Neuronal, stromal, and T-regulatory cell crosstalk in murine skeletal muscle

Abstract

A distinct population of Foxp3⁺CD4⁺ regulatory T (Treg) cells promotes repair of acutely or chronically injured skeletal muscle. The accumulation of these cells depends critically on interleukin (IL)-33 produced by local mesenchymal stromal cells (mSCs). An intriguing physical association among muscle nerves, IL-33⁺ mSCs, and Tregs has been reported, and invites a deeper exploration of this cell triumvirate. Here we evidence a striking proximity between IL-33⁺ muscle mSCs and both large-fiber nerve bundles and small-fiber sensory neurons; report that muscle mSCs transcribe an array of genes encoding neuropeptides, neuropeptide receptors, and other nerve-related proteins; define muscle mSC subtypes that express both IL-33 and the receptor for the calcitonin-gene-related peptide (CGRP); and demonstrate that up- or down-tuning of CGRP signals augments or diminishes, respectively, IL-33 production by muscle mSCs and later accumulation of muscle Tregs. Indeed, a single injection of CGRP induced much of the genetic program elicited in mSCs early after acute skeletal muscle injury. These findings highlight neural/stromal/immune-cell crosstalk in tissue repair, suggesting future therapeutic approaches.

Keywords: CGRP; IL-33; muscle repair; regulatory T cells; stromal cells.

Fig. 1.

Association among Tregs, IL-33⁺ cells, and peripheral nerves within mouse skeletal muscle. Immunofluorescence imaging of whole-mount tissue from hindlimb muscle. (A) Tregs and IL-33⁺ cells, 3 d post-CTX injury, at 10× magnification (Left), 60× (Middle), and 130× (Right). White arrows: Tregs and IL-33⁺ cells within contact distance. (B) Colocalization of IL-33⁺ cells with β3-tubulin⁺ nerve fibers in uninjured muscle (Left) and 3 d post-CTX injury (Right). (C) Minimum distances from either the centers of IL-33⁺ cells or random points to β3-tubulin⁺ area in uninjured muscle. Plotted as kernel density distribution from composite of four images. P value determined by Kolmogorov–Smirnov test. (D) Colocalization of IL-33⁺ cells with Nav1.8⁺ nerve fibers in uninjured muscle (Left), 3 d post-CTX injury (Middle), and 12 h post-CTX injury with additional staining of S100⁺ myelin sheath (Right). Pseudocoloring used for better visualization.

Fig. 2.

MmSCs express genes involved in neural signaling. (A–C) RNA-seq analysis of mSCs isolated from uninjured hindlimb muscle. Triplicate samples. Average expression of select genes encoding neuropeptides (A), neuropeptide receptors (B), and other nerve-related genes involved in neurotrophic signaling, neuromodulation, and axon guidance (C).

Fig. 3.

IL-33–producing MmSCs express the CGRP neuropeptide receptor complex. (A–G) Analysis on uninjured hindlimb muscle. (A) Distinct clusters of MmSCs from single-cell RNA sequencing. (B and C) Heat map representation of gene expression by cluster for Ly6a (B, Left); Pdgfra (B, Right); Il33 (C, Left); Calcrl (C, Middle); and Ramp1 (C, Right). (D) Ramp1 mean fluorescence intensity distribution compared with isotype control (IC) from flow cytometry of MmSCs. (E) Average expression of Il33, Calcrl, Ramp1, and Dpp4 (CD26) by cluster. (F) Ramp1 MFI distribution based on level of CD26 expression on MmSCs. (G) Immunofluorescence imaging of IL-33⁺ cells and CGRP⁺ nerve fibers.

Fig. 4.

CGRP affects IL-33 levels within skeletal muscle and results in Treg modulation. (A–F) Analysis on mice injected once i.p. with CGRP or PBS unless otherwise noted. (A) Muscle IL-33 levels as determined by ELISA. (B) IL-33⁺ mSCs in muscle by flow cytometry. (C) Volcano plot comparisons of mSCs after CGRP vs. PBS injection. Quadruplicate samples. G2M signature highlighted in red with numbers indicating up-regulated and down-regulated genes. Not shown: Gal (4, 8 h; 174, 119 FC), Cxcl5 (8 h; 178 FC). (D) Fold-change by fold-change plot comparing effects of CGRP injection at 8 h and muscle CTX-injury. Numbers indicate genes per quadrant. Not shown: Gal (119, 2 FC), Cxcl5 (178, 3777 FC). (E) Flow cytometry of Tregs after CGRP injection (i.p. every 8 h, for 2 d). (F) Flow cytometry of Tregs in uninjured muscle of nociceptor-ablated mice (Trpv1-Cre^+/−/Dta^+/−; Trpv1⁻) or littermate controls (Trpv1-Cre^−/−/Dta^+/−; Trpv1⁺). All P values represent the unpaired t test, with *P < 0.05; **P < 0.01; ***P < 0.001.