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# CX3CR1 deficiency promotes muscle repair and regeneration by enhancing macrophage ApoE production

Ludovic Arnold, Hélène Perrin, Camille Baudesson de Chanville, Marielle Saclier, Patricia Hermand, Lucie Poupel, Elodie Guyon, Fabrice Licata, Wassila Carpentier, José Vilar, et al.

► To cite this version:

Ludovic Arnold, Hélène Perrin, Camille Baudesson de Chanville, Marielle Saclier, Patricia Hermand, et al.. CX3CR1 deficiency promotes muscle repair and regeneration by enhancing macrophage ApoE production. Nature Communications, 2015, 6, pp.8972. 10.1038/ncomms9972. hal-01514445

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https://hal.science/hal-01514445

Submitted on 3 May 2017

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ARTICLE

Received 8 Jul 2015 | Accepted 21 Oct 2015 | Published 3 Dec 2015

DOI: 10.1038/ncomms9972

OPEN

# CX3CR1 deficiency promotes muscle repair and regeneration by enhancing macrophage ApoE production

Ludovic Arnold<fnref n="1" />,*, Hélène Perrin<fnref n="1" />,*, Camille Baudesson de Chanville<fnref n="1" />, Marielle Saclier²,³,⁴, Patricia Hermand<fnref n="1" />, Lucie Poupel<fnref n="1" />, Elodie Guyon<fnref n="1" />, Fabrice Licata<fnref n="1" />, Wassila Carpentier⁵, José Vilar⁶, Rémi Mounier²,³,⁴, Bénédicte Chazaud²,³,⁴, Nora Benhabiles⁷, Alexandre Boissonnas<fnref n="1" />, Béhazine Combadiere<fnref n="1" /> & Christophe Combadiere<fnref n="1" />

Muscle injury triggers inflammation in which infiltrating mononuclear phagocytes are crucial for tissue regeneration. The interaction of the CCL2/CCR2 and CX3CL1/CX3CR1 chemokine axis that guides phagocyte infiltration is incompletely understood. Here, we show that CX3CR1 deficiency promotes muscle repair and rescues Ccl2⁻/⁻ mice from impaired muscle regeneration as a result of altered macrophage function, not infiltration. Transcriptomic analysis of muscle mononuclear phagocytes reveals that Apolipoprotein E (ApoE) is upregulated in mice with efficient regeneration. ApoE treatment enhances phagocytosis by mononuclear phagocytes in vitro, and restores phagocytic activity and muscle regeneration in Ccl2⁻/⁻ mice. Because CX3CR1 deficiency may compensate for defective CCL2-dependant monocyte recruitment by modulating ApoE-dependent macrophage phagocytic activity, targeting CX3CR1 expressed by macrophages might be a powerful therapeutic approach to improve muscle regeneration.

<footnote n="1">¹ Sorbonne Universités, UPMC Univ Paris 06, Inserm, U1135, CNRS, ERL 8255, Centre d'Immunologie et des Maladies Infectieuses (CIMI-Paris), 91 Boulevard de l'Hôpital, F-75013 Paris, France. ² Inserm, U1016, Institut Cochin, 22 Rue Méchain, F-75014 Paris, France. ³ CNRS, UMR8104, 22 Rue Méchain, F-75014 Paris, France. ⁴ University of Paris Descartes, Sorbonne Paris Cite, F-75006 Paris, France. ⁵ Sorbonne Universités, UPMC Univ Paris 06, Plateforme Post-Génomique de la Pitié-Salpêtrière (P3S), UMS2 Omique, INSERM US029, 91 Boulevard de l'Hôpital, F-75013 Paris, France. ⁶ Paris Centre de Recherche Cardiovasculaire (PARCC) - HEGP, 56 Rue Leblanc, F-75015 Paris, France. ⁷ CEA, List institute CEA Saclay, Digitéo Labs, PC192, F-91191 Gif-sur-Yvette, Cedex, France. * These authors contributed equally to this work. Correspondence and requests for materials should be addressed to C.C. (email: christophe.combadiere@upmc.fr).</footnote>
Monocytes are key players during trauma and inflammation, whereas tissue-resident macrophages have an important role during tissue homeostasis and the resolution of inflammation. However, it is not well established how monocytes and macrophages contribute to both protective/ regenerative features and deleterious pathogenic processes. Therefore, defining the molecular and cellular processes that enhance, limit or resolve inflammation may lead to useful therapy. Muscle damage induces massive macrophage infiltration at the injury site, and macrophages are essential for muscle regeneration¹⁻⁴. Indeed, partial depletion of mononuclear phagocytes impairs muscle regeneration, whereas reconstitution of myeloid lineage restores regeneration²,⁵,⁶. We previously showed that injured skeletal muscle recruits monocytes exhibiting inflammatory profiles that operate phagocytosis and rapidly convert to anti-inflammatory mononuclear phagocytes³. Aside from removing necrotic muscle debris, macrophages promote myogenic cell growth and differentiation by releasing growth factors and cytokines⁷,⁸, protect myogenic cells from apoptosis⁹ and participate in restoring perfusion by promoting collateral artery formation and angiogenesis¹⁰. In mice, infiltrating macrophages differentiate from circulating inflammatory Ly6C<sup>high</sup> monocytes that co-express CCR2 and CX3CR1, two important chemokine receptors involved in monocyte motility²,³,¹¹. In mice lacking CCR2 (Ccr2<sup>-/-</sup>) or its cognate ligand (Ccl2<sup>-/-</sup>), muscle regeneration and macrophage infiltration are impaired, and macrophage activation is modified⁵,⁶,¹²⁻¹⁶. By contrast, the role of CX3CR1 in muscle healing has not been defined yet, despite its importance in monocyte recruitment¹⁷,¹⁸, macrophage polarization¹⁹⁻²², cell survival²⁰,²³ and angiogenesis²⁴,²⁵.

