*3.1. Nfix is Expressed by Anti-Inflammatory MPs*

To understand if the transcription factor Nfix could be involved in MP function, we first analyzed Nfix expression in MPs during normal muscle regeneration in WT mice. We induced muscle injury by cardiotoxin (CTX) injection in the *Tibialis Anterior* (TA) and looked at the number of MPs (F4/80<sup>+</sup> cells) positive for Nfix (Figure 1a). While the number of Nfix-positive MPs was identical between day two (D2) and day four (D4) after injury, we observed an increase of MPs expressing Nfix at D7 after CTX injection (Figure 1a). During muscle regeneration, two populations of MPs are present in the damaged tissue. First, the Ly6C<sup>+</sup> pro-inflammatory MPs appear and then, they switch into Ly6C<sup>−</sup> anti-inflammatory population [3,9,29,34]. Thus, we asked if Nfix could be expressed by one subset of MPs. We sorted MPs after CTX injury at different time points and looked at Nfix expression by immunolabelling. We firstly enriched for CD45<sup>+</sup> cells (mainly composed of MPs and neutrophils) using magnetic beads and then we used the known markers CD64 and Ly6C to separate the pro-inflammatory (CD64+/Ly6C<sup>+</sup> cells) to the anti-inflammatory MPs (CD64+/Ly6C<sup>−</sup> cells) (Figure S1). We observed that the percentage of pro-inflammatory MPs expressing Nfix does not change over the time of the regeneration (Figure 1b). On the contrary, the percentage of CD64+/Ly6C<sup>−</sup> cells positive for Nfix always increased over time (Figure 1b). We also isolated BMDMs (bone marrow derived MPs) from WT mice and polarized them in pro- or anti-inflammatory phenotype. We observed that anti-inflammatory MPs express more Nfix compared to pro-inflammatory MPs (Figure 1c). Therefore, we can conclude that in both in vitro and in vivo analyses, Nfix is more expressed by anti-inflammatory MPs.

**Figure 1.** Nfix is mainly expressed by anti-inflammatory MPs. (**a**) Percentage of F4/80<sup>+</sup> MPs positive for Nfix in *Tibialis Anterior* muscles (TA) of WT mice injected by CTX at D2, D4 and D7, post-injury. Immunostaining for F4/80 (green), Nfix (red) and DAPI (blue) at D4 and D7 after CTX injection; (**b**) Percentage of Ly6C<sup>+</sup> and Ly6C- sorted MPs positive for Nfix in TA muscles of WT mice injected by CTX at D2, D4 and D7 post-injury; (**c**) Percentage of Nfix<sup>+</sup> MPs after M1 and M2c polarization (with IFNγ and IL10, respectively). \* *p* < 0.05; \*\*\* *p* < 0.001; for (b) \* *p* < 0.05 Ly6C+ vs. Ly6C<sup>+</sup> at D4 and D7; # *p* < 0.05 Ly6C<sup>−</sup> D7 vs. D2. Results are means ± SEM of at least three independent experiments. Scale bar = 50 μm.

#### *3.2. Nfix Expression in MPs is Essential for Muscle Regeneration*

We previously demonstrated that Nfix is necessary for the correct differentiation of SCs and, as a consequence, muscle regeneration [27]. In order to understand whether Nfix expression by anti-inflammatory MPs is required for this process, we generated the LysMCRE:Nfixfl/fl mice to obtain an animal model deleted for Nfix only in the myeloid line. Once the proper deletion of Nfix in BMDM and CD45<sup>+</sup> infiltrated cells two days after muscle injury was verified (Figure S2a), we evaluated the overall phenotype of this new animal model, with particular interest in skeletal muscle morphology at one and two months of life (Figure S2b). No differences were observed between the Nfixfl/fl control and the LysMCRE:Nfixfl/fl mice model in terms of general mouse growth, TA/mouse weight and myofiber size (CSA: Cross Sectional Area) (Figure S2b,c). Additionally, we did not observe significant differences in the number of resident MPs expressing Nfix between the Nfixfl/fl and the LysMCRE: Nfixfl/fl mice (Figure S2d,e). Therefore, the specific deletion of Nfix in MPs in the LysMCRE: Nfixfl/fl mice does not influence the general development of the mice. Notably, in the LysMCRE: Nfixfl/fl animals resident MPs expressed Nfix similarly to control mice, meaning that the expression of LysM is not required for the establishment of resident MPs.

