Extracellular vesicles modulate hostmicrobe responses by ligand-dependent inhibition of TLR2 activity

Background: Lactic-acid-bacteria (LABs), including Bifidobacterium and Lactobacillus genera, have been proven beneficial in the maintenance of intestinal homeostasis. Ligation of Toll-like receptors (TLRs) expressed by resident dendritic cells (DC) to cell wall components expressed by LABs contributes to this mechanism of action. Extracellular vesicles (EV), important in cellular communication, originate from a broad range of cell types (including DCs) and can be found in virtually any body fluid. The reported presence of pattern-recognition receptors (including TLRs) on EVs, triggered the hypothesis that EVs can intervene with TLR activity. Method: Heat-inactivated serum-derived EVs were collected using ExoQuick®. Intact human serum (HS), depleted serum (HS-D) and vesicle-containing pellets, reconstituted to the original volume with medium, (HS-EV) were collected. Monocyte-derived dendritic cells (moDC), THP-1 or HEK cells stably transfected with TLR2/TLR6, expressing an NFkB reporter construct were seeded in the presence of HS, HS-D or HS-EVs and stimulated with bacteria, TNFα or specific TLR2 ligands. After 16H NFkB activity (HEK-transfectants, THP-1) or cytokine release (moDC) was measured. Results: Bifidobacterium, in contrast to Lactobacillus strains, induced TLR2 activity which was inhibited by HS or HS-EVs. EVs depletion rescued TLR2 activity. TLR2-heterodimer specific ligands showed that HS-EVs inhibition was TLR2/6 specific. Incubation of bacteria in the presence of HS and HS-EV, in contrast to medium or EV depleted serum, resulted in bacterial aggregation. Both Bifidobacteria and Lactobacilli induced dendritic cell IL-6 and TNFα release, which was either enhanced (Bifidobacteria) or reduced (Lactobacilli) upon EV depletion. Conclusion: EVs modulated TLR2 and moDC responses strain and ligand dependently. Attachment of EVs to bacteria induced bacterial aggregation and either enhanced (Lactobacilli) or reduced (Bifidobacterium) cellular responses.