In the skeletal muscle, recent works indicated that CX3CR1-negative resident macrophages localized in the normal epimysium/perimysium behave as sensors of pathological events, whereas exudate CX3CR1-expressing mononuclear phagocytes may provide immediate inflammatory response in the injured muscle and longstanding immune surveillance until full muscle restoration²⁶. CX3CR1 is not recognized as a key molecule in resident macrophage ontogeny, although it plays a key role in kidney dendritic cells, brain microglial cells and intestinal macrophages colonization²⁷⁻²⁹. Few studies indicate that the survival of monocytes and tissue macrophages may rely on CX3CR1 as shown within the atherosclerotic plaques³⁰ and in the liver²⁰. More recently, CX3CR1 was identified as a regulator of renal macrophage proliferation³¹.

Chemokine (CX3C motif) ligand 1 (CX3CL1) also known as fractalkine or neurotactin is the only known member of the CX3C class of chemokines. It is unique because (i) it is expressed both as a membrane-bound and a soluble form and (ii) it binds specifically to its receptor CX3CR1. It is expressed by human and murine primary myoblasts in culture³²,³³. Whether the CX3CR1/CX3CL1 axis is involved in muscle healing through the recruitment of circulating monocytes or the activation of macrophages, and whether its relation to the CCL2/CCR2 axis is unequivocal, as in atherosclerosis¹⁸,³⁴, or ambiguous, as in age-related macular degeneration (AMD)³⁵, remains to be understood.

In this study, we investigate muscle regeneration and mononuclear phagocyte recruitment in a notexin-induced muscle injury model by comparing C57Bl/6 (wild type, WT), Cx3cr1<sup>-/-</sup>, Ccl2<sup>-/-</sup> and Cx3cr1<sup>-/-</sup> Ccl2<sup>-/-</sup> mice. We show that CX3CR1 deficiency promotes muscle repair and rescues Ccl2<sup>-/-</sup> mice from impaired muscle regeneration. These phenomena result not from altered mononuclear phagocyte infiltration in Cx3cr1<sup>-/-</sup> mice but from functional modifications. Transcriptomic analysis were performed to compare

muscle mononuclear phagocyte subsets between mice with efficient regeneration (WT, Cx3cr1<sup>-/-</sup> and Cx3cr1<sup>-/-</sup> Ccl2<sup>-/-</sup> mice) and those with poor recovery (Ccl2<sup>-/-</sup> mice). Among genes differentially expressed, Apolipoprotein E (ApoE) transcripts are downregulated in Ccl2<sup>-/-</sup> mononuclear phagocyte subsets. Both ApoE administration and ApoE-proficient bone marrow (BM) transfer restore muscle regeneration in Ccl2<sup>-/-</sup> mice. CX3CR1-ApoE-deficient BM transfer did not rescue CCL2-delayed muscle regeneration as effectively as CX3CR1-deficient ApoE-proficient transfer. Finally, ApoE enhances *in vitro* phagocytosis by macrophages, restores phagocytic activity in Ccl2<sup>-/-</sup> mice, and its deficiency leads to incomplete necrotic myofiber removal. Our results suggest that targeting CX3CR1 expressed by mononuclear phagocytes might be useful in alleviating inflammation and promoting muscle regeneration after trauma.

## Results

**CX3CR1 deficiency promotes muscle healing after injury.** We used a mouse model of notexin-induced muscle injury to analyse the muscle reparative process and mononuclear phagocyte recruitment. We show that CX3CR1 (Supplementary Fig. 1a,b) and its ligand CX3CL1 (Supplementary Fig. 1c) are both expressed during muscle regeneration with a maximum reached at day 4. Concomitantly, CCL2 was secreted at days 1 and 4 post injury (Supplementary Fig. 1d). To understand the roles of these chemokines and their functional relationship during muscle repair, we compared muscle regeneration events and mononuclear phagocyte recruitment between C57Bl/6 (WT), Cx3cr1<sup>-/-</sup>, Ccl2<sup>-/-</sup> and Cx3cr1<sup>-/-</sup> Ccl2<sup>-/-</sup> mice. Notexin induced muscle necrosis (day 1, data not shown), which was followed by leukocyte infiltration (day 4), regenerating myofiber formation (day 10) and myofiber growth (day 21; Fig. 1a). Consistent with published data¹⁶, muscle regeneration was impaired in Ccl2<sup>-/-</sup> mice compared with WT mice and was characterized by delays in necrotic fibre removal and myofiber regeneration (Fig. 1a,b and Supplementary Fig. 2), a smaller cross-sectional area (CSA) of regenerating myofibers (Fig. 1c), more intermuscular adipocytes (Fig. 1d), calcification (Fig. 1e) and fibrosis (Fig. 1f), as well as decreased neovascularization (Fig. 1g). CX3CR1 deficiency improved muscle regeneration: Cx3cr1<sup>-/-</sup> mice had fewer necrotic and more regenerating myofibers on day 4 (Fig. 1a,b and Supplementary Fig. 2), higher myofiber CSA on days 10 and 21 post injury (Fig. 1c) and reduced intermuscular calcification (Fig. 1e). Strikingly, muscle regeneration in Cx3cr1<sup>-/-</sup> Ccl2<sup>-/-</sup> mice was better than in Ccl2<sup>-/-</sup> mice and similar to that of WT mice for all the indicators studied; it thus enhanced the muscle regeneration improvement driven by the CX3CR1 deficiency (Fig. 1 and Supplementary Fig. 2). These results were unexpected, as CX3CR1 deficiency usually causes severe disorders in various inflammatory models, associated with cell death³⁶, fibrosis development²⁰, delayed wound healing and neovascularization²⁴,²⁵. The observation of the rescued phenotype in both male and female double-deficient mice (Supplementary Fig. 3) but not in Cx3cr1<sup>-/-</sup> Ccr2<sup>-/-</sup> compared with Ccr2<sup>-/-</sup> mice (Supplementary Fig. 4) suggests that a minimum level of monocyte infiltration is required to compensate for the muscle regeneration impairment (monocyte recruitment is more severely impaired in Ccr2<sup>-/-</sup> than Ccl2<sup>-/-</sup> mice¹²). CX3CR1 deficiency thus appears to promote muscle repair and rescue Ccl2<sup>-/-</sup> mice from impaired muscle regeneration.

