Immune-competent apical-out airway organoids reveal distinct antiviral strategies of macrophages, neutrophils, and monocytes during influenza infection

The respiratory mucosa is the primary entry site for influenza virus and early antiviral immunity is governed there by interactions between epithelial cells and innate immune cells. However, mechanistic insight into these interactions in a human context is limited. Here, we established an immune-competent apical-out human airway organoid model that enables direct epithelial infection and controlled integration of innate immune cells. The organoids recapitulate key features of the human upper airway epithelium and support productive influenza virus replication. Using defined co-cultures, we uncovered distinct innate immune functions: macrophages suppress viral replication and restrict epithelial spread via production of type I and III interferons; neutrophils reduce extracellular virus levels without limiting epithelial infection, consistent with antiviral clearance mechanisms independent of interferon signaling; and monocytes exert modest, transient antiviral effects. Together, we use a human-relevant platform to define how distinct innate immune cells shape mucosal immunity at the respiratory epithelium, with direct implications for antiviral and vaccine development.