Bacterial extracellular vesicles modulate epithelial antiviral responses via macrophage-mediated immunomodulation

Bacterial extracellular vesicles (bEVs) are nano-sized, mostly spherical, double-membrane structures secreted by bacteria throughout their life cycle. In addition to their role in many prokaryotic processes, they can interact with cells of the host immune system, enabling a potential for medical application. This study investigated how bEVs from various pathogenic bacteria modulate human macrophage immune responses and their subsequent ability to protect lung alveolar epithelial cells against SARS-CoV-2 infection.Primary human blood-derived macrophages were stimulated with bEVs derived from Legionella pneumophila (Lp), Klebsiella pneumoniae (Kp), Escherichia coli (Ec), Salmonella Typhimurium (Sal) and Streptococcus pneumoniae (Sp). bEVs from Kp, Ec and Sal strongly induced pro-inflammatory cytokines and interferon-stimulated genes via Toll-like receptor 4-dependent signaling. In contrast, bEVs from Lp or Sp elicited minimal or no immune response. Conditioned media from Kp-, Ec- or Sal- bEV-stimulated macrophages further influenced Calu-3 epithelial cells, leading to the induction of interferon-stimulated genes. Notably, SARS-CoV-2 propagation tended to be reduced in Calu-3 cells pre-stimulated with conditioned media from Kp-bEV- and Ec-bEV-treated macrophages, as demonstrated by decreased infectious virus titers in TCID₅₀ assays.These findings provide new insights into macrophage-bEV interactions, demonstrating that gram-negative Kp- and Ec-derived bEVs induce a potent interferon-dependent antiviral state that can be propagated to lung epithelial cells in vitro. This study highlights a macrophage-driven mechanism of innate immune modulation that extends beyond immune cells to shape antiviral responsiveness in structural cells, providing a conceptual framework for future immunomodulatory or vaccine-oriented strategies.