Single-cell and spatial transcriptomic profiling reveals distinct immune landscapes in murine lungs infected with H1N1 versus H5N1 influenza viruses

Influenza A viruses of divergent pathogenicity elicit distinct host immune responses, yet the underlying cellular and spatial dynamics remain poorly defined. Here, we integrate single-cell RNA sequencing (scRNA-seq) and spatial transcriptomics to systematically compare the immune landscapes in murine lungs following infection with seasonal H1N1 or a highly pathogenic avian influenza H5N1 virus belonging to clade 2.3.2.1. Analysis of 84,162 immune cells identifies 12 distinct cell types, suggesting that H1N1 infection is associated with robust interferon and inflammatory responses, characterized by alveolar macrophage depletion and extensive infiltration of monocytes and neutrophils. In contrast, H5N1 clade 2.3.2.1 infection is characterized by attenuated interferon signaling, delayed monocyte activation, dysregulated neutrophil maturation, and impaired intercellular communication-features that may be distinct from previously characterized H5N1 strains. Spatial mapping indicates distinct distribution and interaction patterns of immune cells between the two infections. Functional validation through myeloid cell depletion supports the critical role of the monocyte-macrophage axis in disease progression. These findings provide a high-resolution atlas of pulmonary immune responses to influenza viruses of differing pathogenicity and highlight cell-type-specific mechanisms of immune modulation.IMPORTANCESeasonal H1N1 influenza virus causes annual epidemics, while highly pathogenic avian H5N1 virus has a high mortality rate and pandemic potential. Understanding why H5N1 causes more severe disease is critical for developing better treatments. In this study, we used advanced single-cell and spatial technologies to create a detailed map of the immune response in the lungs of mice infected with either H1N1 or a clade 2.3.2.1 H5N1 virus. We discovered that H1N1 triggers a strong, well-organized immune response that controls the virus. In contrast, H5N1 infection leads to a disorganized and weakened response where key immune cells fail to activate and communicate properly. These findings suggest that the H5N1 virus may evade and suppress the host's immune system. Our study provides a high-resolution immune atlas and identifies potential targets for new therapies against severe influenza.