Mechanistic insights into the role of extracellular vesicles at the bacterial-host interface

Introduction: Bacterial and human cells produce extracellular vesicles (EVs) in response to diverse stimuli, e.g. toxins, oxidative stress, nutrient depletion, or high cell density. EVs function as transport systems for proteins, enzymes, DNA and RNA, thereby contributing to interkingdom communication. Several studies have characterized isolated EVs from both cell types under distinct conditions.

Objectives: There is currently no platform that enables the simultaneous production of bacterial and host EVs within the same environment. We present a method for bacteria-host co-cultivation using the Transwell® insert system, which allows the exchange of solutes between the two cell types whilst preventing their direct contact.

Methods: We investigated both Gram-positive and Gram-negative bacteria, harboring distinct pathogenicity traits: Streptococcus pyogenes, Staphylococcus aureus, Escherichia coli and Neisseria meningitidis. Bacteria were co-incubated with peripheral blood mononuclear cells (PBMCs) isolated from healthy human donors. The co-cultivation allowed recovery of the shared supernatant and further isolation and characterization of the mixed pool of bacterial and host extracellular vesicles (mix-EVs).

Results: Dynamic light scattering microscopy revealed a negative correlation between the bacterial concentration and the formation of mix-EVs. Exposing healthy human PBMCs to mix-EVs led to an increased expression of pro-inflammatory cytokines (e.g. IL-6, TNF-α) and several chemokines (e.g. MIP-1a, CCL2, CCL5). Additionally, the transcriptome profile of the PBMCs after exposure to mix-EVs revealed an enrichment of IL-1β transcripts, which was further confirmed at the protein level. By utilizing toll-like receptor pathway specific inhibitors, we could show that this increased expression of IL-1β is mediated by IRAK and TRAF6 related pathways.

Conclusions: This work 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.