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.
The human respiratory system and skin are the body?s largest interface exposed to ambient air and environmental particles, allergens, and microbes. This grant aims to identify homologous human and mouse mononuclear phagocytes in lung, skin and lymph nodes based on functional and transcriptional profiles. Identifying and characterizing mononuclear phagocytes cross-species homologies and the key genes conserved across species that are involved in their function, permits both basic and translational advances, including a more accurate picture of which mononuclear phagocyte subtypes to pursue in a given human application, how to identify these cell types in humans, and which specific genes or proteins to target in treatment development.