We then induced muscle injury in control and LysMCRE: Nfixfl/fl mice by CTX injection in TA and we quantified the number of necrotic, phagocyted and regenerating myofibers (Figure 2a,b). Two days after injury, all the myofibers of Nfixfl/fl and LysMCRE: Nfixfl/fl mice were in necrosis or phagocyted (Figure 2b). At D4 in the control mice, some necrotic and phagocyted myofibers were present, but already 60% of the fibers were centronucleated (Figure 2b). On the contrary, in the LysMCRE:Nfixfl/fl mice, we observed a significant decrease in the percentage of centronucleated myofibers (−31%) (Figure 2b). While at D7, almost all myofibers were in regeneration in the control mice, the LysMCRE:Nfixfl/fl mice still exhibited an increase of the percentage of necrotic and phagocyted myofibers and a decrease of centronucleated myofibers (+282%, +150% and −30% respectively), suggesting a delay in the process of muscle regeneration in the absence of Nfix (Figure 2b). We also quantified the CSA of myofibers at D14 and D28 after CTX injury and, in both cases, we observed a decrease of the caliber of myofibers in the LysMCRE:Nfixfl/fl compared to the Nfixfl/fl mice, due to

a decrease of the number of big myofibers and an increase of small myofibers (Figure S3a and Figure 2c). These results demonstrated that the expression of Nfix by MPs is necessary for the proper process of muscle regeneration upon acute injury.

**Figure 2.** Lack of Nfix in MPs induces a delay of skeletal muscle regeneration. (**a**) Hematoxylin-eosin staining of Nfixfl/fl and LysMCRE:Nfixfl/fl TA muscles injected by CTX at D2, D4, D7 postinjury; (**b**) Quantification of necrotic (asterisk), phagocyted (arrowhead) and centrally-nucleated (arrow) myofibers, expressed as percentage out of total myofibers; (**c**) Hematoxylin-eosin staining of Nfixfl/fl

and LysMCRE:Nfixfl/fl TA muscles injected by CTX at D14 and D28 postinjury and repartition in percentage of the cross-sectional area (CSA). \* *p* < 0.05, \*\* *p* < 0.01, \*\*\* *p* < 0.001. Results are means ± SEM of at least three independent experiments. Scale bar = 100 μm.

#### *3.3. Nfix is Required for MP Phenotypical Switch In Vivo and In Vitro*

Defects of muscle regeneration due to MP dysfunction are usually linked to a defect of phenotype acquisition [8,9,35,36]. Thus, we looked at the switch from pro- to anti-inflammatory phenotype after CTX injury at different time points by FACS (Figure 3a). First, we did not observe any differences in neutrophils and MPs infiltration between the two mouse models at all time points analyzed (Figure S3b). At two days after injury, a majority of Ly6C<sup>+</sup> pro-inflammatory MPs was observed in the control Nfixfl/fl mice (Figure 3b). Then, at D4 and D7, the ratio between Ly6C+/Ly6C<sup>−</sup> MPs decreased due to the switch from pro- to anti-inflammatory phenotype (Figure 3b). Interestingly, the ratio between Ly6C+/Ly6C<sup>−</sup> in the LysMCRE:Nfixfl/fl mice was always higher compared to the Nfixfl/fl control, meaning that Nfix is necessary for the switch from the pro- to anti-inflammatory phenotype (Figure 3b).

