The Cellular Landscape Underlying the Heterogeneity of Macrophages in Adult Testes During Normal and Diseased Conditions

• Applicant: Dr. Sudhanshu Bhushan
• Subject Area: Reproductive Medicine, Urology
• Term: since 2023
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 529763762

Macrophages are the dominant immune cell population in the adult testis: They are derived from fetal liver precursors or early neonatal monocytes, but are maintained throughout adulthood without replacement from the blood. Our preliminary single-cell RNA sequencing (scRNA-Seq) analyses of CD45+ leukocytes isolated from healthy murine testes revealed several heterogenous testicular macrophage (TM) populations with distinct phenotypes, functions, transcriptional profiles and localization. Such heterogeneity suggests that niche-specific factors induce distinct macrophage-differentiation programs that drive TM subset evolution. Interestingly, under conditions of inflammation or TM-depletion, macrophages derived from blood monocytes occupy the empty testicular niche and differentiate into TM, suggesting that the testicular microenvironment can determine the TM phenotype. Indeed, our scRNA-Seq analysis revealed a distinct subset of interstitial macrophage populations that express insulin-like growth factor-1 — a growth factor that promotes Leydig cell differentiation — and various immunoregulatory molecules that regulate wound and tissue healing. This population could, therefore, be required to resolve inflammation and promote Leydig cell development. Elucidating the microenvironmental interactions that regulate TM development provides a better basis to understand the biological processes driving conditions such as male infertility. As such, this proposal aims: 1) To spatially resolve the testicular niche and the microenvironmental factors that determine TM phenotypes; 2) to elucidate whether infiltrating monocytes replace resident TM under inflammatory conditions; and 3) to understand the role of interstitial macrophage subsets under normal conditions. We will leverage a multi-omics approach based on scRNA-Seq, spatial transcriptomics, and CODEX multiplexed imaging to interrogate cell type-specific, in vivo knockout TM systems. We will combine these assays with in vitro analyses and functional profiling of the testes during homeostasis and under inflammatory conditions. This will allow us to refine the role of niche-specific factors in determining TM phenotypes and hopefully in the future, design suitable interventions for patients with inflammation-driven infertility.

• DFG Programme: Research Grants