmSCs 12 hrs following CTX-induced injury and performed population-level RNA-seq. To assess tissue-level transcriptional changes in muscle at 7 days following CTX-induced injury in mice depleted of Cxcl5 (via i.p. anti-Cxcl5 antibody injections), whole muscle RNA was isolated and submitted for RNAseq. Muscle tissue isolated at the same timepoint from mice i.p. injected with IgG was used as the control. To survey MmSCs at different disease stages of a muscular dystrophy mouse model, we flow-cytometrically double-sorted MmSCs from the hindlimb muscles of 2.5-, 7- and 12.5-wk-old mdx mice and performed population-level RNA-seq. To determine the importance of Il-1b in the induction of inflammatory transcripts in MmSCs in vivo, we flow-cytometrically double-sorted MmSCs from Il-1b KO or Il-1b WT littermates at 1 day after CTX-induced injury for population-level RNAseq.