**CCL2 but not CX3CR1 deficiency alters macrophage infiltration.** To identify the cellular origin of the muscle regeneration rescue
<figure><img src="image_4.png" /><figcaption>**Figure 1 | CX3CR1 deficiency promotes and rescues Ccl2<sup>-/-</sup> muscle healing after injury.** Muscle regeneration was assessed after notexin injury in WT, Cx3cr1<sup>-/-</sup>, Ccl2<sup>-/-</sup> and Cx3cr1<sup>-/-</sup>Ccl2<sup>-/-</sup> mice on days 4, 10 and 21 post injury. (a) Haematoxylin/eosin staining of uninjured and regenerating muscle (×100 magnification, scale bar, 100 µm). Arrows indicate dense violet structures corresponding to calcified myofibers, and arrowheads mark intermuscular adipocytes (transparent monovacuolar cells). (b) Quantification of regeneration on day 4 post injury assessed by the percentage of necrotic (infiltrated or not) and regenerating (basophilic + centrally nucleated) myofibers, expressed as a percentage of the total number of myofibers. (c) Quantification of the mean cross-sectional area (CSA) of regenerating myofibers, by image J software. (d) Oil Red O staining of lipids within intermuscular adipocytes, (e) Alizarin Red staining of calcified myofibers, (f) Masson Trichrome staining of connective tissue and quantification of the different areas, by setting appropriate thresholds on image J software. Fat, calcification and fibrosis are expressed as percentages of the total injured area (between 60 and 90% of total muscle surface) and results are a mean of three different muscle cross-sections taken at each end and the middle of the muscle. (d, e and f: scale bar, 100 µm). (g) Vascularization was quantified on muscle cross-sections stained with wheat germ/Griffonia simplicifolia agglutinin (scale bar, 50 µm). Data are mean ± s.e.m. of six mice per strain, which represent two distinct experiments. NS, not significant, \*P<0.05, \*\*P<0.01 and \*\*\*P<0.001 for each knock-out strain versus WT and for Ccl2<sup>-/-</sup> versus Cx3cr1<sup>-/-</sup>Ccl2<sup>-/-</sup> mice.</figcaption></figure>

observed in Cx3cr1<sup>-/-</sup> Ccl2<sup>-/-</sup> mice, we performed BM transplantation experiments in sub-lethally irradiated Ccl2<sup>-/-</sup> mice. The control graft produced strongly improved muscle regeneration on day 10 post injury, characterized by increased regenerating myofiber CSA and decreased intermuscular fat accumulation and calcification (Fig. 2a). BM transfer from Cx3cr1<sup>-/-</sup> mice amplified muscle regeneration rescue and increased the regenerating myofiber CSA. This indicates that the muscle regeneration improvement observed in Cx3cr1<sup>-/-</sup> mice relied on BM-derived cells.

As both CX3CR1 and CCL2 are involved in mononuclear phagocyte trafficking, we hypothesized that modulation of mononuclear phagocyte recruitment in the injured muscle might account for the improved regeneration in Cx3cr1<sup>-/-</sup> mice and

