Decoding the language of inflammation between central nervous system resident immune cells

解码中枢神经系统驻留免疫细胞之间的炎症语言

基本信息

批准号：
10050667
负责人：
Iain Clark
金额：
$ 44.17万
依托单位：
UNIVERSITY OF CALIFORNIA BERKELEY
依托单位国家：
美国
项目类别：
财政年份：
2021
资助国家：
美国
起止时间：
2021-06-07 至 2026-05-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10050667
关键词：
Adopted Alzheimer&apos s Disease Anti-Inflammatory Agents Astrocytes Autoantigens Autoimmune Diseases B-Lymphocytes Bar Codes CNS autoimmune disease CRISPR library CRISPR screen CRISPR/Cas technology Cell Communication Cell surface Cells Central Nervous System Infections Clustered Regularly Interspaced Short Palindromic Repeats Communication Custom Demyelinations Dendritic Cells Detection Disease Disease Pathway Dissection Encapsulated Environmental Risk Factor Equilibrium Equipment Experimental Autoimmune Encephalomyelitis Genes Genetic Genetic Screening Genetic Transcription Genomics Health Homeostasis Image Immune Incubated Individual Infection Infiltration Inflammation Inflammatory Inflammatory Response Injury Language Mediating Methodology Methods Microfluidics Microglia Molecular Multiple Sclerosis Mutation Nerve Degeneration Nervous System Physiology Neuraxis Neurodegenerative Disorders Neurologic Neurons Oils Oligodendroglia Pathogenicity Pathway interactions Peripheral Phase Physiological Process RNA Sequences Rabies Regulation Reporter Resolution Structure T-Lymphocyte Technology Therapeutic Tissues Water base cell type cost disability genome wide screen genome-wide high throughput technology in situ sequencing in vivo indexing invention monocyte mouse model nervous system disorder neuroimmunology novel novel strategies novel therapeutic intervention p65 programs rabies viral tracing recruit response single cell sequencing single-cell RNA sequencing spatial relationship tissue repair tool transcriptome

项目摘要

SUMMARY Interactions between central nervous system (CNS)-resident cells are highly heterogeneous; astrocytes and microglia nourish and protect neurons, while inflammatory subsets drive demyelination and neurodegeneration in neurologic diseases. However, the molecular mechanisms that control CNS-resident immune cell interactions remain mostly unknown because methods for defining the specific cell types, pathways and molecules involved are limited. In this project we apply two novel approaches that we developed to study astrocyte-microglia interactions during inflammation: 1) an in vivo barcoded rabies tracing strategy that analyzes cell connections and transcriptomes with single cell resolution, and 2) a droplet-based platform for genome-wide, unbiased CRISPR/Cas9 screening of interacting cell pairs. These methods provide a unique opportunity to study pathways used by CNS-resident immune cells to communicate with each other and control inflammation and neurodegeneration. We propose to: SPECIFIC AIM 1: Define the transcriptomes of single cells in pro-and anti-inflammatory networks. We will simultaneously sequence connections and single cell transcriptomes of CNS cells in the experimental autoimmune encephalomyelitis (EAE) mouse model of multiple sclerosis (MS). These studies will identify pro- and anti-inflammatory cellular networks and the molecular mechanisms that regulate disease-relevant cell-cell interactions within these networks. SPECIFIC AIM 2: Identify novel astrocyte-microglia interactions using a droplet-based platform for CRISPR/Cas9 forward genetic screens. We will perform unbiased genome-wide screens for genes that participate in microglia-astrocyte crosstalk. We will co-incubate and microfluidically sort millions of picoliter water-in-oil droplets containing single microglia harboring a CRISPR/Cas9 library mutation and single astrocytes carrying a fluorescent reporter. Independent droplets do not mix, providing a powerful platform for the identification of immune interactions mediated by cell surface and soluble molecules.

概括中枢神经系统 (CNS) 驻留细胞之间的相互作用具有高度异质性；星形胶质细胞和小胶质细胞滋养和保护神经元，而炎症亚群则导致脱髓鞘和神经退行性变在神经系统疾病中。然而，控制中枢神经系统驻留免疫细胞的分子机制相互作用仍然大多未知，因为定义特定细胞类型、途径和涉及的分子是有限的。在这个项目中，我们应用了两种我们开发的新方法来研究炎症期间星形胶质细胞-小胶质细胞的相互作用：1）体内条形码狂犬病追踪策略以单细胞分辨率分析细胞连接和转录组，2) 基于液滴的平台对相互作用的细胞对进行全基因组、无偏见的 CRISPR/Cas9 筛选。这些方法提供了独特的有机会研究中枢神经系统驻留免疫细胞用于相互通信和控制的途径炎症和神经变性。我们建议：具体目标 1：定义促炎和抗炎网络中单细胞的转录组。我们将在实验中同时对中枢神经系统细胞的连接和单细胞转录组进行测序多发性硬化症（MS）的自身免疫性脑脊髓炎（EAE）小鼠模型。这些研究将确定亲和抗炎细胞网络以及调节疾病相关细胞间的分子机制这些网络内的相互作用。具体目标 2：使用基于液滴的平台识别新型星形胶质细胞-小胶质细胞相互作用 CRISPR/Cas9 正向遗传筛选。我们将对以下基因进行公正的全基因组筛选：参与小胶质细胞-星形胶质细胞的串扰。我们将共同孵化和微流体分选数百万皮升含有带有 CRISPR/Cas9 文库突变的单个小胶质细胞的油包水滴和单个星形胶质细胞携带荧光报告基因。独立的液滴不会混合，为识别细胞表面和可溶性分子介导的免疫相互作用。