In vertebrates, activation of innate immunity is an early response to injury, implicating it in the regenerative process. However, the mechanisms by which innate signals might regulate stem cell functionality are unknown. Here, we demonstrate that type 2 innate immunity is required for regeneration of skeletal muscle after injury. Muscle damage results in rapid recruitment of eosinophils, which secrete IL-4 to activate the regenerative actions of muscle resident fibro/adipocyte progenitors (FAPs). In FAPs, IL-4/IL-13 signaling serves as a key switch to control their fate and functions. Activation of IL-4/IL-13 signaling promotes proliferation of FAPs to support myogenesis while inhibiting their differentiation into adipocytes. Surprisingly, type 2 cytokine signaling is also required in FAPs, but not in myeloid cells, for rapid clearance of necrotic debris, a process that is necessary for timely and complete regeneration of tissues.

In a number of species, tissue regeneration is associated with the presence of the molecular signature for type 2 innate immune response, such as alternatively activated (M2) macrophages and eosinophils (). This observation led us to postulate that signals—such as IL-4 and IL-13—that orchestrate type 2 innate immune responses might be good candidates for mediating the crosstalk between the immune system and skeletal muscle stem cells. Here, we report that muscle injury leads to the recruitment of IL-4 secreting eosinophils, which form an adaptive niche for proliferating stem cells in regenerating muscles. Loss of IL-4/IL-13 signaling or genetic absence of eosinophils severely compromises the ability of injured muscles to regenerate. Unexpectedly, we find that the regenerative effects of IL-4/IL-13 are not mediated by its signaling in myeloid cells but rather in FAPs. In FAPs, IL-4 acts as a molecular switch to control their fate between fibroblasts and adipocytes and to promote the clearance of necrotic debris. Consequently, global or cell-specific loss of IL-4/IL-13 signaling in FAPs severely impairs their functionality, resulting in persistence of necrotic debris and impairment in muscle regeneration.

Muscle injury results in rapid activation of the innate immune system, which exerts pleiotropic effects on regenerating muscle (). Within minutes of injury, neutrophils infiltrate injured skeletal muscle and release tissue-damaging reactive molecules, which exacerbate muscle damage (). This initial burst of collateral damage caused by the innate immune system is followed by a wave of reparative macrophages. For instance, it has been proposed that classically activated (M1) macrophages infiltrate early to facilitate the clearance of necrotic debris, whereas alternatively activated (M2) macrophages infiltrate later to assist with muscle growth (). In support of this idea, impairment in transcriptional programming of M2 macrophages, as in mice with reduced expression of C/EBPβ, results in smaller regenerated myofibers (), potentially reflecting the reduced secretion of myogenic growth factor IGF-1 by these cells (). Although these studies demonstrate a facilitative role of innate immune cells in muscle regrowth after injury, a direct molecular link between the innate immune system and muscle progenitor biology remains to be established.

In addition to satellite cells, recent studies have identified an important role for fibro/adipogenic progenitors (FAPs) in muscle regeneration and its fatty degeneration (). FAPs, which do not arise from the myogenic lineage, are bipotential cells capable of giving rise to fibroblasts and adipocytes. The close association of FAPs with regenerating muscle fibers, along with their expression of factors that influence myogenic differentiation, such as IL-6 and IGF-1, suggests that these stromal cells may play a supportive role in myogenic differentiation (). However, reflecting their adipogenic potential, FAPs can also give rise to ectopic adipocytes that accumulate in degenerating muscles (). Based on these findings, it has been postulated that factors that modulate the proliferation or differentiation of FAPs could potentially influence the muscle’s regenerative response to injury; however, none have been identified to date.

The regenerative response of skeletal muscle to injury is dependent on the quiescent population of skeletal muscle stem cells, termed the satellite cells, which reside beneath the basal lamina of each myofiber (). Upon injury, these quiescent satellite cells become activated and undergo proliferation, giving rise to myogenic progenitors (MPs) that ultimately differentiate into mature myofibers. In this context of injury and repair, a number of factors have been identified that promote proliferation and differentiation of MPs (). For instance, autocrine Notch signaling regulates the activation and proliferation of satellite cells (), whereas paracrine actions of interleukin (IL)-6 and insulin-like growth factors (IGFs) have been implicated in the differentiation of MPs into mature myotubes ().

