Unraveling the Hematopoietic Origins and Functional Heterogeneity of Macrophages

PROJECT SUMMARY Macrophages are highly versatile innate immune cells that are essential for a range of functions, including coordination of inflammatory responses, regeneration, and tissue homeostasis. Heterogeneous subsets of macrophages arise from two primary hematopoietic waves. The primitive wave generates tissue-resident macrophages with specialized functions, whereas the definitive wave generates hematopoietic stem/progenitor cells (HSPCs) that differentiate into traditional circulatory macrophages. Imbalances in macrophage number or activation can lead to significant tissue dysfunction and disease. Unfortunately, the specialized roles of distinct macrophage subsets in immune and non-immune contexts remain poorly understood due to limitations in our ability to isolate and track subsets long-term in vivo. To circumvent these technical challenges, I will capitalize on the advantages of the zebrafish model, specifically their transparency throughout development, to track the dynamics of distinct macrophage subsets from their genesis to destination tissues. This proposal will leverage the transcription factor interferon regulatory factor 8 (Irf8) as a tool to separate macrophage populations based on their hematopoietic origin and will identify their autonomous functions in: (i) establishing tissue macrophage heterogeneity, (ii) mounting responses to challenge, and (iii) maintaining ratios of other blood lineages. Irf8 is required to generate primitive macrophages, but its role in regulating definitive macrophages remains poorly understood. Herein, I identify irf8 expression within a subset of emerging HSPCs, suggesting the presence of distinct irf8+ and irf8- definitive macrophage subsets. As such, I posit that macrophage heterogeneity can be classified by both irf8 expression and hematopoietic origin. I will investigate the functional and transcriptional differences between irf8+ primitive, irf8+ definitive, and irf8- definitive macrophages. In Aim 1, I will determine the heterogeneity of primitive and definitive macrophages across tissues from organogenesis to adulthood, the first longitudinal analysis of tissue macrophage dynamics. To achieve this, I designed a Cre/loxP- based lineage tracing system under the irf8 promoter to label macrophage subsets by their irf8 expression and hematopoietic origin, i.e., primitive or HSPC-derived. Through Aim 1, I will define the dynamics of macrophage subsets in their distribution across tissues, their differential responses to injury or systemic infection, and their unique transcriptional profiles. In Aim 2, I will determine the specialized functions of Irf8 within primitive and definitive macrophages as they relate to the production of macrophages versus other blood cells and colonization in tissues. I will achieve this by rescuing irf8 specifically within irf8+ primitive or irf8+ definitive macrophages in a global irf8 mutant background. By elucidating the ontogeny, heterogeneity, and functional contributions of distinct macrophage subsets, this research will provide new insights into macrophage biology and uncover novel therapeutic targets for manipulating macrophage functions in tissue repair and hematologic diseases.