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.

成纤维细胞在末端器官纤维化中的作用已经确定，但对它们在慢性中的作用有见解 外周组织中的炎症性疾病，如类风湿关节炎（RA）和狼疮性肾炎（LN）仍然是 新兴。我们确定了RA滑膜组织中成纤维细胞的高度扩展的炎症亚群。它 在RA中，占所有成纤维细胞的50％，但它是骨关节炎（OA）的罕见人群。这 扩大的人群的区别是CD90（THY1），HLA-DR和IL-6和IL-6的产生的高表达。 许多趋化因子。我们假设这些CD90+ DR+成纤维细胞是直接驱动炎症的关键 通过招募和激活白细胞来间接分泌炎症因子，以保持慢性 炎。从RA/SLE加速药物合作伙伴关系中分析单细胞RNA-seq数据时 （AMP）财团，我们发现，衬里和升华的成纤维细胞的标记不是绝对的 - 而是代表轨迹分析中基因表达的梯度。我们发现这个转录 梯度对应于从血管产生的滑膜中的解剖空间梯度。我们的数据 表明Notch信号传导是梯度的主要驱动力，从血管周围的成纤维细胞开始 在表达Notch3的升华室中。当我们从活性狼疮肾炎中聚集成纤维细胞时 使用RA Syronovium的成纤维细胞，我们确定了Notch3+成纤维细胞的共簇，也表达JAG1在 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，专门驱动空间 图案化和增额成纤维细胞分化。为此，在AIM 1中，我们使用混合细胞器官 内皮块茎和成纤维细胞比较Notch3的空间图案和分化不足 与WT成纤维细胞相比。在AIM 2中，我们确定CD90+ DR+炎症细胞因子的位置 在滑膜炎和肾脏肾炎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种群与白细胞（T，B和巨噬细胞）最显着相关。 在AIM 3中，我们进一步激活了滑源和肾脏衍生的成纤维细胞系，并具有炎症性细胞因子 存在或不存在缺口配体。我们使用流式细胞仪，RNA-SEQ，LDA和轨迹分析 比较成纤维细胞态在体外诱导的细胞状态与狼疮肾炎中的RA和肾脏中的肾脏中发现的成纤维细胞状态。 然后，我们通过确定是否是否将Notch梯度概念从成纤维细胞扩展到相邻的白细胞。 成纤维细胞衍生的凹口配体激活器官中附着的T细胞。最后，在AIM 4中，我们确定是否 有条件地删除成纤维细胞中缺口信号的有条件删除或有针对性的有条件缺失。 在成纤维细胞上可防止小鼠模型中的炎症性关节炎。这些研究将共同​​发展 了解成纤维细胞如何区分和成为慢性感染和病理的驱动因素 发炎的人体组织，以及如何将其靶向历史。