Human and mouse transcriptome profiling identifies cross-species homology of mononuclear phagocytes

项目总结/摘要 单核巨噬细胞（MP）系统在先天性和适应性两个方面都起着重要作用。 免疫力它包括在小鼠中广泛表征的三大类MP：（1）巨噬细胞， 包括肺泡巨噬细胞、朗格汉斯细胞和三种不同亚型的间质巨噬细胞;（2） 组织运输单核细胞;和（3）树突细胞（DC），其分为两种主要类型（DC 1和DC 2）， DC 2可以进一步细分。所有这些MP，除了AM和LC，这是肺和皮肤所特有的， 存在于多个器官中，包括心脏、皮肤、肝脏和肠道。MP亚型表现出明显的分化， 在先天性和适应性免疫过程中工作，几乎没有功能冗余，这意味着， 它们之间的特异性相互作用对于针对病毒、细菌和真菌的最佳免疫应答至关重要 感染.然而，目前，在确定和理解这些问题如何解决方面存在着许多根本性的差距。 MP在人体器官中的功能限制了我们制定疾病预防和治疗策略的能力。 本项目将研究跨物种和跨组织的同源性在细胞，基因表达 功能水平。我们将从多个器官（肺、皮肤及其周围组织）获得新鲜的人类和小鼠组织。 引流淋巴结），并采用三种广泛的方法。首先，我们将使用批量RNA测序 RNA测序（RNA-seq）和单细胞RNA测序（scRNA-seq）来鉴定跨物种和跨组织同源性。 RNA-seq提供测序深度（即，全转录组覆盖），而scRNA-seq提供了能力 确认大量同源MP亚型并检查先前定义的MP内的异质性 亚型因此，生物信息学分析将识别同源MP细胞类型的簇，并将它们对齐。 跨越物种。其次，对于每个确定的簇，我们将确定跨物种保守的基因， 组织，以及那些对于给定的同源MP亚型是独特的，称为标记基因。的结果 这些分析将为人类MP亚型和遗传治疗靶点提供特异性遗传标记。 一般来说，我们将在功能上研究两类关键标记基因： 在人-小鼠MP对应物中保守，（1）在小鼠中已明确定义，但以前没有 在人类中进行了调查;（2）在两个物种中没有明确定义或广泛研究。 第三，我们将对人-鼠MP中鉴定的关键基因进行严格的功能验证 同行这包括（a）体内鼠模型，其使用 转基因和条件性敲除（KO）小鼠;（B）人MP的体外模型系统，包括测定 抗原获得和加工、细胞相互作用和适应性免疫应答的诱导;以及（c） 用细胞水平显微镜制作延时视频，用于功能和形态表征。

Project Summary/Abstract The mononuclear phagocyte (MP) system plays a fundamental role in both innate and adaptive immunity. It includes three broad classes of MPs extensively characterized in the mouse: (1) macrophages, including alveolar macrophages, Langerhans cells, and three distinct subtypes of interstitial macrophages; (2) tissue-trafficking monocytes; and (3) dendritic cells (DCs), which fall into two main types (DC1 and DC2), though DC2 can be further subdivided. All these MPs, except AMs and LCs, which are unique to lung and skin, reside in multiple organs, including the heart, skin, liver, and gut. MP subtypes demonstrate a clear division of labor during innate and adaptive immunity with little to virtually no functional redundancy, which means that specific interactions among them are crucial for optimal immune responses against viral, bacterial, and fungal infections. Currently, however, multiple fundamental gaps for the identification and understanding of how these MPs function in human organs limit our ability to develop prevention and treatment strategies across diseases. This project will investigate cross-species and cross-tissue homologies at the cellular, gene expression and functional levels. We will obtain fresh human and mouse tissue from multiple organs (lung, skin, and their draining lymph nodes), and employ three broad approaches. First, we will use both bulk RNA sequencing (RNA-seq) and single-cell RNA sequencing (scRNA-seq) to identify cross-species and cross-tissue homology. RNA-seq provides sequencing depth (i.e., whole-transcriptome coverage), and scRNA-seq provides the ability to confirm bulk homologous MP subtypes and examine the heterogeneity within previously defined MP subtypes. Thus, bioinformatics analyses will identify clusters of homologous MP cell types and align them across species. Second, for each cluster identified, we will identify genes conserved across species and tissues, and those that are unique to a given homologous MP subtype, termed marker genes. The results of these analyses will provide specific genetic markers for human MP subtypes and genetic treatment targets. Broadly speaking, there are two categories of key marker genes we will functionally investigate: those conserved in human-mouse MP counterparts that (1) have been well-defined in mice, but not previously investigated in their human counterparts; and (2) not well-defined or extensively studied in either species. Third, we will undertake a rigorous functional validation of the key genes identified in human-mouse MP counterparts. This includes (a) in-vivo murine models with selective depletion of specific genes using transgenic and conditional knockout (KO) mice; (b) in vitro model systems for human MPs, including assays for antigen acquisition and processing, cellular interactions, and induction of adaptive immune responses; and (c) create time-lapse videos with cellular-level microscopy for functional and morphological characterization.