Table_1_Distinct differences in immunological properties of equine orthobiologics revealed by functional and transcriptomic analysis using an activated macrophage readout system.XLSX

IntroductionMultiple biological therapies for orthopedic injuries are marketed to veterinarians, despite a lack of rigorous comparative biological activity data to guide informed decisions in selecting a most effective compound. Therefore, the goal of this study was to use relevant bioassay systems to directly compare the anti-inflammatory and immunomodulatory activity of three commonly used orthobiological therapies (OTs): mesenchymal stromal cells (MSC), autologous conditioned serum (ACS), and platelet rich plasma (PRP).
MethodsEquine monocyte-derived macrophages were used as the readout system to compare therapies, including cytokine production and transcriptomic responses. Macrophages were stimulated with IL-1ß and treated 24 h with OTs, washed and cultured an additional 24 h to generate supernatants. Secreted cytokines were measured by multiplex immunoassay and ELISA. To assess global transcriptomic responses to treatments, RNA was extracted from macrophages and subjected to full RNA sequencing, using an Illumina-based platform. Data analysis included comparison of differentially expressed genes and pathway analysis in treated vs. untreated macrophages.
ResultsAll treatments reduced production of IL-1ß by macrophages. Secretion of IL-10 was highest in MSC-CM treated macrophages, while PRP lysate and ACS resulted in greater downregulation of IL-6 and IP-10. Transcriptomic analysis revealed that ACS triggered multiple inflammatory response pathways in macrophages based on GSEA, while MSC generated significant downregulation of inflammatory pathways, and PRP lysate induced a mixed immune response profile. Key downregulated genes in MSC-treated cultures included type 1 and type 2 interferon response, TNF-α and IL-6. PRP lysate cultures demonstrated downregulation of inflammation-related genes IL-1RA, SLAMF9, ENSECAG00000022247 but concurrent upregulation of TNF-α, IL-2 signaling, and Myc targets. ACS induced upregulation of inflammatory IL-2 signaling, TNFα and KRAS signaling and hypoxia, but downregulation of MTOR signaling and type 1 interferon signaling.
DiscussionThese findings, representing the first comprehensive look at immune response pathways for popular equine OTs, reveal distinct differences between therapies. These studies address a critical gap in our understanding of the relative immunomodulatory properties of regenerative therapies commonly used in equine practice to treat musculoskeletal disease and will serve as a platform from which further in vivo comparisons may build.

引言：尽管目前尚无严谨的对比生物活性数据以指导兽医在遴选最优骨伤治疗制剂时做出科学决策，但市场上已有多款针对骨伤的生物疗法面向兽医群体推广。因此，本研究旨在通过相关生物检测系统，直接对比三种常用骨科生物疗法（orthobiological therapies, OTs）的抗炎与免疫调节活性：间充质基质细胞（mesenchymal stromal cells, MSC）、自体条件血清（autologous conditioned serum, ACS）以及富血小板血浆（platelet rich plasma, PRP）。
方法：本研究以马源单核细胞衍生巨噬细胞作为检测体系，对比各疗法对巨噬细胞的细胞因子分泌与转录组应答的影响。将巨噬细胞经IL-1β刺激后，用三种骨科生物疗法处理24小时，随后洗涤细胞并继续培养24小时以收集细胞上清液。采用多重免疫分析法与酶联免疫吸附试验（ELISA）检测上清液中的分泌型细胞因子水平。为全面评估各处理组的转录组应答特征，本研究从巨噬细胞中提取总RNA，基于Illumina测序平台开展全转录组测序。数据分析环节包括对比处理组与未处理组巨噬细胞的差异表达基因，并进行通路富集分析。
结果：所有处理组均能降低巨噬细胞的IL-1β分泌水平。间充质基质细胞条件培养基（MSC-CM）处理的巨噬细胞中IL-10分泌水平最高；而富血小板血浆裂解液（PRP裂解液）与自体条件血清则对IL-6与IP-10的下调作用更为显著。转录组分析基于基因集富集分析（Gene Set Enrichment Analysis, GSEA）结果显示：自体条件血清可触发巨噬细胞的多条炎症应答通路；间充质基质细胞则显著下调炎症通路；而富血小板血浆裂解液诱导出混合的免疫应答表型。间充质基质细胞处理组的关键下调基因包括I型与II型干扰素应答相关基因、TNF-α以及IL-6。富血小板血浆裂解液处理组中，炎症相关基因IL-1RA、SLAMF9及ENSECAG00000022247均出现下调，但同时TNF-α、IL-2信号通路以及Myc靶基因呈现上调。自体条件血清处理组则上调炎症相关的IL-2信号通路、TNF-α、KRAS信号通路以及缺氧应答通路，但下调MTOR信号通路与I型干扰素信号通路。
讨论：本研究首次全面剖析了临床常用的马源骨科生物疗法的免疫应答通路，结果显示不同疗法之间存在显著差异。当前学界对马临床中常用于治疗肌肉骨骼疾病的再生疗法的相对免疫调节特性尚缺乏系统认知，本研究填补了这一关键认知空白；本研究成果将为后续开展体内对比研究提供重要的研究基础。