Bacterial Nanovesicles as Interkingdom Signaling Moieties Mediating Pain Hypersensitivity

Gut dysbiosis contributes to multiple pathologies, yet the mechanisms of the gut microbiota-mediated influence on systemic and distant responses remain largely elusive. This study aimed to identify the role of nanosized bacterial extracellular vesicles (bEVs) in mediating allodynia, i.e., pain hypersensitivity, in a diet-induced obesity (DIO) gut dysbiosis model. bEVs were enriched from the feces of lean (bEVLean) and DIO (bEVDIO) mice by an approach combining ultracentrifugation and immunoprecipitation and then extensively analyzed for purity and bacterial characteristics. Next, bEVs were injected, either intraplantarly or intravenously, in mice to assess pain sensitivity. Fluorescence-labeled bEVs were injected in mice by enema to assess biodistribution. The effect of bEV on immune cells and inflammation was analyzed by array, immunophenotyping, microscopy, NF-κB activation, and cellular uptake assays. Results showed that bEVDIO administration in wild-type mice replicated the allodynia phenotype observed in DIO mice for both mechanical and thermal stimuli. Importantly, this effect was compromised in TRPA1/TRPV1 double-knockout mice. Biodistribution analyses showed bEV entry into systemic circulation with subsequent localization at distant sites. Multiple analyses revealed that bEVDIO exposure incited systemic inflammation, primarily through modulating the innate immune system. This inflammatory mechanism involved LPS on the bEV surface, activating TLR2- and TLR4-related pathways, as confirmed using TLR2 and TLR4 inhibitors and shaving bEV surface proteins. Interestingly, the enhanced cellular uptake of bEVDIO was contingent on interactions involving LPS and proteins on bEVs and TLR2/TLR4 on monocytes. These findings illuminate the hitherto unexplored role of bEV as pivotal mediators of allodynia and inflammation linked to gut dysbiosis.