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: 10427147
• 负责人: Michael B. Brenner
• 金额: $ 49.84万
• 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/10427147
• 关键词: Abnormal Cell; 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; 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; base; cell type; chemokine; chronic inflammatory disease; cytokine; experimental study; human tissue; in vivo; insight; leukocyte activation; lymph nodes; macrophage; mouse model; notch protein; novel strategies; prevent; recruit; single cell analysis; 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）和狼疮性肾炎（LN）仍然是 正在浮现我们确定了一个高度膨胀的炎症亚群成纤维细胞在RA滑膜组织。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 扩增的群体的特征在于CD 90（Thy 1）、HLA-DR的高表达和IL-6的产生， 许多趋化因子。我们假设这些CD 90 +DR+成纤维细胞是直接驱动炎症的关键， 炎症因子的分泌，并间接通过募集和激活白细胞，以维持慢性 炎症当分析RA/SLE加速药物合作伙伴关系的单细胞RNA-seq数据时 (AMP)联合体中，我们发现滑膜中衬里和亚衬里成纤维细胞的标记物不是绝对的， 而是在轨迹分析中表示基因表达的梯度。我们发现这种转录 梯度对应于从血管发出的滑膜中的解剖学空间梯度。我们的数据 表明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Notch 3的下层隔室中。当我们把活动性狼疮肾炎的成纤维细胞 对于来自RA滑膜的成纤维细胞，我们鉴定了Notch 3+成纤维细胞的共簇，它们也表达Jag 1， 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信号特异性地驱动空间表达。 成纤维细胞分化的模式化和亚衬化。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WT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， 在狼疮性肾炎的滑膜和肾脏中产生成纤维细胞和Notch 3+成纤维细胞， 确定哪种成纤维细胞群与白细胞（T、B和巨噬细胞）最显著相关。 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， Notch配体的存在或不存在。我们使用流式细胞术，RNA-seq，LDA和轨迹分析， 将体外诱导的成纤维细胞状态与RA滑膜和狼疮肾炎肾脏中发现的成纤维细胞状态进行比较。 然后，我们将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， 成纤维细胞中Notch信号传导的靶向条件性缺失或Notch配体的靶向条件性缺失 预防小鼠模型中的炎性关节炎。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炎症和病理学的驱动因素 发炎的人体组织，以及如何将它们作为治疗目标。