Characterisation of LPS+ bacterial extracellular vesicles along the gut‐hepatic portal vein‐liver axis

Gut microbiome dysbiosis is a major contributing factor to several pathological conditions. However, the mechanistic understanding of the communication between gut microbiota and extra‐intestinal organs remains largely elusive. Extracellular vesicles (EVs), secreted by almost every form of life, including bacteria, could play a critical role in this inter‐kingdom crosstalk and are the focus of present study. Here, we present a novel approach for isolating lipopolysaccharide (LPS)+ bacterial extracellular vesicles (bEV LPS ) from complex biological samples, including faeces, plasma and the liver from lean and diet‐induced obese (DIO) mice. bEV LPS were extensively characterised using nanoparticle tracking analyses, immunogold labelling coupled with transmission electron microscopy, flow cytometry, super‐resolution microscopy and 16S sequencing. In liver tissues, the protein expressions of TLR4 and a few macrophage‐specific biomarkers were assessed by immunohistochemistry, and the gene expressions of inflammation‐related cytokines and their receptors ( n = 89 genes) were measured using a PCR array. Faecal samples from DIO mice revealed a remarkably lower concentration of total EVs but a significantly higher percentage of LPS+ EVs. Interestingly, DIO faecal bEV LPS showed a higher abundance of Proteobacteria by 16S sequencing. Importantly, in DIO mice, a higher number of total EVs and bEV LPS consistently entered the hepatic portal vein and subsequently reached the liver, associated with increased expression of TLR4, macrophage markers (F4/80, CD86 and CD206), cytokines and receptors ( Il1rn , Ccr1 , Cxcl10 , Il2rg and Ccr2 ). Furthermore, a portion of bEV LPS escaped liver and entered the peripheral circulation. In conclusion, bEV could be the key mediator orchestrating various well‐established biological effects induced by gut bacteria on distant organs.