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.

肌腱粘连是肌腱损伤后常见的并发症，如果没有有效的抗纤维化治疗，纤维化组织会不断积累，导致严重残疾。巨噬细胞被广泛认为是腱周粘连形成过程中纤维化的触发因素。然而，不同的巨噬细胞簇具有不同的功能并受到多重调节，这些都仍然是未知的。在我们目前的研究中，对肌腱损伤后不同阶段的人和小鼠肌腱粘连组织进行了包括单细胞RNA测序和蛋白质组学在内的多组学分析。对超过 74000 个人类单细胞的转录组进行了分析。结果，我们发现SPP1 ⁺巨噬细胞、RGCC ⁺内皮细胞、ACKR1 ⁺内皮细胞和ADAM12 ⁺成纤维细胞参与肌腱粘附形成。有趣的是，尽管肌腱粘附中存在特定的纤维化簇，但通过使用人体细胞的体外实验，FOLR2 ⁺巨噬细胞被鉴定为抗纤维化簇。此外，通过将Lysm-Cre;R26R ^tdTomato小鼠的骨髓移植到致命照射的Ackr1 ^−/−小鼠（ Ackr1 ^−/−嵌合体；ACKR1 缺陷）和对照小鼠中，确认 ACKR1 可以调节受伤腱周部位的 FOLR2 ⁺巨噬细胞迁移。小鼠（WT嵌合体）。与WT嵌合体相比，还观察到FOLR2 ⁺巨噬细胞的下降，表明ACKR1特异性参与FOLR2 ⁺巨噬细胞的迁移。 综上所述，我们的研究不仅通过多组学分析表征了肌腱粘连的纤维化微环境景观，而且还揭示了一种新型的抗纤维化巨噬细胞簇及其起源。这些结果提供了针对人类肌腱粘连的潜在治疗靶点。