We also silenced Nfix in WT BMDM (Bone Marrow Derived MPs) by using a lentiviral vector carrying a small hairpin RNA targeting Nfix (shNfix), or a scrambled sequence as a control (shScramble) [27]. The decrease of Nfix expression in shNfix infected MPs was confirmed by qRT-PCR (Figure S3c). We polarized transduced MPs in pro-inflammatory (M1) and anti-inflammatory (M2c) phenotype (with IFN-γ and IL-10, respectively), and we looked at the expression of several proand anti-inflammatory markers by immunofluorescence. As expected, M1 shScramble MPs expressed significantly more TNFα, iNOS and CCl3 pro-inflammatory markers than M2c shScramble MPs (Figure 3c). Conversely, M2c shScramble MPs expressed more ArgI, TGFβ, CD163 and CD206 anti-inflammatory markers than M1 shScramble MPs (Figure 3c). Interestingly, in the absence of Nfix we observed an increase of pro-inflammatory markers (except for CCl3) in MPs polarized to M2c phenotype (Figure 3c). We also observed a decrease of MPs positive for anti-inflammatory markers in the polarized M2c MPs lacking Nfix (Figure 3b). These results clearly show that Nfix is necessary for the proper adoption of an anti-inflammatory phenotype and that, without Nfix, MPs remain in a pro-inflammatory status.

#### *3.4. Nfix is Required for Macrophage Function on Mpcs In Vivo and In Vitro*

Since depending on their phenotype MPs act differentially on WT myogenic progenitor cells (mpcs), we set experiments of conditioned medium (CM) coming from pro- or anti-inflammatory MPs [37]. We added CM coming from BMDM derived from Nfixfl/fl and LysMCRE:Nfixfl/fl mice on proliferating or differentiating mpcs. We looked at mpc proliferation by mean of Ki67 staining and at their differentiation by the quantification of the fusion index. As expected, CM coming from M1 Nfixfl/fl MPs stimulated the proliferation of mpcs, while M2c CM had no effect (Figure 4a and Figure S4a). Similarly, CM coming from M1 LysMCRE:Nfixfl/fl MPs stimulated at the same extent the proliferation of mpcs (Figure 4a and Figure S4a). Interestingly, CM from M2c LysMCRE:Nfixfl/fl MPs stimulated the proliferation of mpcs as M1 CM does (Figure 4a and Figure S4b), whereas CM coming from both Nfixfl/fl and LysMCRE:Nfixfl/fl M1 MPs had no effect on the fusion index of mpcs (Figure 4b and Figure S4b). While M2c Nfixfl/fl MPs CM increased the fusion of mpcs compared to M1 Nfixfl/fl CM, the CM from M2c LysMCRE:Nfixfl/fl MPs lost its pro-fusion effect (Figure 4b and Figure S4b ). We also investigated if the lack of Nfix in MPs affects myogenic cells in vivo. To quantify mpcs proliferation, we injured, using CTX, the TA of both Nfixfl/fl and LysMCRE:Nfixfl/fl mice and we injected EdU 12 h before sacrifice. The proliferation of SCs (EdU+/Pax7<sup>+</sup> cells) was identical between the two models at all the time points analyzed (Figure S4c). On the contrary, the percentage of EdU+/MyoD<sup>+</sup> cells at D2 and D4 was higher in the LysMCRE:Nfixfl/fl mice compared to Nfixfl/fl mice (Figure 4c). To quantify the number of newly formed myofibers, we performed an immunofluorescence for eMyHC (embryonic Myosin Heavy Chain) on both injured mouse models (Figure 4d). At D4, the percentage of myofibers positive for eMyHC was around 80%, meaning that almost all myofibers were formed de

novo (Figure 4d). At D7, 20% of the Nfixfl/fl myofibers were positive for the eMyHC and only 6.5% at D14 (Figure 4d). On the contrary, at D7 and D14 we observed an increase in the number of eMyHC<sup>+</sup> myofibers in the LysMCRE:Nfixfl/fl mice (34.7% and 15.8% respectively) (Figure 4d). To conclude, Nfix is necessary to MPs to adopt an anti-inflammatory phenotype and, consequently, function. The defect of the phenotypical switch due to the absence of Nfix results in a persistence of pro-inflammatory MPs in the injured muscle, leading to a continuous proliferation of MyoD<sup>+</sup> cells. The absence of anti-inflammatory MPs induces a delay in the differentiation of new myofibers and, therefore, in the proper muscle regeneration.

