Regulation, function and localization of monocytes in autoimmune tissues

自身免疫组织中单核细胞的调节、功能和定位

• 批准号: 10598099
• 负责人: Nir Hacohen
• 金额: $ 53.05万
• 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/10598099
• 关键词: Address; Affect; Animal Disease Models; Animal Model; Anti-Inflammatory Agents; Antigen-Antibody Complex; Apoptotic; Autoimmune; Autoimmunity; Automobile Driving; B-Cell Activation; B-Lymphocytes; Cell Communication; Cell physiology; Cells; Clinical; Coculture Techniques; Cytotoxic T-Lymphocytes; DIF factor; Data; Dimensions; Disease; Educational process of instructing; Endothelium; Environmental Risk Factor; Extravasation; FCGR3B gene; Fibroblasts; Generations; Genes; Genetic Risk; Genetic study; Human; Image; Image Analysis; Immune; Immune response; Immunology; In Vitro; Infiltration; Inflammation; Inflammatory; Kidney; Lesion; Link; Location; Lupus; Lupus Nephritis; Lymphocyte; Maps; Measures; Methods; Microscopy; Monitor; Necrosis; Nucleic Acids; Pathologic; Pathology; Pathway interactions; Patients; Phagocytosis; Physiological; RNA; Reagent; Regulation; Resolution; Role; Sampling; Signal Transduction; Stains; Structure; System; Systemic Lupus Erythematosus; T-Cell Activation; Testing; Tissues; Transcript; Virulence Factors; Visualization; Work; cell type; cohort; cytokine; differential expression; exhaustion; experimental study; genetic association; genetic variant; genome wide association study; high dimensionality; human tissue; imaging study; kidney cell; monocyte; novel; overexpression; patient stratification; programs; repaired; risk variant; single-cell RNA sequencing; tissue repair; transcription factor; transcriptome sequencing

A central challenge in autoimmunity is to discover the cells and pathways that drive pathological immune responses in humans. Previous studies of human samples were limited by low-dimensional single cell methods or high-dimensional bulk methods confounded by cell mixtures. By sequencing RNA of thousands of single cells from lupus nephritis kidneys, we found 21 unique immune cell states, many of which were also found in synovial tissue of RA patients. In Project 2, we will focus on 3 monocyte states that we observe in lupus nephritis kidneys, which likely arise from patrolling blood monocytes that enter the kidney. While these monocytes share expressed genes with the previously described M1/M2 spectrum of monocyte states, they express distinct functional modules and do not map directly to those states. To better understand these disease-associated monocyte states, we will perform experiments to address three hypotheses. First, we hypothesize that monocyte inflammation, phagocytosis, and tissue repair programs in lupus nephritis kidneys are regulated by fibroblasts and tissue-derived factors. With Project 3, we will co-culture primary monocytes with activated/inflammatory fibroblasts together with known local pathogenic factors and determine changes in cellular functions, including phagocytosis, cytokine secretion, endothelial extravasation, T/B cell activation (with Project 1) and other functions. Preliminary data show that fibroblasts and a proposed pathogenic factor, necrotic cells, strongly induce monocyte differentiation. Second, we hypothesize that TFs expressed in lupus monocytes will induce differentiation and expression of disease-associated gene inflammation and tissue repair programs. We will overexpress candidate TFs (based on their expression and genetic association with lupus) in monocytes and assess inflammation, phagocytosis, and differentiation. Preliminary studies show that the TF overexpression is feasible, impacts differentiation, and if successful, would allow reliable in vitro generation of differentiated monocytes for functional studies. Third, to address monocyte roles in lupus nephritis kidneys, we hypothesize that the proximity of monocytes, fibroblasts and tissue lesions, along with expression of fibroblast-induced monocyte gene programs, will reflect cell-cell interactions and functions of monocytes in patient kidneys. Using automated staining, microscopy and image analysis (with the Computational Systems Immunology Core) of kidney sections from up to 205 clinically-annotated lupus nephritis patients, we will visualize and assess co- localization of monocytes, fibroblasts, tissue structures/lesions. Preliminary data shows close contacts between monocytes and fibroblasts and feasibility of scaling our imaging studies to the full cohort. By building on a more accurate definition of monocytes in human lupus nephritis kidneys, developing methods to study them in vitro and in human tissues, we will define the origin, differentiation pathways, functions and spatial locations of monocytes in lupus nephritis, provide a roadmap for mechanistic studies in animal models of disease and identify approaches to manipulate monocytes as potential therapies for lupus nephritis.

自身免疫的一个中心挑战是发现驱动病理性免疫的细胞和途径。 人类的反应。以前对人体样本的研究受到低维单细胞方法的限制。 或者被细胞混合物混淆的高维散体方法。通过对数千个单细胞的RNA进行测序 从狼疮性肾炎肾脏中，我们发现了21种独特的免疫细胞状态，其中许多也存在于滑膜中。 类风湿关节炎患者的组织。在项目2中，我们将重点关注狼疮性肾炎肾脏中观察到的三种单核细胞状态， 可能是由巡视进入肾脏的单核细胞引起的。而这些单核细胞分享表达的 具有前面描述的单核细胞状态的M1/M2谱的基因，它们表达不同的功能 模块，并且不直接映射到这些状态。为了更好地了解这些与疾病相关的单核细胞 在美国，我们将进行实验来解决三个假设。首先，我们假设单核细胞 狼疮性肾炎肾脏的炎症、吞噬和组织修复程序由成纤维细胞调节 和组织衍生因子。在项目3中，我们将与活化/炎症的原代单核细胞共培养 成纤维细胞与已知的局部致病因素一起决定细胞功能的变化，包括 吞噬、细胞因子分泌、血管内皮细胞外溢、T/B细胞活化(项目1)和其他 功能。初步数据显示，成纤维细胞和一种公认的致病因素--坏死细胞--强烈地诱导 单核细胞分化。第二，我们假设在狼疮单核细胞中表达的转铁蛋白将诱导 疾病相关基因的分化和表达炎症和组织修复程序。我们会 在单核细胞中过表达候选转录因子(基于它们的表达和与狼疮的遗传关联)和 评估炎症、吞噬和分化。初步研究表明，Tf过表达是 可行，影响分化，如果成功，将允许可靠的体外分化 单核细胞用于功能研究。第三，为了说明单核细胞在狼疮性肾炎肾脏中的作用，我们假设 说明单核细胞、成纤维细胞和组织病变的接近程度，以及成纤维细胞的表达诱导 单核细胞基因程序，将反映患者肾脏中细胞与细胞的相互作用和单核细胞的功能。vbl.使用 自动染色、显微镜和图像分析(以计算系统免疫学为核心) 来自多达205名临床注释的狼疮性肾炎患者的肾脏切片，我们将可视化和评估联合 单核细胞、成纤维细胞、组织结构/损伤的定位。初步数据显示，两人之间有密切接触 单核细胞和成纤维细胞，以及将我们的成像研究扩大到全队列的可行性。通过在更多 狼疮性肾炎肾脏中单核细胞的准确定义及其体外研究方法的建立 在人类组织中，我们将定义它的起源、分化途径、功能和空间位置 单核细胞在狼疮性肾炎中的作用，为疾病动物模型的机制研究和鉴定提供了路线图。 操纵单核细胞作为狼疮性肾炎潜在治疗方法的方法。