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. 2012 Oct 16;20(11):2168–2179. doi: 10.1038/mt.2012.189

Proinflammatory Macrophages Enhance the Regenerative Capacity of Human Myoblasts by Modifying Their Kinetics of Proliferation and Differentiation

Maximilien Bencze 1,2,3, Elisa Negroni 1,2,3, Denis Vallese 1,2,3, Houda Yacoub–Youssef 4,5,6, Soraya Chaouch 1,2,3, Annie Wolff 1,2,3, Ahmed Aamiri 7, James P Di Santo 8, Bénédicte Chazaud 4,5,6, Gillian Butler-Browne 1,2,3, Wilson Savino 9, Vincent Mouly 1,2,3,*, Ingo Riederer 9
PMCID: PMC3498804  PMID: 23070116

Abstract

Macrophages have been shown to be essential for muscle repair by delivering trophic cues to growing skeletal muscle precursors and young fibers. Here, we investigated whether human macrophages, either proinflammatory or anti-inflammatory, coinjected with human myoblasts into regenerating muscle of Rag2−/− γC−/− immunodeficient mice, could modify in vivo the kinetics of proliferation and differentiation of the transplanted human myogenic precursors. Our results clearly show that proinflammatory macrophages improve in vivo the participation of injected myoblasts to host muscle regeneration, extending the window of proliferation, increasing migration, and delaying differentiation. Interestingly, immunostaining of transplanted proinflammatory macrophages at different time points strongly suggests that these cells are able to switch to an anti-inflammatory phenotype in vivo, which then may stimulate differentiation during muscle regeneration. Conceptually, our data provide for the first time in vivo evidence strongly suggesting that proinflammatory macrophages play a supportive role in the regulation of myoblast behavior after transplantation into preinjured muscle, and could thus potentially optimize transplantation of myogenic progenitors in the context of cell therapy.

Introduction

Skeletal muscle growth and regeneration are essentially assured by progenitors called satellite cells, located underneath the myofiber basal lamina,1 and identified by the expression of the paired-box transcription factor Pax7, as well as surface markers such as CD56, M-Cadherin, c-met, syndecans 3 and 4, and α7β1 integrin.2 Following activation, satellite cells, now named myoblasts, proliferate, differentiate, and fuse to form multinucleated muscle fibers. During proliferation, MyoD and Myf5 proteins are both expressed, and once cells exit the cell cycle and become committed to differentiate, they express myogenin and subsequently MRF4.3

Myoblasts can be isolated in vitro, amplified and reintroduced into a damaged muscle where they are able to participate in the regeneration of the host's muscle.4 Accordingly, transplantation was initially envisioned as a therapeutic strategy for certain neuromuscular disorders such as Duchenne muscular dystrophy (DMD), in order to allow dystrophin expression by the incorporation of healthy myoblasts into the newly formed fibers, within the host's muscle tissue.

However, these early clinical trials, using local intramuscular injections of heterologous myoblasts, did not result in significant clinical benefit for the patients.2,5 More recently, myoblast transplantation protocols were improved by innovative systems of injection,6 although the overall efficacy clearly needs further optimization.

Among the aspects potentially related to these rather disappointing results, it has been shown in the mouse, that the transplanted myoblasts undergo a massive and early cell death,7 and have a very limited migration within the recipient's muscle.8,9

Muscle degeneration and regeneration involves not only muscle fibers and muscle precursors, but is a very complex process comprising many other cell types, some of which are recruited from the circulation during the process of injury and/or repair: a huge inflammatory infiltrate is established after muscle injury, and is likely to participate in the regulation of muscle regeneration,10,11 a concept reinforced by the observation that depletion of monocytes/macrophages impairs muscle regeneration, influencing muscle-specific gene expression, and myofiber formation.12,13,14 In the case of DMD patients or in the mdx mouse model of the disease, both adaptive and innate immune elements such as cytotoxic lymphocytes,15 neutrophils,16 mast cells,17 eosinophils,18 and macrophages19 have been described to be active during this process.

Macrophages can adopt proinflammatory, anti-inflammatory, or alternatively activated patterns, depending on their microenvironment. Proinflammatory macrophages (also named M1 or classically activated macrophages) have a proinflammatory and microbicidal phenotype, producing reactive oxygen species, and cytokines such as, interferon-γ, interleukin-1β (IL-1β), and tumor necrosis factor-α (TNF-α). Anti-inflammatory macrophages (also named M2c, deactivated or regulatory macrophages) are activated by and then produce themselves IL-10, which in turn down regulates IL-12 production, characteristic of inflammation inhibition. Alternatively activated macrophages, the wound-healing macrophages (also named M2a) are stimulated by IL-4, and regulate extracellular matrix production, thus contributing to wound healing.20,21

Proinflammatory macrophages stimulate myoblast proliferation while inhibiting their differentiation,14 but the impact of the polarized macrophage subpopulations during muscle regeneration in vivo, and more specifically their effects on satellite cells, remain largely unknown. Moreover, no data are available concerning the impact of activated macrophage subpopulations on the engraftment of human myoblasts into an injured muscle. In the present study, we have investigated whether polarized human proinflammatory macrophages, coinjected with human myoblasts could modify in vivo the kinetics of proliferation/differentiation. Our results clearly show that proinflammatory macrophages have a positive impact on the behavior of transplanted human myoblasts during cryodamage-induced muscle regeneration, extending the proliferation phase, increasing migration and delaying differentiation of the myogenic precursors. Conceptually, our data provide for the first time in vivo evidence strongly suggesting that proinflammatory macrophages play a supportive role in the regulation of myoblast behavior after engraftment into preinjured muscle, and could thus potentially optimize transplantation of myogenic progenitors in the context of cell therapy.

