Abstract Rationale Aging markedly increases the risk of severe influenza, yet the underlying cellular mechanisms driving impaired recovery remain poorly defined. Influenza also exhibits a clear sex bias in susceptibility and disease severity. Epidemiological and human challenge studies consistently report that females experience more severe disease than males, suggesting intrinsic biological differences rather than behavioral or social factors. However, how age and sex interact to shape innate immune responses to influenza remains unclear. This study aimed to define how age and sex modulate antiviral and inflammatory responses during influenzas infection. Methods Young (8-12 weeks) and aged (20 months) male and female C57BL/6 mice were intranasally infected with 5 PFU of influenza A/H1N1/PR8 virus. Disease progression was monitored by body weight loss. Lung tissues were collected from male and female mice at days 0,3,7, and 11 post-infection for histopathology, viral RNA quantification, NS1 protein detection, and single-cell RNA sequencing (scRNA-seq). Cell-type specific transcriptional profiles were analyzed to define age- and sex-dependent alterations in immune activation, viral sensing, and resolution pathways. Results Aged mice exhibited significantly greater weight loss, delayed recovery, and persistent pulmonary inflammation compared with young mice. scRNA-seq profiling revealed extensive age-related immune dysregulation across cell types. Aged lungs displayed sustained viral RNA and NS1 protein expression, indicating defective viral clearance. Viral transcripts were broadly distributed among epithelial, endothelial, and myeloid population, with aged neutrophils retaining viral transcripts longer than young counterparts. Aged neutrophils retained viral transcripts longer and failed to activate resolution-associated pathways. Aged monocytes demonstrated reduced Pde4d expression alongside heightened inflammatory gene signatures, while alveolar macrophages displayed sustained antiviral and proinflammatory gene expression instead of reparative programs. Conclusions Aging and sex jointly shape antiviral and inflammatory trajectories during influenza infection. Persistent myeloid-driven inflammation, impaired viral clearance, and sex-specific immune cell activation patterns contribute to differential disease outcomes. These findings identify Pde4d-dependent regulation and myeloid cells functional diversity as key determinants of age- and sex-mediated susceptibility to severe influenza, providing potential therapeutic targets for high-risk populations. This abstract is funded by: Department of Defense, NIH (NIAID)
Kim et al. (Fri,) studied this question.