Hepatocyte CD44 in aged mouse liver correlates with immune modulation and fibrotic priming

Liver cancer incidences increase beyond 55 years of age suggesting that molecular and immunological changes accompanying aging contribute critically to tumor initiation. However, the mechanisms linking aging and liver cancer initiation are not well understood. Our study investigates the role of CD44, a transmembrane glycoprotein implicated in cancer development, in aging-associated liver pathophysiology. CD44 expression was analyzed in young and aged mouse livers, with aged livers showing an accumulation of CD44 expressing hepatocytes. Analyses by single nucleus RNA sequencing reveal that CD44 expressing hepatocytes in aged livers exhibit enrichment of immune modulatory genes and interferon associated pathways. Spatial analyses showed that CD44 expression in hepatocytes co-occurs with T-cell neighborhoods exhibiting reduced cytokine expression. Functional assays showed reduced antigen-specific IFN-? production by adoptively transferred, ex vivo activated young CD8+ T cells in the aged liver environment. Finally, aged livers lacking hepatocyte CD44 showed attenuation in immune-associated and fibrosis-related gene signatures, consistent with a role for hepatocyte CD44 in age-associated inflammation and early fibrosis. Our findings identify an aging-associated hepatocyte CD44 state that co-occurs with immune modulation and early fibrotic cues, overall suggestive of an early cancer-initiating niche signature in aged livers. Overall design: Animals were procured from NIA aging colony and housed at SBP Animal facility. After 2 weeks, recombinant AAV8-TBG-gp33DYDDK80 was used to express LCMV antigen Gp33 in a hepatocyte specific manner. Viral suspension was prepared at a dose of 1 million viral genomes per mouse in 100 microL and injected retro-orbitally. After 2 weeks of AAV8 infection, ex vivo activated (with gp33 peptide) Thy1.1+ P14 T cells from young mice were injected at 1 million cells per 100 microlitre sterile T cell media. After 5 days of adoptive T cell transfer, livers were collected and immune cells from 7 young and 7 old mice were combined to enrich for adoptively transferred P14 CD8+ T cells. Thy1.1+ CD8+ T cells were purified by flow sorting and loaded onto 10X scRNAseq chip. The samples included in this submission are Thy1.1+ CD8+ P14 T cells isolated from young and old Gp33-expressing mouse livers.