rescue of Cx3cr1<sup>-/-</sup> Ccl2<sup>-/-</sup> mice. We assessed mononuclear phagocyte infiltration throughout the injured muscle by flow cytometry (Fig. 2b) and identified four distinct populations (subsets 1–4) expressing typical mononuclear phagocyte phenotypic markers (CD11b<sup>+</sup> Ly6G<sup>-</sup> NK1.1<sup>-</sup> F4/80<sup>+</sup> Mgl1<sup>+</sup>; Fig. 2b and Supplementary Fig. 5). Subset 1 (F4/80<sup>low</sup> Ly6C<sup>high</sup>) has bean-shaped nuclei and may correspond to infiltrating Ly6C<sup>high</sup> blood monocytes. Subset 2 (F4/80<sup>low</sup> Ly6C<sup>low</sup>) expressed dendritic cell markers, such as CD11c and IAb (major histocompatibility complex molecules-class II), and CD64 (FcγRI) at lower levels than the other three subsets. Cells from subsets 3 and 4 were larger and had typical macrophage morphology; subset 3 (F4/80<sup>high</sup> Ly6C<sup>low</sup> Mgl1<sup>low</sup>) expressed lower levels of M2-type macrophage markers (such as
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**Figure 2** | CCL2 but not CX3CR1 deficiency affects macrophage recruitment in muscle. (a) Analysis of muscle regeneration in chimeric Ccl2<sup>-/-</sup> mice engrafted with bone marrow from Ccl2<sup>-/-</sup>, WT and Cx3cr1<sup>-/-</sup> mice, by measuring the mean regenerating myofiber CSA and the areas of intermuscular adipocyte and of calcified myofibers on day 10 post injury. ***P<0.001 for Ccl2<sup>-/-</sup> mice engrafted with bone marrow from WT or Cx3cr1<sup>-/-</sup> mice versus Ccl2<sup>-/-</sup> mice engrafted with Ccl2<sup>-/-</sup> bone marrow. Data are mean of a single experiment + s.e.m. of three mice per strain. (b) Gating strategy for macrophages on day 4 post injury. Macrophages expressed CD11b<sup>+</sup>, F4/80<sup>+</sup> and Mgl1<sup>−/−</sup> but not Ly6G<sup>+</sup> or NK1.1<sup>−/−</sup>. Four subsets of cells corresponding to this phenotype were identified; a subset of CD11b<sup>+</sup> F4/80<sup>+</sup> Ly6C<sup>low</sup> Mgl1<sup>−/−</sup> cells, with morphology typical of eosinophils, was excluded from the analysis. (c) Proportion of each macrophage subset on day 4 post injury. (d) Kinetics of the four macrophage subsets during muscle regeneration in WT, Cx3cr1<sup>-/-</sup>, Ccl2<sup>-/-</sup> and Cx3cr1<sup>-/-</sup> Ccl2<sup>-/-</sup> mice. *P<0.05 for Ccl2<sup>-/-</sup> and Cx3cr1<sup>-/-</sup> Ccl2<sup>-/-</sup> strains versus WT. Data are mean of two to four distinct experiments ± s.e.m. of at least six mice per strain per time point. (e) The four macrophage subsets from injured muscle were sorted by flow cytometry on day 4 post injury according to the gating strategy described in (b). RNA from one cell-sorting experiment, representing a pool of three mice per strain, was amplified and hybridized on the Illumina whole mouse genome oligo microarrays (WG6). Hierarchical clustered heat map of macrophage subsets 1, 2, 3 and 4 based on the gene expression of ten different clusters. Red: increased expression, green: decreased expression.

Mgl1 and CD206) than subset 4 (F4/80<sup>high</sup> Ly6C<sup>low</sup> Mgl1<sup>high</sup>; Fig. 2b and Supplementary Fig. 5). Cells from subsets 1 and 3 were the main mononuclear phagocyte subsets (about 86%) in muscle at the peak of inflammation, on day 4 post injury (Fig. 2c). In a previous study², we showed that Ly6C<sup>high</sup> monocytes in the muscle are recruited from the blood and are non-dividing cells. They switched their phenotype to Ly6C<sup>low</sup> mononuclear

phagocytes and actively proliferated *in situ*, contributing to the large amount of mononuclear phagocytes observed in the muscle at day 4 post injury.

Subset 1 was found in significantly lower numbers on day 1 (70% reduction, *P*<0.0006) as was subset 3 on day 4 (48% reduction, *P*<0.0001) in injured muscles from Ccl2<sup>-/-</sup> and Cx3cr1<sup>-/-</sup> Ccl2<sup>-/-</sup> compared with WT and Cx3cr1<sup>-/-</sup> mice
(Fig. 2d). This finding confirms that monocyte/macrophage
recruitment requires CCL2 (ref. 12). Conversely, CCL2 and
CX3CR1 deficiency did not or only slightly affected the
proportions of subsets 2 and 4. The greater impairment of the
recruitment of subsets 1 and 3 in the injured muscle of Ccr2⁻¹⁻
mice (Supplementary Fig. 6) suggests that their presence in the
lesion is necessary for the CX3CR1-dependent compensatory
effect. Strikingly, the infiltration kinetics of all mononuclear
phagocyte subsets of Ccl2⁻¹⁻ and Cx3cr1⁻¹⁻ Ccl2⁻¹⁻ mice,
as for WT and Cx3cr1⁻¹⁻ mice, were superimposable (Fig. 2d)
despite strongly different pathophysiological conditions. The
apparent lack of association between the muscle regeneration
rescue observed in Cx3cr1⁻¹⁻ Ccl2⁻¹⁻ mice and any change in
the number of monocytes infiltrating/macrophages proliferating
in the injured muscle suggests that the process allowing muscle
recovery in Cx3cr1⁻¹⁻ mice may be related to mononuclear
phagocyte functions. We next investigated the role of BM-derived
macrophages from the various mice strains regarding myogenic
precursor cells (MPCs) using in vitro assays. No differences were
observed in MPC proliferation or fusion (Supplementary Fig. 7).
Neither CX3CR1 nor CCR2 were detected on satellite cells¹³, on
myoblasts or on regenerating myofibers (data not shown).

To address more globally the mononuclear phagocyte func-
tions that may promote muscle regeneration, we performed a
genome-wide microarray analysis of the four mononuclear
phagocyte subsets from WT, Cx3cr1⁻¹⁻, Ccl2⁻¹⁻ and
Cx3cr1⁻¹⁻ Ccl2⁻¹⁻ muscles on day 4 post injury
(GSE73473). One-way analysis of variance analysis identified
434 genes (P<0.05) that distinguished the four subsets (Fig. 2e).
Using hierarchical clustering and pathway analysis, we identified
10 clusters that confirmed the original identity and functions of
each subset. Because muscle regeneration was not rescued
in Cx3cr1⁻¹⁻ Ccr2⁻¹⁻ mice (Supplementary Fig. 4) and
muscle infiltration of subsets 1 and 3 was fully abrogated
only in Ccr2⁻¹⁻ mice (Supplementary Fig. 5), we postulated
that these subsets were mainly responsible for this rescued
phenotype and sought to identify in them the genes that promote
healing.