Results

Leukocyte infiltration in the TA muscle after cryodamage and human myoblast transplantation

Since the recipient's microenvironment can exert an important influence upon the behavior of the myoblasts, we first analyzed, in the host tissue, mouse specific gene transcripts coding for proinflammatory cytokines, namely IL-1β and TNF-α, as well as transcripts for the secretory leukocyte proteinase inhibitor (SLPI), typically expressed by proinflammatory macrophages and neutrophils.22,23 In the first day post-transplantation, proinflammatory (but not anti-inflammatory) gene expression was clearly detected. By days 3–5, gene transcription for anti-inflammatory cytokines was also detected, including IL-10, transforming growth factor-β (TGF-β), together with peroxisome proliferator-activated receptor γ, a powerful deactivator and marker of anti-inflammatory macrophages.24 These findings, summarized in Figure 1a, suggest a sequential appearance of a pro- and then an anti-inflammatory microenvironment, with possible consequences upon the outcome of the transplanted human myogenic precursors.

Figure 1.

Figure 1

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Leukocyte infiltration after injection of human myoblasts into tibialis anterior muscle of Rag2−/−γC−/− mice. (a) The early cytokine inflammatory patterns of the tibialis anteriors (TA) injected with human cells. Reverse transcription-PCR (RT-PCR) using specific murine primers (inflammatory markers interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α), and secretory leukocyte proteinase inhibitor (SLPI) and anti-inflammatory markers IL-10, transforming growth factor-β (TGF-β) and peroxisome proliferator-activated receptor γ (PPARγ)) were performed at different time points (0, 12, 24 hours and 3 and 5 days) after cryodamage and injection of human myoblasts. (b,c) Immunostaining and quantification showing neutrophils, as defined by the Ly6G marker (red) infiltrating muscle tissue 24 hours and 3 days after injection. (d) Quantification revealing the very early appearance of neutrophils peaking at 24 hours post-engraftment. (e,f) Immunostaining and (g) quantification of the recipient's macrophages (identified with the F4/80 marker, red), whose increase was seen 3 days post-transplantation. In all immunostaining human myoblasts are stained with antihuman-specific lamin A/C antibody (green) and nuclei are labeled in blue with Hoechst. The quantification of macrophages and neutrophils was performed counting the number of the inflammatory cells in representative micrographs in the area of the injected cells. Data represent mean ± SEM. n = 3 per point, *P < 0.05; **P < 0.01; ***P < 0.001. Bars (b,c): 100 µm; (e,f): 50 µm.

In the next set of experiments, we phenotyped the host leukocyte populations within and around the niche where human donor cells were settled. We initially found that CD11b+ cells start to infiltrate the transplanted tibialis anterior (TA) muscle 6 hours after cryodamage, and remain present at 5 days post-engraftment. Interestingly, from 12 hours to 5 days post-transplantation, CD11b+ infiltrating leukocytes were frequently found in close contact with the injected human myoblasts (data not shown).

Since both granulocytes and macrophages bear the CD11b integrin chain, we further investigated the alymphoid inflammatory infiltrate by using specific markers for cell subpopulations within the infiltrate. Shortly after cryodamage and human myoblast injection, we found a transient infiltration of neutrophils, herein defined by the membrane expression of the Ly-6G marker. Their peak was observed at 12 and 24 hours post-engraftment, with a subsequent decrease at days 3 and 5 post-transplantation (Figure 1b–d).

The kinetics of macrophage influx (phenotypically defined by the marker F4/80) differed from that of granulocytes. In the first 24 hours post-engraftment, only rare resident cells were seen, scattered throughout the muscle tissue. In contrast, a massive macrophage infiltration was observed in the cryodamaged muscle, including the area of injected human myoblasts, by days 3–5 post-transplantation (Figure 1e–g). These findings suggest that the proinflammatory milieu ascertained by the expression of proinflammatory cytokines, derives initially from neutrophils, rather than proinflammatory M1 macrophages, although M2 macrophages may in turn be involved in the later production of anti-inflammatory cytokines.

To further investigate the populations of macrophages infiltrating the regenerating tissue, we analyzed sections from regenerating TAs at 24 hours, 3 and 5 days postinjury, using inducible nitric oxide synthase (iNOS) as a marker of M1 proinflammatory macrophages and arginase as a general marker of M2 macrophages. At 24 hours, we did not observe any M2 macrophages, whereas M1 macrophages expressing iNOS were present (see Figure 2a and d). M2 macrophages expressing arginase were detected only at day 3 and 5, whereas M1 positive for iNOS were still detectable (see Figure 2b–f). This is in full agreement with the kinetics of detection of pro- and anti-inflammatory cytokines illustrated in Figure 1a.

Figure 2.

Figure 2

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Pro- and anti-inflammatory host macrophages after human myoblasts transplantation into tibialis anterior muscle of Rag2−/− γC−/− mice. Immunofluorescence showing double staining of F4/80 macrophages (green) with arginase (a–c) or inducible nitric oxide synthase (iNOS) (d–f) (red). Arrows show some double positive cells. In (a) no double positive cells are observed. Nuclei were counterstained with Hoechst (blue). Bars: 20 µm.

