Human iPSC-Based Alveolus-on-Chip Reveals Immunometabolic Regulation of Influenza A Virus Infection by Butyrate

Host–microbiota interactions critically shape susceptibility and outcome of viral respiratory infections such as influenza A virus (IAV). The short-chain fatty acid butyrate is a key microbial metabolite with established immunomodulatory properties, yet its pleiotropic effects on viral pathogenesis and immune–metabolic balance remain incompletely understood. Physiologically relevant models that recapitulate the complexity of the alveolar niche remain limited. However, induced pluripotent stem cells (iPSC)-derived cell cultures offer a unique opportunity to investigate these interactions within a structured human microenvironment and to extend them toward patient-specific applications. We developed a multicellular alveolus-on-chip platform derived from isogenic human iPSCs, integrating alveolar type II epithelial cells, endothelial cells, and macrophages under an air–liquid interface and physiological flow. Influenza A virus (IAV) infection was modelled in this system to test the influence of the short-chain fatty acid butyrate on viral replication. Single-cell RNA sequencing and cytokine profiling were used to map transcriptional dynamics and immune–metabolic responses related to viral infection and butyrate treatment. The alveolus-on-chip recapitulated key features of the human distal lung, including epithelial–macrophage crosstalk, barrier integrity, and IAV susceptibility. Macrophage plasticity and epithelial remodeling were central determinants of infection outcome. Butyrate treatment constrained host biosynthetic and metabolic programs exploited by IAV, including ribosome biogenesis, RNA processing, and mitochondrial function, while preserving type I/II interferon signaling. Cytokine profiling further demonstrated reduced IL-1β and TNF-α, elevated IL-10, and increased GM-CSF, suggesting a shift toward regulatory and tissue-supportive immunity. This study establishes an isogenic iPSC-derived alveolus-on-chip as a human-relevant platform to dissect immunometabolic regulation during viral infection. The model reveals a dual role of butyrate in dampening inflammation and metabolic activation while maintaining antiviral defense. Beyond influenza, the approach may serve as a scalable framework for patient-specific modeling of respiratory infections and for assessing nutritional or metabolite-based interventions.