A special population of regulatory T cells potentiates muscle repair

Abstract

Long recognized to be potent suppressors of immune responses, Foxp3(+)CD4(+) regulatory T (Treg) cells are being rediscovered as regulators of nonimmunological processes. We describe a phenotypically and functionally distinct population of Treg cells that rapidly accumulated in the acutely injured skeletal muscle of mice, just as invading myeloid-lineage cells switched from a proinflammatory to a proregenerative state. A Treg population of similar phenotype accumulated in muscles of genetically dystrophic mice. Punctual depletion of Treg cells during the repair process prolonged the proinflammatory infiltrate and impaired muscle repair, while treatments that increased or decreased Treg activities diminished or enhanced (respectively) muscle damage in a dystrophy model. Muscle Treg cells expressed the growth factor Amphiregulin, which acted directly on muscle satellite cells in vitro and improved muscle repair in vivo. Thus, Treg cells and their products may provide new therapeutic opportunities for wound repair and muscular dystrophies.

Figure 1. Treg Accumulation at the Site of Injury

(A–C)Cytofluorometric analysesofhindlimb muscle infiltratesafter Ctx injury. (A) Top: Ly6c expression by CD11b⁺ cells. Middle and bottom: Foxp3 expression by CD4⁺ cells. Numbers depict % of gated cells. Representative of three experiments. (B and C) Summary data on fraction and number of cells. Mean ± SD (n ≥ 4). (D) Immunofluorescence microscopy of muscle sections 7 days after injury. The same section without (top) or with (bottom) DAPI. Arrows show Tregs in close contact with regenerating myofibers. Original magnification ×400. (E) As in (B), but after cryoinjury. See also Figure S1.

Figure 2. A Unique Population

(A–E) Gene-expression analyses on cells 14 days after Ctx injury. (A) Comparison plots of normalized expression values. Numbers indicate the number of genes whose expression differed by more than 2-fold. (B) PCA analysis comparing muscle Tregs 4 or 14 days after Ctx injury with other Treg populations. Each dot represents the meanofthree independentexperiments. (C) Top: volcano plot comparing gene expression ofmuscle versus spleen Tregs. Bottom: same plot after subtraction of the Treg activation signature (Hill et al., 2007). Numbers represent the number of differentially expressed genes (>2-fold). (D) Volcano plots comparing gene expression of Treg versus Tconv cells. Treg signature genes (Hill et al., 2007) are highlighted in red (induced) or blue (repressed). Numbers represent the number of genes from each signature expressed by each population. (E) Fold-change differences in gene expression between Treg and Tconv cells from muscle and spleen. Differentially expressed genes are highlighted in orange, gray, or pink. (F and G) Cytofluorometric analyses of surface and intracellular markers. Histograms depict expression by Foxp3⁺ T cells from muscle (red) or spleen (black). Gray: isotype control. See also Figure S2 and Table S1.

Figure 3. Clonal Expansion of the Treg Population at the Injury Site

(A and B) Ki67 expression (A) and EdU uptake (B) 4 days after Ctx-induced injury. Left: representative dot plotsofcells. Numbers refer to % marker⁺ Treg or Tconv cells. Right: summary plots. n = 5; **p < 0.01; ***p < 0.001. (C) TCR CDR3 α and CDR3 β sequences for individual cells. Each pie chart represents a single mouse. n, number of analyzed sequences per mouse. Total frequency of expanded clones shown at the top right. (D) Summary of clonal TCR frequencies over time. Open circles correspond to mice analyzed later in independent experiments for Figure 7. Mean ± SD (n ≥ 3). (E) Identical paired CDR3-α and β sequences found in multiple individuals in independent experiments. Two CDR3 α sequences from independent mice bear two conserved substitutions. See also Table S2.