Proinflammatory macrophages favor engraftment of human myoblasts

We then investigated the in vivo influence of pro- or anti-inflammatory environment created by exogenously injected human macrophages on the outcome of human myoblasts transplanted into regenerating muscle, using the same immunodeficient mouse model. The rational for these experiments was based on the in vitro demonstration that proinflammatory macrophages stimulate myoblast proliferation while slowing their differentiation.14 At 4 weeks post-transplantation, coinjection of human myoblasts with proinflammatory macrophages generated twice as many fibers expressing human spectrin than injection of myoblasts alone or myoblasts coinjected with anti-inflammatory macrophages (Figure 3a). Furthermore, proinflammatory macrophages increased by 2.5-fold the number of human lamin A/C positive nuclei detected in the fibers (Figure 3b). When macrophages were injected alone, no labeling of human lamin A/C was detected at 1 month, confirming that their eventual participation to myotubes by fusion was negligible (data not shown).

Figure 3.

Figure 3

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Myogenic potential of human myoblasts coinjected with human proinflammatory macrophages into tibialis anterior (TA) muscles of Rag2−/− γC−/− mice. The graphs show the number of (a) human fibers, (b) human nuclei and the area occupied by (c) human cells 1 month after injection of 105 myoblasts alone (CTR) or after the coinjection of 105 human myoblasts with 5 × 105 proinflammatory macrophages (PRO) or anti-inflammatory macrophages (ANTI). (d–f) Representative immunofluorescence micrographs show the distribution pattern of human myoblasts 1 month following transplantation. The number of human cells (lamin A/C, green) and human fibers (spectrin, green) are higher and more dispersed in the group coinjected with proinflammatory macrophages (PRO). Bar: 100 µm. *P < 0.05.

We also found a significant increase in myogenic cell dispersion within the host muscle, as compared to the group injected with anti-inflammatory macrophages or the control group (Figure 3c–f).

In order to further investigate these effects in a murine model closer to dystrophic situations, we have performed the same cell implantations in a new immunodeficient and dystrophic model, i.e., the Rag2−/− Il2rb−/− crossed with a dystrophin knockout mouse. The resulting mouse model has no B and T lymphocytes nor NK cells as the Rag2−/−γC−/− strain used in this study. The introduction of a mutant Dmdmdx-βgeo allele in the Rag2−/− Il2rb−/− background resulted in the generation of Rag2−/− Il2rb−/−Dmd −/− strain. In addition to the features associated with the Rag2−/− Il2rb−/−genotype, the mutant Dmd allele prevents any dystrophin re-expression in revertant fibers. These mice show a phenotype similar to the well-known mdx model, but with a longer period of degeneration/regeneration of their muscle fibers (data not shown). We applied exactly the same experimental procedure, including the cryodamage, since the level of natural degeneration is always limited at a given time-point. We confirmed in this new model the enhancement of engraftment of human myoblasts by the presence of proinflammatory (M1) macrophages, despite the fact that some inflammation is always present in dystrophic situations. At 1 month post-implantation, we observed the expression of human dystrophin, as detected by human specific antibodies and illustrated on Figure 4c and d, in fibers where either human nuclei, identified by human specific anti-lamin A/C, or human proteins, e.g., human spectrin, were detected (Figure 4b). More importantly, we confirmed that up to five times more human nuclei were detected in the coinjected muscles as compared to those injected with myoblasts alone (data not shown). The dispersion of the human nuclei was also greatly enhanced by the presence of proinflammatory (M1) macrophages, as observed in the nondystrophic immunodeficient model. The area containing human nuclei, identified by the expression of human lamin A/C, was increased by a factor of two (data not shown).

Figure 4.

Figure 4

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Expression of human dystrophin in tibialis anterior (TA) muscles of Rag2−/−Il2rb−/−Dmd−/− mice. Representative image of newly formed human fibers after coinjection of 105 human myoblasts with 6 × 105 proinflammatory macrophages. Total nuclei were stained with hoechst, and are shown in (a). Human nuclei (lamin A/C, green) and fibers (spectrin, green) were recognized by human-specific antibodies and are shown in (b). Spectrin positive fibers were also positively stained for dystrophin, whose expression was detected by human-specific antibodies MANDYS102 and MANDYS106 and is shown in (c) (red). A merged picture is shown in (d). Bar: 100 µm.

We next evaluated whether the transplanted myoblasts remained located close to the coinjected macrophages (at least until day 5). Figure 5a and b shows the detection of human CD56+ myoblasts and the nonmyogenic injected cells (human lamin A/C positive nuclei), largely represented by the macrophages. It should be noted that for some CD56+ cells the nucleus is not visible, due to the fact that the section is peripheral to the nuclei in these cells.

Figure 5.

Figure 5

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Localization and survival of cotransplanted human cell populations. Representative immunofluorescence micrographs 5 days after transplantation, with a: anti-inflammatory (ANTI) or b: proinflammatory (PRO) macrophages, respectively. Human cells were stained with antibodies directed against human lamin A/C (green) and human CD56 (red), showing respectively human nuclei from myoblasts and macrophages (lamin A/C), and plasma membrane of only human myoblasts (CD56). Bars: 100 µm. Graphs representing the percentages of pro- or anti-inflammatory human macrophages 5 days post-transplantation, identified by the coexpression of CD68 and lamin A/C, within the total population of human lamin A/C positive cells (c).

As seen in this figure, most of the coinjected human macrophages, whether they are anti-inflammatory (Figure 5a) or proinflammatory (Figure 5b) remained in close proximity to the engrafted myoblasts, at 5 days post-transplantation, for both coinjected groups. It is thus conceivable that at early time points, implanted human myoblasts and macrophages do not migrate away from each other, but stay in close vicinity, allowing cell-to-cell contacts as well as paracrine interactions mediated by soluble secreted factors including cytokines. It should be noted that we did not observe any increase in cell death of either injected myoblasts or macrophages in these experiments. At 5 days after coinjections, we quantified the ratio between human macrophages, by counting cells positive for CD68 and lamin A/C, as compared to lamin A/C only positive cells, i.e., coimplanted myoblasts. This quantification is presented on Figure 5c. The percentage of macrophages among the human cells present at that time point was 81% for coinjections with proinflammatory macrophages, and 83% for anti-inflammatory macrophages, thus very similar to the original ratio between the different cell types at the time of injection (85.7%).