**Figure 3.** MPs lacking Nfix are unable to adopt an anti-inflammatory phenotype in vivo and in vitro. (**a**) Representative FACS (Fluorescence-Activated Cell Sorting) gate of pro- and anti-inflammatory CD64<sup>+</sup> MP populations in TA of Nfixfl/fl and LysMCRE:Nfixfl/fl mice at D2 after CTX injection. (CD64+/Ly6C<sup>+</sup> and CD64+/Ly6C<sup>−</sup> respectively); (**b**) Ratio of Ly6C+/Ly6C<sup>−</sup> MPs sorted from TA of Nfixfl/fl and LysMCRE:Nfixfl/fl mice at D2, D4 and D7, after CTX injection; (**c**) WT BMDM (Bone Marrow Derived Macrophages) were transduced by shScramble and shNfix lentiviral vectors and then polarized into M1 and M2c MPs with IFNγ and IL10 treatment, respectively. MPs were immunolabeled for pro-inflammatory markers (TNFα, iNOS and CCl3) and anti-inflammatory markers (ArgI, TGFβ, CD163 and CD206). The number of positive cells is expressed as percentage out of total cells. \* *p* < 0.05;

\*\* *p* < 0.01; \*\*\* *p* < 0.001 vs. shScramble M1 MPs. # *p* < 0.05, ## *p* < 0.01, ### *p* < 0.001 vs. shScramble M2c MPs. Results are means ± SEM of at least three independent experiments.

**Figure 4.** M2 MPs lacking Nfix display M1 MP features on myoblasts in vitro and in vivo. (**a**) Conditioned medium of M1 or M2c polarized Nfixfl/fl and LysMCRE:Nfixfl/fl BMDM was added on mpcs, and after 24 h, mpc proliferation was measured as a percentage of Ki67<sup>+</sup> cells; (**b**) Conditioned medium of M1 or M2c polarized Nfixfl/fl and LysMCRE:Nfixfl/fl BMDM was added on mpcs and after 72 h, mpcs fusion index was calculated after sarcomeric MyHC staining (% of MyHC+nuclei into myotubes out of the total nuclei). \* *p* < 0.05, \*\* *p* < 0.01 vs. mpcs. # *p* < 0.05, ## *p* < 0.01 versus M1 Nfixfl/fl. \$ p < 0.05 versus same Nfixfl/fl polarization; (**c**) Immunostaining for EdU (red), MyoD (green) and Hoechst (blue) of Nfixfl/fl and LysMCRE:Nfixfl/fl TA injected by CTX, at D2, D4 and D7 post-injury and quantification of EdU+/MyoD<sup>+</sup> cells. EdU was injected in Nfixfl/fl and LysMCRE:Nfixfl/fl mice 8 h before sacrifice; (**d**) Immunostaining for Lam (red), eMyHC (green) and Hoechst (blue) of Nfixfl/fl and LysMCRE:Nfixfl/fl TA injected by CTX, at D4, D7 and D14 post-injury and quantification of eMyHC+/centrally-nucleated myofibers. \* *<sup>p</sup>* <sup>&</sup>lt; 0.05, \*\* *<sup>p</sup>* <sup>&</sup>lt; 0.01 Results are means <sup>±</sup> SEM of at least three independent experiments. Scale bar = 50 μm.

