Regulation, function and localization of monocytes in autoimmune tissues

自身免疫的核心挑战是发现驱动病理性免疫的细胞和途径 人类的反应。以往对人体样本的研究受到低维单细胞方法的限制 或被细胞混合物混淆的高维本体方法。通过对数千个单细胞的RNA进行测序 从狼疮肾炎的肾脏中，我们发现了21种独特的免疫细胞状态，其中许多也在滑膜中发现。 RA患者的组织。在项目2中，我们将重点关注我们在狼疮肾炎肾脏中观察到的3种单核细胞状态， 这可能是由巡逻进入肾脏的血液单核细胞引起的。虽然这些单核细胞共享表达 具有先前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性 模块，并不直接映射到这些状态。为了更好地了解这些疾病相关的单核细胞 我们将进行实验来解决三个假设。首先，我们假设单核细胞 成纤维细胞调节狼疮肾炎肾脏的炎症、吞噬作用和组织修复程序 和组织衍生因子。在项目3中，我们将原代单核细胞与活化/炎症细胞共培养。 成纤维细胞与已知的局部致病因子一起，决定细胞功能的变化，包括 吞噬作用、细胞因子分泌、内皮外渗、T/B细胞活化（项目1）和其他 功能协调发展的初步数据显示，成纤维细胞和一种致病因子坏死细胞强烈诱导 单核细胞分化其次，我们假设狼疮单核细胞表达的TF将诱导 疾病相关基因炎症和组织修复程序的分化和表达。我们将 在单核细胞中过表达候选TF（基于它们的表达和与狼疮的遗传关联）， 评估炎症、吞噬作用和分化。初步研究表明，TF过表达是 可行的，影响分化，如果成功，将允许可靠的体外产生分化的细胞。 单核细胞进行功能研究。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 单核细胞、成纤维细胞和病变组织的接近，沿着成纤维细胞诱导的表达， 单核细胞基因程序将反映患者肾脏中单核细胞的细胞-细胞相互作用和功能。使用 自动染色，显微镜和图像分析（与计算系统免疫学核心）， 肾脏切片从多达205临床注释狼疮肾炎患者，我们将可视化和评估共同的， 单核细胞、成纤维细胞、组织结构/病变的定位。初步数据显示， 单核细胞和成纤维细胞以及将我们的成像研究扩展到整个队列的可行性。通过建立在一个更 准确定义人类狼疮性肾炎肾脏中的单核细胞，开发体外研究方法 在人体组织中，我们将确定的起源，分化途径，功能和空间位置， 单核细胞在狼疮肾炎，提供了一个路线图机制的研究，在动物模型的疾病，并确定 操纵单核细胞作为狼疮性肾炎的潜在疗法的方法。

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.