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

基本信息

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
项目类别：
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

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

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项目摘要

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：白癜风是一种毁容器官特异性的自身免疫性疾病，影响了世界人口的1％，仅在美国就包括300万个人。白癜风没有FDA批准的医疗治疗当前的一线疗法具有边际效率。是什么控制着白癜风的扩散？为什么它的进展速度因患者而异？这些知识不足的问题。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。我们的范式转移假设是炎症与直接在皮肤内的耐受性途径决定了病变的位置及其进展速度。那就是皮肤的局部变化是白癜风进展的核心。我们建议使用技术进步来检验和扩展我们的假设，而技术进步仅使这成为可能。首先，我们开发了一种任务泡得的方法，该方法可提供组织浸润的免疫细胞的可靠抽样不会因发泡本身而混淆炎症。其次，我们建立了一个单细胞RNA-seq （SCRNA-SEQ）方法，并成功地将其应用于水泡流体样品。第三，我们建议开发gridrna-seq，在其中使用激光对白癜风病变的露孔活检的激光微调为网格每个包含10-20个细胞的组织小块，代表三维组织结构。然后我们使用低输入RNA-seq协议来介绍每个立方体的表达水平。这三种技术的结合使我们处于从根本上改变我们的方法的独特立场研究白癜风。使用SCRNA-SEQ，我们将发现白癜风病变中存在的细胞类型并编译基因每个细胞的表达签名。使用配体/受体分析，我们将构建细胞信号传导网络支持疾病进展模型。通过将病变与非复位泡样样品进行比较将在白癜风中识别异常信号传导，尤其是耐受性和炎症信号如何不同在病变和非静态皮肤之间。虽然功能强大，但SCRNA-SEQ序列是细胞混合物，而起源细胞的丢失。为了解决此限制，我们将开发Gridrna-Seq以介绍10-20个单元的“块” 来自皮肤中已知的划界区域。网格中的每个块都有一个特征良好的表型通过组织学外观在块的位置建立。通过集成特定的细胞类型来自Gridrna-Seq的散装RNA-seq数据的签名，我们建议“解除”此混合表达数据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将特定表达和细胞类型组成随疾病表型的变化而变化。总的来说，我们的数据将直接评估单个细胞及其关联的确切耐受性和炎症平衡通过疾病表型，因此既检验了我们的原始假设，又提供了公正的视野在单个细胞水平上的白癜风发病机理。