Monocyte/Macrophage-derived IGF-1 Orchestrates Murine Skeletal Muscle Regeneration and Modulates Autocrine Polarization

Abstract

Insulin-like growth factor 1 (IGF-1) is a potent enhancer of tissue regeneration, and its overexpression in muscle injury leads to hastened resolution of the inflammatory phase. Here, we show that monocytes/macrophages constitute an important initial source of IGF-1 in muscle injury, as conditional deletion of the IGF-1 gene specifically in mouse myeloid cells (ϕIGF-1 CKO) blocked the normal surge of local IGF-1 in damaged muscle and significantly compromised regeneration. In injured muscle, Ly6C+ monocytes/macrophages and CD206+ macrophages expressed equivalent IGF-1 levels, which were transiently upregulated during transition from the inflammation to repair. In injured ϕIGF-1 CKO mouse muscle, accumulation of CD206+ macrophages was impaired, while an increase in Ly6C+ monocytes/macrophages was favored. Transcriptional profiling uncovered inflammatory skewing in ϕIGF-1 CKO macrophages, which failed to fully induce a reparative gene program in vitro or in vivo, revealing a novel autocrine role for IGF-1 in modulating murine macrophage phenotypes. These data establish local macrophage-derived IGF-1 as a key factor in inflammation resolution and macrophage polarization during muscle regeneration.

Figure 1

Structure of the rodent IGF-1 gene. The IGF-1 gene is comprised of six exons and five introns. Exons 3 and 4 encode the mature protein. Differential splicing on the 3′-end of the gene gives rise to a short Ea-peptide (dark gray box) when exon 5 is spliced out (35aa) and a longer Eb-peptide (light gray box), when exon 5 is included. IGF-1 propeptides contain either the Ea- or the Eb-peptide, which can be cleaved to produce the mature IGF-1 protein. Regions spanning exon 3 and exon 5 deleted in ϕIGF-1 CKO mice (ΔIGF-1) and IGF-1Eb KO (ΔIGF-1Eb), respectively, are indicated.

Figure 2

The surge in monocyte/macrophage IGF-1 production correlates with transition from inflammatory to repair macrophage phenotypes following muscle injury. (a) Kinetics of Ly6C+ monocyte/macrophage and CD206+ macrophage accumulation in the quadricep muscle is presented at various time points after CTX injury in WT muscle. (b) For analysis of monocyte/macrophage subpopulations, mononuclear cells were isolated from quadricep muscles and analyzed by flow cytometry. Monocytes/macrophages were defined as CD45+CD11b+F4/80+ as shown in the first two images, and then, the inflammatory monocyte/macrophages were discriminated by Ly6C expression while a mature reparative macrophage population was defined by CD206 expression. Isotype controls were used as negative gating controls to define positive signals and are shown in Supplementary Figure S2. (c) IGF-1Ea and IGF-1Eb propeptide expression was determined by qPCR of whole muscle (black bars) as well as Ly6C+ (red bars) and CD206+ (blue bars) macrophages isolated from CTX-injured muscle at 0, 2, 5, and 10 days after CTX injury. (d) MCSF expression measured by qPCR in whole muscle at 0, 2, 5, and 10 days after CTX injury. n = 7–8. Data represent mean ± SEM. CTX, cardiotoxin; WT, wild type.

