**Marielle Saclier, Michela Lapi, Chiara Bonfanti, Giuliana Rossi** †**, Stefania Antonini and Graziella Messina \***

Department of Biosciences, University of Milan, via Celoria 26, 20133 Milan, Italy; marielle.saclier@unimi.it (M.S.); michela.lapi@unimi.it (M.L.); chiara.bonfanti@unimi.it (C.B.); giuliana.rossi@epfl.ch (G.R.); stefania.antonini@unimi.it (S.A.)

**\*** Correspondence: graziella.messina@unimi.it; Tel.: +3902 503 14800

† Current affiliation: Laboratory of Stem Cell Bioengineering, Institute of Bioengineering, School of Life Sciences and School of Engineering, École Polytechnique Fédérale de Lausanne (EPFL), 1015 Lausanne, Vaud, Switzerland.

Received: 19 February 2020; Accepted: 12 March 2020; Published: 13 March 2020

**Abstract:** Macrophages (MPs) are immune cells which are crucial for tissue repair. In skeletal muscle regeneration, pro-inflammatory cells first infiltrate to promote myogenic cell proliferation, then they switch into an anti-inflammatory phenotype to sustain myogenic cells differentiation and myofiber formation. This phenotypical switch is induced by dead cell phagocytosis. We previously demonstrated that the transcription factor Nfix, a member of the nuclear factor I (Nfi) family, plays a pivotal role during muscle development, regeneration and in the progression of muscular dystrophies. Here, we show that Nfix is mainly expressed by anti-inflammatory macrophages. Upon acute injury, mice deleted for Nfix in myeloid line displayed a significant defect in the process of muscle regeneration. Indeed, Nfix is involved in the macrophage phenotypical switch and macrophages lacking Nfix failed to adopt an anti-inflammatory phenotype and interact with myogenic cells. Moreover, we demonstrated that phagocytosis induced by the inhibition of the RhoA-ROCK1 pathway leads to Nfix expression and, consequently, to acquisition of the anti-inflammatory phenotype. Our study identified Nfix as a link between RhoA-ROCK1-dependent phagocytosis and the MP phenotypical switch, thus establishing a new role for Nfix in macrophage biology for the resolution of inflammation and tissue repair.

**Keywords:** macrophages; Nfix; skeletal muscle; phagocytosis; RhoA-ROCK1