**ApoE production is adversely regulated by CCL2 and CX3CR1.**
Comparison of the mononuclear phagocyte transcriptomic pro-
files from mice with poor regenerative abilities (Ccl2⁻¹⁻) and
from mice with effective muscle healing (WT, Cx3cr1⁻¹⁻ and
Cx3cr1⁻¹⁻ Ccl2⁻¹⁻) identified 22 and 17 genes significantly
differentially expressed in mononuclear phagocyte subsets 1 and
3, respectively (Fig. 3a,b). Among them, only ApoE transcripts
were downregulated in both Ccl2⁻¹⁻ subsets 1 and 3. We con-
firmed severely lower ApoE expression in macrophages isolated
from injured muscle of Ccl2⁻¹⁻ mice at both RNA (Fig. 3c) and
protein levels (Fig. 3d,e and Supplementary Fig. 8). In accordance
with the microarray data, ApoE expression was restored in
Cx3cr1⁻¹⁻ Ccl2⁻¹⁻ mice. Moreover, we found a higher
expression of ApoE in mononuclear phagocytes from CX3CR1-
deficient compared with CX3CR1-proficient mice (Fig. 3c-e). We
also showed that addition of CX3CL1 on WT macrophages
*in vitro* suppressed ApoE expression by about 30%, without
affecting CCL2 expression (Fig. 3f). These results reveal that
CX3CL1/CX3CR1 axis is also directly involved in the regulation
of ApoE expression.

Interestingly, most of the dysregulated genes in mononuclear
phagocytes from Ccl2⁻¹⁻ versus WT, Cx3cr1⁻¹⁻ and
Cx3cr1⁻¹⁻ Ccl2⁻¹⁻ mice can be linked in a network in which
ApoE is central and related to inflammatory response, endocy-
tosis signalling and the LXR/RXR activation pathway involved in
the regulation of inflammation and lipid metabolism³⁷ (Fig. 3g).

We previously published a functional link between ApoE and CX3CR1 in AMD³⁸. Sub-retinal macrophages in AMD patients as well as in sub-retinal inflammation observed in Cx3cr1⁻¹⁻ mice express high levels of ApoE. Previous studies indicate that ApoE is an immunomodulatory agent that polarizes macrophages in a M2 anti-inflammatory phenotype³⁹. We previously showed that M2 macrophages are associated with myofiber growth and myoblast differentiation and fusion³. In addition, ApoE can suppress the pro-inflammatory response mediated by microbial stimuli *in vivo*⁴⁰,⁴¹. Furthermore, the delay in skeletal muscle healing in ApoE⁻¹⁻ mice following hindlimb ischaemia-reperfusion⁴² suggests that this gene plays a major role in the muscle regeneration process.

**ApoE favours phagocytosis and muscle regeneration.** Histological analysis of injured muscle from ApoE⁻¹⁻ mice 10 days after injury revealed strongly impaired muscle regeneration, with a markedly lower CSA of regenerating myofiber and increased intermuscular fat and calcification (Fig. 4a). These results are consistent with previous reports that ApoE⁻¹⁻ mice develop calcification and accumulate lipid-filled foam cells within atheromatous plaque when fed a high-fat diet⁴³,⁴⁴. Using hydrodynamic-based *in vivo* transfection, we next showed that systemic expression of human ApoE3 improved muscle repair in Ccl2⁻¹⁻ mice (Fig. 4b). In addition, Cx3cr1⁻¹⁻ ApoE⁻¹⁻ bone-marrow transfer in irradiated Ccl2⁻¹⁻ mice did not promote myofiber growth as effectively as Cx3cr1⁻¹⁻ bone-marrow transfer (+33% myofiber CSA versus +58%; Fig. 4c). We also observed high reduction of myofiber growth in Cx3cr1⁻¹⁻ Ccl2⁻¹⁻ ApoE⁻¹⁻ mice compared with Cx3cr1⁻¹⁻ Ccl2⁻¹⁻ mice (Fig. 4d). These results demonstrate that the impairment of muscle regeneration in Ccl2⁻¹⁻ mice depends at least partially on the inability of muscle mononuclear phagocytes to produce enough ApoE and that CX3CR1 deficiency promotes and rescues Ccl2⁻¹⁻ muscle healing by regulating ApoE production by macrophages.