Finally, we examined the role of type 2 innate signals in clearance of necrotic debris in injured muscle. Three days after muscle injury with CTX, mice were injected with CFSE-labeled necrotic thymocytes, and the presence of CFSE in FAPs was assessed 90 min later. This time point was chosen because CFSE signal in WT FAPs peaked at 60 min, followed by a decline over the next hour ( Figure S7 C). In mice impaired in type 2 innate signaling, such as ΔdblGATA and Il4rαmice, a higher percentage of FAPs were CFSE, suggesting delayed clearance of necrotic debris in these cells ( Figures 7 G, 7H, and S7 D). Consistent with this notion, administration of IL-4 into Il4/Il13was sufficient to enhance clearance of necrotic muscle fibers, as assessed by deposition of IgG in the necrotic myofibers or staining for calcified myofibers ( Figures 7 I and 7J).

To test this hypothesis, we examined whether PDGFRα is selectively expressed in FAPs of injured muscle. Analysis of Pdgfrαmice, which harbor a knockin of H2B-eGFP fusion gene into the endogenous Pdgfra locus (), showed that GFP was exclusively expressed in FAPs of regenerating muscles ( Figure S7 A). Based on these observations, we crossed Il4rαwith Pdgfrαmice to selectively delete Il-4rα in FAPs (designated Il4rαPdgfrα) (). Flow cytometric analysis of mononuclear cells isolated from regenerating muscles confirmed the absence of IL-4Rα protein in FAPs, but not in CD45or CD31cells ( Figure S7 B). Notably, after CTX-mediated injury, skeletal muscles of Il4rαPdgfrαmice recapitulated many of the histologic features observed in mice lacking type 2 immunity, including persistence of necrotic debris, larger areas of injury, and decreased formation of regenerative muscle fibers ( Figures 7 E and 7F). In aggregate, these findings suggest that regenerative actions of type 2 innate cytokines IL-4 and IL-13 are directly mediated via their actions in FAPs.

(B) Representative flow plots demonstrating the deletion of IL-4Rα in FAPs but not MPs, CD45 + or CD31 + cells isolated from regenerating muscles of Il4rα f/f and Il4rα f/f Pdgfrα Cre mice 3 days after injury with CTX.

We next asked whether FAPs were capable of phagocytizing necrotic cellular debris in the context of injured muscle, a setting in which macrophages, the professional phagocytes implicated in clearance of necrotic debris (), are abundantly present. For these experiments, CFSE-labeled necrotic thymocytes were injected into injured TA muscles, and uptake of necrotic debris by various cellular populations was quantified 1 hr later by flow cytometry. Surprisingly, compared to macrophages, FAPs were ∼4-fold more efficient at phagocytizing necrotic thymocytes ( Figures 7 C and S6 D–S6H). In contrast, no significant uptake of necrotic thymocytes was observed in MPs or CD31endothelial cells. Consistent with this observation, PDGFRαFAPs were observed circumscribing necrotic muscle fibers, which can be distinguished by nonspecific deposition of IgG ( Figure 7 D). Together, these data demonstrate that FAPs are capable of phagocytizing necrotic debris in injured muscle, leading us to postulate that they might be the primary cell type responsible for clearance of necrotic fibers in injured muscle.

Although FAPs can support myogenic differentiation, this observation is unlikely to explain the histologic defects in muscle regeneration observed in mice lacking type 2 innate immunity, including ΔdblGATA, Il4/Il13, and Il4rαmice. In these animals, a common histologic finding is the impaired clearance of necrotic muscle fibers, prompting us to examine whether FAPs might participate in the phagocytosis of necrotic debris. To investigate this idea, we purified FAPs from injured muscles of WT mice on day 3 and fed them CFSE-labeled live, necrotic, apoptotic, or opsonized thymocytes (). Remarkably, FAPs were very efficient at phagocytizing necrotic thymocytes, as assessed by the percentage of FAPs that were positive for CFSE and their mean fluorescence intensity ( Figures 7 A, S6 A, and S6B). This response was specific for necrotic thymocytes because live, apoptotic, and opsonized thymocytes were not phagocytized at any appreciable level by FAPs ( Figures 7 A, S6 A, and S6B). Moreover, confocal microscopy revealed that FAPs, which uniformly express PDGFRα (), had indeed engulfed the necrotic thymocytes ( Figures 7 B and S6 C).

(A) Efficiency of FAPs to phagocytize live (LTs), necrotic (NTs), apoptotic (ATs), and opsonized (OTs) thymocytes in vitro. Mean fluorescence intensity (MFI) of CSFE + FAPs is plotted for the various targets, n = 3 per treatment.

(C and D) FAPs are efficient at phagocytizing necrotic debris in vivo. (C) Three days after CTX-induced injury, TA muscles were injected with CFSE-labeled necrotic thymocytes, and phagocytosis was enumerated 1 hr later. Data are plotted as percent uptake in the indicated cellular population; n = 3. (D) Confocal microscopy of FAPs in regenerating muscle (magnification, 600×). Red, PDGFRα; green, IgG.

