**1. Introduction**

During their lifetime, tissues encounter physiological and non-physiological damages and an effective regeneration is necessary to make these tissues able to continuously sustain their biological functions. Macrophage-mediated inflammation is a fundamental step for tissue recovery. Macrophages (MPs) are immune cells required for tissue regeneration, as their depletion prevents regeneration of different tissues/organs, such as liver [1], spinal cord [2] and skeletal muscle [3]. In several regenerative processes, two populations of MPs have been described. The first population reaching the damaged tissue is the pro-inflammatory population, also called M1 MPs. Pro-inflammatory MPs secrete pro-inflammatory molecules, being the main actors of dead cell clearance. The second population is composed of the anti-inflammatory MPs, named M2 MPs, that come from pro-inflammatory MPs and are involved in the resolution of the inflammation, wound healing and tissue regeneration or repair [4–7]. Several studies have shown that an impaired or a precocious phenotypical switch

from M1 to M2 MPs results in defective tissue regeneration [1,8,9]. Interestingly, it has been observed that phagocytosis is at the basis of the pro to anti-inflammatory phenotypical switch in MPs [3,8,10–13]. Although the process of the induction of phagocytosis is well-known ("find-me", "eat-me" and "don't eat-me" signals), the molecular and transcriptional pathways between phagocytosis and the phenotypical switch are still unexplored [10,11,14,15].

Interestingly, the interplay between MPs and tissue regeneration has been widely documented in skeletal muscle [7,16–20]. In vertebrates, muscle progenitors originate from pre-somitic and cranial mesoderm. In pre-natal period, two myogenic waves are necessary for muscle establishment: the first forms the basic muscle pattern and is called primary or "embryonic" myogenesis, while the second or "fetal" myogenesis is characterized by muscle maturation and growth [21]. Adult skeletal muscle is able to regenerate thanks to resident stem cells called satellite cells (SCs), located under the basal lamina of myofibers [22]. Upon injury, SCs exit from quiescence, proliferate, differentiate in myoblasts and fuse to reform myofibers [23]. Nuclear factor I X (Nfix) is a transcription factor belonging to the highly conserved DNA-binding nuclear factor one family (Nfi) together with Nfia, Nfib and Nfic [24]. Nfix has a key role in prenatal myogenesis by driving the transcriptional switch from embryonic to fetal myogenesis [25,26]. Nfix is also required for adult myogenesis upon injury, since its absence leads to defect of SC differentiation [27]. Finally, we recently demonstrated that the deletion of Nfix in two mouse models of muscular dystrophy induces a significant morphological and functional amelioration of the pathology by slowing-down muscle regeneration and promoting a switch towards a more oxidative musculature [28].

During muscle regeneration, myogenic cells and MPs closely interact [7]. Soon after injury, activated SCs attract blood monocytes that infiltrate damaged muscle and differentiate in pro-inflammatory MPs that stimulate the proliferation of myoblasts. Then, by removing dead cells, MPs switch to an anti-inflammatory phenotype that sustains myogenic differentiation [3,29]. While MPs are required for muscle regeneration, preventing MPs infiltration in dystrophic disease decreases muscle damage [30]. Thus, depending on a context of acute or chronic injury, MPs adopt a complete opposite function toward muscle cells and environment [31,32].

In this study, we address the role of Nfix in MPs during skeletal muscle regeneration, by using a mouse model in which Nfix is deleted specifically in MPs. We report that mice lacking Nfix in myeloid lineages exhibit a delay of muscle regeneration upon acute injury. We demonstrated that the RhoA-ROCK1-dependent phagocytosis induces Nfix, whose expression is necessary for the acquisition of anti-inflammatory phenotype and thus pro-regenerative properties through myogenic cells. Indeed, during the process of muscle regeneration, in the absence of Nfix, MPs are able to phagocyte, but failed to adopt an anti-inflammatory phenotype necessary for the resolution of inflammation and muscle regeneration.

### **2. Materials and Methods**

#### *2.1. Animal Models and In Vivo Experimentations*

WT, Nfixfl/fl and LysMCRE:Nfixfl/fl mice were used in this study. LysMCRE:Nfixfl/fl mice were generated, crossing Nfixfl/fl mice obtained from Prof. Richard M. Gronostajski [33] and LysMCRE mice obtained from Dr. Rémi Mounier [8]. All LysMCRE:Nfixfl/fl mice analyzed were heterozygous for the LysMCRE. Muscle regeneration was realized by the injection of 20 uL of 100 uM cardiotoxin (CTX, Latoxan, L8102) in the *Tibialis anterior* (TA) of 2-month-old mice. For the in vivo analysis of satellite cells and myoblasts proliferation, EdU (5-ethynyl-2'-deoxyuridine) was injected in Nfixfl/fl and LysMCRE:Nfixfl/fl mice in intraperitoneal, 12 h before the sacrifice of the mice (100 μL of 6mg/mL EdU solution for 20 g of mouse weight) (Click-iT EdU Imaging Kits Alexa Fluor 594, Thermo Fisher A10044, Paisley, UK). Mice were kept in pathogen-free conditions and all procedures conformed to Italian law (D. Lgs n◦ 2014/26, implementation of the 2010/63/UE) and approved by the University of Milan Animal Welfare Body and by the Italian Minister of Health.
