Single-cell and spatial transcriptomics define eosinophil heterogeneity during influenza A virus-induced exacerbation of fungal allergic asthma in C57BL/6 mice.

Although eosinophils (EOS) are accepted as transcriptionally heterogeneous in asthma, how allergic inflammation shapes EOS states during influenza A virus (IAV) infection remains undefined. We integrated high-resolution single-cell RNA sequencing, spatial transcriptomics, quantitative immunofluorescence imaging, and cell-cell communication analyses to profile murine lungs across naïve, asthma, influenza (flu), and co-morbid asthma and flu (asthma + flu) conditions. We identified 3 related EOS transcriptional states distributed along a disease-associated continuum. EOS in the flu-only condition preferentially expressed canonical antiviral and interferon-stimulated genes, including Isg15, Gbp2, and Samhd1, whereas asthma + flu EOS had attenuated induction of these programs together with enrichment of genes associated with chemotaxis, adhesion, inflammatory regulation, and metabolic remodeling. Asthma + flu EOS were also enriched for a human asthma-associated high EOS gene signature. A progressive shift in EOS from antigen-presenting programs toward chemotactic and tissue remodeling-associated states, as identified by pseudotime analysis, may imply a changing functional emphasis rather than sustained canonical antiviral activation. Spatial profiling confirmed condition-dependent EOS abundance, increased EOS Alox15 expression in allergic conditions, and disease-specific cellular neighborhoods. Cross-platform analyses identified recurrent predicted interactions between EOS and a distinct Alox15+ alveolar macrophage population. Mast cells adopted infection-specific interleukin-1 and leukocyte-activation transcriptional programs without major compositional changes. Collectively, these findings demonstrate that the type 2 microenvironment reshapes both the transcriptional state and spatial organization of lung EOS during IAV infection, providing an integrated framework for understanding EOS heterogeneity and cellular coordination in the allergic lungs.