FAPs are bipotential cells capable of facilitating skeletal muscle regeneration or contributing to its fatty degeneration (). Because IL-4 serves as a molecular switch for controlling the fate of FAPs in regenerating muscle, we postulated that administration of IL-4 might prevent fatty degeneration of injured skeletal muscle. To test this hypothesis, we injected glycerol into the TA muscles of WT mice to induce fatty degeneration of skeletal muscle (). As reported, glycerol-injected TA muscles were infiltrated by oil-red-O-positive cells (), which stained positive for the adipocyte lipid droplet protein perilipin ( Figures 6 E and S5 D). In contrast, fatty infiltration and perilipin staining was dramatically reduced in TA muscles of all mice (n = 6) that received intraperitoneal injections of IL-4 ( Figures 6 E, 6F, and S5 D). Paralleling the decrease in fatty infiltration, expression of adipocyte-specific genes, such as leptin (Lep) and adiponectin (Adipoq), was reduced by ∼65% in TA muscles of mice treated with IL-4 ( Figure 6 G).

Because IL-4 signaling inhibits adipogenic differentiation of FAPs, we tested whether genetic disruption of IL-4/IL-13 signaling results in fatty infiltration of regenerating muscle. In agreement with published reports (), we failed to detect a significant number of adipocytes in regenerating muscles of WT mice ( Figure 6 D). In contrast, clusters of oil-red-O-positive adipocytes were readily detected in regenerating muscles of Il4/Il13and Stat6mice ( Figure 6 D).

Because IL-4 suppressed spontaneous differentiation of FAPs into adipocytes, we tested whether it could also inhibit adipogenic conversion of FAPs by insulin. As assessed by microscopy and staining for oil red O, treatment with IL-4 potently suppressed insulin-induced differentiation of FAPs into adipocytes ( Figures 6 B and S5 B). This was further affirmed by expression analysis, which revealed a strong repressive effect of IL-4 on expression of adipogenic genes in WT FAPs ( Figure S5 C). Importantly, signaling via IL-4Rα and STAT6 was required because no significant inhibition of adipogenesis was observed in Il4rαor Stat6FAPs ( Figures 6 B and S5 C). Congruent with these observations, treatment with IL-4 repressed expression of adipogenic proteins, such as PPARγ, Acc1, and HSL, by insulin ( Figure 6 C).

FAPs isolated from injured muscle can be induced to differentiate into mature adipocytes in culture (). Because a large number of genes involved in triglyceride synthesis and adipogenesis were repressed by IL-4 in FAPs ( Tables S2 and S4 ), we tested whether IL-4 could prevent differentiation of FAPs into adipocytes. As shown in Figure S5 A, FAPs cultured in growth medium spontaneously differentiated into lipid-laden adipocytes, a process that was completely inhibited by IL-4. Moreover, qRT-PCR analysis revealed that IL-4 suppressed the expression of a number of genes that are normally induced during adipogenesis (), including PPARγ, Lep, Fabp4, Acaca, Cd36, and Dgat2 ( Figure 6 A).

(E) TA muscles were analyzed on day 8 after glycerol injection by oil red O staining. After initiating glycerol-induced muscle damage, mice were injected with vehicle or IL-4 complex on days 1 and 4; n = 4–6 per treatment. Representative sections stained for oil red O are shown.

(C) Quantitative RT-PCR analysis of adipogenic mRNAs in WT and Il4rα −/− FAPs treated with insulin or insulin plus IL-4, n = 3 per genotype and treatment.

In coculture systems, FAPs can promote differentiation of MPs into myofibers (). Therefore, we tested whether stimulation of FAPs with IL-4 might enhance their capacity to support myogenic differentiation. WT MPs were cultured with FAPs-conditioned medium, and myogenic gene expression was measured 5 days later. Treatment of MPs with conditioned medium taken from IL-4 stimulated WT FAPS, but not from Il4rαFAPs, enhanced myogenic differentiation, as evidenced by increased expression of myogenic transcription factors and structural proteins ( Figures 5 G and S4 E). Because conditioned medium taken from IL-4-stimulated FAPs did not stimulate proliferation of MPs in single myofiber preparations ( Figure S4 F), it suggests that factors derived from FAPs likely work in trans to enhance terminal differentiation of myoblasts.

In order to understand how IL-4/STAT6 transcriptional axis regulates proliferation of FAPs, we performed microarray analyses from WT and Il4rαFAPs stimulated with IL-4 for 24 hr. Pathway analysis of differentially expressed genes revealed that gene ontology (GO) terms associated with DNA replication, cell cycle, and mitosis were highly enriched in the upregulated gene set, whereas those associated with triglyceride metabolism and lipid biogenesis were significantly downregulated in WT FAPs treated with IL-4 ( Tables S2 S3 , and S4 ). qRT-PCR analyses further verified that IL-4 induced the expression of a number of cell cycle genes, including Cdc20, Ccnd1, Cdk1, Cdc7, Cdca3, and Cenpa, in WT, but not in Il4rαor Stat6FAPs ( Figure 5 E). In aggregate, these data demonstrate that mitogenic actions of IL-4 enhance the proliferation of FAPs by promoting their entry and progression through the cell cycle.

