Xu Junxiang, Zheng Minzhe, Feng Zongxian, Lin Qiji
Department of Orthopedics, Ningbo Medical Center Lihuili Hospital, Ningbo, Zhejiang Province, 315000, China.
J Orthop Surg Res. 2024 Dec 19;19(1):836. doi: 10.1186/s13018-024-05268-9.
Tendinopathy is very common in clinical practice, which is highly prevalent in athletes, sports enthusiasts and other people involved in high-load weight-bearing activities. Common types of tendinopathy include rotator cuff injury, Achilles tendinitis, tennis elbow and so on. Macrophages (Macs) are key immune cells in the pathogenesis of tendinopathy. In this study, CCL4L2 M1-related signaling pathways were screened by combining single-cell RNA sequencing (scRNA-seq) to explore their significance in tendinopathy treatment.
Immune cell populations were screened by Uniform Manifold Approximation and Projection (UMAP) downscaling, and Mac cell subsets were annotated using cell marker genes. The cellular communication mechanism between different cellular subsets such as Macs and tendon stem/progenitor cells (TSPCs) was demonstrated by cellular communication analysis. Based on cell marker genes of CCL4L2 + M1, Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment were performed to compare the expression differences in M1 and M2 between the Disease and Healthy groups. Associations between CCL4L2 M1 and TSPCs were inferred by cell-cell communication analysis. The effects of CCL4L2 on Mac polarization and TSPCs were verified by enzyme-linked immunosorbent assay (ELISA) and real-time fluorescence quantitative PCR (qPCR).
The proportions of TSPCs, endothelial cells (ECs), smooth muscle cells (SMCs), and immune cells were significantly elevated in the Disease group. The proportion of M1 cells in the Disease group was higher than that in the Healthy group, while the proportion of M2 cells was lower than that in the Healthy group. M1 differentially expressed genes (DEGs) were mainly enriched to disease-related and immunoinflammation-related signaling pathways. Signaling intensities between M1 and TSPCs in pathways related to immunoinflammation and ischemic injury were significantly increased in the Disease group. The proportion of CCL4L2 + M1 in the Disease group was significantly higher than in the Healthy group, and communications between CCL4L2 + M1 and TSPCs varied significantly. Compared with the Control group, the expression levels of inflammatory cytokines were higher in the CCL4L2 group, and the expression levels of tendon differentiation markers (Egr1, Mkx, Scx, Type 1 collagen, Tnmd) were significantly down-regulated.
The present study analyzed the heterogeneous alterations in the Healthy and Disease groups by scRNA-seq data and found that there was a significant inflammatory infiltrate in the Disease group with markedly increased Mac activity, which was associated with activation of the CCL4L2 + M1-associated signaling pathways. CCL4L2 promotes M1 polarization and inhibits TSPC differentiation through activating M1-related inflammatory signaling pathways. These findings contribute to a more comprehensive understanding of tendon injury progression and provide potential targets for tendinopathy treatment.
肌腱病在临床实践中非常常见，在运动员、运动爱好者和其他从事高负荷负重活动的人群中高度流行。常见的肌腱病类型包括肩袖损伤、跟腱炎、网球肘等。巨噬细胞（Macs）是肌腱病发病机制中的关键免疫细胞。在本研究中，通过结合单细胞RNA测序（scRNA-seq）筛选CCL4L2 M1相关信号通路，以探讨其在肌腱病治疗中的意义。
通过均匀流形近似和投影（UMAP）降维筛选免疫细胞群体，并使用细胞标记基因对Mac细胞亚群进行注释。通过细胞通讯分析证明Macs与肌腱干/祖细胞（TSPCs）等不同细胞亚群之间的细胞通讯机制。基于CCL4L2 + M1的细胞标记基因，进行本体论（GO）和京都基因与基因组百科全书（KEGG）通路富集，以比较疾病组和健康组中M1和M2的表达差异。通过细胞-细胞通讯分析推断CCL4L2 M1与TSPCs之间的关联。通过酶联免疫吸附测定（ELISA）和实时荧光定量PCR（qPCR）验证CCL4L2对Mac极化和TSPCs的影响。
疾病组中TSPCs、内皮细胞（ECs）、平滑肌细胞（SMCs）和免疫细胞的比例显著升高。疾病组中M1细胞的比例高于健康组，而M2细胞的比例低于健康组。M1差异表达基因（DEGs）主要富集到与疾病相关和免疫炎症相关的信号通路。疾病组中免疫炎症和缺血损伤相关通路中M1与TSPCs之间的信号强度显著增加。疾病组中CCL4L2 + M1的比例显著高于健康组，且CCL4L2 + M1与TSPCs之间的通讯差异显著。与对照组相比，CCL4L2组中炎性细胞因子的表达水平较高，肌腱分化标志物（Egr1、Mkx、Scx、I型胶原、Tnmd）的表达水平显著下调。
本研究通过scRNA-seq数据分析了健康组和疾病组中的异质性变化，发现疾病组存在明显的炎症浸润，Mac活性显著增加，这与CCL4L2 + M1相关信号通路的激活有关。CCL4L2通过激活M1相关炎症信号通路促进M1极化并抑制TSPC分化。这些发现有助于更全面地了解肌腱损伤进展，并为肌腱病治疗提供潜在靶点。