Proinflammatory macrophages enhance proliferation and delay differentiation of engrafted human myoblasts

In order to identify by which mechanism(s) this general improvement in myoblast regenerative capacity occurred, we analyzed the effect of macrophages on myoblast proliferation and differentiation. Coculture experiments, in medium-containing low serum concentration, demonstrated that proinflammatory macrophages increased the number of KI67+ myoblasts after 3 days (Figure 6a). Conversely, a significant decrease in differentiation (i.e., formation of myotubes) was observed in the presence of proinflammatory macrophage-derived conditioned medium, whereas the opposite was observed when we added conditioned medium from anti-inflammatory macrophage cultures (Figure 6b). This is in agreement with the stimulation of myoblast proliferation by proinflammatory macrophages, as previously reported.14

Figure 6.

Figure 6

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Proliferation and differentiation of human myoblasts in the presence of human pro- and anti-inflammatory macrophages in vitro and in vivo. (a) Percentages of Ki67+ proliferating human myoblasts alone and after coculture with proinflammatory macrophages (PRO) or anti-inflammatory macrophages (ANTI) in a medium-containing low serum concentration. (b) Percentage of differentiated human myotubes in vitro, identified using the human neonatal myosin heavy chain (neoMyH), of human myoblasts cultured alone, and with proinflammatory (PRO) or anti-inflammatory (ANTI) macrophage-derived conditioned medium. (c) Percentages of proliferating human KI67 positive cells after injection into adult immunodeficient Rag2−/−γC−/− mouse recipients, 5 days post-engraftment in the three groups: myoblasts injected alone (CTR), myoblasts coinjected with proinflammatory macrophages (PRO) and myoblasts coinjected with anti-inflammatory macrophages (ANTI). The results were obtained by performing a triple-staining directed against lamin A/C (human nuclei), human CD56 (myoblasts at 5 days) and Ki67. Results are expressed as the percentage of Ki67+ cells of the total number of human CD56+ cells. (d) Percentage of differentiated human CD56 myoblasts derived myotubes in vivo, identified using the human neonatal myosin heavy chain (neoMyHC), a terminal differentiation marker, 5 days after transplantation, in each group: myoblasts alone (CTR), myoblasts coinjected with proinflammatory macrophages (PRO) or coinjected with anti-inflammatory macrophages (ANTI). Data represented as mean ± SEM, with at least three experimental groups per point. *P < 0.05; **P < 0.05.

We then analyzed donor human myoblast proliferation in vivo. When myoblasts were injected in the presence of proinflammatory macrophages, and examined 24 hours later, we found no difference in the number of proliferating human cells (data not shown), as defined by three-color immunofluorescence for detecting the following molecules: Ki67, CD56, and lamin A/C. However, at 5 days, even though the proportion of transplanted myoblasts still proliferating has decreased to <20%, the proportion of proliferating transplanted myoblasts is still 2.5-fold higher in the group coinjected with proinflammatory macrophages (Figure 6c), suggesting that proinflammatory macrophages exert in vivo a proliferative effect on the transplanted myoblasts, as they do in vitro (see Figure 6a).

This effect was not observed when anti-inflammatory macrophages were coinjected with the myoblasts. This is not due to a difference in survival between pro- and anti-inflammatory macrophages in vivo, since the number of CD68+ human cells at 5 days post-implantation did not show any significant difference (Figure 5c). Terminal differentiation of transplanted cells was assessed by the expression of neonatal myosin heavy chain (MyHC), which has been described as an early marker of skeletal muscle differentiation during regeneration.25 Five days post-transplantation the proportion of differentiated neonatal MyHC-positive fibers within the human-specific CD56+ cells was decreased 4.5-fold in the group coinjected with proinflammatory macrophages, when compared to the group coinjected with anti-inflammatory macrophages, and threefold when compared with the group of myoblasts injected alone (Figure 6d), in accordance with an increased proliferation of the transplanted cells shown in Figure 6c.

Myoblasts coinjected with anti-inflammatory macrophages showed a strong tendency to increase their differentiation rate compared to controls. This finding indicates that injection of anti-inflammatory macrophages, also known to stimulate in vitro differentiation,14 is not a good option for in vivo transplantation because they will induce the injected myoblasts to differentiate too early and consequently less fibers will be formed.

Transplanted proinflammatory macrophages switch to an anti-inflammatory phenotype in vivo

Macrophage populations are known to have a versatile phenotype, which is strongly influenced by the microenvironment as well as by their own phagocytic activity: they can switch from a proinflammatory to an anti-inflammatory phenotype after phagocytosis, or under the influence of cytokines present in the inflammatory environment.14,26

To verify whether the human proinflammatory macrophages undergo such a change in phenotype in our experimental system, we double immunostained the injected muscles with an antibody specific to the human CD68 molecule, a pan-macrophage marker,27 together with an antibody specific for the human CD206 molecule, a marker for M2 macrophages.28,29 As expected, at 24 hours post-transplantation, the majority of the CD68+ transplanted macrophages were also CD206+ in muscles that were grafted with anti-inflammatory macrophages (Figure 7a), whereas in those muscles grafted with proinflammatory macrophages, most of the CD68+ cells were negative for CD206 (Figure 7b). However, at 5 days post-transplantation, in the muscles injected with the proinflammatory cells, we observed clusters of CD68+ macrophages also expressing the CD206 marker (Figure 7c), confirming a partial phenotype switch, although some proinflammatory macrophages maintained their CD206– phenotype. In the group injected with the anti-inflammatory macrophages, the CD68+CD206+ phenotype persisted until day 5 post-transplantation (data not shown).