We next examined which pathway was required for IL-4-induced proliferation of FAPs. Consistent with the loss of IL-4 signaling, IL-4 failed to increase cell number or BrdU incorporation in Il4rαFAPs ( Figures 5 B and 5C). Likewise, Stat6FAPs failed to proliferate in response to IL-4, as quantified by the change in cell number and the rate of BrdU incorporation ( Figures 5 B and 5D). In contrast, treatment of FAPs with PI3K or MEK1 inhibitors did not significantly alter the stimulatory effects of IL-4 on BrdU incorporation ( Figure S4 D). Together, these findings suggest that STAT6 predominantly mediates the mitogenic effects of IL-4 in FAPs.

We purified FAPs from CTX-injured muscles (day 1) using the Miltenyi MACS purification system in order to study the signaling pathways activated by IL-4 in these cells. This method of negative selection allowed us to isolate FAPs that were ≥98% pure; i.e., CD31CD45α7-integrinSCA-1 Figure S4 A). Similar to what was observed in vivo, stimulation of WT FAPs with IL-4 induced cell proliferation, as evinced by the increase in cell number (∼2-fold at 48 hr and ∼4-fold at 72 hr, Figure S4 B). To elucidate the pathways that mediate the mitogenic effects of IL-4, we investigated the downstream signaling pathways that are activated by IL-4 in FAPs. Stimulation with IL-4 activated three major signaling pathways in FAPs: (1) signal transducer of transcription 6 (STAT6), (2) the protein kinase Akt, and (weakly) (3) the extracellular signal-regulated protein kinases (ERKs) ( Figure 5 A). As expected, inhibition of PI3K by LY294002 prevented serine phosphorylation of Akt, whereas the MEK1 inhibitor PD98059 potently inhibited phosphorylation of ERK1/2 ( Figure S4 C). Moreover, consistent with the direct tyrosine phosphorylation of STAT6 proteins by Janus kinases (), treatment with PI3K or MEK1 inhibitors did not alter the phosphorylation status of STAT6 ( Figure S4 C). Lastly, activation of these three signaling pathways in FAPs showed an absolute dependence on IL-4Rα, as their phosphorylation and activation were abolished in Il4rαFAPs ( Figure 5 A).

(G) Expression of myogenic genes in WT MPs cultured with FAPs conditioned media. FAPs were stimulated with vehicle or IL-4 for 72 hr prior to collection of conditioned media; n = 3 per treatment.

(F) qRT-PCR analysis of cell cycle genes in WT, Il4rα −/− , and Stat6 −/− FAPs after stimulation with vehicle or IL-4 for 24 hr; n = 4 per genotype and treatment.

(E) Heatmap of differentially expressed genes in WT and Il4rα −/− FAPs treated with vehicle or IL-4 for 24 hr; red, induced; blue, repressed. GO terms associated with DNA replication, cell cycle, and mitosis are enriched in the upregulated gene set, whereas those associated with triglyceride metabolism and lipid biogenesis are enriched in the downregulated gene set.

(B) Quantification of cell number after stimulation of WT, Il4rα −/− , and Stat6 −/− FAPs with vehicle or IL-4 for 48 hr; n = 4 per genotype and time point. For each genotype, cell number is normalized to its vehicle control.

(F) Quantification of satellite cells in single fibers exposed to FAPs conditioned media. Wild-type muscle single fibers were cultured with conditioned media from WT or Il4rα −/− FAPs for 3 days, and MyoD + satellite cells were enumerated.

(E) Quantitative RT-PCR analysis of myogenic genes. Wild-type MPs were cultured with conditioned media from WT or Il4rα −/− FAPs for 5 days, and gene expression was quantified in differentiating MPs, n = 3/genotype and treatment. ∗ p < 0.05.

(D) BrdU incorporation in WT FAPs after inhibition of PI3K or MEK1. FAPs were stimulated PBS or IL-4 (10nM) for 24 hr in the presence or absence of inhibitors for PI3K (LY294002 (30 μM)) or MEK1 (PD98059 (30 μM)). Cells were pulsed with BrdU for 30 min prior to harvest for analysis, n = 3 per treatment. Data are normalized to the PBS sample in each treatment group.