Figure 3

Monocyte/macrophage-derived IGF-1 regulates muscle regeneration. (a) Myeloid deletion of IGF-1 was confirmed in ϕIGF-1 CKO mice by qPCR of monocytes and macrophages isolated from CTX-injured muscle as well as BMM cultures. Monocytes (CD45+CD11b+F480lo/-Ly6G-) and macrophages (CD45+CD11b+F480hi) isolated from injured ϕIGF-1 CKO muscle were compared to IGF-1^Fl/Fl controls, n = 4. BMM prepared from ϕIGF-1 CKO and IGF-1^Fl/Fl controls were stimulated with IL-4 to induce IGF-1 upregulation and mRNA levels measured after 12 hours (n =4). (b) Quadricep muscles of control IGF-1^Fl/Fl and ϕIGF-1 CKO mice were injured with CTX and analyzed for IGF-1 expression by qPCR 0, 2, 5, and 10 days after injury. Data represent mean ± SEM of four muscles. (c) Representative images of Trichrome-stained TA muscle sections from IGF-1^Fl/Fl and ϕIGF-1 CKO mice 0, 2, 5, and 10 days after CTX injury. Bar = 100 μm. (d–f) Quantification of regeneration in the TA at 5 and 10 days postinjury. (d) Regeneration parameters at 5 days after injury; the mean CSA of regenerating (centrally-nucleated) myofibers, the distribution plot of myofiber CSAs and number of fibers per unit area. (e) Myoblasts were isolated from injured muscle 5 days after injury using flow cytometry, identified as shown in Supplementary Figure S1a. The data are presented as the number of myoblasts per milligram of muscle in IGF-1^Fl/Fl and ϕIGF-1 CKO mice. (f) The mean CSA, CSA distribution plot, and number of fibers per square millimeter of section at 10 days after injury. At the very right, the number of nuclei per myofiber is shown for the 10-day time point. *P ≤ 0.05; **P ≤ 0.01 compared to IGF-1^Fl/Fl control by Mann–Whitney test. BMM, bone marrow–derived macrophages; CSA, cross-sectional area; CTX, cardiotoxin; Mϕ, macrophage; TA, tibialis anterior.

Figure 4

Impaired accumulation of CD206+ macrophages in injured ϕIGF-1 CKO mice. (a) Representation of the gating strategy used to identify monocytes (CD45+CD11b+Ly6G-Ly6Chi) and neutrophils (CD45+CD11b+Ly6G+). (b) Monocyte and neutrophil recruitment to quadricep muscles of ϕIGF-1 CKO mice, and IGF-1^Fl/Fl controls was measured 1 day after CTX injury. (c) Accumulation of macrophages (CD45+CD11b+F4/80+) was quantified at 2, 5, and 10 days. (d) Histogram showing F4/80 expression on ϕIGF-1 CKO (red line) and IGF-1^Fl/Fl control (blue line) macrophages (CD45+CD11b+F4/80+) isolated 10 days after injury or from uninjured muscle. (e) To follow changes in macrophage phenotypes, the proportion of Ly6C+ monocytes/macrophages and CD206+ macrophages were measured at 2, 5, and 10 days after CTX injury in ϕIGF-1 CKO and control muscle. (f) Accumulation of nonmyeloid hematopoietic cells (CD45+CD11b-) in injured muscle. (g) Viability of cells isolated from injured muscle showed was determined using a ViCell counter. Data represent mean ± SEM of 4–5 mice. *P ≤ 0.05; ** P ≤ 0.01 compared to IGF-1^Fl/Fl by Mann–Whitney test.

Figure 5

Dysregulated inflammatory gene expression in ϕIGF-1 CKO monocytes/macrophages. (a, b) Monocytes/macrophages were FACS isolated from injured ϕIGF-1 CKO muscle at days 2, 5, and 10 days after induction of CTX injury for gene expression analyses. (a) Inflammatory gene expression was measured in the cells at 2 and 5 days after injury corresponding to peak expression. (b) Expression of M2 genes was measured in isolated ϕIGF-1 CKO monocytes/macrophages 5 and 10 days after injury and compared to IGF-1^Fl/Fl controls. (c) BMM were prepared from ϕIGF-1 CKO and IGF-1^Fl/Fl and analyzed for gene expression changes by qPCR. For the measurement of inflammatory M1 genes, BMM were polarized with IFNγ/LPS. For the measurement of M2 genes, BMM were stimulated with IL-4 for 12 hours. Data represent mean ± SEM of six tests. For cell isolation experiments, n = 4; *P ≤ 0.05, **P ≤ 0.01 compared to IGF-1^Fl/Fl by Mann–Whitney test. BMM, bone marrow–derived macrophages.

Figure 6

Macrophage gene regulation by IGF-1 in vitro. (a) Bone marrow–derived macrophages were stimulated with mature, recombinant IGF-1 for 12 hours and expression of M2 genes assessed by qPCR. Treatment with IL-4 was performed for comparison. *P ≤ 0.05, for IGF-1-treated cells compared to unstimulated cells. (b) Macrophages M1 polarized with LPS and IFNγ were subsequently exposed to recombinant IGF-1 for 8 hours and inflammatory gene expression evaluated by qPCR, normalized to GAPDH. *P ≤ 0.05, for IGF-1/IFNγ-treated cells compared to IFNγ-treated cells. Data represent mean ± SEM of six tests.