Programming of Resident Macrophages by the Brain Environment Following Transplantation

项目总结 小胶质细胞是大脑中驻留的巨噬细胞，它的一个显著特点是能够适应变化 在大脑环境中。小胶质细胞状态变化在发育和几乎所有疾病中都会发生，通常与 有害的或有益的功能。更好地理解小胶质细胞状态的调节机制 因此，变化将提高对大脑疾病的理解，并发现新的治疗靶点。几十个 疾病反应状态已被确定，但对小胶质细胞如何在它们之间转换知之甚少。 即使是小胶质细胞的“动态平衡”，也是一种由大脑环境信号主动维持的状态，在一种文化中消失了。 碟子。我们是分离和操纵小胶质细胞的专家，并创造了独特的颅内模型 遗传性小胶质细胞耗竭后小胶质细胞和其他巨噬细胞的移植。移植后， 巨噬细胞植入大脑，在14天内经历基因表达的戏剧性变化。在预赛中 数据，我们在几个时间点采集了移植的巨噬细胞，并通过单细胞RNA测序 (ScRNAseq)，测量了小胶质细胞身份随着时间的推移而逐渐获得。在高度受控的情况下 在活体模型中，我们将生成移植的小胶质细胞的全面指纹 由大脑环境编程，并用它来识别基因、通路、调控网络 很可能要负责任。在目标1A中，我们将捕捉培养的小胶质细胞的环境编程 移植，使用配对的单细胞RNA/ATACseq来识别中间状态，并预测外部 信号、转录因子、受体、通路和网络负责。在目标1B中，我们将比较 供者和宿主性别的所有组合的移植，以确定其在小胶质细胞鉴定中的作用 规格。最后，由于血液中渗透的巨噬细胞可能类似于小胶质细胞，但仍是一种不同的细胞类型， Aim 2将在移植后测量它们的编程，以更好地理解为什么它们不能成为 小胶质细胞。总之，这些目标的完成将填补有关小胶质细胞状态调节的知识空白 和身份，创建新的基础数据资源，并证实R01建议对身份进行因果测试 监管者在这里被揭发。

PROJECT SUMMARY A striking feature of microglia, the brain's resident macrophages, is their ability to adapt in response to changes in the brain environment. Microglial state changes occur in development and nearly all diseases, often linked to harmful or helpful functions. A better understanding of the regulatory mechanisms underlying microglia state change will therefore improve understanding of brain diseases, and uncover new therapeutic targets. Dozens of disease reactive states have been identified, but little is known about how microglia transition between them. Even microglial “homeostasis” is a state actively maintained by brain environmental signals, and lost in a culture dish. We are experts in the isolation and manipulation of microglia, and created a unique model for intracranial transplantation of microglia and other macrophages following genetic microglia depletion. After transplantation, macrophages engraft the brain and over 14 days undergo dramatic changes in gene expression. In preliminary data, we harvested transplanted macrophages at several timepoints, and by single cell RNA sequencing (scRNAseq), measured the progressive acquisition of microglial identity over time. With this highly controlled in vivo model, we will generate a comprehensive fingerprint of how transplanted microglia are programmed by the brain environment, and use it to identify the genes, pathways, regulatory networks likely to be responsible. In aim 1A, we will capture the environmental programming of cultured microglia after transplant, using paired single cell RNA/ATACseq to identify intermediate states, and to predict the external signals, transcription factors, receptors, pathways and networks responsible. In aim 1B, we will compare transplantation with all combinations of donor and host sex, in order to determine its role in microglia identity specification. Finally, since blood infiltrating macrophages can resemble microglia but remain a distinct cell type, aim 2 will measure their programming after transplantation, to better understand why they cannot become microglia. In summary, completion of these aims will fill knowledge gaps about the regulation of microglial state and identity, create a new foundational data resource, and substantiate an R01 proposal to causally test identity regulators uncovered here.