(C) Activation of signaling pathways by IL-4 in WT FAPs. Cells were stimulated with IL-4 (10nM) for 15 min, and cell lysates were analyzed for the indicated proteins by immunoblotting. PI3K was inhibited by LY294002 (30 μM) and PD98059 (30 μM) was used to inhibit MEK1.

(B) Quantification of cell number after stimulation of WT FAPs with vehicle or IL-4 (10nM) for 48 or 72 hr, n = 4 per treatment and time point.

(A) Flow cytometric analysis of FAPs before and after purification with Miltenyi MACS purification system. FAPs were negatively selected using CD31, CD45, and α7 integrin antibodies. Purified FAPs are SCA1 + and CD45 − CD31 − .

Previous studies have implicated IL-4 signaling in myoblast fusion and muscle fiber growth (), prompting us to examine the role of IL-4Rα in regenerating muscle fibers. Western blot analysis of purified FAPs and MPs revealed robust expression of IL-4Rα and PDGFRα in FAPs, but not in MPs ( Figure S3 F). In contrast to MPs, IL-4Rα protein was detectable in MP-derived myotubes, which displayed fused morphology and higher expression of desmin ( Figure S3 F). This observation prompted us to generate mice (designated Il4rαPax7) in which IL-4Rα could be deleted in Pax7satellite cells in a tamoxifen-inducible manner (). As quantified by quantitative PCR (qPCR), administration of tamoxifen for 5 days induced >90% recombination at the Il4rα locus ( Figure S3 G). Because Pax7satellite cells give rise to all regenerating myofibers (), we investigated the requirement for IL-4Rα in satellite cell-derived myofiber regrowth. Notably, deletion of IL-4Rα in satellite cells did not impair the muscle’s regenerative response to injury by CTX, as assessed by gross and microscopic histology ( Figures 4 G and 4H). In agreement with these observations, we failed to detect expression of IL-4Rα in regenerating myofibers in vivo ( Figure S3 H).

Muscle injury also leads to proliferation of MPs, which are required for myogenesis (). We tested whether absence of eosinophils or loss of IL-4/IL-13 signaling decreased the proliferation of MPs. In contrast to FAPs, the rate of BrdU incorporation in MPs was not significantly different among WT, Il4/Il13, and Il4rαmice ( Figures 4 D, 4E, and S3 B). However, we did observe a modest decrease in the proliferation rate of MPs in eosinophil-deficient ΔdblGATA mice ( Figure 4 F). These data are consistent with the observation that IL-4Rα is not expressed on MPs at any stage of muscle regeneration ( Figure 3 B), thereby suggesting that FAPs are likely the primary target of IL-4/IL-13 signaling in regenerating muscle. Congruent with this idea, we failed to detect significant differences in the proliferation rates of FAPs or MPs in Il4rαand Il4rαLysMmice ( Figures S3 D and S3E).

FAPs, which are present in healthy muscle, undergo rapid proliferation after muscle injury to support myogenic differentiation (). Because IL-4Rα is highly expressed in FAPs ( Figure 3 B), we hypothesized that IL-4/IL-13 might regulate proliferation or differentiation of FAPs. To investigate this postulate, we utilized flow cytometry to identify FAPs and MPs on day 1 after injury and followed their proliferation using 5-bromodeoxyuridine (BrdU) incorporation. Strikingly, absence of IL-4/IL-13 or IL-4Rα decreased BrdU incorporation in FAPs by ∼60% ( Figures 4 A, 4B, and S3 A) and reduced the FAP content of regenerating muscles on day 3 by ∼40%–45% ( Figure S3 C). Albeit to a slightly lower degree, BrdU incorporation was also reduced in FAPs of ΔdblGATA mice (∼40%), which lack IL-4-expressing eosinophils in their regenerating muscles ( Figure 4 C).

(H) Immunostaining for IL-4Rα in regenerating muscle. Sections of WT and Il4rα −/− TA muscles were stained for IL-4Rα. Note expression of IL-4Rα is primarily detected in mononuclear cells 8 days after injury with CTX (magnification, X200).

(D and E) Quantification of BrdU incorporation in FAPs (C) and MPs (D) of Il4rα f/f and Il4rα f/f LysM Cre mice 24 hr after muscle injury, n = 4 per genotype.

(G and H) Signaling via IL-4Rα in satellite cells is dispensable for muscle regrowth after injury. (G) Representative day 8 muscle sections from Il4rα f/f and Il4rα f/f Pax7 CreERT2 mice stained with hematoxylin and eosin; n = 6 per genotype (magnification, 200×). (H) Gross appearance of Il4rα f/f and Il4rα f/f Pax7 CreERT2 TA muscles 8 days after injury with CTX.

