LSC Abstract – Extracellular vesicle release by macrophages during infection with non-typeable Haemophilus influenzae

Introduction: Non-typeable Haemophilus influenzae (NTHi) colonizes the upper airways of most healthy adults and invades the lower airways when innate immunity is compromised. Control over infection is mainly conferred by alveolar macrophages and involves sound intercellular communication, e.g. mediated by extracellular vesicles (EVs). Such EVs, derived from macrophages infected with intracellular pathogens, have been shown to induce pro-inflammatory responses (Bhatnagar et al, 2007). Here we investigated whether macrophages also release immunostimulatory EVs in response to NTHi. Methods: EVs from infected or heat-inactivated (hi) NTHi-stimulated Thp1 macrophages (THP1) were assessed by flow cytometry to determine release kinetics. Moreover, EVs were isolated by ultracentrifugation (UC) for characterization by electron microscopy (EM), western blotting (WB) and the functional activity was assessed. Results: Flow cytometric analysis revealed a dose- and time-dependent release of CD63 + /CD81 + -EVs which peaked at six hours after NTHi (10-fold) or hi-NTHi (6-fold) challenge. EM and WB confirmed the presence of vesicles in the medium. Finally, EVs from both conditions both induced a TNF-α response, which was comparable to an LPS-induced response. Conclusion: Both infection with NTHi and stimulation with hi-NTHi led to an enhanced EV-release. The vesicles released during infection possessed a strong pro-inflammatory character; however, we cannot exclude a contribution from NTHi-derived outer membrane vesicles. Yet, EVs from hi-NTHi stimulated cells possess a similar pro-inflammatory character, suggesting that the pro-inflammatory response is mainly due to macrophage-derived EVs.