Role of fibroblastic stromal cells and notch signaling in tissue inflammation in RA and SLE

成纤维细胞在终末器官纤维化中的作用已经确定，但对其在慢性纤维化中的作用的洞察 周围组织中的炎症性疾病，如类风湿性关节炎(RA)和狼疮性肾炎(LN)仍然存在 正在浮现。我们在RA滑膜组织中发现了高度扩张的炎性成纤维细胞亚群。它 占类风湿性关节炎滑膜成纤维细胞的50%，但在骨性关节炎(OA)中是一个罕见的群体。这个 CD90(Thy1)、HLA-DR的高表达和IL-6、IL-6的产生是扩大人群的显著特征 很多趋化因子。我们推测这些CD90+DR+成纤维细胞是直接通过 分泌炎症因子并间接通过招募和激活白细胞维持慢性 发炎。当分析来自RA/SLE加速药物伙伴关系的单细胞RNA-SEQ数据时 (AMP)，我们发现滑膜内衬里和下层成纤维细胞的标记并不是绝对的。 而是在轨迹分析中代表了基因表达的梯度。我们发现这个转录产物 梯度对应于从血管发出的滑膜的解剖空间梯度。我们的数据 提示Notch信号是从血管周围的成纤维细胞开始的梯度的主要驱动因素 在表达Notch3的次线隔间中。当我们将活动性狼疮性肾炎的成纤维细胞聚集在一起时 对于来自RA滑膜的成纤维细胞，我们鉴定出Notch3+成纤维细胞的共同簇也表达Jag1。 两种疾病都有。 在这里，我们希望确定是否是Notch 3信号在成纤维细胞上特异性地驱动空间 成纤维细胞分化的图案化和亚标化。为了做到这一点，在目标1中，我们使用混合细胞有机体和 血管内皮细胞和成纤维细胞比较Notch3缺陷的空间构型和分化 与WT成纤维细胞相比。在目标2中，我们确定了CD90+DR+炎症细胞因子的位置 狼疮性肾炎和狼疮性肾炎滑膜和肾脏中产生成纤维细胞和Notch3+成纤维细胞 确定哪个成纤维细胞群(S)与白细胞(T、B和巨噬细胞)最相关。 在目标3中，我们进一步用炎症细胞因子激活滑膜和肾脏来源的成纤维细胞系 存在或不存在Notch配体。我们使用流式细胞术、rna-seq、LDA和轨迹分析来 比较体外诱导的成纤维细胞状态与类风湿性关节炎滑膜和狼疮性肾炎肾脏中的成纤维细胞状态。 然后，我们将Notch梯度的概念从成纤维细胞扩展到邻近的白细胞，通过确定 成纤维细胞来源的Notch配体激活器官中附着的T细胞。最后，在目标4中，我们确定是否 靶向、条件性缺失成纤维细胞中的Notch信号或靶向条件性缺失Notch配体 在小鼠模型中，成纤维细胞可预防炎症性关节炎。总之，这些研究将推动我们的 了解成纤维细胞如何分化并成为慢性前列腺癌炎症和病理的驱动因素 发炎的人体组织，以及它们可能如何成为治疗的靶点。

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) and lupus nephritis (LN) is still emerging. We identified a highly expanded inflammatory subpopulation of fibroblasts in 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), HLA-DR and production of IL-6 and many chemokines. We hypothesize that these CD90+DR+ fibroblasts are key in driving inflammation directly by secretion of 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 in the sublining compartment that express Notch3. When we clustered fibroblasts from active lupus nephritis with fibroblasts from RA synovium, we identified co-clusters of Notch3+ fibroblasts that also express Jag1 in both diseases. Here, we wish to determine if it is Notch 3 signaling on fibroblasts that specifically drives spatial pattering and sublining fibroblast differentiation. 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 WT fibroblasts. In Aim 2, we determine the location of the CD90+DR+ inflammatory cytokine producing fibroblasts and Notch3+ fibroblasts in the synovium and in the kidney in lupus nephritis and determine which fibroblast population(s) most significantly associate with leukocytes (T, B and macrophage). In Aim 3 we further activate synovial and kidney-derived fibroblast lines with inflammatory cytokines in the presence or absence of Notch ligands. We use flow cytometry, RNA-seq, LDA, and trajectory analysis to compare fibroblast cell states induced in vitro with those found in synovium in RA and kidney in lupus nephritis. Then, we extend the Notch gradient concept from fibroblasts to adjacent leukocytes by determining if fibroblast-derived Notch ligands activate attached T cell in organoids. Finally, in Aim 4, we determine if targeted, conditional deletion of Notch signaling in fibroblasts or targeted conditional deletion of Notch ligands on fibroblasts prevents inflammatory arthritis in mouse models. Together, these studies will advance our knowledge of how fibroblasts differentiate and become drivers of inflammation and pathology in chronically inflamed human tissues, and how they might be targeted therapeutically.