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

成纤维基质细胞和 Notch 信号在 RA 和 SLE 组织炎症中的作用

• 批准号: 10598101
• 负责人: Michael B. Brenner
• 金额: $ 61.35万
• 依托单位: BRIGHAM AND WOMEN'S HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-06-15 至 2026-03-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10598101
• 关键词: Abnormal Cell; Acceleration; Affect; Anatomy; Automobile Driving; B-Lymphocytes; Blood Vessels; Cells; Characteristics; Chronic; Cytokine Activation; Data; Degenerative polyarthritis; Development; Discriminant Analysis; Disease; Endothelium; Fibroblasts; Fibrosis; Flow Cytometry; Gene Expression; Genetic Transcription; HLA-DR Antigens; Image; Immune; Immune system; In Vitro; Inflammation; Inflammatory; Inflammatory Arthritis; Interferon Type II; Interferon alpha; Interferons; Interleukin-1 beta; Interleukin-6; Joints; Kidney; Knowledge; Leukocytes; Ligands; Location; Lupus; Lupus Nephritis; Lymphoid Tissue; Macrophage; Mediating; Medicine; NOTCH3 gene; Nephritis; Organ; Organoids; Pathology; Pattern; Peripheral; Play; Population; Production; Rheumatoid Arthritis; Role; Signal Transduction; Stromal Cells; Synovial Membrane; System; Systemic Lupus Erythematosus; T-Lymphocyte; TNF gene; Testing; Tissues; cell type; chemokine; chronic inflammatory disease; cytokine; experimental study; human tissue; in vivo; insight; leukocyte activation; lymph nodes; mouse model; notch protein; novel strategies; prevent; recruit; single-cell RNA sequencing; targeted treatment; transcriptome sequencing; transcriptomics

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.

成纤维细胞在终末器官纤维化中的作用已得到充分证实，但对其在慢性纤维化中的作用的深入了解 外周组织的炎症性疾病，如类风湿性关节炎（RA）和狼疮性肾炎（LN）仍然是 我们在 RA 滑膜组织中发现了高度扩张的成纤维细胞炎症亚群。 RA 中滑膜中所有成纤维细胞的 50% 以上，但在骨关节炎 (OA) 中却很少见。 扩大的群体以 CD90 (Thy1)、HLA-DR 的高表达以及 IL-6 和 IL-6 的产生为特征。 我们认为这些 CD90+DR+ 成纤维细胞是直接驱动炎症的关键。 分泌炎症因子并间接通过招募和激活白细胞来维持慢性 分析来自 RA/SLE Accelerated Medicines Partnership 的单细胞 RNA-seq 数据时。 (AMP) 联盟，我们发现滑膜中的内层和下层成纤维细胞的标记并不是绝对的 – 但我们发现这种转录轨迹代表了基因表达的梯度。 梯度对应于我们的数据的滑膜中的解剖空间梯度。 表明Notch信号传导是从血管周围的成纤维细胞开始的梯度的主要驱动因素 当我们对活动性狼疮肾炎的成纤维细胞进行聚集时，在表达Notch3的下层室中。 通过使用来自 RA 滑膜的成纤维细胞，我们发现了 Notch3+ 成纤维细胞的共簇，它们也表达 Jag1 两种疾病。 在这里，我们希望确定是否是成纤维细胞上的 Notch 3 信号传导专门空间驱动 为了实现这一目标，我们在目标 1 中使用了混合细胞类器官。 内皮小管和成纤维细胞比较 Notch3 缺陷的空间模式和分化 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。 在狼疮性肾炎的滑膜和肾脏中产生成纤维细胞和 Notch3+ 成纤维细胞， 确定哪些成纤维细胞群与白细胞（T、B 和巨噬细胞）最显着相关。 在目标 3 中，我们进一步用炎症细胞因子激活滑膜和肾源性成纤维细胞系。 我们使用流式细胞术、RNA-seq、LDA 和轨迹分析来确定是否存在 Notch 配体。 将体外诱导的成纤维细胞状态与 RA 滑膜和狼疮性肾炎肾脏中发现的成纤维细胞状态进行比较。 然后，我们通过确定是否将 Notch 梯度概念从成纤维细胞扩展到相邻的白细胞 成纤维细胞衍生的 Notch 配体激活类器官中附着的 T 细胞最后，在目标 4 中，我们确定是否。 成纤维细胞中 Notch 信号传导的靶向条件删除或 Notch 配体的靶向条件删除 成纤维细胞可预防小鼠模型中的炎症性关节炎，这些研究将共同​​推进我们的研究。 了解成纤维细胞如何分化并成为慢性炎症和病理的驱动因素 发炎的人体组织，以及如何针对它们进行治疗。