#### *3.5. Phagocytosis Induces the Expression of Nfix*

It has been shown in literature that the phagocytosis of apoptotic cells is the process driving the switch from pro- to anti-inflammatory phenotype, and several studies have demonstrated that MPs presenting a switch defect have a decrease of phagocytic capacity [8,9,35,38]. So, we decided to investigate whether the phagocytosis is altered in LysMCRE:Nfixfl/fl MPs compared to Nfixfl/fl MPs. Primary mpcs previously labelled with CellVue-647 were induced to apoptosis and added on Nfixfl/fl or LysMCRE:Nfixfl/fl MPs. After 6 h in culture, we used a CD64 antibody to discriminate MPs from mpcs: apoptotic mpcs are CellVue-647+/CD64- , non-phagocytic MPs are CellVue-647−/CD64<sup>+</sup> and phagocytic MPs are CellVue-647+/CD64<sup>+</sup> (Figure S5a). Surprisingly, we did not observe any difference in the phagocytic capacity of Nfixfl/fl and LysMCRE:Nfixfl/fl MPs (Figure 5a). Interestingly, while Nfixfl/fl MPs in contact with apoptotic mpcs adopted an anti-inflammatory phenotype (Figure S5b), LysMCRE:Nfixfl/fl MPs failed to switch from a pro- to anti-inflammatory phenotype (Figure S5c). Thus, we hypothesized that phagocytosis could induce Nfix expression. To answer to this question, we did the same experiment of phagocytosis using WT MPs and we sorted MPs according to their phagocytic capability (phagocytic and non-phagocytic WT MPs, respectively) (Figure 5b). To verify that no mpcs were sorted with MPs, we first analyzed the expression of myogenin in apoptotic mpcs and in both non-phagocytic and phagocytic MPs. Apoptotic mpcs highly expressed myogenin compared to non-phagocytic and phagocytic MPs and no differences in myogenin expression was observed between the two populations of MPs (Figure S5d). Interestingly, we observed an increase of Nfix expression and MPs positive for Nfix in phagocytic MPs compared to the non-phagocytic ones (Figure 5b). On the contrary, treatment of MPs with cytochalasin D (an inhibitor of phagocytosis) prevents the increase of Nfix positive MPs (Figure 5c and Figure S5e).

We recently demonstrated that the inhibition of the RhoA-ROCK1 pathway induces Nfix expression in fetal myoblasts and numerous studies have shown that the inhibition of RhoA-ROCK1 increases the phagocytosis, while its stimulation prevents phagocytosis [39–43]. Thus, we treated WT MPs with Y27632, an inhibitor of ROCK1, and after 1 h of treatment, the phosphorylation of ROCK1-target Mypt decreased, meaning that the inhibition of RhoA-ROCK1 pathway was effective (Figure S5e). After 16 h of treatment, Y27632-treated WT MPs exhibited an increase of Nfix protein (Figure 5d); most importantly, this increase led to a reduction of pro-inflammatory markers and an increase of anti-inflammatory markers (Figure 5e). On the contrary, this switch through an anti-inflammation phenotype did not occur in LysMCRE:Nfixfl/fl MPs treated with Y27632 (Figure 5f). These results show that RhoA-ROCK-dependent phagocytosis induces the expression of Nfix, which in turn is necessary to promote the phenotypical switch of MPs from pro- to anti-inflammatory.

**Figure 5.** *Cont*.

**Figure 5.** Nfix is expressed after phagocytosis and drive MP phenotypical switch. (**a**) Phagocytosis assay of M1 and M2c Nfixfl/fl and LysMCRE:Nfixfl/fl MPs cocultured 8h with apoptotic mpc. Representative FACS gate of phagocytotic M2c Nfixfl/fl and LysMCRE:Nfixfl/fl MPs (CD64+CellVue+) and percentage of phagocytotic M1 and M2c MPs coming from Nfixfl/fl and LysMCRE:Nfixfl/fl BMDM; (**b**) WT MPs were cocultured 16h with apoptotic mpcs. Representative FACS gate of non-phagocytotic (CD64+CellVue−) and phagocytotic (CD64+CellVue+) WT MPs. Quantification of Nfix expression realized by RT-qPCR on sorted non-phagocytotic and phagocytotic WT MPs and quantification of MPs positive for Nfix (Nfix+/F4/80+) realized by IF on non-phagocytotic and phagocytotic WT MPs; (**c**) WT MPs were cocultured for 16 h with apoptotic mpcs, with or without addition of Cytochalasin D. Quantification of F4/80<sup>+</sup> MPs were positive for Nfix on a total of F4/80<sup>+</sup> MPs; (**d**) Western blot of Nfix expression in WT MPs treated with DMSO (Dimethyl sulfoxide) or Y27632 for 16 h and quantification. Vinculin was used to normalize; (**e**) WT MPs were treated with DMSO or Y27632 for 16 h and were immunolabeled for pro-inflammatory markers (iNOS and TNFα) and anti-inflammatory markers (TGFβ and CD163). The number of positive cells is expressed as percentage out of total cells; (**f**) LysMCRE:Nfixfl/fl MPs were treated with DMSO or Y27632 for 16 h and were immunolabeled for pro-inflammatory markers (iNOS and TNFα) and anti-inflammatory markers (TGFβ and CD163). The number of positive cells is expressed as percentage out of total cells. \* *p* < 0.05, \*\*\* *p* < 0.001. Results are means ± SEM of at least three independent experiments.

