Project Summary/Abstract Prostate organogenesis starts prior to birth, with extensive tissue growth occurring during puberty in response to androgens after the immune system is fully functional. Androgens are generally considered immune- suppressive, so this suggests contradictory roles for immune cells in the prostate, where an immune suppressive microenvironment is needed as new prostate-specific proteins are generated, yet immune cell functions are required to promote morphogenesis and tissue growth during puberty. Our recent single-cell RNA sequencing analyses of normal mouse and human prostates revealed previously unknown heterogeneity in prostate epithelial cell types. These studies form the foundation for a new direction in which I propose to shift my focus from prostate epithelial cells to investigate the function, developmental origins, and androgen-dependence of immune cells in the prostate. The long-term goal of this research is to investigate immune cell regulation of prostate organogenesis so that we can better understand the underpinnings of benign prostate disease. This application specifically seeks to elucidate the function of macrophages during prostate organogenesis and their potential contribution to BPH progression. Our central hypothesis is that heterogeneous populations of prostate macrophages with distinct cellular origins and tissue-specific properties regulate the organogenesis and function of the prostate, and their dysregulation contributes to BPH. To test our hypothesis, Specific Aim 1 will identify the function and heterogeneity of macrophages in the developing prostate. Specific Aim 2 will determine the cellular origins of macrophages in the developing prostate and in BPH. Specific Aim 3 will identify the androgen signaling requirements of macrophage populations in the developing prostate and in BPH. Under Aim 1, we will conduct immunofluorescence analysis of macrophages in prostate tissues, incorporating high-resolution 3D imaging, live imaging, and data from single-cell RNA sequencing. For Aim 2, we will conduct genetic-lineage tracing studies of macrophages of yolk sac, fetal liver, and bone marrow origin. For Aim 3, we will test the role of AR in prostate macrophages by deleting AR in macrophages during prostate organogenesis and in a mouse model of BPH. This proposal is innovative due to the novel investigation into how immune cells contribute to promoting prostate organogenesis both prior to and during puberty in this androgen-regulated organ; the novel investigation into how AR functions in prostate immune cells to regulate organ morphogenesis and immune suppression during puberty; and the use of innovative technology. The proposed research is significant because successful completion of this proposal will elucidate the in vivo function of macrophages during prostate organogenesis; demonstrate the cellular origin of prostate macrophages across the various stages of prostate organogenesis; and clarify the in vivo role of cell-autonomous AR in macrophages during prostate organogenesis. Importantly, this project will further investigate the role of macrophages in the development of BPH, which has the potential to inform the development of improved treatment strategies for BPH.

Project Narrative This study is relevant to public health because it is focused on generating knowledge on immune cells in the prostate. Results from this study have the potential to inform the development of improved treatment strategies for prostate diseases including benign prostatic hyperplasia (BPH). Thus, the proposed research is relevant to the NIDDK mission of advancing scientific knowledge to improve disease treatment for urologic disease.