Because macrophage phagocytic activity is impaired in ApoE⁻¹⁻ (Supplementary Fig. 9) and improved in Cx3cr1⁻¹⁻ mice²¹,⁴⁵, we hypothesized that improved muscle regeneration in Cx3cr1⁻¹⁻ mice and rescue in Cx3cr1⁻¹⁻ Ccl2⁻¹⁻ mice might be linked to increased phagocytosis, and indeed we measured more phagocytic activity in muscles from Cx3cr1⁻¹⁻ compared with WT mice (Fig. 5a). The better necrotic myofiber removal in Cx3cr1⁻¹⁻ Ccl2⁻¹⁻ compared with Ccl2⁻¹⁻ mice (for an equivalent number of infiltrated mononuclear phagocytes) suggests that CX3CR1-deficient mononuclear phagocytes have higher phagocytic activity. *Ex vivo* experiments confirmed that muscle macrophages from Cx3cr1⁻¹⁻ and Cx3cr1⁻¹⁻ Ccl2⁻¹⁻ mice engulfed fluorescent microspheres more effectively than those from Ccl2⁻¹⁻ animals (Fig. 5b). Moreover, addition of exogenous APOE3 improved both phagocytic activity of Ccl2⁻¹⁻ muscle macrophages *in vitro* (Fig. 5c) and necrotic myofibre removal in Ccl2⁻¹⁻ mice (Fig. 5d). Addition of CCL2 restored phagocytic activity of Ccl2⁻¹⁻ macrophages (Fig. 5e, right panel) but not that of WT and Cx3cr1⁻¹⁻ macrophages. A reduction in WT macrophage phagocytic capacity was observed after incubation with CX3CL1 (Fig. 5e, left panel). Finally, the addition of CX3CL1 and CCL2 on WT macrophage-enriched muscular cell suspensions triggered similar expression of molecules involved in inflammatory response such as IL-1β, IL-1RA (IL-1 receptor antagonist) and iNOS (inducible nitric oxide synthase; Fig. 5f). Altogether, our results suggest that impaired muscle healing in Ccl2⁻¹⁻ mice is linked to decreases in monocyte/macrophage recruitment and function, notably phagocytosis.
<figure><img src="image_9.png" /><figcaption>**Figure 3** | ApoE macrophage production is adversely regulated by CCL2 and CX3CR1. (a) Venn diagrams illustrating the comparisons between the set of genes significantly differentially regulated in cell subsets 1 and 3 from Ccl2⁻/⁻ versus WT (in red), Cx3cr1⁻/⁻ (in green) and Cx3cr1⁻/⁻ Ccl2⁻/⁻ (in grey), mice. (b) Heat maps representing the positive (red) and negative (green) log2 ratio of gene expression in subsets 1 (22 genes) and 3 (17 genes) from Ccl2⁻/⁻ mice compared with WT, Cx3cr1⁻/⁻ and Cx3cr1⁻/⁻ Ccl2⁻/⁻ mice. ApoE expression in the global pool of mononuclear phagocytes extracted from injured muscle though magnetic columns (CD11b⁺ Ly6G⁻ NK1.1⁻) was analysed for each mouse strain on day 4 post injury by: (c) Real time-PCR (data are a mean + s.e.m. of three experiments). (d) Western blotting (one experiment representative of two; first lane: size marker in kDa), (e) immunolabelling and quantification of mean fluorescent intensity (MFI), with the appropriate threshold using image J software. (Data are a mean + s.e.m. of at least five fields counted (magnification of ×40) from two cell sorting experiments.) NS, not significant, \*P < 0.05, \*\*P < 0.01 and \*\*\*P < 0.001 for each knock-out strain versus wild type. (f) Modifications in ApoE and Ccl2 RNA expression induced after CX3CL1 (100 nM) *in vitro* stimulation during 16 h was analysed by real-time PCR in the global pool of mononuclear phagocytes extracted from WT mice, compared with Cx3cr1⁻/⁻. Data are a mean + s.e.m. for six mice per strain in three distinct experiments; NS, not significant, \*\*P < 0.01. (g) Network diagram showing the links between most of the genes differentially up- (red) or downregulated (green) in Ccl2⁻/⁻ mononuclear phagocytes compared with Cx3cr1⁻/⁻ Ccl2⁻/⁻ mononuclear phagocytes, inflammatory response, endocytosis signalling, LXR/RXR activation and lipid metabolism (Ingenuity Pathway analysis).</figcaption></figure>

Discussion

Skeletal muscle regeneration is a complex series of events, in
which monocytes/macrophages play a major part in the healing
process. Chemokines and their receptors orchestrate their

distribution and their functions to ensure muscle regeneration.
CX3CR1 and CCL2 deficiencies affect post-lesion blood mono-
cytosis differently: CX3CR1 deficiency is associated with a
reduction in Ly6C<sup>low</sup> monocytes, whereas CCL2 deficiency affects
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<figure><img src="image_14.png" /><figcaption>b</figcaption></figure>

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<figure><img src="image_19.png" /><figcaption>c Host: Ccl2−/− mice</figcaption></figure>

<figure><img src="image_20.png" /><figcaption>d</figcaption></figure>

<figure><img src="image_21.png" /></figure>
the number of Ly6Clow and Ly6Chigh monocytes (Supplementary Fig. 10). Because Ly6Clow blood monocytes have no role in the muscle regeneration process³,⁴⁶, their lack may not impair this process. Few groups including Saederup *et al*.⁴⁷ and our group¹⁸ have shown additive or synergistic effects of CCR2 and CX3CR1 axis on monocyte recruitment or macrophage accumulation. CCR2 and CX3CR1 axis may also lead to the opposite effect, and more specifically CX3CR1 deficiency may promote CCR2-dependent microglial cell accumulation in the retina³⁵. Here we could not detect any changes in monocyte recruitment or in macrophage accumulation consistently with our previous work showing that monocyte recruitment in the injured muscle was exclusively dependent on CCR2 axis³. In these conditions, CX3CR1 may only affect macrophage functions, not monocyte recruitment or macrophage proliferation.

CX3CR1 is an essential pleiotropic chemokine receptor involved in various biological functions, including cell chemo- taxis, apoptosis, cell activation/polarization, organ development and neovascularization. Its deficiency is considered deleterious in a large variety of acute inflammatory responses where it exacerbates inflammation²⁰,²⁴,³⁶. In chronic diseases, such as atherosclerosis, CX3CR1 deficiency also limits monocyte recruitment and macrophage accumulation at inflammatory sites leading to reduced atherosclerosis¹⁸,⁴⁸,⁴⁹. In contrast, CX3CR1 presence is somewhat protective in AMD⁵⁰. The role of CX3CR1 appears to be different depending on the inflammatory model. Our results show for the first time that CX3CR1 deficiency can improve wound healing in an acute injury model without affecting monocyte recruitment or macrophage accumulation. In the mdx mice, a chronic model of muscle injury (a murine model for Duchenne muscular dystrophy), previous studies using gene expression microarrays have demonstrated that dystrophic muscles are characterized by an inflammatory 'molecular signature'⁵¹,⁵², in which CC chemokines are prominent and among which CCL2 and its receptor CCR2 are highly expressed⁵³,⁵⁴. There is no evidence of CX3CR1/CX3CL1 expression in this context and the role of CX3CR1 has not yet been investigated. In a previous work⁹, we showed that four molecular systems including CX3CR1/CX3CL1 are involved in MPC apoptosis inhibition driven by macrophages *in vivo* and *in vitro*. Although apoptosis induction is fairly easy to achieve *in vitro* (through the incubation with various pro-apoptotic drugs), the MPC apoptosis events *in vivo* are rather rare, with apoptotic MPC representing only a very small number of cells in the myoblast population. In contrast, phagocytosis events during muscle repair are ubiquitous, and it is more likely that macrophages will engulf apoptotic MPC *in vivo* than protect them through the CX3CR1/CX3CL1 molecular system.

