Exosomes from M2c macrophages alleviate intervertebral disc degeneration by promoting synthesis of the extracellular matrix via MiR-124/CILP/TGF-β

Immuno-inflammation is highly associated with anabolic and catabolic dysregulation of the extracellular matrix (ECM) in the nucleus pulposus (NP), which dramatically propels intervertebral disc degeneration (IVDD). With the characteristics of tissue remodeling and regeneration, M2c macrophages have attracted great attention in research on immune modulation that rebuilds degenerated tissues. Therefore, we first demonstrated the facilitating effects of M2c macrophages on ECM anabolism of the NP in vitro. We subsequently found that exosomes from M2c macrophages (M2c-Exoss) mediated their metabolic rebalancing effects on the ECM. To determine whether M2c-Exoss served as positive agents protecting the ECM in IVDD, we constructed an M2c-Exos-loaded hyaluronic acid hydrogel (M2c-Exos@HA hydrogel) and implanted it into the degenerated caudal disc of rats. The results of MRI and histological staining indicated that the M2c-Exos@HA hydrogel alleviated IVDD in vivo in the long term. To elucidate the underlying molecular mechanism, we performed 4D label-free proteomics to screen dysregulated proteins in NPs treated with M2c-Exoss. Cartilage intermediate layer protein (CILP) was the key protein responsible for the rebalancing effects of M2c-Exoss on ECM metabolism in the NP. With prediction and verification using luciferase assays and rescue experiments, miR-124-3p was identified as the upstream regulator in M2c-Exoss that regulated CILP and consequently enhanced the activity of the TGF-β/smad3 pathway. In conclusion, we demonstrated ameliorating effects of M2c-Exoss on the imbalance of ECM metabolism in IVDD via the miR-124/CILP/TGF-β regulatory axis, which provides a promising theoretical basis for the application of M2c macrophages and their exosomes in the treatment of IVDD.