DataSheet_1_Transcriptome profiling of osteoclast subsets associated with arthritis: A pathogenic role of CCR2hi osteoclast progenitors.zip

IntroductionThe existence of different osteoclast progenitor (OCP) subsets has been confirmed by numerous studies. However, pathological inflammation-induced osteoclastogenesis remains incompletely understood. Detailed characterization of OCP subsets may elucidate the pathophysiology of increased osteoclast activity causing periarticular and systemic bone resorption in arthritis. In our study, we rely on previously defined OCP subsets categorized by the level of CCR2 expression as circulatory-like committed CCR2hi OCPs, which are substantially expanded in arthritis, and marrow-resident CCR2lo OCPs of immature phenotype and behavior.
MethodsIn order to perform transcriptome characterization of those subsets in the context of collagen-induced arthritis (CIA), we sorted CCR2hi and CCR2lo periarticular bone marrow OCPs of control and arthritic mice, and performed next-generation RNA sequencing (n=4 for each group) to evaluate the differential gene expression profile using gene set enrichment analysis with further validation.
ResultsA disparity between CCR2hi and CCR2lo subset transcriptomes (863 genes) was detected, with the enrichment of pathways for osteoclast differentiation, chemokine and NOD-like receptor signaling in the CCR2hi OCP subset, and ribosome biogenesis in eukaryotes and ribosome pathways in the CCR2lo OCP subset. The effect of intervention (CIA) within each subset was greater in CCR2hi (92 genes) than in CCR2lo (43 genes) OCPs. Genes associated with the osteoclastogenic pathway (Fcgr1, Socs3), and several genes involved in cell adhesion and migration (F11r, Cd38, Lrg1) identified the CCR2hi subset and distinguish CIA from control group, as validated by qPCR (n=6 for control mice, n=9 for CIA mice). The latter gene set showed a significant positive correlation with arthritis clinical score and frequency of CCR2hi OCPs. Protein-level validation by flow cytometry showed increased proportion of OCPs expressing F11r/CD321, CD38 and Lrg1 in CIA, indicating that they could be used as disease markers. Moreover, osteoclast pathway-identifying genes remained similarly expressed (Fcgr1) or even induced by several fold (Socs3) in preosteoclasts differentiated in vitro from CIA mice compared to pre-cultured levels, suggesting their importance for enhanced osteoclastogenesis of the CCR2hi OCPs in arthritis.
ConclusionOur approach detected differentially expressed genes that could identify distinct subset of OCPs associated with arthritis as well as indicate possible therapeutic targets aimed to modulate osteoclast activity.

引言
多项研究已证实存在不同的破骨细胞祖细胞（Osteoclast Progenitor, OCP）亚群，但病理性炎症诱导的破骨细胞生成机制仍未完全阐明。对OCP亚群进行详细表征，或可阐明关节炎中破骨细胞活性增强所引发的关节周围及全身性骨吸收的病理生理学机制。本研究依托此前基于CCR2表达水平划分的OCP亚群分类标准：一类为循环样定向型CCR2高表达（CCR2hi）OCPs，该类亚群在关节炎中显著扩增；另一类为骨髓驻留型CCR2低表达（CCR2lo）OCPs，其表型与功能均未成熟。
方法
为在胶原诱导性关节炎（Collagen-Induced Arthritis, CIA）模型中对上述亚群进行转录组表征，本研究分选了对照组与关节炎模型小鼠的关节周围骨髓CCR2hi及CCR2lo OCPs，并开展二代RNA测序（Next-Generation RNA Sequencing，每组n=4），通过基因集富集分析评估差异基因表达谱，并进行后续验证。
结果
研究检测到CCR2hi与CCR2lo亚群的转录组间存在863个差异基因：CCR2hi OCP亚群富集破骨细胞分化、趋化因子及NOD样受体信号通路，而CCR2lo OCP亚群则富集真核生物核糖体生物发生及核糖体通路。在各亚群中，CIA造模干预对CCR2hi OCPs的影响（涉及92个差异基因）显著大于对CCR2lo OCPs的影响（涉及43个差异基因）。与破骨细胞生成通路相关的基因（Fcgr1、Socs3）以及参与细胞黏附与迁移的若干基因（F11r、Cd38、Lrg1）可作为CCR2hi亚群的特征基因，并可区分CIA模型与对照组小鼠，该结果经实时定量PCR（qPCR，对照组小鼠n=6，CIA模型小鼠n=9）验证。上述细胞黏附与迁移相关基因集与关节炎临床评分及CCR2hi OCPs的占比呈显著正相关。流式细胞术（Flow Cytometry）的蛋白水平验证显示，CIA模型小鼠体内表达F11r/CD321、CD38及Lrg1的OCPs占比升高，提示这些分子可作为疾病标志物。此外，与预培养状态下的表达水平相比，从CIA模型小鼠体外分化得到的前破骨细胞中，破骨细胞生成通路特征基因的表达水平要么维持不变（Fcgr1），要么被诱导上调数倍（Socs3），这表明这些基因在关节炎中CCR2hi OCPs的破骨细胞生成增强过程中发挥重要作用。
结论
本研究通过转录组分析鉴定出可区分与关节炎相关的OCP亚群的差异表达基因，同时也为调控破骨细胞活性的潜在治疗靶点提供了新思路。