An Exploratory Study on the Pathogenic Role of Faecal Extracellular Vesicles in Metabolic Dysfunction-Associated Steatotic Liver Disease Progression and in Drug-Induced Liver Injury.

DILI patients showed a consistent and distinct bacterial profile in faecal extracellular vesicles characterized by specific microbial changes.

• DILI fEVs showed a decrease in Paraprevotella and an increase in AAP99, Acinetobacter, Actinobacillus, Aerococcus, and Anaeroglobus.
• This pattern was described as 'consistent' across DILI patients, distinguishing them from other groups.
• The bacterial profiling was performed using 16S rDNA analysis of faecal extracellular vesicles.

A higher presence of 16S rDNA was observed in plasma extracellular vesicles from MASLD and DILI patients compared to controls.

• 16S rDNA was detected in plasma EVs, indicating that bacterial-derived vesicles enter systemic circulation.
• This finding was observed in both MASLD and DILI patient groups.
• The result supports the hypothesis that gut-derived bacterial EVs can translocate into the bloodstream and potentially modulate systemic and hepatic immune responses.

HepG2 cells treated with DILI and MASLD F≥3 fEVs showed increased expression of inflammatory and apoptotic markers along with lipid droplet accumulation.

• Treatment with DILI and MASLD F≥3 fEVs increased TLR4, TLR5, IL6, and CASP3 expression in HepG2 cells.
• Accumulation of lipid droplets was observed in HepG2 cells treated with these fEVs.
• MASLD patients were stratified into those without significant fibrosis (F≤2) and those with significant fibrosis (F≥3), with the F≥3 group showing these pro-inflammatory effects.

DILI fEVs enhanced the hepatotoxic impact of diclofenac across multiple cellular pathways in HepG2 cells.

• Co-treatment with DILI fEVs and diclofenac enhanced the response to microbial components as evidenced by increased TLR4 and TLR5 expression.
• The inflammatory response was amplified, with increased IL1B and IL6 expression.
• Lipid droplet accumulation was increased compared to diclofenac treatment alone.
• Mitochondrial dysfunction was enhanced, as indicated by altered expression of OPA1 and DNM1L, genes involved in mitochondrial dynamics.

Faecal extracellular vesicles from MASLD patients with significant fibrosis (F≥3) but not those without significant fibrosis (F≤2) drove pro-inflammatory and steatotic effects in hepatocytes.

• MASLD patients were stratified by fibrosis stage: without significant fibrosis (F≤2) and with significant fibrosis (F≥3).
• Only F≥3 fEVs, along with DILI fEVs, increased TLR4, TLR5, IL6, CASP3 expression and lipid droplet accumulation in HepG2 cells.
• This suggests that fEV-mediated hepatotoxic effects are associated with more advanced fibrosis stages in MASLD.

The study investigated the microbiome profiles of faecal extracellular vesicles in MASLD and DILI as part of an exploratory study design.

• The study was described as 'exploratory' in nature.
• fEVs were isolated from faeces of MASLD patients (stratified as F≤2 and F≥3) and idiosyncratic DILI patients.
• Both 16S rDNA microbiome profiling of fEVs and in vitro functional assays using HepG2 cells were employed.
• The role of gut microbiota-secreted extracellular vesicles was assessed in the context of liver disease progression and drug-induced liver injury.