Multi-omics analysis of human tendon adhesion reveals that ACKR1-regulated macrophage migration is involved in regeneration

Tendon adhesion is a common complication after tendon injury with the development of accumulated fibrotic tissues without effective anti-fibrotic therapies, resulting in severe disability. Macrophages are widely recognized as a fibrotic trigger during peritendinous adhesion formation. However, different clusters of macrophages have various functions and receive multiple regulation, which are both still unknown. In our current study, multi-omics analysis including single-cell RNA sequencing and proteomics was performed on both human and mouse tendon adhesion tissue at different stages after tendon injury. The transcriptomes of over 74 000 human single cells were profiled. As results, we found that SPP1⁺ macrophages, RGCC⁺ endothelial cells, ACKR1⁺ endothelial cells and ADAM12⁺ fibroblasts participated in tendon adhesion formation. Interestingly, despite specific fibrotic clusters in tendon adhesion, FOLR2⁺ macrophages were identified as an antifibrotic cluster by in vitro experiments using human cells. Furthermore, ACKR1 was verified to regulate FOLR2⁺ macrophages migration at the injured peritendinous site by transplantation of bone marrow from Lysm-Cre;R26R^tdTomato mice to lethally irradiated Ackr1^-/- mice (Ackr1^-/- chimeras; deficient in ACKR1) and control mice (WT chimeras). Compared with WT chimeras, the decline of FOLR2⁺ macrophages was also observed, indicating that ACKR1 was specifically involved in FOLR2⁺ macrophages migration. Taken together, our study not only characterized the fibrosis microenvironment landscape of tendon adhesion by multi-omics analysis, but also uncovered a novel antifibrotic cluster of macrophages and their origin. These results provide potential therapeutic targets against human tendon adhesion.