A Single-Cell Woodchuck Liver Atlas Identifies Healthy and Disease-related Cellular Programs Conserved in Human [spatial transcriptomics]

Background: The eastern woodchuck (Marmota monax), which can be naturally infected with woodchuck hepatitis virus (WHV), has served as a model for aspects of human hepatitis B virus (HBV) infection, including the establishment of chronic infection and progression from chronic hepatitis to liver cancer. However, the cellular landscape of the woodchuck liver and its parallels to HBV infection remain uncharacterized. Methods: We present a woodchuck single-cell and spatial transcriptomic atlas in health and chronic WHV infection, with a characterization of cell-type and infection-driven processes in hepatic (Healthy: 52,024 cells; Infected: 40,810 cells; n=8 each) and peripheral blood mononuclear cells (Healthy: 25,314 cells, n=7; Infected: 19,518 cells, n=8). We further examined shared WHV–HBV disease pathways transcriptionally and assessed woodchuck liver immune responses functionally using precision-cut woodchuck liver slice stimulation. Results: We applied our generated atlas and found hepatic cellular and immune diversity in woodchuck liver was comparable to human livers. We found that immune cells in the PMA/IONO stimulated PCLS displayed a type I inflammatory response as expected, reinforcing our annotations. Our atlas further demonstrated transcriptional and cellular similarities between the HBV and WHV infected liver, including the activation of dendritic cells in the periportal region of the infected liver, and a restructuring of the T cell compartment in WHV infection from memory towards exhaustion, a hallmark of human HBV. Conclusions: We present a multi-omic atlas of healthy, diseased, and ex vivo–stimulated woodchuck liver. This work identified shared WHV–HBV pathological processes, reinforces the value of this preclinical model and provides a resource to advance HBV pathogenesis studies and therapeutic development. Overall design: Unbiased spatial transcriptomics (Visium) was applied to ascribe a geographical pattern of the different cell types in the woodchuck liver.