The Transcription Factor Nfix Requires RhoA-ROCK1 Dependent Phagocytosis to Mediate Macrophage Skewing during Skeletal Muscle Regeneration

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.

Selective ablation of Nfix in Macrophages preserves Muscular Dystrophy by inhibiting FAPs-dependent fibrosis

“…We observed an increase in the number of MPs expressing Nfix in both TA and Dia muscles of the mdx and Sgca null mice between 2 and 6 months of life (Figure 1a and b). During muscle regeneration after acute injury, we showed that Nfix is required for Ly6Canti-inflammatory phenotype acquisition 27 . Although the concept of pro-and anti-inflammatory MPs does not seem to be conserved between acute and chronic muscle injury context, we still decided to look at the number CD64 + MPs positive for Nfix within the Ly6C + and Ly6Cpopulation sorted from the TA of Sgca null mice.…”

“…Since MPs are linked to MDs 11,20,28 and as we demonstrated a pivotal function of Nfix in MPs function during muscle regeneration 27 , we decided to analyze the number of Nfix + MPs during the progression of MD within the Tibialis Anterior (TA) and diaphragm (Dia) of two different dystrophic murine models (the mdx and the Sgca null mice). We observed an increase in the number of MPs expressing Nfix in both TA and Dia muscles of the mdx and Sgca null mice between 2 and 6 months of life (Figure 1a and b).…”

“…We previously demonstrated that Nfix is expressed upon RhoA-ROCK dependent phagocytosis in a context of muscle acute injury 27 , thus we asked whether this pathway was conserved in dystrophic musculature and if the stimulation of Nfix expression by MPs could accelerate fibrosis. Therefore, we injected Sgca null mice with the ROCK inhibitor Y27632 (then called Y), or its control, 3 times per week for 2 weeks.…”

Myofiber / pro-inflammatory macrophage interplay controls muscle damage in mdx mice

“…Resolution of inflammation is characterized by a progressive switch of the inflammatory profile exhibited by macrophages, that become anti-inflammatory or recovery macrophages. This process is controlled by several molecular effectors, including the metabolic sensor AMPK (Mounier et al, 2013), the IGF1 pathway (Tonkin et al, 2016), the p38 regulator MKP-1 (Perdiguero et al, 2011), and the transcription factors C/EBPβ (Ruffell et al, 2009) and NFIX (Saclier et al, 2020). These anti-inflammatory macrophages exert a variety of effects on surrounding cells: they stimulate the last steps of myogenesis, including differentiation and fusion (Saclier et al, 2013), they activate FAPs to remodel the extracellular matrix (ECM) (Juban et al, 2018; Lemos et al, 2015), and favor angiogenesis, which occurs concomitantly to myogenesis (Latroche et al, 2017).…”

Muscle Homeostasis and Regeneration: From Molecular Mechanisms to Therapeutic Opportunities

“…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. Saclier, et al, showed that Nfix is mainly expressed by anti-inflammatory macrophages [14]. Upon acute injury, mice deleted for Nfix in myeloid line displayed a significant defect in the process of muscle regeneration.…”