Linde Peter, Chow Lyndah, Sabino Isabella, Williams Zoë, Impastato Renata, Dow Steven, Pezzanite Lynn
Department of Clinical Sciences, College of Veterinary Medicine and Biomedical Sciences, Colorado State University, Fort Collins, CO, United States.
Front Bioeng Biotechnol. 2025 Aug 8;13:1605148. doi: 10.3389/fbioe.2025.1605148. eCollection 2025.
Mesenchymal stromal cells (MSCs) have been evaluated as a local therapeutic option to treat osteoarthritis (OA) with conflicting clinical results. Our previous studies have evaluated immune licensing of MSC through activation of Toll-like receptor and cytosolic cGAS-STING pathways, with demonstrated improvement in functional and structural outcomes in a rodent model of OA. The objective of this study was to investigate impact of MSC activation on their interaction with relevant joint target cells to better understand the mechanisms by which pre-activation improves MSC activity for treatment of osteoarthritis.
Equine bone-marrow-derived MSCs (passage 2-3) from 3 healthy donors were stimulated with a TLR3-pathway agonist (polyinosinic:polycytidylic acid) or STING pathway agonist (2'3'-cGAMP) (10 μg/mL, 2 h, 2 × 10 cells/mL in suspension). Cells were plated (100,000 cells/well, 24-well plates) and conditioned media (CM) collected at 24 h. Equine monocyte-derived macrophages, synovial cells, and chondrocytes were stimulated with IL-1ß/TNF-α (20 ng/mL, 2 h), washed and treated 24 h with MSC-CM, TLR-MSC-CM or STING-MSC-CM, washed and cultured 24 h. CM was examined for cytokine secretion by multiplex immunoassay and ELISA (25 cytokines). Bulk RNA sequencing was performed on MSC and joint cell lines via an Illumina based platform.
TLR-MSC-CM decreased IL-1β (p = 0.02), IL-6 (p = 0.02) secretion by synoviocytes and IL-18 secretion by activated chondrocytes (p = 0.002). STING-MSC-CM decreased IL-6, IL-8 secretion (p = 0.08) by synoviocytes, decreased IL-8 (p = 0.05) by activated chondrocytes, increased G-CSF (p = 0.01), IL-4 (p = 0.01) and decreased IL-5 (p = 0.01) by activated macrophages. Transcriptomic analyses indicated differential gene expression in each cell line following CM treatment varied by cell line. STING-MSC-CM vs TLR-MSC-CM induced 38 significantly altered DEGs in synoviocytes, 20 in chondrocytes, and 47 in macrophages.
These findings indicate that joint cells respond differently to factors secreted by TLR or STING pathway activated MSC. The pathways altered were different for each target cell type and no clear pattern of responses was apparent. These results indicate that modeling of target cell responses to "licensed" MSC can provide new information on the MSC and target cell interactions, though ultimately the functional impacts of activated MSC need to be evaluated using models.
间充质基质细胞（MSCs）已被评估为治疗骨关节炎（OA）的一种局部治疗选择，但临床结果相互矛盾。我们之前的研究通过激活Toll样受体和胞质cGAS-STING途径评估了MSCs的免疫许可，结果表明在OA啮齿动物模型中其功能和结构结局得到改善。本研究的目的是调查MSCs激活对其与相关关节靶细胞相互作用的影响，以更好地理解预激活改善MSCs治疗骨关节炎活性的机制。
来自3名健康供体的马骨髓源性MSCs（第2-3代）用TLR3途径激动剂（聚肌苷酸:聚胞苷酸）或STING途径激动剂（2'3'-环鸟苷酸-腺苷酸）（10μg/mL，2小时，悬浮液中2×10个细胞/mL）刺激。将细胞接种（100,000个细胞/孔，24孔板），并在24小时时收集条件培养基（CM）。用IL-1β/TNF-α（20ng/mL，2小时）刺激马单核细胞衍生的巨噬细胞、滑膜细胞和软骨细胞，洗涤后用MSC-CM、TLR-MSC-CM或STING-MSC-CM处理24小时，洗涤后再培养24小时。通过多重免疫测定和ELISA（25种细胞因子）检测CM中的细胞因子分泌。通过基于Illumina的平台对MSCs和关节细胞系进行批量RNA测序。
TLR-MSC-CM降低了滑膜细胞分泌的IL-1β（p = 0.02）、IL-6（p = 0.02）以及活化软骨细胞分泌的IL-18（p = 0.002）。STING-MSC-CM降低了滑膜细胞分泌的IL-6、IL-8（p = 0.08），降低了活化软骨细胞分泌的IL-8（p = 0.05），增加了活化巨噬细胞分泌的G-CSF（p = 0.01）、IL-4（p = 0.01）并降低了IL-5（p = 0.01）。转录组分析表明，CM处理后每个细胞系中的基因表达差异因细胞系而异。与TLR-MSC-CM相比，STING-MSC-CM在滑膜细胞中诱导了38个显著改变的差异表达基因，在软骨细胞中诱导了20个，在巨噬细胞中诱导了47个。
这些发现表明关节细胞对TLR或STING途径激活的MSCs分泌的因子反应不同。每种靶细胞类型改变的途径不同，且没有明显的反应模式。这些结果表明，对靶细胞对“许可”MSCs反应的建模可以提供有关MSCs与靶细胞相互作用的新信息，尽管最终需要使用模型评估活化MSCs的功能影响。
