Dissecting autoimmune cellular and molecular networks in vitiligo

剖析白癜风的自身免疫细胞和分子网络

• 批准号: 9469066
• 负责人: Manuel Garber
• 金额: $ 41.88万
• 依托单位: UNIV OF MASSACHUSETTS MED SCH WORCESTER
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-09-15 至 2019-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9469066
• 关键词: Address; Affect; Alpha Cell; Appearance; Area; Autoimmune Diseases; Autoimmune Process; Autoimmunity; Bar Codes; Biological Assay; Bulla; Cells; Chromosome Mapping; Code; Complex; Cytotoxic T-Lymphocytes; Data; Dimensions; Disease Progression; Disease model; Equilibrium; FDA approved; Failure; Flow Cytometry; Gene Expression; Gene Expression Profile; Genes; Genetic Transcription; Histologic; Immune; Immunofluorescence Immunologic; Incisional Biopsy; Individual; Inflammation; Inflammatory; Interleukin-10; Knowledge; Lasers; Lesion; Ligands; Liquid substance; Location; Maps; Medical; Methodology; Methods; Microdissection; Microfluidics; Modeling; Molecular; Molecular Profiling; Morbidity - disease rate; Organ; Pathogenesis; Pathway interactions; Patients; Pattern; Phenotype; Pigments; Population; Positioning Attribute; Protocols documentation; Regulatory T-Lymphocyte; Resolution; Sampling; Signal Transduction; Skin; Solid; Structure; Suction; T-Lymphocyte; Techniques; Technology; Testing; Therapeutic; Tissue Sample; Tissues; Vitiligo; base; cell type; disease phenotype; disorder control; human tissue; innovation; insight; melanocyte; protein expression; receptor; reconstruction; single cell analysis; skin disorder; skin lesion; therapy design; tissue processing; transcriptome sequencing; treatment strategy

Abstract: Vitiligo is a disfiguring organ-specific autoimmune disease that affects 1% of the world population, including 3 million individuals in the U.S. alone. There are no FDA-approved medical treatments for vitiligo, and current first-line therapies have marginal efficacy. What controls the spreading of vitiligo? Why is its rate of progression different from patient to patient? These are poorly understood problems. In this project we seek to provide the molecular mechanism that explains these fundamental questions. Our paradigm-shifting hypothesis is that the balance between inflammatory and tolerogenic pathways directly within the skin determines the location of lesions and their rate of progression. Thus, local changes in skin are at the core of vitiligo progression. We propose to test and expand our hypothesis using technological advances that only now make this possible. First, we developed a suction blistering method that provides robust sampling of tissue-infiltrating immune cells without confounding inflammation from blistering itself. Second, we have established a single cell RNA-Seq (scRNA-Seq) methodology and have successfully applied it to blister fluid samples. Third, we propose to develop gridRNA-Seq where we use laser microdissection of incisional biopsies of vitiligo lesions into grids of small cubes of tissue containing 10-20 cells each, representing the three dimensional tissue structure. We then use a low input RNA-Seq protocol to profile the expression level of each cube. The combination of these three technologies put us in a unique position to fundamentally change our approach to study vitiligo. With scRNA-Seq we will find the cell types present within vitiligo lesions and compile gene expression signatures for each of these cells. Using ligand/receptor analysis we will build cell signaling networks to support models of disease progression. By comparing lesional with nonlesional blister samples we will identify aberrant signaling in vitiligo, and in particular how tolerogenic and inflammatory signals differ between lesions and nonlesional skin. Although powerful, scRNA-Seq sequences a cell mixture, and the origin of the cells is lost. To address this limitation, we will develop gridRNA-Seq to profile a “block” of 10-20 cells from a known, demarcated region in the skin. Each block in the grid has a well-characterized phenotype as established by the histological appearance at the location of the block. By integrating cell type specific signatures with bulk RNA-Seq data from gridRNA-Seq we propose to “deconvolve” this mixed expression data to estimate the cell composition of each block as well as its expression. We will then use this information to correlate specific expression and cell type composition changes with disease phenotype. Taken together, our data will directly assess the exact tolerogenic and inflammatory balance of individual cells and its association with disease phenotype and hence both test our original hypothesis as well as provide an unbiased view of vitiligo pathogenesis at the individual cell level.

摘要：白癜风是一种毁容性器官特异性自身免疫性疾病，影响着世界 1% 的人口。 仅在美国就有 300 万人。目前还没有 FDA 批准的白癜风治疗方法。 目前的一线疗法疗效有限。 是什么控制了白癜风的扩散？为什么不同患者的进展速度不同？ 在这个项目中，我们试图提供解释的分子机制。 这些基本问题是炎症和炎症之间的平衡。 皮肤内直接的耐受性通路决定了病变的位置及其进展速度。 因此，皮肤的局部变化是白癜风进展的核心。 我们建议利用现在才使这成为可能的技术进步来测试和扩展我们的假设。 首先，我们开发了一种抽吸起泡方法，可以提供组织浸润免疫细胞的可靠采样 其次，我们建立了单细胞 RNA 测序。 （scRNA-Seq）方法并已成功地将其应用于泡罩液体样品。 开发 gridRNA-Seq，我们使用激光显微切割将白癜风病变的切口活检分成网格 每个包含 10-20 个细胞的小组织立方体，代表三维组织结构。 使用低输入 RNA-Seq 协议来分析每个立方体的表达水平。 这三种技术的结合使我们处于独特的地位，可以从根本上改变我们的方法 通过 scRNA-Seq 研究白癜风，我们将找到白癜风病变中存在的细胞类型并编译基因。 使用配体/受体分析，我们将构建细胞信号传导。 通过比较病变和非病变水泡样本，我们可以建立支持疾病进展模型的网络。 将识别白癜风的异常信号传导，特别是耐受性信号和炎症信号的差异 scRNA-Seq 可以对病变和非病变皮肤之间的细胞混合物及其起源进行测序。 为了解决这个限制，我们将开发 gridRNA-Seq 来分析 10-20 个细胞的“块”。 来自皮肤中已知的、划定的区域。网格中的每个块都有一个明确表征的表型： 通过整合特定细胞类型，根据块位置的组织学外观确定。 来自 gridRNA-Seq 的大量 RNA-Seq 数据的签名，我们建议对这种混合表达数据进行“解卷积” 估计每个块的细胞组成及其表达，然后我们将使用此信息来估计每个块的细胞组成及其表达。 将特异性表达和细胞类型组成的变化与疾病表型联系起来。 数据将直接评估单个细胞的确切耐受性和炎症平衡及其关联 与疾病表型，因此既检验了我们最初的假设，也提供了一个公正的观点 白癜风在个体细胞水平上的发病机制。