Project Summary Tissue-resident macrophages (TRMs) play fundamental roles in tissue homeostasis, immunity, and disease. Thus, unlocking their biology is key to gaining a deeper knowledge of many human pathologies. TRMs are unique from other hematopoietic cells, most of which are comparatively short-lived and continually replenished from the bone marrow. Instead, TRMs form from yolk sac and fetal progenitors and persist into adult life through self- renewal. Over time, and with kinetics specific to each tissue, these fetal-derived TRMs are replaced in most tissues by bone marrow-derived monocytes, which may subsequently acquire a similar transcriptional profile to their embryonic-derived counterparts. However, our understanding of universal factors that regulate TRMs across tissues is limited. Cellular metabolism is one such factor that governs the differentiation trajectories of various immune cell subsets, but how it shapes TRM differentiation, persistence, and function has yet to be studied in detail. We previously identified polyamine metabolism, and its role in the synthesis of the amino acid hypusine as a central axis governing macrophage metabolism and activation. We also showed that hypusine synthesis directs the ability of T cells to take on distinct effector fates. These findings illuminated hypusine as a focal coordinator of immune cell fate and effector programs. However, how hypusine contributes to tissue immunity and TRM maintenance remains unknown. The sole protein to contain hypusine is the translation factor eIF5A, in which a conserved lysine is enzymatically converted to hypusine in a two-step process via spermidine. Hypusinated eIF5A promotes the translation of transcripts with specific sequence properties. Our goal in this proposal is to gain deep understanding of TRM biology in homeostasis and disease by addressing hypusine metabolism. Our central hypothesis is that hypusine regulates the differentiation of monocyte-derived cells into TRMs and/or their maintenance in tissues, and that by targeting hypusine we can modulate macrophages to benefit disease. We base this on our published work and striking preliminary data suggesting that hypusine synthesis controls macrophage tissue-residency across multiple organs. Our approach will add new insight into how short-lived precursor cells develop into long-lived TRMs that carry out functions essential for life. Importantly, it will establish if hypusine synthesis is a tractable route to modulate TRMs in contexts where they influence disease, such as with tumor-associated macrophages and cancer. We will test our central hypothesis by, 1) investigating the role of hypusine synthesis in TRM formation and/or maintenance, 2) probing the mechanisms through which hypusine governs macrophage tissue-residency, and 3) examining whether manipulating hypusine synthesis in macrophages benefits anti-tumor immunity.

PROJECT NARRATIVE Tissue-resident macrophages (TRMs) play fundamental roles in health and disease, thus unlocking the biology of these cells is key to gaining a deeper knowledge of many human pathologies. This proposal’s objective is to understand the role of the amino acid hypusine in the maintenance and function of TRMs. Our approach will add new insight into how TRMs carry out functions essential for life, and will establish if hypusine synthesis is a tractable route to modulate TRMs in contexts where they influence disease, such as with tumor-associated macrophages and cancer.