Viral Infection Induces Subset- and Age-specific Gene Expression Patterns in Lymph Node Stromal Cells

West Nile virus (WNV), a flavivirus endemic to the United States, typically causes asymptomatic infection. However, the probability of severe WNV neurological disease increases in the elderly. The immune response to WNV declines with age due to cell-intrinsic and cell-extrinsic defects in the aged lymphocyte compartment. Within the lymph node (LN), hematopoietic immune cells are in contact with structural networks of nonhematopoietic lymph node stromal cells (LNSCs). LNSCs are a diverse, but rare, population of stromal subsets who orchestrate antiviral immune responses. The impact of aging on LNSC function has not been elucidated. In this study, we seek to define the unknown role of LNSCs during WNV infection within adult and aged LNs. Using mouse models of WNV infection, we found that WNV triggers cellular infiltration and LNSC expansion in adult LNs. However, infected old LNs exhibited reduced cellular accumulation, delayed LNSC proliferation, and altered LNSC subset composition. We established an ex vivo culture system to mimic the infected LN microenvironment and showed that adult and old LNSC activation was driven primarily by sensing type I interferon. Bulk RNA sequencing of adult and old LNSCs revealed that WNV infection triggers global antiviral gene signatures. Old LNSCs were found to constitutively upregulate immediate early response (IER) genes due to the enrichment of IER-expressing LNSC subsets in old LNs. To further probe LNSC transcriptional dynamics during WNV infection, we utilized single-cell RNA sequencing (scRNA-seq) to capture this rare and diverse population. Supporting bulk sequencing data, we found that adult and old LNSCs upregulated genes associated with the response to type I interferon. Old LNSCs downregulated genes involved in metabolic pathways. Data presented here is the first to examine age-dependent responses by LNSCs to WNV infection, and age-dependent single cell LNSC dynamics. Findings from these studies can expand our scientific understanding of viral immunity as well as guide strategies that enhance immune responses within older individuals.