Figure 7.

Figure 7

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Partial switch of human proinflammatory macrophages into an anti-inflammatory phenotype after being injected in vivo. Immunofluorescence micrographs of anti-inflammatory macrophages after coinjection of human macrophages with human myoblasts into adult immunodeficient Rag2−/−γC−/− mouse recipients, using a double staining with CD68 (green) and CD206 (red) as markers. Nuclei were stained with Hoechst (blue). (a,b) Patterns 24 hours after coinjection of human myoblasts with either anti-inflammatory (ANTI, a), or proinflammatory macrophages (PRO, b). (c) Five days after coinjection of human myoblasts with proinflammatory macrophages (PRO). (d,e) transforming growth factor-β (TGF-β) expression 5 days post-transplantation of human myoblasts coinjected with anti-inflammatory (ANTI, d) or proinflammatory (PRO, e) macrophages into adult immunodeficient Rag2−/−γC−/− mouse recipients. Sections were triple-stained for the presence of lamin A/C (blue), TGF-β1 (green) and CD206 (red). White arrowheads show lamin A/C+ CD206+ TGF-β1+ macrophages. Yellow arrowheads show lamin A/C+ CD206+ TGF-β1– macrophages. Bars: 200 µm (a–c) and 100 µm (d,f).

In order to confirm the anti-inflammatory phenotype of the CD68+CD206+ human macrophages, we performed a TGF-β1 immunostaining, which showed that in the group injected with anti-inflammatory macrophages, the vast majority of cells labeled for CD206 were also TGF-β1+ (Figure 7d). Among the proinflammatory macrophages that switched to the CD206 anti-inflammatory phenotype, some (but not all) were also TGF-β+ (Figure 7e, white arrows).

Discussion

It has been previously demonstrated that cells from the innate immunity, such as macrophages are involved in the normal process of regeneration in murine skeletal muscle due to their ability to release cytokines29 and to protect myoblasts and myotubes from apoptosis.30,31

In the experimental model described in the present study, when human myoblasts were engrafted in vivo after cryodamage-induced regeneration of the TA in muscle of immunodeficient alymphoid mice, we found early gene transcripts for proinflammatory cytokines, followed by expression of anti-inflammatory genes. During the first day following human myoblast transplantation, the proinflammatory environment observed in the muscle was most likely due to an influx of neutrophils, since only rare proinflammatory M1 macrophages were detected at 24 hours postinjection. Later, by days 3–5, host macrophages (both M1 and M2, as identified by iNOS and arginase expression, respectively) appeared in the inflammatory infiltrate, coinciding with the detection of anti-inflammatory gene transcripts in the muscle.

Since there is no adaptive immune response in this model, the presence of inflammatory cells is most likely due to the cryodamage performed before transplantation. Indeed, after cryodamage and transplantation of human myoblasts, an early and progressive infiltration of host inflammatory cells (detected by the CD11b marker, which stains both neutrophils and macrophages) was observed in the TA muscles of the immunodeficient mice. This infiltrate was first observed at 6 hours and remained close to the injected myoblasts from 12 hours until day 5 post-transplantation. A similar sequential pattern of neutrophil macrophage infiltration after muscle damage has been described in the literature.29,32,33 In fact, when we specifically analyzed the host macrophages, using the specific marker F4/80, they were not found around the injected human cells until 24 hours post-transplantation, but were present at days 3 and 5. It should be noted that expression of F4/80 by the macrophages increases as the cells differentiate within the tissue, thus the low level of infiltrating macrophages observed before day 3 may be underestimated by the immunolabeling technique. In contrast neutrophil infiltration increased progressively until 24 hours, but then subsequently decreased between 3 and 5 days. In fact, the proinflammatory environment observed until 24 hours can be produced by neutrophils, which express the SLPI23 and can produce many inflammatory mediators including TNF-α and IL1-β.34 Thus, enhancement of a proinflammatory microenvironment could be envisioned as a relevant strategy to optimize efficacy of myoblast transplantation. Nevertheless, neutrophils can hardly be envisioned for such an approach since in most experimental conditions they die shortly after arriving in the inflamed tissue.35,36 Alternatively, a more persistent inflammatory microenvironment could be created by exogenous proinflammatory macrophages, coinjected with the myoblasts to be transplanted.

Previous work has shown that, in vitro, macrophages increase myoblast proliferation.37,38 However, it has not been established whether these effects can modulate the efficiency of exogenous myoblasts to be incorporated into regenerated fibers, by cell–cell contact and/or effector cytokine release.

In the present study, we used coinjections of human macrophages with human myoblasts in order to maximize the potential interactions between these two cell types. We showed that the presence of human proinflammatory macrophages increased the efficiency of human myoblast engraftment in vivo, after cryodamage-triggered regeneration of the TAs muscle of immunodeficient mice. Such an improvement was clearly demonstrated by the significantly higher number of muscle fibers expressing human proteins detected within the recipient's muscle 1 month after engraftment, compared to when myoblasts were injected alone or in combination with anti-inflammatory macrophages. These results were further confirmed when the same cell types were implanted in the same ratio into regenerating muscles of Rag2−/−Il2rb−/−Dmd−/− mice, a model generated by crossing the immunodeficient strain with a dystrophin knockout strain. Both the number of cells and their dispersion were increased in the presence of proinflammatory macrophages in this dystrophic environment, and human dystrophin was expressed in the fibers expressing human proteins, e.g., spectrin recognized by a species-specific antibody, and/or containing human nuclei identified by the species-specific anti-lamin A/C.

