Single-Cell and Spatial Transcriptomics Define Eosinophil Heterogeneity during Influenza A Virus-induced Exacerbation...

Although eosinophils are accepted as transcriptionally heterogeneous in asthma, how allergic inflammation shapes eosinophil 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 Asthma+Flu conditions. We identified three related eosinophil transcriptional states distributed along a disease-associated continuum. Eosinophils in the Flu-only condition preferentially expressed canonical antiviral and interferon-stimulated genes including Isg15, Gbp2, Samhd1, whilst Asthma+Flu eosinophils showed attenuated induction of these programs together with enrichment of genes associated with chemotaxis, adhesion, inflammatory regulation, and metabolic remodeling. Asthma+Flu eosinophils were also enriched for a human asthma-associated EosHigh gene signature. A progressive shift in eosinophils 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 eosinophil abundance, increased eosinophil Alox15 expression in allergic conditions, and disease-specific cellular neighborhoods. Cross-platform analyses identified recurrent predicted interactions between eosinophils and a distinct Alox15+ alveolar macrophage population. Mast cells adopted infection-specific IL-1 and leukocyte-activation transcriptional programs without major compositional changes. Collectively, these findings demonstrate that the T2 microenvironment reshapes both the transcriptional state and spatial organization of lung eosinophils during IAV infection, providing an integrated framework for understanding eosinophil heterogeneity and cellular coordination in the allergic lungs.