The newly established Junior Research Group for Bacterial Interface Dynamics led by Dr. Cláudia Vilhena has recently received support from two foundations to advance and sediment the research on…

Bacterial and human cells produce extracellular vesicles (EVs) in response to diverse stimuli, e.g., toxins, oxidative stress, nutrient depletion, or high cell density. Here, we describe a cocultivation platform that allows recovery of mixed extracellular vesicles (mix-EVs) produced simultaneously by both cell types. We investigated interactions between Gram-positive and Gram-negative bacteria (Streptococcus pyogenes, Staphylococcus aureus, Escherichia coli, and Neisseria meningitidis) and human peripheral blood mononuclear cells (PBMCs). The production of the mix-EVs population decreased with higher bacterial concentrations. Exposing PBMCs to mix-EVs repressed the general transcriptomic signature, in contrast with a significant upregulation generated by single bacterial-EVs. However, mix-EVs-derived IL-1β upregulation was confirmed at the protein level. Inhibition experiments showed that IL-1β production involved TLR2 and TLR4 signaling, acting through IRAK-1 and TRAF6 related pathways. This approach provides a new platform for the study of EVs at the pathogen–host interface and presents mechanistic insights into the effect of EVs on an infected host.