Skeletal Muscle Macrophages: Characterization and Regulation by Regulatory T Cells

Abstract

Skeletal muscle regeneration is a highly orchestrated process that relies heavily on the immune system. Efficient regeneration post-acute injury requires both F4/80+ macrophages (MFs) and CD4+Foxp3+ T regulatory (Treg) cells. MFs, licensed antigen-presenting cells, are the most abundant immunocyte in healthy and injured muscle, with effector functions that include phagocytosis of muscle debris, production of inflammatory and pro-regenerative factors, and interactions with immune and stromal cells. However, little is known about their population dynamics and phenotype at steady-state and after injury. Moreover, regulatory mechanisms of their phenotype are ill-described. Here, we used cytofluorimetric, transcriptional and immunofluorescence analyses to profile muscle MFs at steady-state and after injury. We identified two subsets of MFs based on their expression of the class II Major Histocompatibility Complex (MHCII). We found that while MHCII- and MHCII+ subsets had distinct phenotypes at steady-state, their transcriptional profiles behaved similarly after injury and adopted characteristics associated with the timeline of repair. We also demonstrated that antigen presentation by muscle MFs promoted activation of muscle CD4+Foxp3- T conventional (Tconv) cells, but it was dispensable for accumulation of Treg cells. Furthermore, by punctually depleting Treg cells, we showed that Treg cells controlled muscle MF subset composition, proliferation and phenotype. The MHCII- subset was lost, whereas the MHCII+ subset became the dominant subset, with an aberrant phenotype reflecting a response to IFN-γ. We investigated mechanisms governing the resulting disproportion in MF subsets and showed that it was possibly due to an increase in proliferation of the MHCII+ subset and perhaps, partially due to conversion of the MHCII- to MHCII+ subset. We identified Natural Killer (NK) cells as the predominant source of IFN-γ during muscle repair, which was rapidly increased upon loss of Treg cells. In gain-of-function experiments, we showed that excess IFN-γ could mimic some of the effects exhibited in the absence of Treg cells, including the aberrant phenotype in MFs, increased levels of IFN-γ and inflammation in regenerating muscle. In summary, we showed that the environment of muscle repair was strongly regulated by Treg cells, which limited IFN-γ to promote proper MF subset proportion and phenotype, thereby permitting efficient regeneration.