Cathepsin S contributes to influenza-induced lung injury by driving inflammation, promoting apoptosis, and disrupting epithelial barrier integrity.

Influenza virus infection causes significant morbidity and mortality worldwide, with severe cases often driven by excessive host inflammatory responses and disruption of epithelial barrier function. Here, we identified cathepsin S (CTSS), a lysosomal cysteine protease, as a key mediator of influenza-induced lung injury. Influenza virus infection upregulated CTSS in a time-dependent and dose-dependent manner, leading to lysosomal membrane permeabilization and cytoplasmic release of CTSS, which correlated with increased apoptosis and loss of epithelial barrier integrity. Knockdown of CTSS reduced proinflammatory cytokine production, apoptosis, and barrier disruption in A549 cells. Air-liquid interface airway epithelial cultures further validated the essential role of CTSS in preserving epithelial barrier integrity. In vivo, pharmacological inhibition of CTSS alleviated lung inflammation and disease severity in infected mice without reducing viral titers. Tumor necrosis factor-alpha (TNF-α) strongly induced CTSS activation, but failed to restore apoptosis and inflammatory responses in CTSS-knockdown cells, suggesting that CTSS acts downstream of cytokine signaling. These findings reveal a central role for CTSS in linking viral infection, cytokine storm, and epithelial damage and highlight CTSS as a promising target for host-directed therapy against severe influenza.IMPORTANCESevere influenza is often driven by excessive host inflammation and epithelial barrier disruption, yet the molecular mediators connecting viral infection to these pathological processes remain poorly defined. This study identifies cathepsin S (CTSS) as a central driver of influenza-induced epithelial injury, acting downstream of viral replication and cytokine signaling to promote apoptosis, inflammation, and barrier disruption. Using both in vitro and in vivo models, we demonstrate that inhibiting CTSS preserves barrier integrity and attenuates inflammation despite having no beneficial effect on viral clearance. These findings provide mechanistic insight into influenza pathogenesis and support CTSS as a promising target for host-directed interventions to mitigate cytokine storm-driven lung injury in severe respiratory viral infections.