Expanded fibroblast subset drives pathology in rheumatoid arthritis

成纤维细胞在终末器官纤维化中的作用已经得到很好的确立，但是对它们在慢性纤维化中的作用的了解还不够。 外周组织中的炎性疾病如类风湿性关节炎（RA）仍在出现。我们确定了一个 在RA滑膜组织的下层区域中成纤维细胞的高度扩增的炎性亚群。它 占RA滑膜中所有成纤维细胞的>50%，但在骨关节炎（OA）中是罕见的群体。的 扩增群体的特征在于CD 90（Thy 1，一种亚衬标记物）和HLA-DR的高表达， IL-6和许多趋化因子的产生。我们假设这些CD 90 +DR+IL-6+成纤维细胞是 通过分泌炎症因子直接驱动炎症，通过募集和激活 白细胞来维持慢性炎症。当分析来自RA/SLE的单细胞RNA-seq数据时， 加速药物伙伴关系（AMP）联盟，我们发现，标记的内衬和内衬 滑膜中的成纤维细胞不是绝对的，而是代表了基因表达的梯度 分析.我们发现，这种转录梯度对应于一个解剖空间梯度， 从血管中发出的滑膜。我们的数据表明，Notch信号是一个主要的驱动程序， 梯度从血管周围的成纤维细胞开始，并延伸到表达 Notch 3受体和Jagged（Jag）1 Notch配体。 在这里，我们希望确定成纤维细胞上特异性的Notch 3信号是否驱动了空间模式 和成纤维细胞的分化。为了实现这一点，在目标1中，我们使用混合细胞类器官， 内皮小管和成纤维细胞，以比较Notch 3缺陷的空间模式和分化。 与对照成纤维细胞相比。在目标2中，我们确定了CD 90 +DR+炎性细胞因子的位置， 在滑膜中产生成纤维细胞和Notch 3+成纤维细胞，并确定哪种成纤维细胞群体 最显著地与白细胞（T细胞、B细胞和巨噬细胞）相关。在目标3中，我们激活滑膜 在Notch配体存在或不存在的情况下，用在RA中发现的炎性细胞因子对成纤维细胞系进行刺激。 我们使用流式细胞术，RNA-seq，LDA和轨迹分析来比较在不同的细胞中诱导的成纤维细胞状态。 与RA滑膜中发现的那些体外。然后，我们将Notch梯度的概念从成纤维细胞扩展到 成纤维细胞来源的Notch配体如何激活类器官中附着的T细胞。最后，在目标4中， 我们确定成纤维细胞中Notch信号的靶向条件性破坏或靶向条件性缺失 成纤维细胞中Notch配体的表达预防小鼠模型中的炎性关节炎。这些研究将 推进我们对成纤维细胞如何在RA中分化成为炎症和病理学驱动因素的认识 在慢性炎症的滑膜组织中，以及它们如何在小鼠模型中被靶向治疗。

The role of fibroblasts in end organ fibrosis is well established, but insights into their roles in chronic inflammatory diseases in peripheral tissues like rheumatoid arthritis (RA) is still emerging. We identified a highly expanded inflammatory subpopulation of fibroblasts in the sublining region of RA synovial tissue. It accounts for >50% of all fibroblasts in the synovium in RA, but it is a rare population in osteoarthritis (OA). The expanded population is distinguished by high expression of CD90 (Thy1, a sublining marker) and HLA-DR, and the production of IL-6 and many chemokines. We hypothesize that these CD90+DR+IL-6+ fibroblasts are key in driving inflammation directly by secreting inflammatory factors and indirectly by recruiting and activating leukocytes to maintain chronic inflammation. When analyzing single cell RNA-seq data from the RA/SLE Accelerating Medicines Partnership (AMP) Consortium, we found that markers of lining and sublining fibroblasts in synovium were not absolute – but instead represented a gradient in gene expression in trajectory analysis. We found that this transcriptional gradient corresponds to an anatomic spatial gradient in the synovium emanating from blood vessels. Our data suggest that Notch signaling is a dominant driver of the gradient starting with fibroblasts around blood vessels and extending to sublining fibroblasts that express Notch3 receptors and Jagged (Jag)1 Notch ligands. Here, we wish to determine if Notch 3 signaling specifically on fibroblasts drives the spatial pattering and the differentiation of sublining fibroblasts. To accomplish this, in Aim 1 we use mixed cell organoids with endothelial tubules and fibroblasts to compare spatial pattering and differentiation of Notch3 deficient compared to control fibroblasts. In Aim 2, we determine the location of the CD90+DR+ inflammatory cytokine producing fibroblasts and Notch3+ fibroblasts in the synovium and determine which fibroblast population(s) most significantly associate with leukocytes (T cells, B cells and macrophages). In Aim 3 we activate synovial fibroblast lines with inflammatory cytokines that are found in RA, in the presence or absence of Notch ligands. We use flow cytometry, RNA-seq, LDA, and trajectory analyses to compare fibroblast cell states induced in vitro with those found in the synovium in RA. Then, we extend the Notch gradient concept from fibroblast differentiation to how fibroblast-derived Notch ligands activate attached T cell in organoids. Finally, in Aim 4, we determine if targeted, conditional disruption of Notch signaling in fibroblasts or targeted conditional deletion of Notch ligands in fibroblasts prevents inflammatory arthritis in mouse models. Together, these studies will advance our knowledge of how fibroblasts differentiate in RA to become drivers of inflammation and pathology in chronically inflamed synovial tissues, and how they might be targeted therapeutically in murine models.