Pulmonary Macrophage Transplantation for Pulmonary Alveolar Proteinosis

摘要 在理解GM-CSF如何引导肺损伤后巨噬细胞的特性方面存在显著的差距 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， 临床前数据将不足以证明PMT的安全性足以获得在人体上测试PMT的批准。 长期目标是开发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(S) 在PMT过程中指导AM规范。中心假设是AM规范主要由 由肺泡微环境中的因素包括1)GM-CSF，它使AM特异的DNA调节 可获得的元素；和2)表面活性磷脂衍生脂肪酸，激活PPARγ结合新的 可访问的DNA并将AM从LXR驱动切换到PPARγ驱动的规范(和胆固醇代谢)。 我们的理论基础是，通过展示GM-CSF引导巨噬细胞采用正常的AM转录图谱 在单细胞分辨率下(“大量”研究未能表明这一点)将是确定PMT是安全的关键。指导原则 我们强劲的初步数据，这一假设将通过追求三个具体目标来检验，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 移植、规范和PMT后的命运；以及3)管理AM规范的顺法规体系结构。在……里面 目的1、PMT将不同发育阶段的髓系细胞接种于Csf2raKO小鼠体内，以阻止其分化。 挖掘能够赋予治疗益处的细胞的可塑性。在目标2中，正常造血的PMT 有/没有M-CSF抑制的Csf2raKO(或正常)小鼠的干细胞/祖细胞(HSPC)捐赠者将进行 确定供体存活、增殖、克隆扩增、表型和功能。细胞种群动态将 由单细胞RNA-Seq使用Monocle进行时间跟踪。在目标3中，小鼠或人类的HSPC将是 有或没有GM-CSF和表面活性物质或DPPC的培养和基因调控动态将通过 单细胞RNA-SEQ使用Monocle来确定分化的“轨迹”。DNA元件调节细胞 种群动态将通过SCI-ATAC-SEQ和使用CICERO测量染色质可及性来确定 将DNA调控元件与它们调控的靶基因联系起来。建议的研究具有创新性。 因为它支持开发一种基于恢复AM功能而不是 物理去除表面活性剂(目前的方法)，因为它使用了新的方法、软件和 将指导AM规范的要素确定为“上游调节器”或“下游”目标的统计工具； 这是以前可用的方法所不可能实现的。这项拟议的研究具有重要意义，因为 它将确定肺泡决定因素和转录机制(S)调节AM规范在 PMT，支持PMT作为治疗HPAP儿童的有效、疾病特异性细胞疗法的发展， 并为开发以巨噬细胞为基础的治疗其他肺部疾病的新疗法提供了基础。

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.