We tested whether alternatively activated macrophages are required for muscle regeneration using control (Il4rα) and myeloid-cell-specific knockouts of the IL-4Rα (Il4rαLysM). To our surprise, unlike the Il4/Il13and IL-4Rαmice, myeloid cell deletion of Il4rα ( Figure S2 H) did not result in a strong defect in skeletal muscle regeneration ( Figures 3 G–3I). These findings suggest that myeloid cells are not the primary targets for the regenerative effects of IL-4/IL-13 during muscle injury.

First, we examined the time course of macrophage recruitment into injured muscle. In contrast to the previous report (), quantitative RT-PCR analysis of whole TA muscles revealed that expression of messenger RNAs (mRNAs) encoding classical (Nos2, Il6, and Tnfa) and alternative activation (Arg1 and Ym1) genes was rapidly induced during the first two days of muscle injury ( Figures 3 C, 3D, and S2 C–S2F). To exclude the possibility that markers of classical and alternative activation might be concurrently expressed in the same cells, we analyzed expression of arginase 1 (Arg1) and inducible nitric oxide synthase (Nos2) by immunostaining. Arg1 expression was primarily restricted to cells that express the macrophage marker Cd68 ( Figure S2 G). Furthermore, alternative (Arg1) and classical (Nos2) activation markers were expressed in distinct macrophages ( Figure 3 E), which is indicative of concurrent infiltration of damaged muscle by both types of macrophages. This observation was independently verified using YARG mice (), which have a fluorescent reporter introduced into the Arg1 gene ( Figure 3 F). In aggregate, these data demonstrate that muscle injury leads to rapid recruitment of both alternatively (M2) and classically (M1) activated macrophages.

An early event that occurs after muscle injury is the recruitment of macrophages, which exert pleiotropic effects on muscle regeneration (). Classically activated (also referred to as M1) macrophages infiltrate damaged muscle within the first 2 days and have been implicated in the clearance of cellular debris and stimulation of myogenesis. In contrast, alternatively activated (also referred to as M2) macrophages, whose numbers increase during later stages of muscle regeneration (days 4–10), are implicated in the promotion of muscle growth via secretion of paracrine factors. Because IL-4, the dominant regulator of alternative macrophage activation (), is expressed during the early phase of muscle injury ( Figures 2 A and S2 B), we postulated that the observed defects in muscle regeneration might stem from impairment in alternative macrophage activation.

To elucidate the target cells that respond to IL-4/IL-13 in injured muscles, we profiled the expression of IL-4Rα on various cell types that participate in the repair of damaged skeletal muscle. For these studies, we utilized flow cytometry and established gating strategies to identify myogenic progenitors (MPs), FAPs, and myeloid cells ( Figure S2 A) and then evaluated the expression of IL-4Rα on each of these populations. Figure 3 A shows that IL-4Rα is specifically expressed on WT FAPs and myeloid cells and is largely absent from the cell surface of MPs. Because stimulation with IL-4/IL-13 induces the expression of IL-4Rα (), the relative change in its expression serves as a good indicator of target cell responsiveness to these cytokines. Indeed, temporal profiling revealed that IL-4Rα expression was rapidly induced on the surface of FAPs and CD45cells, but not MPs, after injury ( Figure 3 B). This observed increase in IL-4Rα expression coincided with the presence of IL-4-expressing eosinophils in injured muscle ( Figure 2 A), implying that the primary regenerative actions of these cytokines might be restricted to this early time frame after injury.

(H) Flow cytometric analysis of IL-4Rα expression on macrophages infiltrating injured muscle. Mononuclear cells were isolated on day 3 after injury from the TA muscles of Il4rα f/f , Il4rα f/f LysM Cre , and Il4rα −/− mice, and IL-4Rα expression was analyzed on infiltrating macrophages.

(G) Localization of Arg1 positive cells in day 2 regenerating muscle. TA muscle sections were stained for Arg1, a marker of alternative activation, and CD68, a pan-marker of macrophages (magnification, X400). Note, nearly all of the Arg1 + cells co-localize with cells staining positively for CD68 + .

(C–F) Quantitative RT-PCR analysis for macrophage markers during the time course of muscle regeneration, n = 4 per time point. Chi3l3 is a marker of alternatively activated (M2) macrophages; Emr1 encodes the pan macrophage marker F4/80; Il6 and Tnfα are markers of classically activated (M1) macrophages. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001.

(A) Isolated mononuclear cells were gated for live/dead and doublets prior to analysis of myeloid cells, FAPs and MPs. FAPs were defined as SCA1 + α7 inegrin − CD45 − CD31 − ; MPs as SCA1 − α7 inegrin + CD45 − CD31 − ; and myeloid cells as CD45 + CD11b + .