Figure 6 summarizes the main findings of the work and proposes a model of action of macrophages in muscle regeneration via ApoE production. We demonstrated that CX3CR1 deficiency is associated with increased removal of necrotic myofibers, as it is with the phagocytosis of amyloid-beta deposits in the brain of a murine model of Alzheimer's disease²¹. Similarly, CX3CR1-deficient mononuclear phagocytes overexpressed ApoE protein,

which is known to stimulate macrophage phagocytosis and dampen inflammatory response³⁹,⁴⁵. Moreover, a recent study³⁸ demonstrated a link between ApoE and CX3CR1 in AMD. Sub- retinal macrophages in AMD patients as well as in sub-retinal inflammation observed in Cx3cr1⁻/⁻ mice express high levels of ApoE. In addition, ApoE deletion in Cx3cr1⁻/⁻ mice prevents pathogenic- and stress-induced subretinal macrophage accumulation. Here we showed that the exogenous administration of ApoE enhanced CCL2-deficient mononuclear phagocyte phagocytic activity. These results suggest that defective CCL2- dependent monocyte recruitment may be compensated by CX3CR1 deficiency through the modulation of ApoE-dependent macrophage phagocytic activity. Therefore, therapeutic intervention to improve mononuclear phagocyte functions by targeting the CX3CR1 chemokine axis might control inflammation and enhance muscle regeneration in response to injury.

## Methods

**Mice.** Experiments were conducted on adult female and male mice (8–16 weeks old) of the following strains (all on a C57Bl/6J genetic background): C57Bl/6J (WT; purchased from Janvier), Cx3cr1⁻/⁻ (ref. 55), Ccl2⁻/⁻ (‡004434, JAX), Ccr2⁻/⁻ (‡004999, JAX), ApoE⁻/⁻ (‡002052, JAX), Cx3cr1gfp⁺ (‡008451, JAX) were intercrossed to obtain Cx3cr1⁻/⁻ Ccl2⁻/⁻, Cx3cr1⁻/⁻ ApoE⁻/⁻, Cx3cr1⁻/⁻ Ccl2⁻/⁻ ApoE⁻/⁻ and CX3CR1gfp/gfp ApoE⁻/⁻ as described in ref. 18. Mice were bred and maintained in our specific pathogen-free animal facility according to institutional guidelines and used according to the French legislation (no. A-75-1315). All experiment protocols were approved by the local ethics committee and validated by the Service Protection et Santé Animales, Environnement (no. A-75-2065).

**Muscle injury.** Muscle injury was caused by intramuscular injection of 10 µl of notexin (25 µg ml⁻¹ in PBS; Latoxan) into the tibialis anterior (TA) muscle. Muscle and fascia were harvested together for analysis at different time points post injury (1, 4, 7, 10, 14, 21 days). In some experiments, a pLIVE plasmid encoding for human ApoE3 was injected into mouse tail veins 6 h before muscle injury.

**Histological analysis.** TA muscles were removed, snap frozen in nitrogen-chilled isopentane and kept at -80 °C until use for histological analysis. Cryosections of 7 µm thick were prepared for various types of histological staining. Muscle regeneration was analysed quantitatively for the entire injured area (between 60 and 90% of the entire muscle). The percentages of necrotic and regenerating myofibers as well as the CSA of the regenerating myofibers, an established indicator, were quantified on haematoxylin/eosin-stained muscle sections recorded with an Olympus BX51 at ×10 magnification, connected to an evolution VF camera (medicaCybernetics). We used image J software to count three fields always chosen in the same overall injured area (representing 660 ± 150 myofibers) for at least six different mice analysed per time point. Regenerating myofiber density was assessed in the same fields and is expressed as the number of regenerating myofibers per field. Phagocytosis was assessed on day 4 post injury on muscle cross-sections stained with haematoxylin/eosin by counting the ratio of infiltrated to non-infiltrated necrotic myofibers. Vascularization was quantified on muscle cross-sections stained with wheat germ/Griffonia simplicifolia agglutinin. We counted 8 ± 1 fields (at ×20 magnification) chosen randomly over the entire injured area and representing 485 ± 160 myofibers, in three different mice. Areas of fat, calcification and fibrosis were evaluated over the entire injured area of three muscle cross-sections at levels spaced 900 µm apart, stained, respectively, with Oil Red O, Alizarin Red and Masson Trichrome and recorded with a Nikon AZ100 macroscope connected to a DS-Ri1 camera (Nikon). Appropriate thresholds were set with image J software to evaluate these areas. Each parameter was studied on days 4 (except fibrosis), 10 and 21 post injury in at least six different mice.

