Pulmonary Macrophage Transplantation for Pulmonary Alveolar Proteinosis

肺巨噬细胞移植治疗肺泡蛋白沉积症

• 批准号: 9982374
• 负责人: Bruce Colston Trapnell
• 金额: $ 45.97万
• 依托单位: CINCINNATI CHILDRENS HOSP MED CTR
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-05-01 至 2022-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9982374
• 关键词: ATAC-seq; Adopted; Alveolar; Alveolar Macrophages; Architecture; Binding; Biological Markers; Bone Marrow; Bronchoalveolar Lavage; CSF2RA gene; Catabolism; Cell Therapy; Cells; Child; Cholesterol; Cholesterol Homeostasis; Chromatin; Clinical Data; Clinical Research; Clonal Expansion; Computer software; DNA; Data; Data Reporting; Development; Diagnosis; Disease; Elements; Engraftment; Evaluation; Exposure to; Fatty Acids; Fetal Liver; Foundations; Gene Expression Regulation; Genes; Genetic Transcription; Goals; Granulocyte-Macrophage Colony-Stimulating Factor; Granulocyte-Macrophage Colony-Stimulating Factor Receptors; Growth; Health; Hematopoietic stem cells; Histopathology; Homeostasis; Host Defense; Human; Inherited; Kinetics; Knowledge; Lecithin; Link; Lung; Lung diseases; Macrophage Colony-Stimulating Factor; Macrophage Colony-Stimulating Factor Receptor; Measurement; Measures; Messenger RNA; Methods; Mission; Molecular; Mus; Mutation; Myelogenous; Myeloid Cells; Nature; Outcome; PPAR gamma; Pathogenesis; Pathologic; Patients; Pharmacologic Substance; Pharmacology; Pharmacotherapy; Phenotype; Phospholipids; Population Dynamics; Process; Proto-Oncogene Protein c-kit; Public Health; Publishing; Pulmonary Alveolar Proteinosis; Receptor Signaling; Regulator Genes; Regulatory Element; Replacement Therapy; Reporting; Research; Resolution; Respiratory physiology; Role; Safety; Severity of illness; Signal Transduction; Stem Cell Factor; Survanta; Techniques; Testing; Therapeutic; Therapeutic Effect; Time; To specify; Translations; Transplantation; Treatment Efficacy; United States National Institutes of Health; Withdrawal; base; cell type; chromatin remodeling; clinical center; cohort; combinatorial; comparative efficacy; effective therapy; experience; granulocyte; indexing; innovation; macrophage; monocyte; novel; novel therapeutics; pre-clinical; progenitor; programs; protein expression; self-renewal; single-cell RNA sequencing; surfactant; tool; transcription factor; transcriptome; transcriptome sequencing

ABSTRACT There is a significant gap in understanding how GM-CSF directs macrophage specification following pulmonary macrophage transplantation (PMT). This is an important problem, because, without this crucial information, the preclinical data will not adequately demonstrate PMT safety sufficient to gain approval to test PMT in humans. The long-term goal is to develop PMT as therapy for patients with hereditary pulmonary alveolar proteinosis (hPAP) aimed at restoring alveolar macrophage (AM) function. The objective here is to identify mechanism(s) directing AM specification during PMT. The central Hypothesis is that AM specification is determined primarily by factors in the alveolar microenvironment including 1) GM-CSF, which renders AM-specific DNA regulatory elements accessible; and 2) surfactant phospholipid-derived fatty acids, which activate PPARγ to bind newly accessible DNA and switch AMs from LXR-driven to PPARγ-driven specification (and cholesterol metabolism). Our rationale is that by demonstrating GM-CSF directs macrophages to adopt a normal AM transcription profile at single cell resolution (which `bulk' studies failed to show) will be crucial to establish PMT is safe. Guided by our strong preliminary data, this hypothesis will be tested by pursuing three specific aims to determine 1) the relationship between donor cell plasticity and therapeutic efficacy; 2) temporal dynamics of macrophage engraftment, specification, and fate after PMT; and 3) cis-regulatory architecture governing AM specification. In aim 1, myeloid cells of various developmental stages will be administered by PMT to Csf2raKO mice to deter- mine the plasticity of cells capable of conferring therapeutic benefit. In aim 2, PMT of normal hematopoietic stem/progenitor cell (HSPC) donors in Csf2raKO (or normal) mice with/without M-CSF inhibition will be done to determine donor survival, proliferation, clonal expansion, phenotype and function. Cell population dynamics will be tracked temporally by single-cell RNA-seq using Monocle. In aim 3, mouse or human HSPCs will be cultured with/without GM-CSF and surfactant or DPPC and gene regulatory dynamics will be measured by single-cell RNA-seq using Monocle to determine differentiation `trajectories'. DNA elements regulating cell population dynamics will be identified by measuring chromatin accessibility by sci-ATAC-seq and using Cicero to link DNA regulatory elements to the target genes they regulate. The proposed research is innovative because it supports the development of a new therapy for hPAP based on restoring AM function rather than physically removing surfactant (the current approach), and because it uses novel methods, software and statistical tools to identify elements directing AM specification as `upstream regulators' or `downstream' targets, which has not been possible with methods available previously. The proposed research is significant because it will identify the alveolar determinants and transcriptional mechanism(s) regulating AM specification during PMT, support the development of PMT as an effective, disease-specific cell therapy for children with hPAP, and provide a foundation for developing novel macrophage-based therapies for other lung diseases.

