Fibrosis, inflammation, and osteophyte formation in post-traumatic osteoarthritis

创伤后骨关节炎(PTOA)是一种退行性关节疾病，发生在受伤后，影响数百万人。 全世界。目前还没有治疗疾病的方法。摘要PTOA是一种复杂的多组织关节疾病。 以疼痛、软骨退化、滑膜炎症和纤维化以及异位骨形成为特征 生长的东西叫做骨赘。这种疾病固有的复杂性是发展有效的障碍。 治疗，因为人们对PTOA进展背后复杂的组织串扰知之甚少。我们的长- 学期目标是发现并全面描述关键的细胞和分子机制 PTOA的病理后遗症：滑膜纤维化、炎症和骨赘形成。我们将重点关注 典型的Wnt/β-连环蛋白(CWnt)信号转导。最近，cWnt过度激活被认为是一个驱动因素 关节炎的症状。我们的数据显示，cWnt信号激动剂R-响应蛋白2(Rsp2)在多发性骨髓瘤中被强烈诱导。 在PTOA期间的关节组织，仅Rsp2就足以诱导出具有以下特征的病理特征 PTOA。利用单细胞rna-seq，我们对PTOA小鼠的滑膜进行了分析，发现产生了rspO2。 通过滑膜衬里成纤维细胞。我们鉴定了一种新的促纤维化细胞群，它们在损伤后出现，并 表达Rsp2的LGR细胞表面受体。我们发现滑膜成纤维细胞对Rspo2的反应是 分泌细胞因子，进而激活促炎巨噬细胞(已知推动滑膜病理 PTOA)。单细胞图谱还揭示了一种新的损伤诱导、表达LGR的骨软骨细胞亚群 滑膜中的祖细胞，我们认为它能产生骨赘。我们假设Rspo2驱动的cWNT 信号转导联合驻留细胞类型之间的病理性串扰，以加强PTOA。为了测试这一点，我们的 K99阶段的目标是：1)确定Rspo2驱动的cWnt信号在出现和 利用转基因报告小鼠、多组学分析和临床试验研究PTOA中促纤维化滑膜细胞的功能 体外分化试验，以及2)表征cWnt活性的滑膜成纤维细胞和Pro-Wnt之间的串扰。 炎性巨噬细胞，使用基因敲除小鼠和相声分析。来延伸我的分子生物学 和免疫学专业知识，我将从我的不同导师那里接受严格的技术和概念培训 委员会在K99阶段，和宝贵的职业指导。生物信息学方面的专家培训；cWNT 信号；骨、软骨和滑膜生物学；以及多模式成像，将是执行MY的关键 K99的目标是成功启动我的独立职业生涯，这一点尤为关键。这些技能将被利用 在我的R00阶段：3)确定Rspo2/LGR信号如何促进PTOA的骨赘形成，使用 组织特异性缺失和报告小鼠，以及体外分化试验。这项工作将大大扩展 我们对滑膜纤维化、炎症和炎症的细胞和分子机制的理解 PTOA中的骨赘形成。这些见解将对人类健康产生有意义的、切实的结果，通过 加快为PTOA患者开发有效的疾病修正治疗方法。

Post-traumatic osteoarthritis (PTOA) is a degenerative joint disease that arises after injury and affects millions worldwide. There are currently no disease-modifying treatments. PTOA is a complex, multi-tissue joint disease characterized by pain, cartilage degradation, synovial inflammation and fibrosis, and formation of ectopic bone growths called osteophytes. The inherent complexity of this disease is a barrier to developing effective treatments, as little is known about the intricate tissue crosstalk that underlies PTOA progression. Our long- term goal is to uncover and comprehensively characterize cellular and molecular mechanisms central to key pathological sequalae of PTOA: synovial fibrosis, inflammation, and osteophyte formation. We will focus on canonical Wnt/β-catenin (cWnt) signaling. cWnt overactivation has recently been implicated as a driving factor of arthritis. Our data show that the cWnt signaling agonist R-spondin 2 (Rspo2) is strongly induced in multiple joint tissues during PTOA, and that Rspo2 alone is sufficient to induce pathological features characteristic of PTOA. Using single-cell RNA-seq, we profiled synovium of mice with PTOA and found that Rspo2 is produced by synovial lining fibroblasts. We identified a novel population of pro-fibrotic cells that arise after injury and express Lgr cell surface receptors for Rspo2. We showed that synovial fibroblasts respond to Rspo2 by secreting cytokines that in turn activate pro-inflammatory macrophages (known to drive synovial pathology in PTOA). Single-cell profiling also revealed a novel subset of injury-induced, Lgr-expressing osteochondral progenitors in synovium, which we propose give rise to osteophytes. We hypothesize that Rspo2-driven cWnt signaling mediates pathological crosstalk between joint-resident cell types to potentiate PTOA. To test this, our aims in the K99 phase are to: 1) determine the role of Rspo2-driven cWnt signaling in the emergence and function of pro-fibrotic synovial cells during PTOA using transgenic reporter mice, multi-omic analyses, and in vitro differentiation assays, and 2) characterize crosstalk between cWnt-active synovial fibroblasts and pro- inflammatory macrophages, using knockout mice and crosstalk assays. To extend upon my molecular biology and immunology expertise, I will receive rigorous technical and conceptual training from my diverse mentorship committee during the K99 phase, and valuable career guidance. This expert training in bioinformatics; cWnt signaling; bone, cartilage, and synovial biology; and multi-modal imaging, will be crucial for carrying out my K99 aims and especially critical for successfully launching my independent career. These skills will be utilized in my R00 phase to: 3) determine how Rspo2/Lgr signaling promotes osteophyte formation in PTOA, using tissue-specific deletion and reporter mice, and in vitro differentiation assays. This work will significantly extend our understanding of cellular and molecular mechanisms that underpin synovial fibrosis, inflammation, and osteophyte formation in PTOA. These insights will have meaningful, tangible outcomes for human health, by accelerating development of effective disease-modifying treatments for PTOA sufferers.