We then investigated whether such an improvement could be related to the role of proinflammatory macrophages upon distinct biological functions of the transplanted myoblasts, such as cell migration and/or proliferation, which would result in vivo in a delayed and more prolonged phase of myoblast differentiation into myotubes.

It has been consistently reported that myoblasts injected into skeletal muscle remain close to the site of injection.8,9 When we analyzed the early post-transplantation time points, we found at day 5 post-transplantation that proliferation and dispersion were enhanced and differentiation was delayed in the group coinjected with proinflammatory macrophages. This was not due to a difference in survival between pro- and anti-inflammatory macrophages, since the number of human cells negative for myogenic markers was not significantly different between both experimental situations. Human myoblasts were identified by a human-specific CD56 antibody, and although some CD56+ cells could be labeled without showing a nucleus (due to the thickness of the section) these were not considered in the quantification. Although this method may introduce a limited bias toward underestimation, the bias is the same for all the experimental situations compared in this set of experiments. Furthermore, we observed that 5 days after cotransplantation, the ratio between human macrophages, either proinflammatory and anti-inflammatory, and human myogenic cells (respectively 81% for pro- and 83% for anti-inflammatory macrophages), was very similar to the original ratio defined for the injections (85.7%), thus showing that there is no cell-type specific increase by preferential proliferation or decrease by cell death, at least in this experimental setting. Overall, these results suggest that proinflammatory macrophages exert a proproliferative effect upon the transplanted myoblasts, which inhibits their differentiation at that time-point, as shown by the decrease in neonatal MyHC-expressing myotubes in vivo. As a result, the period during which transplanted myoblasts can proliferate and migrate is extended, thus resulting at 1 month post-transplantation in an increase in the total number of human nuclei, but also in fibers expressing human proteins, secondary to the fusion of more transplanted myoblasts which proliferated for an extended period before differentiating at later time-points. These results were reinforced by coculture experiments that actually confirmed previous data.14

Our results demonstrate that when myoblasts are coinjected with proinflammatory macrophages which create a proinflammatory environment, the increased proliferation and the delayed differentiation observed at day 5 post-transplantation will extend the period of myoblast expansion, resulting in the formation of a larger number of human fibers, which is what we observed 1 month after engraftment, as well as a twofold increase in donor cell dispersion.

The presence of human macrophages bearing the CD206+TGF-β+ in the group injected with proinflammatory macrophages, at days 3 and 5 post-transplantation, seems to confirm that there is an in vivo shift, from the proinflammatory toward an anti-inflammatory macrophage phenotype, which would ultimately favor myoblast differentiation. This is important for a long-term effect of proinflammatory macrophages, which delay myoblast differentiation. This delay, although sufficient to enhance the participation of human myoblasts to host's regeneration in our model, will be limited in time due to a change in fate of these proinflammatory macrophages toward an anti-inflammatory phenotype which will then allow myoblast differentiation, and will probably be resolved together with the inflammation of the regenerating muscle.

In conclusion, our results suggest that a proinflammatory environment, such as that generated by proinflammatory macrophages, plays a role in the regulation of the kinetics of proliferation and differentiation of engrafted myoblasts, probably by cell–cell contact and the release of cytokines. More precisely, we propose that these cytokines can modulate the balance between myoblast proliferation and differentiation within the complex microenvironment of a regenerating tissue, and thus orchestrate the different phases of muscle regeneration by cell–cell interactions. In this report, we show that a proinflammatory environment results in an increase in both the proliferation and the dispersion of implanted human cells in a regenerating context, and will thus result in the long term in an increased efficiency of cell therapy, as suggested by the expression of human dystrophin in the immunodeficient and dystrophic model. Consequently, strategies which will extend the period during which injected cells will proliferate and migrate within the host tissue may be instrumental for improving myoblast and stem cell transplantation based cell therapy. In addition the cytokine(s) involved in maintaining the proliferation and dispersion of the myoblasts can be identified and used as tools to modulate temporarily the environment to enhance the regenerative capacity of implanted cells, since this may be easier to set up in a clinical context. In the same vein, the injection of human myoblasts in a serum-containing medium increases the numbers of human fibers, detected 1 month post-transplantation, by decreasing early myoblast differentiation while increasing proliferation.39 The fact that implanted myoblasts are influenced by the environment is in agreement with previous results, showing that coinjections of side-population (SP) cells myoblasts in vivo in a regenerating mouse muscle enhanced the regenerative capacity of these myoblasts, most probably by the release of paracrine factors by SP cells, since SP cells rarely fuse with the regenerating host fibers. Moreover, the same authors showed that SP cells release matrix metalloproteinase-2, which promoted the migration of the implanted myoblasts.40

The availability of blood-derived monocytes from patients that can be further differentiated and activated toward proinflammatory or anti-inflammatory macrophages, together with the positive effect that these cells may have upon the transplanted myogenic precursors, are in favor of this novel strategy for the improvement of cell-based therapy for muscular dystrophies. Moreover, proinflammatory macrophages will not trigger tissue or cell damage because they will spontaneously change their phenotype in vivo during the time course of regeneration, inducing first the resolution of inflammation and eventually the differentiation of myogenic cells.