摘要 在理解GM-CSF如何指导肺移植后巨噬细胞特化方面存在重大差距。 巨噬细胞移植（PMT）。这是一个重要的问题，因为，如果没有这一关键信息， 临床前数据不能充分证明PMT的安全性，不足以获得批准在人体中测试PMT。 长期目标是发展PMT作为遗传性肺泡蛋白沉积症患者的治疗方法 （hPAP）旨在恢复肺泡巨噬细胞（AM）功能。这里的目标是确定机制 在PMT期间指导AM规范。中心假设是AM规格主要由 通过肺泡微环境中的因素，包括1）GM-CSF，其使AM特异性DNA调节 表面活性剂磷脂衍生的脂肪酸，其激活PPARγ以重新结合 可访问的DNA和开关AM从LXR驱动的到PPARγ驱动的规格（和胆固醇代谢）。 我们的基本原理是，通过证明GM-CSF指导巨噬细胞采用正常的AM转录谱， 在单细胞分辨率（“批量”研究未能显示）将是至关重要的，以建立PMT是安全的。指导 我们强大的初步数据，这一假设将通过追求三个具体目标来测试，以确定1） 供体细胞可塑性与疗效的关系; 2）巨噬细胞的时间动力学 PMT后的植入、特化和命运;和3）控制AM特化的顺式调节结构。在 目的1，将通过PMT向Csf 2 raKO小鼠施用不同发育阶段的骨髓细胞以阻止 挖掘细胞的可塑性，使其具有治疗效果。目的2：正常造血细胞PMT 将在有/无M-CSF抑制的Csf 2 raKO（或正常）小鼠中进行干/祖细胞（HSPC）供体， 确定供体存活、增殖、克隆扩增、表型和功能。细胞群体动力学将 通过使用Monocle的单细胞RNA-seq进行时间跟踪。在目标3中，小鼠或人类HSPC将被 在有/没有GM-CSF和表面活性剂或DPPC的情况下培养，基因调节动态将通过以下方式测量 使用Monocle进行单细胞RNA-seq以确定分化“特异性”。DNA调控元件 将通过sci-ATAC-seq测量染色质可及性并使用Cicero 将DNA调控元件与它们调控的靶基因连接起来。该研究具有创新性 因为它支持开发基于恢复AM功能而不是 物理去除表面活性剂（目前的方法），因为它使用新的方法，软件和 统计工具，以确定指导增材制造规范的要素为“上游调节器”或“下游”目标， 这在以前可用的方法中是不可能的。这项研究意义重大，因为 它将确定肺泡决定因素和转录机制，调节AM特化， PMT，支持PMT作为hPAP儿童有效的疾病特异性细胞疗法的开发， 并为开发用于其他肺部疾病的基于巨噬细胞的新疗法提供基础。