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

Abstract 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 TCID 50 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.