Figure 4. Treg Ablation Impairs Muscle Regeneration after Wounding

Eight-week-old Foxp3⁻DTR⁺ or DTRT⁻ littermates were injured with Ctx and treated with DT to induce Treg depletion. (A and B) Flow cytometry of Treg and myeloid-lineage population, 7 days postinjury. Numbers in (A) refer to % CD4⁺ T cells that are Foxp3⁺, and numbers in (B) refer to % Ly6c^−/lo or Ly6c^hi of CD11b⁺ cells. (C) Hematoxylin and eosin (H&E) staining of muscle sections 7 days after Ctx injury. Representative of at least three experiments. Original magnification ×100. (D) Left: fibrosis detection by Gomori trichrome staining of muscle sections 13 days after injury. Collagen stained in blue. Representative examples of seven DTR⁺ and seven DTR⁻ mice. Original magnification ×200. Right: summary quantification. (E) Left: H&E staining of muscle sections 7 days after cryoinjury. Original magnification ×25 (left) and ×100 (middle and right). Representative examples of six samples. Right: quantification of regenerative fibers per mm² of injured area. ***p < 0.001. (F) Clonal myogenesis assays of satellite cells from uninjured or day 4 Ctx-injured muscles. Percent of wells seeded that showed a myogenic colony at day 5. Summary of six clonal assays *p < 0.05; **p < 0.01. (G) Left: FC/FC plot of gene expression values in muscle 4 versus 8 days after Ctx-induced injury in mice without (DTR⁺) or with (DTR⁻) Tregs. Highlighted in different colors are sets of genes described in the text. Right, representative examples of genes in each highlighted set. See also Figure S3 and Table S3.

Figure 5. Treg Cells Are Enriched in Muscles of Dystrophic Mice and Impact Muscle Damage

(A) Frequency of Foxp3⁺ cells for mdx and control C57Bl/10 mice. Left: representative dot plots. Right: summary data. n = 5; **p < 0.01; ***p < 0.001. (B) Treg clonal expansion in mdx muscle measured as described in the legend to Figure 3C. (C) Loss-of-function experiments. Mdx mice treated with anti-CD25 mAb (clone PC61) over 10 days. (D) Gain-of-function experiments. Mice treated with complexes of recombinant IL-2 and anti-iL-2 mAb (clone JES6-1A12). (C and D) Left: representative dot plot numbers refer to % of cells in the indicated gates in total CD4⁺ cells. Middle: summary data. Right: quantification of serum CKe. *p < 0.05; ***p < 0.001. See also Figure S4 and Table S2.

Figure 6. Areg Improves Muscle Repair after Injury

(A) Expression of Areg in ImmGen microarray data sets from hematopoietic-lineage cells. Mo, monocytes; MF, macrophages; DC, dendritic cells; GN, granulocytes; MDSC, myeloid-derived suppressor cells; AU, arbitrary units. (B) Expression of Areg 2 weeks after Ctx-induced injury. Numbers represent % Foxp3⁺ cells that are Areg⁺. (C) Volcano plots of transcriptomes of whole muscle from injured DTR⁺ (Treg-less) mice treated or not treated with Areg. Left: experimental protocol. Right: superimposition of transcript sets highlighted in Figure 4G and identically color-coded. Values refer to the number of genes upregulated (right) ordownregulated (left) in Areg-treated versus PBS-treated muscles. p values from a χ² test. (D) As in (C), except DTRT⁻ mice were used. (E) The same data set shown in (D) plotted as the fold-change for Areg- versus PBS-treated muscles versus the mean expression value for each transcript. Highlighted in orange and purple are genes up- ordownregulated by Areg, respectively. (F) Myogenic clonal assay in the presence of Areg. Results were plotted as the fold-change over the mean of vehicle-control treated samples per experiment. Data represent mean ± SD and five independent experiments. **p < 0.01. (G) Induction of satellite cells differentiation by Areg. Satellite cells were bulk-cultured in the presence of Areg or vehicle control for 12 days. Left: MyHC mRNA titered by quantitative PCR, represented as fold-change expression of Myh2 relative to vehicle control. Center: MyHC protein quantified by immunofluorescence microscopy; differentiation index calculated as the mean ratio of MyHC-positive nuclei to the total nuclei per field of view. Right: sum of DAPI-positive nuclei per well. Data represent mean ± SD and four experiments. *p < 0.05; ***p < 0.0001. See also Figure S5.

Figure 7. Sharing of TCR Sequences between Areg⁺ Splenic and Muscle-Infiltrating Treg Cells

(A) TCRCDR3 α and β sequences for individual Areg⁺ splenic Treg cells. Each pie chart represents a single mouse. n, number of analyzed sequences per mouse. Total frequency of expanded clones shown at the top right. (B) Identical paired CDR3 α (data not shown) and β sequences found in splenic Areg⁺ and muscle Areg⁺ and Areg⁻ Treg cells from the same individual in independent experiments. #: number of times the sequence was found. %: frequency of Tregs expressing the same sequence. (C) As in (A), for muscle Treg cells. Clones represented in Areg⁺, Areg⁻, or both subsets of muscle Tregs were labeled in red, blue, and green, respectively. See also Table S2.