Uncovering Stromal Cell Functions in Acute and Chronic Muscle Injuries

Abstract

Skeletal muscle injuries are highly prevalent, yet current therapies are poor and often limited to symptom management. Inadequate muscle repair can lead to impaired mobility, not only affecting patients’ quality of life but also increasing their propensity for other medical sequelae. This clinical gap is partly because efficient muscle regeneration requires a tightly coordinated response by diverse cell types. Although substantial progress has been made to understanding the roles of certain skeletal muscle subpopulations, in comparison, the stromal compartment is relatively understudied. The overall objective of this thesis was to identify mediators by which stromal cells interact with other cell types in the muscle, with an emphasis on cells of the immune and nervous systems, and mechanistically understand how they contribute to tissue regeneration. Previously, IL-33+ cells in the muscle have been reported to be enriched around nerves and, therefore, can potentially interface with the peripheral nervous system. With closer investigation, we found that Il33-expressing muscle mesenchymal stromal cells (MmSCs) transcribe the genes encoding both subunits of the receptor for CGRP (calcitonin gene-related peptide), a neuropeptide involved in pain transmission, among other functions. Interestingly, intraperitoneal (i.p.) injection of CGRP led to increased IL-33 in the hindlimb muscle, revealing a CGRP:IL-33, nerve:stromal-cells axis. Foxp3+CD4+ regulatory T cells (Tregs), which promote repair of acutely or chronically injured skeletal muscle, depend critically on IL-33. Upon multiple i.p. CGRP injections, Tregs were found to be elevated in the skeletal muscle but not systemically. Conversely, a deficiency in TRPV1+ nociceptor neurons (and thereby CGRP+ neurons) resulted in decreased Tregs in the muscle. Together, these data revealed an interesting nerve:stromal-cell:immunocyte axis in the skeletal muscle and could suggest future therapeutic approaches. We were curious if this axis was also involved during muscle injury and, more broadly, the mechanisms by which stromal cells interact with other cellular compartments to promote muscle regeneration. Time-resolved single-cell RNA sequencing of 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-1beta, TNFalpha, 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. Together, these findings indicate crucial stromal-cell:immunocyte crosstalk in which “early-responder” MmSCs, already in place, permit rapid and coordinated mobilization and amplification of immunocytes, thereby helping facilitate tissue regeneration.