Because eosinophils were the dominant cell type secreting IL-4 in injured muscles, we next investigated their involvement in muscle’s regenerative response using eosinophil-deficient ΔdblGATA mice (). Flow cytometric analyses of 4get-ΔdblGATA mice revealed a near complete absence (>93% reduction) of infiltrating eosinophils in injured TA muscles ( Figure 2 C). Moreover, the absence of eosinophils reduced the total number of GFPcells by ∼75% ( Figure S1 D), decreased the number of IL-4-expressing eosinophils by ∼30-fold ( Figure 2 D), and completely abolished the expression of IL-4 in mononuclear cells derived from ΔdblGATA mice ( Figure S1 E). Consequently, 4get-ΔdblGATA mice were unable to regenerate their injured muscles, as assessed by Evans blue staining of their TA muscles ( Figure 2 E). Paralleling the changes in the gross phenotype, hematoxylin and eosin staining of tissue sections revealed a paucity of centrally nucleated regenerating myofibers ( Figure 2 F), a finding that was confirmed by immunostaining for desmin ( Figure 2 G). These results suggest that eosinophil-derived factors, such as IL-4, are essential for orchestrating skeletal muscle regeneration.

To prospectively identify the cellular source of IL-4 in regenerating muscles, we utilized 4get reporter mice, which express green fluorescent protein (GFP) from the 3′ UTR of the endogenous Il4 gene (). GFPcells, which were readily identified throughout the time course of muscle regeneration ( Figure 2 A and Figure S1 A), were primarily of hematopoietic origin, as they coexpressed CD45 ( Figure S1 B). To characterize the identity of these IL-4-expressing cells, we analyzed the expression of markers for eosinophils (Siglec FCD11b), basophils (FceRICD117), mast cells (FceRICD117), and Th2 cells (CD3CD4) in the GFPpopulation that infiltrated injured muscles of 4get mice. Notably, ∼85%–90% of GFPcells were eosinophils, whereas the remainder were mast cells ( Figure 2 B). Consistent with this, expression of chemokines and cytokines involved in chemotaxis of eosinophils was induced in CTX-injured muscles ( Figure S1 C).

(E) Intracellular staining for IL-4 in WT, ΔdblGATA, and Il4/Il13 −/− mice. Mononuclear cells were isolated from injured muscle on day 2 and cultured in the presence of Brefeldin A for 18 hr prior to staining for IL-4.

(C) Expression of various chemokines and cytokines that have been implicated in the recruitment of eosinophils and macrophages in regenerating muscle, n = 4-6 per time point. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001.

(B) Presence of GFP+ cells in uninjured and injured muscles of 4get mice. CTX-injured muscles were digested on various days and mononuclear cells were isolated for flow cytometric analysis. Note that all GFP+ cells are of hematopoietic origin (CD45 + ).

(A) Detection of IL-4 expressing cells in regenerating muscle. TA muscles from 4get mice were stained for GFP on various days after injury with CTX (magnification, X100). Note that expression of IL-4 is not detected in regenerating myofibers but rather in mononuclear cells.

(B) Flow cytometric gating strategy for identification of IL-4-expressing cells in injured muscles. Eosinophils were defined as being CD11b + and Siglec F + , whereas mast cells were CD117 + and FcεRI + .

Two distinct receptors (IL-4Rα/γc and IL-4Rα/IL-13Rα1) transduce the biologic effects of IL-4 and IL-13 in cells (). Using Il4rαmice, we next investigated whether the canonical IL-4/IL-13 signaling pathways mediate the regenerative responses in injured muscles. Both at a gross and microscopic level, Il4rαmice exhibited similar defects in skeletal muscle regeneration as Il4/Il13mice ( Figures 1 D and 1E). Moreover, immunofluorescence staining for desmin demonstrated an absence of centrally nucleated fibers in the regenerating TA muscles of Il4rαmice ( Figure 1 F). These findings provide strong genetic evidence that the IL-4/IL-13 immune signals play a central role in skeletal muscle regeneration and raise three important questions: (1) what is the cellular source of IL-4 during muscle regeneration, (2) what cells respond to IL-4/IL-13 in regenerating muscle, and (3) how do IL-4/IL-13 promote muscle regeneration?

