Type I IFN stimulates lymph node stromal cells from adult and old mice during a West Nile virus infection

Advanced age is a significant risk factor during viral infection due to an age-associated decline in the immune response. While infection by West Nile virus (WNV) is typically mild, older individuals are at an increased risk of severe neuroinvasive disease. Previous studies have characterized age-associated defects in hematopoietic immune cells during WNV infection that culminate in diminished antiviral immunity. Situated amongst immune cells in the draining lymph node (DLN) are structural networks of nonhematopoietic lymph node stromal cells (LNSCs). LNSCs are comprised of numerous, diverse subsets, whose roles in the coordination of robust immune responses are underappreciated. Like lymphocytes, LNSCs mount tailored responses to viral pathogens and are susceptible to aging. However, the contributions of LNSCs to WNV immunity and immune senescence are unclear. Here, we examine LNSC responses to WNV infection within adult and old DLNs. Acute WNV infection was shown to trigger cellular infiltration and LNSC expansion. Comparatively, aged DLNs exhibited diminished leukocyte accumulation and altered LNSC subset composition. Upon recognition of WNV, adult and old LNSCs were activated in a distinct, type I interferon-driven mechanism that promoted antiviral gene expression. Aged LNSCs were found to constitutively upregulate immediate early response genes, several of which are associated with immune suppression. Collectively, these data suggest LNSCs uniquely respond to WNV infection via a novel activation mechanism. We are the first to report age-associated differences in LNSCs on the population- and gene expression-level during WNV infection. These changes may compromise antiviral immunity, leading to increased WNV disease in older individuals. Overall design: Bulk RNA sequencing (RNA-seq) was performed on adult and old lymph node stromal cells (LNSCs) stimulated with viral inflammatory stimuli for 20 hours to determine differentially expressed transcripts in activated stromal cells. Draining lymph nodes from adult (8-10 weeks) and old (18 month) C57BL/6 mice were digested and plated. LNSCs were then enriched for using CD45-labeled microbeads to magnetically deplete unwanted CD45+ cells. Cultures of adult and old LNSCs were incubated for 20 hours with supernatant from bone marrow derived dendritic cells infected with West Nile Kunjin virus ("BMDC"), recombinant IFNa (2 ng/mL, "Recomb"), or were untreated. RNA was extracted from LNSCs using the RNeasy Mini Kit (QIAGEN) and DNase treated. RNA was quantified using the Qubit™ RNA HS Assay Kit (Invitrogen) on the Quantus Fluorometer (Promega). All RNA samples had a RIN score above 8. Total RNA samples were normalized to 50 ng and libraries were constructed using the Universal Plus mRNA-Seq kit (Tecan), followed by RNA-seq on the Illumina NovaSeq6000 S4 flow cell, obtaining 150bp paired-end reads. Raw RNA-seq reads were assessed using FastQC, aligned to mouse reference genome mm10 with STAR, and quantified using featureCounts. Gene expression was normalized to counts per million units and averaged across sample groups, followed by principal component analysis to assess sample clustering. Differential gene analysis was performed in edgeR.