Materials and Methods

Animals. Rag2−/−γC−/−immunodeficient mice aged 2–3 months were used as recipients for human myoblast transplantation.41 These animals are alymphoid, but do contain circulating granulocytes and monocytes as well as tissue macrophages.42,43 Two month-old Rag2−/−Il2rb−/−Dmd−/− mice were also used for experiments. This new strain is the result of a cross between Rag2−/−Il2rb−/− animals, a strain with the same immunodeficient status as Rag2−/−γC−/−, and Dmdmdx-βgeo mice, which completely lacks dystrophin and in addition has no revertant fibers which is a great improvement over the classic mdx mouse model.44

Animals were anesthetized by an intraperitonial injection of 80 mg/kg of ketamine hydrochloride and 10 mg/kg xylasine (Sigma-Aldrich, St Louis, MO), as described previously.41 Surgical procedures were performed under aseptic conditions and in accordance with the legal regulations in France and with European Union ethical guidelines for animal research.

Cultures of human myoblasts. Myoblasts were isolated from the quadriceps muscle of a 5-day-old infant, as reported previously,45 and in accordance with the French legislation on ethical rules. Cells were expanded in Ham's F10 growth medium supplemented with 5 µg/ml gentamycine and 20% fetal calf serum (Invitrogen, Carlsbad, CA), at 37 °C in a humid atmosphere containing 5% CO2. Since intermediate filament protein desmin is only expressed in myoblasts and not in fibroblasts,46 myogenic purity of each cell preparation was determined by counting the number of desmin-containing cells as a percentage of the total number of nuclei. Immunocytochemistry was performed using an anti-desmin antibody (see Table 1). Specific antibody binding was revealed using an Alexa-488 coupled goat anti mouse secondary antibody. A total of at least 500 cells were counted for each experiment. All cell preparations used had a myogenicity >80%.

Table 1. Antibodies applied in immunohistochemistry and cytochemistry.

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Cell preparation and myoblast transplantation. All myoblast cultures were allowed to proliferate until they reached 80% confluence. Before injection, the cells were trypsinized, centrifuged, and resuspended in phosphate-buffered saline (PBS). The cells were then injected immediately into the TA muscles. Before myoblast engraftment, the TAs of the Rag2−/−γC−/− mice were subjected to three freeze lesion cycles of 10 seconds each, in order to damage the muscle fibers, and trigger regeneration, thus stimulating the implanted myoblasts to fuse and form new muscle fibers.47 Fifteen microliter cell suspensions containing 5 × 105 myoblasts in PBS were injected using a 25-µl Hamilton syringe in a single injection site in the mid belly of the TA. The skin was then closed using fine sutures. At 6, 12, 24 hours, 3 and 5 days and 1 month after engraftment, mice were sacrificed (n = 3 for each time point) and the TAs were dissected. In selected experiments, 105 myoblasts diluted in the F10 culture medium were coinjected, with 6 × 105 proinflammatory or 6 × 105 anti-inflammatory human macrophages, obtained as described below. The same procedure was applied to coimplantations of human macrophages and human myoblasts into regenerating TAs of Rag2−/−Il2rb−/−Dmd−/−. In all experiments, the TAs were mounted in tragacanth gum (6% in water; Sigma-Aldrich), and frozen in isopentane precooled in liquid nitrogen.

Cultures of human macrophages and corresponding supernatants. Peripheral blood mononuclear cells were isolated from human blood samples using Ficoll density gradient. Monocytes were isolated by an adhesion step48 for 1 hour at 37 °C, washed and incubated overnight containing RPMI 1640 medium and 10% decomplemented human AB serum [supplemented with 1% sodium pyruvate, 10 mmol/l Hepes, 50 µmol/l β-mercaptoethanol, 1% nonessential amino acids, 1% vitamins, 1% penicillin/streptamycin (10,000 U)]. In order to differentiate monocytes into macrophages, cells were then seeded on RPMI 1640 medium-containing 15% human AB serum for 7 days at 0.7 × 106 cells/ml in Teflon bags (Cellgenix, Freiburg, Germany) for in vivo experiments or plastic dishes for in vitro experiments. Macrophages were polarized using 1 µg/ml lipopolysaccharide (Sigma-Aldrich) and 10 ng/ml interferon-γ (PeproTech) for classical activation (M1 proinflammatory phenotype) or 80 ng/ml DEX (Sigma-Aldrich) and 10 ng/ml IL-10 (PeproTech, Neuilly sur Seine, France) for 48 hours for anti-inflammatory (M2) phenotype.14 Coculture experiments were performed after washing steps and adding myoblasts in an advanced RPMI medium complemented with 2% or 10% fetal calf serum and 5 µg/ml gentamycine, and dishes were fixed after 24 hours, 3 days, or 5 days of coculture in 100% ethanol.

Cytokine gene expression in the recipient's muscle. Total RNA was extracted from each TA using Trizol reagent (Invitrogen) according to the manufacturer's recommendations. RNA concentration was determined using a NanoDrop 1000 (Thermo Scientific, Illkirch, France post-genomic platform, Hospital Pitié-Salpêtrière), RNA integrity was checked by running 1 µg on an agarose gel (data not shown) and 2 µg of total RNA was reverse transcribed into cDNA using M-MLV Reverse Transcriptase (Invitrogen) according to the manufacturer instructions, in a final volume of 40 µl. One microliter of the reverse transcription products were subjected to PCR amplification in a final volume of 25 µl, using the 2× ReddyMix PCR Master Mix (ABgene; Epsom, Surrey, UK) and the following specific primers (sense and antisense, respectively):

TNFα: 5′-TTCCAGATTCTTCCCTGAGGT-3′ 5′-TAAGCAAAAGA GGAGGCAACA-3′ IL-1β: 5′-TGACGTTCCCATTAGACAACTG-3′ 5′-CCGTCTTTCATTACACAGGACA-3′