#### **4. Discussion**

Skeletal muscle regeneration requires specific temporal steps for the efficacious tissue reconstruction and MPs are the immune cells that are necessary to this process [3,29]. Previous work from our group demonstrated that *Nfix* null mice exhibit a delay of muscle regeneration, due to a defect of SC differentiation [27]. In this study, we show that the transcription factor Nfix is also expressed by MPs and that mice lacking Nfix in the myeloid lineage have defects in muscle regeneration upon acute injury. We observed that Nfix is preferentially expressed by anti-Ly6C− MPs and that its expression increases in time with the progression of the regenerative process. Using an shNfix strategy, we observed that M2c MPs silenced for Nfix express higher levels of pro-inflammatory markers (TNFα and Cox2), while they express lower levels of anti-inflammatory markers (CD163, CD206, ArgI and TGFβ) than polarized M2c control MPs. Importantly, we observed in vitro that LysMCRE:Nfixfl/fl M2c MPs act as M1 MPs on myogenic cells: they stimulate myogenic proliferation and are unable to sustain myogenic differentiation. These two features also occur in vivo since without Nfix, MPs exhibit a defect of phenotypical switch from pro-Ly6C<sup>+</sup> to anti-Ly6C<sup>−</sup> MPs, and within the injured muscle, there is a persistence of myoblast (MyoD<sup>+</sup> cells) proliferation and a delay of newly formed myofibers (eMyHC<sup>+</sup> myofibers). Previous studies showed that the temporal window of the phenotype skewing is a critical step of an effective regeneration. The switch defect [8,35,36] or early appearance of anti-inflammatory MPs impairs muscle regeneration [9]. In line with this evidence, the impairment in the acquisition of an anti-inflammatory phenotype in MPs lacking Nfix leads to a muscle regenerative delay.

So far, the function of Nfix was mainly analyzed in myogenic and neural cells during both development and adult life [25,27,44–46]. Recently, Nfix was also shown to play a positive role in the survival of hematopoietic stem and progenitor cells (HSPC) [47], but also to be involved in the fate decision between early B lymphopoiesis and myelopoiesis from blood HSPC [48]. During development, yolk sac gives rise to tissue resident MPs and fetal liver to HSPC, from which blood monocytes and damaged-infiltrating MPs are derived [49]. In our experiments, no differences in the number of infiltrating MPs between control and LysMCRE:Nfixfl/fl mice were observed, meaning that the delay observed is due to a defect of macrophage features within the damaged muscle, but not in terms of failed HSPC development.

