Pulmonarom® promotes TLR-related and interferon-related antiviral responses during influenza A (H1N1 and H3N2) infection in human monocyte-derived dendritic cells

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Background Bacterial lysates have been associated with improved host defense against respiratory infections; however, their mechanisms of action remain incompletely understood. Based on our previous in vitro work showing that Pulmonarom ® activates human monocyte-derived dendritic cells (moDCs) and induces TLR- and cytokine-related responses consistent with innate immune activation, we evaluated its immunomodulatory effect during influenza A virus infection using two in vitro conditions: pre-exposure and post-infection treatment. Materials and methods Human monocytes obtained from buffy coats were differentiated into moDCs. Lyophilized Pulmonarom ® was quantified as total protein and evaluated at 0.01, 0.1, or 0.5 µg/mL in 24-well cultures containing 1 × 10 6 moDCs in 1 mL medium per well. In the pre-exposure condition, moDCs were incubated with Pulmonarom ® for 1 h before infection with influenza A/H1N1 or A/H3N2 virus. In the post-infection treatment condition, Pulmonarom ® was added after the 1-h infection period. TLRs and cytokine expression were evaluated by flow cytometry. Changes in hemagglutinating activity were evaluated by hemagglutination inhibition assay and virus infectivity was evaluated with a MDCK plaque cell assay. Results Under both pre-exposure and post-infection treatment conditions, Pulmonarom ® enhanced MHC class II expression and improved cell viability following infection with either A/H1N1 or A/H3N2 virus, consistent with moDC activation. In hemagglutination inhibition assays, Pulmonarom ® was associated with reduced hemagglutinating activity in culture supernatants. Similarly, plaque infectivity assays demonstrated that Pulmonarom ® , administered either before or after infection, decreased the number of lysed cells within plaques, with a more pronounced effect observed under post-infection treatment. These immunomodulatory changes were accompanied by increased cytokine secretion and the induction of type I interferon-related responses. Conclusions Pulmonarom ® modulated human moDC responses during in vitro infection with influenza A/H1N1 and A/H3N2 virus. These findings extend our previous observations on Pulmonarom ® -induced innate immune activation in human dendritic cells and support further analysis of this bacterial lysate as a host-directed immunomodulatory strategy. Future studies should be evaluated to determine infectious viral titers, TLRs dependency, dendritic-cell maturation markers, and downstream T-cell responses; using plaque assay with sensible cells, receptor-blocking approaches, expanded maturation and characterization panels, and functional co-culture assays.

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