TGFβ1: 5′-GAGACGGAATACAGGGCTTTC-3′, 5′-TCTCTGTGG AGCTGAAGCAAT-3′ IL-10: 5′-ACCAGCTGGACAACATACTGC-3′, 5′-TCACTCTTCACCTGCTCCACT-3′ secretory leukocyte proteinase inhibitor: 5′-CCTTAAGCTTGAGAAGCCACA-3′, 5′-AGCACTTGTATT TGCCGTCAC-3′ peroxisome proliferator-activated receptor γ: 5′AA GAGCTGACCCAATGGTTG-3′, 5′-GGATCCGGCAGTTAAGATCA-3′ RPLPO: 5′-CTCCAAGCAGATGCAGCAGA-3′, 5′-ATAGCCTTGCGCA TCATGGT-3′.

Amplifications were performed in a thermocycler DNA Engine (Bio Rad, Hercules, CA), initiated by 2 minutes at 95 °C, followed by cycles of amplification, each consisting of 30 seconds at 95 °C, 30 seconds at 60 °C, and 30 seconds at 72 °C. The number of cycles (25 for RPLPO, 30 for peroxisome proliferator-activated receptor γ, secretory leukocyte proteinase inhibitor, IL1-β and TGF-β1, and 32 for TNF-α and IL-10) has been chosen so that the amplification process has not reached the plateau phase. Twelve microliter of the amplification products were subjected to electrophoresis on a 1.5% agarose gel-containing ethidium bromide for visualization using Gel Doc 2000 software (Bio Rad).

Histology and immunofluorescence. Immunofluorescence analyses of grafted TA muscles were performed using mouse monoclonal antibodies specific for human spectrin and human lamin A/C (see Table 1 for details). These antibodies were used to visualize fibers expressing human proteins after 1 month (anti-spectrin) and to detect human nuclei (anti-lamin A/C) at all time points. Antihuman dystrophin MANDYS102 and MANDYS106 were used in combination in the coimplantation of human macrophages and myoblasts in the Rag2−/−Il2rb−/−Dmd−/− model. To evaluate the proliferation and differentiation of human cells during the kinetics of regeneration, double immunofluorescence analyses were performed combining antibodies directed against human lamin A/C with the following antibodies: anti-Ki67 (a panmarker for proliferating cells), anti-myogenin (labeling the early phase of human myoblast differentiation). For MyHC (global marker for early and full differentiation of myoblasts) staining with the antibody neonatal MyHC was used together with an antihuman CD56 (N-CAM), used to detect the injected myoblasts. In addition, we used, anti-Ly-6G or anti F4/80 monoclonal antibody to detect recipient's neutrophils and macrophages that are recruited to the tissue after cryodamage. Although F4/80 can also recognize circulating monocytes, which express this marker but at a lower level than macrophages,49 it is used here to detect cells which infiltrated into the host tissue. To detect M1 and M2 host macrophages, antibodies against iNOS and arginase together with F4/80 were used. Human macrophages were visualized with species-specific reagents, recognizing the molecules CD68 and CD206. For detection of TGF-β1 in human macrophages we also applied a species-specific mAb (see Table 1).

Five micrometer thick transverse cryostat sections were fixed in cold acetone for 10 minutes, washed twice in PBS, blocked in 2% bovine serum albumin/1% sheep serum in PBS during 30 minutes. Sections were then incubated with primary antibodies for 1 hour, at room temperature washed in PBS, and incubated with the appropriate secondary antibodies during 45 minutes. Characteristics of all antibodies are summarized in Table 1.

Nuclei were visualized in the sections by mounting the sections in a medium (Cytomation fluorescent mounting Medium, S3023; Dako, Trappes, France) after a 3 minutes Hoechst staining (bis-benzimide, 0.0001% wt/vol, no. 33258; Sigma-Aldrich).

All images were visualized using an Olympus BX60 microscope (Olympus Optical, Hamburg, Germany), digitalized using a PhotometricsCoolSNAPfx CCD camera (Roper Scientific, Tucson, AZ) and analyzed using the MetaView image analysis software (Universal Imaging, Downington, PA), except images for cell dispersion analysis that were visualized with a motorized fluorescence microscope (Zeiss AxioImager.Z1 (Carl Zeiss, Oberkochen, Germany) and captured using a digital camera (Hamamatsu ORCA-ER; Shizuoka, Japan).

Analysis of muscle samples. TA muscles were cut along their entire length into 5-µm sections, with one section every 500 µm, being used for immunohistochemical analysis. For the quantification experiments concerning the human fibers, the number of spectrin positive profiles in each of these sections was counted and the maximum value was determined for each TA analyzed, as already described.50

Statistical analysis. Data were expressed as the mean ± SE of at least three different animals. All statistical analyses were performed using GraphPad Prism (version 4.0b; GraphPad Software, San Diego, CA). Statistical significance was assessed by one-way ANOVA with the Bonferroni post-test, with P < 0.05 being considered significant.

Acknowledgments

This work was financially supported with grants from PAPES/Fiocruz, CAPES, CNPq (Brazil), Inserm, UPMC, CNRS, Association Françaisecontre les Myopathies (AFM), ANR (Genopath INAFIB), MyoAge (EC 7th FP, contract 223576),Inserm/Fiocruz and CNPq/Inserm Conjoint Programs. This work was developed in the context of the CNPq/Inserm/Fiocruz/UPMC International Associated Laboratory of Cell Therapy and Immunotherapy. The authors thank Lidia Dolle for technical assistance and the MSG study group for discussions.

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