To investigate the functions of IL-4/IL-13 in muscle regeneration, we used cardiotoxin (CTX) to acutely injure the tibialis anterior (TA) muscles of mice. CTX administration induces local muscle necrosis, which is rapidly followed by recruitment of inflammatory cells, clearance of cellular debris, and regeneration of injured muscle (). To evaluate the overall efficacy of the regenerative response, we gave mice an intraperitoneal injection of Evans blue dye, which accumulates in damaged muscle fibers ( Figure 1 ). Wild-type (WT) mice exhibited a robust regenerative response, resulting in the restoration of intact, regenerating fibers, as evidenced by the absence of Evans blue staining of TA muscles ( Figure 1 A). In striking contrast, Evans blue dye uptake was markedly higher in TA muscles of Il4/Il13mice, which is indicative of a failure of the regenerative response to restore mature, intact myofibers ( Figure 1 A). In agreement with this interpretation, histological examination revealed that TA muscles of WT mice contained centrally nucleated regenerative myofibers, which were largely absent from those of Il4/Il13mice ( Figure 1 B). Instead, cellular debris and inflammatory infiltrate persisted in the injured TA muscles of Il4/Il13mice ( Figure 1 B). Moreover, immunostaining for desmin, a marker of mature myofibers, showed a near complete absence of regenerated muscle fibers in Il4/Il13mice ( Figure 1 C). This impairment in muscle regeneration did not result from altered trafficking of immune cells into injured muscles of Il4/Il13mice ( Table S1 available online).

Discussion

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So L.

Wang J.

Rudnicki M.A.

Rossi F.M. Muscle injury activates resident fibro/adipogenic progenitors that facilitate myogenesis. Uezumi et al., 2010 Uezumi A.

Fukada S.

Yamamoto N.

Takeda S.

Tsuchida K. Mesenchymal progenitors distinct from satellite cells contribute to ectopic fat cell formation in skeletal muscle. The functions performed by FAPs in injured muscles are context dependent. In the experimental paradigm of CTX-mediated muscle regeneration, factors synthesized by FAPs support myogenic differentiation of MPs (). In contrast, in the experimental paradigm of glycerol-induced muscle degeneration, FAPs contribute to adipogenic infiltration of injured muscle, resulting in its fatty degeneration (). In both of these experimental models, cell-nonautonomous signals control the ultimate fate adopted by FAPs in injured muscle. In this context, our current work demonstrates that injury leads to the formation of a dynamic niche that provides signals to regulate the functionality of FAPs. Specifically, muscle injury leads to the recruitment of eosinophils, which form the transitional niche for the proliferating FAPs via secretion of IL-4 ( Figure 7 K). In the presence of IL-4, FAPs proliferate as fibroblasts to support myogenesis by facilitating the clearance of necrotic debris. However, in its absence, FAPs fail to clear necrotic muscle fibers and differentiate into adipocytes, contributing to the persistence of cellular debris and fatty degeneration of skeletal muscle.

Stoick-Cooper et al., 2007 Stoick-Cooper C.L.

Moon R.T.

Weidinger G. Advances in signaling in vertebrate regeneration as a prelude to regenerative medicine. Arnold et al., 2007 Arnold L.

Henry A.

Poron F.

Baba-Amer Y.

van Rooijen N.

Plonquet A.

Gherardi R.K.

Chazaud B. Inflammatory monocytes recruited after skeletal muscle injury switch into antiinflammatory macrophages to support myogenesis. Tidball and Villalta, 2010 Tidball J.G.

Villalta S.A. Regulatory interactions between muscle and the immune system during muscle regeneration. −/− mice, which normally harbor necrotic debris, enhances the clearance of necrotic muscle fibers. Taken together, these data broaden the functions performed by FAPs during muscle’s regenerative response to injury and demonstrate that type 2 cytokines (IL-4 and IL-13) control nearly all known aspects of FAPs’ functionality. It is widely recognized that regeneration of injured tissues requires clearance of cellular debris (). Previously, this function has been ascribed to macrophages, which rapidly infiltrate injured muscle (). Although persistence of necrotic debris was observed in mice lacking type 2 cytokines IL-4 and IL-13, deletion of its signaling receptor, IL-4Rα, in myeloid cells did not significantly impair debris clearance or muscle regeneration. These observations suggest the potential involvement of a nonmyeloid cell in the removal of damaged muscle fibers. Indeed, multiple lines of evidence presented here demonstrate that FAPs, which rapidly proliferate after injury, are critically important for the clearance of injured muscle fibers. First, FAPs are capable of rapidly phagocytizing necrotic cellular debris in vitro and can outcompete macrophages (4:1) for engulfment of necrotic debris in vivo. Second, the ability of FAPs to degrade necrotic debris is reduced in animals lacking type 2 innate signals. Third, deletion of Il-4rα in FAPs is sufficient to impair the clearance of muscle debris, delaying muscle regeneration. Fourth, administration of IL-4 to injured Il4/Il13mice, which normally harbor necrotic debris, enhances the clearance of necrotic muscle fibers. Taken together, these data broaden the functions performed by FAPs during muscle’s regenerative response to injury and demonstrate that type 2 cytokines (IL-4 and IL-13) control nearly all known aspects of FAPs’ functionality.