Little is known about Nfix up-stream regulation, but recently our laboratory identified ERK and RhoA-ROCK1 pathways as, respectively, positive and negative regulators of Nfix expression in pre-natal muscle development. The inhibition of RhoA-ROCK1 induces Nfix expression, promoting myoblasts fusion which is a reflect of myogenesis progression [39]. In MPs, the inhibition of the RhoA-ROCK1 pathway increases the clearance of dead cell phagocytosis, while the constitutive activation of RhoA reduces their phagocytic capacity [40,41]. Importantly, phagocytosis is the process responsible for the induction of the pro- to anti-phenotypical switch in MPs. While numerous studies investigated the mechanisms involved in the progression or inhibition of phagocytosis, how apoptotic cells attract MPs and how MPs recognize them is still unknown [10,14,15,50,51]. In our study, phagocytosis of LysMCRE:Nfixfl/fl MPs was not impaired compared to control cells. We observed that upon phagocytosis, MPs exhibit an increase in Nfix expression and, conversely, the inhibition of phagocytosis, by using the inhibitor of actin polymerization cytochalasin D, prevents Nfix expression. The stimulation of phagocytosis using the ROCK1 inhibitor Y27632 increases Nfix protein, therefore decreasing pro-inflammatory markers and increasing anti-inflammatory markers in WT MPs. On the contrary, either after phagocytosis or after ROCK1 inhibitor treatment, we observed that MPs lacking Nfix do not have a decrease of pro-inflammatory markers and an increase of anti-inflammatory markers. Thus, the inhibition of the RhoA-ROCK1 pathway induces phagocytosis, leading to Nfix expression that, in turn, drives the MP phenotypical switch.

This study is particularly relevant in light of the recent role for Nfix in muscular dystrophies (MDs)[28]. We indeed demonstrated that the lack of Nfix in two different dystrophic animal models improves both morphological and functional parameters associated to the disease, by promoting a more oxidative musculature and by slowing down muscle regeneration [28]. Different studies have shown that the improvement of dystrophies correlates with a decrease of MPs infiltration [30,32]. While MPs are necessary for muscle regeneration upon acute injury, they are deleterious in the case of chronic injury. Indeed, in muscle myopathies, as in several chronic injured pathologies, MPs are at the origin of fibrosis [4,32,52,53]. In the context of acute injury regeneration, pro-inflammatory MPs secrete TNFα that stimulates myoblast proliferation and fibroblast apoptosis, whereas anti-inflammatory MPs secrete TGFβ that promotes myoblast fusion, but also fibroblast proliferation [29,54]. In muscular dystrophies, numerous studies demonstrated that the fibrosis establishment is linked to an over-activation of

the TGFβ pathway that stimulates collagen expression by fibroblasts, and in a dystrophic context, more than 75% of MPs express TGFβ [54–62]. Thus, in muscle tissue, MPs closely interact with fibroblasts, promoting normal matrix reformation upon acute injury and fibrosis in chronic injury. With this study, we identified Nfix as a new actor of MPs, demonstrating that Nfix is the link between phagocytosis and the phenotypical switch, a necessary step for the resolution of inflammation and tissue repair. Increasing knowledge about signals and factors controlling MP phenotype and, consequently, functions, will help us to understand and control their function in fibrotic pathologies.

**Supplementary Materials:** The following are available online at http://www.mdpi.com/2073-4409/9/3/708/s1, **Figure S1**: Gating strategy to isolate MPs from CTX-injured muscles. Figure S2: Characterization of the LysMCRE:Nfixfl/fl mice. Figure S3: CSA quantification, NT and MPs infiltration in Nfixfl/fl and LysMCRE:Nfixfl/fl mice after CTX injury and Nfix silencing in WT BMDM. Figure S4: In vitro proliferation and differentiation assay. Proliferation of Pax7<sup>+</sup> cells after CTX injury. Figure S5: Phagocytosis strategy, inhibition and stimulation.

**Author Contributions:** Conceptualization, M.S. and G.M.; methodology, M.S., M.L., C.B. and S.A.; validation, M.S., M.L., C.B. and S.A.; formal analysis, M.S.; investigation, M.S., M.L., C.B, G.R. and S.A.; resources, G.M.; data curation, M.S.; writing—original draft preparation, M.S.; writing—review and editing, G.M.; visualization, M.S.; supervision, G.M.; project administration, G.M.; funding acquisition, G.M. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by the European Community, ERCStG2011 (RegeneratioNfix 280611) and the Association Française contre les Myopathies AFM-Telethon (Grant number 20002).

**Acknowledgments:** We thank Richard Gronostajski for the kind exchange of information and animal models. We are also grateful to Bénédicte Chazaud and Rémi Mounier for helpful discussions and the exchange of animal models.

**Conflicts of Interest:** The authors declare no conflict of interest.