**Isolation of leukocytes and macrophages from muscle.** Mice were flushed with 10 ml of PBS to remove blood. Muscles and fascia were weighed together, minced

**Figure 4 | Macrophage ApoE production improves skeletal muscle regeneration.** (a) Analysis of muscle regeneration of ApoE⁻/⁻ (n = 11) versus WT mice (n = 7) on day 10 post injury, by measurement of mean CSA of regenerating myofibers, myofiber density and areas of fat and calcification. Data are mean ± s.e.m. of two distinct experiments. (b) Analysis on day 10 post injury of muscle regeneration in Ccl2⁻/⁻ mice, transfected (n = 3) or not (n = 3) with a pLIVE plasmid encoding for human ApoE3, by measurement of mean CSA of regenerating myofibers, myofiber density and areas of fat and calcification. In this experimental system, systemic expression of ApoE3 was detected for at least 8 days after transfection (data not shown). Data are mean ± s.e.m. of a single experiment. Scale bar, 100 µm. (c) Analysis of muscle regeneration in chimeric Ccl2⁻/⁻ mice engrafted with Ccl2⁻/⁻ (n = 5), Cx3cr1⁻/⁻ (n = 5) or Cx3cr1⁻/⁻ ApoE⁻/⁻ (n = 4) bone marrow, by measurement of the mean CSA of regenerating myofibers on day 21 post injury. (d) Analysis of muscle regeneration in Cx3cr1⁻/⁻ Ccl2⁻/⁻ mice (n = 4) and Cx3cr1⁻/⁻ Ccl2⁻/⁻ ApoE⁻/⁻ mice (n = 4). Data are mean ± s.e.m.; NS, not significant, \*P < 0.05, \*\*P < 0.01 and \*\*\*P < 0.001.
<figure><img src="image_22.png" /><figcaption>**Figure 5** | Macrophage ApoE production enhances muscle debris phagocytosis. (a) Quantification of *in vivo* phagocytosis, by assessment of the numbers of infiltrated necrotic myofibers versus total necrotic myofibers infiltrated or not on day 4 post injury. Scale bar, 100 µm. Data are means ± s.e.m. of six mice per strain in two independent experiments. (b) Phagocytosis activity of mononuclear phagocytes extracted from injured-muscle of WT, *Cx3cr1*⁻/⁻, *Ccl2*⁻/⁻ and *Cx3cr1*⁻/⁻ *Ccl2*⁻/⁻ mice on day 4 post injury was assessed by incubating muscle cells for 4 h with red fluorescent microspheres. This parameter is expressed as the number of cells that engulfed at least one bead per 10,000 cells. Results are means ± s.e.m. of six mice in three independent experiments. (c) Phagocytosis activity of *Ccl2*⁻/⁻ muscle mononuclear phagocytes in the absence or presence of exogenous human APOE3 protein. Results are mean ± s.e.m. of five mice in two independent experiments. (d) Quantification on day 4 post injury of *Ccl2*⁻/⁻ myofiber removal in the absence (*n* = 2) or presence (*n* = 4) of exogenous human APOE3 protein. (e) Phagocytosis activity of mononuclear phagocytes from injured-muscle of WT, *Cx3cr1*⁻/⁻ and *Ccl2*⁻/⁻ mice on day 4 post injury was assessed 16 h after incubation of muscle cells with CX3CL1 or CCL2 chemokines. The numbers of muscle cells that engulfed at least one fluorescent microsphere were compared between treated and untreated cells for all mice strains. (f) Effect of CX3CL1 and CCL2 on IL-1, IL-1 receptor antagonist (Ra) and iNOS (nitric oxide synthase) expression was measured by real-time PCR in mononuclear phagocytes extracted from WT mice incubated during 16 h with CX3CL1 (100 nM) or CCL2 (100 nM), and compared with untreated cells. Data are mean ± s.e.m. of six mice per strain in three independent experiments. NS, not significant, \*P\<0.05, \*\*P\<0.01 and \*\*\*P\<0.001.</figcaption></figure>

<figure><img src="image_23.png" /><figcaption>**Figure 6** | Macrophage infiltration and function during muscle regeneration. Diagram depicting macrophage infiltration and functions during muscle regeneration in WT, *Cx3cr1*⁻/⁻, *Ccl2*⁻/⁻ and *Cx3cr1*⁻/⁻ *Ccl2*⁻/⁻ mice.</figcaption></figure>

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## Acknowledgements

This work was supported by the European Community's Seventh Framework Project FP7-Health-2009 ENDOSTEM 241440, Association Française contre les Myopathies (AFM) 15775, the Institut National de la Santé et de la Recherche Médicale (INSERM) and the Université Pierre Marie Curie (UPMC). We are deeply grateful to Olivier Levy and Florian Sennlaub from the 'Institut national de la vision' for the generous gift of ApoE3-pLIVE and pLIVE plasmid controls.

## Author contributions

L.A. and H.P. designed and performed the experiments, analysed the data and wrote the manuscript. C.B.deC., M.S., P.H., L.P., E.G., F.L., J.V., R.M. and W.C. performed the experiments and analysed the data. B.Ch. and A.B. gave technical support and conceptual
advice. N.B. conducted all bioinformatics analyses. B.C. and C.C. designed experiments, wrote the manuscript and supervised the project.

**Additional information**

**Accession number:** Microarray data have been deposited in the Gene Expression Omnibus (GEO) database (http://www.ncbi.nlm.nih.gov/gds) under accession code GSE73473.

**Supplementary Information** accompanies this paper at http://www.nature.com/naturecommunications

**Competing financial interests:** The authors declare no competing financial interests.

**Reprints and permission** information is available online at http://npg.nature.com/reprintsandpermissions/

**How to cite this article:** Arnold, L. et al. CX3CR1 deficiency promotes muscle repair and regeneration by enhancing macrophage ApoE production. *Nat. Commun.* 6:8972 doi: 10.1038/ncomms9972 (2015).

<figure><img src="image_24.png" /></figure>

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